Customize coordinates for v4 region

Commands in mothur
1

Hello, I am customizing reference alignment for 16S v4 region. After I align the .fasta file to the silva.seed_v132.align file using “align.seqs” command, there was no .align file being generated and I can’t run the summary.seqs command. Can anyone help me with this issue?

2

Can you post the commands you are running and the names of the files that are being generated along with any warning/error messages?

Thanks,
Pat

3

Hi Dr. Schloss,

This is the my align command:
“align.seqs(fasta=Ecoli.v4.fasta, reference=silva.seed_v132.align)”
There is no .align file generated after this command, maybe something wrong with my v4.fasta file and I can post later.

Alternatively, I just use v4 coordinates 11895 and 25318 to trim the silva.seed_v132.align and it worked fine.
Two quick questions: Should I use the Silva full-length sequence references or the recreated seed database as the alignment reference? Can I use the HMP_MOCK.v35.fasta file to check my own sequencing errors?

Thanks for your time.

4

Hi,

  1. Were there any error messages when you ran align.seqs?

  2. I think the recreated seed should work as well as the non-redundant alignments (they both contain full length sequences)

  3. I doubt you sequenced the HMP Mock community. You would need to get your own fasta file with sequences for the 16S rRNA gene sequences in your mock comunity

Pat

5

Hi Dr. Schloss,

  1. When I ran align.seqs, nothing popped out .

  2. Yes, the recreated seed file worked fine for me now.

  3. I found my own mock file. However, when I used them as reference to run the error.seqs command, the following warning message popped out. Maybe I should screen the mock file to get rid of ambigs?

[WARNING]: We found more than 25% of the bases in sequence AA>1BCAFFAC11FEGFG?FGHCG?AEHHHHF1FCEGEGHGGEFEEEEGECGEC>ECDGGHHFFFGBFHHEG?GCEFGH?EGE<>?GFFGCGG/>@CGCFFHH1C1FGB11GFCGECCG0<0=0DGGFHCCGEHGHGHHGEHHHHHGHFEGFBFGG?.;EFB0.9AB-99////;9FFA–B?FFFF9BB/-@>-999=@@?FFFFFFBBFFFF//;///—?BF99AA–;-BFB–@-@=9A@9>@- to be ambiguous. Mothur is not setup to process protein sequences.
[WARNING]: 417 reference sequences have ambiguous bases, these bases will be ignored.

6

I suspect you haven’t correctly formatted your reference file.

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