ITS analysis -- 2 Questions

Commands in mothur
1

Thanks kmitchell. I would like to use the procedure ( https://github.com/krmaas/bioinformatics/blob/master/mothur.fungal.batch ) you have outlined for ITS analysis.

Question #1 – A special case for skipping make.contigs?

Our lab used a primer set that would amplify ITS1, 5.8S, and ITS2 regions.
This expectation is based on the yeast genome.
It turns out the length of PCR amplicon is about 750 bp.

We are using MiSeq 300 x 2 (PE).
Based on the gene coordinates, we consider that Read1 would have most of ITS1, whereas Read2 would span ITS2 and some portion of 5.8S.
This means there shouldn’t be a place where Read1 and Read2 overlap.
Hence, is it reasonable to skip the make.contigs() step in this situation? Thank you.

Question #2 – If it is okay to skip make.contigs(), can I apply the above procedure, respectively, for Read1 ( ITS1 ) and Read2 ( ITS2 ), in our specific case? Thank you.

2

Have you already done the sequencing? Why attempt to sequence such a big chunk? Your error rate is going to be through the roof. You could still make.contigs but expect that most won’t overlap (I’d check merging the reads anyway just to see). I don’t have any recommendations for how to handle single read ITS because I’d rather spend the money to re sequence a smaller amplicon than months trying to make sense of data with that high of an error rate.

3

Thanks a lot kmitchell for your timely and much helpful advice. Sure, I will talk to our sequencing team to see if they could choose another primer set that would just amplify a single ITS region, rather than the whole region. Thanks again !!

4

If you want to test out the difference, I can help with ITS2 sequencing and don’t have a minimum number of samples.