Package: epiVIA
Description of epiVIAVector Integration Analysis with epigenomic assays.(1) Identify Chimeric reads from bam fileEpiVIA 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 readsAlthough 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.InstalldependenciesMake sure UCSC "bigBedToBed" tool is in your PATHinstall with pippip install epiVIAintall from github pagegit clonehttps://github.com/wangwl/epiVIAcd epiVIApython setup.py installOutputThis 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 filesfilenamedescriptionIntegration_sites.listIdentified integration sitesbarcode_integration.listIngretation sites sorted by cell barcoedsCell_summary.txtNumber of chimeric and viral reads in each cellVector_coverage.csvCoverage of viral reads at each positioin along the proviral genomeIntegration_sites.listColumn nameDescriptionGenomeReference genome version (eg: hg19, hg38, mm9, mm10)ChromChromosome of the integration siteChrStartStart position in host genome where the chimeric reads aligned, or the integration siteChrEndEnd Position in host genome where the chimeric reads aligned, or the integration siteVectorStartStart posision in proviral genome where the chimeric reads alignedVectorEndEnd position in proviral genome where the chimeric reads alignedLTRThe chimeric reads is from 5' LTR or 3' LTRInsertOriIntegration orietation of the proviral genome based on the reference sequenceInsertGeneThe annotated gene at the integration site of the host genomeGeneOriOrientation of the geneExonIntronThe integration is in intron or exon region of the geneEnhancerEnhancer or Promoter information at the integration siteEliteIf the integration site locates in an enhancer based on UCSC annotation, whether it's EliteTEFamilyFamily of transposable elements annotation at the integration siteTEClassClass of transposable elements annotation at the integration siteCellNumberNumber of cells have this integration siteCellBarcodesCell barcodes that have this integration siteReadInCellNumber of reads in each cell that support this integration siteReadNumberTotal number of reads that support this integration siteQualityThe quality of the integration site, adapted from the mapping quality of BWAbarcode_integration.listEach cell that have integration sites. If a cell have multiple integration sites, ".1", ".2", ".3", ..., were appended to the cell barcode.Column nameDescriptionCellBarcodeCell Barcode of the cellChromChromosome of the integration siteChrStartStart position in host genome where the chimeric reads aligned, or the integration siteChrEndEnd Position in host genome where the chimeric reads aligned, or the integration siteVectorStartStart posision in proviral genome where the chimeric reads alignedVectorEndEnd position in proviral genome where the chimeric reads alignedInsertGeneThe annotated gene at the integration site of the host genomeExonIntronThe integration is in intron or exon region of the geneEnhancerEnhancer or Promoter information at the integration siteTEFamilyFamily of transposable elements annotation at the integration siteTEClassClass of transposable elements annotation at the integration siteQualityThe quality of the integration site, adapted from the mapping quality of BWACell_summary.txtColumn nameDescriptionCellBarcodeCell Barcode of the cellChimeraSumTotal number of chimeric reads from the cellPairNumber of 'Pair' chimeric reads (case a) from the cellHostReadNumber of 'HostRead' chimeric reads (case b) from the cellVectorReadNumber of 'VectorRead' chimeric reads (case c) from the cellintegrationNumber of integration sites in the cellVectorSumTotal number of reads from the integrated proviral genomenoLTRNumber of proviral reads that is not from LTR regionsLTRNumber of proviral reads that is from LTR regionsTemporary filesepiVIA 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.ExampleepiVIA --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 --gbdbhttp://hgdownload.soe.ucsc.edu/gbdb/test/data/candidate.bam .