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