How to find primers using biopieces?

[LizBent]

Hi, I'm not much of a bioinformatician, and I am trying to learn how to use biopieces, which our real bioinformatician left behind on our server. Mostly it seems straightforward, but I am stumped by how to generate a sequence file containing only sequences (forward or reverse) that contain a given primer sequence. The reason this is important is that multiple amplicons were mixed together and co-sequenced in lanes of the 454 run, and these amplicons cannot be separated by barcodes, but rather have to be separated based on the primer sequences.

So after I use read_sff, should I look for the primer pattern using patscan_seq? If so, then how do I generate a fastq file containing only sequences containing the primers of interest- do I use write_fastq, and that will collect all the sequences identified by patscan_seq if I pipe them together? I'm a little unsure of how biopieces works.

Thanks for your help,

Liz

[maasha]

Try something like this:

 read_sff -ci data_in.sff |
patscan_seq -ip ATGATCAT[2,1,1] |
grab -p MATCH |
write_fastq -o data_out.fq -x

This will read in the SFF data from the a file called data_in.sff and clip the sequence according to the clipping information in the file.

Next the patscan_seq will find a primer sequence allowing for 2 mismatches and 1 insertion and 1 deletion and report the result per sequence record (the -i switch). You need to play around with the primer and the mismatch/indel information to get the best result. I would start with 14-16 bases from the 3'-end of the primer. Remember that patscan_seq is aware of ambiguity codes.

Next we grab the records where the primer was found.

Finally, you write the output to a FASTQ file.

You need to do this for each primer.