bedgraph-format-parsing-and-validation

bedgraph-format-parsing-and-validation

Summary

Parse and validate UCSC bedGraph files to ensure they meet format requirements for sparse chromatin profiling analysis, particularly for CUT&RUN peak-calling workflows. This skill detects malformed coordinates, missing signal fields, and zero-signal regions that must be filtered before downstream peak-calling.

When to use

You have generated or received bedGraph files from paired-end sequencing (via bedtools genomecov or similar) and need to verify they conform to UCSC bedGraph format before passing them to peak-calling tools like SEACR. Use this when input bedGraphs have unknown provenance, were generated from custom pipelines, or have failed in downstream analysis.

When NOT to use

• Input is already a peak-called BED file with enriched regions; parse as standard BED3 or BED6 instead.
• Input is a single-end BAM or fragment file where read pair structure is unknown; cannot reliably convert to density-based bedGraph without pair information.
• Input is a different wiggle format (e.g., bedWig, variableStep); requires format-specific parser.

Inputs

• UCSC bedGraph file (tab-delimited: chr, start, end, signal)
• Paired-end BAM file (optional, for de novo bedGraph generation)
• Paired-end BED file with 5' and 3' read coordinates (optional)

Outputs

• Validated UCSC bedGraph file with zero-signal lines removed
• Parsed bedGraph dataframe or table (for downstream analysis)
• Coordinate and signal validation report

How to apply

Verify that bedGraph input adheres to UCSC bedGraph format (https://genome.ucsc.edu/goldenpath/help/bedgraph.html), checking that each line contains four tab-delimited fields: chromosome, start coordinate, end coordinate, and signal value. Filter out any bedGraph lines containing zero signal, as SEACR and similar tools expect bedGraphs that omit regions with zero coverage. Validate coordinate ordering (start < end) and signal values as numeric. If converting from BAM files, follow the paired-end fragment workflow: convert to bedpe format with bedtools bamtobed, extract 5' and 3' termini coordinates, sort, and generate the bedGraph using bedtools genomecov with the -bg flag to produce density across read pairs rather than individual reads.