To start with fastq file

[watayako]

Dear Simone,

I want to ask for your help. I've tried to run my data on your pipeline MetONTIIME, but unfortunately, I haven't succeeded yet.

The latest error message that I got is followings:

(MetONTIIME_env) ayako@DESKTOP-B54P3SI:~$ nohup ./MetONTIIME.sh /home/ayako/nanopore/FAP89498_pass_barcode01_a8865088_bigfile.fastq.gz /home/ayako/nanopore/sample-metadata.txt /home/ayako/nanopore/silva-138-99-seqs.qza /home/ayako/nanopore/silva-138-99-tax.qza 5 Vsearch 3 0.8 0.85 & [1] 5254 (MetONTIIME_env) ayako@DESKTOP-B54P3SI:~$ nohup: ignoring input and appending output to 'nohup.out' nohup: failed to run command './MetONTIIME.sh': No such file or directory

So, could you walk me through how to analyze data starting from fastq files? I installed MetONTIIME and activated it , and then what is the next step?

I appreciate your help.

Best regards,

Ayako

[MaestSi]

Hi, based on this error: nohup: failed to run command './MetONTIIME.sh': No such file or directory it looks like you are not in the MetONTIIME directory. So, I think you should just cd to the directory where you downloaded the pipeline. Moreover, from the parameter: /home/ayako/nanopore/FAP89498_pass_barcode01_a8865088_bigfile.fastq.gz I can see you are specifying a file path, while you should only specify working directory as with: /home/ayako/nanopore/

Best, Simone

[watayako]

Dear Simone,

Thank you for the advice. Followed by your instruction, I could solve the problems.

But, I got another trouble. Phred quality score seems to be out of range. I think the MetONTIIME applied SingleEndFastqManifestPhred33V2. Have you met any similar troubles?

Find the attached file.

Best regards,

Ayako

[MaestSi]

Hi, the mean quality score looks like centered around 20, which may be a bit over-optimistic (that would correspond to a accuracy of 99%), but not absurd: with latest chemistries and base-callers you are expected to approach quite high accuracies. So, I don't think it is a matter of wrong PHRED offset, SingleEndFastqManifestPhred33V2 seems right to me. Based on the plot, it looks like the quality distribution has a lower variance for coordinates 1 to 1600 compared to the remaining part of the plot, is 1600 bp the expected amplicons size? Quality values at higher coordinates seem to be due to a lower number of reads, thus the higher variance. If this is the case, I suggest you to apply more stringent read length filtering, in order to discard non-specific amplification products. Moreover, I suggest you to check quality plots produced by pycoQC in the qc folder. Best, Simone

[MaestSi]

Hi, I am closing the issue due to inactivity. Feel free to reopen it, in case you have any other related issues. Simone

[watayako]

Sorry for the long silence.

I have not tried your advice yet, so I will post an issue when I get a problem.

Thanks a lot.

Ayako