Error in pcr.seqs and other errors and warnings

Commands in mothur
#1

Hi,

I have been trying to look at some new MiSeq data (16S rRNA V4 region) using the Mothur pipeline, however I have been getting back different warnings / errors despite having what I think are correctly formatted files (e.g. fastq, oligos etc.). So any help or suggestions would be really great.

So my Oligo file (with a few samples as an example) looks like this (the forward and reverse primers, and dual index barcodes were supplied by the sequencing centre):

forward NNNNNGTGCCAGCMGCCGCGGTAA
reverse GGACTACHVGGGTWTCTAAT
barcode TAAGGCGA TAGATCGC APM_C01_FOP_2014_001
barcode TAAGGCGA AGAGTAGA APM_C01_FOP_2014_004
barcode TAAGGCGA ACTGCATA APM_C01_FOP_2014_006

And my .files file looks like this:

APM_C01_FOP_2014_001 APM_C01_FOP_2014_001_R1.fastq APM_C01_FOP_2014_001_R2.fastq
APM_C01_FOP_2014_004 APM_C01_FOP_2014_004_R1.fastq APM_C01_FOP_2014_004_R2.fastq
APM_C01_FOP_2014_006 APM_C01_FOP_2014_006_R1.fastq APM_C01_FOP_2014_006_R2.fastq




The first problem, is that in the make.contigs command, I get many many warnings (although this does not stop me continuing on with the pipeline, and some files appear to be processed fine). e.g.

[WARNING]: did not find paired read for HWI-M02748_51_000000000-ACVB7_1_1101_10000_9863, ignoring.
[WARNING]: did not find paired read for HWI-M02748_51_000000000-ACVB7_1_1101_10001_18652, ignoring.
[WARNING]: did not find paired read for HWI-M02748_51_000000000-ACVB7_1_1101_10008_15512, ignoring.
[WARNING]: did not find paired read for HWI-M02748_51_000000000-ACVB7_1_1101_10008_2824, ignoring.
[WARNING]: did not find paired read for HWI-M02748_51_000000000-ACVB7_1_1101_10011_12798, ignoring.


Second, in the summary.seqs table the NBases appear to be way too small for the region (i.e. a minimum of 10):

Using 8 processors.

Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 10 10 0 1 1
2.5%-tile: 1 12 12 0 2 179723
25%-tile: 1 27 27 0 4 1797228
Median: 1 297 297 0 4 3594456
75%-tile: 1 297 297 0 4 5391684
97.5%-tile: 1 298 298 1 5 7009189
Maximum: 1 501 500 94 250 7188911
Mean: 1 203.522 203.522 0.0844707 3.90803

of Seqs: 7188911




Third, using pcr.seqs(fasta=silva.bacteria.fasta, oligos=bees_barn.oligos, processors=8) I get this error back:

[ERROR]: cannot mix paired primers and barcodes with non paired or linkers and spacers, quitting.

Cheers and many thanks for any thoughts or suggestions on these problems,

Julia

#2

Can you try this:

primer NNNNNGTGCCAGCMGCCGCGGTAA GGACTACHVGGGTWTCTAAT
barcode TAAGGCGA TAGATCGC APM_C01_FOP_2014_001
barcode TAAGGCGA AGAGTAGA APM_C01_FOP_2014_004
barcode TAAGGCGA ACTGCATA APM_C01_FOP_2014_006

When you have paired barcodes you need to use paired primers.

#3

Hi,
Many thanks for the suggestion.
I did try make.contigs with the oligo file adjusted as you suggest, however I still get many warnings… Do you know what else might be wrong?
Thanks again,
Julia