CiFi Computational Pipeline

1. Create nextflow Conda env and activate:

Conda will be used to install software. The pipeline relies on singularity and nextflow . Installation for both of these as well as any necessary modules are included in the conda environment.

wget https://raw.githubusercontent.com/mydennislab/CiFi/refs/heads/main/CiFi_Nextflow_env.yml 
conda env create -f CiFi_Nextflow_env.yml
conda activate CiFi_Nextflow_env

2. Create paths to tmp files

By default the pipeline will store tmp files in /tmp. If you wish to write to new temporary files for SINGULARITY_TMPDIR and SINGULARITY_CACHEDIR save them here:

export SINGULARITY_TMPDIR=/PATH/TO/TMPDIR
export SINGULARITY_CACHEDIR=/PATH/TO/CACHE

3. Create path to results folder

mkdir /PATH/TO/RESULTS/FOLDER_NAME

When complete this folder will contain these folders:

• bams

• execution

• filtered_out

• ingress_results

• paired_end

As well as this report: wf-pore-c-report.html

4. Run nextflow command and check flags

Check flags:

nextflow run epi2me-labs/wf-pore-c \--help

Run Command:

nextflow run epi2me-labs/wf-pore-c -work-dir ./workflow/  -c  nextflow.config \
--bam <INPUT_BAM> \ 
--ref <PATH_TO_REF_GENOME> \
--cutter <RESTRICTION ENZYME NAME> \
--out_dir <./results/FOLDER_NAME> \
--threads 120 \
--minimap2_settings '-x map-pb' \
--paired_end \ #generates paired-end file that will be converted to Hi-C
--sample <SAMPLE_NAME> # can match folder

5. Filter the paired-end bam by mapping quality (MAPQ)

This software needs to be downloaded separated. See github here

Mapq filter only retains reads where BOTH reads in the pair cross the mapq threshold as well as removing unpaired reads

Download software

Environment Setup

mamba create -n bamfilter bioconda::samtools bioconda::htslib zlib gcc 

Compilation

g++ -O2 -o filter_bam bam_filter.cpp -I$CONDA_PREFIX/include -L$CONDA_PREFIX/lib -lhts -lz -lpthread -Wl,-rpath,$CONDA_PREFIX/lib

Usage

Run the program with the following command:

./filter_bam input.bam output.bam MAPQ_threshold [num_threads]

• input.bam: Path to the input BAM file
• output.bam: Path for the output BAM file
• MAPQ_threshold: Minimum MAPQ value to keep a read pair
• num_threads (optional): Number of threads to use (default is 1)

Run within the paired_end folder within results with a mapq filter of 1:

./filter_bam </RESULTS/FOLDER_NAME/paired_end/input.paired_end.bam> </RESULTS/FOLDER_NAME/paired_end/output.mapq_filtered_paired_end.bam> <1> <num_threads>

6. Convert filtered paired-end bam to pairs

Reactivate CiFi_Nextflow_env conda environment:

conda activate CiFi_Nextflow_env

Generate chromosome sizes file from index fasta reference

cut -f1,2 fasta.fai > sizes.genome

Run bam2pairs to convert filtered paired-end file into .pairs.gz file

bam2pairs -l -c <sizes.genome> <output.mapq_filtered_paired_end.bam> <sample.bam2pairs>

7. Convert pairs file to HiC file

Install java and load

Download juicertools

Juicertools takes in pairs input to output a .hic file. This .hic file can be loaded into Juicebox or UCSC for visualization.

Run juicertools:

java -jar juicer_tools.VERSION_jcuda.0.8.jar pre -t </PATH/TO/WORKING/DIR> <sample.bam2pairsbsorted.pairs.gz> <output_sample_name.hic> <sizes.genome>

Downloading CiFi Data

HG002 with DpnII as RE:

wget https://bioshare.bioinformatics.ucdavis.edu/bioshare/download/rlc692m7tk5cibb/ciFi/HG002_revio/HG002_DpnII_aligned_segments.cs.bam #Aligned coordinate sorted segment bam
wget https://bioshare.bioinformatics.ucdavis.edu/bioshare/download/rlc692m7tk5cibb/ciFi/HG002_revio/HG002_DpnII_aligned_segments.cs.bam.csi #index
wget https://bioshare.bioinformatics.ucdavis.edu/bioshare/download/rlc692m7tk5cibb/ciFi/HG002_revio/HG002_DpnII_paired_end.ns.bam #Mock Paired end bam

GM12878 with DpnII as RE:

wget https://bioshare.bioinformatics.ucdavis.edu/bioshare/download/rlc692m7tk5cibb/ciFi/GM12878_DpnII_aligned_segments.cs.bam #Aligned coordinate sorted segment bam
wget https://bioshare.bioinformatics.ucdavis.edu/bioshare/download/rlc692m7tk5cibb/ciFi/GM12878_DpnII_aligned_segments.cs.bam.csi #index 
wget https://bioshare.bioinformatics.ucdavis.edu/bioshare/download/rlc692m7tk5cibb/ciFi/GM12878_DpnII_paired_end.ns.bam #Mock Paired end bam