While I’m running nf-core/rnasplice pipeline (1.0.4 version: https://nf-co.re/rnasplice/1.0.4/), I’m facing this error relating to “featureCounts:”

Workflow execution completed unsuccessfully!
The exit status of the task that caused the workflow execution to fail was: 255.
The full error message was:
Error executing process > 'NFCORE_RNASPLICE:RNASPLICE:EDGER_DEU:SUBREAD_FEATURECOUNTS (SRX12134688)'

Caused by:
 Process `NFCORE_RNASPLICE:RNASPLICE:EDGER_DEU:SUBREAD_FEATURECOUNTS (SRX12134688)` terminated with an error exit status (255)

Command executed:

 featureCounts \
     -B -C -g gene_biotype -t exon \
     -p \
     -T 6 \
     -a annotation.saf \
     -s 0 \
     -o SRX12134688.featureCounts.txt \
     SRX12134688_sorted.bam
 
 cat <<-END_VERSIONS > versions.yml
 "NFCORE_RNASPLICE:RNASPLICE:EDGER_DEU:SUBREAD_FEATURECOUNTS":
     subread: $( echo $(featureCounts -v 2>&1) | sed -e "s/featureCounts v//g")
 END_VERSIONS

Command exit status:
 255

Command output:
 (empty)

Command error:
 WARNING: Skipping mount /var/singularity/mnt/session/etc/resolv.conf [files]: /etc/resolv.conf doesn't exist in container
 
         ==========    _____ _   _ ____ _____ ______         _____ 
         =====        / ____| | | | _ \| __ \| ____|  /\  | __ \ 
           =====     | (___ | | | | |_) | |__) | |__    / \ | | | |
             ====     \___ \| | | | _ <| _ /|  __|  / /\ \ | | | |
               ====   ____) | |__| | |_) | | \ \| |____ / ____ \| |__| |
         ==========  |_____/ \____/|____/|_| \_\______/_/   \_\_____/
              v2.0.1
 
 //========================== featureCounts setting ===========================\\
 ||                                                                           ||
 ||            Input files : 1 BAM file                                      ||
 ||                          o SRX12134688_sorted.bam                        ||
 ||                                                                           ||
 ||            Output file : SRX12134688.featureCounts.txt                   ||
 ||                Summary : SRX12134688.featureCounts.txt.summary           ||
 ||             Annotation : annotation.saf (GTF)                            ||
 ||     Dir for temp files : ./                                              ||
 ||                                                                           ||
 ||                Threads : 6                                               ||
 ||                  Level : meta-feature level                              ||
 ||             Paired-end : yes                                             ||
 ||     Multimapping reads : not counted                                     ||
 || Multi-overlapping reads : not counted                                     ||
 ||  Min overlapping bases : 1                                               ||
 ||                                                                           ||
 ||         Chimeric reads : not counted                                     ||
 ||       Both ends mapped : required                                        ||
 ||                                                                           ||
 \\============================================================================//
 
 //================================= Running ==================================\\
 ||                                                                           ||
 || Load annotation file annotation.saf ...                                   ||
 ERROR: no features were loaded in format GTF. The annotation format can be specified by the '-F' option, and the required feature type can be specified by the '-t' option..
 The porgram has to terminate.

Work dir:
 /home/tud03125/pipeline/normal_human_liver_rnasplice/work/19/6265861010e98b14767ab01dab7af7

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

Has anyone had “featureCounts” issues before, esp. in Singularity profile (what I'm currently using)? I’ve asked ChatGPT about what’s going on, and it gave me list of suggestions. One is checking if I have the GTF file: “Homo_sapiens.GRCh38.108.gtf,” which I do have. The other suggestion is to “Update Pipeline Configuration,” specifically “this adjustment needs to be applied directly in the Nextflow pipeline or in the rnasplice pipeline's configuration files.” When looking further into it, the specific configuration file is “main.nf,” specifically this directory: “nf-core-rnasplice_1.0.4/1_0_4/modules/nf-core/subread/featurecounts/main.nf,” and to add “-F GTF” or “-F SAF” line in it. Is that allowed or how you’d approach it?

Since it is looking for

gene_biotype

. it looks like this is just for the QC. The rnaseq pipeline has a parameter

--skip_biotype_qc

, but rnasplice apparently not. You could, however, set

errorStrategy = 'ignore'

for this particular process. The root of the problem seems to be that

featureCounts

does not like your GTF. Make sure it is not corrupted or malformed. The 3rd column should contain a bunch of

exon

entries, and ideally the 9th column will have

gene_biotype

entries. (Ensembl GTFs have that, at least for mammalian genomes).

@Thomas Danhorn yeah, I was working on

featureCounts

. The thing is that the only GTF is the Homo Sapiens reference genome:

Homo_sapiens.GRCh38.108.gtf

. The rest of my samples are FASTQ. I have checked all of these files; none are corrupted or malformed (plus, been using them endlessly).

exon

and other entries are present in there. During this investigation,

featureCounts

is looking for the

annotation

file of each sample that

rnasplice

automatically creates. The problem is that 1)

annotation

is in SAF form, or

annotation.saf

, and 2)

featureCounts

is searching for the GTF file (like

annotation.gtf

), not the SAF one. And so, for both `main.nf`:

/home/tud03125/pipeline/nf-core-rnasplice_1.0.4/1_0_4/modules/nf-core/subread/featurecounts/main.nf

and the

.command.sh

scripts automatically made in each work directory, I have to manually insert

-F SAF

inside each

featureCounts

section on either of these scripts. The

main.nf

one would be for future runs. But still, is that right, given what you have told me before, about changing or editing scripts, particularly

main.nf

ones? Because if I do not fix this missing

-F SAF

issue, I feel like there would not be any other way around it, like no alternatives or better plans.

I ran version 1.0.3 of this pipeline not long ago, and it worked just fine, including

featureCounts

(and that module has not been touched in a year), so I have doubts that the problem is in the pipeline (and the solution would be to fix the code). Does your GTF have

gene_biotype

entries? If not, just download a GTF from Ensembl and try with that.

@Thomas Danhorn regarding

gene_biotype

, not me! I did not touch that! I was too focused on

featureCounts

and

-F SAF

, that's all. Where that came from? Good question!

--subread_threads

is not a parameter in the current version (1.04) of the pipeline, as far as I can see, nor are your

--skip_*

options. This makes me think you are running a rather old version, which may well have a bug with the

featureCounts

process. My recommendation would be to run the newest version. If you really, really need the old one to be compatible with prior work (and can't redo the prior work with the new version), you would have to use the exact same command as was used previously (and that should then work, if it did before). But I would use the newest version if at all possible.

@Thomas Danhorn I was looking into why I'm using these

--skip_*

options, plus

--subread_threads

,

--ribo_database_manifest

,

--kallisto_index

and so forth. It was actually due to these warning messages inside my output log when running without them:

N E X T F L O W ~ version 23.04.0
Launching `/home/tud03125/pipeline/rnasplice/main.nf` [gigantic_minsky] DSL2 - revision: 93d808d3e7
WARN: Access to undefined parameter `bbsplit_index` -- Initialise it to a default value eg. `params.bbsplit_index = some_value`
WARN: Access to undefined parameter `kallisto_index` -- Initialise it to a default value eg. `params.kallisto_index = some_value`
WARN: Access to undefined parameter `rsem_index` -- Initialise it to a default value eg. `params.rsem_index = some_value`
WARN: Access to undefined parameter `hisat2_index` -- Initialise it to a default value eg. `params.hisat2_index = some_value`
WARN: Access to undefined parameter `additional_fasta` -- Initialise it to a default value eg. `params.additional_fasta = some_value`
WARN: Access to undefined parameter `gene_bed` -- Initialise it to a default value eg. `params.gene_bed = some_value`
WARN: The following invalid input values have been detected:

* --contrasts: /home/tud03125/pipeline/contrast_NORMAL.csv
* --source: fastq
* --clip_r1: null
* --clip_r2: null
* --three_prime_clip_r1: null
* --three_prime_clip_r2: null
* --trim_nextseq: null
* --skip_trimgalore_fastqc: false
* --rmats: true
* --rmats_splice_diff_cutoff: 0.0001
* --rmats_paired_stats: true
* --rmats_read_len: 40
* --rmats_novel_splice_site: false
* --rmats_min_intron_len: 50
* --rmats_max_exon_len: 500
* --dexseq_exon: true
* --save_dexseq_annotation: false
* --gff_dexseq: null
* --alignment_quality: 10
* --aggregation: true
* --save_dexseq_plot: true
* --n_dexseq_plot: 10
* --edger_exon: true
* --save_edger_plot: true
* --n_edger_plot: 10
* --dexseq_dtu: true
* --dtu_txi: dtuScaledTPM
* --sashimi_plot: true
* --miso_genes: ENSG00000004961, ENSG00000005302, ENSG00000147403
* --miso_genes_file: null
* --miso_read_len: 75
* --fig_width: 7
* --fig_height: 7
* --min_samps_feature_expr: 2
* --min_samps_feature_prop: 2
* --min_samps_gene_expr: 4
* --min_feature_expr: 10
* --min_feature_prop: 0.1
* --min_gene_expr: 10
* --suppa: true
* --suppa_per_local_event: true
* --suppa_per_isoform: true
* --suppa_tpm: null
* --generateevents_pool_genes: true
* --generateevents_event_type: SE SS MX RI FL
* --generateevents_boundary: S
* --generateevents_threshold: 10
* --generateevents_exon_length: 100
* --psiperevent_total_filter: 0
* --diffsplice_local_event: true
* --diffsplice_isoform: true
* --diffsplice_method: empirical
* --diffsplice_area: 1000
* --diffsplice_lower_bound: 0
* --diffsplice_gene_correction: true
* --diffsplice_paired: true
* --diffsplice_alpha: 0.05
* --diffsplice_median: false
* --diffsplice_tpm_threshold: 0
* --diffsplice_nan_threshold: 0
* --clusterevents_local_event: true
* --clusterevents_isoform: true
* --clusterevents_sigthreshold: null
* --clusterevents_dpsithreshold: 0.05
* --clusterevents_eps: 0.05
* --clusterevents_metric: euclidean
* --clusterevents_separation: null
* --clusterevents_min_pts: 20
* --clusterevents_method: DBSCAN

I don't get these warnings -- have you found out what version of the pipeline you have? If you look in the

.nextflow.log

, it should be under the nf-core logo:

------------------------------------------------------
                                        ,--./,-.
        ___     __   __   __   ___     /,-._.--~'
  |\ | |__  __ /  ` /  \ |__) |__         }  {
  | \| |       \__, \__/ |  \ |___     \`-._,-`-,
                                        `._,._,'
  nf-core/rnasplice v1.0.3
------------------------------------------------------

I've deleted the output and error logs. They kept piling up since

rnasplice

is new to me and I've kept running into problems constantly. 😆 But to answer your question, those errors I've mentioned, both

v1.0.3

and

v1.0.4

I'm getting them. Whenever I get error and output logs, I can open up JupyterLab and visually look at them. It’s easier for me than using

nano

or

vim

. But also, whenever I'm executing

./nextflow

command, I can see that logo and their descriptions, including which version. Anyway, yeah, these list of warning messages I'm getting, they're in both versions on my end. Btw, I just tried

v1.0.3

one, and

featureCounts

issue still pops up. So, it’s unlikely a version issue and most likely me: Temple HPC or what I’ve put in there, in my own directories. Since you’ve suggested to stick with the newest version given your reasons, I’m going back to

1.0.4

one.

I find this very strange: • You are using parameters that don't exist in the pipeline to suppress warnings about other, supposedly uninitialized. parameters (which I am not getting), some of which (

additional_fasta

) don't even exit in the pipeline code. • Your error stems from an invocation of

featureCounts

which is missing

-F SAF

(apparently because the

ext.args

from the

module.config

got overwritten) but contains

-g gene_biotype

. •

gene_biotype

only occurs in one Groovy utility function in the entire pipeline code, and I doubt that is even used. When I run the pipeline, I only get

-g gene_id

, nothing ever with any

biotype

, so I have no idea where that comes from in your run ... I am curious what is in

/home/tud03125/pipeline/rnasplice

. What does

grep -e " version *= *'" /home/tud03125/pipeline/rnasplice/nextflow.config

show? And can you run the pipeline "on the fly" (automatically downloaded) with

nextflow run nf-core/rnasplice -r 1.0.4 ...

?

Inside

nextflow.config

, when looking for

version

, these are what I could find:

…
…
// Boilerplate options
   outdir                    = null
   publish_dir_mode          = 'copy'
   email                     = null
   email_on_fail             = null
   plaintext_email           = false
   monochrome_logs           = false
   hook_url                  = null
   help                      = false
   version                   = false

   // Config options
   config_profile_name       = null
   config_profile_description = null
   custom_config_version     = 'master'
   custom_config_base        = "<https://raw.githubusercontent.com/nf-core/configs/${params.custom_config_version}>"
   config_profile_contact    = null
   config_profile_url        = null
…
…
manifest {
   name           = 'nf-core/rnasplice'
   author         = """Ben Southgate, James Ashmore"""
   homePage       = '<https://github.com/nf-core/rnasplice>'
   description    = """Alternative splicing analysis using RNA-seq."""
   mainScript     = '<http://main.nf|main.nf>'
   nextflowVersion = '!>=23.04.0'
   version        = '1.0.4'
   doi            = '10.5281/zenodo.8424632'
}
…
…

It’s funny that `Nextflow`’s version is

23.04.0

. I’ve just installed

24.04.04

recently. In addition, inside

modules.config

, searching

ext.arg

and focusing on

featureCounts

, I found these:

…
…
if (params.source == "fastq" | params.source == "genome_bam") {

   if (params.edger_exon) {
       process {
           withName: 'SUBREAD_FLATTENGTF' {
               ext.args  = [
                   "-t exon",
                   "-g ${params.gtf_group_features}",
                   '-C',
               ].join(' ').trim()
               publishDir = [
                   path: { "${params.outdir}/${params.aligner}/featurecounts" },
                   mode: params.publish_dir_mode,
                   saveAs: { filename -> filename.equals('versions.yml') ? null : filename }
               ]
           }
           withName: 'SUBREAD_FEATURECOUNTS' {
               ext.args  = [
                   '-F SAF',
                   "-t exon",
                   "-g ${params.gtf_group_features}",
                   '-f',
                   '-O',
                   '-B',
                   '-C'
               ].join(' ').trim()
               publishDir = [
                   path: { "${params.outdir}/${params.aligner}/featurecounts" },
                   mode: params.publish_dir_mode,
                   saveAs: { filename -> filename.equals('versions.yml') ? null : filename }
               ]
           }
       }
…
…

And, what’s inside “/home/tud03125/pipeline/rnasplice” is this:

(base) [tud03125@compute ~]$ ls -l pipeline/rnasplice
total 249
drwxr-xr-x 2 tud03125 eng   13 Aug 24 21:44 assets
drwxr-xr-x 2 tud03125 eng   21 Aug 24 21:44 bin
-rw-r--r-- 1 tud03125 eng 1881 Aug 24 21:44 CHANGELOG.md
-rw-r--r-- 1 tud03125 eng 8468 Aug 24 21:44 CITATIONS.md
-rw-r--r-- 1 tud03125 eng 13562 Aug 24 21:44 CODE_OF_CONDUCT.md
drwxr-xr-x 3 tud03125 eng   16 Aug 24 22:03 conf
drwxr-xr-x 3 tud03125 eng    7 Aug 24 21:44 docs
drwxr-xr-x 2 tud03125 eng    7 Aug 24 21:44 lib
-rw-r--r-- 1 tud03125 eng 1080 Aug 24 21:44 LICENSE
-rw-r--r-- 1 tud03125 eng 2868 Aug 24 21:44 <http://main.nf|main.nf>
drwxr-xr-x 4 tud03125 eng    4 Aug 24 21:44 modules
-rw-r--r-- 1 tud03125 eng 8853 Aug 24 21:46 modules.json
-rw-r--r-- 1 tud03125 eng 14104 Aug 24 21:44 nextflow.config
-rw-r--r-- 1 tud03125 eng 43985 Aug 24 21:44 nextflow_schema.json
-rw-r--r-- 1 tud03125 eng  359 Aug 24 21:44 pyproject.toml
-rw-r--r-- 1 tud03125 eng 9562 Aug 24 21:44 README.md
drwxr-xr-x 4 tud03125 eng    4 Aug 24 21:44 subworkflows
-rw-r--r-- 1 tud03125 eng  165 Aug 24 21:44 tower.yml
drwxr-xr-x 2 tud03125 eng    3 Aug 24 21:44 workflows
(base) [tud03125@compute ~]$

About this question: “And can you run the pipeline "on the fly" (automatically downloaded) with

nextflow run nf-core/rnasplice -r 1.0.4 ...

?,” I don’t know what you mean by that.

Looks like the installed version is

1.0.4

, which does not do anything to clear up why you are getting these weird messages. (BTW,

nextflowVersion = '!>=23.04.0'

mean the minimum version is 23.04, i.e. it will most likely fail if you try with an older one.) What meant by my last sentence is, instead of running with

./nextflow run /home/tud03125/pipeline/rnasplice <other parameters>

in your script, try

./nextflow run nf-core/rnasplice -r 1.0.4 <other parameters>

. This should guarantee that the specified version is run with code downloaded from GitHub (i.e. no chance of any modification like there could be in a local repo).

@Thomas Danhorn Sorry it took a long time. I was re-running the pipeline from scratch (removing

work

directory), all the way at the beginning, using

./nextflow run nf-core/rnasplice -r 1.0.4 <other parameters>

rather than

./nextflow run /home/tud03125/pipeline/rnasplice <other parameters>

as you’ve suggested. Anyway, just ran it, and I’m getting this same warning message:

Starting Nextflow pipeline...                                                                                                                      [95/1969]

 N E X T F L O W  ~ version 24.04.4

Launching `<https://github.com/nf-core/rnasplice>` [confident_wozniak] DSL2 - revision: 1d0494ae34 [1.0.4]

WARN: Access to undefined parameter `bbsplit_index` -- Initialise it to a default value eg. `params.bbsplit_index = some_value`
WARN: Access to undefined parameter `kallisto_index` -- Initialise it to a default value eg. `params.kallisto_index = some_value`
WARN: Access to undefined parameter `rsem_index` -- Initialise it to a default value eg. `params.rsem_index = some_value`
WARN: Access to undefined parameter `hisat2_index` -- Initialise it to a default value eg. `params.hisat2_index = some_value`
WARN: Access to undefined parameter `additional_fasta` -- Initialise it to a default value eg. `params.additional_fasta = some_value`
WARN: Access to undefined parameter `gene_bed` -- Initialise it to a default value eg. `params.gene_bed = some_value`
WARN: The following invalid input values have been detected:

* --contrasts: /home/tud03125/pipeline/contrast_NORMAL.csv
* --source: fastq
* --clip_r1: null
* --clip_r2: null
* --three_prime_clip_r1: null
* --three_prime_clip_r2: null
* --trim_nextseq: null
* --skip_trimgalore_fastqc: false
* --rmats: true
* --rmats_splice_diff_cutoff: 0.0001
* --rmats_paired_stats: true
* --rmats_read_len: 40
* --rmats_novel_splice_site: false
* --rmats_min_intron_len: 50
* --rmats_max_exon_len: 500
* --dexseq_exon: true
* --save_dexseq_annotation: false
* --gff_dexseq: null
* --alignment_quality: 10
* --aggregation: true
* --save_dexseq_plot: true
* --n_dexseq_plot: 10
* --edger_exon: true
* --save_edger_plot: true
* --n_edger_plot: 10
* --dexseq_dtu: true
* --dtu_txi: dtuScaledTPM
* --sashimi_plot: true
* --miso_genes: ENSG00000004961, ENSG00000005302, ENSG00000147403
* --miso_genes_file: null
* --miso_read_len: 75
* --fig_width: 7
* --fig_height: 7                                                                                                                                  [49/1969]
* --min_samps_feature_expr: 2
* --min_samps_feature_prop: 2
* --min_samps_gene_expr: 4
* --min_feature_expr: 10
* --min_feature_prop: 0.1
* --min_gene_expr: 10
* --suppa: true
* --suppa_per_local_event: true
* --suppa_per_isoform: true
* --suppa_tpm: null
* --generateevents_pool_genes: true
* --generateevents_event_type: SE SS MX RI FL
* --generateevents_boundary: S
* --generateevents_threshold: 10
* --generateevents_exon_length: 100
* --psiperevent_total_filter: 0
* --diffsplice_local_event: true
* --diffsplice_isoform: true
* --diffsplice_method: empirical
* --diffsplice_area: 1000
* --diffsplice_lower_bound: 0
* --diffsplice_gene_correction: true
* --diffsplice_paired: true
* --diffsplice_alpha: 0.05
* --diffsplice_median: false
* --diffsplice_tpm_threshold: 0
* --diffsplice_nan_threshold: 0
* --clusterevents_local_event: true
* --clusterevents_isoform: true
* --clusterevents_sigthreshold: null
* --clusterevents_dpsithreshold: 0.05
* --clusterevents_eps: 0.05
* --clusterevents_metric: euclidean
* --clusterevents_separation: null
* --clusterevents_min_pts: 20
* --clusterevents_method: DBSCAN                                                                                                                   [13/1969]




------------------------------------------------------
                                       ,--./,-.
       ___    __  __  __  ___    /,-._.--~'
 |\ | |__ __ / ` / \ |__) |__        } {
 | \| |      \__, \__/ | \ |___    \`-._,-`-,
                                       `._,._,'
 nf-core/rnaseq v3.14.0-g1d0494a
------------------------------------------------------
Core Nextflow options
 revision             : 1.0.4
 runName              : confident_wozniak
 containerEngine      : singularity
 launchDir            : /home/tud03125/pipeline
 workDir              : /home/tud03125/pipeline/normal_human_liver_rnasplice_2/work
 projectDir           : /home/tud03125/.nextflow/assets/nf-core/rnasplice
 userName             : tud03125
 profile              : singularity
 configFiles          :

Input/output options
 input                : /home/tud03125/pipeline/normal_human_liver_design_rnasplice_2.csv
 outdir               : /home/tud03125/pipeline/normal_human_liver_rnasplice_2

Reference genome options
 fasta                : /home/tud03125/pipeline/Homo_sapiens.GRCh38.dna_sm.primary_assembly.fa
 gtf                  : /home/tud03125/pipeline/Homo_sapiens.GRCh38.108.gtf
 star_index           : /home/tud03125/STAR_index
 salmon_index         : /home/tud03125/salmon_index

Read filtering options
 ribo_database_manifest: /home/tud03125/ribo_database_manifest.txt

Alignment options
 min_mapped_reads     : 5

Optional outputs
 save_reference       : true

Quality Control
 rseqc_modules        : bam_stat, inner_distance, infer_experiment, junction_annotation, junction_saturation, read_distribution, read_duplication

!! Only displaying parameters that differ from the pipeline defaults !!

And, this same error message (you can see

gene_biotype

shown here, even with using GitHub's):

-[nf-core/rnaseq] Pipeline completed with errors-                                                             [53/1969]
ERROR ~ Error executing process > 'NFCORE_RNASPLICE:RNASPLICE:EDGER_DEU:SUBREAD_FEATURECOUNTS (SRX12134673)'

Caused by:
 Process `NFCORE_RNASPLICE:RNASPLICE:EDGER_DEU:SUBREAD_FEATURECOUNTS (SRX12134673)` terminated with an error exit stat$s (255)


Command executed:

 featureCounts \
     -B -C -g gene_biotype -t exon \
     -p \
     -T 6 \
     -a annotation.saf \
     -s 0 \
     -o SRX12134673.featureCounts.txt \
     SRX12134673_sorted.bam

 cat <<-END_VERSIONS > versions.yml
 "NFCORE_RNASPLICE:RNASPLICE:EDGER_DEU:SUBREAD_FEATURECOUNTS":
     subread: $( echo $(featureCounts -v 2>&1) | sed -e "s/featureCounts v//g")
 END_VERSIONS

Command exit status:
 255

Command output:                                                                                                                                     [6/1822]
 (empty)

Command error:
 WARNING: Skipping mount /var/singularity/mnt/session/etc/resolv.conf [files]: /etc/resolv.conf doesn't exist in container

         ==========    _____ _   _ ____ _____ ______         _____
         =====        / ____| | | | _ \| __ \| ____|  /\  | __ \
           =====     | (___ | | | | |_) | |__) | |__    / \ | | | |
             ====     \___ \| | | | _ <| _ /| __|  / /\ \ | | | |
               ====   ____) | |__| | |_) | | \ \| |____ / ____ \| |__| |
         ==========  |_____/ \____/|____/|_| \_\______/_/   \_\_____/
         v2.0.1

 //========================== featureCounts setting ===========================\\
 ||                                                                           ||
 ||            Input files : 1 BAM file                                      ||
 ||                          o SRX12134673_sorted.bam                        ||
 ||                                                                           ||
 ||            Output file : SRX12134673.featureCounts.txt                   ||
 ||                Summary : SRX12134673.featureCounts.txt.summary           ||
 ||             Annotation : annotation.saf (GTF)                            ||
 ||     Dir for temp files : ./                                              ||
 ||                                                                           ||
 ||                Threads : 6                                               ||
 ||                  Level : meta-feature level                              ||
 ||             Paired-end : yes                                             ||
 ||     Multimapping reads : not counted                                     ||
 || Multi-overlapping reads : not counted                                     ||
 ||  Min overlapping bases : 1                                               ||
 ||                                                                           ||
 ||         Chimeric reads : not counted                                     ||
 ||       Both ends mapped : required                                        ||
 ||                                                                           ||
 \\============================================================================//

 //================================= Running ==================================\\
 ||                                                                           ||
 || Load annotation file annotation.saf ...                                   ||
 ERROR: no features were loaded in format GTF. The annotation format can be specified by the '-F' option, and the required feature type can be specified by the '-t' option..                                                                                                                                             The porgram has to terminate.                                                                                                                                                                                                                                                                                          Work dir:                                                                                                                                                     /home/tud03125/pipeline/normal_human_liver_rnasplice_2/work/3d/7e5e1a1591904e68a4d0fa324b5eb0                                                                                                                                                                                                                          Tip: you can try to figure out what's wrong by changing to the process work dir and showing the script file named `.command.sh`

 -- Check '.nextflow.log' file for details

Processing complete.
Job ended on Sun Aug 25 13:20:11 EDT 2024

OK, let's see if the developers have any ideas. I am stumped, the only thing I can suggest is to try a minimal run on a different system, to make sure no weird configurations, etc., get carried over, but that may not be practical for you ... I don't get any of these parameter warnings, here is my command for reference:

nextflow run /path/to/nf-core-rnasplice-1.0.3/1_0_3 \
        -profile my_cluster -ansi-log false \
        --input "$samplefile" \
        --contrasts "$contrastfile" \
        --source fastq \
        --outdir "$pipeoutdir" \
        --gtf "$gtf" \
        --fasta "$fasta" \
        --star_index "$star_genome" \
        --save_trimmed \
        --aligner star_salmon \
        --pseudo_aligner false \
        --star_ignore_sjdbgtf \
        --seq_center "$seqcenter" \
        --save_unaligned \
        --skip_bigwig \
        --rmats \
        --rmats_read_len 135 \
        --dexseq_exon \
        --n_dexseq_plot 50 \
        --edger_exon \
        --n_edger_plot 50 \
        --sashimi_plot false \
        --suppa \
        -c "$project/dexseq_exon.config"

The (locally installed) profile I'm using just contains generic cluster-specific settings. The

dexseq_exon.config

sets a much longer run time for the

DEXSEQ_EXON

proess, because the default didn't cut it for my samples. The bash variables just contain various appropriate paths, etc. You could see if you can find the minimal set of parameters that reproduces the issue for you, and maybe even find a parameter set (perhaps by not running some tool, skipping so steps, etc) that does not run into this.

🤞

To run the pipeline, all you need is Nextflow and Singularity/Apptainer. All other software is in the containers and thus as old or new as the pipeline. Pipelines normally require some minimum Nextflow version, but since you can install that yourself, this should not be an issue. And for Singularity it matters much less, a version from 2021 should be fine (I used 3.7.4 until recently). In short, don't worry too much about software versions, as long as the software does what it needs to do.

Got it!