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  • Manipulating the quality file during fasta-demultiplexing with QIIME

    Hi!

    I am using QIIME to process my dataset of 2 million joined PE-reads (~500 bp).

    I want to use cd-hit as my OTU picker. cd-hit requires a fastq file with every sequence startsing with the 5'-F-amplification-primer.

    With QIIME, i could convert the fastq to fasta and a quality file.
    I did a split_libraries.py:

    split_libraries.py -f in.fasta -m mapping.txt -q qualityfile -l 200 --keep-primer -a 0 -H 6 -o Split_lib -b 8 -M 2

    The --keep-primer option keeps the primer sequence inside the sequence, -l deletes short reads, -a keeps the number of Ns at zero, and -H removes long stretches of homopolymers.

    The problem is that, while this command runs smoothly, the corresponding quality file is not processed, so i cannot convert back the resulting fasta to fastq to run cd-hit.

    As far as i can see it, there are several options:

    1) Run QIIME's split_libraries_fastq.py instead, but all these nice options available in the fasta-script are not available.
    What would be the most equivalent command line for split_libraries_fastq.py with:
    - removal of ambigous base calls
    - removal of short reads
    - removal of barcodes
    - removal of reads with long homopolymer calls
    - keeping the primers

    ?

    2) Compare the headers in the resulting fasta to the headers in the qual.file, and do some whitelisting process. I have no idea how to do that, though.

    3) Find a way telling split_libraries.py to process the quality file as well. I though it would be -q in the split_libraries.py, but that did actually nothing.


    Any help is really, really appreciated.
    Last edited by nouse; 01-15-2015, 04:01 AM.

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