# Sequencing orientation problem

**URL:** <https://forum.mothur.org/t/sequencing-orientation-problem/1888>\
**Category:** Theory behind mothur\
**Created:** [June 11, 2014, 3:05pm UTC](https://forum.mothur.org/t/sequencing-orientation-problem/1888 "2014-06-11T15:05:40Z")\
**Posts on this page:** 2\
**Page:** 1

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**Author:** ![mengmengmumu](https://avatars.discourse-cdn.com/v4/letter/m/e9bcb4/32.png) [@mengmengmumu](https://forum.mothur.org/u/mengmengmumu)\
**Post date:** [June 11, 2014, 3:05pm UTC](https://forum.mothur.org/t/sequencing-orientation-problem/1888/1 "2014-06-11T15:05:40Z")

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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=\*\*\*’?

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<div class="post-metadata">

**Author:** ![pschloss](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/pschloss/32/4_2.png) [@pschloss](https://forum.mothur.org/u/pschloss)\
**Post date:** [June 17, 2014, 8:24pm UTC](https://forum.mothur.org/t/sequencing-orientation-problem/1888/2 "2014-06-17T20:24:19Z")

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> 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