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

– 100-200bp 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 2000• 50 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 Promise The current reality

Billions of reads ~80,000 reads

10,000 – 50,000 read length 30bp, up to 1000bp

High accuracy > 5% error rate

Branton et al (2008) Nat. Biotech. 26(10):1146Schadt 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

THANK YOU!