Sequencing orientation problem

Theory behind mothur
1

Hi there,
I am working on Mothur according to 454 SOP. And I am confused about the sequencing orientation.
It seemed the SOP sequenced from the reverse end (3’ end) of the 16S rRNA gene [See the “Logistics” section, around the 4th line.] , but why the GQY1XT001.oligos file (content listed below) used forward primer for processing?

forward CCGTCAATTCMTTTRAGT
barcode AATGGTAC F003D000
barcode AACCTGGC F003D002
barcode TTCGTGGC F003D004
barcode TTCTTGAC F003D006
barcode TTCGCGAC F003D008
barcode TCCAGAAC F003D142
barcode AAGGCCTC F003D144
barcode TGACCGTC F003D146
barcode AGGTTGTC F003D148
barcode TGGTGAAC F003D150
barcode AACCGTGTC MOCK.GQY1XT001

Furthermore, we sequenced from the forward end (5’ end), do we need to change the command due to the different orientation of sequencing?
e.g… when it comes to the ‘screen.seqs’ step, [mothur > screen.seqs(fasta=GQY1XT001.shhh.trim.unique.align, name=GQY1XT001.shhh.trim.unique.names, group=GQY1XT001.shhh.groups, end=27659, optimize=start, criteria=95, processors=2) in 454 SOP], would I change the ‘end=***’ to be ‘start=***’?

2

why the GQY1XT001.oligos file (content listed below) used forward primer for processing?

Because that was the primer that sequencing was done from.

Furthermore, we sequenced from the forward end (5’ end), do we need to change the command due to the different orientation of sequencing?

Correct, you do not need to use the flip option and in screen.seqs you will set the start position and optimize the end position.

Pat