Hi Everyone, I’m right now attempting to re-run

nf-core/rnasplice

pipeline with using

USCS

files. Last time I successfully ran them, the

GTF

and

FASTA

files were both

Ensembl

format. However, 1) my boss ran hers with

USCS

(last year, older's:

mm10

instead of

mm39

) and 2) we are getting different results. So, for better reproducibility, we think it’s best if I restart mine but with `USCS`’s

FASTA

and

GTF

files. I’ve 1) downloaded `USCS`’s genome

FASTA

and

GTF

files through these: FASTA:

# Download the genome FASTA file 
wget <https://hgdownload.soe.ucsc.edu/goldenPath/mm39/bigZips/mm39.fa.gz>
# Uncompress the FASTA file 
gunzip mm39.fa.gz

GTF:

# Download the RefSeq GTF file
wget <https://hgdownload.soe.ucsc.edu/goldenPath/mm39/bigZips/genes/mm39.ncbiRefSeq.gtf.gz>

# Uncompress the GTF file
gunzip mm39.ncbiRefSeq.gtf.gz

# Rename the file for clarity (optional)
mv mm39.ncbiRefSeq.gtf mm39_RefSeq.gtf

And, rebuild both STAR and salmon index through these:

Rebuild STAR index:
STAR --runMode genomeGenerate \
    --genomeDir /home/tud03125/STAR_index_mouse_ucsc \
    --genomeFastaFiles /home/tud03125/pipeline/mm39.fa \
    --sjdbGTFfile /home/tud03125/pipeline/mm39_RefSeq.gtf \
    --sjdbOverhang 100

Rebuild salmon index:
# Generate transcriptome FASTA using gffread
gffread /home/tud03125/pipeline/mm39_RefSeq.gtf \
   -g /home/tud03125/pipeline/mm39.fa \
   -w /home/tud03125/pipeline/mm39_transcripts.fa

# Build the Salmon index using the generated transcriptome FASTA file
salmon index \
      -t /home/tud03125/pipeline/mm39_transcripts.fa \
      -i /home/tud03125/salmon_index_mouse_ucsc

So after gathering `USCS`’s

FASTA

and

GTF

files, and rebuilding indexes from `USCS`’s files, I re-ran the pipeline using that format:

#!/bin/bash

# Debugging: Print environment information
echo "Job started on $(date)"
echo "Running on $(hostname)"
echo "Current directory: $(pwd)"

# Output directory
OUTPUT_DIR="/home/tud03125/pipeline/mouse_liver_rnasplice_UCSC"

# Create the output directory if it doesn't exist
mkdir -p "$OUTPUT_DIR"

# Load necessary modules
module load gcc/11.2.0
module load singularity

# Change directory to your pipeline directory
cd /home/tud03125/pipeline

# Set Nextflow version
export NXF_VER=24.04.4

# Install Nextflow if not already installed
if [ ! -f ./nextflow ]; then
   echo "Nextflow not found, downloading..."
   curl -s <https://get.nextflow.io> | bash
   chmod +x nextflow
fi

# Set Nextflow work directory to avoid quota issues
export NXF_WORK="$OUTPUT_DIR/work"

# Extract gene versions for MISO_SASHIMI
#GTF_FILE="/home/tud03125/pipeline/Mus_musculus.GRCm39.112.gtf"
#GENE_VERSIONS=$(awk -F '[\t ;"]+' '/gene_id "(ENSMUSG00000019836|ENSMUSG00000020887)"/ {for (i=1; i<=NF; i++) if ($i=="gene_id") {gid=$(i+1)} else if ($i=="gene_version") {print gid"."$(i+1)}}' $GTF_FILE | sort | uniq | paste -sd "," -)

# The top part's for this code line (if there ever comes trouble with it): --miso_genes "$GENE_VERSIONS"

# Run Nextflow with your local rnasplice pipeline and include the mouse liver data
echo "Starting Nextflow pipeline..."
./nextflow run nf-core/rnasplice -resume -profile singularity \
   --input /home/tud03125/pipeline/mouse_liver_2.csv \
   --fasta /home/tud03125/pipeline/mm39.fa \
   --gtf /home/tud03125/pipeline/mm39_RefSeq.gtf \
   --save_reference \
   --outdir "$OUTPUT_DIR" \
   --star_index /home/tud03125/STAR_index_mouse_ucsc \
   --salmon_index /home/tud03125/salmon_index_mouse_ucsc \
   --rmats \
   --rmats_novel_splice_site \
   --suppa \
   --contrasts /home/tud03125/pipeline/contrast_mouse_liver_2.csv

# Debugging: Print completion message and end timestamp
echo "Processing complete."
echo "Job ended on $(date)"

So, I started this Shell script, and I ran into this problem, currently:

ERROR ~ Error executing process > 'NFCORE_RNASPLICE:RNASPLICE:DEXSEQ_DEU:DEXSEQ_ANNOTATION (mm39_RefSeq.gtf)'

Caused by:
 Process `NFCORE_RNASPLICE:RNASPLICE:DEXSEQ_DEU:DEXSEQ_ANNOTATION (mm39_RefSeq.gtf)` terminated with an error exit status (1)


Command executed:

 dexseq_prepare_annotation.py mm39_RefSeq.gtf DEXSeq.gff

 cat <<-END_VERSIONS > versions.yml
 "NFCORE_RNASPLICE:RNASPLICE:DEXSEQ_DEU:DEXSEQ_ANNOTATION":
     htseq: $(python -c "import pkg_resources; print(pkg_resources.get_distribution('htseq').version)")
 END_VERSIONS

Command exit status:
 1

Command output:
 (empty)

Command error:
 Traceback (most recent call last):
   File "/home/tud03125/.nextflow/assets/nf-core/rnasplice/bin/dexseq_prepare_annotation.py", line 130, in <module>
     raise ValueError("Same name found on two chromosomes: %s, %s" % (str(l[i]), str(l[i + 1])))
 ValueError: Same name found on two chromosomes: <GenomicFeature: exonic_part 'Erdr1x' at chrY: 90827617 -> 90827734 (strand '+')>, <GenomicFeature: exonic_part 'Erdr1x' at chrX: 168793521 -> 168794059 (strand '+')>

Work dir:
 /home/tud03125/pipeline/mouse_liver_rnasplice_UCSC/work/a7/c2272e12426b7d7b882261db579079

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

Anyone had this issue before? I think I also attempted with

GENCODE

files and indexes too, and the error’s very similar to this.