SEQUENCING – THE BENCHTOPS

Roche 454 Junior Same technology as 454 FLX Read length: 400 bases Paired-end 100,000 reads 12 hours (instrument time) Output 35-70Mb

LifeTech: Ion Torrent Semi conductor based sequencing Records the release of H+ atoms when bases are incorporated 2 hours of sequencing Need extra kit for library prep – bp reads – 1 million reads on 316 chip (100Mb) – Aim for 1Gb out of 318 chip – Homopolymer problems – No paired-end yet, although Broad….

Illumina MiSeq Same technology as HiSeq or 150bp read kits Paired-end 1 x 35bp is 4 hours, total run time 8 hours 2 x 150bp is 27 hours 3.4M paired reads 1-2Gb of data

SEQUENCING – SINGLE MOLECULES

Helicos Heliscope First SMS produced 25-55bp reads (avg 35bp) 600M to 1b reads per run 21 – 35Gb per run Raw error rate – Substitution 0.2% – Insertion 1.5% – Deletion 3.0%

Pacific Biosciences RS PacBio get mean 2.3Kb read lengths, max 17kb Others get 1.7Kb, maximum 6kb Raw read 85% accurate Circularize: – 95% accurate – Shorter read length Errors are again indels

APPLICATIONS

Applications of NGS Novel genome sequencing Re-sequencing – Discover genome variation Targetted re-sequencing Exome sequencing High-throughput pathogen sequencing – Including metagenomics RNA-Seq ChIP-Seq microRNA-Seq Methylated DNA sequencing

Future Technologies Third-generation sequencing (TGS) Single molecule sequencing Challenges – Each observation relates to zero, one or more realities – Technology-aware software The PromiseThe current reality Billions of reads~80,000 reads 10,000 – 50,000 read length30bp, up to 1000bp High accuracy> 5% error rate Branton et al (2008) Nat. Biotech. 26(10):1146 Schadt et al (2010) Hum. Mol. Gen. 19(R2):R227

Personal / individual genomics 1000 genomes data – petabyte scale TGS will enable entire scans of genomes, transcriptomes and epigenomes in minutes Huge data potential – exabyte scale

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