LeafCutter Error

[AlessandraLongo]

Hello everyone,
I'm encountering an issue while running a Baltica workflow (version 1.2.4, python 3.7.12) with unstranded RNA-seq data. The execution halted at LeafCutter differential splicing analysis step. Here’s a summary of the error message:

Command: baltica all path/to/config.yaml - - use-singularity - -verbose - -cores 10

Executing subworkflow leafcutter.
Building DAG of jobs...
Using shell: /usr/bin/bash
Provided cores: 18
Rules claiming more threads will be scaled down.
Job stats:
job count

---

leafcutter_differential_splicing 1
total 1

Select jobs to execute...

[Thu Oct 31 15:34:34 2024]
rule leafcutter_differential_splicing:
input: leafcutter/exons.gtf.gz, leafcutter/RGNEF-vs-CTR/RGNEF-vs-CTR_perind_numers.counts.gz, leafcutter/RGNEF-vs-CTR/diff_introns.txt
output: leafcutter/RGNEF-vs-CTR/RGNEF-vs-CTR_cluster_significance.txt
log: logs/leafcutter/leafcutter_differential_splicing/RGNEF-vs-CTR.log
jobid: 0
reason: Missing output files: leafcutter/RGNEF-vs-CTR/RGNEF-vs-CTR_cluster_significance.txt
wildcards: comp_names=RGNEF-vs-CTR
threads: 10
resources: tmpdir=/tmp

Building DAG of jobs...
Using shell: /usr/bin/bash
Provided cores: 18
Rules claiming more threads will be scaled down.
Select jobs to execute...
Activating singularity image /home/cappelli/.baltica/singularity/2f0497e2227c782b99d020de15ba0db2.simg
/bin/bash: /root/.bashrc: Permission denied
Loading counts from leafcutter/RGNEF-vs-CTR/RGNEF-vs-CTR_perind_numers.counts.gz
Loading metadata from leafcutter/RGNEF-vs-CTR/diff_introns.txt
Encoding as CTR =0, RGNEF =1
Settings:
$output_prefix
[1] "leafcutter/RGNEF-vs-CTR/RGNEF-vs-CTR"
$max_cluster_size
[1] Inf
$min_samples_per_intron
[1] 2
$min_samples_per_group
[1] 2
$min_coverage
[1] 5
$timeout
[1] 30
$num_threads
[1] 10
$exon_file
[1] "leafcutter/exons.gtf.gz"
$init
[1] "smart"
$seed
[1] 12345
$help
[1] FALSE

Running differential splicing analysis...
[Thu Oct 31 15:34:36 2024]
Error in rule leafcutter_differential_splicing:
jobid: 0
input: leafcutter/exons.gtf.gz, leafcutter/RGNEF-vs-CTR/RGNEF-vs-CTR_perind_numers.counts.gz, leafcutter/RGNEF-vs-CTR/diff_introns.txt
output: leafcutter/RGNEF-vs-CTR/RGNEF-vs-CTR_cluster_significance.txt
log: logs/leafcutter/leafcutter_differential_splicing/RGNEF-vs-CTR.log (check log file(s) for error details)
shell:

    leafcutter_ds_pair.R --exon_file=leafcutter/exons.gtf.gz         --min_coverage 5          leafcutter/RGNEF-vs-CTR/RGNEF-vs-CTR_perind_numers.counts.gz leafcutter/RGNEF-vs-CTR/diff_introns.txt --num_threads 10 --output_prefix=leafcutter/RGNEF-vs-CTR/RGNEF-vs-CTR         -i 2 -g 2 2> logs/leafcutter/leafcutter_differential_splicing/RGNEF-vs-CTR.log
    
    (one of the commands exited with non-zero exit code; note that snakemake uses bash strict mode!)

Shutting down, this might take some time.
Exiting because a job execution failed. Look above for error message
Shutting down, this might take some time.
Exiting because a job execution failed. Look above for error message
Complete log: .snakemake/log/2024-10-31T153434.446038.snakemake.log<<

From the RGNEF-vs-CTR.log I got this:
Loading required package: Rcpp
Loading required package: doMC
Loading required package: foreach
Loading required package: iterators
Loading required package: parallel
Error in colnames<-(*tmp*, value = c("chr", "start", "end", "clu")) :
attempt to set 'colnames' on an object with less than two dimensions
Calls: differential_splicing -> get_intron_meta -> colnames<-
Execution halted

Plus, also the intron_clustering.log reported a strange output:
scanning /home/cappelli/Desktop/Baltica_RGNEF/leafcutter/CTR_1.junc...
scanning /home/cappelli/Desktop/Baltica_RGNEF/leafcutter/CTR_2.junc...
scanning /home/cappelli/Desktop/Baltica_RGNEF/leafcutter/CTR_3.junc...
scanning /home/cappelli/Desktop/Baltica_RGNEF/leafcutter/RGNEF_1.junc...
scanning /home/cappelli/Desktop/Baltica_RGNEF/leafcutter/RGNEF_2.junc...
scanning /home/cappelli/Desktop/Baltica_RGNEF/leafcutter/RGNEF_3.junc...
Parsing...

Wrote 0 clusters..
Split into 0 clusters...
Sorting /home/cappelli/Desktop/Baltica_RGNEF/leafcutter/CTR_1.junc..
Sorting /home/cappelli/Desktop/Baltica_RGNEF/leafcutter/CTR_2.junc..
Sorting /home/cappelli/Desktop/Baltica_RGNEF/leafcutter/CTR_3.junc..
Sorting /home/cappelli/Desktop/Baltica_RGNEF/leafcutter/RGNEF_1.junc..
Sorting /home/cappelli/Desktop/Baltica_RGNEF/leafcutter/RGNEF_2.junc..
Sorting /home/cappelli/Desktop/Baltica_RGNEF/leafcutter/RGNEF_3.junc..
merging 6 junction files...
merging 6 files done.

I am not sure if it is an upstream input file problem (I used an annotation.gtf file converted from gff3 using Agat) cause I checked the format and seems to be correct.

Any insights or suggestions would be greatly appreciated!
Thank you for your help!

Alessandra

[tbrittoborges]

The origin of the problem here is on this line: Wrote 0 clusters.. This means no clusters (splicing events) were selected. Which GTF/GFF annotation are you using? Is it the same you used to align the BAM files?