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  • Two sizes in sequence length

    I'm working with Illumina Miseq data. After BAM to fastq extraction I run fastqc to generate reports. With many of my samples I see two different sequence lengths as shown in the picture below:



    Also want to add that if I see this in the sequence length distribution, it also means the sequence composition will be skewed:



    Any idea what is causing this?
    Single End Data
    Adapters are removed off the sequencer

    Thanks!

  • #2
    ... you have single end Illumina data? Is that a thing?

    We use Ion Torrent technology, but if I see that it's usually a size selection problem.. something went wrong with my bead ratios.

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    • #3
      This is odd. Illumina does not support read lengths greater than 300bp as far as I know (and even 300bp lengths are not well-supported), so I'm not sure what you are doing.

      Can you explain the library-prep and sequencing methodology in more detail?

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      • #4
        It is possible to run strange lengths e.g. 2 x 300 kit can be run as 600 single-end. Not sure why anyone would want to but one can.

        @jetjr: Can you attach a complete fastqc analysis file?
        Last edited by GenoMax; 04-19-2017, 11:41 AM.

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        • #5
          In addition to questions in above posts, I wonder if these plots are from the same sample or file. Sequence length distribution plot shows a flat 0 count on Y axis for sequence length over ~275 while Per base sequence plot indicates that read length are up to 370 base.

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          • #6
            Probably from merged reads from a 500 cycle run. (The plots are from fastqc, not SAV.) The shorter peak from reads that didn't merge.

            BTW, can I add that fastqc's variable width bins make me feel uneasy?
            --
            Phillip

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            • #7
              Originally posted by pmiguel View Post
              BTW, can I add that fastqc's variable width bins make me feel uneasy?
              --
              Phillip
              Easily fixed by adding --nogroup to command.

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              • #8
                Thanks all for the replies! I stand corrected it is Ion Torrent data >.< We are looking into some of the things mentioned in this thread. I will add full QC reports soon...

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