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Update on Next-Generation Sequencing

Update on Next-Generation Sequencing. Mick Watson. Next-generation sequencing. Ultra- highthroughput Characterised by Many millions of… … short reads Dominated by 3 companies Roche 454 FLX pyrosequencing Illumina / Solexa (SBS) LifeTech SOLiD / Ion Torrent. Sequencing – the big boys.

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Update on Next-Generation Sequencing

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  1. Update on Next-Generation Sequencing Mick Watson

  2. Next-generation sequencing • Ultra-highthroughput • Characterised by • Many millions of… • … short reads • Dominated by 3 companies • Roche 454 FLX pyrosequencing • Illumina / Solexa (SBS) • LifeTechSOLiD / Ion Torrent

  3. Sequencing – the big boys

  4. Roche 454 Each DNA molecule attached to a single bead Emulsion PCR – molecule amplified to several million per bead PicoTiterPlate - ~29um wells Adapters added to SS DNA Nucleotides washed across the plate, emitting a flash as the base is incorporated Each bead into a single well Image analysis – each spot represents a flash of light; a base being incorporated

  5. Roche 454 • Complex emulsion PCR library prep • Read lengths average 400-500bp • Typically 1 million reads per run • Run takes 10 hours • 400Mb output • FLX+ out now • Read length average 700bp • 700Mb output • Both have paired-end • Homo-polymer problems

  6. 454: Homopolymer problems • 454 suffers from homopolymer problems • Can introduce frameshifts etc

  7. ABI SOLiD • More complex system – “colour space” • Bases are incorporated as di-nucleotides • Four colours represent many different di-nucleotides • After each round, the primer is moved back to position n-1 • Therefore each base is sampled twice • Therefore with the first base, and a colour chart, you can work out the sequence of bases from the colours DNA attached to beads Beads attached to slide

  8. ABI SOLiD • Reads are “colourspace” • Colours indicate incorporation of dinucleotide • Current machine is 5500xl • Read lengths are 35bp, 60bp or 75bp • 75 bp (fragment) • 75 bp x 35 bp (paired-end) • Up to 60 bp x 60 bp (mate-paired) • 4.8 billion paired-end reads per run • A run takes 7 days

  9. Illumina Solexa technology Fluorescently labelled nucleotides are added Laser captures image to determine first base Fluorescently dyed nucleotides are added Laser captures image to determine second base

  10. Illumina GA IIx • Read lengths are 36, 57, 78, 101 and 150bp • Paired-end available • 30M reads per lane • 9Gb per lane • 8 lanes • 14 days for 150 pe

  11. Illumina HiSeq 2000 • Read lengths are 35, 50, 75 and 100bp • Paired-end available • 150M reads per lane • 30Gb per lane • 16 lanes (2*8) • 9 days for one flow cell • 11 days for two

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