RNA_seq read separation help

[umamayil]

Hi to everyone,

I am new member to this forum. I have 100bp single read illumina fastq files. When we looked at the reads we saw some interesting sequences. We want to separate those reads and write it in separate fastq file for analysis. For example we want to separate "ATTTTTTTTAGAAAAAAAA" containing reads (we saw something around 2million reads out of 9million reads). Can you please give me guidance how to do it. IF there is program or any unix commands will be helpful. I am not a unix person. please give me commands to execute.

Thanks a lot.
Mayil

[GenoMax]

bbduk.sh from BBMap package can do this. If that sequence is at the end of the reads then,

$ bbduk.sh -Xmx1g in=reads.fq outm=matched.fq outu=unmatched.fq restrictleft=19 k=19 literal=ATTTTTTTTAGAAAAAAAA

In this case, all reads starting with "ATTTTTTTTAGAAAAAAAA" will end up in "matched.fq" and all other reads will end up in "unmatched.fq". Specifically, the command means "look for 19-mers in the leftmost 19 bp of the read", which will require an exact prefix match, though you can relax that if you want.

So you could bin all the reads with your known sequence, then look at the remaining reads to see what they have in common. You can do the same thing with the tail of the read using "restrictright" instead, though you can't use both restrictions at the same time.

[umamayil]

Hi,
Thanks. The sequence will be either in the middle or in the end. How to separate if the interested sequence is in the middle.

Thanks again
Mayil

[GenoMax]

Just remove the "restrictleft/right" directive and the entire sequence will be searched.

[umamayil]

Hi,

Thanks a lot. I will try the commands you have given to me.

Thanks again and have a nice weekend.

Mayil