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  • How can I tell if an Illumina run is gone bad?

    Sorry for the very basic question. I am dealing with "raw" Illumina data for the first time and I am not sure how to pre-process the reads.

    The *sequence.txt file for each lane contains reads that have tons of 'N' at the end, with quality 'B'. (see below)

    How can I tell if this is normal or if something went wrong with the sequencing run?

    For example:
    @HWI-EAS413_0021:2:1:1159:4065#0/1
    TCGTGCCCGGGTAGCTCTGACTGGGCTGACTGTGGCTGAATACTTNAGNGACNANGAAGGTCANGAGATCGG
    +HWI-EAS413_0021:2:1:1159:4065#0/1
    dddb\dddcd^aaccdcdd\cdd^dadddc^c`ccdcd`d^`b``B\ZB`UYB]BUUU^][]_B_VWWWYT^
    @HWI-EAS413_0021:2:1:1159:11764#0/1
    GTGTGCCTGGTCATGCTGTGGTGGATCACCGTCCCAGGGCATTGGNGANTTNNGNATTTACGANATCGGAAG
    +HWI-EAS413_0021:2:1:1159:11764#0/1
    fffefffffffffeffffdff_ffefffffffeff`eedd`a^b`B\VB`]BB]B_aa\^_`_BaO[[Y]dc

  • #2
    GAP >= 1.5 (or OLB...) call bases in a slightly different way, so that if you have a bad quality read, instead of having low scores on each base, you'll see a series of at least 3 consecutive 'B' (or '#' if you converted in sanger) in the "bad zone" (but you'd better check the manual about the number of consecutives). If you see a number of reads mostly called with 'B' qual, you may have an issue. The reads you posted look good.
    This is an example of a bad run:

    @ILLUMINA-F3E58E_10:5:1:2798:3037
    ATGGGCCCTTTATTTCCCATCTTTTCCCAACTTTTT
    +
    A@=>@###############################
    @ILLUMINA-F3E58E_10:5:1:2798:15617
    CATGTATCATGGTCATTTAGCATTATCAAATGTCCT
    +
    EDEABBB@BA:@:??B@:555:@@::5:??@,<B:A
    @ILLUMINA-F3E58E_10:5:1:2798:10376
    TCCCAACAGAACAGCCCCAAGGGGCAAGGGGTTAAA
    +
    A7@#################################
    @ILLUMINA-F3E58E_10:5:1:2798:1024
    TNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
    +
    ####################################
    @ILLUMINA-F3E58E_10:5:1:2798:1640
    ATTGTTCCATCACCTCTGACTTTTTCCCCCACTTTT
    +
    @?##################################

    Comment


    • #3
      This 'B' quality (PHRED 2) is the Illumina 1.5+ Read Segment Quality Control Indicator (RSQCI), see also:
      Discussion of next-gen sequencing related bioinformatics: resources, algorithms, open source efforts, etc

      Comment

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