AGAIN

Genome-wide detection of human intronic AG-gain variants between the splicing branchpoint and acceptor site

Introduction

• Human variants that introduce an AG in the intronic region between the branchpoint (BP) and the canonical splice acceptor site (ACC) of protein-coding genes can disrupt pre-mRNA splicing.

• Based on our previously developed BPHunter (GitHub and webserver), we precisely delineate the BP-ACC segments of all human introns of protein-coding genes, for the first time. We find an extreme depletion of AG/YAG in the high-risk [BP+8, ACC-4] region.

• AGAIN is a genome-wide approach to pinpoint intronic AG-gain variants (SNVs, insertions, deletions) in the BP-ACC region of human genomes. AGAIN also predicts protein-level outcomes resulting from two major missplicing consequences (new acceptor site & complete exon skipping).

• This standalone version can be implemented into NGS analysis by a one-line command. We also provide the AGAIN webserver with a user-friendly interface.

News

• Feb 2023: AGAIN webserver & GitHub was launched.
• Sep 2022: AGAIN prototype was completed.
• Jan 2022: Inspired by the BPHunter study, AGAIN idea was conceived.

Usage

Current version: version-1

Dependency

The code is written in python3, and requires bedtools installed.

Reference datasets

Due to the data limit in GitHub, please download the AGAIN reference datasets and put them into your AGAIN folder.

File Format

Input: Variants in VCF format, with 5 mandatory and tab-delimited fields (CHROM, POS, ID, REF, ALT), (Sample_variants.vcf)

Output: AGAIN-detected variants will be output with the following annotations.

• SAMPLE (only for AGAIN_VCF_batch.py)
• CHROM, POS, ID, REF, ALT (exactly the same as input)
• STRAND: +/-
• VAR_TYPE: snv, x-nt del, x-nt ins
• GENE: gene symbol
• TRANSCRIPT_IVS: ENST123456789_IVS10
• CANONICAL: canonical transcript_IVS, or '.'
• AGAIN_ZONE: ZONE1/ZONE2, ZONE1 is from 1st BP to ACC, ZONE2 is from 2nd BP to 1st BP
• AGAIN_YAG: YES/NO, if the AG-gain variant also fits YAG
• AGAIN_BP_DIST: the distance from the created AG to BP
• AGAIN_ACC_DIST: the distance from the created AG to ACC
• AGAIN_HIGHRISK: YES/NO, if the AG-gain variant falls inside the high-risk [BP+8, ACC-4] region
• AGAIN_SCORE: score of the AG-gain variant (suggested cutoff >= 3, max = 5)
• PROT_SEQ_WT: wild-type protein sequence
• PROT_SEQ_NEW_ACC: consequent protein sequence if a new acceptor site is created
• HGVS_NEW_ACC: protein-level HGVS annotation if a new acceptor site is created
• PROT_SEQ_EXON_SKIP: consequent protein sequence if exon skipping occurs
• HGVS_EXON_SKIP: protein-level HGVS annotation if exon skipping occurs

Command & Parameters (AGAIN_VCF.py)

python AGAIN_VCF.py -i Sample_variants.vcf

python AGAIN_VCF.py -g GRCh37 -t all -i Sample_variants.vcf

Parameter	Type	Description	Default
-g	str	human reference genome assembly (GRCh37 / GRCh38)	GRCh37
-t	str	all / canonical transcripts?	all
-i	file	variants in VCF format, with 5 fields (CHROM, POS, ID, REF, ALT)	N.A.

Command & Parameters (AGAIN_VCF_batch.py)

python AGAIN_VCF_batch.py -d directory -s samplelist.txt -o output.csv

python AGAIN_VCF_batch.py -g GRCh37 -t all -d directory -s samplelist.txt -o output.csv

Parameter	Type	Description	Default
-g	str	human reference genome assembly (GRCh37 / GRCh38)	GRCh37
-t	str	all / canonical transcripts?	all
-d	str	directory of VCF files	N.A.
-s	file	sample list (without .vcf extension) in the above directory	N.A.
-o	str	output filename in csv	N.A.

AGAIN Scoring Scheme

Reference

• Zhang P. et al. Genome-wide detection of human intronic AG-gain variants located between the splicing branchpoint and canonical splice acceptor site. 2023.

Contact

Developer: Peng Zhang, Ph.D.

Email: [email protected]

Laboratory: St. Giles Laboratory of Human Genetics of Infectious Diseases

Institution: The Rockefeller University, New York, NY, USA