Sequencing – the Benchtops
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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!