Error in pcr.seqs and other errors and warnings

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

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.

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