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BED (Browser Extensible Data) format is a simple text format for location-oriented data (genomic regions) developed to support UCSC Genome Browser (GenBrowse) tracks. Standard BED files have 3 to 6 Tab-separated columns, although up to 12 columns are defined. (Read more about the UCSC Genome Browser's official BED format.)
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The BEDTools suite is a set of utilities for manipulating BED and BAM files. We call it the "Swiss army knife" for genomic region analyses because its sub-commands are so numerous and versatile. Some of the most common bedtools operations perform set-theory functions on regions: intersection (intersect), union (merge), set difference (subtract) – but there are many others. The table below lists some of the most useful sub-commands along with applicable use cases.
| Sub-command | Description | Use case(s) |
|---|---|---|
| bamtobed | Convert BAM files to BED format. | You want to have the contig, start, end, and strand information for each mapped alignment record in separate fields. Recall that the strand is encoded in a BAM flag (0x10) and the exact end coordinate requires parsing the CIGAR string. |
| bamtofastq | Extract FASTQ sequences from BAM alignment records. | You have downloaded a BAM file from a public database, but it was not aligned against the reference version you want to use (e.g. it is hg19 and you want an hg38 alignment). To re-process, you need to start with the original FASTQ sequences. |
| getfasta | Get FASTA entries corresponding to regions. | You want to run motif analysis, which requires |
| FASTA sequences, on a set of regions of interest. |
| In addition to |
| a BED or BAM file, you must provide FASTA file(s) for the genome/reference used for alignment (e.g. the FASTA file used to build the aligner index). | ||
| genomecov | Generate per-base genome-wide signal trace | Produce a per-base genome-wide signal (in bedGraph format), for example for a ChIP-seq or ATAC-seq experiment. After conversion to binary bigWig format, such tracks can be visualized in the Broad's IGV (Integrative Genome Browser) application, or configured in the UCSC Genome Browser as custom tracks. |
| coverage | Compute coverage of your regions |
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| multicov | Count overlaps between one or more BAM files and a set of regions of interest. | Count RNA-seq alignments that overlap a set of genes of interest. While this task is usually done with a specialized RNA-seq quantification tool (e.g. featureCounts or HTSeq), bedtools multicov can provide a quick estimate, e.g. for QC purposes. |
| merge | Combine a set of possibly-overlapping regions into a single set of non-overlapping regions. | Collapse overlapping gene annotations into per-strand non-overlapping regions before counting (e.g with featureCounts or HTSeq). If this is not done, the source regions will potentially be counted multiple times, once for each (overlapping) target region it intersects. |
| subtract | Remove unwanted regions. |
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| intersect | Determine the overlap between two sets of regions. | Similar to multicov, but can also report the overlapping regions, not just count them. |
| closest | Find the genomic features nearest to a set of regions. | For a set of significant ChIP-seq |
| Transcription Factor (TF) binding regions ("peaks") that have been identified, determine nearby genes that may be targets of TF regulation. |
We will explore a few of these functions in our exercises.
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