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  • Help understanding FastQC output

    Hello,

    I recently attempted to align some MiSeq reads using Bowtie2 and found that only about 50-55% of my reads aligned to the reference genome. When I look at the FastQC graphs of the sequencing files, the Adapter Content graph stands out to me. But I'm not sure exactly what it means (image attached). Clearly there's some Illumina Adapter in my sequences, but I'm a little confused. The plots for both read pairs look the same with the adapter showing up at the 3' end of the read. I'm trying to understand this. Can anyone help?
    Attached Files

  • #2
    When you have DNA fragments that are shorter than the read length you end up reading into the adapter sequence, and you will get the adapter sequence towards the 3' end of both R1 and R2.

    See the University of Texas at Austin's genome centre webpages - they have a diagram of what the sequencing constructs look like - adapters, primers, barcodes, and DNA fragment inserts.

    See

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    • #3
      Yeah, I sort of figured this might be the case, but just wanted to confirm to be sure.

      Is there a universal adapter sequence I can feed in to clip the adapter sequence from the reads before alignment?

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      • #4
        Popular trimming programs (trimmomatic, BBDuk etc) come with the relevant adapter sequence files bundled in. Search for the threads here and you will find information on how to use them.

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        • #5
          Thanks to you both for your help!

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          • #6
            BBDuk does adapter trimming and comes with truseq and nextera adapter sequences, the most commonly used in Illumina sequencing. They are in the /resources/ directory.

            If you have paired-end reads, it is also capable of trimming without specifying an adapter sequence, in case some custom adapters were used.

            Edit: Wow, I typed too slowly =)

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