The screencasts under "Metagenomic Analyses with Galaxy" specifically
use 454 data and would likely be helpful, maybe even if you have already
resolved your prior issue.
Apologies for the delay in reply, we were a bit backed up with questions
in March and a few slipped through.
On 3/10/11 8:14 AM, Jackie Lighten wrote:
I have a question for you guys regarding quality filtering.
I have a data set of double MID tagged 454 amplicons, from which I wish
to select high quality sequences above Q20.
The 454 quality filtering system seems to work differently from that
given for the Illumina sequencing i.e. 454 filtering takes high quality
segments, while Illumina (FASTQ) can select high quality full reads
based on certain parameters.
OK, so I know that the total length of my amplicon, including primers
and barcodes is around 260bp. If I then set the 454 quality filtering
tool to extract contiguous high quality sequence of >260, it gives me
back around 45% of my raw data as hitting this criterion i.e. All 260bp
are above Q20. I don’t necessarily need this high stringency as most
bases may not be informative.
But if I convert my 454 data to FASTQ format and then run the Illumina
filtering system which also allows me to set the number of bases allowed
to deviate from the Q20 criteria, I get back over 90% of my data
(allowing 10bp to deviate from Q20).
I then need to go ahead and convert back to 454 format.
Can you tell me if this is OK?
Will I loose /confuse information somewhere along these conversions?
It seems that if I do this, my barcodes are removed, as amplicons do not
sort properly when I parse them through my barcode filtering program.
Does anyone know of a program to filter 454 data based on average
sequence quality score, which doesn’t involve Linux and the Roche off
instrument program (I have no experience in Linux! )
Department of Biology,
Halifax, NS, B3H 4J1
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