Sequencing technology and assembly Sanger sequencing Sanger - - PowerPoint PPT Presentation

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sequencing technology and assembly sanger sequencing
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Sequencing technology and assembly Sanger sequencing Sanger - - PowerPoint PPT Presentation

Apr 19, 2023 394 likes •585 views

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

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SLIDE 1

Sequencing technology and assembly

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 Sanger sequencing with radioactivity  High throughput Sanger sequencing

with fmuorescence

Sanger sequencing

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Roche/454 sequencing

  • Yield: 500,000,000 bp
  • Cost: $5,000
  • Time: ~1 min per bp
  • Read length: 450 bp - > 1kb
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Pyrosequencing

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 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

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Illumina sequencing

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

Ion Torrent

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Ion Torrent

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PacBio

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

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Minion

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

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Errors

Difgerent technologies have difgerent error rates:

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 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

Base calling

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 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

Quality values

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Homopolymeric errors

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

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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