Help with RSEM: how to change default phred33 to phred64

[LearningBIOINFO]

Hi all,

When I was using the RSEM to align my reads.fastq to my transcripts.fasta (trinityrnaseq_r2013-02-25/util/RSEM_util/run_RSEM_align_n_estimate.pl), I happened to see this:

bowtie -q --phred33-quals -n 2 -e 99999999 -l 25 -I 1 -X 1000 --chunkmbs 200 -p 4 -a -m 200 -S TRANS -1

But my fastq used phred64, so is there a way to change the default setting (phred33)? Does it affect much if I used the default setting?

Thanks,
He

[Brian Bushnell]

It shouldn't matter much. But if you want to change the quality, you could download BBTools and do this:

reformat.sh in=reads.fastq out=fixed.fastq qin=64 qout=33

[yueluo]

From the trinity page--

http://trinityrnaseq.sourceforge.net/analysis/abundance_estimation.html

# To pass additional parameters to rsem-calculate-expression,
# type ' -- ' followed by additional pass-through params

You could try <original command> --phred64-quals
Other parameters can be passed in the same way.

[LearningBIOINFO]

Hi Yueluo,
I tried typing --phred64-quals behind my command (../run_RSEM_align_n_estimate.pl/ --transcripts) but it said: Unknown option: phred64-quals.
He

[LearningBIOINFO]

Brian,
I will try it and see if it works.
Thanks,
He

[yueluo]

Well I guess you could follow what Brian suggested. But it is strange you can't use the '--phred64-quals' option as it is mentioned on both trinity's and RSEM's websites.

Converting the phred-qual isn't too difficult, but it is time-consuming when you process large data sets and adds pressure to the system's I/O and storage -- which is a concern to myself and probably some others.

[Brian Bushnell]

Converting the phred-qual isn't too difficult, but it is time-consuming when you process large data sets and adds pressure to the system's I/O and storage -- which is a concern to myself and probably some others.

So you would think! But no, reformat.sh is extremely fast, clocked at over 500MB/s (note that a single HDD won't be able to deliver this). And you can pipe it, like this:

reformat.sh in=reads.fq out=stdout.fq qin=64 qout=33 | someOtherProcess

...which will add no load to the system's I/O or storage, as long as the other process accepts stdin. For, you see, I am also concerned with these things!

[yueluo]

Good to hear Brian ! I'll give it a go soon and see how well that goes