Sequencing technology and assembly Sanger sequencing Sanger - PowerPoint PPT Presentation

Sequencing technology and assembly

Sanger sequencing  Sanger sequencing with radioactivity  High throughput Sanger sequencing with fmuorescence

Roche/454 sequencing ● Yield: 500,000,000 bp ● Cost: $5,000 ● Time: ~1 min per bp ● Read length: 450 bp - > 1kb

Pyrosequencing

Illumina sequencing  Yield: 8,000,000,000 – 80,000,000,000 bp  Time: ~1 hour per bp  Read length: ~150 bp  Cost: – Sample Extraction, $14.00/sample – Automated Sample Library, $90.00/sample – MiSeq (2x250), 1 lane 8-10Gb/lane, $1,700.00/sample – MiSeq (2x300), 1 lane, 10-12Gb/lane, $2,100.00/sample – HiSeq2500 (2x150), 1 lane, ~40Gb/lane, $2,500.00/lane – HiSeq2500 (2x250), 1 lane, ~65Gb/lane, $3,500.00/lane

Illumina sequencing

Ion Torrent  Yield: 50,000,000 bp  Time: 2 hours  Read length: 500bp  <1 min per bp  Cost: $500

Ion Torrent

PacBio ● Long reads (5-10kb) ● High error, but read 150x coverage ● Library prep: $600 ● Sequencing: $300

PacBio

Minion ● Quick sample prep ● Long reads (~50kb) ● High error ● $150 per run ●

Minion

Errors Difgerent technologies have difgerent error rates:

Base calling  Need to be sure which base you have identifjed  Depends on the technology  Each machine includes software  Phred is an historical package developed by at U. Washington  Phred scores are probability that the base is correct

Quality values  Phred 10: 1 x 10 1 chance that the base is wrong  Phred 20: 1 x 10 2 chance that the base is wrong  Phred 30: 1 x 10 3 chance that the base is wrong  Phred 40: 1 x 10 4 chance that the base is wrong  Phred 99: the base is correct!  Fastq scores are the score + 33 then converted to ascii text

Homopolymeric errors Homopolymeric runs: Signal is not linear Not clear if 5 or 6 bases

Errors ● Difgerent technologies have difgerent error rates: ● ● Pyrosequencing/Ion Torrent – homopolymeric tracts ● Illumina – substitution errors ● PacBio – Machines can not keep up with biology ● Minion – noise coming through the membrane