BED

Overview

BED (Browser Extensible Data) is a tab-delimited format for describing genomic intervals. It is used throughout bioinformatics to represent regions of interest such as:

Genes, exons, and promoter regions
ChIP-seq peaks and regulatory elements
Target capture regions for exome sequencing
Blacklisted or masked regions
Structural variant breakpoints

BED files are compact, human-readable, and supported by nearly every genomic tool. They are the primary input for bedtools and are accepted by IGV, UCSC Genome Browser, samtools, GATK, and many other programs.

Important

BED uses 0-based, half-open coordinates. The start position is zero-based (the first base of a chromosome is 0) and the end position is exclusive. An interval covering the first 1 000 bases of chr1 is written as chr1 0 1000. This differs from GFF/GTF and VCF, which use 1-based, fully-closed coordinates.

Structure

BED files come in several levels of detail. The first three columns are mandatory; additional columns add information.

BED3 – minimal interval

# BED3: chromosome, start, end
chr1  1000  2000
chr1  3000  4500

Three columns: chrom, chromStart (0-based, inclusive), and chromEnd (exclusive). The interval chr1 1000 2000 covers bases 1000 through 1999 (1 000 bases total).

BED6 – adding name, score, and strand

# BED6: adds name, score, strand
chr1  1000  2000  peak1  500  +
chr1  3000  4500  peak2  300  -

Col	Field	Description
4	name	Feature name or identifier.
5	score	Integer 0–1000 (e.g. peak enrichment score).
6	strand	`+` (forward), `-` (reverse), or `.` (unstranded).

BED12 – transcript models

# BED12: adds thickStart, thickEnd, itemRgb, blockCount,
#         blockSizes, blockStarts (used for transcript models)
chr1  11873  14409  NR_046018  0  +  11873  11873  0  3  354,109,1189,  0,739,1347,

Col	Field	Description
7	thickStart	Start of the thick drawing region (e.g. CDS start).
8	thickEnd	End of the thick drawing region (e.g. CDS end).
9	itemRgb	RGB colour value for display (`0` = black).
10	blockCount	Number of blocks (exons).
11	blockSizes	Comma-separated list of block sizes.
12	blockStarts	Comma-separated list of block start positions relative to chromStart.

In the example above the transcript NR_046018 has 3 exons of sizes 354, 109, and 1189 bp. The exon start positions relative to the transcript start (11873) are 0, 739, and 1347.

Coordinate system comparison

Format	System	First base	Interval [1, 100]
BED	0-based, half-open	0	`chr1 0 100`
GFF / GTF	1-based, closed	1	`chr1 1 100`
VCF	1-based	1	`POS = 1`
SAM	1-based	1	`POS = 1`

Working With

Basic bedtools operations

# Find overlapping intervals between two BED files
bedtools intersect -a peaks.bed -b promoters.bed > overlap.bed

# Subtract one set of intervals from another
bedtools subtract -a regions.bed -b blacklist.bed > clean.bed

# Merge overlapping intervals
sort -k1,1 -k2,2n regions.bed | bedtools merge > merged.bed

# Compute the complement (gaps) relative to a genome
bedtools complement -i regions.bed -g chrom.sizes > gaps.bed

Extracting sequences

# Get FASTA sequences for BED intervals
bedtools getfasta -fi reference.fa -bed regions.bed -fo regions.fa

Coverage and depth

# Count reads overlapping each interval
bedtools coverage -a targets.bed -b aligned.sorted.bam > coverage.bed

# Compute genome-wide coverage histogram
bedtools genomecov -ibam aligned.sorted.bam -bg > coverage.bedgraph

Sorting BED files

# Sort by chromosome and start position
sort -k1,1 -k2,2n unsorted.bed > sorted.bed

Slop (extend) intervals

# Extend each interval by 500 bp on both sides
bedtools slop -i peaks.bed -g chrom.sizes -b 500 > extended.bed

# Extend 2 kb upstream only (strand-aware)
bedtools slop -i genes.bed -g chrom.sizes -l 2000 -r 0 -s > promoters.bed

Filtering BAM by BED regions

# Keep only reads overlapping target regions
samtools view -b -L targets.bed aligned.sorted.bam > on_target.bam