Align two regions

I have in one MiSeq run the same samples sequenced by two different set of primers (16S). The first region sequenced is amplified using the 341F/805R primer set and the same samples were sequenced also by the 909F/1391R primer set.

I follow the MiSeq SOP example and I have run the commands up to pcr.seqs.

With this command you can provide the primer sequences in order to trim the database that you are going to use based on the region that you have amplified. In my case in the MiSeq sequences I have 2 different regions.

Which is the best approach in this case? Can I use the 341/1391R primer sequences in order to trim the silva.align file?

Looking forward for your reply.
George

You should separate the data and process them in parallel.

Pat

Hi,

I have a related question - in this case, after you have processed the files in parallel, what is the best way to compare OTUs across these different runs? For example, is there a way that could make the names given to each OTU (e.g. OTU01, OTU02, OTU03, etc) to be the same in the (separate) final fasta files, regardless of what 16S primers were used? I understand that I could generate a database file with representative sequences for each OTU and then manually compare their identity and adjust names accordingly so that OTU01 in the sequences generated with primer “A” is the same OTU01 in the sequences generated with primer “B”. In my specific case, I would like to process sequences downloaded from NCBI that correspond to gut microbes of an insect, but different 16S primers were used in each study. I am also not sure if the rationale of my strategy is correct, I would really appreciate your opinion.

Thank you in advance!

I don’t think you can compare different primer sets to each other. They each have different biases and what not. At best you can maybe compare them at a very broad taxonomic level.

Pat