Hi
I am using mothur v.1.47.0 on MacOS. I had 90 samples in total including the mock files as well. I have a few questions regarding the analysis.
- At this step when i ran the following command i got some warnings
mothur > classify.seqs(fasta=stability.trim.contigs.unique.good.filter.unique.precluster.denovo.vsearch.fasta, count=stability.trim.contigs.unique.good.filter.unique.precluster.denovo.vseach.count_table, reference=trainset9_032012.pds.fasta, taxonomy=trainset9_032012.pds.tax)
Using 8 processors.
Generating search database… DONE.
It took 6 seconds generate search database.
Reading in the /users/hiraabid/desktop/mothur/M06339_Run_192-Semrau_Soil_Microcosm/trainset9_032012.pds.tax taxonomy… DONE.
Calculating template taxonomy tree… DONE.
Calculating template probabilities… DONE.
It took 13 seconds get probabilities.
Classifying sequences from /users/hiraabid/desktop/mothur/M06339_Run_192-Semrau_Soil_Microcosm/stability.trim.contigs.unique.good.filter.unique.precluster.denovo.vsearch.fasta …
[WARNING]: M06339_192_000000000-KBLJK_1_1111_27355_17100 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M06339_192_000000000-KBLJK_1_2107_16345_28171 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M06339_192_000000000-KBLJK_1_1102_26823_10250 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M06339_192_000000000-KBLJK_1_2108_8133_24247 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M06339_192_000000000-KBLJK_1_1109_17267_18577 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M06339_192_000000000-KBLJK_1_1108_9142_3033 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M06339_192_000000000-KBLJK_1_1110_23375_19566 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M06339_192_000000000-KBLJK_1_2104_12019_12043 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M06339_192_000000000-KBLJK_1_2104_12545_20141 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M06339_192_000000000-KBLJK_1_1113_11938_14950 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
**** Exceeded maximum allowed command warnings, silencing warnings ****
[WARNING]: M06339_192_000000000-KBLJK_1_2107_21484_25899 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M06339_192_000000000-KBLJK_1_2101_19780_9518 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
A lot of warnings like these showed up. Someone please what these mean? I did run the remove.lineage command after this.
- And in the following command it says “Your file does NOT contain sequences from the groups you wish to get.”
mothur > get.groups(count=stability.trim.contigs.unique.good.filter.unique.precluster.denovo.vsearch.pick.count_table, fasta=stability.trim.contigs.unique.good.filter.unique.precluster.denvo.vsearch.pick.fasta, groups=Mock)
Your file does NOT contain sequences from the groups you wish to get.
Selected 0 sequences from your count file.
Your file does NOT contain sequences from the groups you wish to get.
Selected 0 sequences from your fasta file.
Output File names:
/users/hiraabid/desktop/mothur/M06339_Run_192-Semrau_Soil_Microcosm/stability.trim.contigs.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.fasta
The resulting “stability.trim.contigs.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.fasta” is empty.
- As in the above step get.groups command did not work. The following two commands were for accessing error rates:
i) get.groups(count=stability.trim.contigs.unique.good.filter.unique.precluster.denovo.vsearch.pick.count_table, fasta=stability.trim.contigs.unique.good.filter.unique.precluster.denovo.vsearch.pick.fasta, groups=Mock)
ii)
seq.error(fasta=stability.trim.contigs.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.fasta, count=stability.trim.contigs.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count_table, reference=zymo.mock_.16S_fasta.txt, aligned=F)
I skipped the above two because they produced empty output files. So I used the input fasta and count file given in the get.groups command for the next step. The next command i ran was as follows
dist.seqs(fasta=stability.trim.contigs.unique.good.filter.unique.precluster.denovo.vsearch.pick.fasta, cutoff=0.03)
This command is taking too long. its been four days and it has still not completed. I reduced the number of processors but it still doesn’t work. I also tried this on windows computer with 128GB ram and it still did not complete there as well. on windows its been two days now. Can someone tell me any solution. I need to generate an OTU table as soon as possible but it is taking too long.
I hope I was able to explain my questions clearly. I am using this software for the first time that’s why I got a lot of questions. Thanks.
Hira