Pre.cluster error

Good Morning,

I am using mothur to analyze my latest set of MiSeq data. We used V3 chem, which I realize causes issues with distance matrices and sequences not aligning correctly. However, I have been able to work through the MiSeq SOP up to the pre.cluster command. When I run pre.cluster it runs through a large portion of the sequences but eventually produces this error message:

Total number of sequences before pre.cluster was 10120.
pre.cluster removed 3917 sequences.

It took 10 secs to cluster 10120 sequences.
[ERROR]: seq M00780_85_0000TR-B-5 is not in your count table. Please correct.
[ERROR]: M00780_85_0000TR-B-5 is not in your count table. Please correct.

I have checked the count table but the sequence names provided in the error message are lacking a complete sequence name. Here is an example:

Sequence from count table: M00780_85_000000000-AF4Y6_1_1103_8140_12625
Sequence from error: M00780_85_0000TR-B-5

As you can see the error is not providing a full sequence name. When I look at the column associated with group TR-B-5 in the count file there is data for every sequence so I’m lost as to where the error is coming.

I have posted a link to the logfile if anyone has any advice or workarounds for this issue it would be greatly appreciated.

From the summary after the alignment it looks like you have some sequences that aligned poorly removing all the bases. Did you try setting the flip=t parameter?

Start End NBases Ambigs Polymer NumSeqs
Minimum: -1 -1 0 0 1 1

The error messages are starting here.

mothur > filter.seqs(fasta=FireStability.trim.contigs.good.unique.good.align, vertical=T, trump=.)

Using 2 processors.
Creating Filter…
[ERROR]: Sequences are not all the same length, please correct.

Running Filter...
The process that reports that error would have quit, and errors continue to be reported on the following commands. Can you try rerunning the filter.seqs command, but run it in debug mode. It looks like you have some bad seqs you need to remove.

mothur > set.dir(debug=t)
mothur > filter.seqs(fasta=FireStability.trim.contigs.good.unique.good.align, vertical=T, trump=.)

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