trim.flow, trim.seqs, and reverse primer


I want to screen only sequences having both forward and reverse primer sequences.

For reverse primer, I found that only the sequences having the extreme end that matches the reverse primer sequence are screened by the trim.flow or trim.seqs commands.

But in most cases, the ends of the sequences are extended beyond the reverse primer sequence due to Fusion primer region, and the lenth of the extended region is variable.

Thus, I wand to know whether there is a method to screen the sequences including a specific sequence in any position within the sequences in the trim.flow or trim.seqs commands.

Thank you.


There is not at this time.

Around 40% of my seqs has an ambiguous call in the forward primer. All these seqs are called N at a particular position where it is supposed to be A. However, the quality of the seqs are good (except it is 0 where the N is). I would like these seqs to pass the trim.flow command. Do you have a parameter for that or any suggestion on how I should go about this?

Thank you

Have you tried pdiffs=1?

I did and now 83% of the seqs went to scrap…

What version of the 454 software/chemistry/whatever are you using?

GS FLX Titanium

Is that with the A or B flow pattern?

If you go into mac/linux can you run the following?

cut -f 1 -d " " scrap.flow | cut -f 2 -d “|” | sort | uniq -c

Instead of scrap.flow inset the name of your *scrap.flow file.

Hi Patt,
Do you mean lib-A or lib-B? Because we used lib-L. I’m not sure what do you mean by flow patterns. I used the command you suggested and below is the result:
sort | uniq -c
1 450
149971 l

Thank you,

Is this the FLX+ Titanium or just FLX? What you’re getting gout suggests that your sequences are just too short. THis could be becaus of the new way the sequencer works or because your data are bad.