Seqanswers Leaderboard Ad

Collapse

Announcement

Collapse
No announcement yet.
X
 
  • Filter
  • Time
  • Show
Clear All
new posts

  • 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

      Latest Articles

      Collapse

      • seqadmin
        Current Approaches to Protein Sequencing
        by seqadmin


        Proteins are often described as the workhorses of the cell, and identifying their sequences is key to understanding their role in biological processes and disease. Currently, the most common technique used to determine protein sequences is mass spectrometry. While still a valuable tool, mass spectrometry faces several limitations and requires a highly experienced scientist familiar with the equipment to operate it. Additionally, other proteomic methods, like affinity assays, are constrained...
        04-04-2024, 04:25 PM
      • seqadmin
        Strategies for Sequencing Challenging Samples
        by seqadmin


        Despite advancements in sequencing platforms and related sample preparation technologies, certain sample types continue to present significant challenges that can compromise sequencing results. Pedro Echave, Senior Manager of the Global Business Segment at Revvity, explained that the success of a sequencing experiment ultimately depends on the amount and integrity of the nucleic acid template (RNA or DNA) obtained from a sample. “The better the quality of the nucleic acid isolated...
        03-22-2024, 06:39 AM

      ad_right_rmr

      Collapse

      News

      Collapse

      Topics Statistics Last Post
      Started by seqadmin, 04-11-2024, 12:08 PM
      0 responses
      22 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, 04-10-2024, 10:19 PM
      0 responses
      24 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, 04-10-2024, 09:21 AM
      0 responses
      19 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, 04-04-2024, 09:00 AM
      0 responses
      50 views
      0 likes
      Last Post seqadmin  
      Working...
      X