Sequencing technology and assembly 1

Sanger sequencing Sanger sequencing with radioactivity High throughput Sanger sequencing with fluorescence 2

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 5

Illumina sequencing

Ion Torrent Yield: 50,000,000 bp Time: 2 hours Read length: 500bp <1 min per bp Cost: $500 7

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 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 14

Quality values Phred 10: 1 x 101 chance that the base is wrong Phred 99: the base is correct! Fastq scores are the score + 33 then converted to ascii text 15

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 PacBio – Machines can not keep up with biology Minion – noise coming through the membrane