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Old 01-15-2015, 03:28 AM   #1
nouse
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Location: Germany

Join Date: Sep 2013
Posts: 11
Default 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 at 04:01 AM.
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