I. Quality Control

1. Input Data Format

First load the HiCRep R package into R console.

library(hicrep)

The main function that we will use is get.scc which calculates the stratum-adjusted correlation coefficient for each pair of contact matrices on one chromosome. Try ?get.scc to check the input data format.

2. Data Preparation

HiCRep has embedded smoothing function hence it recommends using raw contact count. Therefore, we will utilize bin pair files, such as AMSI_3Dgenomics/results/Ring/step5_bin/Ring_chr7.binPairs, for input matrix generation in R.

library(data.table)
library(dplyr)
hicData1 <- fread("~/AMSI_3Dgenomics/results/Ring/step5_bin/Ring_chr7.binPairs") %>% select(V2, V4, V5)
hicData2 <- fread("~/AMSI_3Dgenomics/results/Trophozoite/step5_bin/Trophozoite_chr7.binPairs") %>% select(V2, V4, V5)

hicDataMerge <- full_join(hicData1, hicData2, by = c("V2", "V4"))
hicDataMerge[is.na(hicDataMerge)] <- 0
head(hicDataMerge)

3. Stratum-adjusted Correlation Coefficient

get.scc(hicDataMerge, 10000, 1000000)$scc

  • Get the SCC score for Ring vs Trophozoite

  • Get the SCC score for Ring vs Schizont

  • Get the SCC score for Trophozoite vs Schizont

II. Differential Chromatin Interaction Detection

1. Install Selfish in Bash

Selfish, i.e., Self-fishing, is a tool for finding differential chromatin interactions between two Hi-C contact maps.

First, we will need to install Selfish in Terminal:

git clone https://github.com/ay-lab/selfish

2. Data Preparation

Our bin pair files, such as AMSI_3Dgenomics/results/Ring/step5_bin/Ring_chr7.binPairs, perfectly match the input data foramt for Selfish:

Chromosome Midpoint 1 Chromosome Midpoint 2 Contact Count
chr1 5000 chr1 65000 438
chr1 5000 chr1 85000 12
… … … … …

3. Differential Detection

mkdir -p AMSI_3Dgenomics/analysis/Selfish
selfish/selfish/selfish.py -f1 AMSI_3Dgenomics/results/Ring/step5_bin/Ring_chr7.binPairs -f2 AMSI_3Dgenomics/results/Trophozoite/step5_bin/Trophozoite_chr7.binPairs -ch 7 -r 10kb -o AMSI_3Dgenomics/analysis/Selfish/Ring_Trophozoite_chr7.npy

Output of Selfish is a matrix of p-values indicating the probability of differential conformation (Smaller values mean more significant.).Convert numpy object into readable text file.

python3 AMSI_3Dgenomics/scripts/selfish_npyToCSV.py AMSI_3Dgenomics/analysis/Selfish/Ring_Trophozoite_chr7.npy AMSI_3Dgenomics/analysis/Selfish/Ring_Trophozoite_chr7.selfish

To control False Discovery Rate, Benjamini-Horchberg multiple testing correction is usually applied before determining the significant differential chromatin interactions.

  • Get the differential interactions for Ring vs Trophozoite.

  • Get the differential interactions for Ring vs Schizont.

  • Get the differential interactions for Trophozoite vs Schizont.