ONTMethCalling

This repository contains scripts to call DNA methylation from ONT data and perform statistical comparisions between a smoker and non-smoker. These scripts contain the analyses presented in

Robert Flynn, Sam Washer, Aaron R Jeffries, Alexandria Andrayas, Gemma Shireby, Meena Kumari, Leonard C Schalkwyk, Jonathan Mill, Eilis Hannon, Evaluation of nanopore sequencing for epigenetic epidemiology: a comparison with DNA methylation microarrays, Human Molecular Genetics, Volume 31, Issue 18, 15 September 2022, Pages 3181–3190, https://doi.org/10.1093/hmg/ddac112**

Notes:

• The unix scripts are run in concert with a config file that contains the relevant filepaths as bash variables. An example config file can be found in

Config/example.txt

• Scripts are separated into job submission scripts which are submitted to the HPC scheduler and the processing scripts which are called/executed by the job submission script. These can be found in

Scripts/

and

JobSubmission/

respectively.

Description of scripts to process ONT data into DNA methylation levels

1_Alignment.sh

Takes fastq files from ONT and merges, aligns with minimap2. Performs downstream prcosessing with samtools including, sorting, fitering to high quality primary alignments and indexing.

2_CalcCoverage.sh

Takes aligned, filtered reads in bam file and use BEDtools to summarise read coverage across genome.

3_DNAMcallingNanopolish.sh

Takes fast5 files from ONT and aligned, indexed bam file from 1_Alignment.sh and uses nanopolish to call DNA methylation and summarise as frequencys per position.

Description of R scripts to perform statistical comparisions

These scripts contain the code to create the figures included in the manuscript.

4_PlotCoverage.r

Takes bedgraph files from BEDtools (2_CalcCoverage.sh) and table of guide positions to generate figures and summary statistics of sequencing coverage across genome and within target regions.

5_CompareArrayONT.r

Takes nanopolish DNA methylation frequencys and compares to matched data generated with the EPIC array.

6_CompareSamples.r

Takes nanopolish DNA methylation frequencys and performs statistical comparision between samples. Compares these results to smoking EWAS from large population cohort.

7_plotMethylationPhasingTargetRegions.r

Calculates D' statistics between pairs of sites within targetted regions and creates plots.

8_plotMethylationPhasingSignificantRegions.r

Calculates D' statistics between pairs of sites within regions with significant differentially methylated sites and creates plots.