Good day,
Are there ARCTIC v4 amplicon info and v4 bed files to use for SARS-CoV-2 analysis. We recently started using the ARCTIC V4 primers. I had to try running the sequence data using the V3 primer set and bed on the SARS-CoV-2 workflow but it doesn’t generate any info.
This maybe?
Hi @Kathleen_Subramoney,
we’ve updated our shared covid-19 resources history on usegalaxy.eu a while ago to include more primer schemes. Galaxy | Europe | Published History | COVID-19 resources has ARTIC v4 primers (and amplicon info) and others since then.
Cheers,
Wolfgang
Thank you Wolfgang 
Yes this looks like it. Thank you.
Hi Wolfgang,
So I used the v4 arctic primers but when I ran the workflow on my fastq files the fasta file that was generated just comprised of N’s. I ran the same samples on genome detective and it works fine. I’m not sure what I am doing wrong because it worked well when needed to run the workflow using V3.
@Kathleen_Subramoney I guess I need a bit more details if you want me to debug this. All Ns in the consensus sequence means that along the way you lost all your genome coverage somehow and since it’s happening only with the new v4 primer scheme the ivar trim step sounds like a plausible candidate.
Look for empty (or nearly empty) BAM datasets generated as part of the workflow.
I’m afraid that’s all I can say without seeing how you invoked the workflow or even which workflow exactly.
Dear Wolfgang,
I am using the workflows as per attachments. I can take screenshots of how I invoke the workflow if that will be more useful, let me know.
The ivar trim step gives the following info:
Found 198 primers in BED file
No affected primer binding sites found! Found 198 primers in BED file Writing to removed_reads.bam Number of references: 1 Reference Name: NC_045512.2 Reference Length: 29903 Using Region: NC_045512.2 Sorted By Coordinate
Kind Regards
Kathleen
(Attachment Galaxy-Workflow-2)COVID-19__consensus_construction(imported_from_uploaded_file).ga is missing)
(Attachment Galaxy-Workflow-1)COVID-19__variation_analysis_on_ARTIC_PE_data_(imported_from_uploaded_file).ga is missing)
(Attachment Galaxy-Workflow-3)COVID-19__variation_analysis_reporting(imported_from_uploaded_file).ga is missing)
Sorry my attachments were rejected. These are the names of the workflows I am using for COVID.
Galaxy-Workflow-2)COVID-19__consensus_construction(imported_from_uploaded_file).ga
Galaxy-Workflow-1)COVID-19__variation_analysis_on_ARTIC_PE_data_(imported_from_uploaded_file).ga
Galaxy-Workflow-3)COVID-19__variation_analysis_reporting(imported_from_uploaded_file).ga
Hi Kathleen,
This doesn’t seem to be the output of ivar trim but of ivar removereads. What we would need is all the details of the ivar trim step, in particular, the input files and standard error and output.
Good day,
I am attempting to run fast files generated from MiNION platform. I wanted to find out if the “COVID-19: variation analysis of ARTIC ONT” is the correct workflow to use?
My aim is to generate an output that will provide the frequencies of each of the amino acids variations across the reads similar to the variant frequency generated from the Illumina workflow.
Kind Regards
Kathleen
Yes, that’s the right workflow. Make sure you’re using version 0.3 of it (or 0.3.1, which is functionally equivalent).
This workflow is compatible with the IWC variant reporting and consensus workflows (just like the Illumina one).
Keep in mind, that though well tested and running stably, the ONT WF probably has a lot more room for improvement on the analytical side. So feel encouraged to propose enhancements.
Best,
Wolfgang
Perfect. Thanks Wolfgang.
Kind Regards
Kathleen
Good day,
I am trying to upload my fast files via FTP. I downloaded FileZilla and entered the ftp.usegalaxy.eu and my login details and completed uploads as seen below. I went to galaxy.eu and selected “choose remote files” and selected the ftp directory for the first 3 fastq files as a test. The upload seemed to have failed (second screenshot below). The error is "Failed to fetch url gxftp://Illumina fastq/K018630_S2_L001_R2_001.fastq.gz. [Errno 2] No such file or directory: '/data/jwd/incoming/kathleen.subramoney@nhls.ac.za/Illumina fastq/K018630_S2_L001_R2_001.fastq.gz’
I selected fastqsanger.gz faster choosing the ftp files but it still does not recognise the files. Kindly assist if possible.
Kind Regards
Kathleen
(Attachment PastedGraphic-2.tiff is missing)
(Attachment PastedGraphic-1.tiff is missing)
Your screenshots are missing @Kathleen_Subramoney
The server is aware of your files it seems. What happens if, in the upload manager, you go into the “Illumina fastq” folder, select all in there, then upload? Do you again get that error? Or was this temporary?
Sorry about the screenshots. Galaxy emails seems to remove them after I send the email. I managed to resolve the issue. I tried this morning with more recent fastq files and seems to upload from FTP into galaxy perfectly. I think it was the format of the fastq that was incorrect. The extension was .fastq.gz but when I changed it to just .gz it worked.
Dear Wolfgang,
I used the “COVID-19: variation analysis of ARTIC ONT” workflow earlier in March just after our previous communication, and it worked well with no errors. I have been trying to run a few fastq files this past 2 weeks and keep getting the same error under adjusted variant calls within primer binding sites: "parameter ‘genome_file_opts_selector’: an invalid option (None) was selected, please verify”. I thought maybe it was my fastq input so a tried running a files that I ran previously that was successful but the same error occurs and the jobs afterwards are paused automatically and won’t continue even if I resume paused jobs.
I tried both 0.3 and 0.3.1 but the same error occurs. Is there an issue with the workflow?
Kind Regards
Kathleen
Hmm, could it be that you don’t have the right version of bedtools installed and the WF is silently falling back to an older version?
I use the same bed tools for the Illumina workflow and it works. I will double check if the ones I am using are the latest versions and try running the workflow again.