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  • Amplicon Sequencing Quality control and downstream analysis

    Hello everyone,

    I'm posting this thread because I'm having a hard time trying to do some quality control on our sequencing data.

    We are doing amplicon sequencing on an Ion PGM using a multiplex PCR for amplicon generation. I tried using FastQC for a fast and easy assassment of run quality but it doesn't seem to be suitable for this kind of application.

    The program tells me that more than 80% of my reads are duplicates which in my opinion is an artifact created by the fact that only the ends of the reads are compared to each other. These ends are characterised by the PCR primer regions used in the multiplex PCR and are therefore (largely) identical. Are there any tools for duplicate detection that ignore the outermost base pairs?

    As our reads have a length between 30 and 200 bp (mean 72) I am currently using bwasw for alignment. Base quality values are pretty good up to bp 100 but than they start to slope down rapidly. As I wasn't able to find this somewhere else I would like to ask here if anybody knows what happens to these low quality bases at the end of the reads in the bwasw algorythm? In the standard bwa algorythm a -q trim command can be used but there is no such thing for bwasw, does anybody know why?`

    Thanks beforehand for any help provided.

    Greatings,
    Mucki

  • #2
    I would also like to know why as well. Any help would be appreciated.

    Jamie =]

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