Problem with SUPPA2 Psi calculation

Question

[achanda]

Hi community,

I have been trying to perform alternative splicing using SUPPA2. Briefly, I have 28 human samples on which RNAseq was performed. From those I have generated the count files with Kallisto using gencode.v44 indices. Using the same gencode.v44 gtf file, I have generated the different .ioe files for alternate splicing events. Now, my original abundance.tsv file generated by kallisto looked like this:

$ head abundance.tsv
target_id       length  eff_length      est_counts      tpm
ENST00000456328.2|ENSG00000290825.1|-|OTTHUMT00000362751.1|DDX11L2-202|DDX11L2|1657|lncRNA|     1657    1658    0       0
ENST00000450305.2|ENSG00000223972.6|OTTHUMG00000000961.2|OTTHUMT00000002844.2|DDX11L1-201|DDX11L1|632|transcribed_unprocessed_pseudogene|       632     633     0       0
ENST00000488147.1|ENSG00000227232.5|OTTHUMG00000000958.1|OTTHUMT00000002839.1|WASH7P-201|WASH7P|1351|unprocessed_pseudogene|    1351    1352    0       0
ENST00000619216.1|ENSG00000278267.1|-|-|MIR6859-1-201|MIR6859-1|68|miRNA|       68      69      0       0
ENST00000473358.1|ENSG00000243485.5|OTTHUMG00000000959.2|OTTHUMT00000002840.1|MIR1302-2HG-202|MIR1302-

Now I processed it to look like this:

$ head abundance_preprocessed.tsv
ENST00000456328.2       0.000000
ENST00000450305.2       0.000000
ENST00000488147.1       0.000000
ENST00000619216.1       0.000000
ENST00000473358.1       0.000000
ENST00000469289.1       0.000000
ENST00000607096.1       0.000000
ENST00000417324.1       0.000000
ENST00000461467.1       0.000000
ENST00000606857.1       0.000000

One of the generated ioe files looks like this:

$ head gencode.v44_A3_strict.ioe
seqname gene_id event_id        alternative_transcripts total_transcripts
chr1    ENSG00000237491.10      ENSG00000237491.10;A3:chr1:779092-803919:779092-803951:+        ENST00000655765.1,ENST00000670700.1,ENST00000657896.1,ENST00000658648.1,ENST00000656571.1,ENST00000669749.1,ENST00000666217.1,ENST00000665719.1       ENST00000665719.1,ENST00000412115.2,ENST00000666217.1,ENST00000658648.1,ENST00000669749.1,ENST00000657896.1,ENST00000670700.1,ENST00000655765.1,ENST00000656571.1
chr1    ENSG00000237491.10      ENSG00000237491.10;A3:chr1:779092-803919:779092-803922:+        ENST00000655765.1,ENST00000670700.1,ENST00000657896.1,ENST00000658648.1,ENST00000656571.1,ENST00000669749.1,ENST00000666217.1,ENST00000665719.1       ENST00000665719.1,ENST00000443772.2,ENST00000666217.1,ENST00000658648.1,ENST00000669749.1,ENST00000657896.1,ENST00000670700.1,ENST00000655765.1,ENST00000656571.1
chr1    ENSG00000237491.10      ENSG00000237491.10;A3:chr1:807323-809622:807323-809658:+        ENST00000412115.2,ENST00000670700.1,ENST00000657896.1,ENST00000658648.1 ENST00000665719.1,ENST00000658648.1,ENST00000588951.5,ENST00000669749.1,ENST00000657896.1,ENST00000670700.1,ENST00000412115.2,ENST00000656571.1

So now I am trying to generate Psi results using the slurm:

#!/bin/bash
#SBATCH --job-name=suppa_psi_calculation
#SBATCH --output=/home/achanda/%x_%j.out
#SBATCH --error=/home/achanda/%x_%j.err
#SBATCH --time=04:00:00
#SBATCH --cpus-per-task=16
#SBATCH --mem=128G

# Load Python or any required modules
# module load python3

# Directories
SUPPA_DIR=~/software/SUPPA-2.3
EVENT_DIR=~/AS_events
PROCESSED_DIR=~/Processed  # Correct directory for processed files
OUT_DIR=~/psi_results

# Create the output directory
mkdir -p ${OUT_DIR}

# Iterate over each event type
for EVENT_FILE in ${EVENT_DIR}/*.ioe; do
    EVENT_TYPE=$(basename ${EVENT_FILE} .ioe)  # Correct extraction of the event type

    # Iterate over each sample's processed file in the Processed directory
    for PROCESSED_FILE in ${PROCESSED_DIR}/*_abundance_preprocessed.tsv; do
        SAMPLE_NAME=$(basename ${PROCESSED_FILE} _abundance_preprocessed.tsv)  # Correct extraction of the sample name
        OUTPUT_PREFIX="${OUT_DIR}/${SAMPLE_NAME}_${EVENT_TYPE}"

        echo "Starting processing of ${SAMPLE_NAME} for event type ${EVENT_TYPE}..."
        python3 ${SUPPA_DIR}/suppa.py psiPerEvent -i ${EVENT_FILE} -e ${PROCESSED_FILE} -o ${OUTPUT_PREFIX}

        if [ $? -eq 0 ]; then
            echo "Successfully processed ${SAMPLE_NAME} for event type ${EVENT_TYPE}."
        else
            echo "Failed to process ${SAMPLE_NAME} for event type ${EVENT_TYPE}. Check error logs for details."
        fi
    done
done

However,

$ head suppa_psi_calculation_28165525.err
INFO:lib.tools:File ~/Sample1_abundance_preprocessed.tsv opened in reading mode.
INFO:psiCalculator:Buffering transcript expression levels.
ERROR:lib.tools:1, in line 2. Skipping line...
ERROR:lib.tools:2, in line 3. Skipping line...
ERROR:lib.tools:3, in line 4. Skipping line...
ERROR:lib.tools:4, in line 5. Skipping line...
ERROR:lib.tools:5, in line 6. Skipping line...
ERROR:lib.tools:6, in line 7. Skipping line...
ERROR:lib.tools:7, in line 8. Skipping line...
ERROR:lib.tools:8, in line 9. Skipping line...

Can someone help with what is going wrong? I can provide further details if needed.

[Ram]

1. It's alternative splicing, not alternate splicing. The former means "the other option", the latter means "change".
2. Please use the formatting bar (especially the code option) to present your post better. You can use backticks for inline code (`text` becomes text), or use one of (a) the option highlighted in the image below/ (b) fenced code blocks for multi-line code. Fenced code blocks are useful in syntax highlighting. If your code has long lines with a single command, break those lines into multiple lines with proper escape sequences so they're easier to read and still run when copy-pasted. I've done it for you this time.
   

[achanda]

I appreciate it. I am new here, so learning the tropes!

[Ram]

I hope you're learning the ropes, we don't have a ton of tropes here :-)