Hi everyone! Hope you’re all doing great. I’m currently writing a module for CIRI2. The input/output channels are ready, but I’ve hit a roadblock with writing the

nf-test

. CIRI2 strictly requires a SAM file generated by BWA-MEM using the

-T 19

flag. Since the

nf-core/test-datasets

repo doesn't host raw, uncompressed SAM files for Homo sapiens, I'm weighing my options according to nf-core guidelines: 1. Chain modules in `setup`: If I run

BWA_MEM

in the setup block, I will also need to chain

BWA_INDEX

before it since pre-computed BWA index directories aren't available for the human test genome. This feels quite heavy for a single module unit test. 2. Local asset: Should I just include a tiny, heavily-truncated mock SAM file directly inside the module's

tests/assets/

directory? Has anyone encountered a similar tool dependency issue during testing? What is the recommended or preferred approach by maintainers in this scenario? Any guidance would be highly appreciated. Thanks in advance!

Hi everyone I have a cross-pipeline question about *mapping-quality filtering in nf-core pipelines. W*hile reviewing the source code, I found that different chromatin pipelines appear to use different default MAPQ thresholds: • nf-core/atacseq: MAPQ ≥1 atacseq/conf/modules.config at 2.1.2 · nf-core/atacseq • nf-core/chipseq: MAPQ ≥1 chipseq/conf/modules.config at master · nf-core/chipseq • nf-core/cutandrun: MAPQ ≥20 The filtering command uses:

samtools view -b -q ${params.minimum_alignment_q_score}

cutandrun/conf/modules.config at master · nf-core/cutandrun The default value of

minimum_alignment_q_score

is defined as 20 here: cutandrun/nextflow_schema.json at master · nf-core/cutandrun • nf-core/hic: MAPQ ≥10 hic/nextflow.config at master · nf-core/hic For nf-core/rnaseq, I could not find one pipeline-wide MAPQ cutoff. I may have missed it I understand that one universal MAPQ threshold may not be appropriate across different aligners and assays. However, ATAC-seq, ChIP-seq and CUT&RUN can all use Bowtie2, yet ATAC-seq and ChIP-seq use MAPQ ≥1 while CUT&RUN uses MAPQ ≥20. Could someone clarify: 1. Are these differences based on assay-specific benchmarking, downstream analysis requirements or historical pipeline decisions? 2. When the same aligner, such as Bowtie2, is used, what is the rationale for MAPQ ≥1 in ATAC-seq and ChIP-seq versus MAPQ ≥20 in CUT&RUN? 3. In ATAC-seq and ChIP-seq, when

keep_multi_map

is false, the pipelines apply

-q 1

. Does MAPQ ≥1 truly restrict the BAM to uniquely mapped reads? My understanding is that

-q 1

removes MAPQ 0 alignments, but does not necessarily prove that every retained read has only one possible genomic alignment. Would it therefore be more accurate to describe this as removing MAPQ 0 or highly ambiguous alignments rather than removing all multimapping reads? 4. When a pipeline has no explicit MAPQ-filtering parameter, does that mean filtering is left to the aligner or downstream tool? 5. Do the final BAM files, bigWigs, peak calls and count matrices all use the same MAPQ-filtered read set within each pipeline? 6. Would it be useful to document this consistently for every alignment-based nf-core pipeline, including: • the default MAPQ cutoff;where it is applied; • whether it is configurable; and • which downstream outputs are affected? Please correct any thresholds or interpretations above that are inaccurate. Thank you @Phil Ewels @Maxime Garcia

Hi, this is a bug report. I tried searching for other channels to put this in but found no other obvious candidates so I'm defaulting to here. Please redirect me if appropriate. I have a fresh conda env containing nextflow v26.04.6, nf-core v4.0.2 and python v3.14.6. I tried

nf-core pipelines launch nf-core/rnaseq -r 3.26.0

. This produced the error found in the attached txt file. I also tried replacing rnaseq with sarek, and this produced the same result. Given the fresh install, repeatable nature across pipelines, and latest versions of everything, I believe this is a genuine bug. Thanks!

I have a question about parameter types. I attempt to add parameter types in nf-core/ampiseq in github.com/nf-core/ampliseq/pull/1027. I did check out the bytesize talk & blog post. I have two questions though: (1) Contrary to the blog post it appears to me that the nested parameters in github.com/nf-core/ampliseq/blob/…/ref_databases.config (similar to igenomes, I thought) seems still to work. Am I here missing something and I should convert it to a Map structure or is that fine for now? (2) I am unsure about defaults for parameter types. Boolean seems to default to false as of nextflow 26.04. Is it safer to still define the

Boolean = false

as in airrflow? What about default for

String?

, is it advised to use

String? = null

? Is there a role model implementation in nf-core already of parameter types? I had a look but didnt find any other than airrflow (but I likely missed something). Edit: trying to lure @Nicolas Vannieuwkerke here 🙂

Hi! I am trying to use the scrnaseq pipeline but I am getting an error when connecting to iGenome. I have tried to ignore igenomes but the error persists. Command:

NXF_VER=25.10.4 nextflow run nf-core/scrnaseq \
    -profile test \
    --aligner cellranger \
    --fasta ~/Shared-MarcelCosta/apps/cellranger-10.0.0/refdata-gex-GRCh38-2024-A/fasta/genome.fa \
    --cellranger_index ~/Shared-MarcelCosta/apps/cellranger-10.0.0/refdata-gex-GRCh38-2024-A/fasta/genome.fa.fai \
    --gtf ~/Shared-MarcelCosta/apps/cellranger-10.0.0/refdata-gex-GRCh38-2024-A/genes/genes.gtf.gz \
    --outdir PROCESSING
    --genome null \
    --igenomes_ignore

Error:

* --igenomes_base (<s3://ngi-igenomes/igenomes/>): could not validate file format of '<s3://ngi-igenomes/igenomes/>': Access Denied (Service: S3, Status Code: 403, Request ID: 4F61R9KM8W39WSMT, Extended Request ID: qa4D4BHgJMfa2KLuSmPYARTuZnKR/ObNHgeSSM6ur226/3vZVhr/BYGvg77an3ssxHERDxzWFZ20SBSWl1oofjWAUIeRrTvz) (SDK Attempt Count: 1)

I don't need the iGenomes access because I already have the genome in local.

I'm getting an error using the

setup-nextflow

action in a few of my pipelines:

Input version '25.10.0' resolved to Nextflow undefined

It seems it just started failing today even though my gha workflow yaml hasn't changed. Perhaps this is a bug in nf-core/setup-nextflow with version resolution? yaml snippet:

- name: Install nextflow
        uses: nf-core/setup-nextflow@v3
        with:
          version: 25.10.0

error:

Input version '25.10.0' resolved to Nextflow undefined
Error: Cannot read properties of undefined (reading 'includes')
Error: Could not run 'nextflow help'. Error: Unable to locate executable file: nextflow. Please verify either the file path exists or the file can be found within a directory specified by the PATH environment variable. Also check the file mode to verify the file is executable.
Error: Process completed with exit code 1.

full github actions log: github.com/CCBR/CHAMPAGNE/…/88735982565#…

#CE6SDBX2A with #CE8SSJV3N error: I am getting the error (see attached screengrab) when I use GTF from NCBI. It like it was fixed in this issue : github.com/nf-core/rnaseq/pull/1889. but still come up when I run rnaseq 3.26.0 . Is there a way of getting around this problem?

Hi everyone! I have a quick question. I recently opened my first PR in

nf-core/test-datasets

and it was just approved! Is it okay for me to go ahead and merge it myself, or should I wait for a maintainer to do it? Thanks in advance for the help!

I am trying to convert #C06R4FC4C1E to the new typed params with the automatic converter, but I am getting

Pipeline cannot be converted due to script errors

.

nextflow lint

gives no errors and 3 very minor warnings. AITA? And how can I find the errors? I don't see a way to see the full log

This message was deleted.

I know this is extremely non-specific, but after converting the pipeline to typed params I am consistently getting a

StackOverflowError

when trying a test run no matter what I do. Upon checking the log, I saw that the reason is an infinite loop that starts like this:

nextflow.file.http.XPath.iterator(XPath.groovy:242)
    at groovy.json.DefaultJsonGenerator.writeObject(DefaultJsonGenerator.java:206)
    at groovy.json.DefaultJsonGenerator.writeObject(DefaultJsonGenerator.java:168)
    at groovy.json.DefaultJsonGenerator.writeIterator(DefaultJsonGenerator.java:416)
    at groovy.json.DefaultJsonGenerator.writeObject(DefaultJsonGenerator.java:206)

Has anyone encountered ths before? The error messages are entirely unhelpful, and I cannot pinpoint where things go wrong. AI suggests bogus theories that don't match the code

I'm having problems signing in from my computer. When I ask slack to reset my password or send a sign-in link I get messages that there's no account associated with my email address (daniel.lundin@lnu.se). So, a catch 22. I was going to change my email address to matricaria.suaveolens@gmail.com anyway -- are any of you admins able to do that so I can continue? Or should I create a new account with this email?

If you want to change your email address, got to: http://my.slack.com/account In the top right, select the right workspace. More details here: https://slack.com/help/articles/207262907-Change-your-email-address

My PR is approved but I don't get a merge button. Not sure what d

*

happened. https://github.com/nf-core/configs/pull/1152

Hi, we have built a nextflow pipeline that is mostly working as expected. Using version 26.04.6 but same was true with previous 26.xx.x versions and we just updated everything to latest version. We are facing 2 issues. # Issue 1: We use a custom.config file to feed parameters to the pipeline. When we change a parameter in that file (with a bash sed command outside the pipeline), we expect that the modules that use this parameter do not use the cached results from the previous runs. Unfortunately it does. We can use the 'cache false' option but we would like to use the cache when no parameter change is made. Is there something we do not understand about this caching mechanism? Would you have suggestions to investigate this in our setup? # Issue 2: We enjoy the short performance summary displayed at the end of each run, ie: Completed at: 28-Jul-2026 193051 Duration : 1h 47m 8s CPU hours : 1.9 Succeeded : 13 But for some reasons it is not always displayed. Some server display it at the end of each run, some do not. The code seems the same (git clone). We believe it is a configuration in nextflow but we can't find where this is set up. Can you help?

Hi! This GHaction has been stuck since this morning with the error:

Canceling since a higher priority waiting request for Run nf-test-759 exists

. Any ideas why is happening?

Hey, I am reviewing the new rnastructurome pipeline (github.com/nf-core/rnastructurome), and am not sure if this is a valid way to organise the main.nf (hopefully its fine). But the pipeline allows all columns in a samplesheet to be filled in by params given at command line. E.g. Organism, if not set in the samplesheet, then adding --organism ‘Homo sapiens’, will use that for all samples in the sheet. github.com/nf-core/rnastructurome/pull/5/changes#… It is set in a subworkflow. So that if there is content in the samplesheet for a particular attribute the param will not overwrite it. It only overides with a param if the field is empty. rnastructurome/subworkflows/local/utils_nfcore_rnastructurome_pipeline/main.nf at 2678cdd9f6a82c25d6… Not sure if this is a useful feature, but if it is, is this allowed in an nf-core pipeline?

Hi everyone! I'm stuck on a local lint issue for my PR. My module uses both ciri2 and samtools (for SAM file conversion). I generated the Docker and Singularity container links directly from Seqera Containers using the exact tool versions specified in environment.yml. However, nf-core lint continuously fails with Unable to connect to container registry (404) and Container versions do not match. I suspect this might also be causing the automated CI test to fail, but I'm not 100% sure. Would anyone mind taking a quick look or helping out? Thanks in advance! github.com/nf-core/modules/pull/12224

Hi! Does anyone know if it's possible for institutions to purchase access to Seqera AI (co-scientist)? Or could someone point me to the appropriate channel to ask about Seqera AI? Thanks!

Hi everyone. My colleague raised an interesting question about usage of process substitution for concatenation tasks rather than using CAT_FASTQ , to avoid storing the large intermediate files. I don't see that pattern used much over in nf-core/modules, apart from one module. Are there any downsides to this? In my specific case most of the tools in the pipeline require the concatenated input, so I'm not sure if I should be worried about the potential time and I/O cost of process substitution in each tools process, versus the storage cost of using CAT_FASTQ once for all tools.

Hi, I'm trying to fix the environment issue for phyloflash with conda. The environment can be create locally but fail on the github action with the following error:

>       ERROR conda.core.link:_execute(1014): An error occurred while installing package 'bioconda::spades-3.15.5-h95f258a_1'.
      >       Rolling back transaction:
      >       LinkError: post-link script failed for package bioconda::spades-3.15.5-h95f258a_1
      >       location of failed script: /home/runner/work/modules/modules/.nf-test/tests/769e57f0e7d8bc77bee19f97fef1c9d9/work/conda/env-27f8de4e77d219b103e2d1302f904167/bin/.spades-post-link.sh
      >       ==> script messages <==
      >       <None>
      >       ==> script output <==
      >       stdout:
      >       stderr: /home/runner/work/modules/modules/.nf-test/tests/769e57f0e7d8bc77bee19f97fef1c9d9/work/conda/env-27f8de4e77d219b103e2d1302f904167/etc/conda/deactivate.d/deactivate-gxx_linux-64.sh: line 68: CONDA_BACKUP_CXX: unbound variable

I've tried to specify additional libraries to match the singularity container, but I always came back to this error. Does anybody know how to solve this ? github.com/nf-core/modules/pull/12577

Any idea what these singularity errors here are about: github.com/nf-core/proteinfamilies/…/92642599648?pr=183

> Command error:
      >   WARNING: DEPRECATED USAGE: Forwarding SINGULARITYENV_NXF_TASK_WORKDIR as environment variable will not be supported in the future, use APPTAINERENV_NXF_TASK_WORKDIR instead
      >   WARNING: DEPRECATED USAGE: Forwarding SINGULARITYENV_NXF_DEBUG as environment variable will not be supported in the future, use APPTAINERENV_NXF_DEBUG instead
      >   ERROR  : Failed to create container process: Operation not permitted

I think I broke the template-merge pr (again). I tried rebasing it onto dev, but this gives me hours of merge conflicts to resolve, and I think there has to be a better way? github.com/nf-core/genomeassembler/pull/208

When updating an nf-core module, how do I get the conda lockfile required by meta.yml, e.g.

modules/nf-core/vuegen/.conda-lock/linux_amd64-bd-9eebdec0448f6563_1.txt

I'm able to get all the other information from: seqera.io/containers For reference, here is my container. There is a conda lockfile block there but it is a format that I don't really recognise and doesn't seem to match other modules with the lockfile

I'm running the ChIP-Seq pipeline and wanted to change a couple of parameters, which I'm assuming are to be provided by a config file. Is this the correct way to provide additional arguments?

process {
    withName: 'BOWTIE2_ALIGN' {
        ext.args = {
            [
                meta.read_group ? "--rg-id ${meta.id} --rg SM:${meta.id - ~/_T\\d+$/} --rg PL:ILLUMINA --rg LB:${meta.id} --rg PU:1" : '',
                params.seq_center ? "--rg CN:${params.seq_center}" : '',
                '-X 1000'
            ].join(' ').trim()
        }
    }

    withName: 'BAMTOOLS_FILTER' {
        ext.args = {
            [
                meta.single_end ? '-F 0x004' : '-F 0x004 -F 0x0008 -f 0x001',
                params.keep_dups ? '' : '-F 0x0400',
                params.keep_multi_map ? '' : '-q 30'
            ].join(' ').trim()
        }
    }

Hi everyone, I'm very new to bioinformatics and I'm currently working with the nf-core/fastquorum pipeline for Duplex Sequencing data. I'm analyzing a dataset with approximately 94 million reads from a 376 bp target region containing 10 nt UMIs. The pipeline runs successfully through alignment, but repeatedly struggles during the fgbio GroupReadsByUmi step. A 1% subset of the data (~0.94 million reads) completes successfully, but the full dataset either runs for a very long time or fails due to resource limitations. I've already spent some time troubleshooting memory allocation and runtime settings, and I've been able to resolve some scheduling/resource issues, but GroupReadsByUmi remains the bottleneck. Has anyone encountered similar scaling issues with Duplex-Seq data or extremely deep sequencing of a very small target region? Any suggestions would be greatly appreciated. Thanks!

Hi everyone , I'm currently working on the development of a pipeline to process the BLISS ( break labelling in situ sequencing) data, but I have a bug to fix. I launch it with as input a csv of 2 samples paired-end, it finish without error but losing one sample and then when I relaunch it with -resume , the lost sample is process without error. At the beggining of the pipeline I used the subworkflow fastq_fastqc_umitools_trimgalore , then bwa_mem and umitools_dedup and it seems that the lost of one of two samples Can someone help me ?

Hi everyone, I'm Arash, a PhD student in Ola Larsson's group at SciLifeLab. We're hosting the TRANSLACORE Bioinformatics Training School in Scilifelab Solna from August 24–26 for trainees working on translational control and Ribo-seq/Polysome profiling. It would be awesome to have someone who knows the pipeline come give a ~30 min talk to introduce the nf-core/riboseq pipeline to researchers in this field. I'd really appreciate it if anyone could help or point me to someone who could. If there's no candidate available in person at SciLifeLab Solna, a Zoom talk would work as well.