I expect 170 bp for several reasons: a) approximate sequence length for S. cerevisiae using these primers that was published in a paper cited on the EMB website b) I made a reference alignment using database sequences for the organisms in my mock community, aligned using MEGA and trimmed to primers, and they were 174 bp without gaps, and c) that is the length of sequences I sampled from my ‘stability.trim.contigs.fasta’ file, once I trimmed them down to the primers.
I forgot to label the sequences above, my bad! The longer sequence comes from ‘stability.trim.contigs.fasta.’ However, the screen.seqs max length I used was large enough that it didn’t screen much out - essentially ALL of my contigs were 420-450, so I don’t think using “YourName.trim.contigs.good.fasta” would have made much difference. The shorter sequence is from 'stability.trim.contigs.good.unique.align.
I did this comparison for several sets of sequences and I think the stuff before my forward primer is the adapter, linker etc from the sequencing process. So my contigs are larger because of that. When I go through the MiSeq SOP, the sequences are getting cut off too soon, as you can see in the short sequence from my example above.
I’ve taken several sequences, both from my mock reference alignment (the one I made on MEGA) and from my ‘stability.trim.contigs.fasta’ file (trimmed to primers), and aligned them 2 ways, (1) using mothur and reference file ‘silva.nr_v132.align’ (2) using https://www.arb-silva.de/aligner/ and viewing on Wasabi (Thanks for that Rene, by the way!).
For one example sequence:
(1) Run on mothur: Start= 41,791 End= 43,116 NBases= 131
(2) Run online: Start= 41,790 End= 43,275 Counted the #bases in alignment = 174
When I use the values from (2) for the pcr.seqs step in mothur, my alignments still come to 100-130 bp. Pat, any advice for me on that front?
Or maybe it doesn’t matter that much. My main concern is what I would be losing using truncated sequences, but you’re right Rene, and I’m not losing a ton of sequence length anyway.
Sorry for all the posts lately - I really appreciate all the help!