I don’t know if it’s a bug of the program or a problem with my oligo file - I’ve checked it and cannot find anything wrong with it. I’m running mothur V39.5 on Ubuntu 14.03 LTS, and there’s enough free space on the hard disk. This is the sixt time I’m using mothur as the best pipeline ever for metabarcoding - and I can’t understand what’s going on. The sequences are MiSeq V3 paired ends and the length of the amplicons is 300-400. Both F and R primers were barcoded.
This is the command:
make.contigs(ffastq=Oom11F_R1.fastq, rfastq=Oom11F_R2.fastq, oligos=OligoOom.txt, pdiffs=1, bdiffs=0, processors=12, rename=T, checkorient=T)
This is the oligo file, tab-separated:
primer GCGGAAGGATCATTACCAC TCTTCATCGDTGTGCGAGC
barcode GCTTCTAG ATAGCTTG AEF001
barcode GCTTCTAG CCTTAATG AEF002
barcode GCTTCTAG CACATGCT AEF003
barcode GCTTCTAG TGTCATGC AEF004
barcode GCTTCTAG CGATAAGG AEF005
barcode GCTTCTAG TTACGCGA AEF006
barcode GCTTCTAG TGGAGCTT AEF007
barcode GCTTCTAG GATACTGC AEF008 etc. (150 samples + mock community)
After make contig (which works well, good overlap, 13 millions assembled sequences), in the fasta file all sequences have been renamed according to the sample name but not all have the barcodes and primers removed!
This one is OK, it starts after the F primer and ends before the reverse:
The following still has both barcodes corresponding to HEF041- first and last 8 characters ( and the F and R primers):
If I run trim.seqs it doesn’t fix the problem, it discards all the sequences that were cut, of course, which are the majority. I end up with 5 millions sequences only. Just let me know if I should send the oligo file and part of the fatsq files… Any idea of what’s going on and any solution?
I will really appreciate your answer… I have 8 runs to analyze!