Problems about the oligo file

Commands in mothur
1

Hi, all!
I’m a new user to the mothur. And now I got a problem. When I run the trim.flows command, it imput a blank flow.files file, I saw that all the data were in the scrap.flow file. I don’t know if there are something wrong with my oligo file.

What’s the oligo file look like:
forward CATGCTGCCTCCCGTAGGAGT
reverse TCAGAGTTTGATCCTGGCTCAG
barcode ACGAGTGCGT P01
barcode ACGCTCGACA P02
barcode AGACGCACTC P03
barcode AGCACTGTAG P04
barcode ATCAGACACG P05
barcode ATATCGCGAG P06
barcode CGTGTCTCTA P07
barcode CTCGCGTGTC P08
barcode TCTCTATGCG P09
barcode TGATACGTCT P10

If anyone who would like to help me, you’ll receive my great thanking!!! :smiley: I’m waiting for the answers! Thanks!

songer
8/29

2

Try it without the reverse line. If you include the reverse primer then every sequence is required to have the reverse primer at the very end of the sequence - this is highly unlikely with 454 data…

Pat

3

Very thanks for your suggestion! :slight_smile: It worked when I ignored the reverse primer. But I still want to know ，why it is unlikely that 454 data have the reverse primer at the very end of the sequence. what’s the reasons?

songer
8/30

4

Because the reads aren’t long enough to get to the distal end of the fragment