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  • High % "N" in first 35bp using MultiQC

    Hi all,
    Just did some sequencing for a client and got a little odd plot using MultiQC for the "FastQ per base N-Content". It looks like the first 35 bp had problems with "N" then suddenly stopped. The percentage isn't high, but I don't understand what happened. Using trimmomatic removed these which appeared to be runs of "N". Anyone seen something like this before, or have an explanation for this?



    Thanks,
    -pete
    Edit: We made these libraries using KAPA tagmentation kits, and there were no bad tiles (none) using FastQC.
    Last edited by hoytpr; 01-07-2019, 10:34 AM. Reason: clarity

  • #2
    What'd the metrics graphs (intensity, %base, %Q30, error rate) look like?

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    • #3
      Most everything looked okay. There is a little glitch at 35bp in the data by Cycle Error rate

      Last edited by hoytpr; 01-07-2019, 12:58 PM.

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      • #4
        The % NoCall looks a little weird:

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        • #5
          Might be due to the decrease in base diversity at the beginning of the reads. Maybe you've got some primer dimer in there?

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          • #6
            Must just be primer dimers. I talked with Illumina today and they looked at it. Basically said it's normal for a NextSeq to have these runs of "N" (they call "N-masking when multiplexing") when primer-dimers are present, and gave me a couple flags for bcl2fastq to get rid of them:

            --minimum-trimmed-read-length 0
            --mask-short-adapter-reads 0

            Trimmomatic also does a good job of cleaning them up.

            java -jar trimmomatic-0.35.jar SE -phred33 input.fq.gz output.fq.gz ILLUMINACLIP:TruSeq3-SE:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36

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