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Cutting-Edge Sequencing Technologies for Precision Genome Analysis

Explore the latest in sequencing technology and assembly methods, including Sanger sequencing, Pyrosequencing, Illumina sequencing, Ion Torrent, PacBio, and Minion. Understand error rates, base calling, quality values, homopolymeric errors, and more for accurate genetic analysis.

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Cutting-Edge Sequencing Technologies for Precision Genome Analysis

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  1. Sequencing technologyand assembly

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

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

  4. Pyrosequencing

  5. 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

  6. Illumina sequencing

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

  8. Ion Torrent

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

  10. PacBio

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

  12. Minion

  13. Errors Different technologies have different error rates:

  14. 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

  15. 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

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

  17. 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

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