Pipeline of analysis for recombination of organellar genomes

Introduction

This pipeline was initialy built for Chevigny et al., 2022 and aims to analyze recombination of organellar genomes from Illumina paired-end data (2 × 150 bp). To have an overview of this pipeline, please refer to Graphical_summary_PlantOrgRec.pdf

Methods

Data pre-processing

Illumina paired-end reads (2 × 150 bp) were trimmed with trimmomatic and overlapping reads joined with fastq-join. Then reads were mapped on the three genomes: nuclear, plastid and mitochondrial and only reads mapped in pair were kept using BWA mem.

Organellar genome coverage analysis

This part is mandatory because the recombination part of the study require coverage file "mean_cov.csv".From the bam file containing reads mapped in proper pair, coverage data were extracted with Bedtools as well as the number of reads mapped using Samtools view. Then, using JGAF-Plant_organellar_coverage.R coverage data are normalized to 1'000'000 mapped per genomes, and plot of the means coverage per kb are drawned.

Organellar genome recombination analysis

Reads properly pairing to the cpDNA and mtDNA were filtered to only keep those showing a soft-clipping sequence (threshold 20 nucleotides) and no indel (looking for the presence of an S in the CIGAR string without any I, D and H). The soft-clipping sequences were then extracted with SE-MEI/extractSoftclipped (github.com/dpryan79/SE-MEI) and aligned using bowtie2 against the corresponding genome. The positions of the soft-clipping sequences mapping the cpDNA and of their relative read were rounded down to the upper kb to analyze the localization of the rearrangement (see JGAF-Plant_organellar_recombination.R). Those corresponding to the isomerization that results from the recombination involving the large inverted repeats were filtered out.