I am currently analysing 18 samples on MiSeq, which I followed the MiSeq tutorial.
First I made a stability.file and proceed to the the align.seqs step.
However, I used primers 27F/519R to target the V1-V3 region.
Therefore, should I use the customized silva.bacteria.fasta file as in the MiSeq SOP tutorial or should I use the silva.bactera.align file of version 119?
When I was aligning the 18 samples using the silva.bacteria.align file using a supercomputer, it still runs out of memory.
Should I try to use the one provided in the tutorial?
I have this very same question. And once in this release there is a very big .aling file, can I run the pcr.seqs with this silva.nr_v119.aling? To run pcr.seqs I saw that I need a fasta file… so I have this question too.
Thanks!
It’s running out of memory because the way you are doing your sequencing you are generating a lot of sequencing errors within the sequencer. To get adequate denoising so you can make OTUs, you need to have fully overlapping sequence reads. Otherwise you’ll just get out a lot of uniques and an impossibly large distance matrix.