hi all mewhiq the pipeline is pretty much completed, besides some todo comments to be changed. I have tested with test config, test data also uploaded to nf-core/test_datasets. Also ran 'nf-core pipelines lint' without any failed test. Should I follow the instruction here (https://nf-co.re/docs/tutorials/adding_a_pipeline/move_to_nf-core_org) and transfer the ownership of the pipeline to nf-core?

I realised there were some caching issues due to the way how I pipe between the subworkflows. I changed the scripts now and have merged this into the

dev

branch in nf-core

would anyone be able to review my pipeline? mewhiq

Hi! I think we are pretty close to our first release. In terms of the test dataset, I have 3 samples, (homo sapiens - ONT) and (arabidopsis - ONT and PacBio) as the minimal test dataset. Is this sufficient for the test?

I noticed that the pseudo PR is from

dev

to

TEMPLATE

, in this case the tests were not trigger, should I open another PR from

dev

to

master

?

Helloo, my CI tests failed and I was wondering if this is due to some docker permissions error? the below is the error shown: permission denied

Run nextflow run ${GITHUB_WORKSPACE} -profile test,docker docker --outdir ./results
N E X T F L O W  ~  version 25.02.3-edge
Launching `/home/runner/work/methylong/methylong/main.nf` [disturbed_khorana] DSL2 - revision: cccd09db65
Downloading plugin nf-schema@2.1.1

------------------------------------------------------
                                        ,--./,-.
        ___     __   __   __   ___     /,-._.--~'
  |\ | |__  __ /  ` /  \ |__) |__         }  {
  | \| |       \__, \__/ |  \ |___     \`-._,-`-,
                                        `._,._,'
  nf-core/methylong 1.0.0
------------------------------------------------------
Input/output options
  input                     : <https://raw.githubusercontent.com/nf-core/test-datasets/refs/heads/methylong/test_data/test_samplesheet.csv>
  outdir                    : ./results

Institutional config options
  config_profile_name       : Test profile
  config_profile_description: Minimal test dataset to check pipeline function

Core Nextflow options
  runName                   : disturbed_khorana
  containerEngine           : docker
  launchDir                 : /home/runner/work/methylong/methylong
  workDir                   : /home/runner/work/methylong/methylong/work
  projectDir                : /home/runner/work/methylong/methylong
  userName                  : runner
  profile                   : test,docker
  configFiles               : 

!! Only displaying parameters that differ from the pipeline defaults !!
------------------------------------------------------
* The nf-core framework
    <https://doi.org/10.1038/s41587-020-0439-x>

* Software dependencies
    <https://github.com/nf-core/methylong/blob/master/CITATIONS.md>

WARN: nf-core pipelines do not accept positional arguments. The positional argument `docker` has been detected.
HINT: A common mistake is to provide multiple values separated by spaces e.g. `-profile test, docker`.

WARN: The `when` process section is deprecated -- use conditional logic in the calling workflow instead
WARN: Directive `process.shell` cannot contain new-line characters - offending value: [bash

set -e # Exit if a tool returns a non-zero status/exit code
set -u # Treat unset variables and parameters as an error
set -o pipefail # Returns the status of the last command to exit with a non-zero status or zero if all successfully execute
set -C # No clobber - prevent output redirection from overwriting files.
]
[da/36deee] Submitted process > NFCORE_METHYLONG:METHYLONG:FASTQ_UNZIP:GUNZIP (Col-CEN_v1.2.Chr1_5MB-7MB.fasta.gz)
[a3/f441fe] Submitted process > NFCORE_METHYLONG:METHYLONG:FASTQ_UNZIP:GUNZIP (Col-CEN_v1.2.Chr1_5MB-7MB.fasta.gz)
ERROR ~ Error executing process > 'NFCORE_METHYLONG:METHYLONG:FASTQ_UNZIP:GUNZIP (Col-CEN_v1.2.Chr1_5MB-7MB.fasta.gz)'

Caused by:
  Process `NFCORE_METHYLONG:METHYLONG:FASTQ_UNZIP:GUNZIP (Col-CEN_v1.2.Chr1_5MB-7MB.fasta.gz)` terminated with an error exit status (126)


Command executed:

  # Not calling gunzip itself because it creates files
  # with the original group ownership rather than the
  # default one for that user / the work directory
  gzip \
      -cd \
       \
      Col-CEN_v1.2.Chr1_5MB-7MB.fasta.gz \
      > Col-CEN_v1.2.Chr1_5MB-7MB.fasta
  
  cat <<-END_VERSIONS > versions.yml
  "NFCORE_METHYLONG:METHYLONG:FASTQ_UNZIP:GUNZIP":
      gunzip: $(echo $(gunzip --version 2>&1) | sed 's/^.*(gzip) //; s/ Copyright.*$//')
  END_VERSIONS

Command exit status:
  126

Command output:
  (empty)

Command error:
  9f0d519e5362: Pulling fs layer
  2fc3b27fe229: Pulling fs layer
  4f4fb700ef54: Waiting
  17dc7ea432cc: Waiting
  92dc97a3ef36: Waiting
  403f74b0f85e: Waiting
  bb36d6c3110d: Waiting
  10b8c00c10a5: Waiting
  030a47592a0a: Waiting
  0ea1a16bbe82: Waiting
  9f0d519e5362: Waiting
  2fc3b27fe229: Waiting
  dec6b097362e: Verifying Checksum
  dec6b097362e: Download complete
  f88da01cff0b: Verifying Checksum
  f88da01cff0b: Download complete
  dafa2b0c44d2: Verifying Checksum
  dafa2b0c44d2: Download complete
  4f4fb700ef54: Download complete
  92dc97a3ef36: Download complete
  403f74b0f85e: Verifying Checksum
  403f74b0f85e: Download complete
  10b8c00c10a5: Verifying Checksum
  10b8c00c10a5: Download complete
  17dc7ea432cc: Download complete
  bb36d6c3110d: Verifying Checksum
  bb36d6c3110d: Download complete
  0ea1a16bbe82: Verifying Checksum
  0ea1a16bbe82: Download complete
  dafa2b0c44d2: Pull complete
  030a47592a0a: Download complete
  9f0d519e5362: Verifying Checksum
  9f0d519e5362: Download complete
  2fc3b27fe229: Verifying Checksum
  2fc3b27fe229: Download complete
  dec6b097362e: Pull complete
  f88da01cff0b: Pull complete
  4f4fb700ef54: Pull complete
  92dc97a3ef36: Pull complete
  403f74b0f85e: Pull complete
  10b8c00c10a5: Pull complete
  17dc7ea432cc: Pull complete
  bb36d6c3110d: Pull complete
  0ea1a16bbe82: Pull complete
  030a47592a0a: Pull complete
  9f0d519e5362: Pull complete
  2fc3b27fe229: Pull complete
  Digest: sha256:63c2c6b22e83b2f656e88fbb1553e595da4e9e58794e3bfcb98b20b3837f328a
  Status: Downloaded newer image for <http://community.wave.seqera.io/library/coreutils_grep_gzip_lbzip2_pruned:838ba80435a629f8|community.wave.seqera.io/library/coreutils_grep_gzip_lbzip2_pruned:838ba80435a629f8>
  /bin/bash: line 10: .command.run: Permission denied

Work dir:
  /home/runner/work/methylong/methylong/work/a3/f441fe3ce24371e2940f1e402a266b

Container:
  <http://community.wave.seqera.io/library/coreutils_grep_gzip_lbzip2_pruned:838ba80435a629f8|community.wave.seqera.io/library/coreutils_grep_gzip_lbzip2_pruned:838ba80435a629f8>

Tip: you can replicate the issue by changing to the process work dir and entering the command `bash .command.run`

 -- Check '.nextflow.log' file for details
Execution cancelled -- Finishing pending tasks before exit
ERROR ~ Pipeline failed. Please refer to troubleshooting docs: <https://nf-co.re/docs/usage/troubleshooting>

 -- Check '.nextflow.log' file for details
-[nf-core/methylong] Pipeline completed with errors-
Error: Process completed with exit code 1.

heres the PR: https://github.com/nf-core/methylong/pull/7

i followed the instruction from this page: https://nf-co.re/docs/usage/troubleshooting/docker_permissions but it doesnt seems to work 😕

Hi all, thanks for all the great advices 🌷 I believe I have answered/ responded to all reviews and comments. All CI tests were passed ✅ are there any parts where I missed to correct? or can we push to run full test on aws sooon? confused dog

Hi, still need a review/approval from the <!subteam^S016E36N7TQ> on the actual PR (not pseudo): https://github.com/nf-core/methylong/pull/6 woohoo

Hii, I ran into an issue while running the ci test using singularity with disk issue:

Caused by:
  Failed to pull singularity image
    command: singularity pull  --name depot.galaxyproject.org-singularity-fastqc-0.12.1--hdfd78af_0.img.pulling.1745409665295 <https://depot.galaxyproject.org/singularity/fastqc:0.12.1--hdfd78af_0> > /dev/null
    status : 255
    hint   : Try and increase singularity.pullTimeout in the config (current is "20m")
    message:
      INFO:    Downloading network image
      INFO:    Cleaning up incomplete download: /home/runner/.apptainer/cache/net/tmp_4018132622
      FATAL:   write /home/runner/.apptainer/cache/net/tmp_4018132622: no space left on device

didnt have this issue while testing with docker. any suggestions how I could fix this? woohoo

will it be fine for merging if only all the tests were passed on using docker, but not singularity?

Workflow execution completed unsuccessfully

Error executing process > 'NFCORE_METHYLONG:METHYLONG:ONT:ONT_TRIM_REPAIR:PORECHOP_PORECHOP (HG002)'

Caused by:
  Essential container in task exited


Command executed:

  porechop \
      -i HG002_ont.fastq.gz \
      -t 3 \
      --discard_middle --extra_end_trim 10 \
      -o HG002.fastq.gz \
      > HG002.log
  cat <<-END_VERSIONS > versions.yml
  "NFCORE_METHYLONG:METHYLONG:ONT:ONT_TRIM_REPAIR:PORECHOP_PORECHOP":
      porechop: $( porechop --version )
  END_VERSIONS

Command exit status:
  -

Command output:
  (empty)

Command error:
  10:07PM INF instance metadata ami-id=ami-082efb76d7dd52982 instance-id=i-001da517311a473fb instance-type=r6id.32xlarge
  Fusion Info:
      max_open_files: 1048576
      fusion_version: 2.4.11-8ead802
      clone_namespace: false
      kernel_version: 6.1
      disk_cache_size: 7076Gb

Work dir:
  <s3://nf-core-awsmegatests/work/methylong/work-f48b21e6a41757689b3951e23c28d70e0b274004/40/48a5519838ee5fb37d95d4148163df>

Container:
  <http://wave.seqera.io/wt/0f1b6e7ec6c7/library/porechop_pigz:d1655e5b5bad786c|wave.seqera.io/wt/0f1b6e7ec6c7/library/porechop_pigz:d1655e5b5bad786c>

Tip: when you have fixed the problem you can continue the execution adding the option `-resume` to the run command line

hello all, do u know what was the reason for this error? This was from the full test running on aws.

Hello all, all CI tests and aws tests completed successfully ✅ anything else that is missing to complete? or we are good to go for the first release? 🚀

Just have to mention that I've been following this progress for the last 3 months or so, eager to try it out with some human data as soon as you get the first release out 👍

Hi, I am the main developer of methflow, my colleague Peng and I have joined the channel. We've already read the contributing guidelines. Should I create a new issue about our planned work and merge our modules in a forked repository? And Is there anything else I should pay attention to?

I tried to run some human data through this pipeline as a test run. There seems to be an issue with MULTIQC. This is the error code I am receiving: Error executing process > 'NFCORE_METHYLONGMETHYLONGMULTIQC' Caused by: Path value cannot be null

Any ideas as to the cause of this error?

Hii, I plan to add

dorado basecaller

at the beginning of ONT workflow and take pod5 files as input, but I'm not sure if that's a good idea... Can anyone give me some advice?loadingtux waddling

Hi! I'm interested in understanding nf-core/methylong output from ONT data. Could you suggest some tools or tutorials to help me get started with analyzing methylation profiles? ty in advance!

Hi! I'm testing methylong with some ONT data from a plant sample. I have passed the process parameters using -c as I have done with several other nf-core pipelines but I get this warning WARN: The following invalid input values have been detected: * --max_cpus: 30 * --max_memory: 58 GB * --max_time: 3d I have next made a nextflow.config file in the working directory, but I get the same warning. The content of config file is the following: params { max_cpus = 30 max_memory = 58.GB max_time = 72.h } singularity { enabled = true autoMounts = true cacheDir = '/pathto/singularity_cache' } Anyone has experienced the same problem or know how to fix it? Thanks!

Hi, I have solved the previous issue, but now during execution of the pipeline in one of the last steps I get this

java.lang.NullPointerException: Cannot get property 'containerEngine' on null object

it seems is related to

containerEngine

of the MULTIQC process. Anyone that can help me? I'm using apptainer and in the main.nf file of multiqc module I can see

process MULTIQC {                                                                       label 'process_single'

    conda "${moduleDir}/environment.yml"
    container "${ workflow.containerEngine == 'singularity' && !task.ext.singularity_pull_docker_container ?
        '<https://depot.galaxyproject.org/singularity/multiqc:1.28--pyhdfd78af_0>' :
        'biocontainers/multiqc:1.28--pyhdfd78af_0' }"

can the issue be here because instead of apptainer is finding 'singularity' as profile?

Hello developers! In the dev version, what is the difference between DMR analysis for haplotype level and population scale? Thanks!

Hello! When I use the

dev

version and use

sup

model instead of the default

hac

i.e.,

--dorado_model sup

then I get the error below,

[error] Must set --data when using automatic model detection

. Any tips to fix this please, thanks!! 🙏

👋 Hello, team!

Hello! I am using the methylong pipeline for a few weeks now and I do not understnd how does the it handles barcodes in the pod5 file. I think it is quite common to have all your sequencing data mixed in the same files. But having different barcode corresponding to different experimental condition. Then if one want to make a differerential analysis study (with dmr pair for instance), one need to be able to specify to the program which barcode to select. What I want to do is that each barcode corresponds for instance to a different sample for instance. It is also needed to specify to dorado demux which barcode kit has been used. I do not understand how this is done in your pipeline.)

sample,group,path,ref,method,barcode
Project_23_24_DAM_S288c,DAM_S288c,pod5/,ref.fasta,ont,barcode23
Project_23_24_NoDAM_S288c,NoDAM_S288c,pod5/,ref.fasta,ont,barcode24

I have a question about DMR calling with three cell lines (groups), each with 4–5 replicates. In the documentation, I saw the options

--dmr_population_scale

and

--population_dmrer

, but I’m unsure whether these are meant for much larger “population-scale” experiments and if they are appropriate for my setup. I’d also like to compare DMRs in one cell line against all others. Is there a way to do this within a single run, or do I need to specify

--dmr_a

and

--dmr_b

for each pair (e.g. three separate runs to cover all combinations)? In other words, can the pipeline perform all pairwise comparisons between 3 groups in one go, or must they be run separately? And what options would I have to set for that in my script? Any guidance on recommended settings for this design would be greatly appreciated.

Hi all! Here because I'm starting FiberSeq experiments and want to see what the methylong pipeline can do for me 😉

Hi all. I just ran methylong pipeline (v2.0.0) and bumped into an issue. Would like to know your thoughts on this. The bedgraph output files it makes are empty. I ran it for 4 samples and they came out blank for every sample. I only caught it because I opened the files and checked. I ran the pileup step with

--combine-mods

( which combines 5mC and 5hmC into one call). And I think that changes how one column in the output looks. And the bedgraph step is expecting the old format, so its filter never matches anything, and it drops every row.