ref_genome parameter

[achang44]

Ask away!

Hello. I am attempting to run my workflow, but I need a ref_genome parameter. I need guidance on what the correct input is for this. I have a reference genome and am trying to analyze cDNA data. In addition, what is the correct input for nextflow to run pychopper first?

nextflow run epi2me-labs/wf-transcriptomes \ --fastq differential_expression_fastq \ --transcriptome-source precomputed \ --de_analysis \ --ref_genome burtoni/GCF_018398535.1_NCSU_Asbu1_genomic.fna\ --ref_annotation burtoni/genomic.gff \ --pychopper_opts '-k LSK114' \ --ref_transcriptome burtoni/rna.fna \ --sample_sheet induction_samplesheet.csv

[sarahjeeeze]

Hi @achang44 thanks for your query. What is the error message you get when running that command? To run pychopper you would set the the --direct_rna parameter to false, which is the default so you shouldn't need to change it.

[achang44]

Hi @sarahjeeeze ERROR ~ As transcriptome source is reference guided you must include a ref_genome parameter.

Got it. I will try that for pychopper.

[sarahjeeeze]

Hi, thanks. I'm not entirely sure why its not working but I think it might be to do with your newline placement, could you try without them eg.

nextflow run epi2me-labs/wf-transcriptomes --fastq  differential_expression_fastq  --transcriptome-source precomputed --de_analysis --ref_genome burtoni/GCF_018398535.1_NCSU_Asbu1_genomic.fna  --ref_annotation burtoni/genomic.gff   --pychopper_opts '-k LSK114'   --ref_transcriptome burtoni/rna.fna   --sample_sheet induction_samplesheet.csv

[achang44]

Yes, that seemed to work. I'm now getting this error:

ERROR ~ Error executing process > 'pipeline:preprocess_ref_annotation'

[sarahjeeeze]

Hi, is there any more error info in the .nextflow.log file, did you download the reference annotation from a public source? It should be in GFF2 or GFF3 format. If possible could you share the link to the public file or your annotations file so I could check we have handling for the format.

[achang44]

Hi - This is the error info from the nextflow.log: Caused by: Process pipeline:preprocess_ref_annotation terminated with an error exit status (1)

Command executed:

sed -i -e 's/transcript_id "";//g' genomic.gff mv genomic.gff "ammended.genomic.gff"

Command exit status: 1

Command output:

The command 'docker' could not be found in this WSL 2 distro. We recommend to activate the WSL integration in Docker Desktop settings.

For details about using Docker Desktop with WSL 2, visit:

https://docs.docker.com/go/wsl2/

Command error:

The command 'docker' could not be found in this WSL 2 distro. We recommend to activate the WSL integration in Docker Desktop settings.

For details about using Docker Desktop with WSL 2, visit:

Yes, I downloaded by reference annotation from NCBI https://www.ncbi.nlm.nih.gov/datasets/taxonomy/8153/ The file format listed is just GFF.

[sarahjeeeze]

Hi, It looks like you don't have docker in your environment. I suggest either following these instructions https://labs.epi2me.io/nextflow-on-windows/ or trying our EPI2ME application https://labs.epi2me.io/downloads/ to run this workflow locally on a windows environment.

[achang44]

Thank you! I was able to run my transcriptomes with the EPI2ME labs