Genome Sequencing in MicrofabricatedHigh-density Picolitre Reactors

Presented by Colin RussellJanuary 26, 2007

EECE491c

Marcel Margulies, Michael Egholm, William E. Altman, Said Attiya, Joel S. Bader, Lisa A. Bemben, Jan Berka, Michael S. Braverman, Yi-Ju Chen, Zhoutao Chen, Scott B. Dewell, Alex de Winter, James Drake, Lei Du,

Joseph M. Fierro, Robin Forte, Xavier V. Gomes, Brian C. Godwin, Wen He, Scott Helgesen, Chun Heen Ho, Stephen K. Hutchison, Gerard P. Irzyk, Szilveszter C. Jando, Maria L. I. Alenquer, Thomas P. Jarvie, Kshama

B. Jirage, Jong-Bum Kim, James R. Knight, Janna R. Lanza, John H. Leamon, William L. Lee, Steven M. Lefkowitz, Ming Lei, Jing Li, Kenton L. Lohman, Hong Lu, Vinod B. Makhijani, Keith E. McDade, Michael P. McKenna, Eugene W. Myers, Elizabeth Nickerson, John R. Nobile, Ramona Plant, Bernard P. Puc, Michael

Reifler, Michael T. Ronan, George T. Roth, Gary J. Sarkis, Jan Fredrik Simons, John W. Simpson, Maithreyan Srinivasan, Karrie R. Tartaro, Alexander Tomasz, Kari A. Vogt, Greg A. Volkmer, Shally H. Wang, Yong Wang,

Michael P. Weiner, David A. Willoughby, Pengguang Yu, Richard F. Begley& Jonathan M. Rothberg

Nature, 437: 376-380 (2005)

Background: DNA Sequencing

• Sequencing is…Determining nucleotide ordering in DNA– Useful in pure and applied research on

how organisms function

• Field dominated by ‘Sanger sequencing’ technique, aka the chain termination method for last 30 years

• New methods desired to reduce cost ($20K - $25M, weeks – months per genome)

Sequencing in FFW

• The 454 method vastly reduces time required for sequencing

• (a) μL-scale Sanger sequencing and electrophoresis

• (b) pL-scale massively parallel 454 method

Smaller Footprint

• Other benefits include:

– Less support equipment, facility space needed

– Less labour required

– Smaller sample volumes resulting in lower cost (assumed, although no estimates stated in paper…)

Sample Preparation

• Genome fragmented• Fragment ends augmented with A and B

adaptors (to facilitate binding, amplification, identification)

DNA Amplification

• Each fragment is bound to a unique microbead (~28 μm di.)

• Beads kept separate in water/oil emulsion which contains nutrients for amplification by standard polymerase chain reaction (PCR)

• Results in ~10 million DNA strand copies on a bead

Bead Binding

• Streptavidin coated beads

• Biotin tag on single stranded DNA (on adptor B) binds to bead

• Allows DNA containment in well

Sample Insertion

• Immobolized enzymes deposited in wells for pyrophosphate sequencing

• Beads placed into pL reactor wells on slide (only one bead fits per well)

Sample Images: Emulsion, Microreactors

Sequencing

• Reactor slide washed sequentially with nucleotides

• Nucleotide incorporation within a well releases inorganic pyrophosphate and photons

• Timescale: diffusion in/out of wells ~10 s, signal generating enzymatic reaction ~0.02–1.5 s

• CCD coupled fibre-optically to base of slide records light intensity associated with each well, to determine ‘growth’ sequence of complementary DNA

System Overview

• (A) Fluidic assembly, delivering nucleotide washes

• (B) Slide (60mm x 60mm) with microreactor wells

• (C) CCD to detect nucleotide incorporation, and attached computer for data storage/processing

Results: Flowgram (M. genitalium)

Results: Statistics (M. genitalium)

Error Correction

• Several sources of error:– Optical and chemical cross-talk between wells– Asynchronicity of beaded DNA template

response within a well– Background noise

• Errors accounted for in processing algorithm

• Corrected and normalized signal is linear in number of nucleotides absorbed per wash (up to at least 8-mer repetition)

But it is it really any good?

• Wall Street Journal's Gold Medal for Innovation in 2005

Analysis Software

• Optical signal record, clearly showing hexagonal wells

• ‘Flowgram’: nucleotide sequence for a single well

Critique Summary

Pros Cons

•Scalable system leaves room for improvement•Relatively simple and cost effective setup could enable smaller players to begin sequencing•Bacterial and viral genomes can be very quickly sequenced (100x faster than status quo Sanger sequencing)•Admit some limitations

•Only short read lengths possible (80-120 bases compared to 1K)•Accuracy degrades in reads with repeated single bases•Sample preparation process complexity is a limiting factor in usability•Single-stranded DNA library limits de novo sequencing (lacks paired-end reads)•Comparative cost estimate???

•Sexy diagrams•Enough authors to start a genomic revolution

•Article somewhat disjointed – often refers to supplementary details and figures (50+ pages!), hindering readability•Data shown not very interesting

Major

Minor