CLC Germline workflow

• fastq files are imported to clc.

  • Reads that did not pass a quality filter are ignored.

• Trimming sequence ends.

  • Quality trimming:

    • Phred score > 20
    • trim ambiguous nucleotides

  • Sequence filtering:

    • Remove 1 3’ terminal nucleotide
    • number of residues of reads must be in that range: [30, 500]; short and long reads are discarded.

• Map reads to reference (hg19). For algorithm see: http://www.clcbio.com/files/whitepapers/whitepaper-on-CLC-read-mapper.pdf

  • Mapping parameters:

    • match score 1
    • mismatch cost 2
    • linear gap cost
    • insertion cost 3
    • deletion cost 3
    • length fraction 0.95
    • similarity fraction 0.95
    • global alignment
    • ignore non-specific matches.

• Local Realignment.

  • Realign unaligned ends
  • perform local realignment 2 times.

• Variant Detection. See: http://www.clcbio.com/files/whitepapers/whitepaper-probabilistic-variant-caller-1.pdf

  • Minimum coverage:10
  • min count: 3
  • min frequency: 25.
  • Restrict calling to target region: as given by SureSelect.