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Input File Preparation

The main input files to the pVACsplice pipeline are an annotated VCF file and a RegTools output file (tsv).

Step 1: Annotate VCF file

Please see the pVACseq Input File Preparation for instructions on how to prepare an annotated VCF file with the following differences:

  • pVACsplice does not require annotations with the VEP Frameshift and Wildtype plugins so those parameters can be omitted when running VEP.

  • pVACsplice does not use a phased VCF of proximal variants so that step can be skipped.

Step 2: Run RegTools

RegTools is a set of tools that integrate DNA-seq and RNA-seq data to help interpret mutations in a regulatory and splicing context. To run pVACsplice, you must first run RegTools cis-splice-effects identify to generate a list of non-canonical splicing junctions created from cis-acting regulatory variants. The output tsv file (-o option) is an input to pVACsplice. Here is an example command:

regtools cis-splice-effects identify \
-o <output tsv file> \
-s <rna_strand> \
annotated_variants.vcf \
rna_alignments.bam \
ref.fa \
annotations.gtf

Please see the RegTools documentation for more information.