Scribd Logo
Upload

Welcome to Scribd!

  • 4 views

    Lecture13 2022

    Uploaded by

    tulitahkay

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd

    Lecture13 2022

    Uploaded by

    tulitahkay
    4 views20 pages

    Original Title

    Lecture13_2022

    Copyright

    © © All Rights Reserved

    Available Formats

    PDF, TXT or read online from Scribd

    Did you find this document useful?

    Is this content inappropriate?

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    4 views20 pages

    Lecture13 2022

    Uploaded by

    tulitahkay

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    of 20

    LECTURE 13

    What I want you to know

    How the different sequencing technologies work


    The Different generations of sequencing technology
    How the different sequencing technologies are used
    The differences in the data that are produced from the different technologies

    What is Big Data in genomics and its influence on your future.


    PacBio Sequencing
    PacBio technology

    Two fundamental Innovations


    Fluorescent molecule at terminal phosphate
    Other NGS technologies on base
    DNA polymerase cleaves fluorescent label when base incorporated
    Natural DNA strand left over

    Nanophotonic visualisation chamber called the Zero Mode Waveguide (ZMW)


    50 nm wide
    Small detection volume
    1 Nucleotide can be detected
    Millisecond fluorescent pulses collected
    1. A DNA template-polymerase complex is immobilized at the bottom of
    ZMW chamber.

    2. Different fluorescently labelled nucleotides are introduced into the ZMW


    chamber.

    3. DNA is not fractionated as seen in the second generation technologies.

    4. During incorporation, the phosphate chain is cleaved, releasing the attach-


    ed fluorophore which produces a light pulse.

    5. The nucleotide that is incorporated into the new strand will be present for
    milliseconds while the polymerase is catalyzing the new phosphodiester
    bond. This time difference provides sufficient signal to enable accurate base-
    calling. ZMW provides a tiny detection volume which enables observation of
    the individual molecules against the required background of labelled
    nucleotides, while maintaining high signal to noise.

    6. This process occurs in parallel in every ZMWs which are monitored


    simultaneously, in real time.
    Process occurs
    in parallel.
    Nanopore Sequencing
    (MinION)
    MinION technology

    Innovation
    Protein nanopore (nanoscale diameter)
    As a Ionic flow is applied continuously any particle movement (DNA, RNA
    or even Protein) can disrupt the voltage across the channel which is detected
    by standard electrophysiological technique. Readout is relied on the size
    difference between all dNTP.
    Reading speed is 30 bases per second per nanopore.

    The lack of a polymerase implies


    that nanopore sequencing is less
    sensitive to temperature.

    DNA is not fractionated as seen for


    second generation technologies.
    Process occurs
    in parallel.
    SHOTGUN SEQUENCING
    All second generation NGS relies on shotgun sequencing
    Reference-based
    assembly
    De novo assembly
    Reference-based assembly
    De novo assembly
    Genome comparisons
    Species Complex
    100 F. graminearum 31084
    100 F. asiaticum 26156
    Fusarium phylogeny 100 F. culmorum 25475
    100
    F. pseudograminearum 28062
    19 genes
    F. praegraminearum 39664
    55,140 bp 23,668 PIC 100 F. sporotrichioides 3299
    100
    best ML tree: -927514.615 100 F. langsethiae 53436

    84
    F. armeniacum 6227 sambucinum
    100 F. sambucinum 13708
    100 100
    F. venenatum 66329
    F. poae 26941
    100 F. compactum 13829
    100 100 F. transvaalense 31008
    100
    F. longipes 13317
    F. longipes 13368-1
    100 F. chlamydosporum 13444
    100 100 F. nelsonii 13338 chlamydosporum
    F. aywerte 25410
    100 F. scirpi 66328
    100 F. equiseti 66338 incarnatum-
    100
    F. irregulare 31160
    94 equiseti
    F. hainanense 66475
    100 F. avenaceum 54939
    100 F. tricinctum 25481 tricinctum
    99 F. torulosum 22747
    100 F. nurragi 36452

    Phylogenetics
    F. graminum 20692
    100
    F. heterosporum 20693
    heterosporum
    100 F. fujikuroi 5538
    100 F. mangiferae 25226
    F. sacchari 66326
    100 F. thapsinum 22049
    100
    100 F. verticillioides 20956
    F. xylarioides 25486
    fujikuroi
    86 F. circinatum 25331
    100

    100
    100 F. subglutinans 66333
    99 90 F. guttiforme 22945
    Fusarium sp. 52700
    100 F. gaditjirrii 45417
    100 F. lyarnte 54252
    100
    89
    96
    F. miscanthi 26231 nisikadoi
    F. nisikadoi 25179
    F. commune 28387

    Prokaryotic
    Eukaryotic
    Genomics and you
    Golden State Killer – Joseph James DeAngelo
    First convicted offender based on genome seq (August 2020)

    You might also like