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Sequencing – the Benchtops

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

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Sequencing – the Benchtops

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  1. Sequencing – the Benchtops

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

  3. 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….

  4. 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

  5. Sequencing – single molecules

  6. HelicosHeliscope • 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%

  7. 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

  8. Applications

  9. 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

  10. Future Technologies • Third-generation sequencing (TGS) • Single molecule sequencing • Challenges • Each observation relates to zero, one or more realities • Technology-aware software Branton et al (2008) Nat. Biotech. 26(10):1146 Schadt et al (2010) Hum. Mol. Gen. 19(R2):R227

  11. 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

  12. Thank you!

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