Code & Resources

Single-cell Repli-seq analysis script
(Dileep and Gilbert, 2018)

This is an R pipeline for analyzing DNA replication in single cells using Whole Genome Amplification (WGA). The pipeline starts with read counts from single-cell WGA binned into 50 kb windows. Sample data is provided along with the code. Read counts of all cells are converted to reads per million to control for variable sequencing depth. To control for amplification and mappability biases, we also sorted G1 and G2 cells, which contain a relatively uniform DNA content. Regions of low mappability and over-amplification were removed based on the G1 and G2 controls. Read counts were normalized by dividing the coverage data of each single cell by the coverage data of the G1 and G2 control cells. A median filter was applied to smooth the data, producing Copy Number Variation (CNV) profiles in 50 kb bins. Next, a novel binarization strategy is used to convert the CNV profile to replicated or unreplicated. The binarization algorithm is based on iterating through multiple thresholds for binarization and calculating the difference between the CNV profile and binarized profile for each given threshold. The threshold with the least difference is chosen as the ideal binarization.