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

Holy Grail: the Human Genome

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

- 3x109 base pairs (nucleotides)

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 GenomeHow 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 WadePublished: 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 Genome

August 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

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

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Sanger or di-deoxy- method of sequencing

Sequencing TechnologiesCommercially 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……….

Fluorescent chain termination sequencing

See video

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…..

Output from Fluorescent Chain Termination Sequencing

Fluorescent chain termination sequencing: dominates market until ~ 2005:

Next Generation (NextGen) Sequencing

First out of the blocks:

Roche 454 FLX Genome Sequencer

Genome Sequencer FLX System Customer Training Technical Overview

400 million bases/ day (5th floor, Sanger Hall)(equal to 2 years output from cap sequencer!!)

www.roche-applied-science.com

Based on Pyrosequencing…..

Roche 454 Flx Technologies

- Based on Pyrosequencing –

- Pyrosequencing video

- Roche 454 FLX workflow video

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

Pyrosequencing – 454/Roche

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

Current Market Leader: Illumina Genome Analyzer

Solexa/Illumina

Reversible Terminator Chemistry

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Cycle 1: Add sequencing reagents

Remove unincorporated bases

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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

Solexa/Illumina

Base Calling

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

(sequencing genomes with the Illumina video)

Solexa/Illumina

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 109 in 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 109 bases/run

- ~ 10 X 1012 bases/year

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

- >3,000 human genomes!

Applied Biosystems: Solid 4/HQ Sequencing by Ligation…

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

See video…..

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

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 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

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

http://www.iontorrent.com/technology/

http://www.iontorrent.com/technology/

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

Nanopore

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?)

Thank you!