sequencing error and make.contigs

Theory behind mothur
1

Hi again,

what’s the range of the expected sequencing error from seq.error? I’ve started using a commercial microbial community standard and I was wondering about what I should expect. So far a few orders of magnitude worse than what it’s shown in the MiSeq SOP…

Is make.contigs able to handle properly ‘very overlapping’ pairs? The sequencing centre does 2x250 for 16S-V4 and make.contigs seems to have problems. Using FLASH, modifying the defaults, clearly improves the amount of successfully assembled reads, the proportion of chimeras detected diminish (values around 50% with make.contigs vs around 10% with FLASH) and the sequencing error improves (not enormously but it does).
Not sure if it’s because of the full overlap of the reads or something else. Any idea?

Thanks,
Xabi

2

Hmmm. Not sure what to tell you. We do 2x250 with the V4 region and that works best. That’s also what we use in the MiSeq SOP. What problems are you having with make.contigs? After pre.cluster, you should get an error rate around or below 0.01%. You might want to double check that you have the right fasta reference file for your mock.

Pat