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    Sequencing Technologies

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    Ohhh Okay
    This document summarizes several key techniques in DNA sequencing. It describes how DNA can be replicated in vitro, fragmented into libraries, and amplified. It also discusses genetic mapping, genomic libraries, hybridization, Sanger sequencing including its steps and applications. DNA arrays and how they work to reveal sequences are explained. Next generation sequencing techniques like Illumina and nanopore sequencing are introduced along with sample preparation, cluster generation, sequencing and data analysis steps. Nanopore sequencing's unique scalable approach and advantages are highlighted.

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    Sequencing Technologies

    Uploaded by

    Ohhh Okay
    72 views25 pages
    This document summarizes several key techniques in DNA sequencing. It describes how DNA can be replicated in vitro, fragmented into libraries, and amplified. It also discusses genetic mapping, genomic libraries, hybridization, Sanger sequencing including its steps and applications. DNA arrays and how they work to reveal sequences are explained. Next generation sequencing techniques like Illumina and nanopore sequencing are introduced along with sample preparation, cluster generation, sequencing and data analysis steps. Nanopore sequencing's unique scalable approach and advantages are highlighted.

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    This document summarizes several key techniques in DNA sequencing. It describes how DNA can be replicated in vitro, fragmented into libraries, and amplified. It also discusses genetic mapping, genomic libraries, hybridization, Sanger sequencing including its steps and applications. DNA arrays and how they work to reveal sequences are explained. Next generation sequencing techniques like Illumina and nanopore sequencing are introduced along with sample preparation, cluster generation, sequencing and data analysis steps. Nanopore sequencing's unique scalable approach and advantages are highlighted.

    Copyright:

    © All Rights Reserved
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    72 views25 pages

    Sequencing Technologies

    Uploaded by

    Ohhh Okay
    This document summarizes several key techniques in DNA sequencing. It describes how DNA can be replicated in vitro, fragmented into libraries, and amplified. It also discusses genetic mapping, genomic libraries, hybridization, Sanger sequencing including its steps and applications. DNA arrays and how they work to reveal sequences are explained. Next generation sequencing techniques like Illumina and nanopore sequencing are introduced along with sample preparation, cluster generation, sequencing and data analysis steps. Nanopore sequencing's unique scalable approach and advantages are highlighted.

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    © All Rights Reserved
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    of 25

    From fragments to reads

    Replication
    • Replication can be achieved in
    vitro if template DNA, Primer,
    Polymerase enzyme and
    nucleotides are available.
    • Nucleotides are added
    one at a time
    Fragmentation and amplification
    • Isolated DNA can be sheared into fragments
    either with known ends or unknown ends
    • The fragments can be separated into libraries
    • Amplified into millions of copies by cloning or
    polymerase chain reaction (PCR)
    • PCR involves Denaturation, Annealing and
    Elongation
    Genetic map
    Type of
    Chromosome map that shows
    the relative locations
    of genes and other important
    features.
    Knowledge of it
    ensures that the
    sequencing
    process can be
    tailored
    Genomic library
    A genomic library is
    usually stored as a set of
    bacteria, each carrying
    a different fragment of
    human/any species DNA.
    Hybridization

    Single strand DNA can


    pairup with any
    complementary strand.
    This forms basis of
    sequencing techniques
    like southern blotting,
    Illumina, DNA Array
    Sanger Sequencing
    • Dideoxy or chain terminataion method
    • Fred Sanger, 1977, X174 virus, 5386bp
    • Small genome like that of virus succesfully
    sequenced
    • Based on in vitro DNA synthesis performed in
    the presence of chain-terminating nucleotides
    (dideoxy nucleoside triphosphates)
    • ddNTPs: ddATP, ddGTP, ddCTP, and ddTTP
    dideoxy nucleoside triphosphates
    Sanger Sequencing Steps
    • DNA fragments separated into single strand
    • Primer attached to the sequence
    • Polymerase solution, four types of dNTPs
    (nucleotides) & ddNTP are added.
    • Replication process in fragments are stopped as
    soon as one of the ddNTP attaches.
    • Fragments are separated by gel electrophoresis
    and the sequence is determined.
    Fragment and ddNTP
    • Very accurate, 99.99% base accuracy
    • Considered “gold standard” for validating
    DNA sequences
    • Smaller genome sequenced, cost effective
    • Cheap 3$ per cycle
    • Most widely used sequencing method for
    approximately 40 years
    • Read length maximum upto 500 bp
    • Slow, becomes expensive for larger and
    complex genome
    DNA arrays
    • Radoje Drmanac, Andrey Mirzabekov, and
    Edwin Southern, 1988
    • Goal of cheaply generating a genome’s k-mer
    composition
    • A smaller read length k approx 10bp
    Working DNA arrays
    • First synthesize all 4k possible DNA k-mers. For
    a k mer of length 10bp, the possible k mers to
    be synthesized is 4 = 1,048, 576
    • Attach them to a DNA array, which is a grid on
    which each k-mer is assigned a unique position
    • Solution with unknown single strand DNA
    fragment with fluorescent label was applied to
    the DNA array.
    • Unknown fragment hybridizes with the
    complementary k mer on array
    Reading the array
    • Fluoresence was analyzed
    with spectroscopy
    • The reverse complements
    of k-mers corresponding
    to these sites belong to
    the (unknown) DNA
    fragment
    • k-mers on the array
    reveals the sequence of a
    DNA fragment
    Array technology application
    • Could not be applied to genome sequencing because
    Fidelity of DNA for hybridization with the array was too
    low and value of k was too small
    New Application
    • arrays are used to measure gene expression
    • Analyze genetic variations.
    • DNA arrays is a multi-billion dollar industry that
    included Hyseq, founded by Radoje Drmanac (one of
    the original inventors of DNA arrays.)
    NGS – Next generation sequencing
    • Illumina
    • Nanopore techniques
    Sample preparation
    1. Breaking and adaptor attachment

    2. Fragment
    with adaptor

    3. Achment of
    bridging group
    Cluster Generation
    • Clustering is amplification process in flow cell
    • Each fragment is first attached onto glass
    channels on a flow cell and then amplified into
    millions of copy
    Sequencing
    • Begins by adding one nucleotides at a time
    which generates a signal
    • The reads are generated for forward as well as
    reverse strand
    • Illumina generates
    paired reads
    that is two reads for
    each fragment
    Data Analysis
    • Preliminary data analysis is done
    • Data is locally clustered based on indices given to
    each cluster
    • Contiguous sequence prepared
    • Contig aligned to reference genome for
    verification and identification.
    Nanopore Sequencing
    • Unique & scalable technology
    • Enables direct, real-time analysis of long DNA or
    RNA fragments.
    • It works by monitoring changes to an electrical
    current as nucleic acids are passed through a
    protein nanopore.
    • The resulting signal is decoded to provide the
    specific DNA or RNA sequence.
    • Advantage is fast and cost effective &
    disadvantage is error in reads

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