Using pcr.seqs without a forward primer in the oligo file results in first four bases being removed

1

Hi everyone,

I have been playing around with the pcr.seqs command and ran into an issue that I am trying to solve. It seems for some reason when I call pcr.seqs if I input my forward primer as NONE the first four base pairs in the input sequence still get removed in the output.

I looked through the documentation and couldn’t find an obvious reason as to why this is the case. For reference my oligo file is:

primer NONE GCTGCCTCCCGTAGGAGT V1V2

Here is an example input sequence:

>DLJKDFLD_03460_16S_ribosomal_RNA|M1160910797
GACGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAGCGGAAAGGCCCTTCGGGGTACTCGAGCGGCGAACGGGTGAGTAACACGTGAGTAACCTGCCCCTGACTCTGGGATAAGCCTGGGAAACTGGGTCTAATACCGGATATGACACACGATGGCATCGTCTGTGTGTGGAAAGTTTTTTCGGTCAGGGATGGGCTCGCGGCCTATCAGCTTGTTGGTGGGGTAGTGGCCTACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCGCAATGGGCGGAAGCCTGACGCAGCGACGCCGCGTGGGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCGGCAGGGACGAAGTTGACGTGTACCTGTAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGTGGCTTGTCGCGTCTGCCGTGAAAGCCCATGGCTTAACTATGGGTCTGCGGTGGATACGGGCAGGCTAGAGGCTGGTAGGGGCAAGCGGAATTCCTGGTGTAGCGGTGAAATGCGCGGATATCAGGAGGAACACCGGTGGCGAAGGCGGCTTGCTGGGCCAGTTCTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGTTGGGCGCTAGGTGTGGGGTGCTTCCACGTGTCCCGTGCCGTAGCTAACGCATTAAGCGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTTGCTTAATTCGACGCAACGCGAAGAACCTTACCAAGGTTTGACATCACCCGGAAACGGCCAGAGATGGTCGCCTCTTCGGACTGGGTGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTCCATGTTGCCAGCAACCTCTTCGGAGGGTTGGGGACTCATGGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGTCTTGGGCTGCAAACATGCTACAATGGCCGGTACAGAGGGTTGCGATACCGTGAGGTGGAGCGAATCCCTAAAAGCCGGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCACGAAAGTCGGCAACACCCGAAGCCCGTGGCCCAACCAGCAATGGGGGGAGCGGTCGAAGGTGGGGCTGGCGATTGGGACG

And here is the matching output:

>DLJKDFLD_03460_16S_ribosomal_RNA|M1160910797   fpdiffs=0(match) rpdiffs=0(match)       
AACGCTGGCGGCGTGCTTAACACATGCAAGTCGAGCGGAAAGGCCCTTCGGGGTACTCGAGCGGCGAACGGGTGAGTAACACGTGAGTAACCTGCCCCTGACTCTGGGATAAGCCTGGGAAACTGGGTCTAATACCGGATATGACACACGATGGCATCGTCTGTGTGTGGAAAGTTTTTTCGGTCAGGGATGGGCTCGCGGCCTATCAGCTTGTTGGTGGGGTAGTGGCCTACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAG

On quick inspect you can see that the first 4 bases of the output seem to be missing. This seems constant across all sequences that were processed.

Cheers,
Jacob Nearing

2

Thanks Jacob - that is weird.

Can you try doing this instead?

reverse GCTGCCTCCCGTAGGAGT V1V2
3

Hi @pschloss,

Thanks for the reply appreciate it. Sorry for the slow response on my end. Only including the reverse primer as you indicated did seem to fix this issue (no longer cutting off the first 4 bases).

Perhaps there is some odd behavior when you include NONE as one of the primer options (maybe interpreting them as base characters?).

Thanks,
Jacob Nearing