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  • Do my FASTQC results indicate sequencing bias?

    Hello,
    I used the Illumina Nextera Flex kit to sequence bacteria (WGS) and I got two warning that I usually do not get when using Nextera XT:

    1. overrepresented sequences in the first read (R1) but not the second read (R2).

    2. warning/error k-mer content

    Why do I get such errors? Does this indicate sequencing bias?
    Attached Files

  • #2
    This data must have been sequenced on NextSeq (or NovaSeq). That string of "G" is probably no-signal which is interpreted as G base.

    The bias at the beginning of the reads is likely due to the Nextera transposase sequence bias (similar to bias seen in "random priming") .

    If these are the only two "issues" then your data looks ok. Proceed with your normal analysis.

    Comment


    • #3
      Thank you for helping me!
      We use a MiseQ and it was the first time that we used Nextera Flex instead of XT. And it`s also the first time we got such a string of G.

      Beside these issues, I also got a warning for "per base sequence content". However, using Nextera XT always gives such issues.

      Comment

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