Predicting splice-altering variants

Usage

Example

  nextflow run main.nf --i sample.vcf --pcv --gpu --indels spliceai_scores.raw.indel.hg38.vcf.gz --snvs spliceai_scores.raw.snv.hg38.vcf.gz --sdb 2203_hg38 --ref grch38 --fa hg38.fa.gz --t 10 --o results/ -resume

Execution parameters

Parameter	Type	Description
--i	file path/expression	Input VCF file path or expression
--pcv	standalone parameter	Enables usage of precalculated scores
--gpu	standalone parameter	Enables SpliceAI GPU annotation
--indels	file path	Precomputed SpliceAI indels scores
--snvs	file path	Precomputed SpliceAI snvs scores
--sdb	folder path	SQUIRLS database folder
--fa	file path	FASTA reference file
--ref	string	Gene annotation file (either 'grch37', 'grch38' or a custom file)
--t	integer	CPU threads to use in multithreading-compatible tasks
--o	folder path	Output folder path

SQUIRLS database folder

SQUIRLS's database can be downloaded from it's documentation page. Make sure to download the adequate files according to the genome build being used.

Illumina's SpliceAI precalculated scores

Illumina made available annotations for all possible substitutions, 1 base insertions, and 1-4 base deletions within genes for download at their online platform. These annotations are free for academic and not-for-profit use; other use requires a commercial license from Illumina, Inc. To use them in our pipeline, you should execute it with the --pcv parameter and use the parameters indels and snvs to indicate the score file paths.

Glob expressions

To input multiple files at once, glob expressions can be utilized as described in the Nextflow documentation. Expressions must be enclosed in quotes while individual file paths do not.

Custom gene annotation files

It's possible to create custom gene annotation files using the files here as a template.

CPU threads

Some tasks in the pipeline allow for the usage of multithreading, and the amount of CPU threads used for those will be determined by the --t parameter

Results

Predictions are annotated on the the VCF file info field with the general format:

SQUIRLS_SCORE=SCORE;SpliceAI=ALLELE|SYMBOL|DS_AG|DS_AL|DS_DG|DS_DL|DP_AG|DP_AL|DP_DG|DP_DL;SplicePrediction=PREDICTION

SQUIRLS

SQUIRLS provides a single score that is the maximum predicted splicing pathogenicity score among the ones calculated for each variant. More information can be found at the SQUIRLS documentation

SpliceAI

SpliceAI provides a series of scores and information separated by |, in the order presented in the table below. More information can be found at the SpliceAI Github repository

Field	Description
ALLELE	Alternate allele
SYMBOL	Gene symbol
DS_AG	Delta score (acceptor gain)
DS_AL	Delta score (acceptor loss)
DS_DG	Delta score (donor gain)
DS_DL	Delta score (donor loss)
DP_AG	Delta position (acceptor gain)
DP_AL	Delta position (acceptor loss)
DP_DG	Delta position (donor gain)
DP_DL	Delta position (donor loss)

Prediction

The SplicePrediction is the final annotation of the pipeline for each variant according to the cutoffs and annotation mode utilized. It indicates P for a pathogenic prediction and N for a non-pathogenic prediction.

References

Jaganathan K, Kyriazopoulou Panagiotopoulou S, McRae JF, et al. Predicting Splicing from Primary Sequence with Deep Learning. Cell. 2019;176(3):535-548.e24. doi:10.1016/j.cell.2018.12.015

Danis D, Jacobsen JOB, Carmody LC, et al. Interpretable prioritization of splice variants in diagnostic next-generation sequencing [published correction appears in Am J Hum Genet. 2021 Nov 4;108(11):2205]. Am J Hum Genet. 2021;108(9):1564-1577. doi:10.1016/j.ajhg.2021.06.014