trim.seqs, trouble locating primers and barcodes

Commands in mothur
1

I’m having trouble in getting Mothur to locate my primer(s) and barcodes when using trim.seqs. Thus only 8 of my >20000 sequences went into trim.fasta and the rest went into scrap. The reason for scrapping most sequences seems to be unlocated primer and barcode.

I was using command: trim.seqs(fasta=H88XYFD01.fasta, oligos=arch.oligos, qfile=H88XYFD01.qual, maxambig=0, maxhomop=10, minlength=50)

My .oligos file looks like this:

forward GYGCASCAGKCGMGAAW
#reverse GCBGGTDTTACCGCGGCGGCTGRCA
barcode AGCGCGCGC Tit-B-Arch349F_101_SB4
barcode AGCTCTATC Tit-B-Arch349F_102_CC2
barcode ATAGACATC Tit-B-Arch349F_103_MM3

And here is an example of a sequence that went into the .trim.fasta file:

H88XYFD01D45JJ xy=1581_3261
AGCGCGCGCGCGCACCAGTCGAGAAAATTACCCAATCCCGACACGGGGAGGTAGTGACAATAAATAACAATATAGGGCTCTTTCGGGTCTTATAATTGGAATGAGTACAATTCAAATCTCTTAACGAGGAACGATTGGAGGGCAAGTCTGGTGCCAGCCGCCGCGGTAACACCCGATGGCGCGAGGGAGGCGATA

And here are 2 examples of sequences that went into the scrap file because barcode and primer were not located (although I was able to locate them manually).

H88XYFD01CLSNL|br
TAGCTCTATCGTGCAGCAGTCGCGAATCTGCCGTTACTGCCTCTGATGCCAGCCGCCGCGGTAACACCTGATGGCGCGAGGGAGGCGATA

H88XYFD01BA06O|br
TAGCTCTATCGTGCACCAGGCGCGAATCACCAGCCTGTCAGCCGCCGCGGTAACACCAGATGGCGCGAGGGAGGCGATA

How should I modify my command so that the primer and bar code can be located. Allow mismatches?

2

trim.seqs expects the sequence to start with your barcode and then your primer. So, the initial T is throwing things off. Double check that you have the correct barcode sequences - I suspect that you really want those barcodes to start with a T. In general we allow 1 mismatch to the barcode and 2 to the primer.

Pat

3

Thanks, Pat!

Johanna