Multiple Matches sequences retained in pcr.seqs

Commands in mothur
1

I use pcr.seqs to trim primers from my sequences.
In one dataset (fungal ITS) I have quite a few unique sequences that are not scrapped but give the information ‘multiple matches’ to one of the primers. I had thought perhaps it was a concatenated primer… But I can’t find the multiple match to that primer in any of the sequences. I’ve pasted an example below.

Either way, I’d like to either remove these sequences from my dataset or remove the primer from the sequences, as they still appear to have a primer in them…
Below is an example, showing that reverse primer was removed, but forward primer wasn’t and gives the info (multipleMatches)

post pcr.seqs (in the good file)

M03033_9_000000000-AALWV_1_1101_24378_5923 fpdiffs=1(multipleMatches) rpdiffs=0(match)
TCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCCTTGGTATTCCGAGGGGCATGCCTGTTCGAGCGTCATTGCAACCCTCAAGCACTGCTTGGTATTGAGCGCTCGCCTCCCTGTCGCAGGGGCGGCGTGCTCGAAAACTAGTGGCGGTCTCGGAAGACTTCAAGCGCAGTTAAGCCGTCGCTTTGGAAGCTTCTGCCCGGGACCTAGCCGAGAAACCCCCTACGAATTCCAATGATTGACCTCGGATCAGGCAGGGATACCCGCTGAACTTAA

before pcr.seqs

M03033_9_000000000-AALWV_1_1101_24378_5923
TCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCCTTGGTATTCCGAGGGGCATGCCTGTTCGAGCGTCATTGCAACCCTCAAGCACTGCTTGGTATTGAGCGCTCGCCTCCCTGTCGCAGGGGCGGCGTGCTCGAAAACTAGTGGCGGTCTCGGAAGACTTCAAGCGCAGTTAAGCCGTCGCTTTGGAAGCTTCTGCCCGGGACCTAGCCGAGAAACCCCCTACGAATTCCAATGATTGACCTCGGATCAGGCAGGGATACCCGCTGAACTTAAGCATATTAATAAGCGGAGGA

Oligos were:
primer GCATCGATGAAGAACGCAGC TCCTCCGCTTATTRATATGC

and pdiffs=1

Also, strangely, when the ‘multiple match’ is the reverse primer, the sequence that is retained after pcr.seqs is actually blank in the fasta file.

after pcr.seqs

M03033_9_000000000-AALWV_1_2110_4473_19010 fpdiffs=0(match) rpdiffs=1(multipleMatches)

before pcr.seqs

M03033_9_000000000-AALWV_1_2110_4473_19010
GCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTACACCTCTCAAGCTCAGCTTGGTATTGGGTCTTCGTCCCTCGTGGACGGGCCTGAAAATCAGTGGCGGTGCAACCGGTCCTCAAGCGTAACAGCATTGAATATCCCGCTTTGGAGTGACCGCGTGGACCCGCCAGATAACCCCTAATATATTTTCAATGATTGACCTCGGATCAGGTAGGGATATCCGCTGAACTTAAGCATATCAATAAGCGGAGGC



Thanks for your help,
2

Hi,

It’s pretty weird to me that you could have a primer that is the length of your primers match twice (about 1 in 4^20). That makes me worry that there’s something deeper going on with the data that you’re using. I’d rather not change the functionality of the function if it’s a problem with your data rather than a problem with our features.

Pat