samples/SU008/scATAC/Gen_seg_table.md

In seasoncloud/Alleloscope: Allele-specific copy number genotyping for single cell sequencing data

Alleloscope (WES/WGS)

Chi-Yun Wu, Zhang Lab, University of Pennsylvania

Description

Alleloscope provides a function to generate the segmentation table (seg_table) for the downstream analysis from matched WGS or WES data.

For more information about the method, please check out the github and the paper.

Prepare input files

Two BED files (for tumor and normal WGS/WES) with each row a genomic bin. The first column is chromosomes; the second column is start sites; thee third column is end sites; the fourth column is summed read counts. This can be generated using bedtools with the following command. * For GRCh38 reference genome and 100kb bins, the 'hg38.100Kb.windows.sorted.bed' file here can be used.

# for tumor sample
bedtools intersect -a ~/hg38.100Kb.windows.sorted.bed \
-b ~/sample.bam \
-c -sorted \
> ~/hg38.100Kb.windows.counts.tumor.bedg

# for germline sample
bedtools intersect -a ~/hg38.100Kb.windows.sorted.bed \
-b ~/sample.bam \
-c -sorted \
> ~/hg38.100Kb.windows.counts.germline.bedg

Step0. Load the input files

• In R, set up the environment and read files

library(Alleloscope) # load the library
setwd("~/Alleloscope/") # set path to the github folder

dir_path <- "./WGS/"; dir.create(dir_path) # set up output directory

size=read.table("data-raw/sizes.cellranger-GRCh38-1.0.0.txt", stringsAsFactors = F) # read size file
size=size[1:22,]

WGSt=read.table("~/hg38.100Kb.windows.counts.tumor.bedg", stringsAsFactors = F, sep='\t')
WGSn=read.table("~/hg38.100Kb.windows.counts.germline.bedg", stringsAsFactors = F, sep='\t')
WGSt=WGSt[which(WGSt$V1 %in% paste0('chr',1:22)), ]
WGSn=WGSn[which(WGSn$V1 %in% paste0('chr',1:22)), ]
WGSt[,2]=as.numeric(WGSt[,2])+1; WGSt[,3]=as.numeric(WGSt[,3])+1
WGSn[,2]=as.numeric(WGSn[,2])+1; WGSn[,3]=as.numeric(WGSn[,3])+1
rownames(WGSn)=rownames(WGSt)=paste0(WGSt$V1,"-", WGSt$V2,'-', WGSt$V3)
WGSt=WGSt[,4, drop=F]; WGSn=WGSn[,4, drop=F]

Step1. Creating a Alleloscope object for the analysis

• First, create an Alleloscope object

Obj_filtered=Createobj(samplename=name, genome_assembly="GRCh38", dir_path=dir_path, size=size, assay='WGS', cov=TRUE)

Step2. Unbiased segmentation based on matched WES/WGS data

Obj_filtered=Segmentation(Obj_filtered=Obj_filtered, 
                          raw_counts=WGSt, # from matched DNA sequencing (bulk/single)
                          ref_counts=WGSn, # from matched DNA sequencing (bulk/single)
                          plot_seg = TRUE)

* The segmentation plot and seg_table.rds is stored in the dir_path.

Citation

Wu, C.-Y. et al. Integrative single-cell analysis of allele-specific copy number alterations and chromatin accessibility in cancer. Nature Biotechnology (2021): https://doi.org/10.1038/s41587-021-00911-w

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seasoncloud/Alleloscope documentation built on March 17, 2023, 1:14 a.m.