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
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