BEDTools

Overview

BEDTools is the standard toolkit for genomic interval arithmetic. It provides fast, set-theoretic operations – intersection, union, complement, merging, and more – on BED, BAM, VCF, and GFF/GTF files. BEDTools is essential for tasks such as identifying overlapping genomic features, filtering regions against blacklists, computing genome-wide coverage, and measuring similarity between interval sets with the Jaccard statistic.

Installation

mamba install -c bioconda bedtools

Basic Usage

Intersect peaks with gene promoters

bedtools intersect -a peaks.bed -b promoters.bed -wa -wb > overlap.bed

Find peaks that do NOT overlap with blacklist regions

bedtools intersect -a peaks.bed -b blacklist.bed -v > clean_peaks.bed

Merge overlapping intervals

bedtools merge -i sorted_peaks.bed -d 100 > merged.bed

Find the closest gene to each peak

bedtools closest -a peaks.bed -b genes.bed -d > closest.bed

Compute genome-wide coverage from a BAM file

bedtools genomecov -ibam sample.bam -bg > coverage.bedgraph

Generate windows across the genome

bedtools makewindows -g genome.sizes -w 10000 > windows.bed

Compute Jaccard similarity between two sets of intervals

bedtools jaccard -a set1.bed -b set2.bed

Key Parameters

Flag / option	Description
`-a`	The “query” file (BED/BAM/VCF/GFF).
`-b`	The “subject” file(s) to compare against.
`-wa`	Write the original entry from `-a` for each overlap.
`-wb`	Write the original entry from `-b` for each overlap.
`-v`	Report entries in `-a` that have no overlap with `-b`.
`-d INT`	Maximum distance between features to merge (used with `merge`); features within this distance are combined into a single interval.
`-f FLOAT`	Minimum overlap required as a fraction of `-a` (e.g., `0.50` for 50 %).
`-r`	Require the overlap fraction to be reciprocal for both `-a` and `-b`.
`-s`	Require that features are on the same strand.
`-bg`	Report depth in bedGraph format (used with `genomecov`).
`-g`	Genome file providing chromosome sizes (required by `makewindows`, `complement`, etc.).
`-w INT`	Window size in bp (used with `makewindows`).

Expected Output

intersect – a BED file listing features (or feature pairs with -wa -wb) that overlap between the two inputs. With -v, only non-overlapping entries from -a are reported.
merge – a BED file of merged intervals where overlapping or nearby features have been collapsed.
closest – a BED file pairing each -a feature with the nearest -b feature; the -d flag appends the distance as an extra column.
genomecov – a bedGraph file of per-base or per-bin coverage across the genome.
makewindows – a BED file of fixed-size, non-overlapping (or sliding) windows tiling the genome.
jaccard – a single-line summary with the intersection size, union size, and Jaccard index.