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PDCB BioC for HTS topic Understanding the tech. 02

PDCB BioC for HTS topic Understanding the tech. 02. LCG Leonardo Collado Torres lcollado@wintergenomic.com lcollado@ibt.unam.mx September 2 nd , 2010. Topics. Basecalling Quality Filtering FASTQ format Error rates A gamma of problems / reports

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PDCB BioC for HTS topic Understanding the tech. 02

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  1. PDCB BioC for HTS topicUnderstanding the tech. 02 LCG Leonardo Collado Torres lcollado@wintergenomic.com lcollado@ibt.unam.mx September 2nd, 2010

  2. Topics • Basecalling • Quality Filtering • FASTQ format • Error rates • A gamma of problems / reports • Fragment of James Huntley’s ppt on best practices

  3. Basecalling: Illumina

  4. Cross-talk

  5. SWIFT: cross-talk correction

  6. Phasing and Prephasing options

  7. Some warnings!

  8. Describe each case

  9. Quality Filtering: Purity and Chastity

  10. What artifact can be derived from this step?

  11. FASTQ format @ is the seq id sequence + is the qual id Quality in ASCII chars

  12. Originally…

  13. Q to error probability (p) formulas Qphred Qsolexa1.3

  14. FASTQ types What is the quickest way to distinguish fastq-sanger from fastq-illumina? Tip: Check the ASCII table 

  15. phred.R

  16. It is NOT clear what quals of 1 and 2 mean in Illumina (version 1.5+)

  17. FASTQ in CS Base 1 does not include a quality value! (It’s a 0)

  18. Error rates

  19. IlluminavsSOLiD: % per cycle

  20. IlluminavsSOLiD: num of errs

  21. Understanding 454 (GS20) a bit more

  22. 454 error types

  23. 454 errors

  24. Presence of Ns correlates with error rate (454)

  25. IlluminavsSOLiD

  26. Helicos

  27. A gamma of problems / reports • Aligned to the wrong reference • Did not use the correct quality encoding • Barcodes are trimmed or have mismatches • Trimming the 1st and last base  losing barcodes • GC bias • Sample degradation will affect your data!

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