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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