slide1
Download
Skip this Video
Download Presentation
Gregory A. Buck, Ph.D. Director, Center for the Study of Biological Complexity

Loading in 2 Seconds...

play fullscreen
1 / 35

Gregory A. Buck, Ph.D. Director, Center for the Study of Biological Complexity - PowerPoint PPT Presentation


  • 53 Views
  • Uploaded on

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.

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about ' Gregory A. Buck, Ph.D. Director, Center for the Study of Biological Complexity' - megan-kirk


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
slide1
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)
slide5

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

slide6
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

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

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...

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…..

slide13

Fluorescent chain termination sequencing:

dominates market until ~ 2005:

Next Generation (NextGen) Sequencing

First out of the blocks:

Roche 454 FLX Genome Sequencer

slide14

www.roche-applied-science.com

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
  • Based on Pyrosequencing –
    • Pyrosequencing video
    • Roche 454 FLX workflow video
slide17

Pyrosequencing – 454/Roche

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

roche 454 flx output
Roche 454 FLX Output:
  • 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
slide19

Solexa/Illumina

Current Market Leader: Illumina Genome Analyzer

slide20

Reversible Terminator Chemistry

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
slide21

3’

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
slide22

5

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
  • 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!

slide25

See video…..

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

solid sequencing technology
SOLiD Sequencing Technology

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

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

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

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
slide32

Nanopore

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

wish list
Wish list:
  • 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?)
ad