Introduction to DNA Sequencing Technologies
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Introduction to DNA Sequencing Technologies Advanced Genetic Epidemiology and Statistical Molecular Genetics Workshop October 22, 2010. Gregory A. Buck, Ph.D. Director, Center for the Study of Biological Complexity Professor, Microbiology and Immunology Virginia Commonwealth University.

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Introduction to DNA Sequencing TechnologiesAdvanced Genetic Epidemiology and Statistical Molecular Genetics WorkshopOctober 22, 2010

Gregory A. Buck, Ph.D.

Director, Center for the Study of Biological Complexity

Professor, Microbiology and Immunology

Virginia Commonwealth University


Holy grail the human genome
Holy Grail: the Human Genome

Complexity (number of bases per haploid genome) of the human genome:

- 3x109 base pairs (nucleotides)


Human genome
Human Genome

How much does it cost to sequence?

  • First genome: $3-5 billion

    - James Watson: ~$300,000

    - Today: $5,000 - $100,000

    - Goal: $1000 (soon < $100?)


Human genome1
Human Genome

How much time to sequence?

- First genome sequenced (2004):

. Estimated - 15 years (1990’s)

. Actual - 13 years (capillary sequencing)

  • James Watson (2008): ~ 2 months

    . So-called ‘next generation’ sequencing

  • Now: two weeks?

  • Goal: tricorder (Star Trek)


X Prize:

$10 million award is set for faster DNA maps (2006)

By Nicholas Wade

Published: THURSDAY, OCTOBER 5, 2006

A $10 million prize for cheap and rapid sequencing of the human genome was announced by the X Prize Foundation of Santa Monica, California.

The terms of the prize require competitors to sequence 100 human genomes of their choice within 10 days, and within six months, those of a further 100 people chosen by the foundation.

http://www.iht.com/articles/2006/10/05/news/genome.php


NHGRI Grants Support for 'Revolutionary' Sequencing for $1,000 GenomeAugust 5, 2008 By a GenomeWeb staff reporter

Under one program, NHGRI may grant as much as $5 million in fiscal 2009 to between two and seven awardees. Applicants for these funds may seek up to $1.5 million per year for a period of up to five years.  

A parallel grant program would give up to $2 million over three years to between two and seven grantees, for direct costs of up to $200,000 per year.  

A Small Business Innovation Research Grant from NHGRI will grant between four and six small businesses up to a total of $3.6 million in fiscal 2009 to propose novel technologies to bring down the cost of sequencing. Phase I of this program will give up to $250,000 of total costs per year for up to two years, and Phase II applicants may seek up to $1.5 million total costs per year for up to three years.  

A parallel Small Business Technology Transfer program will spend up to $2 million in fiscal 2009 to support between two and five awards to small businesses investigating the development of new sequencing methods. This program will award up to $250,000 total costs per year for up to two years for Phase I programs, and it will support up to $1.5 million in total costs per year for up to three years for Phase II programs.


Sequencing technologies
Sequencing Technologies $1,000 Genome

1977: Fred Sanger (Cambridge, England) and Walter Gilbert (Harvard University)

  • Chemical sequencing (Gilbert)

  • Dideoxy Nucleotide Triphosphate chain termination sequencing (Sanger)

  • Both used for 8-10 years (different strengths/drawbacks)

    Chain termination sequencing proves most versatile, robust

  • Applicable to automation

  • First automated sequencers commercially available ~1985



Sequencing technologies1
Sequencing Technologies $1,000 Genome

Commercially available (1985):

  • Dideoxy- (Sanger, enzymatic, termination method)

    • Applied Biosystems, Inc., uses fluorescent primers

    • Requires four primers (four dyes) per sequence read

    • Requires four reactions (one for each primer)

    • Works, but expensive, laborious

  • DuPont: Genesis 1000 DNA Sequencer

    • Fluorescent chain termination sequencing

    • One primer, four terminators (one for each base, A, G, C, T)

    • One reaction per sequence read

    • Very efficient

    • Sells IP to ABI……….



  • High throughput genome sequencing the main player
    High Throughput Genome Sequencing: The main player... $1,000 Genome

    The PE/ABI 3700 Prism:

    - automated, easy to use

    - capillaries (not slab gel)

    - 10 runs per day

    - 96 sequences per run

    - ~1000 sequences/day

    - >300,000 sequences/ year

    - >150 million bases/ year

    - $300,000 per machine

    First truly automated high throughput sequencing

    Sequenced the first human genome…..



    Fluorescent chain termination sequencing: $1,000 Genome

    dominates market until ~ 2005:

    Next Generation (NextGen) Sequencing

    First out of the blocks:

    Roche 454 FLX Genome Sequencer


    www.roche-applied-science.com $1,000 Genome

    Genome Sequencer FLX System Customer Training Technical Overview 400 million bases/ day (5th floor, Sanger Hall)

    (equal to 2 years output from cap sequencer!!)



    Roche 454 flx technologies
    Roche 454 Flx Technologies $1,000 Genome

    • Based on Pyrosequencing –

      • Pyrosequencing video

      • Roche 454 FLX workflow video


    Pyrosequencing – 454/Roche $1,000 Genome

    http://454.com/products-solutions/how-it-works/sequencing-chemistry.asp


    Roche 454 flx output
    Roche 454 FLX Output: $1,000 Genome

    • Based on Pyrosequencing –

      • Currently:

        • ~400 base maximum read length

        • ~1 X 106 reads

        • ~ 400 X 106 bases per run

        • 1 run ~ 8 hours (1 day)

          • [compare to 200 X 106 / year for capillary sequencing]

      • Good for de novo sequencing, assembly

      • Cost: $10,000 per run


    Solexa/Illumina $1,000 Genome

    Current Market Leader: Illumina Genome Analyzer


    Reversible Terminator Chemistry $1,000 Genome

    OH

    X

    O

    O

    HN

    HN

    5’

    cleavage

    site

    fluor

    O

    O

    N

    N

    DNA

    O

    O

    O

    PPP

    3’

    3’

    block

    Incorporation

    Detection

    Deblock; fluor removal

    free 3’ end

    Next cycle

    Solexa/Illumina

    • All 4 labeled nucleotides in 1 reaction


    3’ $1,000 Genome

    5’

    A

    A

    A

    A

    A

    A

    A

    A

    T

    T

    T

    T

    T

    T

    T

    T

    G

    G

    G

    G

    G

    G

    G

    C

    C

    C

    C

    C

    C

    C

    C

    C

    5’

    Solexa/Illumina

    Cycle 1: Add sequencing reagents

    First base incorporated

    Remove unincorporated bases

    Detect signal

    Cycle 2-n: Add sequencing reagents and repeat

    • All four labelled nucleotides in one reaction

    • High accuracy

    • Base-by-base sequencing

    • No problems with homopolymer repeats


    5 $1,000 Genome

    1

    2

    3

    4

    TGCTACGAT …

    6

    8

    9

    7

    T T T T T T TGT …

    Solexa/Illumina

    Base Calling

    The identity of each base of a cluster is read off from sequential images

    (sequencing genomes with the Illumina video)


    Illumina solexa technologies
    Illumina/Solexa Technologies $1,000 Genome

    • Based on Sequencing by Synthesis (bridge PCR) –

      • Currently:

        • ~100 base maximum read length

        • ~ 500 X 106 reads/run

        • ~ 50 X 109 bases per run (100 X 109in paired end reads)

        • 1 run ~ 10 days

      • Good for re-sequencing, CHiP Seq, RNA seq

      • Cost: $10 – 20,000 per run

        New Illumina HiSeq2000: 200 X 109bases/run

        - ~ 10 X 1012bases/year

        - 100,000 fold increase over fluor. chain termination seq

        - >3,000 human genomes!



    See video….. $1,000 Genome

    http://marketing.appliedbiosystems.com/images/Product_Microsites/Solid_Knowledge_MS/video/SOLiD_video_final.wmv


    Solid sequencing technology
    SOLiD Sequencing Technology $1,000 Genome

    Currently:

    • 50 base reads (75?)

    • Up to 400 Giga bases (billion bases) per run

    • >20 X 1012 bases per year (~2X Illumina)

    • Reduced costs (<50%/base cost)

      Best for applications where short reads are sufficient:

      CHiP seq, RNA Seq…. (not de novo sequencing)


    Single molecule technologies
    Single Molecule Technologies $1,000 Genome

    Holy Grail:

    • No bias (due to replication, amplification)

    • Should work with limiting amounts of template

    • Long reads: for de novo sequencing

      Contenders:

    • true Single-Molecule Sequencing (tSMS) – Helicos

    • SMRT (Single Molecule, Real Time Sequencing) – Pacific BioSciences

      see videos


    Single molecule technologies1
    Single Molecule Technologies $1,000 Genome

    Advantages:

    • No amplification, cloning biases

    • Use small quantities of substrate (DNA)

    • Fast (rate of replication)

      Challenges

    • Signal to noise ratios

    • Sensitivity

    • Error rates

      To date: still largely experimental:

      • Short reads (Helioscope)

      • Low output; e.g., < 100,000 reads/run (Pac Bio)


    Other technologies
    Other Technologies $1,000 Genome

    Looming:

    • Ion Torrent: based on release of H+ ions

      • requires emulsion PCR

      • Inherent biases

      • Current read length < 100 bp; high error rate

    • Oxford Nanopore Technologies: passage of bases through a nanopore in a lipid bilayer

      • No data available

    • Others coming




    Nanopore $1,000 Genome

    http://www.nanoporetech.com/sections/first/14


    Wish list
    Wish list: $1,000 Genome

    • Longer reads

      • Today: 25 - 800 bases

      • Looming: 1 – 20 Kbases?

      • Ideal: entire chromosome [metagenomics]

    • Low amounts DNA required

      • No amplification bias

      • No replication bias

      • Can sequence hard to get DNA

    • High accuracy and fidelity

    • Rapid (currently over a week per run)

    • Lower cost ($100/human genome?)


    Thank you
    Thank you! $1,000 Genome


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