250bp or 300bp miseq?


I have used 250bpPE miseq successfully before, but now I tried 300bp PE with problems. After make.contigs the resulting aligned fasta are around 350 long (should have been 250 according to SOP). Thought the alignment should solve that (make.contigs). After screen.seqs and unique.seqs, almost none are unique???

Are make.contigs command specifically written for 250bp?

I managed to solve it by removing the last 50bp of the fastaq files (using seqtk), and now everything is back to normal with around 250 bp long sequences and few unique compared to total number of fasta.


when you run make.contigs try using trimoverlap=T. your reads are running through the primers and causing problems. this will trim the reads to overlap the same bases.


Thanks, will try that