Sequencing technology and assembly Sanger sequencing with radioactivity

Sequencing technology and assembly

Sanger sequencing with radioactivity High throughput Sanger sequencing with fluorescence

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

Pyrosequencing

Illumina sequencing Yield: 8, 000, 000 – 80, 000, 000 bp Time: ~1 hour per bp Read length: ~150 bp Cost: – Sample Extraction, $14. 00/sample – Automated Sample Library, $90. 00/sample – Mi. Seq (2 x 250), 1 lane 8 -10 Gb/lane, $1, 700. 00/sample – Mi. Seq (2 x 300), 1 lane, 10 -12 Gb/lane, $2, 100. 00/sample – Hi. Seq 2500 (2 x 150), 1 lane, ~40 Gb/lane,

Illumina sequencing

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

Ion Torrent

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

Pac. Bio

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

Minion

Errors Different technologies have different error rates:

Base calling Need to be sure which base you have identified 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 101 chance that the base is wrong Phred 20: 1 x 102 chance that the base is wrong Phred 30: 1 x 103 chance that the base is wrong Phred 40: 1 x 104 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 Different technologies have different error rates: Pyrosequencing/Ion Torrent – homopolymeric tracts Illumina – substitution errors Pac. Bio – Machines can not keep up with biology Minion – noise coming through the membrane