mothur

merge files from outside of mothur

Dear Pat,
I got DNA sequences from Illumina Miseq V3-V4 region-paired reads (now I know sequencing that region was a disaster) and the reverse reads had poor quality and using mothur I went up to cluster.split and then stuck due to large distance matrix. I know I can re-analyse using only forward reads of those reads. But before that I tried to merge my FWD and REV reads from outside the mothur (using PEAR) and then made a stability file using merge.file (14 DNA samples) and produced a group file using make.group file. What I wanted here, use my all data without discarding them. This way I went up to pre.cluster and then once pre clustering starts the error messages came up saying some specific reads (with their sequenced names) were not in count file or fasta file and mothur asked to correct it. So, from here I cant go forward.

My questions.

  1. Is this a bad idea (merging FWD and REV reads from outside and and start rest of analysis within mothur)
  2. If so, can i expect a good result if I only use FWD reads?
  3. Can I cut only V4 region from v3-v4 sequences and used those filtered V4 sequences for my analysis?

Thank you.
Regards,
Dilhanide

  1. Is this a bad idea (merging FWD and REV reads from outside and and start rest of analysis within mothur)

It’s not necessarily a bad idea; however, I haven’t seen anything to suggest that PEAR will give you as good or better assemblies as what we do in mothur.

  1. If so, can i expect a good result if I only use FWD reads?

You could do that, but you’d have to come up with a way to do the quality trimming to only look at the best part of the forward read.

  1. Can I cut only V4 region from v3-v4 sequences and used those filtered V4 sequences for my analysis?

If your forward read went from V3->V4, then I suspect the V4 data will be a poorer quality than the V3 data.

Pat