epiVIA 1.1.2

Virial Integration Analysis with epigenetic data

Description of epiVIA

Vector Integration Analysis with epigenomic assays.

(1) Identify Chimeric reads from bam file

EpiVIA parses each read and identifies the chimeric fragments, which are further classified into three different categories based on how the read pair is aligned to the combined reference genome: (1) “pair-chimeric” are the fragments with one read mapped to the host genome and the other mapped to the provirus (or vector) genome; (2) “host-chimeric” are the fragments that both ends mapped to the host genome, with a small soft-clipped fragment at one end which can exactly match the start or end of LTR sequence of provirus; (3) “viral-chimeric” are the fragments that are properly pair mapped to either end of the provirus genome, with a soft-clipped fragment at the end which can be mapped to the host genome with a non-zero mapping quality. Since the real chimeric fragment won’t be soft-clipped at 3’ end of 5’ LTR and 5’ end of 3’ LTR, while these cases can present in the alignment results, we corrected the aligned position for these fragments.

(2) Identify integration site with chimeric reads

Although there is pair rescue during the alignment of BWA, we still found some pair-chimeric fragments which have soft-clipped ends from the other genome. Hence, we intend to identify the integration site with the soft-clipped read. If the read mapped to host genome is a soft-clipped alignment, we can determine the precise integration site if the clipped oligonucleotide exactly match either end of the LTR of provirus. In the other case, if the viral read that is aligned to the end of LTR is soft-clipped, we try to search the clipped fragment in the context of up or down 200 bp where the host read aligned, and an exact match can tell us the precise integration site. For host-chimeric and viral-chimeric fragments, we identify the integration site while classifying these fragments. These integration sites are further annotated with genes, transposable elements and enhancer annotation from UCSC genome browser database.

Install

dependencies

Make sure UCSC "bigBedToBed" tool is in your PATH

install with pip

pip install epiVIA

intall from github page

git clone https://github.com/wangwl/epiVIA

cd epiVIA

python setup.py install

Output

This pipeline has multiple output files for the downstream analysis of integration sites, cells with integration sites and with proviral reads. The following tables are detailed descriptions of the output files and their file formats.

Result files

filename	description
Integration_sites.list	Identified integration sites
barcode_integration.list	Ingretation sites sorted by cell barcoeds
Cell_summary.txt	Number of chimeric and viral reads in each cell
Vector_coverage.csv	Coverage of viral reads at each positioin along the proviral genome

Integration_sites.list

Column name	Description
Genome	Reference genome version (eg: hg19, hg38, mm9, mm10)
Chrom	Chromosome of the integration site
ChrStart	Start position in host genome where the chimeric reads aligned, or the integration site
ChrEnd	End Position in host genome where the chimeric reads aligned, or the integration site
VectorStart	Start posision in proviral genome where the chimeric reads aligned
VectorEnd	End position in proviral genome where the chimeric reads aligned
LTR	The chimeric reads is from 5' LTR or 3' LTR
InsertOri	Integration orietation of the proviral genome based on the reference sequence
InsertGene	The annotated gene at the integration site of the host genome
GeneOri	Orientation of the gene
ExonIntron	The integration is in intron or exon region of the gene
Enhancer	Enhancer or Promoter information at the integration site
Elite	If the integration site locates in an enhancer based on UCSC annotation, whether it's Elite
TEFamily	Family of transposable elements annotation at the integration site
TEClass	Class of transposable elements annotation at the integration site
CellNumber	Number of cells have this integration site
CellBarcodes	Cell barcodes that have this integration site
ReadInCell	Number of reads in each cell that support this integration site
ReadNumber	Total number of reads that support this integration site
Quality	The quality of the integration site, adapted from the mapping quality of BWA

barcode_integration.list

Each cell that have integration sites. If a cell have multiple integration sites, ".1", ".2", ".3", ..., were appended to the cell barcode.

Column name	Description
CellBarcode	Cell Barcode of the cell
Chrom	Chromosome of the integration site
ChrStart	Start position in host genome where the chimeric reads aligned, or the integration site
ChrEnd	End Position in host genome where the chimeric reads aligned, or the integration site
VectorStart	Start posision in proviral genome where the chimeric reads aligned
VectorEnd	End position in proviral genome where the chimeric reads aligned
InsertGene	The annotated gene at the integration site of the host genome
ExonIntron	The integration is in intron or exon region of the gene
Enhancer	Enhancer or Promoter information at the integration site
TEFamily	Family of transposable elements annotation at the integration site
TEClass	Class of transposable elements annotation at the integration site
Quality	The quality of the integration site, adapted from the mapping quality of BWA

Cell_summary.txt

Column name	Description
CellBarcode	Cell Barcode of the cell
ChimeraSum	Total number of chimeric reads from the cell
Pair	Number of 'Pair' chimeric reads (case a) from the cell
HostRead	Number of 'HostRead' chimeric reads (case b) from the cell
VectorRead	Number of 'VectorRead' chimeric reads (case c) from the cell
integration	Number of integration sites in the cell
VectorSum	Total number of reads from the integrated proviral genome
noLTR	Number of proviral reads that is not from LTR regions
LTR	Number of proviral reads that is from LTR regions

Temporary files

epiVIA has options of "--candidate_bam" and "--chimera" for writing the bam records used for identifying chimeric reads and those that are classified as chimeric between host genome and viral genome.

Example

epiVIA --Vector data/p746.fa --ltr5_start 17 --ltr5_end 693 --ltr3_start 9149 --ltr3_end 9825 --HostIndex GRCh38 --Host2bit hg38.2bit --tempdir . --chimera_bam chimera.bam --gbdb http://hgdownload.soe.ucsc.edu/gbdb/ test/data/candidate.bam .