Package ‘isolateR’

August 11, 2024

Type Package
Title Automated processing of Sanger sequencing data, taxonomic profiling, and generation of micro-
bial strain libraries
Version 1.0.1
Date 2024-08-11
Author Brendan Daisley, Sarah J Vancuren, Dylan J.L. Brettingham, Jacob Wilde, Simone Ren-
wick, Christine V Macpherson, David A. Good, Alexan-
der J. Botschner, Sandi Yen, Janet E. Hill, Matthew T. Sorbara, Emma Allen-Vercoe.
Maintainer Brendan Daisley <bdaisley@uoguelph.ca>
Description isolateR aims to enhance microbial isolation workflows and support the identifica-
tion of novel taxa. It addresses the challenges of manual Sanger sequencing data process-
ing and limitations of conventional BLAST searches, crucial for identifying microorgan-
isms and creating strain libraries. The package offers a streamlined three-step process that auto-
mates quality trimming Sanger sequence files, taxonomic classification via global align-
ment against type strain databases, and efficient strain library creation based on customizable se-
quence similarity thresholds. It features interactive HTML output tables for easy data explo-
ration and optional tools for generating phylogenetic trees to visualize microbial diversity.
Citation
Daisley et al. (2024). isolateR: an R package for generating microbial libraries from Sanger se-
quencing data. Bioinformatics 40(7):btae448. (https://doi.org/10.1093/bioinformatics/btae448)
License GPL (>= 2)
Encoding UTF-8
LazyData true
Imports ape, BiocManager, Biostrings, crosstalk, cowplot, dataui, dplyr, getPass, ggtree, gg-
beeswarm, ggiraph, IRanges, plotly, htmltools, patchwork, LPSN, methods, msa, pan-
der, R.utils, reactable, reactablefmtr, rentrez, S4Vectors, sangeranalyseR, sangerseqR, scales, se-
qinr, shiny, stringr, svMisc, xmlconvert
Depends R (>= 4.0), Biostrings, dplyr
Remotes timelyportfolio/dataui, glin/reactable, thomasp85/patchwork
Roxygen list(markdown = TRUE)
RoxygenNote 7.3.2
Suggests knitr, rmarkdown
VignetteBuilder knitr
Additional_repositories
http://R-Forge.R-project.org https://bioconductor.org/packages/3.18/bioc

1
2 class-isoLIB

Contents
class-isoLIB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
class-isoQC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
class-isoTAX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
df_to_isoLIB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
df_to_isoTAX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
export_html . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
get_db . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
get_os . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
get_sanger_date . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
get_vsearch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
isoALL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
isoLIB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
isoQC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
isoTAX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
make_fasta . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
make_tree . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
method-isoLIB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
method-isoQC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
method-isoTAX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
S4_to_dataframe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
sanger_assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
search_db . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
show . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
valid_tax_check . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Index 28

class-isoLIB isoLIB Class Object

Description
S4 wrapper for isoLIB function. Access data via S4 slot functions.

Value
Returns an class-isoLIB object.

Slots
input Character string containing input directory information.
sequence_group Character string containing list of group representative filenames.
date Character string containing run date from each of the input Sanger sequence .ab1 files ("YYYY_MM_DD"
format).
filename Character string containing input filenames.
phred_trim Numeric string containing mean Phred scores after trimming.
Ns_trim Numeric string containing count of N’s after trimming.
length_trim Numeric string containing sequence length after trimming.
class-isoLIB 3

seqs_trim Character string containing sequence after trimming.

closest_match Character string containing species + type strain no. of closest match from refer-
ence database.
NCBI_acc Character string containing NCBI accession number associated with closest match from
reference database.
ID Numeric string containing containing pairwise similarity value for query vs database reference
sequence. Calculation of ID is determined by isoTAX ’iddef’ parameter (0-4, Default=2). See
VSEARCH documentation for more details.
• (0) CD-HIT definition: (matching columns) / (shortest sequence length).
• (1) Edit distance: (matching columns) / (alignment length).
• (2) Edit distance excluding terminal gaps (default definition).
• (3) Marine Biological Lab definition counting each gap opening (internal or terminal)
as a single mismatch, whether or not the gap was extended: 1.0- ((mismatches + gap
openings)/(longest sequence length)).
• (4) BLAST definition, equivalent to –iddef 1 for global pairwise alignments.
rank_phylum Character string containing Phylum rank taxonomy
rank_class Character string containing Class rank taxonomy
rank_order Character string containing Order rank taxonomy
rank_family Character string containing Family rank taxonomy
rank_genus Character string containing Genus rank taxonomy
rank_species Character string containing Species rank taxonomy
phylum_threshold Numeric string containing Phylum-level sequence similarity threshold for rank
demarcation
class_threshold Numeric string containing Class-level sequence similarity threshold for rank
demarcation
order_threshold Numeric string containing Order-level sequence similarity threshold for rank
demarcation
family_threshold Numeric string containing Family-level sequence similarity threshold for rank
demarcation
genus_threshold Numeric string containing Genus-level sequence similarity threshold for rank
demarcation
species_threshold Numeric string containing Species-level sequence similarity threshold for
rank demarcation

See Also

isoLIB
4 class-isoQC

class-isoQC isoQC Class Object

Description

S4 wrapper for isoQC function. Access data via S4 slot functions.

Value

Returns an class-isoQC object.

Slots

date Character string containing run date from each of the input Sanger sequence .ab1 files ("YYYY_MM_DD"
format).
filename Character string containing input filenames.
trim.start.pos Numeric string containing trimming position start point.
trim.end.pos Numeric string containing trimming position end point.
phred_spark_raw List containing per nucleotide Phred score values for each sequence
phred_raw Numeric string containing mean Phred scores before trimming.
phred_trim Numeric string containing mean Phred scores after trimming.
Ns_raw Numeric string containing count of N’s before trimming.
Ns_trim Numeric string containing count of N’s after trimming.
length_raw Numeric string containing sequence length before trimming.
length_trim Numeric string containing sequence length after trimming.
seqs_raw Character string containing sequences before trimming.
seqs_trim Character string containing sequence after trimming.
decision Character string containing decision (PASS/FAIL) information based on isoQC ’min_phred_score’
and ’min_length cutoffs’.
input Character string containing input directory information.

See Also

isoQC
class-isoTAX 5

class-isoTAX isoTAX Class Object

Description
S4 wrapper for isoTAX function. Access data via S4 slot functions.

Value
Returns an class-isoTAX object.

Slots
input Character string containing input directory information.
warning Character string containing list filenames of sequences that had poor alignment during
taxonomic classification step.
date Character string containing run date from each of the input Sanger sequence .ab1 files ("YYYY_MM_DD"
format).
filename Character string containing input filenames.
phred_spark_raw List containing per nucleotide Phred score values for each sequence
phred_raw Numeric string containing mean Phred scores before trimming.
phred_trim Numeric string containing mean Phred scores after trimming.
Ns_raw Numeric string containing count of N’s before trimming.
Ns_trim Numeric string containing count of N’s after trimming.
length_raw Numeric string containing sequence length before trimming.
length_trim Numeric string containing sequence length after trimming.
seqs_raw Character string containing sequences before trimming.
seqs_trim Character string containing sequence after trimming.
closest_match Character string containing species + type strain no. of closest match from refer-
ence database.
NCBI_acc Character string containing NCBI accession number associated with closest match from
reference database.
ID Numeric string containing containing pairwise similarity value for query vs database reference
sequence. Calculation of ID is determined by isoTAX ’iddef’ parameter (0-4, Default=2). See
VSEARCH documentation for more details.
• (0) CD-HIT definition: (matching columns) / (shortest sequence length).
• (1) Edit distance: (matching columns) / (alignment length).
• (2) Edit distance excluding terminal gaps (default definition).
• (3) Marine Biological Lab definition counting each gap opening (internal or terminal)
as a single mismatch, whether or not the gap was extended: 1.0- ((mismatches + gap
openings)/(longest sequence length)).
• (4) BLAST definition, equivalent to –iddef 1 for global pairwise alignments.
rank_phylum Character string containing Phylum rank taxonomy
rank_class Character string containing Class rank taxonomy
6 df_to_isoLIB

rank_order Character string containing Order rank taxonomy

rank_family Character string containing Family rank taxonomy
rank_genus Character string containing Genus rank taxonomy
rank_species Character string containing Species rank taxonomy
phylum_threshold Numeric string containing Phylum-level sequence similarity threshold for rank
demarcation
class_threshold Numeric string containing Class-level sequence similarity threshold for rank
demarcation
order_threshold Numeric string containing Order-level sequence similarity threshold for rank
demarcation
family_threshold Numeric string containing Family-level sequence similarity threshold for rank
demarcation
genus_threshold Numeric string containing Genus-level sequence similarity threshold for rank
demarcation
species_threshold Numeric string containing Species-level sequence similarity threshold for
rank demarcation

See Also

isoTAX

df_to_isoLIB Convert isoLIB .CSV output to isoLIB class object

Description

Helper function to convert isoLIB .CSV output to a class-isoLIB class object.

Usage

df_to_isoLIB(df)

Arguments

df Dataframe in same format as .CSV output file from isoLIB step.

Value

Returns an S4 class-isoLIB object that can be used to generate interactive HTML output tables.
df_to_isoTAX 7

df_to_isoTAX Convert isoTAX .CSV output to isoTAX class object

Description
Helper function to convert isoTAX .CSV output to a class-isoTAX class object.

Usage
df_to_isoTAX(df)

Arguments
df Dataframe in same format as .CSV output file from isoTAX step.

Value
Returns an S4 class-isoTAX object that can be used to generate interactive HTML output tables.

export_html Export HTML for isoQC > isoTAX > isoLIB class objects

Description
S4 wrapper functions to export interactive HTML tables from isoQC, isoTAX, or isoLIB class
objects. Saves to HTML to current working directory and automatically opens.

Usage
## S4 method for signature 'isoQC'
export_html(
obj,
min_phred_score = NULL,
min_length = NULL,
sliding_window_cutoff = NULL,
sliding_window_size = NULL
)

## S4 method for signature 'isoTAX'

export_html(obj, quick_search = NULL, db = NULL)

## S4 method for signature 'isoLIB'

export_html(obj, method = NULL, group_cutoff = NULL)

Arguments
obj An S4 class object generated from one of isoQC, isoTAX, or isoLIB steps

Value
HTML output file saved to working directory.
8 get_os

get_db Download taxonomic reference database

Description

This function downloads taxonomic reference database and formats them for use.

Usage

get_db(db = "16S_bac", force_update = FALSE, add_taxonomy = FALSE)

Arguments

db Database selection. One of "16S", "16S_arc", "18S", "ITS", or "cpn60"

force_update Forces new databases to be downloaded.
add_taxonomy Add full taxonomy to header to enable classification of sequences offline. This
is primarily for cases of operation with no internet or when computing within an
HPC cluster without administrator access. Results in a semicolon (;) delimited
FASTA header as follows: Accession_no;d__Domain;p__Phylum;c__Class;o__Order;f__Family;g__

Value

Returns file path for database of interest

Examples
db.path <- get_db(db="16S", force_update=FALSE)

get_os Determine user operating system.

Description

Determines the type of operating system being used.

Usage

get_os()

Value

Returns sysname as one of windows/osx-mac/linux

get_sanger_date 9

Examples

#Example 1 on a Windows-based operating system

os.index <- get_os()
print(os.index)

#Example 2 on a Mac operating system

os.index <- get_os()
print(os.index)

#Example 3 on a Linux operating system

os.index <- get_os()
print(os.index)

get_sanger_date get_sanger_date function

Description

Helper function to automatically retrieve run date from Sanger sequencing .ab1 files.

Usage

get_sanger_date(file = NULL)

Arguments

file The .ab1 file in from which to retrieve the date information. (Must be in S4 abif
format)

Value

Returns date in "YYYY_MM_DD" format

Examples

#Path to the first listed .ab1 file in example directory

fpath <- file.path(system.file("extdata/abif_examples/rocket_salad", package = "isolateR"),
list.files(system.file("extdata/abif_examples/rocket_salad", package = "isolateR"))[1])
#Read in the ab1 file to S4 format
ab1.S4 <- sangerseqR::read.abif(fpath)

#Retrieve date
get_sanger_date(ab1.S4)
10 isoALL

get_vsearch Download VSEARCH software reference database

Description
This function downloads the VSEARCH software used querying sequences against taxonomic
databases of interest.

Usage
get_vsearch(os = NULL)

Arguments
os Operating system, one of: "windows", "osx-mac", or "linux". If blank (os=NULL)
then will try to automatically determine operating system.

Value
Returns path for VSEARCH executable

Examples
#Example for automatically detecting operating system and downloading VSEARCH software
vsearch.path <- get_vsearch()

isoALL Perform all commands in one step.

Description
This function effectively wraps isoQC, isoTAX, and isoLIB steps into a single command for con-
venience. Input can be a single directory or a list of directories to process at once. If multiple
directories are provided, the resultant libraries can be sequentially merged together by toggling
the parameter ’merge=TRUE’. All other respective parameters from the wrapped functions can be
passed through this command. . The The respective input parameters from the wrappred can be
passed through this command with exception of the .creates a strain library by grouping closely
related strains of interest based on sequence similarity. For adding new sequences to an already-
established strain library, specify the .CSV file path of the older strain library using the ’old_lib_csv"
parameter.

Usage
isoALL(
input = NULL,
export_html = TRUE,
export_csv = TRUE,
export_fasta = TRUE,
export_fasta_revcomp = FALSE,
export_blast_table = FALSE,
isoALL 11

quick_search = FALSE,
db = "16S",
iddef = 2,
phylum_threshold = 75,
class_threshold = 78.5,
order_threshold = 82,
family_threshold = 86.5,
genus_threshold = 94.5,
species_threshold = 98.7,
include_warnings = FALSE,
method = "dark_mode",
group_cutoff = 0.995,
keep_old_reps = TRUE,
merge = FALSE
)

Arguments
input Directory path(s) containing .ab1 files. If more than one, provivde as list (e.g.
’input=c("/path/to/directory1","/path/to/directory2")’)
export_html (Default=TRUE) Output the results as an HTML file
export_csv (Default=TRUE) Output the results as a CSV file.
export_fasta (Default=TRUE) Output the sequences in a FASTA file.
export_fasta_revcomp
(Default=FALSE) Output the sequences in reverse complement form in a fasta
file. This is useful in cases where sequencing was done using the reverse primer
and thus the orientation of input sequences needs reversing.
quick_search (Default=FALSE) Whether or not to perform a comprehensive database search
(i.e. optimal global alignment). If TRUE, performs quick search equivalent to
setting VSEARCH parameters "–maxaccepts 100 –maxrejects 100". If FALSE,
performs comprehensive search equivalent to setting VSEARCH parameters "–
maxaccepts 0 –maxrejects 0"
db (Default="16S") Select database option(s) including "16S" (for searching against
the NCBI Refseq targeted loci 16S rRNA database), "ITS" (for searching against
the NCBI Refseq targeted loci ITS database. For combined databases in cases
where input sequences are dervied from bacteria and fungi, select "16S|ITS".
iddef Set pairwise identity definition as per VSEARCH definitions (Default=2, and is
recommended for highest taxonomic accuracy) (0) CD-HIT definition: (match-
ing columns) / (shortest sequence length). (1) Edit distance: (matching columns)
/ (alignment length). (2) Edit distance excluding terminal gaps (default defini-
tion). (3) Marine Biological Lab definition counting each gap opening (internal
or terminal) as a single mismatch, whether or not the gap was extended: 1.0-
((mismatches + gap openings)/(longest sequence length)). (4) BLAST defini-
tion, equivalent to –iddef 1 for global pairwise alignments.
phylum_threshold
Percent cutoff for phylum rank demarcation
class_threshold
Percent cutoff for class rank demarcation
order_threshold
Percent cutoff for order rank demarcation
12 isoALL

family_threshold
Percent cutoff for family rank demarcation
genus_threshold
Percent cutoff for genus rank demarcation
species_threshold
Percent cutoff for species rank demarcation
include_warnings
(Default=FALSE) Whether or not to keep sequences with poor alignment warn-
ings from Step 2 ’isoTAX’ function. Set TRUE to keep warning sequences, and
FALSE to remove warning sequences.
method Method used for grouping sequences. Either 1) "dark_mode", or 2) "closest_species"
(Default="dark_mode").
• Method 1 ("dark_mode") performs agglomerative hierarchical-based clus-
tering to group similar sequences based on pairwise identity (see ’id’ pa-
rameter) and then within each group, attempts to assign the longest se-
quence with the most top hits as the group representative. This method
is tailored for capturing distinct strains which may represent novel taxa (i.e.
microbial dark matter) during isolation workflows. As such, the sequence
representatives chosen in each group will not always have the highest %
identity to the closest matching type strain. In some cases, sequence mem-
bers within a group may also have different taxonomic classifications due
to them having close to equidistant % identities to different matching type
strain material – indicative of a potentially novel taxonomic grouping.
• Method 2 ("closest_species") groups similar sequences based on their clos-
est matching type strain. For each unique grouping, this results in all se-
quence members having the same taxonomic classification. The longest se-
quence with the highest % identity to the closest matching type strain will
be assigned as the group representative. Note: The "id" parameter is only
used for Method 1 ("dark_mode") and otherwise ignored if using Method 2
("closest_species").
group_cutoff (Default=0.995) Similarity threshold based on pairwise identity (0-1) for de-
lineating between sequence groups. 1 = 100% identical/0.995=0.5% differ-
ence/0.95=5.0% difference/etc. Used only if method="dark_mode", otherwise
ignored.
keep_old_reps (Default=TRUE) If TRUE, original sequence representatives from old library
will be kept when merging with new library. If FALSE, sequence group repre-
sentatives will be recalculated after combining old and new libraries. Ignored if
old_lib_csv=NULL.
merge If TRUE, combines isoLIB output files consecutively in the order they are listed.
Default=FALSE performs all the steps (isoQC/isoTAX/isoLIB) on each direc-
tory separately.
verbose (Default=FALSE) Output progress while script is running.
files_manual (Default=NULL) For testing purposes only. Specify a list of files to run as file-
names without extensions, rather than the whole directory format. Primarily
used for testing, use at your own risk.
exclude (Default=NULL) For testing purposes only. Excludes files of interest from input
directory.
min_phred_score
(Default=20) Do not accept trimmed sequences with a mean Phred score below
this cutoff
isoLIB 13

min_length (Default=200) Do not accept trimmed sequences with sequence length below
this number
sliding_window_cutoff
(Default=NULL) Quality trimming parameter (M2) for wrapping SangerRead
function in sangeranalyseR package. If NULL, implements auto cutoff for Phred
score (recommended), otherwise set between 1-60.
sliding_window_size
(Default=15) Quality trimming parameter (M2) for wrapping SangerRead func-
tion in sangeranalyseR package. Recommended range between 5-30.
date Set date "YYYY_MM_DD" format. If NULL, attempts to parse date from .ab1
file

Value
Returns a list of class-isoLIB class objects.

See Also
isoQC, isoTAX, isoLIB

Examples
#Set path to directory containing example .ab1 files
fpath1 <- system.file("extdata/abif_examples/rocket_salad", package = "isolateR")

#Run isoALL function with default settings

isoALL(input=fpath1)

isoLIB Generate new strain library or add to existing one.

Description
This function creates a strain library by grouping closely related strains of interest based on se-
quence similarity. For adding new sequences to an already-established strain library, specify the
.CSV file path of the older strain library using the ’old_lib_csv" parameter.

Usage
isoLIB(
input = NULL,
old_lib_csv = NULL,
method = "dark_mode",
group_cutoff = 0.995,
keep_old_reps = TRUE,
export_html = TRUE,
export_csv = TRUE,
include_warnings = TRUE,
vsearch_path = NULL,
phylum_threshold = 75,
class_threshold = 78.5,
14 isoLIB

order_threshold = 82,
family_threshold = 86.5,
genus_threshold = 94.5,
species_threshold = 98.7
)

Arguments
input Path of CSV output file from isoTAX step.
old_lib_csv Optional: Path of CSV output isoLIB file or combined isoLIB file from previous
run(s)
method Method used for grouping sequences. Either 1) "dark_mode", or 2) "closest_species"
(Default="dark_mode").
• Method 1 ("dark_mode") performs agglomerative hierarchical-based clus-
tering to group similar sequences based on pairwise identity (see ’id’ pa-
rameter) and then within each group, attempts to assign the longest se-
quence with the most top hits as the group representative. This method
is tailored for capturing distinct strains which may represent novel taxa (i.e.
microbial dark matter) during isolation workflows. As such, the sequence
representatives chosen in each group will not always have the highest %
identity to the closest matching type strain. In some cases, sequence mem-
bers within a group may also have different taxonomic classifications due
to them having close to equidistant % identities to different matching type
strain material – indicative of a potentially novel taxonomic grouping.
• Method 2 ("closest_species") groups similar sequences based on their clos-
est matching type strain. For each unique grouping, this results in all se-
quence members having the same taxonomic classification. The longest se-
quence with the highest % identity to the closest matching type strain will
be assigned as the group representative. Note: The "id" parameter is only
used for Method 1 ("dark_mode") and otherwise ignored if using Method 2
("closest_species").
group_cutoff (Default=0.995) Similarity threshold based on pairwise identity (0-1) for de-
lineating between sequence groups. 1 = 100% identical/0.995=0.5% differ-
ence/0.95=5.0% difference/etc. Used only if method="dark_mode", otherwise
ignored.
keep_old_reps (Default=TRUE) If TRUE, original sequence representatives from old library
will be kept when merging with new library. If FALSE, sequence group repre-
sentatives will be recalculated after combining old and new libraries. Ignored if
old_lib_csv=NULL.
export_html (Default=TRUE) Output the results as an HTML file
export_csv (Default=TRUE) Output the results as a CSV file.
include_warnings
(Default=FALSE) Whether or not to keep sequences with poor alignment warn-
ings from Step 2 ’isoTAX’ function. Set TRUE to keep warning sequences, and
FALSE to remove warning sequences.
vsearch_path Path of VSEARCH software if manually downloaded in a custom directory. If
NULL (Default), will attempt automatic download.
phylum_threshold
Percent sequence similarity threshold for phylum rank demarcation
isoQC 15

class_threshold
Percent sequence similarity threshold for class rank demarcation
order_threshold
Percent sequence similarity threshold for order rank demarcation
family_threshold
Percent sequence similarity threshold for family rank demarcation
genus_threshold
Percent sequence similarity threshold for genus rank demarcation
species_threshold
Percent sequence similarity threshold for species rank demarcation

Value

Returns an isoLIB class object. Default taxonomic cutoffs for phylum (75.0), class (78.5), order
(82.0), family (86.5), genus (94.5), and species (98.7) demarcation are based on Yarza et al. 2014,
Nature Reviews Microbiology (DOI:10.1038/nrmicro3330)

See Also

isoTAX, isoLIB

Examples
#Set path to directory containing example .ab1 files
fpath1 <- system.file("extdata/abif_examples/rocket_salad", package = "isolateR")

#Step 1: Run isoQC function with default settings

isoQC.S4 <- isoQC(input=fpath1)

#Step 2: Run isoTAX function with default settings

fpath2 <- file.path(fpath1, "isolateR_output/01_isoQC_trimmed_sequences_PASS.csv")
isoTAX.S4 <- isoTAX(input=fpath2)

#Step 3: Run isoLIB function with default settings

fpath3 <- file.path(fpath1, "isolateR_output/02_isoTAX_results.csv")
isoLIB.S4 <- isoLIB(input=fpath3)

#Show summary statistics

isoLIB.S4

isoQC Perform automated quality trimming of input .ab1 files

Description

This function loads in ABIF files (.ab1 extension) and performs automatic quality trimming in batch
mode.
16 isoQC

Usage
isoQC(
input = NULL,
export_html = TRUE,
export_csv = TRUE,
export_fasta = TRUE,
export_fasta_revcomp = FALSE,
verbose = FALSE,
exclude = NULL,
min_phred_score = 20,
min_length = 200,
sliding_window_cutoff = NULL,
sliding_window_size = 15,
date = NULL,
files_manual = NULL
)

Arguments
input Path to directory with .ab1 files.
export_html (Default=TRUE) Output the results as an HTML file
export_csv (Default=TRUE) Output the results as a CSV file.
export_fasta (Default=TRUE) Output the sequences in a FASTA file.
export_fasta_revcomp
(Default=FALSE) Output the sequences in reverse complement form in a fasta
file. This is useful in cases where sequencing was done using the reverse primer
and thus the orientation of input sequences needs reversing.
verbose (Default =FALSE) Output progress while script is running, FALSE for simpli-
fied progress, TRUE for file-by-file details
exclude (Default=NULL) For testing purposes only. Excludes files of interest from input
directory.
min_phred_score
(Default=20) Do not accept trimmed sequences with a mean Phred score below
this cutoff
min_length (Default=200) Do not accept trimmed sequences with sequence length below
this number
sliding_window_cutoff
(Default=NULL) Quality trimming parameter (M2) for wrapping SangerRead
function in sangeranalyseR package. If NULL, implements auto cutoff for Phred
score (recommended), otherwise set between 1-60.
sliding_window_size
(Default=15) Quality trimming parameter (M2) for wrapping SangerRead func-
tion in sangeranalyseR package. Recommended range between 5-30.
date Set date "YYYY_MM_DD" format. If NULL, attempts to parse date from .ab1
file
files_manual (Default=NULL) For testing purposes only. Specify a list of files to run as file-
names without extensions, rather than the whole directory format. Primarily
used for testing, use at your own risk.
isoTAX 17

Value

Returns quality trimmed Sanger sequences in FASTA format.

See Also

isoTAX, isoLIB

Examples
#Set path to directory containing example .ab1 files
fpath1 <- system.file("extdata/abif_examples/rocket_salad", package = "isolateR")

#Step 1: Run isoQC function with default settings

isoQC.S4 <- isoQC(input=fpath1)

#Show summary statistics

isoQC.S4

isoTAX Classify taxonomy of sequences after quality trimming steps.

Description

This function performs taxonomic classification steps by searching query Sanger sequences against
specified database of interest. Takes CSV input files, extracts FASTA-formatted query sequences
and performs global alignment against specified database of interest via Needleman-Wunsch algo-
rithm by wrapping the –usearch_global command implemented in VSEARCH. Default taxonomic
rank cutoffs for 16S rRNA gene sequences are based on Yarza et al. 2014, Nat Rev Microbiol.

Usage

isoTAX(
input = NULL,
export_html = TRUE,
export_csv = TRUE,
export_blast_table = FALSE,
quick_search = FALSE,
db = "16S_bac",
db_path = NULL,
vsearch_path = NULL,
iddef = 2,
phylum_threshold = 75,
class_threshold = 78.5,
order_threshold = 82,
family_threshold = 86.5,
genus_threshold = 94.5,
species_threshold = 98.7
)
18 isoTAX

Arguments
input Path of either 1) CSV output file from isoQC step, or 2) a FASTA formatted
file. If input is a FASTA file, the sequence(s) will be converted and saved as an
isoQC-formatted output file in the current working directory ("isolateR_output/01_isoQC_mock_table
Sequence date, name, length, and number of ambiguous bases (Ns) will be cal-
culated from the input file and used to populate the relevant columns. Phred
quality scores (phred_trim) will be set to the maximum value (60) and the re-
maining columns will be populated with mock data to allow compatibility with
the isoTAX function. The main purpose of this output file is for flexibility and
to allow users to edit/modify the sequence metadata before continuing with sub-
sequent steps.
export_html (Default=TRUE) Output the results as an HTML file
export_csv (Default=TRUE) Output the results as a CSV file.
export_blast_table
(Default=FALSE) Output the results as a tab-separated BLAST-like hits table.
quick_search (Default=FALSE) Whether or not to perform a comprehensive database search
(i.e. optimal global alignment). If TRUE, performs quick search equivalent to
setting VSEARCH parameters "–maxaccepts 100 –maxrejects 100". If FALSE,
performs comprehensive search equivalent to setting VSEARCH parameters "–
maxaccepts 0 –maxrejects 0"
db (Default="16S_bac") Select database option(s) including "16S" (for searching
against the NCBI Refseq targeted loci 16S rRNA database), "ITS" (for searching
against the NCBI Refseq targeted loci ITS database. For combined databases in
cases where input sequences are derived from bacteria and fungi, select "16S|ITS".
Setting to anything other than db=NULL or db="custom" causes ’db.path’ pa-
rameter to be ignored.
db_path Path of FASTA-formatted database sequence file. Ignored if ’db’ parameter is
set to anything other than NULL or "custom".
vsearch_path Path of VSEARCH software if manually downloaded in a custom directory. If
NULL (Default), will attempt automatic download.
iddef Set pairwise identity definition as per VSEARCH definitions (Default=2, and is
recommended for highest taxonomic accuracy) (0) CD-HIT definition: (match-
ing columns) / (shortest sequence length). (1) Edit distance: (matching columns)
/ (alignment length). (2) Edit distance excluding terminal gaps (default defini-
tion). (3) Marine Biological Lab definition counting each gap opening (internal
or terminal) as a single mismatch, whether or not the gap was extended: 1.0-
((mismatches + gap openings)/(longest sequence length)). (4) BLAST defini-
tion, equivalent to –iddef 1 for global pairwise alignments.
phylum_threshold
Percent sequence similarity threshold for phylum rank demarcation
class_threshold
Percent sequence similarity threshold for class rank demarcation
order_threshold
Percent sequence similarity threshold for order rank demarcation
family_threshold
Percent sequence similarity threshold for family rank demarcation
genus_threshold
Percent sequence similarity threshold for genus rank demarcation
species_threshold
Percent sequence similarity threshold for species rank demarcation
make_fasta 19

Value
Returns taxonomic classification table of class isoTAX. Default taxonomic cutoffs for phylum
(75.0), class (78.5), order (82.0), family (86.5), genus (94.5), and species (98.7) demarcation are
based on Yarza et al. 2014, Nature Reviews Microbiology (DOI:10.1038/nrmicro3330)

See Also
isoQC, isoLIB, search_db

Examples
#Set path to directory containing example .ab1 files
fpath1 <- system.file("extdata/abif_examples/rocket_salad", package = "isolateR")

#Step 1: Run isoQC function with default settings

isoQC.S4 <- isoQC(input=fpath1)

#Step 2: Run isoTAX function with default settings

fpath2 <- file.path(fpath1, "isolateR_output/01_isoQC_trimmed_sequences_PASS.csv")
isoTAX.S4 <- isoTAX(input=fpath2)
#Show summary statistics
isoTAX.S4

make_fasta Convert CSV file containing sequences to FASTA format

Description
This function extracts sequences from a table in CSV format and converts them to FASTA format.
Requires two columns, one with sequences and one with sequence names.

Usage
make_fasta(
csv_file = NULL,
col_names = "ID",
col_seqs = "Sequence",
output = "output.fasta"
)

Arguments
csv_file Filename (or path and filename if not in working directory) of the table from
which you would like to generate a FASTA file.
col_names Column name with the unique names/identifiers. (Default="ID")
col_seqs Column name with the sequences. (Default="Sequence")
output Desired filename for output FASTA file (Default = "output.fasta")

Value
Returns sequences in FASTA format.
20 make_tree

Examples
#Set path to directory containing example .ab1 files
fpath1 <- system.file("extdata/abif_examples/rocket_salad", package = "isolateR")

#Run isoQC function with default settings to generate CSV file

isoQC.S4 <- isoQC(input=fpath1)

#Set path of CSV output file from isoQC step

csv.path <- file.path(fpath1, "isolateR_output/01_isoQC_trimmed_sequences_PASS.csv")

#Run make_fasta function

make_fasta(csv_file= csv.path, col_names="filename", col_seqs="seqs_trim", output="output.fasta")

make_tree Generate a phylogenetic tree from an isoLIB output file

Description
This script will help the user make a simple phylogenetic tree from a strain library. It will allow the
user to colour the tree by taxonomic rank only. See ggtree documentation for more information on
customization options available.

Usage
make_tree(input = NULL)

Arguments
input Full path to isoLIB strain library output file in .CSV format.

Value
Returns a ggtree class object

See Also
isoLIB

Examples
#Set path to directory containing example .ab1 files
fpath1 <- system.file("extdata/abif_examples/rocket_salad", package = "isolateR")

#Step 1: Run isoQC function with default settings

isoQC.S4 <- isoQC(input=fpath1)

#Step 2: Run isoTAX function with default settings

fpath2 <- file.path(fpath1, "isolateR_output/01_isoQC_trimmed_sequences_PASS.csv")
isoTAX.S4 <- isoTAX(input=fpath2)

#Step 3: Run isoLIB function with default settings

fpath3 <- file.path(fpath1, "isolateR_output/02_isoTAX_results.csv")
isoLIB.S4 <- isoLIB(input=fpath3)
method-isoLIB 21

#Step 4: Make a tree from isoLIB output CSV file

fpath4 <- file.path(fpath1, "isolateR_output/03_isoLIB_results.csv")
make_tree(input= fpath4)

method-isoLIB setMethod functions for isoLIB

Description
Initiation of isoLIB functions.

Usage
## S4 method for signature 'missing'
isoLIB(
input = NULL,
old_lib_csv = NULL,
method = "dark_mode",
group_cutoff = 0.995,
keep_old_reps = TRUE,
export_html = TRUE,
export_csv = TRUE,
include_warnings = TRUE,
vsearch_path = NULL,
phylum_threshold = 75,
class_threshold = 78.5,
order_threshold = 82,
family_threshold = 86.5,
genus_threshold = 94.5,
species_threshold = 98.7
)

method-isoQC setMethod functions for isoQC

Description
Initiation of isoQC functions.

Usage
## S4 method for signature 'missing'
isoQC(
input = NULL,
export_html = TRUE,
export_csv = TRUE,
export_fasta = TRUE,
export_fasta_revcomp = FALSE,
verbose = FALSE,
exclude = NULL,
22 S4_to_dataframe

min_phred_score = 20,
min_length = 200,
sliding_window_cutoff = NULL,
sliding_window_size = 15,
date = NULL,
files_manual = NULL
)

method-isoTAX setMethod functions for isoTAX

Description
Initiation of isoTAX functions.

Usage
## S4 method for signature 'missing'
isoTAX(
input = NULL,
export_html = TRUE,
export_csv = TRUE,
export_blast_table = FALSE,
quick_search = FALSE,
db = "16S_bac",
db_path = NULL,
vsearch_path = NULL,
iddef = 2,
phylum_threshold = 75,
class_threshold = 78.5,
order_threshold = 82,
family_threshold = 86.5,
genus_threshold = 94.5,
species_threshold = 98.7
)

S4_to_dataframe Converts S4 objects (isoQC, isoTAX, or isoLIB) to dataframe

Description
Helper function to convert S4 class objects (isoQC, isoTAX, or isoLIB) to dataframe

Usage
S4_to_dataframe(obj)

Arguments
obj S4 object generated from isoQC, isoTAX, or isoLIB steps
sanger_assembly 23

Value
Returns a dataframe containing sequence information in columns.

sanger_assembly Overlap multiple paired Sanger sequences in batch.

Description
This function loads in the CSV results table from isoQC and merges related sequences based on
user input. Original file names before isoQC step need to have a common prefix and differentiating
suffixes. (e.g. SAMPLE_01_F.ab1, SAMPLE_01_R.ab1). After aligning paired sequences, the
consensus sequence is extracted and priority is given to the read with higher quality. Phred quality
scores are reassigned in the final output table in a basic way by taking the mean of both input
sequences.
Note: This function is designed to be used after the isoQC step and before the isoTAX step.

Usage
sanger_assembly(input = NULL, suffix = "_F.ab1|_R.ab1")

Arguments
input Path of CSV output file from isoQC step.
suffix Regex-friendly suffix for denoting filename groupings. Default="_F.ab1|_R.ab1"
for the common scenario of Sanger sequencing a marker gene in forward and
reverse. Direction of sequences including reverse complements will be auto-
matically detected.

Value
Returns merged pairs of Sanger sequences in FASTA format.

See Also
isoQC, isoTAX

Examples
#Load package
library(isolateR)

#Step 1: Set path to directory containing paired .ab1 files

fpath <- system.file("extdata/abif_examples/drosophila_paired", package="isolateR")

#Step 2: Run isoQC function to trim poor quality regions (Phred score <20) before assembly
isoQC.S4 <- isoQC(input=fpath, sliding_window_cutoff = 20)

#Step 3: Assemble paired sequences

sanger_assembly(input = file.path(fpath,"isolateR_output", "01_isoQC_trimmed_sequences_PASS.csv"),
suffix = "_F.ab1|_R.ab1")

#Detected 3 unique group(s) with suffix provided.

24 search_db

#Group Individual filenames

#----- --------------------
# 1 DRO-1-isolate_F.ab1 | DRO-1-isolate_R.ab1
# 2 DRO-2-isolate_F.ab1 | DRO-2-isolate_R.ab1
# 3 DRO-3-isolate_F.ab1 | DRO-3-isolate_R.ab1

search_db Perform global alignment pairwise identity search using VSEARCH

and type strain database of interest.

Description
Performs global alignment between FASTA-formatted query sequences and the specified database
of interest. Uses the Needleman-Wunsch algorithm by wrapping the –usearch_global command
implemented in VSEARCH.

Usage
search_db(
query.path = NULL,
uc.out = "VSEARCH_output.uc",
b6.out = "VSEARCH_output.b6o",
path = getwd(),
quick_search = FALSE,
db = NULL,
db_path = NULL,
vsearch_path = NULL,
keep_temp_files = FALSE,
iddef = 2
)

Arguments
query.path Path of FASTA-formatted query sequence file.
uc.out Path of UC-formatted results output table.
b6.out Path of blast6-formatted results output table.
path Working path directory (Default is set to current working directory via ’getwd()’
quick_search (Default=FALSE) Whether or not to perform a comprehensive database search
(i.e. optimal global alignment). If TRUE, performs quick search equivalent to
setting VSEARCH parameters "–maxaccepts 100 –maxrejects 100". If FALSE,
performs comprehensive search equivalent to setting VSEARCH parameters "–
maxaccepts 0 –maxrejects 0" Note: This option is provided for convenience and
rough approximation of taxonomy only, set to FALSE for accurate % pairwise
identity results.
db Optional: Select any of the standard database option(s) including "16S" (for
searching against the NCBI Refseq targeted loci 16S rRNA database), "ITS" (for
searching against the NCBI Refseq targeted loci ITS database. For combined
databases in cases where input sequences are dervied from bacteria and fungi,
select "16S|ITS". Setting to anything other than db=NULL or db="custom"
causes ’db.path’ parameter to be ignored.
search_db 25

vsearch_path Path of VSEARCH software if manually downloaded in a custom directory. If

NULL (Default), will attempt automatic download.
keep_temp_files
Toggle (TRUE/FALSE). If TRUE, temporary .uc and .b6o output files are kept
from VSEARCH –uc and –blast6out commands, respectively. If FALSE, tem-
porary files are removed.
iddef Set pairwise identity definition as per VSEARCH definitions (Default=2, and is
recommended for highest taxonomic accuracy) (0) CD-HIT definition: (match-
ing columns) / (shortest sequence length). (1) Edit distance: (matching columns)
/ (alignment length). (2) Edit distance excluding terminal gaps (default defini-
tion). (3) Marine Biological Lab definition counting each gap opening (internal
or terminal) as a single mismatch, whether or not the gap was extended: 1.0-
((mismatches + gap openings)/(longest sequence length)). (4) BLAST defini-
tion, equivalent to –iddef 1 for global pairwise alignments.
db.path Path of FASTA-formatted database sequence file. Ignored if ’db’ parameter is
set to anything other than "custom"

Value
Returns a dataframe matching the UC-formatted output table from VSEARCH. Query sequences
are automatically added to the final column. Summary of column information. See VSEARCH
documentation for more details.

• V1 = Record type of hit (H) or no hit (N)

• V2 = Ordinal number of the target sequence (based on input order, starting from zero). Set to
’*’ for N.
• V3 = Sequence length. Set to ’*’ for N.
• V4 = Percentage of similarity with the target sequence. Set to ’*’ for N.
• V5 = Match orientation + or -. . Set to ’.’ for N.
• V6 = Not used, always set to zero for H, or ’*’ for N.
• V7 = Not used, always set to zero for H, or ’*’ for N.
• V8 = Compact representation of the pairwise alignment using the CIGAR format (Compact
Idiosyncratic Gapped Alignment Report): M (match/mismatch), D (deletion) and I (insertion).
The equal sign ’=’ indicates that the query is identical to the centroid sequence. Set to ’*’ for
N.
• V9 = Label of the query sequence. Equivalent to ’filename’ slot of isolateR class objects (e.g.
isoQC, isoTAX, isoLIB).
• V10 = Label of the target centroid sequence. Set to ’*’ for N.

See Also
isoTAX

Examples
#Set path to directory containing example .ab1 files
fpath1 <- system.file("extdata/abif_examples/rocket_salad", package = "isolateR")

#Run isoQC function with default settings

isoQC.S4 <- isoQC(input=fpath1)
26 valid_tax_check

#Set path of CSV output file containing PASS sequences from isoQC step
fasta.path <- "01_isoQC_trimmed_sequences_PASS.fasta"

#Set paths
output.path <- file.path(fpath1, "isolateR_output")

#Run search_db function

uc.df <- search_db(query.path=fasta.path, path=output.path, quick_search=TRUE, db="16S")

#Inspect results
uc.df[1:10,1:10]

show Generic show method for S4 class objects

Description
Generic show method for S4 class objects.

Usage
## S4 method for signature 'isoQC'
show(object)

## S4 method for signature 'isoTAX'

show(object)

## S4 method for signature 'isoLIB'

show(object)

valid_tax_check Validate species name via API client of LPSN

Description
This function will determine if each species in a CSV file is validly published or not. Result file
will be a CSV with the results appended to the input data. This function requires the user to have
an LPSN API account setup. For more details and to register, see here: https://api.lpsn.dsmz.de/)

Usage
valid_tax_check(input = NULL, col_species = "rank_species", export_csv = TRUE)

Arguments
input CSV file path. Expects full path if CSV file is not in the current working direc-
tory.
col_species Specify the column containing the binomial species names (e.g. "Akkermansia
muciniphila")
export_csv Toggle (TRUE/FALSE). Set TRUE to automatically write .CSV file of results to
current directory. (Default=TRUE)
valid_tax_check 27

Value
Returns a CSV saved in working directory
Index

class-isoLIB, 2
class-isoQC, 4
class-isoTAX, 5

df_to_isoLIB, 6
df_to_isoTAX, 7

export_html, 7
export_html-isoLIB (export_html), 7
export_html-isoQC (export_html), 7
export_html-isoTAX (export_html), 7

get_db, 8
get_os, 8
get_sanger_date, 9
get_vsearch, 10
ggtree, 20

isoALL, 10
isoLIB, 2, 3, 6, 7, 13, 13, 15, 17, 19, 20, 22
isoQC, 4, 7, 13, 15, 19, 22, 23
isoTAX, 5–7, 13, 15, 17, 17, 22, 23, 25

make_fasta, 19
make_tree, 20
method-isoLIB, 21
method-isoQC, 21
method-isoTAX, 22

S4_to_dataframe, 22
sanger_assembly, 23
search_db, 19, 24
show, 26

valid_tax_check, 26

28