Dias nummer 1 - CBS

Dias nummer 1 - CBS

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 Often requires cloning (>40bp)

Centre for GeoGenetics What is first generation sequencing? Centre for GeoGenetics Introduction second generation sequencing What is second generation sequencing? 1977 2006

x very big number x 1 1 - 384 1998 2007 9 105 - >10 x even bigger number 2008

x gigantic number x 384 2011 x big number Centre for GeoGenetics Introduction second generation sequencing Defining 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 sequencing The Illumina (GAIIx) platform Clonal Amplification Massively parallel sequencing Centre for GeoGenetics

Illumina reaction flow P5 Insert Index P7 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 Helicos Oxford Nanopore 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) Company/ technology Current machine, some key characteristics 454 GS FLX+, 7-800bp length, 1M reads Illumina

HiSeq 2000, 1.5B reads, up to 2x 100bp SOLiD (Life) 5500XL, 2B reads, up to 2x 60bp PGM (Life) Ion Proton, uncertain Helicos

HeliScope, >1B reads, 25-35bp PacBio SMRT, ~75k reads, long reads (110kb) Oxford nano GridION, 2-8000 pores, long reads (150kb?) Centre for GeoGenetics

Current status part 2 Company/ technology Current machine, some key characteristics 454 GS junior, 4-500bp reads, 100k reads

Illumina MiSeq, 7M reads, up to 2x 250bp PGM (Life) Ion Torrent, 100k reads, 400bp Oxford Nano MinION, uncertain

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