trims.flow doesn't work

mothur bugs
1

I think it’s not a bug. I’m just here asking for help.
So I’m working on sequencing data from GX-FLX Titanium. With ssffinfo comand, I got a fasta, a .flow file. I made a .oligos as described with my primers and barcodes. Next I tried trim.flows function and got no split file named with barcode groups. Then I re-check my primers, barcodes in the fasta file. And found that all the reads begin with the same fragment “ACACGTAGTAT”, followed by my barcodes, then the primers. It seemed these “adaptors” hindered trim.flows recognize barcodes. I wonder if the “header” here is so-called “linker” or “spacer” (I found no explaination), and tried to add “linker ACACGTAGTAT” or “spacer ACACGTAGTAT” to the .oligos files. But it still didn’t work. I even tried to add “ACACGTAGTAT” to the head of my barcodes to form new barcodes. Still no good news.
I wondered if I can just give “ACACGTAGTAT” as primers in the .oligos file and run trim.flows,followed by trim.seqs. In this way, Iwould get a fasta file instead of a .flow file, which can be used for denoising in mothur.
Why it didn’t work?
How to deal with my data?

Thank you!

2

The linker is what mothur looks for before the barcode, so you want to label the fragment you are referring to as a linker in your oligos file. Have you tried using ldiffs=1?

3

It is solved.
The order should be B. But I’m still confused that order showed by sffinfo is the same as order A.
Thank you!

4

I think the first few bases are the same, so if you look closer you’ll probably see some differences