pdiffs in pcr.seqs

mothur bugs
1

Hello

It seems that pdiffs does not work as expected…

Here is a fasta file.

radiolaria.fasta

AB101540.1.1726_U
TGAAACTGCGAATGGCTCATTAAAACAGTTATGGTTTATTTGATGGTTCATTGCTACTTGGATAACCGTAGTAATTCTAGAGCTAATACATGCACGAAGGGGCGACTCACGAAGCCCTGTATT
TATTAGATTAAAGCCAACCCCTTCATTGGGTTCCTGGTGAATCATAGTAACTTCTCGGATCGCCGGCCCTGCGCCAGCGACGTGTCATTCAAATTTCTGCCCTATCAGCTTTAGATGGTAGGA
TATGGGCCTACCATGGCGGTGACGGGTGACGGAGAATTAGGGTTCGATTCCGGAGAGGGAGCCTGAGAAACGGCTACCACTTCTAAGGAAGGCAGCAGGCGCGCAAATTACCCAATCCTGA
TTCAGGGAGGTAGTGACAAGAAATAACAATAGTTCGCCCATTGGGTAAGCTAATTGGAATGAGTACAATTCAAACATCTTAACGAGCACCAATTGGAGGGCAAGTCTGGTGCCAGCAGCCGC
GGTAATTCCAGCTCCAATAGCGTATACTAAAGTTGTTGCAGTTAAAAAGCTCGTAGTTGGATTTCTGCGCGGGTGGGGCGCCTTCCGGCCACTGGTCGGGTTGTGCAGCAACTCCCGCGCAT
CCTGCCGCGAACCACGCTACCATTAACTTGGTGGCGTCTGGGGACCGGCTCGTTTACTTTGAGAAAAATAGAGTGTTCAAAGCAGGCCTGATGCCTTGAATAATGTAGCATGGAATAATGATA
AAGGACTTTGGTTCTATTTTGTTGGTGTTCGGGACCGAAGTAATGATTGATAGGGACAGTTGGGGGCGTCTGTATTCTGAAGCGAGAGGTGAAATTCTTAGACCTTCGGAAGACAAACAGCT
GCGAAAGCGTTCGCCAAGGATGTTCCCTTTGATCAAGAACGAAAGTTGGGGGATCGAAGACGATCAGATACCGTCGTAGTCTCAACTATAAACTATACCAGCTAGGGATCGGCCAACGTTGAT
CTACAAAGACTTGGTCGGCACCTTATGAGAAATCAAAGCATCTGGGTTCCGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGTG
GCTTAATTTGACTCAACACGGGGAAACTTACCAGGTCCAGACATGAGAAGGATTGACAGATTGACAGCTCTTTCTTGATTTTATGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGAT
TTGTCTGGTTAATTCCGTTAACGAACGAGACCCCAGCCTGCTAAATAGCGCGCGTGGCCTCTGGCCGCGGCGAGCTTCTTAGAGGGACTATCGCCAGTTATATGCGAGGGAAGTTTGGGGCA
ATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGCACACGGGCTACACTGACGCGTGCAACGAGCATACCAGCTGGCCCGACAGGGCGTAGGCTAATCTTTTGAACTCGCGTCGTGCTAG
GGATTGACCATTGCAATTGTTGGTCATGCAACGAGGAATGCCTTGTAAACGCGGGTCATCAACCCGCATTGAATACGTCCCTGCCCTTTGTACACACCGCCCGTCGCTCCTACCGATGAATGTT
CCGATGAAATTTTCGGAGGGCGCCTGTGAAGCGGTGCAAACCGCGGAACGGGAGGCCCGAAGTTGCTTAAATCTGAACATTTCGAGGAAGGAGAAGTCGTAACAAGGTTTCCGTAGGTGAAC
CTGCAGAAGGA

Here is a first oligo file which has one mismatch with the sequence

Oligos_V4_Chlo.txt

#Type Sequence Name
forward CCAGCASCYGCGGTAATTCC V4_Chlo
reverse CTTCGAGCCCCCAACTTTC

When I run
pcr.seqs(fasta=radiolaria.fasta, oligos, Oligos_V4_Chlo.txt, pdiffs=1, keepdots=F, processors=8)

It returns 0 sequences while it should return the one sequence since there is a single mismatch.

As a proof if modify the Oligo file to (change in bold)

Oligos_V4_Radio.txt

#Type Sequence Name
forward CCAGCASCYGCGGTAATTCC V4_Chlo
reverse CTTCGATCCCCCAACTTTC

And run
pcr.seqs(fasta=radiolaria.fasta, oligos=Oligos_V4_Radio.txt, pdiffs=0, keepdots=F, processors=8)

It returns the sequence which proves that there is really a single mismatch

Any idea ?

2

The pcr.seqs command only allow pdiffs in the forward primer, not the reverse. We will modify this in our next release to allow diffs in reverse primers as well.

3

Thanks a lot for the answer. Maybe this should indicated in the wiki ?