Genome Sequencing in Microfabricated High-density Picolitre Reactors Presented by Colin Russell January 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)
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Genome Sequencing in Microfabricated High-density Picolitre Reactors Presented by Colin Russell January 26, 2007 EECE491c Marcel Margulies, Michael Egholm,
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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
• (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