I’m not sure if this is a bug as such, but I can’t figure out what’s causing my count_file to suddenly become blank after a remove.lineage command.
My samples were sequenced using Illumina but the reverse reads were pretty shoddy, so I decided to use only the forward reads for my Mothur analysis. The commands I used were as follows:
fastq.info(fastq=Sample1.fastq) (for each of my 50-something samples)
trim.seqs(fasta=Sample1.fasta, qfile=Sample1.qual, qwindowaverage=20, minlength=250)
merge.files(input=Sample1.trim.fasta-Sample2.trim.fasta-Sample3.trim.fasta- (plus many more samples), output=merge.fasta)
make.group(fasta=Sample1.trim.fasta-Sample2.trim.fasta-Sample3.trim.fasta- (plus many more samples), groups=1-2-3-etc-etc)
(merge.groups is generated)
(merge.names is generated)
(merge.count_table is generated)
classify.seqs(fasta=merge.unique.fasta,template=silva.nr_v132.align, taxonomy=silva.nr_v132.tax, count=merge.count_table, cutoff=80)
The resulting output files all look good - the reads have various taxonomic assignments i.e. they’re not all “unknown” or anything strange like that. And then I attempt to remove some of the sequences based on their taxonomic assignments and my count_table is suddenly blank:
mothur > remove.lineage(fasta=merge.unique.fasta, count=merge.count_table, taxonomy=merge.unique.nr_v132.wang.taxonomy, taxon=unknown-Eukaryota-Mitochondria-Chloroplast)
[NOTE]: The count file should contain only unique names, so mothur assumes your fasta, list and taxonomy files also contain only uniques.
[ERROR]: merge.pick.count_table is blank. Please correct.
had a look at the count table and it is very much blank - not so much as a header is present. I’ve been using Mothur for ages and I’ve never run into this problem before, but then I’ve never processed this many samples before either. Is there something I’m doing incorrectly? Or is it a matter of available memory or something like that? Any help/guidance would be hugely appreciated!