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    Application of NGS To Pathogen Genome

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    Application of NGS To Pathogen Genome

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    Julia Martinez
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    Application of NGS to pathogen genome

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    7 views3 pages

    Application of NGS To Pathogen Genome

    Uploaded by

    Julia Martinez

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    © All Rights Reserved
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    Application of Genomics in Infectious Disease.

    Week 1
    Bacterial genomics and antimicrobial resistance

    The application of NGS technologies to pathogen genomes


    By: Elita Jauneikaite

    DNA and DNA Sequencing timeline

    1977- Invention of dideoxy chain terminator sequencing by Fred Sanger


    1981- First human mitochondrial genome sequence
    1995- First complete genome sequences obtained of free-living bacteria (H. influenza)
    1997- First genome sequenced from E. coli and B.subtilis
    1998- First genome sequenced from M. tuberculosis
    1999- Evidence for lateral gene transfer between Archaea and bacteria
    2000- Meningococcal genome sequence kick-starts reverse vaccinology
    2005- First next-generation sequencer: the 454 GS20
    2007- Release of the Illumina Genetic Analyzer 2
    2008- First genomics super-project sequencing of 100 strains of S. Typhi
    2012- CRISPR-Cas adopted for programmable genome editing
    2014- Oxford Nanopore launch MiniION Access Programme
    2015- Genome sequencing adopted for routine surveillance of salmonellosis and tuberculosis

    Nowadays: Databases: NCBI, SRA, Genbank...


    Large-scale genome sequencing projects: 20,000 Global S.pneumoniae genomes, 20,000 HIV
    genomes, 100,000 HGP

    Sanger Sequencing (Chain termination sequencing)


     Sanger sequencing involves a single stranded DNA template, primer, polymerase,
    deoxynucleosidetriphosphates (dNTPs) and modified (ddNTPs). The ddNTPs are
    radioactively or fluorescently labeled, and are responsible for DNA template termination .
     Following the DNA template extension, resulting DNA fragments are denaturated and
    separated based on size by electrophoresis on acrylamide gel .
     The gel is read under UV light and DNA sequence is extrapolated.

    Illumina sequencing (Sequencing-by-synthesis)


     Adapter ligated DNA fragments are attached to a primer on the slide; DNA is then amplified to
    form DNA clusters.
     Four reverse termination bases with different fluorescent labels are added.
     The Illumina sequencer reads the fluorescently modified nucleotides as they are incorporated

    PacBio RS Sequencing (SMRT)


     Single molecule real time sequencing (SMRT) adapted by Pacific Biosciences utilizes a
    determined primer and fluorescent nucleotide analogues that provide a fluorescent signal to
    the machine as they are added for DNA synthesis.
     This is done using a zero mode wavelength (ZMW), which allows only a single light wave to
    pass through the bottom of the well, which is then read by a laser.
     A single SMRT chip consists of 150,000 ZMW wells, each of which has a single polymerase
    attached at the bottom of the well.

    Nanopore sequencing (Strand sequencing)

     Nanopore DNA sequencing is based on detection of nucleotide specific ionic current during
    DNA amplification.
     A single stranded DNA (usually in KCl solution) is driven through a microscopic nanopore.
     The nanopore has a DNA polymerase attached and the DNA molecule blocks it.
     Each of the nucleotides as they pass through the nanopore will produce an ionic current that
    will fluctuate.
     This ionic current fluctuation provides information on length and composition of the DNA
    molecule

    QUIZ

    Q1: Name two sequencing technologies currently able to sequence long reads (>1000bp).
    a) PacBio and Nanopore (around 15Kb but up to 40kb) and (5.4-10kb)

    Q2: Which of the sequencing technologies is know as sequencing-by-synthesis?

    a) Illumina sequencing

    Q3: What does it mean 99.99% accuracy?

    b) 1 error in 10,000 bases sequenced

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