trim.seqs output blank file (sends to scrap file)

Commands in mothur
1

I know I’m not the first to have this problem, but I had some trouble understanding previous posts.
I’m running trim.seqs on my MiSeq data, and the output trim file is blank. It looks like all my reads went to the scrap file.

Here’s the command I ran (I’m omitting file structure for simplicity) :

trim.seqs(fasta=current, qfile=current, oligos=16S_V1_3.oligos)

I also tried running:

trim.seqs(fasta=16S_V1_3_R1.fasta, qfile=16S_V1_3_R1.qual, oligos=16S_V1_3.oligos)

Here’s a line from the scrap file, but I don’t know how to interpret it:

>HWI-M03023_95_000000000-AD3U0_1_1101_14072_1989|br bdiffs=1000(noMatch) fpdiffs=0(match) rpdiffs=1000(noMatch)  
GAGTTTGATCATGGCTCAGTGTAACCTCCGCCTCCTGGGTTCAAGTTTTTCTCATGCCTCAGCTTCCTGAGTATCTTGTATTACATTTTCCTGCCACCATACCCGTCTCCCTTTTTTTTTATTTTCCTTAGTTATGTTTTCTTGCCTTTTTGTCCATTCTTGTCTCTTACTCCTTACCTCCATTTTTCCTCCTTCTTTCTCCTCCCTCTTTTCTTACCTTCCTCTCCTTATCCACCTCACCCTTCTCTTCCTTCCCCCTTCCTCCCTCCCTTCTCTCCTCTCCCCTTCCCCCCTTCTCTC

Here’s the first 10 lines of my oligos file:

forward GAGTTTGATCNTGGCTCAG
reverse GTNTTACNGCGGCKGCTG
#primer GAGTTTGATCNTGGCTCAG GTNTTACNGCGGCKGCTG
barcode ACAGTCATAT CU117B
barcode ACATAGTATC CU172GD2
barcode ACATATACGT CU409B

I would appreciate any help in getting this fixed.

2

The br mean the sequence failed due to the barcode and the reverse primer. bdiffs=1000(noMatch) means noMatch for the barcode was found and since you did not allow for any bdiffs, the default return fail value is 1000. Likewise rdiffs=1000(noMatch) means noMatch for the reverse was found and since you did not allow for any pdiffs, the default return fail value is 1000. fpdiffs=0(match) means there was a match for the forward primer with 0 diffs.

Mothur searches for linkers, then barcodes, then spacers, then forward primers on the left end. On the right, if paired barcodes, then reverse primers.

HWI-M03023_95_000000000-AD3U0_1_1101_14072_1989|br bdiffs=1000(noMatch) fpdiffs=0(match) rpdiffs=1000(noMatch)
GAGTTTGATCATGGCTCAGTGTAACCTCCGCCTCCTGGGTTCAAGTTTTTCTCATGCCTCAGCTTCCTGAGTATCTTGTATTACATTTTCCTGCCACCATACCCGTCTCCCTTTTTTTTTATTTTCCTTAGTTATGTTTTCTTGCCTTTTTGTCCATTCTTGTCTCTTACTCCTTACCTCCATTTTTCCTCCTTCTTTCTCCTCCCTCTTTTCTTACCTTCCTCTCCTTATCCACCTCACCCTTCTCTTCCTTCCCCCTTCCTCCCTCCCTTCTCTCCTCTCCCCTTCCCCCCTTCTCTC