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)
LifeTech SOLiD / 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
Adapters added to SS DNA
PicoTiterPlate - ~29um wells
Image analysis – each spot represents a flash of light; a base being incorporated
Nucleotides washed across the plate, emitting a flash as the base is incorporated
Each bead into a single well

5. Roche 454 Complex emulsion PCR library prep FLX+ out now
Read lengths average bp
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. Beads attached to slide
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” Current machine is 5500xl
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