Missing exons in SpliceWiz.ref.gz

[akikuno]

Thank you for developing SpliceWiz.

I used SpliceWiz for a differential analysis between a sample with exon 2 of the Strap gene deleted as a positive control and wild-type. However, the positive control (exon 2 of the Strap gene) was not detected. I suspect this may be due to the absence of Strap's exon in the SpliceWiz.ref.gz generated by the buildRef function. Is this the case? If so, why is Strap's exon not included in SpliceWiz.ref.gz, and how can I include it? I would appreciate your guidance.

Below are the steps to reproduce.

Setup environment by conda

conda create -y -n splicewiz python=3.10
conda install -y -n splicewiz -c conda-forge -c bioconda -c defaults bioconductor-splicewiz
conda activate splicewiz

conda list | grep splicewiz
# bioconductor-splicewiz    1.4.0             r43hf17093f_0    bioconda

Download reference genome and annotation

mkdir -p data/genome
wget -O - https://ftp.ensembl.org/pub/current_fasta/mus_musculus/dna/Mus_musculus.GRCm39.dna.primary_assembly.fa.gz |
gzip -dc > data/genome/mm39.fa

wget -O - https://ftp.ensembl.org/pub/current_gtf/mus_musculus/Mus_musculus.GRCm39.111.gtf.gz |
gzip -dc > data/genome/mm39.gtf

Build reference

library(SpliceWiz)
ref_path <- "data/genome/splicewiz"
buildRef(
    reference_path = ref_path,
    fasta = "data/genome/mm39.fa",
    gtf = "data/genome/mm39.gtf",
    ontologySpecies = "Mus musculus"
)

Grep Strap

zcat data/genome/splicewiz/SpliceWiz.ref.gz | grep Strap

[alexchwong]

Hi @akikuno

SpliceWiz.ref.gz is a data reference to inform the program which introns should be evaluated for IR. It is not used for other forms of alternative splicing.

It is best if you peruse the reference for the alternative splicing reference using viewASE(). For your specific task, I suspect you are looking for skipped exon event of Strap exon 2.

[akikuno]

@alexchwong

Thank you so much for your guidance! I will look into it.