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Second and third generation sequencing technologies. Morten Rasmussen, PhD. Outline. What was before? All the new players 454 Illumina SOLiD Helicos PGM Pacific Biosciences Oxford Nanopores Many more to come. What is first generation sequencing?. Sequencing = read DNA code, A-C-G-T
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Centre for GeoGenetics Second and third generation sequencing technologies Morten Rasmussen, PhD
Centre for GeoGenetics Outline • What was before? • All the new players • 454 • Illumina • SOLiD • Helicos • PGM • Pacific Biosciences • Oxford Nanopores • Many more to come
Centre for GeoGenetics What is first generation sequencing? • Sequencing = read DNA code, A-C-G-T • Sanger sequencing, 1977 • Dideoxy chemistry • Read length 700-1000bp • Requires PCR primer sites (>40bp) • Often requires cloning
Centre for GeoGenetics What is first generation sequencing?
Centre for GeoGenetics Introduction – second generation sequencingWhat is second generation sequencing? 1977 2006 x very big number x 1 2007 1 - 384 105 - >109 x even bigger number 1998 2008 x gigantic number x 384 2011 x big number
Centre for GeoGenetics Introduction – second generation sequencingDefining features of second generation sequencing • Library • Clonal Amplification • Massively Parallel Sequencing DNA from extract Polished DNA Library Adaptors Library molecule
Centre for GeoGenetics Second generation sequencing platforms 454 Illumina SOLiD Ion Torrent (PGM)
Centre for GeoGenetics The GS FLX (454) platform • Clonal Amplification • Massively parallel sequencing
Centre for GeoGenetics The GS FLX (454) platform
Centre for GeoGenetics Ion Torrent and Proton
Centre for GeoGenetics Introduction – second generation sequencingThe Illumina (GAIIx) platform • Clonal Amplification • Massively parallel sequencing
Centre for GeoGenetics Illumina reaction flow P5 Index P7 Insert Read1 Read2 ReadIndex
Centre for GeoGenetics Sequencing by Oligo Ligation and Detection
Centre for GeoGenetics SOLiD 2-base encoding
Centre for GeoGenetics Third generation sequencing • Defined by no amplification • Simple sample preparation Oxford Nanopore Helicos PacBio
Centre for GeoGenetics Helicos
Centre for GeoGenetics Pacific Biosciences - SMaRT
Centre for GeoGenetics Oxford Nanopore Video
Centre for GeoGenetics Oxford Nanopore
Centre for GeoGenetics Current status (Mar 2012)
Centre for GeoGenetics Current status part 2