ERROR: Could not determine shovill version using '--version

[mabouelk]

Hi, I tried to install the nullarbor on python 3.7 but it says this conflict with the current python so I created an environment for it and activate it but upon check I got this error:

ERROR: Could not determine shovill version using '--version

I checked the shovill version separately and it is working "1.0.9"

[JamesZlosnik]

Hi,

I don't know if this helps, but I have this same error on my mac conda installation of nullarbor (own environment also). However, on a linux installation --check is working fine. I've yet not figured out what it is on the mac side that has caused it. Installed python on both is 3.6.7. Perl is 5.26.2 on both but obviously the mac and linux installs respectively. Shovill --version on its own works ok for me too.

James

[mabouelk]

Thank you so much James for your help, Please how I can install linux on mac? can I use virtualbox for that?

[JamesZlosnik]

Yes, I believe so for virtualbox. However I have not tried this - sorry I should have said that my linux installation is running on a linux node at out institution. Sorry to not be more help.

[peflanag]

Hi,

I just installed Nullabar on my iMac Pro in a conda environment. I am running macOS 10.15.3 I have gotten the same error.

When I type "shovill --version" into the shell it works but not when I do "nullabar.pl --check"

Is there a way of fixing it? Does it matter

Cheers,

P

[mabouelk]

exactly what I got, I am still waiting for Torsten Seemann's help.

[peflanag]

Cool I'll wait and see what @tseemann says and in the mean time see how I go about installing Linux on my Mac

[jb2cool]

The easiest way could likely to be use something like VirtualBox, VMware Fusion or Parallels and create a virtual machine on your Mac.

[peflanag]

Oh Thats cool. I thought I'd have to partition some of the SDD and boot up into a linux install that way which I really dont want to do cause all the files are on the macOS side and ideally if it works like parallels that I can access those files while running it would be great! wouldn't want copies on both partitions.

I'll have a google and read but ideally having Nullarbor work on macOS would be the best solution!

[peflanag]

So I installed parrelles and unbuntu on my iMac Pro and after installing Nullarbor I'm not getting the attached error. But when I cd into the directory the file is there! Any thoughts?

Cheers

[jb2cool]

what does ls -al show? I wonder what the file ownership is like.

[peflanag]

Cool, I've attached the screenshot. Not sure what it means.

[jb2cool]

Hmm, to me that looks ok, you are the owner and it has read access.

[peflanag]

I assume so? I just installed ubuntu into the parelles app and set up one account in my name

[jb2cool]

There is a typo in your environment variable, look at the dates. Your environment variable says 20171019 and the folder is really called 20171013

[peflanag]

How do I check on Linux if I am the owner and have read access?

[peflanag]

Oh, how do i change that? I literally just followed the commands on the install page by copy and paste into a shell

[jb2cool]

How do I check on Linux if I am the owner and have read access?

This is what la -al shows. The first bit shows the permission and whether read, write or execute is allowed. The next bit shows the owning user of the file. The third bit shows the owning group of the file.

Easiest for you would be to change the folder name to match the environment variable.

cd ~
mv minikraken_20171013_4GB minikraken_20171019_4GB

[peflanag]

Oh cool didnt know if i could just do that! I'll try now!

[jb2cool]

Yeah, the folder doesn't care what it's called, so long as the KRAKEN_DEFAULT_DB variable points to the folder it doesn't matter

[peflanag]

You're a legend!! Thank you so much that worked!

[mabouelk]

can you please tell me how to install ubuntu into the parelles app? as I did it using virtual box and I got the same errors.

[peflanag]

Virtual box just woudnet work for me. I just installed paralles and downloaded ubuntu .dgm file and when I started parelles pointed to the .dmg to install unbuntu and it worked

[jb2cool]

This should walk you through it. The virtualization platform shouldn't make a difference though, once Ubuntu is installed it shouldn't really matter how you installed it. The video is 5 years old but the process should be similar.

https://www.youtube.com/watch?v=S314TH48-ss

[mabouelk]

Thank you so much for both of you. I will try it right now, as I can't wait to see nullarbor working on my mac pro.

[peflanag]

Sorry, last question I hope! Im not sure how to run it! This is where I am stuck.

Cheers

[peflanag]

Hey so I seemed to have left it running over night in the linux parelles machine but Snippy seemed to fail this morning on all samples. Any idea what I should do? It mentions something about version but when I did --version I have the correct version. I'm at a loss!

[09:30:31] This is snippy 4.4.3 [09:30:31] Written by Torsten Seemann [09:30:31] Obtained from https://github.com/tseemann/snippy [09:30:31] Detected operating system: linux [09:30:31] Enabling bundled linux tools. [09:30:31] Found bwa - /home/peter/miniconda3/envs/nullarbor/bin/bwa [09:30:31] Found bcftools - /home/peter/miniconda3/envs/nullarbor/bin/bcftools [09:30:31] Found samtools - /home/peter/miniconda3/envs/nullarbor/bin/samtools [09:30:31] Found java - /home/peter/miniconda3/envs/nullarbor/bin/java [09:30:31] Found snpEff - /home/peter/miniconda3/envs/nullarbor/bin/snpEff [09:30:31] Found samclip - /home/peter/miniconda3/envs/nullarbor/bin/samclip [09:30:31] Found seqtk - /home/peter/miniconda3/envs/nullarbor/bin/seqtk [09:30:31] Found parallel - /home/peter/miniconda3/envs/nullarbor/bin/parallel [09:30:31] Found freebayes - /home/peter/miniconda3/envs/nullarbor/bin/freebayes [09:30:31] Found freebayes-parallel - /home/peter/miniconda3/envs/nullarbor/bin/freebayes-parallel [09:30:31] Found fasta_generate_regions.py - /home/peter/miniconda3/envs/nullarbor/bin/fasta_generate_regions.py [09:30:31] Found vcfstreamsort - /home/peter/miniconda3/envs/nullarbor/bin/vcfstreamsort [09:30:31] Found vcfuniq - /home/peter/miniconda3/envs/nullarbor/bin/vcfuniq [09:30:31] Found vcffirstheader - /home/peter/miniconda3/envs/nullarbor/bin/vcffirstheader [09:30:31] Found gzip - /bin/gzip [09:30:31] Found vt - /home/peter/miniconda3/envs/nullarbor/bin/vt [09:30:31] Found snippy-vcf_to_tab - /home/peter/miniconda3/envs/nullarbor/bin/snippy-vcf_to_tab [09:30:31] Found snippy-vcf_report - /home/peter/miniconda3/envs/nullarbor/bin/snippy-vcf_report [09:30:31] Need samtools --version >= 1.7 but you have 1.10 - please upgrade it. cp: cannot stat 'E6268/snippy/snps.tab': No such file or directory cp: cannot stat 'E6268/snippy/snps.aligned.fa': No such file or directory cp: cannot stat 'E6268/snippy/snps.raw.vcf': No such file or directory cp: cannot stat 'E6268/snippy/snps.vcf': No such file or directory cp: cannot stat 'E6268/snippy/snps.bam': No such file or directory cp: cannot stat 'E6268/snippy/snps.bam.bai': No such file or directory cp: cannot stat 'E6268/snippy/snps.log': No such file or directory snippy-core --ref ref.fa E6426 E6236 E6264 E6277 E6137 E6284 E6275 E6214 E6312 E6272 E6254 E6206 E6199 E6268 This is snippy-core 4.4.3 Obtained from http://github.com/tseemann/snippy Enabling bundled tools. Found any2fasta - /home/peter/miniconda3/envs/nullarbor/bin/any2fasta Found samtools - /home/peter/miniconda3/envs/nullarbor/bin/samtools Found minimap2 - /home/peter/miniconda3/envs/nullarbor/bin/minimap2 Found bedtools - /home/peter/miniconda3/envs/nullarbor/bin/bedtools Found snp-sites - /home/peter/miniconda3/envs/nullarbor/bin/snp-sites Saving reference FASTA: core.ref.fa This is any2fasta 0.4.2 Opening 'ref.fa' Detected FASTA format Read 48378 lines from 'ref.fa' Wrote 1 sequences from FASTA file. Processed 1 files. Done. Loaded 1 sequences totalling 2902619 bp. Will mask 0 regions totalling 0 bp ~ 0.00% ERROR: Could not find .aligned.fa/.vcf in E6426 make: *** [Makefile:108: core.aln] Error 2 make: Leaving directory '/home/peter/Documents/Nullarbor_Test/Output' (nullarbor) peter@ubuntu:~$ samtools --version samtools 1.10 Using htslib 1.10.2 Copyright (C) 2019 Genome Research Ltd. (nullarbor) peter@ubuntu:~$

[mabouelk]

any help, I installed the unbutu on virtual box and parellel and still have the same issue

[peflanag]

Hi @mabouelk I'm still waiting so cant help you. Im trying to install it on a college cluster to see if it works better than on my local iMac and parallels running ubuntu.

[peflanag]

Having said that, I've managed to install on ubuntu but it doesnt run the whole program. Files are missing when the program finishes.

[mabouelk]

Hi @peflanag I tried to install it on a college cluster and got the same issue. Thank you for your quick response and I HOPE somebody can help us.

[peflanag]

No worries, I have this tread open on one of my tabs waiting for a reply so I can sort it out! I am trying to compare programs to validate what is best going forward for our needs here!

Hopefully @tseemann will have an update for use soon!

[ajmerritt1]

Hi @peflanag I've watched you trying to get nullarbor running unable to provide any help but I may have some advice on your most recent problem. My experience with this fault is there may be a poor reference or a low quality, mis-identified or highly divergent sample that is nuking snippy.

1) are all your samples what you think they should be? Running kraken (or other metagenomics / k-mer ID eg kmerfinder) by itself can help with this. If something is mis- or poorly identified or contaminated remove it and run again.

2) do all the samples consist of high quality reads and sufficient coverage to the reference to make a good assembly. Some QC and read mapping can help with this. If any are poor remove them run again. 3) are all the samples a closeish match to the selected reference? If there any highly divergent ones remove them and try again. If the reference is too distant try with a closer one. Do you have any other typing info to check against? Already have MLST data ? If so are samples and ref same ST?

Hope this helps

[peflanag]

Hi @ajmerritt1 thanks for the reply. I don't know how to check these things individually so will have a play around and google. The sampels are what I think they are and the funny thing is when I run them a second time I get less of an output than I did the first time!

So for instance the first time I got a resistome and viruome in the main out put folder and second time I didn't . Just in the individual samples folders as posted in the screenshot here

https://github.com/tseemann/nullarbor/issues/87

I will run again with FastQC but as far as I remember they where fine the first time I checked.

[ajmerritt1]

Preview command in nullarbor is a useful tool to ID outliers. You can also brute force the problem by dropping samples until you find the problem one. When starting out with nullarbor I found it easier to delete result folders from failed analysis until i became familiar with the --force switch and make again command.

[peflanag]

Oh I didnt realise there was a preview command. And I assume the --force command means the program will run and any samples taht aren't outputted means that they where the outliers (poor quality/contamination/dont map to reference)?

So should my script be

nullarbor.pl --name PROJNAME --mlst saureus --ref Path/to/GenBank/file --input samples.tab --outdir OUTDIR make preview

[ajmerritt1]

Force in nullarbor.pl cmd line allows it to overwrite old files.

Re: preview Yesss..as long as

PROJNAME is a variable your supplying OUTDIR is a real path to where you want results to go.

I belive you run: nullarbor.pl --name PROJNAME --mlst saureus --ref Path/to/GenBank/file --input samples.tab --outdir OUTDIR Then cd to your OUTDIR then run Make preview

[ajmerritt1]

Force is just for rerunning the entire analysis. Again, I recommend just deleting the old folder. While you are experimenting/troubleshooting a smaller number of samples will run much faster.

[peflanag]

Ok, so i tried that and got the following output which I dont understand. There is still an error.

[11:18:33] Run the full pipeline with: [11:18:33] nice make all -j 3 -l 8 -C ../../media/psf/Home/IMRL_Sequencing/Tanya_MRSA/Nullarbor 2>&1 | tee -a ../../media/psf/Home/IMRL_Sequencing/Tanya_MRSA/Nullarbor/nullarbor.log [11:18:33] Done. (nullarbor) peter@ubuntu:~$ cd '/media/psf/Home/IMRL_Sequencing/Tanya_MRSA/Nullarbor' (nullarbor) peter@ubuntu:/media/psf/Home/IMRL_Sequencing/Tanya_MRSA/Nullarbor$ make preview ln -s -f '/media/psf/Home/IMRL_Sequencing/Tanya_MRSA/Fastq/E6284_S20_L001_R1_001.fastq.gz' 'E6284/R1.fq.gz' ln -s -f '/media/psf/Home/IMRL_Sequencing/Tanya_MRSA/Fastq/E6284_S20_L001_R2_001.fastq.gz' 'E6284/R2.fq.gz' mash sketch -m 5 -s 10000 -r -o E6284/sketch -I E6284 -C E6284/R1.fq.gz E6284/R1.fq.gz Estimated genome size: 2.71221e+06 Estimated coverage: 46.6812 Writing to E6284/sketch.msh... ln -s -f '/media/psf/Home/IMRL_Sequencing/Tanya_MRSA/Fastq/E6275_S19_L001_R1_001.fastq.gz' 'E6275/R1.fq.gz' ln -s -f '/media/psf/Home/IMRL_Sequencing/Tanya_MRSA/Fastq/E6275_S19_L001_R2_001.fastq.gz' 'E6275/R2.fq.gz' mash sketch -m 5 -s 10000 -r -o E6275/sketch -I E6275 -C E6275/R1.fq.gz E6275/R1.fq.gz Estimated genome size: 2.81214e+06 Estimated coverage: 34.9866 Writing to E6275/sketch.msh... ln -s -f '/media/psf/Home/IMRL_Sequencing/Tanya_MRSA/Fastq/E6242_S18_L001_R1_001.fastq.gz' 'E6264/R1.fq.gz' ln -s -f '/media/psf/Home/IMRL_Sequencing/Tanya_MRSA/Fastq/E6242_S18_L001_R2_001.fastq.gz' 'E6264/R2.fq.gz' mash sketch -m 5 -s 10000 -r -o E6264/sketch -I E6264 -C E6264/R1.fq.gz E6264/R1.fq.gz Estimated genome size: 2.75648e+06 Estimated coverage: 46.475 Writing to E6264/sketch.msh... ln -s -f '/media/psf/Home/IMRL_Sequencing/Tanya_MRSA/Fastq/E6199_S11_L001_R1_001.fastq.gz' 'E6199/R1.fq.gz' ln -s -f '/media/psf/Home/IMRL_Sequencing/Tanya_MRSA/Fastq/E6199_S11_L001_R2_001.fastq.gz' 'E6199/R2.fq.gz' mash sketch -m 5 -s 10000 -r -o E6199/sketch -I E6199 -C E6199/R1.fq.gz E6199/R1.fq.gz Estimated genome size: 2.71974e+06 Estimated coverage: 45.9941 Writing to E6199/sketch.msh... ln -s -f '/media/psf/Home/IMRL_Sequencing/Tanya_MRSA/Fastq/E6268_S17_L001_R1_001.fastq.gz' 'E6268/R1.fq.gz' ln -s -f '/media/psf/Home/IMRL_Sequencing/Tanya_MRSA/Fastq/E6268_S17_L001_R2_001.fastq.gz' 'E6268/R2.fq.gz' mash sketch -m 5 -s 10000 -r -o E6268/sketch -I E6268 -C E6268/R1.fq.gz E6268/R1.fq.gz Estimated genome size: 2.76646e+06 Estimated coverage: 60.7504 Writing to E6268/sketch.msh... ln -s -f '/media/psf/Home/IMRL_Sequencing/Tanya_MRSA/Fastq/E6312_S15_L001_R1_001.fastq.gz' 'E6312/R1.fq.gz' ln -s -f '/media/psf/Home/IMRL_Sequencing/Tanya_MRSA/Fastq/E6312_S15_L001_R2_001.fastq.gz' 'E6312/R2.fq.gz' mash sketch -m 5 -s 10000 -r -o E6312/sketch -I E6312 -C E6312/R1.fq.gz E6312/R1.fq.gz Estimated genome size: 2.77483e+06 Estimated coverage: 44.7048 Writing to E6312/sketch.msh... ln -s -f '/media/psf/Home/IMRL_Sequencing/Tanya_MRSA/Fastq/E6214_S22_L001_R1_001.fastq.gz' 'E6214/R1.fq.gz' ln -s -f '/media/psf/Home/IMRL_Sequencing/Tanya_MRSA/Fastq/E6214_S22_L001_R2_001.fastq.gz' 'E6214/R2.fq.gz' mash sketch -m 5 -s 10000 -r -o E6214/sketch -I E6214 -C E6214/R1.fq.gz E6214/R1.fq.gz Estimated genome size: 2.72746e+06 Estimated coverage: 46.2822 Writing to E6214/sketch.msh... ln -s -f '/media/psf/Home/IMRL_Sequencing/Tanya_MRSA/Fastq/E6426_S23_L001_R1_001.fastq.gz' 'E6426/R1.fq.gz' ln -s -f '/media/psf/Home/IMRL_Sequencing/Tanya_MRSA/Fastq/E6426_S23_L001_R2_001.fastq.gz' 'E6426/R2.fq.gz' mash sketch -m 5 -s 10000 -r -o E6426/sketch -I E6426 -C E6426/R1.fq.gz E6426/R1.fq.gz Estimated genome size: 2.76187e+06 Estimated coverage: 54.1224 Writing to E6426/sketch.msh... ln -s -f '/media/psf/Home/IMRL_Sequencing/Tanya_MRSA/Fastq/E6236_S13_L001_R1_001.fastq.gz' 'E6236/R1.fq.gz' ln -s -f '/media/psf/Home/IMRL_Sequencing/Tanya_MRSA/Fastq/E6236_S13_L001_R2_001.fastq.gz' 'E6236/R2.fq.gz' mash sketch -m 5 -s 10000 -r -o E6236/sketch -I E6236 -C E6236/R1.fq.gz E6236/R1.fq.gz Estimated genome size: 2.78533e+06 Estimated coverage: 46.9005 Writing to E6236/sketch.msh... ln -s -f '/media/psf/Home/IMRL_Sequencing/Tanya_MRSA/Fastq/E6137_S12_L001_R1_001.fastq.gz' 'E6137/R1.fq.gz' ln -s -f '/media/psf/Home/IMRL_Sequencing/Tanya_MRSA/Fastq/E6137_S12_L001_R2_001.fastq.gz' 'E6137/R2.fq.gz' mash sketch -m 5 -s 10000 -r -o E6137/sketch -I E6137 -C E6137/R1.fq.gz E6137/R1.fq.gz Estimated genome size: 2.77586e+06 Estimated coverage: 52.1383 Writing to E6137/sketch.msh... ln -s -f '/media/psf/Home/IMRL_Sequencing/Tanya_MRSA/Fastq/E6206_S21_L001_R1_001.fastq.gz' 'E6206/R1.fq.gz' ln -s -f '/media/psf/Home/IMRL_Sequencing/Tanya_MRSA/Fastq/E6206_S21_L001_R2_001.fastq.gz' 'E6206/R2.fq.gz' mash sketch -m 5 -s 10000 -r -o E6206/sketch -I E6206 -C E6206/R1.fq.gz E6206/R1.fq.gz Estimated genome size: 2.76646e+06 Estimated coverage: 31.4446 Writing to E6206/sketch.msh... ln -s -f '/media/psf/Home/IMRL_Sequencing/Tanya_MRSA/Fastq/E6277_S24_L001_R1_001.fastq.gz' 'E6277/R1.fq.gz' ln -s -f '/media/psf/Home/IMRL_Sequencing/Tanya_MRSA/Fastq/E6277_S24_L001_R2_001.fastq.gz' 'E6277/R2.fq.gz' mash sketch -m 5 -s 10000 -r -o E6277/sketch -I E6277 -C E6277/R1.fq.gz E6277/R1.fq.gz Estimated genome size: 2.71965e+06 Estimated coverage: 62.5908 Writing to E6277/sketch.msh... ln -s -f '/media/psf/Home/IMRL_Sequencing/Tanya_MRSA/Fastq/E6254_S16_L001_R1_001.fastq.gz' 'E6254/R1.fq.gz' ln -s -f '/media/psf/Home/IMRL_Sequencing/Tanya_MRSA/Fastq/E6254_S16_L001_R2_001.fastq.gz' 'E6254/R2.fq.gz' mash sketch -m 5 -s 10000 -r -o E6254/sketch -I E6254 -C E6254/R1.fq.gz E6254/R1.fq.gz Estimated genome size: 2.72897e+06 Estimated coverage: 54.2187 Writing to E6254/sketch.msh... ln -s -f '/media/psf/Home/IMRL_Sequencing/Tanya_MRSA/Fastq/E6272_S14_L001_R1_001.fastq.gz' 'E6272/R1.fq.gz' ln -s -f '/media/psf/Home/IMRL_Sequencing/Tanya_MRSA/Fastq/E6272_S14_L001_R2_001.fastq.gz' 'E6272/R2.fq.gz' mash sketch -m 5 -s 10000 -r -o E6272/sketch -I E6272 -C E6272/R1.fq.gz E6272/R1.fq.gz Estimated genome size: 2.78215e+06 Estimated coverage: 47.6832 Writing to E6272/sketch.msh... mash paste preview E6284/sketch.msh E6275/sketch.msh E6264/sketch.msh E6199/sketch.msh E6268/sketch.msh E6312/sketch.msh E6214/sketch.msh E6426/sketch.msh E6236/sketch.msh E6137/sketch.msh E6206/sketch.msh E6277/sketch.msh E6254/sketch.msh E6272/sketch.msh Writing preview.msh... cat isolates.txt | wc -l > preview.mat mash dist -p 4 -t preview.msh preview.msh | grep -v '^#' | sed 's/\/R1.fq.gz//' >> preview.mat quicktree -in m -out t preview.mat | nw_order -c n - > preview.nwk nw_display -S -s -w 1024 -l 'font-size:12;font-family:sans-serif;' -i 'opacity:0' -b 'opacity:0' -v 16 preview.nwk > preview.svg fq --ref ref.fa E6284/R1.fq.gz E6284/R2.fq.gz > E6284/yield.tab [fq] running command: cat E6284/R1.fq.gz E6284/R2.fq.gz | seqtk fqchk -q0 - [fq] processed 1209298 reads. [fq] calculating depth, using size (via --ref ref.fa) Illegal division by zero at /home/peter/miniconda3/envs/nullarbor/bin/fq line 67, <$IN> line 254. make: [Makefile:136: E6284/yield.tab] Error 2 make: Deleting file 'E6284/yield.tab' (nullarbor) peter@ubuntu:/media/psf/Home/IMRL_Sequencing/Tanya_MRSA/Nullarbor$

[ajmerritt1]

Mmm perhaps try take E6284 out and run again? Interested to see if div 0 error changes to another sample.

[peflanag]

Cool, I am trying that now!

[peflanag]

yup, seems to have failed again!

Writing preview.msh... cat isolates.txt | wc -l > preview.mat mash dist -p 4 -t preview.msh preview.msh | grep -v '^#' | sed 's/\/R1.fq.gz//' >> preview.mat quicktree -in m -out t preview.mat | nw_order -c n - > preview.nwk nw_display -S -s -w 1024 -l 'font-size:12;font-family:sans-serif;' -i 'opacity:0' -b 'opacity:0' -v 16 preview.nwk > preview.svg fq --ref ref.fa E6206/R1.fq.gz E6206/R2.fq.gz > E6206/yield.tab [fq] running command: cat E6206/R1.fq.gz E6206/R2.fq.gz | seqtk fqchk -q0 - [fq] processed 785666 reads. [fq] calculating depth, using size (via --ref ref.fa) Illegal division by zero at /home/peter/miniconda3/envs/nullarbor/bin/fq line 67, <$IN> line 254. make: [Makefile:136: E6206/yield.tab] Error 2 make: Deleting file 'E6206/yield.tab' (nullarbor) peter@ubuntu:/media/psf/Home/IMRL_Sequencing/Tanya_MRSA/Nullarbor$

[ajmerritt1]

See https://github.com/tseemann/nullarbor/issues/244 Div 0 error at fq 67 was reported as missing ref.fa file in OUTDIR. A workaround was suggested.

[peflanag]

Hey @ajmerritt1 that seems to have worked. This is the output on the shell and the files in the OUTDIR.

[09:44:40] Hello peter [09:44:40] This is nullarbor-report.pl 2.0.20191013 [09:44:40] Send complaints to Torsten Seemann [09:44:40] Making folder --outdir /media/psf/Home/IMRL_Sequencing/Tanya_MRSA/Nullarbor/report [09:44:40] Identified 13 isolates. [09:44:40] Generating: jobinfo [09:44:40] Generating: mashtree [09:44:40] Generating: seqdata [09:44:40] Generating: mashdist [09:44:40] Generating: about [09:44:40] Results in: /media/psf/Home/IMRL_Sequencing/Tanya_MRSA/Nullarbor/report/index.html [09:44:40] Done. (nullarbor) peter@ubuntu:/media/psf/Home/IMRL_Sequencing/Tanya_MRSA/Nullarbor$

So I assume that is all correct. Now do I just run the command

nice make all -j 3 -l 8 -C ../../media/psf/Home/IMRL_Sequencing/Tanya_MRSA/Nullarbor 2>&1 | tee -a ../../media/psf/Home/IMRL_Sequencing/Tanya_MRSA/Nullarbor/nullarbor.log

While in the OUTDIR?

[peflanag]

hey @ajmerritt1 so I've another update. I tried the nice make etc from my last post and didnt work. But since the preview pumped out whats in my image above I deleted the folder and ran nullarbor without the preview mode and it failed. Which I thought was odd if the preview worked?

Rather than paste the whole shell I'm just pasting the end of it

[14:36:12] Fixed 2 duplicate /gene - hlb_1 hlb_2 [14:36:12] Fixed 2 duplicate /gene - rluD_1 rluD_2 [14:36:12] Fixed 2 duplicate /gene - ssl7_1 ssl7_2 [14:36:12] Fixed 2 duplicate /gene - catE_1 catE_2 [14:36:12] Fixed 2 duplicate /gene - pepA_1 pepA_2 [14:36:12] Fixed 2 duplicate /gene - tcyC_1 tcyC_2 [14:36:12] Fixed 2 duplicate /gene - yciC_1 yciC_2 [14:36:12] Fixed 2 duplicate /gene - ywqD_1 ywqD_2 [14:36:12] Fixed 2 duplicate /gene - cap8A_1 cap8A_2 [14:36:12] Fixed 2 duplicate /gene - pepF1_1 pepF1_2 [14:36:12] Fixed 2 duplicate /gene - cspA_1 cspA_2 [14:36:12] Fixed 3 duplicate /gene - bceB_1 bceB_2 bceB_3 [14:36:12] Fixed 101 colliding /gene names. [14:36:12] Adding /locus_tag identifiers [14:36:12] Assigned 2556 locus_tags to CDS and RNA features. [14:36:12] Writing outputs to /tmp/tmp.9F7csFXU3T/ [14:36:12] Could not run command: tbl2asn -V b -a r10k -l paired-ends -M n -N 1 -y 'Annotated using prokka 1.14.5 from https://github.com/tseemann/prokka' -Z \/tmp\/tmp.g7KyjjsKrS\/prokka.err -i \/tmp\/tmp.g7KyjjsKrS\/prokka.fsa 2> /dev/null make: [Makefile:130: E6254/contigs.gff] Error 2 [14:36:12] Generating annotation statistics file [14:36:12] Generating Genbank and Sequin files [14:36:12] Running: tbl2asn -V b -a r10k -l paired-ends -M n -N 1 -y 'Annotated using prokka 1.14.5 from https://github.com/tseemann/prokka' -Z \/tmp\/tmp.9F7csFXU3T\/prokka.err -i \/tmp\/tmp.9F7csFXU3T\/prokka.fsa 2> /dev/null [14:36:14] Could not run command: tbl2asn -V b -a r10k -l paired-ends -M n -N 1 -y 'Annotated using prokka 1.14.5 from https://github.com/tseemann/prokka' -Z \/tmp\/tmp.eb7Bf5M5Rt\/prokka.err -i \/tmp\/tmp.eb7Bf5M5Rt\/prokka.fsa 2> /dev/null make: [Makefile:130: E6277/contigs.gff] Error 2 [14:36:16] Could not run command: tbl2asn -V b -a r10k -l paired-ends -M n -N 1 -y 'Annotated using prokka 1.14.5 from https://github.com/tseemann/prokka' -Z \/tmp\/tmp.9F7csFXU3T\/prokka.err -i \/tmp\/tmp.9F7csFXU3T\/prokka.fsa 2> /dev/null make: *** [Makefile:130: E6264/contigs.gff] Error 2 (nullarbor) peter@ubuntu:/media/psf/Home/IMRL_Sequencing/Tanya_MRSA/Nullarbor_Output$

So I decided to try make preview right after the fail and it ran generating the files seen in the image in my last post. But there is no SNP matrix or resitome or virulome reports. I am still at a lost to what it is that I am doing wrong. The quality of the coverage appears to be all over 50X according to the report.

Cheers,

Peter

[tseemann]

@peflanag tbl2asn expired (not my software so I can't control it). you need to upgrade Prokka or replace tbl2asn from NCBI FTP site to 25.8 version.

[peflanag]

Hi @tseemann cheers for the reply. I updated prokka and it is now running on version 1.14.6 and I also updated tbl2asn but there was no version 25.8 it was just 25.7

I ran nullarbor again but I've yet to get a report output folder or ristome and virulome results. I have pasted the end of my shell below. Maybe there is something I am missing in it?

[09:54:02] Fixed 2 duplicate /gene - bsaA_1 bsaA_2 [09:54:02] Fixed 2 duplicate /gene - aldC_1 aldC_2 [09:54:02] Fixed 2 duplicate /gene - ywqN_1 ywqN_2 [09:54:02] Fixed 2 duplicate /gene - nhaK_1 nhaK_2 [09:54:02] Fixed 2 duplicate /gene - mleN_1 mleN_2 [09:54:02] Fixed 2 duplicate /gene - mecA_1 mecA_2 [09:54:02] Fixed 2 duplicate /gene - aroA_1 aroA_2 [09:54:02] Fixed 2 duplicate /gene - lysP_1 lysP_2 [09:54:02] Fixed 2 duplicate /gene - cap8A_1 cap8A_2 [09:54:02] Fixed 2 duplicate /gene - entA_1 entA_2 [09:54:02] Fixed 3 duplicate /gene - brnQ_1 brnQ_2 brnQ_3 [09:54:02] Fixed 2 duplicate /gene - ftsK_1 ftsK_2 [09:54:02] Fixed 2 duplicate /gene - cls_1 cls_2 [09:54:02] Fixed 2 duplicate /gene - lipA_1 lipA_2 [09:54:02] Fixed 101 colliding /gene names. [09:54:02] Adding /locus_tag identifiers [09:54:02] Assigned 2585 locus_tags to CDS and RNA features. [09:54:02] Writing outputs to /tmp/tmp.ygBPTMStYh/ [09:54:02] Generating annotation statistics file [09:54:02] Generating Genbank and Sequin files [09:54:02] Running: tbl2asn -V b -a r10k -l paired-ends -M n -N 1 -y 'Annotated using prokka 1.14.6 from https://github.com/tseemann/prokka' -Z \/tmp\/tmp.ygBPTMStYh\/prokka.err -i \/tmp\/tmp.ygBPTMStYh\/prokka.fsa 2> /dev/null [tbl2asn-forever] Found Prokka input, correcting dates in .gbf|.sqn files. [tbl2asn-forever] Correcting dates in /tmp/tmp.BMJnGM0z1O/prokka.gbf [tbl2asn-forever] Correcting dates in /tmp/tmp.BMJnGM0z1O/prokka.sqn [tbl2asn-forever] Dates changed from 01-JAN-2019 to 24-FEB-2020 [09:54:04] Deleting unwanted file: /tmp/tmp.BMJnGM0z1O/errorsummary.val [09:54:04] Deleting unwanted file: /tmp/tmp.BMJnGM0z1O/prokka.dr [09:54:04] Deleting unwanted file: /tmp/tmp.BMJnGM0z1O/prokka.fixedproducts [09:54:04] Deleting unwanted file: /tmp/tmp.BMJnGM0z1O/prokka.ecn [09:54:04] Deleting unwanted file: /tmp/tmp.BMJnGM0z1O/prokka.val [09:54:04] Repairing broken .GBK output that tbl2asn produces... [09:54:04] Running: sed 's/COORDINATES: profile/COORDINATES:profile/' < \/tmp\/tmp.BMJnGM0z1O\/prokka.gbf > \/tmp\/tmp.BMJnGM0z1O\/prokka.gbk [09:54:04] Deleting unwanted file: /tmp/tmp.BMJnGM0z1O/prokka.gbf [09:54:04] Output files: [09:54:04] /tmp/tmp.BMJnGM0z1O/prokka.tsv [09:54:04] /tmp/tmp.BMJnGM0z1O/prokka.gff [09:54:04] /tmp/tmp.BMJnGM0z1O/prokka.fna [09:54:04] /tmp/tmp.BMJnGM0z1O/prokka.faa [09:54:04] /tmp/tmp.BMJnGM0z1O/prokka.log [09:54:04] /tmp/tmp.BMJnGM0z1O/prokka.fsa [09:54:04] /tmp/tmp.BMJnGM0z1O/prokka.txt [09:54:04] /tmp/tmp.BMJnGM0z1O/prokka.tbl [09:54:04] /tmp/tmp.BMJnGM0z1O/prokka.sqn [09:54:04] /tmp/tmp.BMJnGM0z1O/prokka.ffn [09:54:04] /tmp/tmp.BMJnGM0z1O/prokka.err [09:54:04] /tmp/tmp.BMJnGM0z1O/prokka.gbk [09:54:04] Annotation finished successfully. [09:54:04] Walltime used: 0.93 minutes [09:54:04] If you use this result please cite the Prokka paper: [09:54:04] Seemann T (2014) Prokka: rapid prokaryotic genome annotation. Bioinformatics. 30(14):2068-9. [09:54:04] Type 'prokka --citation' for more details. [09:54:04] Thank you, come again. '/tmp/tmp.BMJnGM0z1O/prokka.gff' -> 'E6214/contigs.gff' '/tmp/tmp.BMJnGM0z1O/prokka.gbk' -> 'E6214/contigs.gbk' removed '/tmp/tmp.BMJnGM0z1O/prokka.tsv' removed '/tmp/tmp.BMJnGM0z1O/prokka.gff' removed '/tmp/tmp.BMJnGM0z1O/prokka.fna' removed '/tmp/tmp.BMJnGM0z1O/prokka.faa' removed '/tmp/tmp.BMJnGM0z1O/prokka.log' removed '/tmp/tmp.BMJnGM0z1O/prokka.fsa' removed '/tmp/tmp.BMJnGM0z1O/prokka.txt' removed '/tmp/tmp.BMJnGM0z1O/prokka.tbl' removed '/tmp/tmp.BMJnGM0z1O/prokka.sqn' removed '/tmp/tmp.BMJnGM0z1O/prokka.ffn' removed '/tmp/tmp.BMJnGM0z1O/prokka.err' removed '/tmp/tmp.BMJnGM0z1O/prokka.gbk' removed directory '/tmp/tmp.BMJnGM0z1O' [tbl2asn-forever] Found Prokka input, correcting dates in .gbf|.sqn files. [tbl2asn-forever] Correcting dates in /tmp/tmp.ygBPTMStYh/prokka.gbf [tbl2asn-forever] Correcting dates in /tmp/tmp.ygBPTMStYh/prokka.sqn [tbl2asn-forever] Dates changed from 01-JAN-2019 to 24-FEB-2020 [09:54:06] Deleting unwanted file: /tmp/tmp.ygBPTMStYh/errorsummary.val [09:54:06] Deleting unwanted file: /tmp/tmp.ygBPTMStYh/prokka.dr [09:54:06] Deleting unwanted file: /tmp/tmp.ygBPTMStYh/prokka.fixedproducts [09:54:06] Deleting unwanted file: /tmp/tmp.ygBPTMStYh/prokka.ecn [09:54:06] Deleting unwanted file: /tmp/tmp.ygBPTMStYh/prokka.val [09:54:06] Repairing broken .GBK output that tbl2asn produces... [09:54:06] Running: sed 's/COORDINATES: profile/COORDINATES:profile/' < \/tmp\/tmp.ygBPTMStYh\/prokka.gbf > \/tmp\/tmp.ygBPTMStYh\/prokka.gbk [09:54:06] Deleting unwanted file: /tmp/tmp.ygBPTMStYh/prokka.gbf [09:54:06] Output files: [09:54:06] /tmp/tmp.ygBPTMStYh/prokka.tsv [09:54:06] /tmp/tmp.ygBPTMStYh/prokka.gff [09:54:06] /tmp/tmp.ygBPTMStYh/prokka.fna [09:54:06] /tmp/tmp.ygBPTMStYh/prokka.faa [09:54:06] /tmp/tmp.ygBPTMStYh/prokka.log [09:54:06] /tmp/tmp.ygBPTMStYh/prokka.fsa [09:54:06] /tmp/tmp.ygBPTMStYh/prokka.txt [09:54:06] /tmp/tmp.ygBPTMStYh/prokka.tbl [09:54:06] /tmp/tmp.ygBPTMStYh/prokka.sqn [09:54:06] /tmp/tmp.ygBPTMStYh/prokka.ffn [09:54:06] /tmp/tmp.ygBPTMStYh/prokka.err [09:54:06] /tmp/tmp.ygBPTMStYh/prokka.gbk [09:54:06] Annotation finished successfully. [09:54:06] Walltime used: 0.93 minutes [09:54:06] If you use this result please cite the Prokka paper: [09:54:06] Seemann T (2014) Prokka: rapid prokaryotic genome annotation. Bioinformatics. 30(14):2068-9. [09:54:06] Type 'prokka --citation' for more details. [09:54:06] Thank you, come again. '/tmp/tmp.ygBPTMStYh/prokka.gff' -> 'E6268/contigs.gff' '/tmp/tmp.ygBPTMStYh/prokka.gbk' -> 'E6268/contigs.gbk' removed '/tmp/tmp.ygBPTMStYh/prokka.tsv' removed '/tmp/tmp.ygBPTMStYh/prokka.gff' removed '/tmp/tmp.ygBPTMStYh/prokka.fna' removed '/tmp/tmp.ygBPTMStYh/prokka.faa' removed '/tmp/tmp.ygBPTMStYh/prokka.log' removed '/tmp/tmp.ygBPTMStYh/prokka.fsa' removed '/tmp/tmp.ygBPTMStYh/prokka.txt' removed '/tmp/tmp.ygBPTMStYh/prokka.tbl' removed '/tmp/tmp.ygBPTMStYh/prokka.sqn' removed '/tmp/tmp.ygBPTMStYh/prokka.ffn' removed '/tmp/tmp.ygBPTMStYh/prokka.err' removed '/tmp/tmp.ygBPTMStYh/prokka.gbk' removed directory '/tmp/tmp.ygBPTMStYh' (nullarbor) peter@ubuntu:/media/psf/Home/IMRL_Sequencing/Tanya_MRSA/Nullarbor_Output$ prokka --version prokka 1.14.6 (nullarbor) peter@ubuntu:/media/psf/Home/IMRL_Sequencing/Tanya_MRSA/Nullarbor_Output$ tbl2asn -v [real-tbl2asn] You must supply either an input file (-i) or an input directory (-p). Use -p . to specify the current directory.

Hit Return
(nullarbor) peter@ubuntu:/media/psf/Home/IMRL_Sequencing/Tanya_MRSA/Nullarbor_Output$

[tseemann]

type make -C /path/to/nulla/folder again and then re-run the 'again' rule will delete cruft and try and continue on properly. type make help for other options.

[peflanag]

Just to note I managed to solve all issues by uninstalling the nullarbor env and reinstalling nullarbor from scratch in the (base) shell rather than an env. Works fine in the Linux OS running in Parallels Desktop.

However, running on macOS the same error still persists where it cannot determine shovill version using --version

I am assuming this will be addressed in a future update?

[peflanag]

type make -C /path/to/nulla/folder again and then re-run the 'again' rule will delete cruft and try and continue on properly. type make help for other options.

Thanks, i never got notification of this till I posted above!

[mabouelk]

@peflanag I am so happy for you that you was able to run nullarbor successfully and get output. I managed to get startup allocation on our institution cluster and installed that on centos 7 and the installation worked fine with no issue but I got the same problem like you that there is no output. Can you tell me what you have done to make it work and get output. BTW, I installed it in the shell not in an env.