Allele-specific junction

Allele-Specific Junction Quantification

To quantify allele-specific junction based on the LongcallR-phased BAM, run the following command:

python longcallR-asj.py
-b <phased_bam>
-a <annotation>
-f <reference>
-o <output_prefix>
-t <threads>
-g <gene_types>
-m <min_sup>

+ -b, --bam_file: longcallR phased bam file + -a, --annotation_file: Annotation file GTF for GFF3 format + -f, --reference: Reference genome file + -o, --output_prefix: Prefix of output files + -t, --threads: Number of threads + -g, --gene_types: Gene types to be analyzed. Default: ["protein_coding", "lncRNA"] + -m, --min_sup: Minimum support of phased reads for counting event. Default: 10

High-Confidence ASJ Using Known Genomic SNPs

To increase confidence by requiring allele-specific junction to be supported by both longcallR's RNA SNPs and known genomic SNPs, use the following command:

python longcallR-asj.py
-b <phased_bam>
-a <annotation>
-f <reference>
--dna_vcf <genomic_vcf>
--rna_vcf <rna_vcf>
-o <output_prefix>
-t <threads>
-g <gene_types>
-m <min_sup>

+ --dna_vcf: DNA vcf file + --rna_vcf: longcallR phased RNA vcf file

Store Allele-Specific Junctions in BED Format for IGV Visualization

To visualize allele-specific junctions in IGV, extract regions with a p-value below a specified threshold (default: 1e-10) and save them in BED format:

python asj_to_bed.py output.asj.tsv [p_value_threshold] > output.asj.bed

- output.asj.tsv: Input file containing allele-specific junctions - p_value_threshold (optional): Significance threshold (default: 1e-10) - output.asj.bed: Output BED file for IGV visualization

Output format

Running longcallR-asj.py will generate three output files output.asj.tsv, output.asj_gene.tsv and output.gene_coverage.tsv. The formats of these files are described below:

1.output.asj.tsv

Column	Description
`#Junction`	Junction coordinates in the format `chr:start-end`
`Strand`	Strand of the junction (`+` or `-`)
`Junction_set`	Junction set identifier (cluster of related junctions)
`Phase_set`	Phase set ID
`Hap1_absent`	Number of reads from haplotype 1 where the junction is absent
`Hap1_present`	Number of reads from haplotype 1 where the junction is present
`Hap2_absent`	Number of reads from haplotype 2 where the junction is absent
`Hap2_present`	Number of reads from haplotype 2 where the junction is present
`P_value`	P-value for allele-specific junction
`SOR`	Symmetric odds ratio
`Novel`	Whether the junction is novel (`TRUE` / `FALSE`)
`GT_AG`	Whether junction has canonical GT-AG splice sites (`TRUE` / `FALSE`)
`Gene_name`	Associated gene symbol

2.output.asj_gene.tsv

Column	Description
`#Gene_name`	Gene symbol that containing allele-specific junctions
`Chr`	Chromosome
`P_value`	P-value for the most significant allele-specific junction in the gene
`SOR`	Symmetric odds ratio for the most significant allele-specific junction in the gene

3.output.gene_coverage.tsv

Column	Description
`#Gene_name`	Gene symbol
`Chr`	Chromosome
`Start`	start position of gene, 1-based, inclusive
`End`	end position of gene, 1-based, inclusive
`Num_reads`	Number of reads in the gene