How to screen reverse reads from raw illumine data?

Commands in mothur
#1

Hi Every body.

I am using illumine hi-seq pair-end 100 strategy to sequence my V6 amplicons (20 barcode primers were utilized to encode sample information).

Now the problem is that， after joining paired reads into contigs
The contigs contained both “forward” and “reverse” sequence. Unlike 454, the pair reads seemed to be randomly sequenced in either direction in illumine hiseq.

I know mother have a simple command “reverse.seqs” to reverse compliment reads that are in reverse direction with 5->3 16S RNA gene.

But how to screen out the reverse contigs from large raw data.


Regards
#2

Hmmm… I’m not sure how that would happen.

A couple of options…

  1. When you run trim.seqs to remove the barcodes and primers, you can use the checkorient option (http://www.mothur.org/wiki/Trim.seqs#checkorient)
  2. When you run align.seqs with flip=T the sequence will flip it into the correct orientation and in the next step it will be in the correct orientation.
#3

Thank you

I find RDP pipeline can fix this problem ether

just exchange the forward and reverse primer, RDP will separate the reads of different directions.