Miseq/Miseq FGX System: Installationguide

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The document provides guidance on installing, setting up, testing and verifying the MiSeq/MiSeq FGx sequencing system. It details the components, installation process, setup and configuration steps, and quality control tests to ensure proper functioning.

The main components include the sequencing instrument, computer, flow cell, reagent cartridge, chiller and other accessories. It provides an inventory of required items for setup.

The installation steps include uncrating, leveling and positioning the instrument, installing electrical and network connections, setting up the computer environment, loading software and performing initial tests.

MiSeq/MiSeq FGx System

InstallationGuide

Document # 15027239 v03 ILLUMINA PROPRIETARY


March 2022 Internal Use Only
FOR RESEARCH USE ONLY
Revision History

Version Date Description of Change

03 March 2022 • Changed super flat beaded flow cell PN from 15031390S to
15031390.
• Removed Superman as an MTS password.
• Removed password for Windows to reduce security risk.
• Updated name of sample sheet under Set Up a Run Using Local
Run Manager.
• Updated section Record the Software and Firmware Versions:
changed section title to Record the Software Information; revised text
to be more generic; added instruction for FSE to refer to IQ/OQ
for required information; removed instruction to take screen
capture.
• Added new section, After Leaving the Customer Site.
• Updated section Equipment Required for Setup and Testing:
Changed section name to Items Required; added EDS wristband
and cable ties; changed Flow cell, beaded to Flow cell, super flat
beaded; deleted separate rows for beaded and wash flow cells.
Added note that beaded and wash flow cells are in the MiSeq
alignment toolkit.
• Revised UPS installation instructions under Install the Electrical and
Network Connections. Changed network cable to Ethernet cable.
• Added instructions for chilled wash tray to section Check the Chiller
TEC Temperature.
• Moved section Set Up the Computer Environment to end of Setup
chapter.
• Added section to reference optional MiSeq IQOQ.
• Updated Monitor and Analyze the Run on page 76:
• Analyzing the Run section: added step to verify that the
verification run was uploaded to BaseSpace under the correct
instrument serial number.

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ii
Version Date Description of Change

02 April 2018 • Updated these procedures :


• Adjust Lane Center procedure: Select camera 2, channel G. Also
changed screen captures from yellow to green.
• Z-Motor Rough Position Adjustment.
• Revised the section Run BoltPAC or the Full Optics Test.
• Section heading changed to Test the Optics. Instructions
reorganized based on which test is run: Full Optics Test or
BoltPAC.
• Subsection Determine the Best Z Value –0.035 changed because
the software bug in MCS/MiSeq FGx Control Software has been
fixed. No longer need to subtract 0.035 to determine best Z
value prior to running the full optics test.
• New step added to section Analyze the Full Optics Test Results. Step
3 describes what to do if the bottom-top Z-offset is out of spec.
• Verification chapter updated as follows:
• Revised this section to accommodate separate instructions for
using MiSeq Reporter and Local Run Manager to perform the
verification run.
• Changed the word validation to verification.
• Changed run cycles from 200 (2 x 100) to 2 x 75.
• Added analysis guidance to compare output to published
specifications.
• Saturated pixels and the section, Full Optics Test Results
(BoltPAC.exe):
• Removed: If the BoltPAC results file indicates that the number
of saturated pixels is > 1000, decrease the exposure even more
for the saturated channels.
• Removed Saturated pixels section of screen capture Figure 30,
and annotation B.
• Added: Disregard saturation errors and warnings in script
results and the control software.
• Section Chromstism Results, removed this note: If the number of
saturated pixels for a particular channel is > 1000, lower the
exposure and repeat BoltPAC (displayed by channel under
Saturated Pixels in the report file).
• Changed all references to the installation checklist to IQ/OQ. The
MiSeq/MiSeq FGx Installation Checklist (15027242) is no longer
used.
• Updated keyboard and mouse part number from 15010839 to
20018455.
• Changed type of water required for MiSeq-RUO from deionized to
laboratory-grade.

iii MiSeq/MiSeq FGx Installation Guide


Document # 15027239 v03 Current as of March 2022
Revision History
Version Date Description of Change

01 August 2016 • Corrected super flat beaded flow cell part number to 15031390
(was 15031390S).
• Added procedure for bug in MCS software (finding best focus Z -
0.035) prior to running Full Optics Test
• Changed install workflow to improve efficiency and reduce time
to install (see summary of workflow in section "Installation
Workflow").
• Corrected all instances of 0.002 - 0.005 to 0.002 - 0.0005
• Changed instructions to run the MCS Thermal Ramp Test instead
of using MTS to check the flow cell TEC.
• Made the instructions for using the OTT more flexible.

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iv
Version Date Description of Change

J January 2015 Revised the document so that it can be used for both MiSeq and
MiSeq FGx installations.
Document title: changed to MiSeq/MiSeq FGx Installation Guide
MiSeq FGx-specific information added to these sections:
• Chapter 1:
• About this Guide
• Logins and Passwords
• Equipment Required for Setup and Testing
• About the PhiX Qualification Run (new section added to
Chapter 1; also in Chapter 4)
• Chapter 2: MiSeq/MiSeq FGx Safety
• Chapter 3:
• Inspect the Lab
• Inventory and Inspect the System Components
• Install the Electrical and Network Connections
• Follow the First-Time Setup Screens
• Set Up the Computer Environment
• Final Set Up Activities (also reordered the steps).
• Chapter 4:
• Prepare the PhiX Library
• Start the Run
• Perform a Post-Run Wash
Updated the following information:
• Appendix D: Field Service Repair Kit section: updated the reagent
valve listed the v3 valve.
• Chapters 1 and 4: Table 3 and table 8: Updated the v3 MiSeq
Reagent Kit part number from 15043762 to 15046996 (kit for
internal use only by FSEs/FASs).
• Chapter 1:
• Installation and Qualification Workflow section: swapped the
position of the last two blue rectangles to Perform final installation
tasks followed by Train customer.
• Software Required section: updated the list to include only the
software used by the FSE/FAS to install and service the
instrument.
• Chapter 3:
• Inspect the Lab section: removed the list of laboratory
requirements.
• Combined the instructions under Remove the Protective Cover
with Prepare the Reagent Compartment.
• Removed the section titled: Perform the Remaining Tests.
Instead, the content that was cross referenced by this section
now appears in sequential order.
• Renamed chapters 5, 6 and 7, and reorganized the content. No
changes to the content. Content now organized as Chapter 5:
Adjustment Procedures; Chapter 6: Using MTS; and Chapter 7:
Reagent Delivery.
• Changed the reagent delivery test from manual to using the VCL
function in MTS.
• Updated the procedure Camera 2 Center Adjustment based on the
image now generated by the BoltCameraCenteringD50.exe script.

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Document # 15027239 v03 Current as of March 2022
Revision History
Version Date Description of Change

I May 2014 • Qualification Run: added back the step removed in error from rev
H (step 2, page 44: combine 5 ul of 0.2N NaOH with 5 ul of 4 nM
PhiX library).
• Removed references to the quick reference cards. No longer
shipped with the instrument.
• Revised the appendix, Returning a MiSeq to ILMN, to be more
generic. Also changed the title to Shipping a MiSeq to facilitate
reuse in other documents.
• Chapter 3 changes:
• Removed information under "Inspect the Lab" in Chapter 3.
Readers are now instructed to see the MiSeq Site Preparation
Guide for this information.
• Revised instrument set up instructions in chapter 3. Rev H
included both ship preparation methods (using red lab tape or
white dots). Deleted text and graphics that referred to the red
tape.

H August 2013 • Updated qualification run for v3 chemistry, and the flow cell
description for 19 tiles.
• Updated camera center procedure.
• Added completion of install certification to final tasks.
• Updated art and text to reflect the new appearance of instruments
when removed from the crate.
• Removed the shipping lockdown release video.
• Added extraction tool and level to the alignment toolkit; removed
15027882.
• Updated instrument leveling for the level in the toolkit.

G June 2013 • Added Tween 20 to post run wash list of items required.
• Added reference to WI, Uncrating an Illumin MiSeq or MiSeqDx
System (part # 15041141).
• Updated operating temperature to match site prep guide rev C
(15027615).
• Changed instructions for leveling the instrument.
• Updated the section "About the Instrument Control Computer"
• Updated instructions for moving to center of flow cell in "Adjust
the Rough Z-Motor Position"
• Removed reference to spec under "Determine the Best Focus Z-
Position"
• Added screen capture of focal plane Z position to annotated "Full
Optics Test Results".
• Updated environmental considerations.
• Renamed the System Setup and Qualification chapter to
Instrument Set Up and Testing. Reorganized the content to
improve usability.
• Updated tool kit names and added Fluke meter as separate tool.

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vi
Version Date Description of Change

F February 2013 • Added instructions for checking the LED power (mW) using MTS
to chapter 3.
• Removed Autofocus test information.
• Revised the instructions for the qualification run and post-run
wash. Added example of sample sheet.
• Updated start up screens to MCS 2.1.13.
• Updated DiagConfig file appendix (updated SW version and
removed autofocus test).
• Added chiller thermistor to repair kit.
• Updated MiSeq Alignment Toolkit (new manifold alignment tool
and fluidics wrench).
• Removed the section "What's New with MiSeq II".
• Removed note re bug in MCS for tip/tilt test results.

E December 2012 • Added guidelines for moving the instrument.


• Hot surface warning: max temp changed to 95 deg C.
• Lane center: changed from tile 6 to tiles 2 and 11.
• Specified the use of a super flat beaded flow cell for all inspection
and alignment procedures except those that call for an OTT.
• Updated PhiX preparation for the qualification run to match
15036175 (except for NaOH dilution. Stock concentration is 2.0 N;
not 1.0 N).

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Revision History
Version Date Description of Change

D 6 September • Updated rough Z-stage position instructions based on video.


2012 • Added instructions to update recipe fragment per FSB433 (adds
the MiSeq AutoWash recipe).
• Updated reagent valve part number in MiSeq Repair Toolkit.
• Revised flow cell TEC calibration procedure.
• Updated relevant sections to reflect cut over to MiSeq II (Shock).
• Updated safety and site preparation information.
• Removed instructions for calibrating chiller temperature.
• Updated DiagConfig file appendix.
• Added summary of changes from MiSeq to MiSeq II.
• Added compensator adjustment procedure.
• Miscellaneous edits.
• Removed FSB 428.
• Removed instructions for exiting kiosk mode.
• Updated PhiX concentration and dilution instructions.
• Added an appendix with alignment and inspection specifications.
• Updated appendix for crating the instrument.
• Corrected error in procedure to focus an OTT.
• Chapter 1: fixed -0.17; size of HD changed to 700 GB.
• Chapter 3: Modified instructions for set up of Log On user for
MiSeq Reporter.
• Chapter 4: Added make sure SRE-2 reagent thawed. Changed
location of Experiment Manager for download to SFDC. Changed
9 pM to 12.5 pM under “Load Sample Libraries . . .”
• Removed step to change mirror position for checking illumination
footprint.
• Updated system block diagram and added fluidics system
diagram from training Power Points.
• Added short description of Z-stage travel and the compensator
from training Power Points.
• Updated syringe and reagent valve info in repair kit.
• Updated list of instrument accessories.
• Changed adjustment for rough Z-motor position to use tile 1.
• Updated annotated BoltPAC/Full Optics Test results file.

C 7 May 2012 • Added more clarification to Z-Motor Rough Position Adjustment


procedure.
• Made changes required due to new MCS software (MCS 1.2).
• Updated Illumination Edge check, which is now called
Illumination Footprint.

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Version Date Description of Change

B 27 March 2012 • Updated setup and qualification instructions based on changes in


MCS 1.1.1.
• Removed XY Offset spec from Manual Adjustments chapter.
• Removed instructions for manually adjusting camera rotation.
• Added back “how to check camera centering” to the same section
that includes adjusting camera centering.
• Changed Illumination Uniformity to Illuminated Area.
• Y offset - changed 1 turn = 20 μm to 1 turn = 40 μm
• Changed default Snap current for Green LED to 2000 mA.
• Changed the value used to generate a through-focus model for
MTS
• BoltPAC (14 + actualedgeofslide).
• Updated the MTS qualification activities.
• Added separate section for determining the best focus z-position
using a super flat beaded flow cell.
• Updated Y-position when generating a through-focus image set
to run BoltPAC (actualedgeofslide + 14 mm).
• Updated installation and qualification workflow flowchart.
• Updated MTS flow cell TEC qualification.
• Updated MiSeq toolkit name and contents. Added the generic and
repair toolkits.
• Updated screen captures for flow cell TEC calibration to reflect the
correct temps.
• Updated qualification run to recommended 9 pM PhiX
concentration and new denaturation protocol.
• Added more guidance to Illuminated Area adjustments.
• Removed the word “Service” from the cover. A separate service
guide will be produced.
• Added instructions to change the Green LED snap current before
generating a through-focus image set to: 2000 for a beaded flow
cell; 6500 for an OTT
• Added instructions for changing the Start-Up Option to MCS and
Windows in the Common Procedures appendix.
• Added an appendix that describes how to crate the instrument for
return shipment.
• Added guidelines for X-offset adjustments.
• Chromatism-added note that script results recommend
adjustments in the wrong direction for L1 relative to Camera 1.
Fixed figure 13 - micrometer marks and clarifying text.
• Added info on how to focus bead and OTT images.
• Updated “Determine the Best Focus Z-Position” procedure.
• Added FSB 428 (patch to v1.4) as an appendix. Added step in
instrument qualification section to install the patch.
• Added MiSeq Repair Toolkit contents.
• Removed placeholder for LED calibration. Will not be done in the
field.
• Fixed errors in the Chromatism Adjustment section. Added more
guidance on which direction to turn the micrometer on the L1
adjustment tool.
• Removed extra backslash that was in this path:
D:\MiSeqFirstTimeSetup.xml
• Updated photo of wash tray to the gray version.
• Removed specifications from procedures to avoid future errors
when specs change. Installation checklist is source for specs.
• Updated instructions for generating a through-focus image set
and for running scripts in MTS.

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Revision History
Version Date Description of Change

B 27 March 2012 • Updated flow cell TEC alignment in the lane rotation adjustment
procedure.
• Added reference in Common Procedures chapter regarding work
instruction that contains instructions for logging cases [Logging a
Standard Case in SFDC WI (15004045)] on SFDC Content.
• Changed type of qualification run to 2 x 101.
• Changed the C channel exposure time from 5000 to 200 ms now
that we don’t need to make thru focus image sets using an OTT.
Added the following:
• Info from installation reports such as a table listing Windows-
based tasks (set time zone; sbsuser password expiration,
windows defender and update disabled).
• Rear adjuster to imaging module component table (item 20),
and changed description of items 18 and 19.
• A note that it’s important to remove droplet from tip of PR2
sipper tube during volume check in MCS.
• Note to chromatism results section regarding saturated pixels.
• New style Z-stage to the Z-Motor Rough Position Adjustment
procedure. Also added instructions to check flow cell surface.
• Appendix describing the DiagConfig file.
• Index.
• Networking guidelines.

A 3 October 2011 Initial release.

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xi MiSeq/MiSeq FGx Installation Guide
Document # 15027239 v03 Current as of March 2022
Table of Contents

Revision History ii
Table of Contents xii

Chapter 1 Before You Begin 1


About this Guide 2
About Installation and Verification 4
Logins and Passwords 4
Items Required 4
Software Required 6
About the Instrument Control Computer 7

Chapter 2 Safety 8
Safety Information 9

Chapter 3 Setup 10
Items Required 11
Inspect the Lab 12
Inventory and Inspect the System Components 13
Set Up the Instrument on the Bench 14
Position the Instrument 14
Remove the Shipping Bracket and Release the Shipping Lockdowns 14
Prepare the Reagent Compartment 17
Install the Electrical and Network Connections 18
Level the Instrument 18
Install a Keyboard and Mouse 19
Install Workflow 20
First-time Setup Screens 21
First Time Set Up Screens 22
Run the Motion Tests 26
Check Lane Center and Rotation 27
Focus an OTT 27
Check Lane Center and Rotation 30
Test the Optics 32
Run The Full Optics Test 32
Run BoltPAC.exe 36
About Test Results 38
Determine the Best Focus Z–Position 42
Check the LED Power 44
Illumination Footprint Check 45
Check the Chiller TEC Temperature 49
Record the Software Information 50
Check the Reagent Line Delivery Volumes 51
Set Up the Computer Environment 52

Chapter 4 Verification 54
Verification Run Requirements 55
Prepare the Reagent Cartridge 56
Prepare the PhiX Library 58
Thaw and Mix the Reagents 58
Prepare a Fresh Dilution of NaOH 58
Denature and Dilute the PhiX Control 58
Load the v3 PhiX Library into the Reagent Cartridge 59
Perform a Verification Run Using Local Run Manager 60

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Set Up a Run Using Local Run Manager 60
Clean the Flow Cell 63
Load the Flow Cell 64
Load the Reagents 66
Start the Run —Local Run Manager 67
Monitor and Analyze the Run 68
Perform a Run Using MiSeq Reporter 70
Set Up a Run Using MiSeq Reporter 70
Clean the Flow Cell 71
Load the Flow Cell 72
Load the Reagents 74
Start the Run—MiSeq Reporter 75
Monitor and Analyze the Run 76
Perform a Post-Run Wash 78
Perform a Post-Run Wash 78
Post Run Wash Activities 79

Chapter 5 Final Installation Tasks 80


Final Installation Tasks at the Customer Site 81
After Leaving the Customer Site 82

Appendix A Troubleshooting 84
Imaging Module Components 85
Camera 2 Center Adjustment 87
Check Camera 2 Center 87
Adjust Camera 2 Center 88
Verify the Camera Center Adjustment 90
Camera XY Offset Adjustment 91
Adjust the Y Offset 91
Adjust the X-Offset 93
Verify the XY Offset Adjustment 95
Chromatism Adjustment 96
Run BoltChromatism.exe 96
Adjust L1 97
Compensator Adjustment 99
Flow Cell TEC Thermal Calibration 101
Prepare the Instrument for Flow Cell TEC Calibration 101
Calibrate the Flow Cell TEC 103
Qualify the Flow Cell TEC 105
Illuminated Area Adjustment 107
Prepare the Instrument for Illuminated Area Adjustment 107
Adjust L8 in X 108
Adjust L8 in Y 110
Qualify the Adjustment 112
Lane Center Adjustment 113
Lane Center Adjustment 113
Lane Rotation Adjustment 116
Adjust Lane Rotation 116
Qualify Lane Rotation 118
Check Tip/Tilt 119
Check Tip/Tilt Using MTS 119
Check Tip/Tilt Using the Instrument Control Software 120
Review Tip/Tilt Results 120
Tip/Tilt Adjustment—Camera 2 125
Make Sure that a Tip/Tilt Adjustment is Required 125
Adjust Tip/Tilt 126
Tip/Tilt Adjustment—Camera 1 129
Adjust Tip/Tilt on Camera 1 129
Z-Motor Rough Position Adjustment 131
About Z-Stage Travel and the Compensator 131
Adjust the Rough Z-Motor Position 131
Confirm Focus is on Top of Flow Cell 134
Generate Through Focus Image Sets (Stacks) 136
Move to the Center of a Super Flat Beaded Flow Cell 136

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Table of Contents
Find Best Focus on the Top or Bottom Surface of the Flow Cell 137
Generate the Through Focus Image Stack 139
About MTS 141
Views (Tabs) in Left Pane 142
Image Tab in the Right Pane 143
Running Scripts in MTS 144
Manually Check Reagent Delivery 149
Items Required for Reagent Delivery Test 149
Prime the Instrument 149
Measure Reagent Delivery 150

Appendix B DiagConfig File 152


About the DiagConfig.xml File 153

Appendix C Service Tools 162


Service Tools 163
MiSeq Alignment Toolkit 163
MiSeq Generic Tool Box 166
Field Service Repair Kit 166

Appendix D Alignment and Inspection Specifications 168


Instrument Specifications 169

Appendix E Instrument Returns to Illumina 170


Returning an Instrument to Illumina 171
Ordering the Crate Kit 171
Gather the Instrument Accessories 171
Prepare the Instrument for Crating 172
Crate the Instrument 175

Appendix F Instrument Engineering Drawings 180


Cable Interconnect Diagram for the MiSeq 181
Subsystem Block Diagram for the MiSeq 182
Optical System for the MiSeq 183
Fluidics System for the MiSeq 184

Appendix G Common Procedures 186


Adjust the Clock, Date, and Time (Windows 7) 187
Cleaning the Imaging Module Bench 189
Flow Cell Cleaning, Loading, and Imaging 190
Clean the Flow Cell 190
Load the Flow Cell 192
Imaging Flow Cells 194
Removing and Reinstalling the Instrument Skins 195
Skin Removal 195
Skin Reinstallation 201
Rebooting the Computer 203
Shutting Down the Computer 204
Turning the Instrument On and Off 205

Index 206
Technical Assistance 208

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xv MiSeq/MiSeq FGx Installation Guide
Document # 15027239 v03 Current as of March 2022
  Chapter 1  Before You Begin

Before You Begin

Chapter 1
About this Guide 2
About Installation and Verification 4
About the Instrument Control Computer 7

MiSeq/MiSeq FGx Installation Guide


Document # 15027239 v03 Current as of March 2022
1
Before You Begin
About this Guide

Purpose
This guide describes how to install the MiSeq and MiSeq FGx instruments at customer
sites. It includes instructions for unpacking, setting up, calibrating, and verifying the
instrument.

Intended Audience
This guide is intended for qualified Field Service Engineers (FSEs) and Field Application
Scientists (FASs). Qualified FSEs and FASs have received the appropriate training, and are
authorized to carry out the procedures described in this guide.

Reference Documents
Use these documents to perform the installation. Refer to these documents for instrument
specifications, to record test results, and for other pertinent information.
Table 1 IQ/OQ Required
Instrument IQ/OQ Required

MiSeq • MiSeq/MiSeq FGx Installation Guide (document # 15027239)


• MiSeq System Installation Qualification and Operation Qualification
(document # 15032822)

MiSeq FGx • MiSeq/MiSeq FGx Installation Guide (document # 15027239)


• MiSeq FGx Instrument Installation Qualification and Operation
Qualification (document # 15067076)

Customer receives IQ/OQ certification from Illumina.

Table 2 No IQ/OQ Required (Not Purchased by Customer)


Instrument IQ/OQ Required

MiSeq • MiSeq System Installation Qualification and Operation Qualification


(document # 15032822)
• MiSeq System Installation Certification (document # 15041059)
• MiSeq/MiSeq FGx Installation Guide (document # 15027239)

MiSeq FGx • MiSeq FGx Instrument Installation Qualification and Operation


Qualification (document # 15067076)
• MiSeq FGx Instrument Installation Certification (document #
15058931)
• MiSeq/MiSeq FGx Installation Guide (document # 15027239)

FSE completes and gives customer only the installation certification; customer does not receive
the IQ/OQ.

Additional reference documents for the MiSeq:


} MiSeq System User Guide (document # 15027617)
} MiSeq System Site Prep Guide (document # 15027615)
} MiSeq System Safety and Compliance Guide (document # 15027616)

2 MiSeq/MiSeq FGx Installation Guide


Document # 15027239 v03 Current as of March 2022
About this Guide
Additional reference documents for the MiSeq FGx:

} MiSeq FGx Instrument User Guide (document # 15050524)


} MiSeq FGx Instrument Site Prep Guide (document # 15050525)
} MiSeq FGx Instrument Safety and Compliance Guide (document # 15050819)

MiSeq/MiSeq FGx Installation Guide


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3
Before You Begin
About Installation and Verification
This section contains information about the logins and passwords; and the equipment,
reagents, and software required for system installation and verification.

Logins and Passwords


The preconfigured logins and passwords are listed in this table.

Table 3 Field Service Logins and Passwords


Application Login Password

MiSeq Control sbsuser sbs123


Software
MiSeq FGx Control Two precofigured accounts:
Software
admin@forenseq.uas adminDefaultPwFGx

fas@forenseq.uas fasDefaultPwFGx

Note: Use the fas@forenseq.uas


account only to add population
groups to the system. Do not
use it for anything else.
MiSeq Test Software — password
(MTS)
Windows sbsuser Not listed here to
reduce security risk

Items Required
Besides the accessories included with the instrument, the items listed here are required for
installation. See Verification Run Requirements on page 55 for verification run requirements.
Table 4 Equipment Required
Item Quantity Part Number

ESD wristband with ground strap 1 20000856

Cable ties approximately 5 Not available

Flow cell, super flat beaded (SFBFC) 1 15031390

Fluke Meter, 54-II, or equivalent, calibrated 1 Not available

Wireless keyboard-mouse combo w/ 1 20010844


batteries, (MTS; it is not accessible using the
touch screen)

MiSeq Alignment Toolkit 1 15026398S


NOTE: Includes a wash flow cell and a
beaded flow cell

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Document # 15027239 v03 Current as of March 2022
About Installation and Verification
Item Quantity Part Number

MiSeq Generic Tool Box 1 15030145S

USB (thumb) drive 1 Not available

Type T thermocouples 2 Not available

MiSeq FGx only: Water, DNase-free, RNase- As required Customer supplied


free

Verification Run Requirements


A PhiX run confirms proper hardware and software performance of the instrument. The
PhiX control library provided by Illumina is used for the verification run. Perform a 2 x 75
cycle v3 run using one of these applications (only one is installed on the instrument): Local
Run Manager or MiSeq Reporter. To analyze the run, compare the output to published
specifications.

Table 5 Reagent Kit for MiSeq and MiSeq FGx Runs


Instrument Instrument and Reagent Kit Quantity Part Number

MiSeq MiSeq Reagent Kit version 3, 600 cycles 1 15046996

Table 6 Additional Items Required for a PhiX Verification Run


Item Quantity Part Number

PhiX Control Kit, version 3 (10 μl at 10 nM) 1 tube 15017666

HP3 (2 N NaOH) 1 tube 11324596

Elution Buffer (EBT) 1 tube 15025394


(or 10 mM Tris-Cl, pH 8.5 with 0.1% Tween
20)

PW1, 250 ml (or laboratory-grade water) 1 bottle 11298922

Other Items Required

Alcohol wipes, 70% isopropyl As required Customer supplied

Gloves, disposable, powder-free Customer supplied

Ice bucket with ice 1 Customer supplied

Lab tissue, low-lint As required Customer supplied

Lens paper, 4 x 6 in As required Customer supplied

Lint free lens tissue As required Customer supplied

Microcentrifuge tubes, 1.5 ml to 2 ml 3 Customer supplied

Pipettes and pipette tips (P10 or P20, P200, As required Customer supplied
and P1000)

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5
Before You Begin

Item Quantity Part Number

MiSeq Reporter: PhiXInstallSampleSheet.csv 1 ICE


Local Run Manager: PhiX-verification-run-
MiSeq-RUO-LRM.csv

Tweezers 1 pair Customer supplied

Water, deionized As required Customer supplied

Water bath 1 Customer supplied

Guidelines for Laboratory-Grade Water


Always use laboratory-grade water to perform instrument procedures. Never use tap water.
The following are examples of acceptable water:
} Illumina PW1
} 18 Megohms (MΩ) water
} Milli-Q water
} Super-Q water
} Molecular biology grade water

Software Required
In addition to the software shipped on the instrument, the software listed here are required
for installation. Make sure that the latest versions are installed.
NOTE
Consult with the customer and determine their requirements before making any software
changes. Do not automatically install Local Run Manager on instruments with MiSeq
Reporter. The customer may require MiSeq Reporter. If the instrument has Local Run
Manager installed, but the customer requires MiSeq Reporter, downgrade the instrument to
MiSeq Reporter.

Software Required

MiSeq Test Software (MTS)

Scripts for MTS (latest version)

Sequencing Analysis Viewer (SAV)

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About the Instrument Control Computer
About the Instrument Control Computer

Single Hard Drive Configuration


The computer that controls the instrument is a single board system with one 700 GB hard
drive. The hard drive is partitioned into 2 parts: C: (146 GB) and D: (552 GB).
} 64-bit Windows 7 operating system
} Touch screen and on-screen keyboard
} Three USB ports—1 on the right side of the monitor; 2 on the rear of the instrument
} FSE software such as MTS is on the C: drive
The hard drive is not designed for storing data. Run data are saved to the D drive first;
then copied to the network or another hard drive during the run. Because the image data
are automatically deleted, real output for a 2 x 151 cycle run is approximately 5.5 GB.
If the hard drive ceases to function during a run, the data from that run is lost.
Replacement hard drives are imaged and partitioned before shipment.

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  Chapter 2  Safety

Safety

Chapter 2
Safety Information 9

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Safety
Safety Information
Refer to the appropriate guide listed here for complete instrument safety and compliance
information:
} MiSeq System Safety and Compliance Guide (document # 15027616)

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  Chapter 3  Setup

Setup

Chapter 3
Items Required 11
Inspect the Lab 12
Inventory and Inspect the System Components 13
Set Up the Instrument on the Bench 14
Install Workflow 20
First Time Set Up Screens 22
Run the Motion Tests 26
Check Lane Center and Rotation 27
Test the Optics 32
Determine the Best Focus Z–Position 42
Check the LED Power 44
Illumination Footprint Check 45
Check the Chiller TEC Temperature 49
Record the Software Information 50
Check the Reagent Line Delivery Volumes 51
Set Up the Computer Environment 52

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Setup
Items Required
} MiSeq/MiSeq FGx Installation Guide (document # 15027239)
} The appropriate IQ/OQ document:
• MiSeq System Installation Qualification and Operation Qualification (document #
15032822)
• MiSeq FGx Instrument Installation Qualification and Operation Qualification
(document # 15067076)
} OTT
} PR2 bottle and wash tray
• MiSeq: filled with DI water
• MiSeq FGx: filled with DNase and RNase-free water
} Super flat beaded flow cell
} Wash (open) flow cell
} Waste bottle

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Inspect the Lab
Inspect the Lab
The freight company contracted by Illumina is responsible for uncrating and placing the
instrument on a lab bench.
If the instrument is still in a crate, follow the instructions in this document to uncrate and
place instrument: WI, Uncrating an Illumina MiSeq or MiSeqDx System (part # 15041141).
Inspect the lab and system placement to make sure that it meets the specifications detailed
in the instrument site prep guide. Information in this guide includes:
} Laboratory requirements
} Electrical requirements
} Environmental considerations
} Network considerations
} Antivirus software

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Setup
Inventory and Inspect the System Components
To perform an inventory check:
} Make sure that all system components are present, and that there are no missing parts.
Refer to the IQ/OQ for a list of parts.
} Examine the instrument and all the components for damage.
} Record any damaged or missing parts on the IQ/OQ. Include the serial numbers of
damaged parts.

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Set Up the Instrument on the Bench
Set Up the Instrument on the Bench
Follow the instructions in this section to set up the instrument on the lab bench.

Position the Instrument


Position the instrument as described in the instrument site prep guide. To move the
instrument, grip the rails that run lengthwise under the front and back of the chassis.
CAUTION
To avoid damaging the instrument, never grip or pull on the flow cell
compartment to lift or move it.

Figure 1 Grip Rails Under Front and Back of Instrument to Move

A Rails run lengthwise under the front and back of the instrument
B Never grip the flow cell compartment to move or lift the instrument

Remove the Shipping Bracket and Release the Shipping Lockdowns


Three lockdowns are engaged, and 1 shipping bracket is installed to protect the instrument
during shipping.
To disengage the lockdowns, use the 2 T-handle Allen wrenches included in the accessory
box (M6 and 5/64 in hex). Leave the skins on the instrument while disengaging the
lockdowns. A red label affixed to the instrument skin indicates the location of a lockdown.
CAUTION
Make sure that all lockdowns are fully disengaged, and the shipping bracket is removed
before turning on the instrument.
Disengage the lockdowns:
• In alphabetical order as indicated on the labels (A, B, then C).
• By turning the Allen wrench in the direction indicated on the label.
1 On the left side of the instrument:
a Insert the M6 T-handle Allen wrench through the hole in the label marked A.
b Turn the wrench clockwise approximately 13 full turns until it stops
(A in Figure 2).
c Insert the M6 T-handle Allen wrench through the hole in the label marked B.
d Turn the wrench counterclockwise approximately 1/2 turn until it stops
(B in Figure 2).

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Setup
Figure 3 illustrates the lockdowns when fully disengaged.
Figure 2 Left Side Lockdowns Engaged — Red Labels A and B

A Label marked A
B Label marked B

Figure 3 Left Side Lockdowns Fully Disengaged

2 Remove the red labels and replace them with the white cosmetic labels taped to the left
side of the instrument.
3 Facing the instrument, unlock the Z-stage by inserting the 5/64 in hex
T-handle Allen wrench through the hole in the red label marked C, and turning it
counterclockwise approximately 2-1/2 turns until it stops.

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Set Up the Instrument on the Bench
Figure 4 Disengaging the Z-stage Lockdown — Red Label C

4 Remove the white dot from the flow cell compartment door and open the door.
5 Use a Phillips head screwdriver to remove the shipping bracket.
6 Close the flow cell compartment door.
Figure 5 Y-Stage Shipping Restraint Bracket

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Setup

Prepare the Reagent Compartment


1 Remove the white dot from the chiller compartment door.
2 Remove the protective sheet from the touch screen.
Tape this sheet to the side of the instrument until installation is complete.
Figure 6 Instrument with White Dots and Protective Covers

3 Open the reagent compartment door.


4 Lift the PR2 sipper handle, and remove the protective sheath from the sipper.
5 Lower the sipper handle.
Figure 7 Sheath Covering PR2 Sipper Tube

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Set Up the Instrument on the Bench
Install the Electrical and Network Connections
An uninterruptible power supply (UPS) is not included with the instrument. The site prep
guide recommends connecting this instrument to a UPS.
1 Connect the appropriate instrument power cord to the back of the instrument.
The instrument is shipped with one domestic and 3 international power cords.
2 If a UPS is available:
a Plug the UPS power cord into an appropriate power receptacle, and turn it on.
b Plug the power cord from the instrument into the battery backup enabled outlet on
the UPS.
3 Connect the Ethernet cable to the network port on the back of the instrument, and to the
network port designated for this instrument.
Figure 8 Back of Instrument

A Hard drive access panel


B Power switch
C Power cord receptacle
D USB ports
E Network port
F Connector for optional VGA monitor

Level the Instrument


Make sure that the instrument is stable on the benchtop and does not rock. If necessary,
adjust the screw-on feet to level the instrument.

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Setup

Install a Keyboard and Mouse


A keyboard and mouse must be temporarily installed to use MTS. You can use the
keyboard and mouse recommended under Items Required on page 4. See Install the Electrical
and Network Connections on page 18 for USB port locations.

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Install Workflow
Install Workflow
1 Turn on the instrument, and proceed with the first-time setup (FTS) wizard through the
folder locations screen.
2 Run the motion tests in MCS/MiSeq FGx Control Software:
• Y-stage test
• M3 mirror test
• Compensator test
• Z-stage test
3 In MTS, check lane center and rotation.
4 Test the optics in the imaging module by running BoltPAC.exe (MTS) or the Full Optics
Test (MCS/MiSeq FGx Control Software).
If running the Full Optics Test in MCS/MiSeq FGx Control Software, first determine the
top surface best Z value minus 0.035 (super flat beaded flow cell; tile 1 Top best focus
Z-position)Enter this value in the MiSeqOverride.fg file. This value is measured at the
super flat position of a super flat beaded flow cell.
5 Make adjustments as necessary until all the BoltPAC.exe or Full Optics Tests pass.
6 In MTS:
a Determine the best focus Z-position.
b Update the MiSeqOverride.cfg file with the ZOffsetToFocusLED and Best Focus Z-
Position values.
c Check the LED power.
d Check illumination footprint.
e Check the chiller temperature.
f Set up the computer environment.
7 Test the reagent line volumes by running 1 of these tests:
• Volume Test in MCS/MiSeq FGx Control Software
• VCL in MTS
8 Run the Thermal Ramp Test (MCS/MiSeq FGx Control Software).
9 Perform a verification run.

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Setup

First-time Setup Screens


When the instrument control software is launched for the first time, the first-time setup
screens are automatically displayed.

About the Setup Screens


NOTE
The first-time setup screens are run one time only. To run the wizard again, delete this file on
the D drive:
D:\MiSeqFirstTimeSetup.xml
Network settings, the machine name, the domain name, and the start-up option can be
changed later in the sequencing instrument.
Table 7 Interactive First time Set Up Screens
Screen Required Optional

MiSeq FGx only: Login: admin@forenseq.uas No other options.


ForenSeq Server Password: adminDefaultPwFGx
login and password

IP Settings IP Address: default OK Enter an IP address,


subnet mask, and default
gateway

DNS server address: default is to obtain Specify a preferred and


the address automatically alternate DNS server
address

Machine, Domain, Machine name: default OK unless it is Change the instrument


Workgroup, and MISEQ or MISEQ FGX. Change to name.
Start-Up Option instrument number.

Domain: default OK Enter a domain or


workgroup

Start-up option: use Windowed Mode. No other options


Change to Kiosk mode (no Windows
access) when installation and
verification are finished.

Domain and user Default OK (local system account) Enter a domain\user


name name and a password

Folder locations Default locations OK Change the default


folder locations

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First Time Set Up Screens
First Time Set Up Screens
This edit is just a test.
1 Turn on the instrument.
The first FTS screen is automatically displayed.
2 If a User Account Control warning is displayed, click Yes.
3 MiSeq FGx only:
Log in to the MiSeq FGx Control Software through the ForenSeq Universal Analysis
Software:
• Login = admin@forenseq.uas
• Password = adminDefaultPwFGx
Figure 9 ForenSeq Universal Analysis Software Log In

4 Select Save and Continue to accept the defaults, or optionally enter an IP address or
DNS server information.
Figure 10 Network Settings

5 Select Save and Continue to accept the defaults, or optionally change any of the
following fields:
NOTE
Changing any setting on this screen will force the instrument to reboot after the
next screen, where you are prompted to enter a user name and password.

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Setup
• Machine Name
NOTE
If MISEQ or MISEQ FGX is displayed as the machine name, change it to the
instrument serial number.
• Domain or Workgroup
• Start-Up Option
Do not change the default. Windows access is required during installation. When
installation is complete, change the start-up option to Kiosk Mode.
Figure 11 Machine Name, Domain/Workgroup Name, and Start-up Option

6 Select Save and Continue to accept the defaults, or optionally enter a


domain/username and password.
Figure 12 Enter User Name and Password

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First Time Set Up Screens
7 Select from the following:

If the lockdowns and shipping Then …


bracket …

Have been disengaged/removed Select Yes, initialize device.


The instrument initializes.

Have not been 1. Disengage and remove the lockdowns/shipping


disengaged/removed bracket now. See Remove the Shipping Bracket and
Release the Shipping Lockdowns on page 14 for
more information. Or you can click No, show
me how.
2. Select Yes, initialize device.
The instrument initializes.

NOTE
The shipping lockdowns must be disengaged, and the shipping bracket removed
before initializing the instrument. If they are not disengaged and removed, severe
damage to the instrument occurs. See Remove the Shipping Bracket and Release the
Shipping Lockdowns on page 14 for more information.

Figure 13 Confirm Shipping Lockdowns/Bracket Disengaged before Initializing

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Setup
8 Select Save and Continue to accept the defaults, or optionally change the folder
locations.
Figure 14 MiSeq Folder Locations

Figure 15 MiSeq FGx Folder Locations

9 Select Stop Test on this screen, then select Yes and Done.
Figure 16 Stop Test

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Run the Motion Tests
Run the Motion Tests
1 Insert a USB drive into 1 of the instrument USB ports.
2 On the Welcome screen, select Manage Instrument | System Check.
3 Select these tests, then select Next.
• Y-stage test
• M3 mirror test
• Compensator test
• Z-stage testf
Figure 17 Motion Tests

4 At the prompt, insert a super flat beaded flow cell, and select Next.
5 Select from the following:

If … Then …

All the tests pass 1. Select Export Results, and save the results
to the USB drive. Then select Done.
2. Enter the results on the IQ/OQ.

Any of the tests fail 1. Troubleshoot the instrument, and repeat


the test until it passes.
2. Save the results to the USB drive.
3. Enter the results on the IQ/OQ.

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Setup
Check Lane Center and Rotation
Use an OTT and MTS to check lane center and rotation.

Focus an OTT
NOTE
If you see nothing in tiles 1 through 3, the OTT is loaded backwards.
1 Close the instrument control software, and launch MTS.
2 Initialize and home the instrument.
3 Load an OTT.
4 Go to the Motor tab.
5 Open the Tile drop-down list and select tile 6; then click Move to Tile.
6 Set the Mirror Position to Normal for Imaging.
Clicking the Flip button toggles between Normal for Imaging and Tilted for Focusing.
7 Enter –0.17 in the Z Motor Position field, and hit Enter.
This value represents the optimal Z-motor position.
Figure 18 Motor tab

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Check Lane Center and Rotation
8 Go to the LED tab and make sure that the Green LED Default Snap Curr is 6500, and
the Red LED Default Snap Curr is 1500.
Figure 19 LED Tab

9 Go to the Camera tab and:


a Select Camera 2; deselect Camera 1.
b Select the G channel, and click Continuous (the default exposure of 200 is OK).
Figure 20 Camera Tab

10 In the right pane, click the Image tab and:


a Select the channel used in the previous step; deselect the other channels.
Channel buttons fade upwards to white when deselected.
Figure 21 G Channel Selected; A, C, and T Channels Deselected

b Click Zoom All, then click Contrast to view the image, and look for a small black
crosshair.
— If the image is not usable, try the following:
— Adjust the exposure time (for example, 400, 600).
— If the G channel is not usable, try other channels and exposure times until
the image quality is acceptable. You can also try using Camera 1.
— If no black crosshair is visible, try moving the upper and lower contrast bars.

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Setup
Figure 22 Small Crosshair in OTT

NOTE
The Contrast button in the Image pane works as follows:
1. It measures the foreground and background based on the Z-motor position.
2. It adjusts the contrast to what it calculates as best focus.
If the Z-motor position changes by a large value, or changes several times, click the
Contrast and the Zoom All buttons again to “reset” the software for the new position.
Also click these buttons if you adjusted the position and no image is displayed.
11 Select the Motor tab, and focus the image as follows:
a Zoom in on the cross.
b Move the Z-motor up or down to bring the cross into good focus.
Increments of 0.002–0.0005 typically work well.

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Check Lane Center and Rotation
c To fine-tune the focus, zoom in on the squares in the center of the image, and move
the Z-motor up and down.
Figure 23 Focused OTT

Check Lane Center and Rotation


1 In the Motors tab, select tile 2, and click Move to Tile.
2 Refocus the image if necessary.
3 Place the cursor on the left edge of the vertical axis of the cross; note the X value.
4 Move to tile 11, and refocus the image if necessary.
5 Place the cursor on the left edge of the vertical axis of the cross; note the X value.

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Figure 24 Lane Centering—OTT Tile 2

6 Select from the following:

If the X value for tiles 2 and 11 Then …


are …

Within spec for lane center Lane center is OK.


1. Record the lane center values on the IQ/OQ.
2. Proceed to the next step.

Out of spec for lane center 1. Adjust lane center by following the instructions
under Lane Center Adjustment on page 113.
2. Record the lane center values on the IQ/OQ.
3. Proceed to the next step.

7 Determine the delta between the lane center values.


Delta = [tile 11 lane center] – [tile 2 lane center]. -172< Delta < +172
8 Select from the following:

If the delta is … Then …

Within spec for lane Lane rotation is OK.


rotation 1. Record the lane rotation value on the IQ/OQ.
2. Change the exposure value back to 200 (Camera tab).

Out of spec for lane 1. Adjust lane rotation by following the instructions under
rotation Lane Rotation Adjustment on page 116.
2. Record the lane rotation value on the IQ/OQ.
3. Change the exposure value back to 200 (Camera tab).

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Test the Optics
Test the Optics
Test the instrument optics using one of these tests:
Option 1 Full Optics Test in MCS/MiSeq FGx Control Software (Run The Full Optics Test
on page 32).
Option 2 BoltPAC.exe in MTS (Run BoltPAC.exe on page 145).

Run The Full Optics Test


Follow this procedure to run the Full Optics Test.

Determine the Best Z Value


Before running the Full Optics test, determine the best Z-value. This procedure is required
to make sure that the correct surface of the flow cell is being imaged. It is not required
when running BoltPAC.exe.

Move to the Center of a Super Flat Beaded Flow Cell


1 Launch MTS, initialize the software, and home the instrument.
2 Load a super flat beaded flow cell.
3 Move to the center of the flow cell as follows:
a Open the MiSeqOverride.cfg file and find the value for actualedgeofslide.
b Add 14.0 mm to the actualedgeofslide value (example: 57.4 + 14.0 = 71.4).
Do NOT change the value for actualedgeofslide in the MiSeqOverride.cfg file.
Figure 25 actualedgeofslide in MiSeqOverride.cfg File

c In MTS, open the Motor tab.

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d Type this value into the Y Motor Position field, and hit Enter.
Figure 26 Actualedgeofslide Value and Y Motor Position

Find Best Focus on the Top Surface of the Flow Cell


1 On the Motor tab, set the Mirror Position to Normal for Imaging.
2 Type –0.17 in the Z Motor Position field, and hit Enter.
3 Open the LED tab, change the Green LED Default Snap Curr to 2000, and click Set.
The default snap current of 1500 for the red LED is OK.
Figure 27 LED Tab

4 Open the Camera tab and:


a Deselect Camera 1; select Camera 2.
b Click Continuous.
5 In the right pane, click the Image tab and:
a Click the A channel (deselect the C, G, and T channels).
b Click Zoom All and Contrast (unfocused beads appear).

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Test the Optics
Figure 28 Unfocused Beads

6 Using the wheel on the mouse and the sliders, zoom in on an area of beads.
See Image Tab in the Right Pane on page 143 for additional information.
Figure 29 Example of Well Focused Beads

7 On the Motor tab, bring the beads into sharp focus by moving the Z Motor up or down.
Increments of 0.002–0.0005 typically work well.
8 Record the value, and confirm that it is within spec.
9 Turn off the camera.

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Setup
10 If using MCS/MiSeq FGx Control Software:
a Open the MiSeqOverride.cfg, and enter the best Z value into these fields:
— coarsefocuscenterz
— throughfocuscenterz
— tiltuntiltfocuscenterz
b Save and close the MiSeqOverride.cfg file.

Start the Full Optics Test


1 Close MTS, and launch MCS or MiSeq FGx Control Software.
2 Navigate to Manage Instrument | System Check.
Figure 30 System Tests

3 Select Full Optics Test.


4 Select Next, and follow the prompts to start the test.

Analyze the Full Optics Test Results


An overview of test results is provided under About Test Results on page 38.
1 Open the Detailed_Report results files for the top and bottom surface of the flow cell,
and confirm the bottom-top Z-offset:
Bottom [Focal-Plane Z-position – GTAC average (mm)] – Top [Focal-Plane Z-position –
GTAC average (mm)] ≥ 0.035 mm
2 If the Bottom-top Z-offset is in spec, then proceed to 4.

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Test the Optics
3 If the bottom-top Z-offset is out of spec, then
a Recheck the best Z value (Determine the Best Z Value on page 32).
b If the best Z value is correct, then
1. Subtract 0.035 mm from the best focus Z-position.
— Example: –0.17 mm (best focus Z-position) minus 0.035 mm = –0.205 mm
(calculated value)
2. Open the MiSeqOverride.cfg file, and enter the calculated value into these fields:
— coarsefocuscenterz
— throughfocuscenterz
— tiltuntiltfocuscenterz
3. Save and close the MiSeqOverride.cfg file.
4. Run the Full Optics test again (Start the Full Optics Test on page 35).
c If the bottom-top Z-offset is still out of spec, then repeat Run The Full Optics Test on
page 32 using a different, bubble-free super flat beaded flow cell.
d If the bottom-top Z-offset is still out of spec, then check the compensator. Confirm
that it is installed and operating correctly.
4 Refer to the specifications listed in the IQ/OQ, and review the results for all the other
tests.
5 Troubleshoot any tests that failed.
6 Run the Full Optics Test or BoltPAC.exe again until all tests pass.

Run BoltPAC.exe
1 Generate through-focus image stacks for both surfaces of the flow cell using the center
of a super flat beaded flow cell (see Generate Through Focus Image Sets (Stacks) on page
136).
2 Minimize MTS.
3 Go to C:\Illumina | MiSeq Control Software | MatlabScripts.
Or go to the release scripts folder on the D drive.
4 Double-click the script to open a window with a command prompt.
5 Go to the D drive and open the folder you created for the top surface through-focus
image set.

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Setup
6 Drag and drop the numbered and dated folder onto the command prompt.
Figure 31 Drag and Drop the Through Focus Image Set Folder Onto the Command Prompt

7 Hit Enter.
A BoltPAC status bar is displayed while the script is running. The script runs for
approximately 40 min.
NOTE
If BoltPAC repeatedly fails, make sure that the proper file naming convention is being
applied to the 41x4 image generated. Sometimes the software can name one or more of
the files incorrectly, causing the script to fail.
8 Run BoltPAC again using the bottom surface through-focus image set.

Analyze the BoltPAC Test Results


An overview of test results is provided under About Test Results on page 38.
1 Assess the test results for each surface of the flow cell.
File naming convention:
Detailed_Report_<year.month.day>-<hours-minutes-seconds>.csv
2 Select from the following:

If the results are … Then …

Within spec 1. Enter the ZOffsetToFocusLED value in the


MiSeqOverride.cfg file.
2. Save and close the Override.cfg file.
3. Indicate on the IQ/OQ that these values have been entered
into the MiSeqOverride.cfg file.

Out of spec 1. Make the necessary adjustments.


2. Run BoltPAC again.

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Test the Optics
About Test Results
IMPORTANT
Always refer to the IQ/OQ for the instrument specifications.
This section contains a brief description of the following result files.
} Full Optics and FullOpticsBot detailed results (ie, BoltPAC)
} Full Optics summary file
} Thermal ramp test
} Volume test

Full Optics Test Results (BoltPAC.exe)


The full optics test in the sequencing instrument and the BoltPAC.exe script are the same
series of tests. It checks tip/tilt, uniformity, chromatism, camera offset and rotation, and
D50.
} Always use the center of a super flat beaded flow cell to generate the top and bottom
through-focus image sets, and to run the script.
} Approximately 40 min is required to run this script and analyze the data.
} The Full Optics test in the instrument control software automatically generates the two
through-focus image sets required, and runs BoltPAC.exe on both surfaces of the flow
cell.
} Disregard saturation errors, and warnings in script results and the control software.
} If the test repeatedly fails, check that the proper folder naming convention is being
used.
• The folder name cannot contain any spaces. Use an _ (underscore). Some scripts do
not run if there are spaces in the folder name.
• Add Top or Bottom to the folder name to distinguish between image stacks for each
surface of the flow cell.
• Full optics test results are written to two folders: one for results from the top surface
of the flow cell; and one for results from the bottom surface. The file naming
convention is:
Detailed_Report_<year.month.day>-<hours-minutes-seconds>.csv
The information in these files is annotated in the following figures.
Figure 32 Data Path

A Surface of flow cell must be specified in file name (top or bottom)

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Figure 33 Focal-Plane Z-Position

Figure 34 ZOffset, Chromatism, and Tip/Tilt

A ZOffsetToFocusLED (mm)—Enter this value into the MiSeqOverride.cfg file as a


negative number.
B Chromatism result
C Tip/Tilt results for cameras 1 and 2

Figure 35 Camera XY Offset and Rotation

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Test the Optics
Figure 36 Illuminated Area and Max D50

A Illuminated area results (uniformity)


B Max D50 results

Full Optics Test Summary


The full optics test summary file includes the results for the top and bottom flow cell
surfaces.
Figure 37 Example of Full Optics Test Result Summary

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Thermal Ramp Test Results


The measured temperatures for the flow cell TEC (not the target temperatures) are checked
every 5 seconds for 30 seconds. Make sure that the temperature readings are within spec.
Figure 38 Example of ThermalRamp.csv

Volume Test Results


NOTE
Some columns are not shown in this example.

Figure 39 Example of Volume.csv File

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Determine the Best Focus Z–Position
Determine the Best Focus Z–Position
1 If not open, launch MTS, then initialize and home the instrument.
2 Load a super flat beaded flow cell.
3 Click the Motor tab.
4 Open the Tile menu, select Tile 1, and click Move to Tile.
Figure 40 Move to Tile 1

5 Enter –0.17 in the Z Motor Position field, and hit Enter.


6 Go to the LED tab, change the Green LED Default Snap Curr to 2000, and click Set.
7 Click the Camera tab and:
a Deselect Camera 1; select Camera 2.
b Click Continuous.
8 In the right pane, click the Image tab and:
a Click the A channel button (deselect the C, G, and T channels).
Inactive channel buttons fade to white. Active channel buttons are solid.
b Click Contrast and Zoom All.
c Using the wheel on the mouse and the sliders, zoom in on the beads.
See Image Tab in the Right Pane on page 143 for additional information.
9 On the Motor tab, bring the beads into sharp focus by moving the Z Motor up or down.
Increments of 0.002–0.0005 typically work well.

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10 Open the MiSeqOverride.cfg file and enter the best focus value into these fields:
• coarsefocuscenterz
• throughfocuscenterz
• tiltuntiltfocuscenterz
Figure 41 Focus Params in MiSeqOverride.cfg File

11 Save and close the MiSeqOverride.cfg file.


12 If you are installing the instrument, record this value on the IQ/OQ.

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Check the LED Power
Check the LED Power
1 In MTS, click the LED tab.
2 One LED at a time, do the following:
a Click Enable (this button toggles between Disable and Enable).
b For the green LED only, change the Current to 2000 mA, and click Set.
The default value for the red LED is OK.
c Record the value displayed for Power [mW] on the IQ/OQ.
Figure 42 Example of Green LED Reading

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Setup
Illumination Footprint Check
1 Load a super flat beaded flow cell.
2 In MTS, click the Motor tab and move to any tile.
3 Enter –0.17 in the Z-Motor Position field, and hit Enter.
4 Click the Camera tab and:
a Select Camera 2; deselect Camera 1.
b Click Continuous.
5 In the right pane on the Image tab:
a Turn on channel A; turn off all other channels.
b Click Zoom All and Contrast.
Figure 43 Initial Image of Bead Mass

6 Zoom in and focus the beads (Motor and Image tabs).


Figure 44 Focused Beads

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Illumination Footprint Check
7 Check the left and right sides of the image as follows:
a Use the mouse to zoom out a little, and move the bottom image slider to the left
until you see the left side of the image.
b Place the cursor on the left edge of the beads, and note the X value in pixels.
Figure 45 X Value on Left Edge of Beads

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Setup
c Move the bottom slider to the right until you see the right edge of the beads.
d Place the cursor on the right edge of the beads, and note the X value in pixels.
Figure 46 X Value on Right Edge of Beads

8 Check the top and bottom of the image as follows:


a Move the lower contrast bar all the way down.
b Center the bottom slider.
c Move the right slider all the way up.

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Illumination Footprint Check
d Make sure that beads appear across the top of the image (no blank strip across the
top of the image).
Figure 47 Beads Across Top of Image

e Move the right slider all the way down, and make sure that beads appear across
the bottom of the image (no blank strip across the bottom of the image).
9 Select from the following:

If the illumination Then …


footprint is …

Within spec The illumination footprint is OK, record the result on the
IQ/OQ.

Out of spec Adjust the illuminated area until it is within spec (See
Illuminated Area Adjustment on page 107).

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Setup
Check the Chiller TEC Temperature
NOTE
The chiller cools by air circulation, and can take 3 to 4 hours to reach the set point for testing.
To simulate operational conditions, place a chilled wash cartridge filled with DI water into
reagent compartment before TEC testing.

Prepare for the Chiller TEC Temperature Test


To simulate operational conditions:
1 Fill a wash cartridge with DI water.
2 Chill the cartridge at 4 °C for a minimum of 30 minutes.
3 Place the cartridge into the reagent compartment at least 10 minutes before testing the
chiller TEC.

Check the Chiller TEC Temperature


1 Using MTS v1.0.7.0 or higher, click the Temperature tab.
NOTE
Be sure to use MTS v1.0.7.0 or higher. Earlier versions have an incorrect chiller voltage
which can cause this test to fail.
2 Open the Controller drop-down.

If the temperature
Then …
is …

Within spec The chiller TEC is OK.


1. Record the Actual value on the IQ/OQ.
2. Deselect Enable.

Out of spec 1. Check the ambient temperature of the lab. If it is out of spec, ask
the customer to adjust the lab temperature. Then requalify the
chiller temperature.
2. If the ambient temperature of the lab is within spec, contact
Illumina Technical Support.

Figure 48 Chiller Temperature–Actual

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Record the Software Information
Record the Software Information
1 On the appropriate IQ/OQ, record all of the software information required.
• MiSeq System Installation Qualification and Operation Qualification (document #
15032822)
• MiSeq FGx Instrument Installation Qualification and Operation Qualification
(document # 15067076)
2 For Local Run Manager installations (referred to as the Illumina MiSeq System Suite):
a Open the Start menu, and navigate to Control Panel | Program and Features.
b If Illumina MiSeq System Suite is present, then record the version in the Software
Suite Version field.
3 For MiSeq Reporter installations:
a Navigate to C:\Illumina\MiSeqSuiteInstaller.
b Hover over MiSeqUpdater.exe and record the version in the Software Suite Version
field.
MiSeq Reporter software suite information is also located on the instrument control
software home page. Select the question mark (?), then select About.
Versions shown here are examples; actual installed versions can differ.
Figure 49 Example of Software Listed for MiSeq Reporter Installations

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Setup
Check the Reagent Line Delivery Volumes
You can use MCS/MiSeq FGx Control Software or MTS to check the reagent line delivery
volumes.

If using … Then …

MTS 1. Follow the instructions listed under Manually Check Reagent Delivery on
page 149.
2. Troubleshoot any lines with volumes that are out of spec.
3. When delivery for each line meets spec, note the result on the IQ/OQ.

MCS/MiSeq FGx 1. Fill the wash tray and the PR2 bottle with DI water, and load onto the
Control instrument.
Software 2. In the control software, run Prime Reagent Lines and Conduct
Volume Test.
3. Troubleshoot any lines with volumes that are out of spec.
4. When delivery for each line meets spec, note the result on the IQ/OQ.

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Set Up the Computer Environment
Set Up the Computer Environment
1 Launch MTS, then initialize and home the instrument.
Figure 50 MTS Launch Screen

Figure 51 MTS Initialize Button

Figure 52 MTS Home Button

2 Exit out of MTS after Initialize and Home successfully completes.


3 Perform these tasks to prepare the computer working environment:
Table 8 Computer Environment Preparation
Task How To

CAUTION Speak with the on-site IT professionals. Advise them that the
Make sure that the network setting for Windows Updates must be turned off.
network setting for If turned on, Windows Updates override the instrument and
Windows Updates is forces it to reboot. If a run is in progress, the run fails.
turned off.

Make sure that the From the Windows Control Panel:


sbsuser password is set 1. Open Administrative Tools | Computer Management.
not to expire. 2. On the left, open Local Users and Groups | Users.
3. Right-click sbsuser and select Properties.
4. Select the Password never expires checkbox.
5. Select OK.

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Setup

Task How To

If Local Run Manager is 1. Launch Chromium from the desktop, and log in to Local
installed on the Dx SSD, Run Manager (User Name=admin; Password=password).
create an 2. In the Local Run Manager dashboard navigation bar,
administrative select System | User Management.
customer account.
3. Select Create User in the User Management page.
4. In the Create New User field, enter first and last names,
user name, and password information. The password is
temporary, and is reset at initial login.
5. Toggle to Admin in the Role field by clicking User, then
select Create User and Continue.
6. Log out of Local Run Manager, and close the Chromium
browser.
7. Provide the customer the newly created user name and
temporary password and have them test the account log
in. Direct them to the Local Run Manager Software Reference
Guide for MiSeqDx (1000000011880).

Make sure that the Open folder and make sure that the CloudAPIKey.xml file is
CloudAPIKey.xml file present:
is present and ensure Dx: C:\ProgramData\Illumina\MiSeq
the serial number in RUO: C:\ProgramData\Illumina\MiSeqControlSoftware
this file matches the
serial number of the Note: Program Data may not show--select Hidden Items
instrument. from View tab of file explorer

Synchronize the See Synchronize the Instrument with an Internet Time Server
instrument with an on page 1.
internet time server

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  Chapter 4  Verification

Verification

Chapter 4
Verification Run Requirements 55
Prepare the Reagent Cartridge 56
Prepare the PhiX Library 58
Perform a Verification Run Using Local Run Manager 60
Perform a Run Using MiSeq Reporter 70
Perform a Post-Run Wash 78

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Verification
Verification Run Requirements
A PhiX run confirms proper hardware and software performance of the instrument. The
PhiX control library provided by Illumina is used for the verification run. Perform a 2 x 75
cycle v3 run using one of these applications (only one is installed on the instrument): Local
Run Manager or MiSeq Reporter. To analyze the run, compare the output to published
specifications.

Table 9 Reagent Kit for MiSeq and MiSeq FGx Runs


Instrument Instrument and Reagent Kit Quantity Part Number

MiSeq MiSeq Reagent Kit version 3, 600 cycles 1 15046996

Table 10 Additional Items Required for a PhiX Verification Run


Item Quantity Part Number

PhiX Control Kit, version 3 (10 μl at 10 nM) 1 tube 15017666

HP3 (2 N NaOH) 1 tube 11324596

Elution Buffer (EBT) 1 tube 15025394


(or 10 mM Tris-Cl, pH 8.5 with 0.1% Tween
20)

PW1, 250 ml (or laboratory-grade water) 1 bottle 11298922

Other Items Required

Alcohol wipes, 70% isopropyl As required Customer supplied

Gloves, disposable, powder-free Customer supplied

Ice bucket with ice 1 Customer supplied

Lab tissue, low-lint As required Customer supplied

Lens paper, 4 x 6 in As required Customer supplied

Lint free lens tissue As required Customer supplied

Microcentrifuge tubes, 1.5 ml to 2 ml 3 Customer supplied

Pipettes and pipette tips (P10 or P20, P200, As required Customer supplied
and P1000)

MiSeq Reporter: PhiXInstallSampleSheet.csv 1 ICE


Local Run Manager: PhiX-verification-run-
MiSeq-RUO-LRM.csv

Tweezers 1 pair Customer supplied

Water, deionized As required Customer supplied

Water bath 1 Customer supplied

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Prepare the Reagent Cartridge
Prepare the Reagent Cartridge
1 Record the lot number of the reagent cartridge on the lab tracking form.
2 Remove the reagent cartridge from –15 to –25° C storage.
3 Place the reagent cartridge in a room temperature water bath containing only enough
water to submerge the base without reaching the clear cartridge cover.
Deionized water is preferred.
Figure 53 Maximum Water Line

4 Allow the reagent cartridge to thaw in the room temperature water bath for at
approximately 60 minutes or until thawed.
5 Remove the cartridge from the water bath and gently tap it on the bench to dislodge
water from the base of the cartridge.
6 If not using the reagent cartridge immediately, place on ice or in 4° C refrigerator.
7 Invert the reagent cartridge ten times to mix the thawed reagents.
8 Visually inspect the USM-2 and IMT-1 reagents to make sure that they are fully mixed
and free of precipitates.
9 Gently tap the cartridge on the bench to remove air bubbles in the reagents.
WARNING
A component in this set of reagents contains formamide, an aliphatic amide that is a
probable reproductive toxin. Personal injury can occur through inhalation, ingestion,
skin contact, and eye contact.
Dispose of containers and any unused contents in accordance with the governmental
safety standards for your region.
The SDSs for this kit are available at www.illumina.com/sds.
Table 11 Reagent Cartridge Map
Position Reagent Position Reagent

1 IMT-1, Incorporation Mix 13 HP12-13, Index Primer


Mix

2 USM-2 or blank, Scan Mix 14 HP11-13, R2 Primer Mix

4 CLF-4, Cleavage Mix 15 PW1, Water

5 AMX1-S, Amplification Mix 16 PW1, Water

6 AMX2-6, R2 Amp Mix 17 TMP-17, Template

7 LPM-7, Lin Pre Mix 18 NULL, Empty

8 LDR-8, Formamide 19 NULL, Empty

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Position Reagent Position Reagent

9 LMX1-9, Linearization Mix 20 NULL, Empty

10 LMX2-10 R2 Lin Mix 21 PW1, Water

11 RMF-11, Resynthesis Mix 22 NULL, Empty

12 HP10-12, R1 Primer Mix

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Prepare the PhiX Library
Prepare the PhiX Library
Follow these instructions to denature and dilute the 10 nM PhiX library to a final
concentration of 20.0 pM.

Thaw and Mix the Reagents


1 Thaw one tube each of the following reagents at room temperature:
• PhiX control library
• EBT
• HP3 (2.0 N NaOH)
• HT1 buffer
To speed up thawing, place the HT1 buffer in a container of room temperature
water. Keep the water level well beneath the cap of the HT1 tube.
2 After the reagents are thawed:
a Vortex the PhiX and HP3 tubes to mix.
b Pulse spin to collect contents at bottom of tube.
3 Place the PhiX on ice, and leave HP3 and EBT at room temperature until ready to use.
4 Mix HT1 by inverting the tube at least 10 times.
5 Place the HT1 on ice until ready to use.

Prepare a Fresh Dilution of NaOH


CAUTION
Using freshly diluted NaOH is essential to denature samples for cluster generation on
the instrument. The NaOH stock solution in this protocol is 2.0 N.
To prepare a fresh dilution of 0.2 N NaOH, follow these instructions.
1 To a new microcentrifuge tube, add:
a 900 μl water (or PW1)
— MiSeq: laboratory-grade
— MiSeq FGx: DNase-free and RNase-free water
b 100 μl stock 2.0 N NaOH (HP3)
2 Invert the tube several times to mix.
3 Set aside at room temperature.

Denature and Dilute the PhiX Control


Follow these steps to denature and dilute the 10 nM PhiX library to 20.0 pM.
1 Combine the following volumes to dilute the PhiX library to 4 nM:
• 2 μl of 10 nM PhiX library
• 3 μl of EBT (or use Tris-Cl, pH 8.5 with 0.1% Tween 20)
2 Combine the following volumes of 4 nM PhiX library and 0.2 N NaOH in a
microcentrifuge tube to result in a 2 nM PhiX library.
• 5 μl of 4 nM PhiX library
• 5 μl of 0.2 N NaOH

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3 Discard the remaining dilution of 0.2 N NaOH.
4 Mix the combined 10 μl volume by vortexing briefly.
5 Centrifuge the solution to 280 × g for 1 minute.
6 Incubate for 5 minutes at room temperature to denature the PhiX library.
7 To a new microcentrifuge tube, add the following volumes to result in a 20.0 pM PhiX
library:
• 10 μl of denatured PhiX library
• 990 μl of prechilled HT1
NOTE
You can store denatured 20.0 pM PhiX library up to 3 weeks at –15° to –20°C. After 3
weeks, cluster numbers tend to decrease.
8 Invert the tube 10 times to mix.

Load the v3 PhiX Library into the Reagent Cartridge


1 Spin down the denatured, diluted 20.0 pM PhiX library.
2 Pipette 600 μl of the library into the reservoir on the reagent cartridge labeled Load
Samples.
Figure 54 Load the Sample

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Perform a Verification Run Using Local Run
Perform a Verification Run Using Local Run Manager
If Local Run Manager is installed on the instrument, follow these instructions to perform
the verification run. If MiSeq Reporter is installed, follow the instructions under Perform a
Run Using MiSeq Reporter on page 70.

Set Up a Run Using Local Run Manager


1 Download or create a sample sheet.
• On ICE, download this file: PhiX-verification-run-MiSeq-RUO-LRM.csv
• Or create a sample sheet the same as the example shown here. Change 4/17/2018 to
the current date. Do not modify any other values (eg, cell 1A must read [Header])
Figure 55 Sample Sheet for PhiX Verification Run—GenerateFASTQ in Local Run Manager

2 Upload the sample sheet to this folder on the instrument:


D:\Illumina\MiSeq\SampleSheets
3 Download and install Local Run Manager Generate FASTQ Analysis Module from
support.illumina.com.
4 Launch the Chromium browser and push Ctrl-Shift-R to perform a hard refresh of the
browser.
5 Enter http://localhost in the address bar.

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6 If you see this screen, log in with a valid Local Run Manager user name and
password.
Figure 56 Local Run Manager Home Page

7 Select the RUN DASHBOARD tab, then select:


a Create Run.
b GenerateFASTQ.
Figure 57 Create Run

8 Select Import Sample Sheet, then


a Navigate to and select the sample sheet file.
b Double-click the file or select Open.
9 Make sure that the parameters are set as shown here:
Run Name: PhiX verification run
Run Description Installation
Library Prep Kit: Custom
Index Reads: 0 (PhiX is not indexed. Selecting 1 or 2 index reads can
cause the run to terminate prematurely.)
Read Type: Paired End
Read Length: 75 cycles
Custom Primers None
Module-Specific Settings None
Sample ID PhiX
Sample Description PhiX_sample

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Sample Project OK to leave blank

Figure 58 Run Parameters

10 Select Save Run, and close the browser.


11 From the instrument control software Home screen, select Sequence.
The Sequence Mode Selection screen opens.
12 Select:
a Local Run Manager.
b Optional: Use BaseSpace from the BaseSpace Options screen.
You can optionally run secondary analysis using BaseSpace or BaseSpace Onsite.
c Select Next.
d Select the verification run from the list of available runs.
e Select Next.

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Verification

Clean the Flow Cell


The flow cell is immersed in storage buffer in a flow cell container. Clean the flow cell as
follows before loading it on the instrument.
1 Put on a new pair of powder-free gloves.
2 Using plastic forceps, grip the flow cell by the base of the plastic cartridge and remove
it from the container.
Figure 59 Remove Flow Cell from Container

3 Lightly rinse the flow cell with labaoratory-grade water until both the glass and plastic
cartridge are thoroughly rinsed of excess salts.
Excess salts can affect flow cell seating on the instrument. If salt drys in the imaging
area, imaging can also be affected.
Figure 60 Rinse the Flow Cell

4 Using care around the black flow cell port gasket, thoroughly dry the flow cell and
cartridge with lint-free lens cleaning tissue; gently pat dry in the area of the gasket and
adjacent glass.

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Figure 61 Flow Cell Ports and Gasket

5 Clean the flow cell glass with an alcohol wipe, making sure that the glass is free of
streaks, fingerprints, and lint or tissue fibers.
NOTE
Do not use the alcohol wipe on the flow cell port gasket. Make sure that the
cartridge is securely closed before use.

Figure 62 Dry the Flow Cell

6 Dry excess alcohol with a lint-free lens cleaning tissue.


7 Make sure that the flow cell ports are free of obstructions and that the gasket is well-
seated around the flow cell ports.
If the gasket appears to be dislodged, gently press it bakc into place until it sits securely
around the flow cell ports.inspect it to make sure that no tissue particles are obstructing
the ports.

Load the Flow Cell


NOTE
The instrument must be initialized and homed before loading a flow cell.
1 In MCS, select Sequence from the Welcome screen.
If you are using MTS, click the Motor tab, then click Home Y.
2 Raise the flow cell compartment door, and press the release button to the right of the
flow cell clamp.

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The flow cell clamp opens.
Figure 63 Flow Cell Latch Button

3 Make sure that the flow cell stage is free of lint.


If lint or other debris is present, clean the flow cell stage using an alcohol wipe or a
lint-free tissue moistened with ethanol or isopropanol. Carefully wipe the surface of the
flow cell stage until it is clean and dry.
4 Holding the flow cell (or OTT) by the edges, place it on the flow cell stage.
• Align the top notch around the top pin, and the 2 holes on the right onto the 2
right-hand pins.
• Make sure that the label is facing upward.
Figure 64 Loading a Flow Cell or OTT

A Three alignment pins

5 Gently press down on the flow cell clamp to close it over the flow cell.
As the flow cell clamp closes, alignment pins position the flow cell. An audible click
indicates that tthe flow cell clamp is secure.

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Figure 65 Close the Flow Cell Clamp

6 Close the flow cell compartment door.


7 In MCS, select Next on the Load Flow Cell screen. The Load Reagents screen opens.
In MTS, select a tile, then click Move to Tile.

Load the Reagents


1 Remove the bottle of PR2 from 2° to 8°C storage.
2 Invert to mix, then remove the lid.
3 Open the reagent compartment door.
4 Remove the wash bottle and load the PR2 bottle.
5 Remove the waste bottle, empty the contents into an appropriate waste container, and
replace the waste bottle.
6 Slowly lower the sipper handle, making sure that the sippers lower into the PR2 and
waste bottles.
7 Make sure that the PR2 bottle barcode displays.
8 Select Next.
9 Load the reagent cartridge, and wait for the barcode to display.

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Figure 66 Reagent Cartridge, PR2 Bottle, and Waste Bottle Placement

A Reagent cartridge
B PR2 bottle
C Waste bottle

10 Close the reagent compartment door.

Start the Run —Local Run Manager


NOTE
The run starts automatically if the Start run after pre-run check option is selected in Run
Settings.

Review Run Parameters


1 Review the run parameters one more time (specified in the sample sheet).
2 Optional: When in Local Run Manager or Manual mode, select Edit to make changes,
then select Save.
3 Select Change Folder to review the folder locations.
Modify if needed, then select Save and Continue. To change folder locations, select
Change Folder and browse to a preferred location. Using this option from the Review
screen changes folder locations for all subsequent runs.
4 Select Next.

Review Pre-Run Check


The system performs a check of all run components, disk space, and network connections
before starting the run. If any items do not pass the pre-run check, a message appears on
the screen with instructions on how to correct the error. When all items successfully pass
the pre-run check, select Start Run.
CAUTION
The instrument is sensitive to vibration. Touching the instrument after starting a run could
adversely affect sequencing results. After selecting Start Run, do not open the flow cell
compartment or the reagent compartment doors, or touch the instrument monitor except to
pause the run. Refer to the MiSeq System User Guide (document # 15027617) for information
on runs including how to pause.

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Monitor and Analyze the Run

Monitoring the Run


Monitor run progress, intensities, and quality scores displayed on the Sequencing screen
(view only). To monitor the run in greater detail, use the Sequencing Analysis Viewer (SAV
installed on another computer independent of the instrument computer. A network
connection is required.
} Run Progress: Shows the run progress in a status bar and lists the number of cycles
complete.
} Intensity: The value of cluster intensities of the 90th percentile for each tile.The graphic
in the intensity area represents the number of tiles and number of surfaces being
imaged.
• If the flow cell is imaged on the top surface only, a single-column graphic appears.
• If the flow cell is image on the top surface and bottom surface, a 2 column graphic
appears.

A 2 tiles, top surface only


B 4 tiles, top and bottom surface
} Q-Score All Cycles: The average percentage of bases greater than Q30, which is a
quality score (Q-score) measurement. A Q-score is a prediction of the probability of a
wrong base call. Q-scores are calculated after cycle 25.
} Flow Cell: The current temperature of the flow cell indicated by color. Blue indicates
cooler temperatures, while orange and red indicate warmer temperatures. During
imaging, imaged tiles appear dark gray. During chemistry, wavy lines indicate that
reagents are pumping through the flow cell.
} Cluster Density (K/mm²): The number of clusters per square millimeter for the run.
} Clusters Passing Filter (%): The percentage of clusters passing filter based on the
Illumina chastity filter, which measures quality. This data appears only after cycle 25.
} Estimated Yield (Mb): The projected number of bases called for the run, measured in
megabases. This data appears only after cycle 25.

Analyzing the Run


When the run is complete, the Next button appears.
1 Review the results on the Sequencing screen before proceeding.
The Sequencing screen remains viewable until Next is selected. After selecting Next,
you cannot return to this screen.
2 Compare the output to these specifications:
Specifications for a 2x75 PhiX Verification Run
• Quality Scores: > 85% bases higher than Q30 at 2x75 bp
• Output: 3.3–3.8 Gb
3 Verify the verification run has uploaded to BaseSpace under the correct instrument
serial number, if applicable.

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NOTE
If the serial number is not correct, that likely indicates an issue with the
CloudAPIkey.xml file.

4 Select Next to exit the Sequencing screen and proceed to the post-run wash.

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Perform a Run Using MiSeq Reporter
Perform a Run Using MiSeq Reporter
If MiSeq Reporter is installed on the instrument, follow these instructions to perform the
verification run. If Local Run Manager is installed, follow the instructions under Perform a
Verification Run Using Local Run Manager on page 60.

Set Up a Run Using MiSeq Reporter


Follow the prompts in MiSeq Reporter to set up and start a run.
NOTE
If the instrument is unable to register the barcode of the flow cell, PR2 bottle, or reagent
cartridge, you can manually enter the barcode using the online keyboard. For the flow
cell or PR2 bottle, select the keyboard icon in the lower right corner of the screen. For
the reagent cartridge, press the Enter Reagent Kit Barcode button.
1 Download or create a sample sheet.
• If available on ICE, download the sample sheet for MiSeq Reporter.
• Or create a sample sheet following the example shown here.
Change <date> to the current date. Do not modify any other values (eg, 1A must
read [Header])
Figure 67 Example of Sample Sheet

2 Upload the sample sheet to this folder on the instrument:


D:\Illumina\MiSeq\SampleSheets
3 Log in to the sequencing instrument.
4 Select Sequence.
5 Press Next—Do not select the BaseSpace option.
This option might not be displayed.
6 Select Run.

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Verification

Clean the Flow Cell


The flow cell is immersed in storage buffer in a flow cell container. Clean the flow cell as
follows before loading it on the instrument.
1 Put on a new pair of powder-free gloves.
2 Using plastic forceps, grip the flow cell by the base of the plastic cartridge and remove
it from the container.
Figure 68 Remove Flow Cell from Container

3 Lightly rinse the flow cell with labaoratory-grade water until both the glass and plastic
cartridge are thoroughly rinsed of excess salts.
Excess salts can affect flow cell seating on the instrument. If salt drys in the imaging
area, imaging can also be affected.
Figure 69 Rinse the Flow Cell

4 Using care around the black flow cell port gasket, thoroughly dry the flow cell and
cartridge with lint-free lens cleaning tissue; gently pat dry in the area of the gasket and
adjacent glass.

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Figure 70 Flow Cell Ports and Gasket

5 Clean the flow cell glass with an alcohol wipe, making sure that the glass is free of
streaks, fingerprints, and lint or tissue fibers.
NOTE
Do not use the alcohol wipe on the flow cell port gasket. Make sure that the
cartridge is securely closed before use.

Figure 71 Dry the Flow Cell

6 Dry excess alcohol with a lint-free lens cleaning tissue.


7 Make sure that the flow cell ports are free of obstructions and that the gasket is well-
seated around the flow cell ports.
If the gasket appears to be dislodged, gently press it bakc into place until it sits securely
around the flow cell ports.inspect it to make sure that no tissue particles are obstructing
the ports.

Load the Flow Cell


NOTE
The instrument must be initialized and homed before loading a flow cell.
1 In MCS, select Sequence from the Welcome screen.
If you are using MTS, click the Motor tab, then click Home Y.
2 Raise the flow cell compartment door, and press the release button to the right of the
flow cell clamp.

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The flow cell clamp opens.
Figure 72 Flow Cell Latch Button

3 Make sure that the flow cell stage is free of lint.


If lint or other debris is present, clean the flow cell stage using an alcohol wipe or a
lint-free tissue moistened with ethanol or isopropanol. Carefully wipe the surface of the
flow cell stage until it is clean and dry.
4 Holding the flow cell (or OTT) by the edges, place it on the flow cell stage.
• Align the top notch around the top pin, and the 2 holes on the right onto the 2
right-hand pins.
• Make sure that the label is facing upward.
Figure 73 Loading a Flow Cell or OTT

A Three alignment pins

5 Gently press down on the flow cell clamp to close it over the flow cell.
As the flow cell clamp closes, alignment pins position the flow cell. An audible click
indicates that tthe flow cell clamp is secure.

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Figure 74 Close the Flow Cell Clamp

6 Close the flow cell compartment door.


7 In MCS, select Next on the Load Flow Cell screen. The Load Reagents screen opens.
In MTS, select a tile, then click Move to Tile.

Load the Reagents


1 Remove the bottle of PR2 from 2° to 8°C storage.
2 Invert to mix, then remove the lid.
3 Open the reagent compartment door.
4 Remove the wash bottle and load the PR2 bottle.
5 Remove the waste bottle, empty the contents into an appropriate waste container, and
replace the waste bottle.
6 Slowly lower the sipper handle, making sure that the sippers lower into the PR2 and
waste bottles.
7 Make sure that the PR2 bottle barcode displays.
8 Select Next.
9 Load the reagent cartridge, and wait for the barcode to display.

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Figure 75 Reagent Cartridge, PR2 Bottle, and Waste Bottle Placement

A Reagent cartridge
B PR2 bottle
C Waste bottle

10 Close the reagent compartment door.

Start the Run—MiSeq Reporter


1 Specify a sample sheet as follows:
a Select Change Sample Sheet.
b Browse to and select the sample sheet.
c Select Save and Continue, then press Next.
Figure 76 Change Sample Sheet Button

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2 Review your run setup, then press Next.
The information in this screen capture is an example.
Figure 77 Review Run Setup

3 If the pre-run check is successful, select Start Run.


Figure 78 Pre-run Check Successful

Leave the used flow cell in the instrument when the run has finished. Use it for the post-
run wash.

Monitor and Analyze the Run

Monitoring the Run


Monitor run progress, intensities, and quality scores displayed on the Sequencing screen
(view only). To monitor the run in greater detail, use the Sequencing Analysis Viewer (SAV
installed on another computer independent of the instrument computer. A network
connection is required.
} Run Progress: Shows the run progress in a status bar and lists the number of cycles
complete.

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} Intensity: The value of cluster intensities of the 90th percentile for each tile.The graphic
in the intensity area represents the number of tiles and number of surfaces being
imaged.
• If the flow cell is imaged on the top surface only, a single-column graphic appears.
• If the flow cell is image on the top surface and bottom surface, a 2 column graphic
appears.

A 2 tiles, top surface only


B 4 tiles, top and bottom surface
} Q-Score All Cycles: The average percentage of bases greater than Q30, which is a
quality score (Q-score) measurement. A Q-score is a prediction of the probability of a
wrong base call. Q-scores are calculated after cycle 25.
} Flow Cell: The current temperature of the flow cell indicated by color. Blue indicates
cooler temperatures, while orange and red indicate warmer temperatures. During
imaging, imaged tiles appear dark gray. During chemistry, wavy lines indicate that
reagents are pumping through the flow cell.
} Cluster Density (K/mm²): The number of clusters per square millimeter for the run.
} Clusters Passing Filter (%): The percentage of clusters passing filter based on the
Illumina chastity filter, which measures quality. This data appears only after cycle 25.
} Estimated Yield (Mb): The projected number of bases called for the run, measured in
megabases. This data appears only after cycle 25.

Analyzing the Run


When the run is complete, the Next button appears.
1 Review the results on the Sequencing screen before proceeding.
The Sequencing screen remains viewable until Next is selected. After selecting Next,
you cannot return to this screen.
2 Compare the output to these specifications:
Specifications for a 2x75 PhiX Verification Run
• Quality Scores: > 85% bases higher than Q30 at 2x75 bp
• Output: 3.3–3.8 Gb
3 Verify the verification run has uploaded to BaseSpace under the correct instrument
serial number, if applicable.
NOTE
If the serial number is not correct, that likely indicates an issue with the
CloudAPIkey.xml file.

4 Select Next to exit the Sequencing screen and proceed to the post-run wash.

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Perform a Post-Run Wash
Perform a Post-Run Wash
Always perform a post-run wash after completing a sequencing run. If a wash was not
performed after the last run, a reminder is displayed.

Items Required
Table 12 Items Required for Post-Run Wash
Items Quantity Part Number

Wash tray 1 15027895

Wash bottle, 500 ml (empty PR2 bottle) 1 11230581

DI water As required Customer supplied

Tween 20 As required Customer supplied

Flow cell used for the verification run 1 N/A

Perform a Post-Run Wash


NOTE
Use the flow cell from the verification run for the post-run wash.
1 Prepare fresh wash solution with Tween 20 and laboratory-grade water:
a Add 5 ml 100% Tween 20 to 45 ml laboratory-grade water (or DNase-free and
RNase-free water if MiSeq FGx). The result is 10% Tween 20.
b Add 25 ml 10% Tween 20 to 475 ml laboratory-grade water (or DNase-free and
RNase-free water if MiSeq FGx). The result is a 0.5% Tween 20 wash solution.
c Invert 5 times to mix.
2 Prepare the wash components:
a Add 6 ml wash solution to each reservoir of the wash tray.
b Add 350 ml wash solution to the 500 ml wash bottle.
3 When the run is complete, select Start Wash.
The sippers are raised in the reagent chiller.
4 Follow the prompts to load the flow cell (use the flow cell from the verification run),
wash tray, and wash bottle.
a Remove the PR2 bottle and replace it with the wash bottle.
b Remove the waste bottle, discard the contents appropriately, and return the waste
bottle to the reagent compartment.
c Slowly lower the sipper handle, making sure that the sippers lower into the wash
bottle and waste bottle.
d Close the reagent and chiller compartment doors.
5 Select Next to start the wash.
6 When the post-run wash is complete, select Done.
The Welcome screen is displayed, and you are ready to start another sequencing run.
7 Leave the used flow cell, wash tray, and wash bottle containing the remaining wash
solution on the instrument.

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The sippers remain in the down position. Leaving the unused wash solution in the
wash tray and wash bottle prevents the sippers from drying out and air from entering
the system.

Post Run Wash Activities


When the post-run wash is complete, follow these steps.
1 Do one of the following:
• For installations: leave the flow cell and wash tray, and PR2 bottle on the
instrument.
• For other maintenance: remove the flow cell, wash tray, and PR2 bottle. Clean as
appropriate.
2 Remove the waste bottle.
3 Discard the contents of the waste bottle appropriately and according to site standards.
WARNING
A component in this set of reagents contains formamide, an aliphatic amide that is a
probable reproductive toxin. Personal injury can occur through inhalation, ingestion,
skin contact, and eye contact.
Dispose of containers and any unused contents in accordance with the governmental
safety standards for your region.
The SDSs for this kit are available at www.illumina.com/sds.
4 Empty the wash tray.
5 Using an alcohol wipe or a lint-free tissue moistened with ethanol or isopropanol,
clean the floor of the reagent compartment and any visible spills or condensation.
6 Return the empty waste bottle to the compartment.
7 Using an alcohol wipe or a lint-free tissue moistened with ethanol or isopropanol,
carefully wipe the surface of the flow cell holder until it is clean.

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  Chapter 5  Final Installation Tasks

Final Installation Tasks

Chapter 5
Final Installation Tasks at the Customer Site 81
After Leaving the Customer Site 82

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Final Installation Tasks
Final Installation Tasks at the Customer Site
1 Be sure that you have entered all the required information on the IQ/OQ.
2 Delete all through-focus image stacks and other test data, including these files from
your Top and Bottom folders:
• TIFF files
• .mat files
Do not delete the PNG files.
3 Copy these folders and file into the Install Diagnostics folder:
• Top and Bottom folders
• Software-Firmware.png file
4 Copy these files and folder onto a USB drive:
• Install Diagnostics folder
• MiSeqOverride.cfg file
• CloudAPIKey.xml file
NOTE
The sequencing instrument automatically includes a copy of the MiSeqOverride.cfg file
and the CloudAPIKey.xml file in every run folder.
5 Optional: If preferred by the customer, change the display mode on the instrument to
Kiosk mode.
6 If no IQ/OQ was required, complete the installation certification, and leave a copy with
the customer (see Reference Documents on page 2).

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After Leaving the Customer Site
After Leaving the Customer Site
After leaving the customer site:

} Update the Field Asset Page.


} Complete a Field Visit Report.
} Close the case.
1 In SFDC:
a Optional: Designate at least one contact at the account as appropriate to receive
quality notifications.
Set the Role drop-down to Lab Manager or Principal Investigator.
b Guidelines for updating the Field Asset page.
— First select MSR Not Applicable or LRM Not Applicable. After making your
selection, N/A is written in the nonapplicable section.
— Update firmware
— Update software
— Enter the Software/Firmware Verification Date
— Select N/A for all fields that are not applicable
Figure 79 Field Asset Page Settings (example only)

2 Upload these files to the asset page in SFDC:


IMPORTANT
For compliance, it is critical that you upload these files to the asset page in SFDC.
• Install Diagnostics folder (test results)
• MiSeqOverride.cfg file
• CloudAPIKey.xml file
• Verification run results

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— If Local Run Manager: Field QC report output file
— If MiSeq Reporter: InterOp folder, Runparameters.xml, and Runinfo.xml
• Completed IQ/OQ
• Completed MiSeq System Performance Qualification (document # 15070408) (Local Run
Manager only)
3 Complete a Field Visit Report.
4 Close the case by following the instructions in WI, Installation Process (1000000009934).

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Appendix A  Troubleshooting

Troubleshooting

Appendix A
Imaging Module Components 85
Camera 2 Center Adjustment 87
Camera XY Offset Adjustment 91
Chromatism Adjustment 96
Compensator Adjustment 99
Flow Cell TEC Thermal Calibration 101
Illuminated Area Adjustment 107
Lane Center Adjustment 113
Lane Rotation Adjustment 116
Check Tip/Tilt 119
Tip/Tilt Adjustment—Camera 2 125
Tip/Tilt Adjustment—Camera 1 129
Z-Motor Rough Position Adjustment 131
Generate Through Focus Image Sets (Stacks) 136
About MTS 141
Manually Check Reagent Delivery 149

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Imaging Module Components
The imaging module components are illustrated in this figure.
Figure 80 Imaging Module Components

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Imaging Module Components
Optics that are moveable and are used for adjustments are listed purple.
Table 13 Imaging Module Components
Number Part Number Part

1 Camera 1 11 F3 beam splitter


(Sensor 1; Camera offset)

2 Camera 2 12 L8 Collimator (uniformity)


(Sensor 2; Camera
centering)

3 M3 Focus 13 F4
(Sensor 1 X/Y tilt)

4 Red LED 14 M4

5 Green LED 15 M1

6 F1, emissive 16 Objective

7 F8 double bandpass filter 17 F5 prism (not visible)

8 L1 focus 18 Spring loaded screw assembly


(S1 focus, chromatism) (X and Y adjustments)

9 F2, emissive 19 X tip/tilt adjuster


(Camera 2/S2)

10 L2 focus 20 Y tip/tilt adjuster


(Camera 1/S1)

21 Compensator

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Camera 2 Center Adjustment
Always check and, if necessary, adjust camera 2 center when:
} Camera 2 is replaced
} The Z-stage is adjusted or replaced

Check Camera 2 Center


1 Load a super flat beaded flow cell.
2 Using MTS, move to the center of the flow cell.
Center = actualedgeofslide + 14 mm
3 Generate a through-focus image set for the top surface of the flow cell only.
4 Run BoltCameraCenteringD50.exe.
5 Determine if the results displayed for top-bottom and left-right are ≤ 0 ± 0.20 μm.
Figure 81 Example of Good Camera Center

6 Select from the following:

If the … Then …

Top-bottom value and/or the left-right Adjust camera 2 (see Adjust Camera 2
value is > 0.20 μm Center on page 88).

Top-bottom and the left-right values are Camera center is OK.


≤ 0 ± 0.20 μm

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Adjust Camera 2 Center
Follow this procedure to adjust camera 2 center.

Equipment Required
} Indelible marker or other type of marker
} Vertical adjustment tool

Procedure
TIP
Using an indelible marker or other type of marker, draw a line to use as a reference before
making an adjustment.
1 Remove the instrument skins.
2 Adjust camera 2 in the X direction as follows:
a Loosen the X-axis lockdown screw (Figure 82 on page 89).
b Move the camera left or right towards the highest left-right value.
Example Figure 83 on page 89:
Because 1.91 is the highest left-right value; you would move the camera left.
c Tighten the X-axis lockdown screw.
d Run BoltCameraCenteringD50.exe and review the image.
e Continue adjusting the camera position and rerunning
BoltCameraCenteringD50.exe until camera center is within spec.
3 Adjust camera 2 vertically as follows:
a Install the vertical adjustment tool shown in Figure 82.
b Loosen the Y-axis lockdown screw.
c Turn the micrometer on the adjustment tool so that the camera moves in the
direction of the highest top-bottom value.
NOTE
If turning the micrometer counterclockwise, move up the camera flush against the
tool before tightening the Y-axis lockdown screw.
Example Figure 83:
Because 1.97 is the highest top-bottom value; you would move the camera down.
d Tighten the Y-axis lockdown screw.
e Run BoltCameraCenteringD50.exe and review the image.
f Continue adjusting the camera position and rerunning
BoltCameraCenteringD50.exe until camera center is within spec.

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Figure 82 Vertical Adjustment Tool and Screws

A Vertical adjustment tool


B Y-axis lockdown screw
C X-axis lockdown screw

Figure 83 Direction to Move the Camera

NOTE
In this particular image, the camera is already centered. The image is being used to
illustrate the direction in which you would move the camera when center is out of spec.

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Camera 2 Center Adjustment
Verify the Camera Center Adjustment
After camera centering is adjusted, follow these steps:
1 Generate a new through-focus image set.
2 Run the following scripts concurrently:
• BoltCameraCenteringD50.exe
• BoltCameraOffset.exe
The camera centering script indicates whether the adjustment improved camera
centering. The camera offset script indicates whether the XY offset was maintained
between the cameras.
3 Review the data and make adjustments as necessary. Rerun the scripts until all of the
tests pass.
4 Attach CSV files and an image file for both surfaces to the work order.
5 Complete the post-service call tasks (Completing Post-Service Call Tasks on page 1).

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Camera XY Offset Adjustment
Camera XY offsets are measured as part of the BoltPAC.exe and the BoltCameraOffset.exe
scripts. Follow these instructions to adjust camera XY offsets when they are out of spec.
Figure 84 Example of Camera XY Offset Results File

Equipment Required
} Cam adjustment tool (for camera X-offset)
} Camera vertical adjustment tool

Adjust the Y Offset


1 Remove the instrument skins.
2 Load a super flat beaded flow cell.
3 Using MTS, move to any tile.
4 Attach the vertical adjustment tool to Camera 1.

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Camera XY Offset Adjustment
Figure 85 Vertical Adjustment Tool

A Vertical adjustment tool


B Y-offset lockdown screw

5 Loosen the Y-offset lockdown screw.


NOTE
Your instrument might have 2 adjustment screws. Loosen both for Y offset adjustments.
6 Select from the following:

If moving the camera Then …


Down 1. Turn the micrometer counterclockwise (1 turn = 40 μm).


(increases the Y offset 2. Make sure that the screw on the adjustment tool makes
value) contact with the plate. Manually move the camera if
necessary.
3. Cover the imaging module with a blackout cloth.
4. View your image and make further adjustments until the
target is on the cursor.
Be sure that the appropriate camera is turned off.
5. Tighten the Y-offset lockdown screw when finished.

Up 1. Turn the micrometer clockwise (1 turn = 40 μm).


(decreases the Y offset 2. Cover the imaging module with a blackout cloth.
value) 3. View your image and make further adjustments until the
target is on the cursor.
Be sure that the appropriate camera is turned off.
4. Tighten the Y-offset lockdown screw when finished.

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Adjust the X-Offset


If the X-offset is out of spec, adjust this position first.
1 Load a super flat beaded flow cell.
2 Using MTS, move to any tile.
3 Select a target as follows:
a Moving the contrast bar upward dims the beads, thus helping you to identify the
brightest beads and align them more easily.
b Identify an easily recognizable bead or group of beads (target) on the camera 2
image.
c Overlay that with the camera 1 image and locate the target.
d Check the difference in pixels in X between the target on camera 2 and the target on
camera 1.
e If the difference is as indicated from the results file, turn off the image on camera 2.
f While looking at the camera 1 image, move the cursor the appropriate number of
pixels to where the target is on camera 2, and leave it positioned there.
NOTE
Always make your adjustments based on the number of pixels recommended in
the results file. Do not base your adjustment solely on visualization of a target
that you have identified in the image.

Figure 86 Camera 2 Target

A Point on camera 2 identified

Figure 87 Overlay of Camera 2 and Camera 1 Images

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Camera XY Offset Adjustment
A Confirm that the difference between the target locations in each image is the same as
indicated by the results file.

4 Loosen the X-offset lockdown screw.


Figure 88 X-Offset Lockdown Screw

5 Select from the following:


Guidelines
• Offset too negative: move the camera toward the back of the instrument
• Offset too positive: move the camera toward the front of the instrument
• Turning the cam tool counterclockwise moves the C beads to the right on the
camera image (ie, moving the camera to the left moves the C beads to the right on
the camera image).

If moving the camera Then …


To the left 1. Place the cam adjustment tool in 1 of the holes to the right
of the camera (Figure 89 on page 95).
2. Turn the tool clockwise to push the camera to the left.
3. Cover the imaging module with a blackout cloth.
4. View the image, and make further adjustments until the
target is on the cursor.
Be sure that the appropriate camera is turned off.
5. Tighten the lockdown screw.

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If moving the camera Then …


To the right 1. Place the cam adjustment tool in 1 of the holes to the right
of the camera.
2. Turn the tool counterclockwise 1/4 to 1/2 turn to create a
space, leaving the tool in place.
3. Move the camera to the right up against the tool.
4. Cover the imaging module with a blackout cloth.
5. View your image and make further adjustments until the
target is on the cursor.
Be sure that the appropriate camera is turned off.
6. Tighten the lockdown screw.

Figure 89 Holes for Cam Tool—Camera 1

Verify the XY Offset Adjustment


1 Generate 2 through-focus image sets (top and bottom surfaces of the flow cell).
See Generate Through Focus Image Sets (Stacks) on page 136.
2 In MTS, run these scripts concurrently:
• BoltTileTilt.exe (top and bottom surfaces)
Offset adjustments affect tip/tilt on camera 1; ideally camera 2 is not affected.
• BoltChromatism.exe (top and bottom surfaces)
If tip/tilt is affected, chromatism can be affected as well.
• BoltCameraOffset.exe (top surface only)
See Running Scripts in MTS on page 144 for instructions.
3 Review the results of each script, and make additional adjustments accordingly.
4 Attach CSV files for both surfaces to the work order.
5 Complete the post-service call tasks (Completing Post-Service Call Tasks on page 1).

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Chromatism Adjustment
Chromatism Adjustment

Equipment Required
} L1 adjustment tool
} Phillips screwdriver

Run BoltChromatism.exe
1 Load a super flat beaded flow cell.
2 Using MTS, move to any tile.
3 Generate 2 through-focus image sets (top and bottom of flow cell).
You can use any tile to extend the life of the flow cell. See Generate Through Focus Image
Sets (Stacks) on page 136.
4 Run BoltChromatism.exe for each image set.
See Running Scripts in MTS on page 144 for instructions.
5 View the results files.
A in Figure 90 indicates that the instrument has 15 microns of chromatism.
6 If chromatism is out of spec, adjust L1 as recommended in the last line of the results
file (B in Figure 90).
IMPORTANT
BoltPAC.exe chromatism results include only the information in the row labeled A in
Figure 90. The direction (negative or positive sign) of the recommended correction is the
opposite of the correct direction. BoltChromastism.exe results include the information in
rows A and B. The direction of the adjustment in the row labeled B is correct.

Figure 90 Example of the BoltChromatism.exe Results File

A BoltChromatism.exe result
B Adjustment recommended to improve chromatism

7 Attach CSV files for both surfaces to the work order.

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8 Complete the post-service call tasks (Completing Post-Service Call Tasks on page 1).

Adjust L1
1 Remove the instrument skins.
2 Install the L1 adjustment tool as follows:
a Make sure that the micrometer is installed as shown in Figure 91 so that you can
use the horizontal line on the barrel as a reference when turning the knob. Loosen
the nut to reposition the barrel.
b Position the tool in groove on base plate.
c Loosely tighten the adjustment tool lockdown screw.
d Tighten the micrometer to the point where it touches the lens and the ball spring is
slightly compressed.
e Lock down the tool.
Figure 91 L1 Adjustment Tool Installed

A Groove and screw hole for L1 adjustment tool


B Ball spring slightly compressed
C Micrometer tightened so that it touches the lens
D Lens lockdown screw
E 10 μm marks on lockdown screw

3 Loosen the L1 lockdown screw.

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Chromatism Adjustment
4 Turn the micrometer the appropriate number of minor tick marks clockwise or
counterclockwise (moving the micrometer from 20 to 30 = a 100 μm adjustment).
Example based on Figure 90:
Adjust L1 by: 15 μm towards S1 = turn the micrometer 15 μm counterclockwise.

If the numerical adjustment value Then…


is…

Positive 1. Turn the micrometer counterclockwise.


(Increases the chromatism offset values.)
2. Butt up the lens against the micrometer.

Negative Turn the micrometer clockwise.


(Decreases the chromatism offset values.)

5 Tighten the lens lockdown screw, and back out the micrometer.
6 Reinstall the optical covers (no need to tighten them down).
7 Cover the imaging module with the blackout cloth.
8 Generate new through-focus image sets.
9 Run BoltChromatism.exe and assess the results.
10 Select from the following:

If the BoltChromastism.exe results Then …


are …

Within spec 1. Remove the L1 adjustment tool.


2. Generate a new through-focus image set.
3. Run BoltPACnoD50.exe (checks tip/tilt,
camera offset, and chromatism).
4. Assess the results and make adjustments
accordingly.

Out of spec 1. Continue adjusting L1 and rerunning


BoltChromatism.exe until chromatism is
within spec.
2. Focus the bead image and generate a new
through-focus image set.
3. Run BoltPACnoD50.exe (checks tip/tilt,
camera offset, and chromatism).

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Compensator Adjustment
For a brief description of Z-stage travel and the compensator, see About Z-Stage Travel and
the Compensator on page 131.
Adjusting the compensator affects only the X value of tile tilt for the bottom surface of the
flow cell. If the problem is common for both the top and bottom flow cell surfaces, you
typically adjust the optics plate. Follow these instructions to adjust the compensator.
1 Loosen the 2 M4 screws using a 3 mm Allen wrench.
CAUTION
Loosen the 2 M4 screws first
2 Loosen the M3 screw using a 2.5 mm Allen wrench.
3 Slide the compensator tilt adjuster to the left or right.
Left = increases the X value
Right = decreases the X value
4 Tighten the M3 screw.
Figure 92 Compensator Adjustment

A Compensator adjuster
B M3 screw
C M4 screws

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Compensator Adjustment
5 Firmly push up the compensator against the Z-stage by applying forward pressure and
diagonal pressure to the left.
Figure 93 Apply Forward and Diagonal Pressure From Right to Left

6 Holding the compensator firmly against the Z-stage, tighten down the M4 screws.
7 Make sure that the compensator is firmly in place by trying to move it.
8 Using MTS or the sequencing instrument, check tip/tilt on the bottom surface of a super
flat beaded flow cell.

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Flow Cell TEC Thermal Calibration
The Peltier heaters in this instrument are referred to as thermoelectric coolers (TECs).
NOTE
For the best accuracy:
• Make sure that the skins are on the instrument.
• Close the flow cell compartment door so that no outside air can flow around it and affect
the calibration.

Equipment Required
} Flow cell, open end
} Fluke meter
} Kapton tape

Prepare the Instrument for Flow Cell TEC Calibration

Decontaminate the Instrument


Decontaminate the instrument before performing any repair, part removal or replacement,
or other alteration. Follow the decontamination procedure described in part # 15028850,
Instrument Decontamination.

Set Up the Instrument for Flow Cell TEC Calibration


1 Make sure that the skins are on the instrument.
2 Using MTS, initialize and home the instrument.

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Flow Cell TEC Thermal Calibration
3 Affix a thermocouple to the flow cell using Kapton tape as follows:
a Position the filament at the end of the thermocouple to the area of the flow cell that
sits between the 2 pressure pins of the flow cell clamp.
This area has the best surface contact between the flow cell and the thermal plate.
b Using one piece of tape, attach the thermal filament to the flow cell only (not the
plastic sheathing of the wire), and make sure that no air bubbles are present.
Air bubbles skew temperature readings.
c Secure the sheath part of the wire for stability using another piece of tape.
d Using 1 of your fingernails, press out as much of the air between the tape and
thermal filament as possible.
Figure 94 Thermocouple Affixed to Flow Cell

4 Load the flow cell onto the instrument.


CAUTION
Proper seating of the flow cell is critical for accurate temperature calibration.
5 Pool a few inches of thermocouple line in the stage area, then close the flow cell
compartment door.
IMPORTANT
Make sure that you leave a few inches of thermocouple line in the stage area. If you do
not leave extra line, it can be pulled out from under the Kapton tape as the stage moves
in—Ruining your best tape job ever.
6 In the chiller, make sure that there is ample reagent or water in position 3 (PR2 bottle).
7 Lower the sipper into the PR2 bottle, and close the chiller door.
8 Fill the flow cell with liquid as follows:
a On the Pump tab, open the Solution drop-down menu and select [PR2, 3].
b In the Volume field, enter 500, then click Pump.

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Calibrate the Flow Cell TEC


1 On the Motor tab, type 74 in the Y Motor Position field, and hit Enter to move the flow
cell stage all the way into the instrument.
2 On the Temperature tab, reset the slope and offset to 1 and 0 as follows:
a Open the Controller drop-down menu, and select FlowCellTempControl.
b In the Calibration pane, enter 16 and 96 into the Low and High Measured
temperature fields respectively.
c Click Calculate; then click To E-Box.
Figure 95 Reset the Slope and Offset

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Flow Cell TEC Thermal Calibration
3 Determine the low measured temperature as follows:
a In the Temperature Adjustment pane, enter 16 in the Target field, and click Set.
b Select Enable.
Figure 96 Temperature Adjustment Pane

c When the graph has stabilized, record the Fluke meter reading.
d Deselect Enable.
4 Determine the high measured temperature as follows:
a Enter 96 in the Target field, and click Set.
b Select Enable.
c When the graph has stabilized, record the Fluke meter reading.
d Deselect Enable.
5 Calibrate the flow cell TEC as follows:
a In the Calibration pane, make sure that 16 and 96 are the values displayed in the
Low and High Target fields.
b Enter the Fluke meter readings in the Low and High Measured fields.

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c Click Calculate, then click To E-Box.
Figure 97 Calibration Pane

6 Do the following:
a Repeat step 3. Make sure that the value is 16° C ± 2° C.
b Repeat step 4. Make sure that the value is 96° C ± 2° C.
c If the either of the values is not within spec, the TEC was not calibrated. Repeat
this procedure.
7 Enter these values in the MiSeqOverride.cfg file:
a The high measured temperature from the Fluke meter that you entered into MTS to
calibrate the flow cell TEC (step 4).
b The low measured temperature from the Fluke meter that you entered into MTS to
calibrate the flow cell TEC (step 3).
c Save and close the file.
Figure 98 Measured High and Low Temperatures in the MiSeqOverride.cfg File

8 Check the calibration as follows:


d Launch MTS.
e Run the Thermal Ramp.
f If the test passes, the flow cell TEC has been successfully calibrated.

Qualify the Flow Cell TEC


1 Click the Temperature tab.
2 Open the Controller drop-down and select FlowCellTempControl.
3 In the Temperature Adjustment box:
a Select the Enable checkbox.
b Enter 16 in the Target field, and click Set.
c Wait 30 seconds while observing the temperature graph.
d Note the value displayed as the Actual temperature.

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Flow Cell TEC Thermal Calibration
You can use the mouse to drag the temperature graph (click and drag).
e Now enter 96 in the Target field, and click Set.
f Wait 30 seconds while observing the temperature graph.
g Note the value displayed as Actual temperature.
h Deselect Enable.
4 Select from the following:

If the results are … Then …

Within spec The flow cell TEC is OK.


Record the actual values on theIQ/OQ.

Out of spec Calibrate the flow cell TEC as described under Flow
Cell TEC Thermal Calibration on page 101.

Figure 99 Flow Cell Thermal Test

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Illuminated Area Adjustment
A good illuminated area shows a sphere of beads centered or mostly centered in the
camera area. Beads are visible along every edge of the image. Figure 100 is an example of a
good illuminated area. To adjust the illuminated area, move L8 in X and/or Y.
Figure 100 Examples of Good Illuminated Area in X and Y

A Borders of camera image area

Prepare the Instrument for Illuminated Area Adjustment


1 Remove the instrument skins.
2 Load a super flat beaded flow cell.
3 Launch MTS, initialize, and home the instrument.
4 Focus the beads (see Find Best Focus on the Top or Bottom Surface of the Flow Cell on page
1).
5 In the Image tab, click Zoom All.

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Illuminated Area Adjustment
Adjust L8 in X
The image in Figure 101 shows the illuminated area out of spec in X.

Camera Image X Value Maximum and Minimum

Left side of image < 680

Right side of image > 3620

Notice the X value on the right side of the image is 3212, which is < 3620.
Figure 101 Illuminated Area Out of Spec in X

Follow these steps to adjust L8 in X (side-to-side; Figure 102):


1 Loosen screw A using a 2.5 mm Allen wrench.
2 Remove screw B using a 1.5 mm Allen wrench.
3 Gently move L8 slightly to the left or right as needed.
4 Cover the imaging module with the blackout cloth.
5 View the image, and measure the X value on right and left.
6 Continue making small adjustments to L8 until X is > 3620 on the right, and < 680 on
the left.
7 Gently tighten down screw B.

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8 Reinstall screw A.
Figure 102 X Adjustment Screws

A Loosen this screw.


B Remove this screw.

Figure 103 Illuminated Area Adjusted in X

A Left edge 664 (X < 680)


B Right edge 3698 (X > 3620)

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Adjust L8 in Y
NOTE
Adjusting the contrast helps show the camera edges.
When Y is properly aligned, the top and bottom edges of the illuminated area are outside
the imaged area, ie no blank strip on either the top or bottom of the image.
Figure 104 Illuminated Area Out of Spec in Y

A Borders of camera image


B No beads between bottom of camera image and bead clump
To adjust L8 in Y (up and down; Figure 106):
1 Loosen screw A using a 2.5 mm Allen wrench.
2 Adjust screw B using a 1.5 mm Allen wrench.
3 Gently move the lens up or down as needed.
4 Cover the imaging module with the blackout cloth.
5 View and assess the image.

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6 Continue making small adjustments to L8 until the beads run off the top and bottom of
the camera imaging area.
Figure 105 Illuminated Area Adjusted in Y

7 Tighten down screw B.


8 Reinstall screw A.
Figure 106 X Adjustment Screws

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Illuminated Area Adjustment
Qualify the Adjustment
1 Refocus the beads and generate a new through-focus image set.
2 Run the BoltUniformity.exe and review the results.
Figure 107 Example of BoltUniformity.exe Results

3 If you are unable to bring the illumination area into spec by adjusting L8:
a Measure the LED power as described under Check the LED Power on page 44.
b If the power is too low, replace the LED assembly.
4 Attach CSV files for both surfaces to the work order.
5 Complete the post-service call tasks (Completing Post-Service Call Tasks on page 1).

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Lane Center Adjustment
To check and adjust lane center, follow these procedures:
} Check Lane Center and Rotation on page 30
} Lane Center Adjustment on page 113

Lane Center Adjustment


Follow this procedure to adjust the lane center.

Equipment Required
} Blackout cloth
} OTT

Prepare the Instrument for Lane Center Adjustment


1 Remove the instrument skins.
2 Loosen the screws on the front and back of the instrument as indicated here.
Figure 108 Optical Stage Lockdown Screws for Lane Center Adjustments

3 Cover the imaging module with the blackout cloth, making sure that the cloth does not
cover the rear fan.

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Lane Center Adjustment
Adjust Lane Center
1 Load an OTT.
2 Using MTS, move to tile 6.
3 Go to the Camera tab and:
• Select Camera 2; deselect Camera 1
• Select the G channel, and adjust the exposure time until the image quality is
acceptable.
If the G channel image is not usable, try other channels and exposure times until
the image quality is acceptable.
• Click Continuous.
Figure 109 Camera Tab

4 Bring the crosshair into sharp focus.


5 Move the cursor to the left or right until the X value reads 2141.
Figure 110 Cursor at X = 2141

6 Move the lane into spec as follows:


a Leaving the cursor at 2141, turn both adjustment screws at the same time either
towards you or away from you.
Towards you = lane moves to the right
Away from you = lane moves to the left

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Figure 111 Adjustment Screws for Lane Centering

b Read the X value.


c Continue turning the front adjustment screws and reading the X value until the left
edge of the lane is at 2141.
Figure 112 Edge of Lane at X = 2141

7 Tighten the optical stage lockdown screws as follows:


a Turn the front and rear lockdown screws until they are barely tight.
b View the image and make sure that the lane is still at 2141.
c Tighten the lockdown screws a little more and view the image again.
d If the edge of the lane is still at 2141, finish tightening down the screws.
If the lane moves while tightening down the optical stage, loosen the screws,
readjust the lane center position, and tighten the lockdown screws again.
8 Confirm that lane center is within spec as described under Check Lane Center and
Rotation on page 30.
9 If necessary, continue adjusting lane center until it is within spec.
10 Reinstall the instrument skins.

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Lane Rotation Adjustment
Lane Rotation Adjustment
When lane rotation is out of spec, the 2 guide pins on the right side of the flow cell holder
are either bent or are rotated relative to each other. To remove rotation, adjust the position
of these pins, which are on the flow cell TEC.
To check and adjust lane rotation, follow these procedures:
} Check Lane Center and Rotation on page 30
} Adjust Lane Rotation on page 116

Equipment Required
} OTT
} Beaded flow cell
} Y-stage alignment tool
} 2.5 mm and 3 mm Allen wrenches
} Phillips head screwdriver

Adjust Lane Rotation


1 Remove the instrument skins.
2 Open the flow cell compartment door.
3 Using a Phillips-head screwdriver, remove the 2 flow cell holder plate screws, and lift
off the plate.
Figure 113 Flow Cell Holder Plate Pins and Cover Plate Screws

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4 Using a 3 mm Allen wrench, loosen the 4 outer screws.
Figure 114 Four Outer Screws

5 Install the Y-stage alignment tool as follows:


a Place the tool on the Y-stage.
b Position the tool by gently pushing it forward and to the left.
c Secure the tool to the Y-stage by turning the knob on the side of the tool.
Figure 115 Y-Stage Alignment Tool Positioning

6 Using a flat head screwdriver, carefully move the TEC so that the 3 TEC alignment
pins are seated up against the alignment tool.

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Lane Rotation Adjustment
Figure 116 Seating TEC Pins Against Alignment Tool

A Carefully insert the flat head screwdriver in these slots to move the TEC

7 Tighten the 4 outer screws.


8 Remove the Y-stage alignment tool.
9 Reinstall the flow cell holder cover plate.
10 Check both lane center and rotation ( Check Lane Center and Rotation on page 30).

Qualify Lane Rotation


Adjusting lane rotation affects the alignment of other optics such as tip/tilt. Check the
following specs in the order shown. Make adjustments as necessary.
1 Best focus Z-position
2 Tip/Tilt
3 Chromatism
4 Camera XY offset

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Check Tip/Tilt
Use the sequencing instrument or the sequencing instrument to check tip/tilt (also referred
to as tile tilt). Always use a super flat beaded flow.

Check Tip/Tilt Using MTS


1 Generate 2 through-focus image sets using a super flat beaded flow cell (see Generate
Through Focus Image Sets (Stacks) on page 136).
NOTE
Move up the compensator before running this script (Compensator tab).
2 Minimize MTS.
3 Go to C:\Illumina | MiSeq Control Software | MatlabScripts
Or go to the release scripts folder on the D: drive.
4 Double-click BoltTileTilt.exe.
A window with a command prompt opens Figure 117.
5 Go to the D drive and open the folders you created for the through-focus image sets.
6 Do the following:
a Drag and drop the numbered and dated folder for one image set onto the
command prompt.
b Click in the command prompt window, and hit Enter.
BoltTileTilt.exe executes in < 1 minute.
c Repeat these steps for the second set of images.
Figure 117 Drag and Drop the Through Focus Image Set Folder Onto the Command Prompt

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Check Tip/Tilt
7 Hit Enter in the BoltTileTilt.exe window.
A status bar is displayed while the script is running. When finished, a results file
(BoltTileTilt_Report_<date–time>.csv) and a tip/tilt image for each camera is generated.
CAUTION
If an error occurs and the script does not execute, generate a new through-focus image
set before running BoltTileTilt.exe again.

Check Tip/Tilt Using the Instrument Control Software


The Tip/Tilt test in the sequencing instrument is an automated version of BoltTileTilt.exe. It
automatically generates the 2 through-focus images sets, and checks tip/tilt on the top and
bottom surfaces of the flow cell.
1 From the Welcome screen, click or touch Manage Instrument.
2 Click or touch System Check.
3 Select the Tip/Tilt Test checkbox.
4 Click or touch Next.
5 At the prompt, load a super flat beaded flow cell.
The test begins.
Figure 118 Tip/Tilt Test in the Instrument Control Software

Review Tip/Tilt Results


Select from the following:

If using … Then …

MTS Navigate to the results folder that you created earlier for each
through-focus image set, double-click the image for Camera 1,
and assess the results.

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If using … Then …

Instrument Select Show Results.


Control Software

The circle is essentially a level. The point on the end of the line represents the bubble. If the
bubble falls outside of the circle, the instrument is out of spec for tile tilt. Here are examples
of what you might see and the type of adjustment required.
Figure 119 Tip/Tilt Images and Results File

A Tip/Tilt out of spec


B Tip/Tilt in spec
Table 14 Examples of Tip/Tilt Results
Tip/Tilt In Spec X Adjustment Y Adjustment X and Y
Required Required Adjustments
Required

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Check Tip/Tilt
Select from the following:

If tip/tilt is … Then …

within spec for cameras 1 You are done.


and 2

out of spec and a manual 1. Review the Brenner Metrics images to make sure that
adjustment are required the through-focus image set was generated
successfully (Review the Brenner Metrics Images on
page 122).
2. If tip/tilt must be adjusted, follow the instructions
listed under:
• Tip/Tilt Adjustment—Camera 2 on page 125 or
• Tip/Tilt Adjustment—Camera 1 on page 129

Review the Brenner Metrics Images


Successful generation of a through-focus image set is shown in Figure 120. Both cameras
are tested (Green = G-channel on camera 2; Blue = T-channel on camera 1). Each plot on the
bell curves represents an image from the through-focus image set (1 through 41).
Figure 120 Good Through Focus Image Set Generation Graph

A Y-axis = image sharpness metric


B X-axis = image number
C Best focus image on each camera

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If the beaded flow cell is focused on the wrong (bottom) surface of the flow cell, a Brenner
graph similar to the one shown here is generated.
Figure 121 Beaded Flow Cell Focused on Wrong (Bottom) Surface

Figure 122 illustrates failed through-focus image set generation. A graph with this
appearance can be caused by not optimizing the Z-motor position before generating the
through-focus image set. Repeat the procedure listed under Generate Through Focus Image
Sets (Stacks) on page 136 to optimize the Z-motor position and generate a new set of
through-focus images. Then run BoltTileTilt.exe again.
Figure 122 Failed Through Focus Image Set Generation Due to Instrument Vibration

A Sharp dips in the bell curves caused by instrument vibration

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Check Tip/Tilt
A graph such as the one shown here can be caused by an incorrect value in the Z-Step
field. Repeat the procedure listed under Generate Through Focus Image Sets (Stacks) on page
136 to optimize the Z-motor position and generate a new set of through-focus images. Then
run BoltTileTilt.exe again.
Figure 123 Failed Through Focus Image Set Generation — Incorrect Z-Step Value

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Tip/Tilt Adjustment—Camera 2
When manually adjusting tip/tilt for camera 2, you essentially change the focal plane of the
sample as you make your adjustments. Loosening and tightening the lockdown screws for
the optical stage causes the Y-stage to shift.

Make Sure that a Tip/Tilt Adjustment is Required


If your test results indicate that tip/tilt is out of spec the first time the script is run, rerun the
script.
1 Follow the instructions for checking tip/tilt as listed under Check Tip/Tilt on page 119.
2 View the results.
Table 15 Examples of Tile Tilt Results
Tile Tilt In Spec X Adjustment Y Adjustment X and Y
Required Required Adjustments
Required

3 Select from the following:

If … Then …

the results are within spec tip/tilt is OK.

the results indicate that tip/tilt is out of continue to Adjust Tip/Tilt on page 126.
spec

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Adjust Tip/Tilt
1 Remove the instrument skins.
2 Make sure that the spring loaded screw and the micrometer are touching the optical
stage.
Figure 124 Micrometer and Spring Loaded Screw Just Touching the Optical Stage

A Micrometer (front actuator) touching the optical stage


B Spring loaded screw just touching the optical stage

3 Engage the back actuator.


4 Loosen the front and back adjustment (lock) screws.
Figure 125 Front and Rear Actuators and Adjustment Screws

A Back actuator
B Front actuator

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5 Adjust the actuators per the recommendations in the Camera 2 image.
NOTE
When making X adjustments, be sure that the spring loaded screw is touching the
optical stage. There must be opposing pressure on the stage when the front actuator is
turned to make the adjustment.

Figure 126 Example of Tilt Adjustment Instructions

6 Tighten all of the lock screws to barely snug (do not fully tighten).
a Gently tighten down each screw the same amount until barely snug.
b Gently tighten down each screw a little more by the same amount.
c Finish tightening down each screw.
IMPORTANT
Never fully tighten down one screw at a time. Tilt is reintroduced if the screws are fully
tightened down 1 at a time.
7 Refocus the beads (Motor tab in MTS). If the rough focus is out of spec, adjust the Z-
motor rough position (Adjust the Rough Z-Motor Position on page 131.
8 Enter the new Z-motor value in the Focus tab.
9 Run the tip/tilt test again for both surfaces of the flow cell (Check Tip/Tilt on page 119).
10 If tip/tilt is still out of spec, select from the following:

If tip/tilt … Then …

Is out of spec for the top surface of the flow Return to step 3 and repeat this procedure.
cell

Passes for the top surface of the flow cell, but 1. Adjust the compensator (Compensator
is out of spec for the bottom surface Adjustment on page 99).
2. Repeat this procedure for the bottom
surface only.

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11 When tip/tilt is in spec, tighten down the adjustment screws as follows:
a Gently tighten down each screw the same amount until barely snug.
b Gently tighten down each screw a little more by the same amount.
c Finish tightening down each screw.
IMPORTANT
Never fully tighten down one screw at a time. Tilt is reintroduced if the screws are fully
tightened down 1 at a time.
12 Finish this procedure as follows:
a Run the tip/tilt test again for both surfaces of the flow cell (Check Tip/Tilt on page
119), and make sure that tip/tilt is still within spec.
b Remove the front actuator.
c Back off the spring loaded screw from the optical stage.
d If any settings were modified (eg, camera exposure, LED current), restore the default
values now.
e Reinstall the instrument skins.

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Tip/Tilt Adjustment—Camera 1
NOTE
Before adjusting tip/tilt on Camera 1, make any necessary adjustments to Camera 2 as
described under Tip/Tilt Adjustment—Camera 2 on page 125.
Table 16 Examples of Tip/Tilt Results
Tile Tilt In Spec X Adjustment Y Adjustment X and Y
(point inside the Required Required Adjustments
circle) Required

Adjust Tip/Tilt on Camera 1


1 Remove the instrument skins.
2 Turn each actuator as recommended in the Camera 1 image.
• Screw actuator: Turn using a 2 mm Allen wrench; this actuator has no lock (A).
• Cam actuator: loosen the locking screw using a 2 mm Allen wrench (C). Use a flat-
head screwdriver to turn the cam actuator (B) in the direction recommended in the
Camera 1 image.
IMPORTANT
When rotating the cam actuator counterclockwise, push the mirror back against the
cam. The mirror does not move back on its own. After pushing the mirror against the
actuator, the motor registers the new M3 position.

Figure 127 Camera 1 Actuators on the M3 Mirror

A Screw actuator
B Cam actuator
C Locking screw

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Figure 128 Movement of Mirror Relative to the Direction the Cam is Turned

3 Load a super flat beaded flow cell and move to the center of the flow cell.
Center = actualedgeofslide + 14 mm
4 Cover the imaging module with a blackout cloth.
5 Run the test again on both surfaces of the flow cell (Check Tip/Tilt on page 119).
6 Repeat steps 2 through 5 until tip/tilt is within spec.
7 If the cam actuator was rotated, lock it down now.
8 Run the tip/tilt test again for both surfaces of the flow cell, and make sure that tip/tilt is
still within spec.
9 If any settings were modified (eg, camera exposure, LED current), restore the default
values now.
10 Reinstall the instrument skins.

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Z-Motor Rough Position Adjustment
Follow this procedure to adjust the rough Z-motor position. See Appendix 1: Alignment
and Inspection Specifications for the most current spec.

About Z-Stage Travel and the Compensator


Z-stage travel is 300 microns. The uppermost position is –300 μm; the lowest position is
0 μm. Clusters at the top of the flow cell are at –0.17 mm (upgraded instruments). Clusters
at the bottom of the flow cell are 0.072 mm beneath the top surface. To focus and image
beads on the bottom surface of the flow cell, the compensator is moved into position and
the Z-stage is lowered 0.035 mm.
Figure 129 Z-Stage Travel

Adjust the Rough Z-Motor Position

Prepare the Instrument


1 Remove the instrument skins.
2 Cover the imaging module with a blackout cloth.
3 Launch MTS, initialize, and home the instrument.

Adjust the Rough Z-Motor Position


1 Load a super flat beaded flow cell.
2 Move to the center of the flow cell as follows (Figure 131 on page 132):
a Set the Z-motor position to –0.17.
Figure 130 Z-motor Position

b Open the MiSeqOverride.cfg file and find the value for actualedgeofslide.

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c Add 14.0 mm to the actualedgeofslide value (example: 57.4 + 14.0 = 71.4).
Do NOT change the value for actualedgeofslide in the MiSeqOverride.cfg file.
Figure 131 actualedgeofslide in MiSeqOverride.cfg File

d In MTS, open the Motor tab.


e Type this value into the Y Motor Position field, and hit Enter.
Figure 132 Center of Flow Cell (actualedgeofslide + 14)

3 Visualize the beads on the flow cell as follows:


a Open the Camera tab.
b Deselect Camera 1; select Camera 2.
c Click Continuous.
d In the right pane, click the Image tab and:
e Click the A channel (deselect the C, G, and T channels).
f Click Zoom All and Contrast.
An image of the beads appears.
4 Remove the panel over the Z-stage.
5 Using a 1.5 mm Allen wrench, make sure that the adjustment screw is engaged (A in
Figure 133).
The Z-stage can drop if there is no tension on the adjustment screw before loosening
the lockdown screw.

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6 Loosen the lockdown screws using a 3 mm Allen wrench (B).
Figure 133 Z-Stage without Retaining Bracket

A Adjustment screw
B Straight and angled lockdown screws
C Insert Allen wrench through this opening to loosen the angled lockdown screw

Figure 134 Z-Stage with Retaining Bracket/Screw

7 Adjust the Z-stage height as follows:


a Cover the imaging module with the blackout cloth.
b Keeping the imaging module covered, turn the Z-stage adjustment screw using a
1.5 Allen wrench.
CAUTION
Never loosen or remove the retaining bracket/screw.
Turn the screw clockwise to move the objective farther away from the flow cell.
c As you move the adjustment screw, watch the image to determine if it is more or
less in focus. Change direction if the image is more out of focus.
d Continue turning the adjustment screw and viewing the image until it is in focus.
e Tighten the adjustment lockdown screws (do not over tighten).
8 Visually make sure that the image is still in good focus.

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9 Confirm that you are focused on the top surface of the flow cell (see Confirm Focus is on
Top of Flow Cell on page 134).
10 To determine the effect of this adjustment on the imaging module:
a Generate through-focus image stacks, and run BoltPACnoD50.exe.
b Make adjustments as necessary.
c Generate new through-focus image stacks, and run BoltPACnoD50.exe again.
d Continue making adjustments and running BoltPACnoD50.exe until all tests pass.
e Generate new through-focus image stacks, and run BoltPAC.exe.

Confirm Focus is on Top of Flow Cell


Confirm that the adjusted best focus Z-motor position correlates to the top surface of a flow
cell. The difference in focal plane between the top and bottom surfaces of a MiSeq flow cell
is 0.072 mm (72 microns).
1 In MTS on the Motor tab, enter 0.072 in the Step field.
Figure 135 Step Size Field

2 Click the down arrow.


Correct result: You still see focused bead clusters.
3 Select from the following:

If you see … Then …

Beads that are in You are on the bottom surface of the flow cell. Confirm by
relatively good focus 1. Click the up arrow one time to move back to the top
surface (correct result: you still see focused bead clusters.).
2. Click the up arrow 1 more time. Correct result: No bead
clusters are visible.
The Z-motor position is OK.

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If you see … Then …

Nothing 1. Click the up arrow twice.


If you see beads at this position, you adjusted the Z-motor
position to the bottom of the flow cell. Continue to the
next step.
2. Readjust the rough Z-position as follows:
• Click the down arrow one time to return to -0.17.
• Unlock the Z-stage, and turn the adjustment screw
clockwise until the image is in good focus.
• Lock down the Z-stage.
• Enter 0.072 in the Step field, and click the down arrow.
Correct result: you see beads that are in relatively good
focus (bottom surface).
• Click the up arrow. Correct result: you see beads that
are in good focus (top surface).
• Click the up arrow one time. Correct result: no bead
clusters are visible.

If the beaded flow cell is focused on the wrong (bottom) surface of the flow cell, a Brenner
graph similar to Figure 136is generated.
Figure 136 Brenner Graph Indicating Z-Stage Focused on Wrong (Bottom) Surface

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Generate Through Focus Image Sets (Stacks)
Generate Through Focus Image Sets (Stacks)
To run some of the tests in MTS, you first need to generate 2 through-focus image stacks.
One set is for the top surface of the flow cell, and 1 set is for the bottom surface. Using a
super flat beaded flow cell, you:
} Move to the center of the flow cell
} Focus the beads on the top or the bottom of the flow cell
} Generate a through-focus image stack
NOTE
When an adjustment is made to an imaging module component, generate new image stacks
before repeating the test associated with the adjustment.

Move to the Center of a Super Flat Beaded Flow Cell


1 Launch MTS, initialize the software, and home the instrument.
2 Load a super flat beaded flow cell.
3 Move to the center of the flow cell as follows:
a Open the MiSeqOverride.cfg file and find the value for actualedgeofslide.
b Add 14.0 mm to the actualedgeofslide value (example: 57.4 + 14.0 = 71.4).
Do NOT change the value for actualedgeofslide in the MiSeqOverride.cfg file.
Figure 137 actualedgeofslide in MiSeqOverride.cfg File

c In MTS, open the Motor tab.


d Type this value into the Y Motor Position field, and hit Enter.

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Figure 138 Actualedgeofslide Value and Y Motor Position

Find Best Focus on the Top or Bottom Surface of the Flow Cell
NOTE
When generating an image stack for the bottom surface of the flow cell, it is good practice to
focus the beads on the top surface first. Then move the compensator in, and focus the beads
on the bottom surface of the flow cell.
1 On the Motor tab, set the Mirror Position to Normal for Imaging.
2 Type –0.17 in the Z Motor Position field, and hit Enter.
3 Open the LED tab, change the Green LED Default Snap Curr to 2000, and click Set.
The default snap current of 1500 for the red LED is OK.
Figure 139 LED Tab

4 Open the Camera tab and:


a Deselect Camera 1; select Camera 2.
b Click Continuous.
5 In the right pane, click the Image tab and:
a Click the A channel (deselect the C, G, and T channels).
b Click Zoom All and Contrast (unfocused beads appear).

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Generate Through Focus Image Sets (Stacks)
Figure 140 Unfocused Beads

6 Using the wheel on the mouse and the sliders, zoom in on an area of beads.
See Image Tab in the Right Pane on page 143 for additional information.
Figure 141 Example of Well Focused Beads

If focusing on the … Then …

Top surface Continue to the next step.

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If focusing on the … Then …

Bottom surface 1. Open the Compensator tab.


2. Select the Move Z Motor Simultaneously checkbox.
3. Click Move In (the compensator moves into position, and the Z-
stage is lowered 0.035 mm).
If you do not see beads when the compensator is moved in, you
were focused on the bottom surface. See Confirm Focus is on Top of
Flow Cell on page 134.

Figure 142 Compensator Controls on Compensator Tab

7 On the Motor tab, bring the beads into sharp focus by moving the Z Motor up or down.
Increments of 0.002–0.0005 typically work well.
8 Confirm that the value for best focus is within spec.
9 Turn off the camera.

Generate the Through Focus Image Stack


1 Copy the best focus value in the Z Motor Position field.
2 Open the Focus tab and setup the Through Focus (Mirror Detilted) fields as follows:
You might have to open the tab drop-down list.
Figure 143 Focus Tab Parameters

• Z-Center field: paste the Z-Motor Position value copied from the Camera tab
• Select the Save Images checkbox.
• Deselect Compute Best Focus.
• Output Directory field: browse to the D drive and create a folder for the image
stack (eg, D:\Bottom\20July2012_OutputFolder)

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NOTE
The folder name cannot contain any spaces. Use an _ (underscore). If the folder name
includes spaces, some of the scripts will not run. Add Top or Bottom to the folder name
to distinguish between image stacks for each surface of the flow cell.
• Channels: select all 4 channels
• Exp. Time (ms): change the exposure times to 100, 50, 50, 100 (A, G, T, C
respectively)
• Z-Step field: enter 0.00025
• Number of Steps field: 41
3 Click the Start button.
If the Start button is inactive, make sure that the camera is turned off. The software
generates 41 images and saves them to the designated output directory.
CAUTION
Do not touch the instrument or the bench while through-focus image stacks are being
generated. Avoid walking around near the instrument. Vibration negatively affects the
images and the subsequent tests run with these images.
4 If applicable, move out the compensator (Compensator tab).
5 Repeat this procedure so that you have 2 sets of images (top and bottom surfaces).

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About MTS

MTS Known Issues


IMPORTANT
MTS has the following known issues:
Camera Tab and Continuous
On the Camera tab, when you click Continuous the software turns on both cameras by
default. If you do not turn off one camera, the following issues occur:
• The image moves 600 pixels to the left.
• The colors for the A pixels and the C pixels are switched. C is displayed as red; A is
displayed as yellow.
• The Z-stage becomes unstable. If you manually move the Z-stage, it bounces and might
cause errors.
BoltUniformity.exe and Bad F4 Filter Message
A message that the F4 filter might be bad might be displayed when running the
BoltUniformity.exe script. The F4 filter is typically good.

Using MTS
MTS is used for some instrument installation activities, and for service procedures. This
section includes a brief description of MTS, and includes instructions on how to run the
MATLAB scripts. You can use MTS to run the Bolt scripts used to check instrument
alignment (automated in the first time setup screens). Most of these tests can also be run
from the sequencing instrument (Welcome screen | Manage Instrument).
To begin using MTS:
1 Double-click the MTS icon on the desktop.

2 Click the Initialize button


Observe the activity messages displayed at the bottom left of the screen. The instrument
makes clicking noises. The status indicator changes from Not Initialized to Initialized.
Initialization is complete when the Home button is active.
Figure 144 Instrument Activity Area

A Instrument activity area


B Status indicator changed from not initialized to initialized

3 Click the Home button .


This operation moves the Y-stage to the front of the instrument so that you can load a
flow cell. Again, the activities that occur during homing are displayed in the status bar.
4 Run scripts as described under Confirm Focus is on Top of Flow Cell on page 134.

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About MTS
Views (Tabs) in Left Pane
Views available in the left pane include:
• About • Balance • Camera • Compensator
• Compensator Settling Time • Devices • Flow Rate • Focus
• FPGA • LED • LED Cal • Motor
• Optical Test Target • Pump • Sample
• YSettling Time • Temperature • VCL
• ZSettlingTime • Sensors • Scan

Each view has a tab. However, all the tabs are not visible across the top of the left pane. If
a tab is not displayed, you can access it as follows:
1 Open the quick access view drop-down menu and selecting the tab.
2 Click the Views button and selecting the tab.
You can also hide tabs by deselecting them in the Views drop-down. If you hide a tab, it is
not displayed in the tab drop-down list.
Figure 145 Accessing Views (Tabs) in MTS

A Quick access drop-down for tabs


B Deselect a tab to remove it from the quick access drop-down list

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Image Tab in the Right Pane


Use the Image tab to view images of flow cells and the optical test target. Active channel
buttons are a solid color; inactive channel buttons fade to white at the top.
Figure 146 Image Tab — Beaded Flow Cell

A Active channel button (solid color)


B Inactive channel buttons (color fades to white at top of button)
C Slider bar for foreground contrast adjustment (image brightness)
D Slider bar for background contrast adjustment
Table 17 Working with Images
To … Then …

Adjust the foreground Move the top slider (C in Figure 146).


contrast

Adjust the background Move the top slider (D in Figure 146).


contrast

Zoom in and out Turn the wheel on the USB mouse.

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About MTS
To … Then …

Select a channel Click the channel button so that the color of the button is
solid (A channel). A channel is off when the top of the
button fades to white (G channel).

Running Scripts in MTS


NOTE
For many of the alignment scripts, both surfaces of the flow cell must be checked. To check
both surfaces, you will:
• Generate 2 through-focus image stacks: one for the top and 1 for the bottom surface of
the flow cell.
• Run tests twice: one time for the top and one time for the bottom surface of the flow cell.
• Name your results folders such that you can easily distinguish between the results for the
top and for the bottom of the flow cell.
Most of these scripts can also be run from the sequencing instrument (Welcome screen |
Manage Instrument). Scripts are located in the MatlabScripts folder (C:\Illumina | MiSeq
Control Software | MatlabScripts). On some instruments, they can also be located in a
release scripts folder on the D: drive (Release v.XX).
Follow these instructions to run scripts in MTS using a super flat beaded flow cell.
1 Generate 2 through-focus image stacks.
See Generate the Through Focus Image Stack on page 139.
2 Minimize MTS.
3 Go to C:\Illumina | MiSeq Control Software | MatlabScripts (or the release scripts
folder on the D: drive).
Figure 147 Script Folder Contents

4 Double-click the script to open a window with a command prompt (Figure 148).
5 Go to the D drive and open the folder with the top surface through-focus image stack.

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6 Drag and drop the numbered and dated folder onto the command prompt.
Figure 148 Drag and Drop the Image Stack Folder Onto the Command Prompt

7 Click in the command prompt window, and hit Enter.


A status bar is displayed while the script is running. When finished, a results file
(<script_name>.csv) is generated. For some scripts such as tip/tilt, image files are also
generated.
CAUTION
If an error occurs and the script does not execute, generate a new through-focus image
stack before running the script again.
8 Repeat this procedure using the image stack for the bottom surface of the flow cell.
9 Assess the results for each surface of the flow cell that was tested.

About BoltPAC.exe
This script checks tip/tilt, uniformity, chromatism, camera offset and rotation, and D50.
NOTE
• Always use the center of a super flat beaded flow cell to generate the top
and bottom through-focus image sets, and to run the script.
• The BoltPAC script runs for approximately 40 min.
• Disregard saturation errors, and warnings in script results and the control
software.
• The Full Optics test in the sequencing instrument automatically generates the
2 through-focus image sets required, and runs BoltPAC.exe on both surfaces
of the flow cell.

Run BoltPAC.exe
1 Generate through-focus image stacks for both surfaces of the flow cell using the center
of a super flat beaded flow cell (see Generate Through Focus Image Sets (Stacks) on page
136).
2 Minimize MTS.
3 Go to C:\Illumina | MiSeq Control Software | MatlabScripts.
Or go to the release scripts folder on the D drive.

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About MTS
4 Double-click the script to open a window with a command prompt.
5 Go to the D drive and open the folder you created for the top surface through-focus
image set.
6 Drag and drop the numbered and dated folder onto the command prompt.
Figure 149 Drag and Drop the Through Focus Image Set Folder Onto the Command Prompt

7 Hit Enter.
A BoltPAC status bar is displayed while the script is running. The script runs for
approximately 40 min.
NOTE
If BoltPAC repeatedly fails, make sure that the proper file naming convention is being
applied to the 41x4 image generated. Sometimes the software can name one or more of
the files incorrectly, causing the script to fail.
8 Run BoltPAC again using the bottom surface through-focus image set.

Chromatism Results
In the BoltPAC_Report file, the chromatism result is called Camera 1 – Camera 2 Offset. A
perfect chromatism value is 0 µm.
Figure 150 Example of Perfect Chromatism as Displayed in BoltPAC_Results File

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Figure 151 Example of BoltChromatism.exe Results File with Adjustment Recommendation

A BoltChromatism.exe indicating a chromatism value of -15.438


B Recommendation for improving chromatism

Camera Offset Results


BoltCameraOffset.exe is a script that looks at where the image on camera 2 is in terms of
where the outer pixels are located. It then compares the camera 2 image with the camera 1
image in the same terms. In an XY plane and rotation, the software determines how well
the 2 images can be perfectly overlaid.
Look at C to A measurements (if good, the T to G values are almost the same). An example
of camera offset results is shown in Figure 152. When an adjustment is required, adjust
camera 1 to camera 2 (C to A). Instructions for making camera offset adjustments are listed
under Camera 2 Center Adjustment on page 87.
Figure 152 Example of Camera Offset Results

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About MTS
Illuminated Area Results
If the illuminated area results are out of spec, adjust the illuminated area as described
under Illuminated Area Adjustment on page 107.
Figure 153 Example of Illuminated Area Results

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Manually Check Reagent Delivery
This section describes how to perform a manual check of the volume delivered by each
fluidics line using MTS.

Items Required for Reagent Delivery Test


Table 18 Items required for Reagent Delivery Test
Item Quantity

Eppendorf tubes or equivalent, 15 ml 22

Water As required
• MiSeq: Laboratory-grade water
• MiSeq FGx: DNase-free, RNase-free water

Gravimetric scale 1

Prime the Instrument


The prime recipe delivers 2 × 500 µl from each reagent position. For the best results, run the
recipe twice.
1 Load an empty flow cell.
2 Fill each position in the wash tray and the PR2 bottle with water.
• MiSeq: laboratory-grade water
• MiSeq FGx: DNase-free, RNase-free water
3 Using MTS, raise the sippers (Pump tab |Sipper| Move Up), load the wash tray in the
chiller, and lower the sippers.
4 Load the PR2 bottle.
5 Click the Sample tab and select a tile.
Figure 154 Wash Tray

6 Click the Scan tab and configure the parameters as follows:


• Select the Use Recipe checkbox.
• Recipe name: Prime/Wash.
7 Click Scan to being priming the instrument.

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Manually Check Reagent Delivery
8 When the recipe is finished, click Scan again to prime the system twice.
Figure 155 Scan Tab Setup for Instrument Prime

Measure Reagent Delivery


1 In MTS, click the VCL tab (Figure 156 on page 151).
2 Select the reagent positions that you want to test.
3 Click Start.
The default values are OK.
4 Select from the following:
• If the test passes, record the information on the IQ/OQ (Figure 157 on page 151).
• If the test fails, troubleshoot the failure.

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Figure 156 VCL Tab

Figure 157 VCL Test Pass

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Appendix B  DiagConfig File

DiagConfig File

Appendix B
About the DiagConfig.xml File 153

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DiagConfig File
About the DiagConfig.xml File
This DiagConfig.xml file contains the settings and pass/fail specs used by the MiSeq and
MiSeq FGx sequencing instrument for the first time startup screens and the diagnostic tests.
It is on the D drive in the DiagResults folder. In the future, you might need to modify this
file if a spec changes.
Figure 158 Location of DiagConfig.xml

Here is a brief description of the contents of this file. Because the file is long, it is described
by section in linear order.

Table 19 DiagConfig.xml File for the MiSeq and the MiSeq FGx
Comments DiagConfig.xml Content

< ?xml version="1.0" encoding="utf-8" ?>


< DiagConfig xmlns:xsd="http://www.w3.org/2001/XMLSchema"
xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance">

<MCSVersion>2.1.13/MCSVersion>

<CoarseThroughFocusSettings>

Initial coarse <OTTStepSize>0.002</OTTStepSize>


focus settings <OTTNumSteps>51</OTTNumSteps>
to determine <BFCStepSize>0.002</BFCStepSize>
the correct
start point for <BFCNumSteps>41</BFCNumSteps>
through-focus
image set
generation for
OTT and bead
flow cell

<ScanSettings>

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About the DiagConfig.xml File
Comments DiagConfig.xml Content

<ExposureTimeMS_
ChA>
100</ExposureTimeMS_
ChA>
<ExposureTimeMS_
ChC>
100</ExposureTimeMS_
ChC>
<ExposureTimeMS_
ChG>
50</ExposureTimeMS_
ChG>
<ExposureTimeMS_
ChT>
50</ExposureTimeMS_
ChT>
<GreenLEDCurrent>
2000</GreenLEDCurrent>
<RedLEDCurrent>
1500</RedLEDCurrent>
<OTTExposureTimeMS_
ChA>
5000
</OTTExposureTimeMS_
ChA>
<OTTExposureTimeMS_
ChC>
2000
</OTTExposureTimeMS_
ChC>
<OTTExposureTimeMS_
ChG>
200
</OTTExposureTimeMS_
ChG>
<OTTExposureTimeMS_
ChT>
200
</OTTExposureTimeMS_
ChT>
<OTTGreenLEDCurrent>
6500
</OTTGreenLEDCurrent>
<OTTRedLEDCurrent>
1500
</OTTRedLEDCurrent>

</ScanSettings>

</CoarseThroughFocusSettings>

<FineThroughFocusSettings>

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DiagConfig File

Comments DiagConfig.xml Content

Through focus <StepSize>0.00025</StepSize>


image set <NumSteps>31</NumSteps>
settings <ScanSettings>

Exposures and <ExposureTimeMS_


LED current ChA>
settings for 100</ExposureTimeMS_
the full optics ChA>
test and tip/tilt <ExposureTimeMS_
when using a ChC>
flow cell 100</ExposureTimeMS_
ChC>
<ExposureTimeMS_
ChG>
50</ExposureTimeMS_
ChG>
<ExposureTimeMS_
ChT>
50</ExposureTimeMS_
ChT>
<GreenLEDCurrent>
2000</GreenLEDCurrent>
<RedLEDCurrent>
1500</RedLEDCurrent>

Exposures and <OTTExposureTimeMS_


LED current ChA>
settings for 5000
the full optics </OTTExposureTimeMS_
test and tip/tilt ChA>
when using an <OTTExposureTimeMS_
OTT ChC>
5000
</OTTExposureTimeMS_
ChC>
<OTTExposureTimeMS_
ChG>
200
</OTTExposureTimeMS_
ChG>
<OTTExposureTimeMS_
ChT>
200
</OTTExposureTimeMS_
ChT>
<OTTGreenLEDCurrent>
6500
</OTTGreenLEDCurrent>
<OTTRedLEDCurrent>
1500
</OTTRedLEDCurrent>

</ScanSettings>

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About the DiagConfig.xml File
Comments DiagConfig.xml Content

</FineThroughFocusSettings>

<FullOpticsTestOptions>

<Camera12ZOffset>0</Camera12ZOffset>
Chromatism <Camera12ZOffsetDelta>
specification 40</Camera12ZOffsetDelta>
Focal plane <Camera12ZOffsetDeltaBot>
specification 60</Camera12ZOffsetDeltaBot>
<FocalPlaneZPosition>-
0.15</FocalPlaneZPosition>
<FocalPlaneZPositionDelta
>0.05</FocalPlaneZPositionDelta>
<MaxSaturatedPixels>1000</MaxSaturatedPixels>
<CameraOffsetTileCenter>
0</CameraOffsetTileCenter>
<CameraOffsetTileCenterXDelta>75</
CameraOffsetTileCenterXDelta>
<CameraOffsetTileCenterYDelta>40</
CameraOffsetTileCenterYDelta>
<CameraRotation>0</CameraRotation>
<CameraRotationDelta>
1</CameraRotationDelta>
<IlluminatedArea>0.54</IlluminatedArea>
<MaxD50>2.9</MaxD50>

<FullOpticsTestOptions>

LED power <LEDPowerOptions>


test

<GreenLEDCurrent>2000</GreenLEDCurrent>
<RedLEDCurrent>1500</RedLEDCurrent>
<GreenADC>550</GreenADC>
<RedADC>8500</RedADC>

<LEDPowerOptions>

Prime reagent <PrimeReagentOptions>


lines

<PumpCommands>

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DiagConfig File

Comments DiagConfig.xml Content

Each line <PumpToFlowcell


primed twice AspirationRate="2000"
Solution="1"
DispenseRate="2500"
Volume="500" />
<PumpToFlowcell
AspirationRate="2000"
Solution="2"
DispenseRate="2500"
Volume="500" />
<PumpToFlowcell
AspirationRate="2000"
Solution="3"
DispenseRate="2500"
Volume="500" />
<PumpToFlowcell
AspirationRate="2000"
Solution="4"
DispenseRate="2500"
Volume="500" />
<PumpToFlowcell
AspirationRate="2000"
Solution="5"
DispenseRate="2500"
Volume="500" />
<PumpToFlowcell
AspirationRate="2000"
Solution="6"
DispenseRate="2500"
Volume="500" />
<PumpToFlowcell
AspirationRate="2000"
Solution="7"
DispenseRate="2500"
Volume="500" />
<PumpToFlowcell
AspirationRate="2000"
Solution="8"
DispenseRate="2500"
Volume="500" />
<PumpToFlowcell
AspirationRate="2000"
Solution="9"
DispenseRate="2500"
Volume="500" />
<PumpToFlowcell
AspirationRate="2000"
Solution="10"
DispenseRate="2500"
Volume="500" />
<PumpToFlowcell
AspirationRate="2000"
Solution="11"
DispenseRate="2500"
Volume="500" />
<PumpToFlowcell
AspirationRate="2000"
Solution="12"
DispenseRate="2500"
Volume="500" />
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AspirationRate="2000"
Solution="13"
DispenseRate="2500"
About the DiagConfig.xml File
Comments DiagConfig.xml Content

<PumpToFlowcell
AspirationRate="2000"
Solution="17"
DispenseRate="2500"
Volume="500" />
<PumpToFlowcell
AspirationRate="2000"
Solution="18"
DispenseRate="2500"
Volume="500" />
<PumpToFlowcell
AspirationRate="2000"
Solution="19"
DispenseRate="2500"
Volume="500" />
<PumpToFlowcell
AspirationRate="2000"
Solution="20"
DispenseRate="2500"
Volume="500" />
<PumpToFlowcell
AspirationRate="2000"
Solution="21"
DispenseRate="2500"
Volume="500" />
<PumpToFlowcell
AspirationRate="2000"
Solution="22"
DispenseRate="2500"
Volume="500" />

<PumpCommands>

<PrimeReagentOptions>

<ThermalRampOptions>

High and low <OverrideCalibrationHighMeasured>0


measured <OverrideCalibrationHighMeasured>
values are <MaxDeviationHighTemp>
checked; not 2</MaxDeviationHighTemp>
target values <WaitTimeHighTemp>45</WaitTimeHighTemp>
in the MiSeq
Override.cfg <MonitorTimeHighTemp>
file 30</MonitorTimeHighTemp>
<OverrideCalibrationLowMeasured>0
<OverrideCalibrationLowMeasured>
<MaxDeviationLowTemp>
2</MaxDeviationLowTemp>
<WaitTimeLowTemp>45</WaitTimeLowTemp>
<MonitorTimeLowTemp>
30</MonitorTimeLowTemp>

<ThermalRampOptions>

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DiagConfig File

Comments DiagConfig.xml Content

<TipTiltTestOptions>

Tip/Tilt <useOTT>false</useOTT>
specifications <UpperLimit>400</UpperLimit>
for bead flow <LowerLimit>--400</LowerLimit>
cell
<BotUpperLimit>500</BotUpperLimit>
<BotLowerLimit>--500</BotLowerLimit>

<TipTiltTestOptions>

<VolumeCheckOptions>

<PrimeVolume>200</PrimeVolume>
<AirVolume>12</AirVolume>
<PreMeasurementVolume>
200</PreMeasurementVolume>
<TargetVolume>100</TargetVolume>
<PreAirDeliveryDelay>
10000</PreAirDeliveryDelay>
<PostPumpDelay>2000</PostPumpDelay>
<AirDeliveryRate>750</AirDeliveryRate>
<MeasurementDeliveryRate>
500</MeasurementDeliveryRate>
<PassFailSpecPerCent>5</PassFailSpecPerCent>
<PR2PrimeVolume>250</PR2PrimeVolume>
<PR2TargetVolume>100</PR2TargetVolume>
<PR2PreMeasurementVolume>
700</PR2PreMeasurementVolume>

<VolumeCheckOptions>

<VolumeCheckPositions>

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About the DiagConfig.xml File
Comments DiagConfig.xml Content

<int>1</int>
<int>2</int>
<int>3</int>
<int>4</int>
<int>5</int>
<int>6</int>
<int>7</int>
<int>8</int>
<int>9</int>
<int>10</int>
<int>11</int>
<int>12</int>
<int>13</int>
<int>14</int>
<int>15</int>
<int>16</int>
<int>17</int>
<int>18</int>
<int>19</int>
<int>20</int>
<int>21</int>
<int>22</int>
<int>23</int>
<int>24</int>

<VolumeCheckPositions>

</DiagConfig>

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Appendix C  Service Tools

Service Tools

Appendix C
Service Tools 163

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Service Tools
Service Tools

Toolkits
The following toolkits are used to install, refresh, and service MiSeq instruments:
} MiSeq Alignment Toolkit (part # 15026398S)
} MiSeq Generic Tool Box (part # 15030145S)
} Field Service Repair Kit (part # 15028821S)

Super Flat Beaded Flow Cell


The super flat beaded flow cell (SFBFC) is not included in a toolkit.

Fluke Meter and Thermocouples


Use the following Fluke meter and thermocouples when required.
} Fluke Meter, 54-II, or equivalent, calibrated
} Type T thermocouples

MiSeq Alignment Toolkit


The following table lists the contents of the MiSeq Alignment Toolkit.
Table 20 MiSeq Alignment Toolkit (part # 15026398S)
Item Image Part Qty
Description Number

Interlock 15029220 1
override cable

Blackout cloth 15027879 1

Camera X offset 15024104 1


tool

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Service Tools
Item Image Part Qty
Description Number

Lens spring pliers 15026194 1

Camera Y offset 15023894 1


tool

Fluidics line tool 15032994 1

Chromatism 15023706 1
adjustment tool

Manifold 15032247 1
alignment tool

Y-stage 15029808 1
alignment tool

Extraction Tool 15032848 1

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Service Tools

Item Image Part Qty


Description Number

Kapton tape 15023907 1 roll

Level 15042044 1

Flow cells and


OTT:

• MiSeq 15026129 1
Beaded Flow
Cell, open

• MiSeq 15026717 1
Beaded Flow
Cell, sealed

• Open Flow 15026863 1


Cell
(fluidics tests)

This flow cell is


not in a holder.
Use the spare
holder in this kit.

• Optical test 15029331 1


target, v2

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Service Tools
Item Image Part Qty
Description Number

Flow cell holder– 15019050 1


manifold (manifold)
Flow cell holder–
cartridge 15020142
(cartridge)

Tool, Set IM 15072883 1 set


Isolator
Extensions

MiSeq Generic Tool Box


The MiSeq Generic Tool Box (part # 15030145S) includes some of the tools required to
install the MiSeq. Some of the tools included are:
} Hex wrenches
} Screwdrivers
} Optical cleaning cloths

Field Service Repair Kit


The Field Service Repair Kit (part # 15028821S) includes the most frequently replaced FRUs
for the instrument.
Table 21 Field Service Repair Kit
Item Description Image Qty

M3 Mirror Motor Cable Assembly 1


(SPARE, CABLE ASSY, MOTOR
M3 MIRROR)

Manifold Assembly, 2 port 1


(INA-SPARE, ASSY, MANIFOLD, 2
PORT, TALL, ULTEM)

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Service Tools

Item Description Image Qty

Reagent Valve with Bracket 1


(MISEQ v3 REAGENT SELECTOR
VALVE)

Syringe 1
(SPARE, SYRINGE, XC PUMP,
500 μl)
NOTE: Old style syringe here.
New style syringe in Syringe
Replacement on page 1.

Syringe Pump 1
(SPARE, SYRINGE PUMP, Tecan
XC, CERAMIC 3-WAY
DISTRIBUTION)
NOTE: Old style pump here. New
style pump in Syringe Pump
Replacement on page 1.

Flow Cell TEC (Peltier heater) 1


(SPARE, ASSY, FCH TEC, PROD
MODS)

Chiller TEC (Peltier heater) 1


(ASSY, TEC, CHILLER,
SHIELDED)

Chiller Thermistor 1

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Appendix D  Alignment and Inspection Specifications

Alignment and Inspection

Appendix D
Specifications
Instrument Specifications 169

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Alignment and Inspection Specifications
Instrument Specifications
The specifications listed here are the same for the MiSeq and MiSeq FGx.
Alignment specifications: Use these specs to realign the instrument when parts are
replaced. These specs are used by manufacturing to build
and test the instrument.
Inspection specifications: Use these specs for instrument verification.

NOTE
The following specs are different for the top and bottom surface of a lane:
• Camera 1–Camera2 Z Offset
• Tip/Tilt

Table 22 MiSeq and MiSeq FGx Alignment and Inspection Specifications


Specification Lane Top Lane Top Lane Bottom Lane Bottom
Alignment Inspection Alignment Inspection

Camera 1– 0 ± 25 μm 0 ± 40 μm 0 ± 40 μm 0 ± 60 μm


Camera 2 Z
Offset
(chromatism)

Tip/Tilt 0 ± 150 nM 0 ± 400 nM 0 ± 300 nM 0 ± 500 nM

Camera Offset 0 ± 13 pixels X: 0 ± 75 N/A N/A


at Tile Center (X pixels
and Y) Y: 0 ± 40
pixels

Camera 0 ± 1 degree 0 ± 1 degree N/A N/A


Rotation (C to
A)

Illuminated ≥ 0.54 ≥ 0.54 N/A N/A


Area

Max D50 < 2.90 pixels < 2.90 pixels < 2.90 pixels < 2.90 pixels

Focal Plane Z- –0.17 mm ± -0.135 to N/A N/A


position 0.025 -0.195

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Appendix E  Instrument Returns to Illumina

Instrument Returns to Illumina

Appendix E
Returning an Instrument to Illumina 171

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Instrument Returns to Illumina
Returning an Instrument to Illumina
This section describes how to return a MiSeq to Illumina.

Last Updated 18 December 2014

Estimated Time Crating the instrument: 1 hour

FRU Search Criteria Crate kit: 10567S

Prerequisites
For instructions on the following prerequisites, see Ordering the Crate Kit on page 171.
} Place a service order
} Order the crate kit

Items Required
} Allen wrenches, M6, and 5/64 inch T-handle
} Box for instrument accessories
} Crate kit
} Lab tape
} Shipping request form

Ordering the Crate Kit


NOTE
To order a crate, refer to Ordering Platform Crate Kits FSB450 (part # 15044391).
To ensure timely delivery, order crates with sufficient lead time. To find lead times, refer to
the work instruction Global Parts Ordering, PN 15042146.
1 Generate a service order on the relevant case. For more information, refer to the work
instruction Logging a Standard Case in SFDC (part # 15004045).
2 Complete the Shipping Request and Site Requirements forms and attach them to the
return or relocation case.
3 Email the Shipping Request and Site Requirements forms to Customer Service.

Gather the Instrument Accessories


NOTE
The instrument accessories have changed over time (for example, the user
documentation). Therefore, the accessories you gather might differ from the
following list.

Gather the instrument accessories and place them in a box. Ship the accessory box in the
crate with the instrument. Accessories include:
} Instrument safety and compliance guide
} Instrument user guide
} Wash tray (1 or 2 depending upon when the instrument was shipped)
} PR2 bottle
} Waste bottle with cap
} M6 T-handle hex wrench

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} 5/64 inch T-handle hex wrench
} Power cord
} Network Ethernet cable

Prepare the Instrument for Crating


1 Disconnect the power cord and network cable.
2 Remove any reagents or the wash tray.
3 Empty the waste bottle (C), seal with the cap, and place back inside the instrument.
4 Cover the PR2 sipper tube (A) with the protective sheath (B).
Figure 159 Protective Sheath for PR2 Sipper Tube

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5 Retract the Y-stage all the way into the instrument by manually turning the shaft.
Figure 160 Retract Y-Stage Into Instrument

6 Make sure that the Y-stage shipping restraint bracket can be properly installed
(Figure 163).
The Y-stage can be moved back too far, causing problems later when trying to install
the bracket.
7 Using an M6 T-handle Allen wrench, reengage the shipping lockdowns on the left side
of the instrument.
• Turn the lockdown marked B clockwise approximately 1/2 turn until it stops.
• Turn the lockdown screw marked A counterclockwise approximately 13 full turns
until it stops.
Figure 161 Left Side Shipping Restraints Re-Engaged

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8 Reengage the Z-stage lockdown as follows:
a Face the instrument, and insert the 5/64 in T-handle Allen wrench through the hole
where the top skin and reagent compartment skins meet.
You can also reengage the lockdown with the skins off the instrument.
b Turn the wrench clockwise approximately 2-1/2 turns until it stops.
Figure 162 Reengaging the Z-stage Lockdown

9 Open the flow cell compartment, and install the Y-stage shipping restraint bracket.
Figure 163 Y-Stage Shipping Restraint Bracket

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10 Using lab tape:
a Tape the flow cell compartment and the chiller doors closed.
b Tape a protective cover sheet over the touch screen.
Figure 164 Compartments Tape Shut and Protective Sheet Over Touch Screen

Crate the Instrument


NOTE
The protective cover and tape or dots on the instrument can differ from what is shown in
these instructions.
1 Place the bottom of the crate next to the bench with the instrument.
Figure 165 Bottom of Shipping Crate

2 Using a 2 person lift:


a Grip the rails that run lengthwise under the front and back of the chassis (never
grip the flow cell compartment).
b Lift and place the instrument on the bottom of the crate.

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NOTE
Note the FRONT and BACK markings on the crate and position the instrument
accordingly.

Figure 166 Positioning the Instrument on the Bottom of the Crate

A Front of instrument facing FRONT lettering on the crate

Figure 167 Side View of Instrument on Bottom of Crate

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3 Place the plastic bag over the instrument.
Figure 168 Plastic Bag Over Instrument

4 Place the sides of the crate in position and lock them in place.
Figure 169 Position and Lock Sides of Crate

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5 Insert the hard plastic packing foam.
Figure 170 Hard Foam Packing Installed

6 Place the accessory box inside the crate.


7 Place the top on the crate and lock it in place.

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Appendix F  Instrument Engineering Drawings

Instrument Engineering Drawings

Appendix F
Cable Interconnect Diagram for the MiSeq 181
Subsystem Block Diagram for the MiSeq 182
Optical System for the MiSeq 183
Fluidics System for the MiSeq 184

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Instrument Engineering Drawings
Cable Interconnect Diagram for the MiSeq

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Subsystem Block Diagram for the MiSeq
Subsystem Block Diagram for the MiSeq

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Instrument Engineering Drawings
Optical System for the MiSeq

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Fluidics System for the MiSeq
Fluidics System for the MiSeq

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Appendix G  Common Procedures

Common Procedures

Appendix G
Adjust the Clock, Date, and Time (Windows 7) 187
Cleaning the Imaging Module Bench 189
Flow Cell Cleaning, Loading, and Imaging 190
Removing and Reinstalling the Instrument Skins 195
Rebooting the Computer 203
Shutting Down the Computer 204
Turning the Instrument On and Off 205

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Common Procedures
Adjust the Clock, Date, and Time (Windows 7)
Follow these instructions to adjust for daylight savings time, and change the time zone if
necessary (the default is US PDT) on Windows 7.

1 Select the time/date displayed in the lower right corner of the screen.
2 Select Change date and time settings.
Figure 171 Open the Clock

3 Select Change time zone to open the time zone settings window.
4 Open the drop-down menu and select the appropriate time zone.
5 Deselect this checkbox: Automatically adjust clock for Daylight Saving Time.
6 Select OK.
Figure 172 Automatic Adjustment for Daylight Saving Time Off

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Adjust the Clock, Date, and Time (Windows 7)
7 If the date and time are not correct:
a Select Change date and time
b Set the correct date and time.
c Select OK; then select OK again.
Figure 173 Date and Time Settings Window

NOTE
If the date and time are not correct, the connection to BaseSpace Broker fails.

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Common Procedures
Cleaning the Imaging Module Bench
Use only central clean dry air (CDA), nitrogen, or hand puffer to clean the imaging module
bench. Do not use canned air.

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Flow Cell Cleaning, Loading, and Imaging
Flow Cell Cleaning, Loading, and Imaging
This section includes information on how to clean and load flow cells, and information on
flow cell imaging.

Clean the Flow Cell


The flow cell is immersed in storage buffer in a flow cell container. Clean the flow cell as
follows before loading it on the instrument.
1 Put on a new pair of powder-free gloves.
2 Using plastic forceps, grip the flow cell by the base of the plastic cartridge and remove
it from the container.
Figure 174 Remove Flow Cell from Container

3 Lightly rinse the flow cell with labaoratory-grade water until both the glass and plastic
cartridge are thoroughly rinsed of excess salts.
Excess salts can affect flow cell seating on the instrument. If salt drys in the imaging
area, imaging can also be affected.
Figure 175 Rinse the Flow Cell

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Common Procedures
4 Using care around the black flow cell port gasket, thoroughly dry the flow cell and
cartridge with lint-free lens cleaning tissue; gently pat dry in the area of the gasket and
adjacent glass.
Figure 176 Flow Cell Ports and Gasket

5 Clean the flow cell glass with an alcohol wipe, making sure that the glass is free of
streaks, fingerprints, and lint or tissue fibers.
NOTE
Do not use the alcohol wipe on the flow cell port gasket. Make sure that the
cartridge is securely closed before use.

Figure 177 Dry the Flow Cell

6 Dry excess alcohol with a lint-free lens cleaning tissue.


7 Make sure that the flow cell ports are free of obstructions and that the gasket is well-
seated around the flow cell ports.
If the gasket appears to be dislodged, gently press it bakc into place until it sits securely
around the flow cell ports.inspect it to make sure that no tissue particles are obstructing
the ports.

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Flow Cell Cleaning, Loading, and Imaging
Load the Flow Cell
NOTE
The instrument must be initialized and homed before loading a flow cell.
1 In MCS, select Sequence from the Welcome screen.
If you are using MTS, click the Motor tab, then click Home Y.
2 Raise the flow cell compartment door, and press the release button to the right of the
flow cell clamp.
The flow cell clamp opens.
Figure 178 Flow Cell Latch Button

3 Make sure that the flow cell stage is free of lint.


If lint or other debris is present, clean the flow cell stage using an alcohol wipe or a
lint-free tissue moistened with ethanol or isopropanol. Carefully wipe the surface of the
flow cell stage until it is clean and dry.

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Common Procedures
4 Holding the flow cell (or OTT) by the edges, place it on the flow cell stage.
• Align the top notch around the top pin, and the 2 holes on the right onto the 2
right-hand pins.
• Make sure that the label is facing upward.
Figure 179 Loading a Flow Cell or OTT

A Three alignment pins

5 Gently press down on the flow cell clamp to close it over the flow cell.
As the flow cell clamp closes, alignment pins position the flow cell. An audible click
indicates that tthe flow cell clamp is secure.
Figure 180 Close the Flow Cell Clamp

6 Close the flow cell compartment door.


7 In MCS, select Next on the Load Flow Cell screen. The Load Reagents screen opens.
In MTS, select a tile, then click Move to Tile.

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Flow Cell Cleaning, Loading, and Imaging
Imaging Flow Cells
The flow cells used on the instrument consist of one column of 19 rows on the top surface,
and one column of 19 rows on the bottom surface. The column is referred to as a lane. Each
row is referred to as a tile. Tile 0 is used for through-focus image set generation. The
distance between the top and bottom surfaces of the lane is 0.072 mm.
The instrument images the top and bottom surfaces of the flow cell using red and green
LEDs as the light source. The red LED flashes first, then the green. The red LED is > 145 W;
the green LED is > 160 mW. Each tile is first autofocused, then imaged. Two cameras are
utilized to capture the images: S1 for the C and T channels; S2 for the A and G channels.
When the flow cell is properly loaded, tile 0 is closest to the rear of the instrument.
Figure 181 Flow Cell Map

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Common Procedures
Removing and Reinstalling the Instrument Skins
The instructions in this section describe how to remove and reinstall the instrument skins.
NOTE
MiSeq and MiSeq FGx skins differ in color. The examples shown here depict the MiSeq.
Removal and installation are the same for both instruments.

Equipment Required
} Allen wrench, 4 mm
} Screwdrivers, Phillips-head, and flat-head

Skin Removal
Remove the skins (also referred to as panels) from the instrument in the order indicated
here.
Figure 182 MiSeq Skin Removal

A Right side skin


B Left side skin
C Top skin
D Flow cell compartment skin

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Removing and Reinstalling the Instrument
Right Side Skin
Figure 183 Right Side Skin Captive Screw

1 Using a 4 mm Allen wrench, remove the captive screw, centered in bottom of skin
(panel).
2 Pull the panel out and up.
Two clips are on the bottom, inner side of the panel. These clips are the same on the
left and right panels.

Left Side Skin


Figure 184 Left Side Skin —Inner and Outer Views

1 Using a 4 mm Allen wrench, remove the captive screw, centered in bottom of panel.
2 Pull the panel out and up.
Two clips are on the bottom, inner side of the panel.

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Common Procedures

Top Skin
Figure 185 Screw Locations for Top Skin

1 Using a Phillips-head screwdriver:


• Remove the screw the left side of the instrument in the lower rear corner.
• Remove the screw on the right side of the instrument in the upper rear corner.
2 Standing in front of the instrument, pivot the cover towards the back of the instrument,
then lift it up and off.

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3 Set the cover on the floor.
NOTE
To avoid damaging the top skin, rest it on the floor.

Figure 186 Pivot Cover Towards Back of Instrument

Figure 187 Proper Position to Rest the Top Cover on the Floor

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Common Procedures

Flow Cell Compartment Skin Removal


1 Open the reagent chiller door.
2 Loosen the inner captive screw at top right corner of the skin (you might need to use a
flat-head screwdriver to loosen the screw).
3 Using a Phillips-head screwdriver, remove the outer screw on the bottom left corner of
the skin.
Figure 188 Reagent Compartment Skin Screws

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Removing and Reinstalling the Instrument
4 Slide the skin to the left to move the shoulder screws out of the slide lock holes, then
carefully move it forward and off the instrument.
CAUTION
An interlock cable is attached to this skin (Figure 191). Keep the skin close to the
instrument to avoid damaging the cable.

Figure 189 Flow Cell Compartment Skin—Shoulder Screws and Slide Lock Holes

5 Disconnect the interlock cable attached to the skin.


Figure 190 Interlock Cable Attached to Flow Cell Compartment Skin

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Common Procedures
6 Install the interlock defeat connector included in the MiSeq toolkit.
Figure 191 Interlock Defeat Connector Installed

Skin Reinstallation
Reinstall the skins in the reverse order in which they were removed.
NOTE
The skins are designed to reseat easily. Do not try to force any skins back on. Forcing the
skins can create torque on internal components, and can affect optical alignment. If the skins
do not seat easily, especially the top skin, check the cabling in the affected area. Cables can
shift or be moved during repair or alignment.
TIP
Moving the flow cell stage to tile 12 makes it easier to reinstall the top skin and the flow cell
compartment skin.
} Flow cell compartment skin—Remember to:
• Remove the interlock defeat cable and reattach the cable on the flow cell
compartment skin to the connector on the chassis.
• Open the reagent chiller door when reinstalling this skin.
TIP
Curl one hand under the instrument, and guide both shoulder lock screws into the slide
lock holes (Figure 189 on page 200).
} Top skin—Fit it onto the back of the instrument first:
• Slide the slot on the back of the cover over the captive screw on the back of the
instrument.
• Position the bottom of the cover in the track, and slide it to the left so that the
cutout fits around the cables.
CAUTION
Be careful not to pinch any cables when installing the top skin.

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Removing and Reinstalling the Instrument
Figure 192 Slot on the Back of the Top Skin

Figure 193 Bottom of Cover in Track—Slide to the Left

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Common Procedures
Rebooting the Computer
1 From the Welcome screen in the sequencing instrument, click or press Manage
Instrument.
2 Select Reboot.

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Shutting Down the Computer
Shutting Down the Computer
This procedure shuts down the computer only; it does not shut down the instrument.
1 From the Welcome screen in the sequencing instrument, click or press Manage
Instrument.
2 Select Shutdown.

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Common Procedures
Turning the Instrument On and Off
Figure 194 On/Off Switch on the Back of the Instrument

Turning the Instrument On


1 Turn the on/off switch on the back of the instrument to the ON position.
2 Allow the instrument to initialize.

Turning the Instrument Off


CAUTION
To prevent hard drive corruption, always turn off the PC before turning off the instrument.
1 Close all applications that are running.
2 Shut down the PC first.
3 Then, turn the on/off switch on the back of the instrument to the OFF position.

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Index

Index
B
BoltPAC.exe, how to run 36, 145
Brenner graph examples
failed through focus image set
generation 124
focus on wrong surface of flow L
cell 123 lanes on a flow cell 194
good through focus image set 122 leveling the instrument 18
logins and passwords 4
C
calibration, flow cell TEC thermal 101 M
camera 2 moving the instrument 14
adjust centering 87 MTS
check centering 87 channel buttons 143
camera offset test results 147 Image tab 143
camera XY offset, how to adjust 91 running scripts 144
chromatism test results 146 views available 142
CloudAPIKey 53 working with images 143
crating instrument for return to
Illumina 171 N
customer support 208 network connections 18
D O
DiagConfig.xml file, about 153 optics, cleaning 189
documentation 208
documents, reference 2 P
E panels. See skins. 195
passwords and logins 4
equipment required 4 PhiX Control DNA preparation 58
F Q
flow cell qualification run 5, 55
lanes and tiles 194 cleaning flow cell 63, 71, 190
flow cell cleaning 63, 71, 190 items required 5, 55
flow cell TEC thermal calibration 101 loading sample libraries 59
flow cells PhiX Control DNA preparation 58
handling, loading, and imaging 190 reagent cartridge map 56
reagent cartridge preparation 56
H
help, technical 208 R
reagent cartridge map 56
I reagent cartridge preparation 56
illuminated area test results 148 reagent compartment setup 17
illuminated area, how to adjust 107 reagent delivery test, manual 149
imaging module bench reagents required 4
cleaning 189 recipes required 6
installation
system component inventory 13 S
instrument sample libraries, loading onto reagent
crating for return 171
turn off 205 cartridge 59
instrument leveling 18 sbsuser password expiration 52
instrument qualification 22 shipping bracket removal 14
inventory of system components 13 shipping lockdown release 14
skins, removal and reinstallation 195
K software required 6
startup screens 22
keyboard and mouse installation 19 about 21

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Index

T
TEC, chiller
how to qualify 49
TEC, flow cell
how to qualify 105
technical assistance 208
thermal ramp test results, example
of 41
through focus image set, generate using
a beaded flow cell 136
tile tilt
adjusting on camera 1 129
adjusting on camera 2 125
tile tilt. See tip tilt test. 119
tiles on a flow cell 194
tip tilt test
how to run 119
U
uncrating the instrument 12
UPS recommendations 18
W
wash, post run 78-79
Z
z-motor, adjusting rough position 131
z-position, determining best focus 42

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Technical Assistance

Index
Region Toll Free International

Netherland +31 800 022 +31 20 713


For technical assistance, contact Illumina s 2493 2960
Technical Support.
New +64 800 451
Website: www.illumina.com Zealand 650
Email: techsupport@illumina.com
Norway +47 800 16 +47 21 93 96
Illumina Technical Support Telephone 836 93
Numbers
Philippines +63
18001651079
Region Toll Free International 8
Australia +61 1800 775 Singapore 1 800 5792
688 745
Austria +43 800 +43 1 9286540 South +82 80 234
006249 Korea 5300
Belgium +32 800 77 +32 3 400 29 Spain +34 800 300 +34 911 899
160 73 143 417
Canada +1 800 809 Sweden +46 2 +46 8
4566 00883979 50619671
China +86 400 066 Switzerland +41 800 200 +41 56 580 00
5835 442 00
Denmark +45 80 82 01 +45 89 87 11 Taiwan, +886 8
83 56 China 06651752
Finland +358 800 918 +358 9 7479 Thailand +66 1800 011
363 0110 304
France +33 8 05 10 +33 1 70 77 04 United +44 800 012 +44 20 7305
21 93 46 Kingdom 6019 7197
Germany +49 800 101 +49 89 3803 United +1 800 809 +1 858 202
4940 5677 States 4566 4566
Hong +852 800 960 Vietnam +84 1206
Kong, 230 5263
China

India +91 Safety data sheets (SDSs)—Available on


8006500375 the Illumina website at
Indonesia 007803651004 support.illumina.com/sds.html.
8
Product documentation—Available for
Ireland +353 1800 +353 1 695 download from support.illumina.com.
936608 0506

Italy +39 800 +39


985513 236003759

Japan +81 0800 111


5011

Malaysia +60 1800 80


6789

MiSeq/MiSeq FGx Installation Guide


Document # 15027239 v03 Current as of March 2022
208
Index

209 MiSeq/MiSeq FGx Installation Guide


Document # 15027239 v03 Current as of March 2022

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