MetONTIIME is giving blank output

Hello
I am trying to use MetONTIIME for analysing nanopore data.
Though, am not getting any error, the collapsed feature table is showing blank frequency tables.tsv and I am not getting any other output as well or progression of the analysis is not seen. All the files are appropriately labelled as BC–.fastq.gz in the files folder.
Following is the command I entered- nohup ./MetONTIIME.sh /home/mim/MetONTIIME/files /home/mim/MetONTIIME/covidmetadata.tsv /home/mim/MetONTIIME/silva_132_99_16S_sequence.qza /home/mim/MetONTIIME/silva_132_99_16S_taxonomy.qza 4 Vsearch
Also, do we have to prepare manifest files?It got generated, but it was blank again.
Kindly suggest me the possibilities as to why I am not getting any output.
Thank you

Hi @kkcool,

I never personally used this plug in, I just tagging @MaestSi , the developer, to see if he could give a hand on this.

Cheers

Hi! <max accepts>, <query coverage> and <id thr> parameters are missing!
Just try removing the empty manifest file that was generated and run, for example:

nohup ./MetONTIIME.sh /home/mim/MetONTIIME/files /home/mim/MetONTIIME/covidmetadata.tsv /home/mim/MetONTIIME/silva_132_99_16S_sequence.qza /home/mim/MetONTIIME/silva_132_99_16S_taxonomy.qza 4 Vsearch 3 0.8 0.85 &

Simone

Thank you so much. I had initially put the values.
A joining question. How do we decide these values for these parameters?

You can decide the values for those parameters based on the quality of your sequencing run and your need for higher sensitivity or precision.
For example, increasing the <id thr> will result in a higher number of “Unassigned” reads, and this value should be tuned based on base-caller version used and the expected error rate. Assuming about 5-10% error rate and a 3% intraspecies variability, for me a value in the range 0.85-0.90 seems reasonable.
A smaller value of <max accepts> (e.g. 1) will result in a higher number of sequences classified at species level, while increasing this value will result in more “confident” assignments, but frequently at a lower taxonomic level. Mind that all taxa surviving the <id thr> and <query coverage> are hits, and the top (up to) <max accepts> will be used for consensus taxonomy assignment (namely the read is assigned at the taxonomic level at which at least 50% of accepted hits agree).
<query coverage> doesn’t probably have a big impact, 0.8 (80%) seems a reasonable value to me, meaning that at least 80% of the read should be aligned to the hit.
Simone

Thank you so much for explaining so well.

Hello. The analysis stopped after I got sequences.qza file.
How do I proceed now? I was under the notion that we would be getting feature table and taxonomy table as well. Should I proceed with the conventional QIIME2 pipeline after this step or is there some error that I might be facing?

It’s difficult to answer without seeing the error. Could you please post the first lines of the nohup.out file?
P.s.: it may be better to open an Issue on the Github page.
Simone

Yes.sure. I will open an issue on Github. There is nothing in the nohup.out file though

You may have an empty nohup.out file if you forgot to put ‘&’ at the end of the command.
First, I would advise to remove all generated files with extension .qz*, and rerun the pipeline. Then, if it stops again, please open an Issue reporting the error.
Thanks,
Simone

Hello
I didn’t forget to put & after my command.
Also the only file that has got generated is sequences.qza
Should I delete that file?

Yes. I saw you opened an issue on Github, let’s continue the discussion there.
Simone