Issue running preprocessing pipeline in MOP2

[luciabserra]

Hi,

I have been trying unsuccessfully to run the Master of Pores 2, mop_preprocess pipeline. I installed Docker and Guppy as mentioned in the installation doc. My input folder (2xpA-293T) includes several fast5 files. The FASTA reference file is in a separate folder as well as the GTTF annotation file. I defined my parameters in a command line:

./nextflow run /home/lucia/MOP2/mop_preprocess/mop_preprocess.nf -with-docker --fast5_files /home/lucia/MOP_rawdata/2xpA-293T/*/.fast5 --annotation /home/lucia/annotation/ --reference /home/lucia/reference/ --basecalling guppy --mapping minimap2 --count nanocount --filtering nanofilt --output /home/lucia/MOP_preprocessed/2xpA-293T/ > log.txt

However, the command is not working. I have no error message appearing but I have no output, so I think the command might not be going through all the fast5 files in the input folder. I tried multiple variations of this command line to see if it was a syntax error but the result is always the same. I also reinstalled MOP2. How can I fix this issue?

Here's the log.txt doc:

conffile. : final_summary_01.txt fast5 : /home/lucia/MOP2/mop_preprocess/../data/*/.fast5 fastq : reference : /home/lucia/reference/ annotation : /home/lucia/annotation/ granularity. : 1 ref_type : transcriptome pars_tools : drna_tool_splice_opt.tsv output : /home/lucia/MOP_preprocessed/2xpA-293T/ GPU : OFF basecalling : guppy demultiplexing : NO demulti_fast5 : NO filtering : nanofilt mapping : minimap2 counting : nanocount discovery : NO cram_conv : YES subsampling_cram : 50 saveSpace : NO email : lucacozzuto@crg.es /home/outdirs.nf (/home/outdirs.nf)

-- Check script 'MOP2/mop_preprocess/mop_preprocess.nf' at line: 69 or see '.nextflow.log' file for more details

Thank you!

[gkanogiannis]

I think that you have not correctly defined the annotation and reference. You need to give as parameter the actual file (gtf and fa) of annotation and reference and not just the folder they are in.

[lucacozzuto]

Yes please provide good annotation and reference.

[luciabserra]

Thank you for your help. I managed to resolve the issue. If anyone has the same problem, I altered the following parameters:

• I defined the reference and annotation files
• I used singularity instead of docker
• I provided my input path not in my command, but in the params.config file
• I used the profile standard (to control for memory used).

So at the end, my command was: /home/lucia/nextflow run mop_preprocess.nf -with-singularity -profile standard --annotation /home/lucia/annotation/gencode.v40.annotation.gtf --reference /home/lucia/reference/GRCh38.p13.genome.fa --basecalling guppy --mapping minimap2 --count nanocount --filtering nanofilt --output /home/lucia/MOP_preprocessed/

And my params.config file:

params { conffile = "final_summary_01.txt" fast5 = "/home/lucia/MOP_rawdata/*/.fast5" fastq = ""

reference           = "$baseDir/../anno/yeast_rRNA_ref.fa.gz"
annotation          = ""
ref_type            = "transcriptome"

pars_tools          = "drna_tool_splice_opt.tsv"
output              = "$baseDir/output"
qualityqc           = 5
granularity         = 1

basecalling         = "guppy"
GPU                 = "OFF"
demultiplexing      = "NO"
demulti_fast5       = "NO"

filtering           = "nanoq"

mapping             = "graphmap"
counting            = "nanocount"
discovery           = "NO"

cram_conv           = "YES"
subsampling_cram    = 50