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

Roche 454

Adapters added to SS DNA Each DNA molecule attached to a single bead

Emulsion PCR – molecule amplified to several million per

bead

PicoTiterPlate - ~29um wells

Each bead into a single wellNucleotides washed across the plate, emitting a flash as

the base is incorporated

Image analysis – each spot represents a flash of light; a base

being incorporated

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

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

ABI SOLiD • More complex system – “colour space”

DNA attached to beads Beads attached to slide

• 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

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

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

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

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