BAM-to-BigWig conversion
Summary
Convert bias-corrected ATAC-seq signal data from BAM format to BigWig format for visualization and downstream footprinting analysis. This transformation enables efficient storage, scalable genome browser display, and preparation for footprint scoring.
When to use
After running TOBIAS ATACorrect to generate bias-corrected signal tracks from aligned ATAC-seq reads. Use this skill when you have corrected cutsite signal (as .bw or equivalent intermediate format) that must be visualized across the genome or fed into footprint scoring tools like ScoreBigwig.
When NOT to use
- Input is already in BigWig format—no conversion needed.
- Analysis goal is to call peaks or identify open chromatin regions; use peak-calling tools (e.g., MACS2) directly on aligned BAM instead.
- Working with raw, uncorrected ATAC-seq cutsite data for motif discovery without bias correction; apply ATACorrect first.
Inputs
- Bias-corrected ATAC-seq signal data (as .bw output from TOBIAS ATACorrect, or intermediate cutsite BAM/bedGraph)
- Reference genome index or chrom.sizes file (for bedGraph-to-bigWig conversion if needed)
Outputs
- BigWig (.bw) file containing bias-corrected signal track
- IGV/genome-browser compatible signal visualization
How to apply
Following TOBIAS ATACorrect bias correction, the corrected signal output (stored initially as a bigWig file by ATACorrect itself) is ready for visualization and downstream analysis. ATACorrect natively outputs bias-corrected signal as a .bw file alongside uncorrected, bias model, and expected signal tracks. No additional conversion step is strictly required—ATACorrect produces the BigWig directly. However, if working with intermediate cutsite signals or custom corrected BAM files, conversion to BigWig can be achieved through standard genomic tools (e.g., bedtools, deeptools, or UCSC tools) that bin aligned reads into fixed-width windows and store normalized signal values. The resulting BigWig tracks preserve positional cutsite resolution while enabling rapid querying by genome browsers and footprinting downstream tools.
Related tools
- TOBIAS ATACorrect (Produces bias-corrected signal as primary bigWig output; generates _corrected.bw file from aligned BAM and reference genome) — https://github.com/loosolab/TOBIAS
- TOBIAS ScoreBigwig (Consumes corrected bigWig tracks to calculate footprint scores within regulatory regions) — https://github.com/loosolab/TOBIAS
- bedtools genomecov / bedGraphToBigWig (Alternative tools for converting intermediate bedGraph or BAM-derived signal to bigWig if not using TOBIAS ATACorrect directly)
Examples
TOBIAS ATACorrect --bam reads.bam --genome genome.fa --peaks peaks.bed --outdir ./corrected_tracks
Evaluation signals
- Output file exists and is valid bigWig format (check header and chrom extent with
bigWigInfo or similar utility).
- BigWig signal values align with corrected cutsite positions from the source BAM or intermediate track—spot-check a known footprint region.
- BigWig can be loaded into a genome browser (IGV, UCSC) without errors and displays expected ATAC-seq signal distribution.
- Downstream footprinting tools (TOBIAS ScoreBigwig, PlotAggregate, PlotHeatmap) successfully ingest the BigWig without format errors.
- Signal magnitude and range are consistent with the bias-corrected BAM read depth—no anomalous scaling artifacts.
Limitations
- BigWig is a lossy format for some use cases; exact per-base read counts may be lost if binned at low resolution. For precise analysis, retain the original BAM.
- Conversion does not add new biological information—it is purely a format transformation for visualization and downstream tool compatibility.
- TOBIAS ATACorrect outputs multiple bigWig files (_uncorrected.bw, _bias.bw, _expected.bw, _corrected.bw); ensure you use the _corrected.bw for downstream footprinting, not the uncorrected or bias tracks.
- Single-cell ATAC-seq analysis requires pseudobulk aggregation (via SC-Framework) before conversion, as individual cell BAMs lack sufficient coverage for meaningful footprinting.
Evidence
- [readme] Output files: - /_uncorrected.bw - /_bias.bw - /_expected.bw - /_corrected.bw: "Output files:
- /_uncorrected.bw
- /_bias.bw
- /_expected.bw
- /_corrected.bw"
- [other] Convert corrected signal to bigWig format for visualization and downstream footprinting analysis.: "Convert corrected signal to bigWig format for visualization and downstream footprinting analysis."
- [readme] ATACorrect corrects the cutsite-signal from ATAC-seq with regard to the underlying sequence preference of Tn5 transposase.: "ATACorrect corrects the cutsite-signal from ATAC-seq with regard to the underlying sequence preference of Tn5 transposase."
- [intro] the local distribution of Tn5 insertions contains information about transcription factor binding due to the visible depletion of insertions around sites bound by protein - known as footprints: "the local distribution of Tn5 insertions contains information about transcription factor binding due to the visible depletion of insertions around sites bound by protein - known as footprints"
1---2name: bam-to-bigwig-conversion3description: Use when after running TOBIAS ATACorrect to generate bias-corrected signal tracks from aligned ATAC-seq reads. Use this skill when you have corrected cutsite signal (as .4license: CC-BY-4.05---67# BAM-to-BigWig conversion89## Summary1011Convert bias-corrected ATAC-seq signal data from BAM format to BigWig format for visualization and downstream footprinting analysis. This transformation enables efficient storage, scalable genome browser display, and preparation for footprint scoring.1213## When to use1415After running TOBIAS ATACorrect to generate bias-corrected signal tracks from aligned ATAC-seq reads. Use this skill when you have corrected cutsite signal (as .bw or equivalent intermediate format) that must be visualized across the genome or fed into footprint scoring tools like ScoreBigwig.1617## When NOT to use1819- Input is already in BigWig format—no conversion needed.20- Analysis goal is to call peaks or identify open chromatin regions; use peak-calling tools (e.g., MACS2) directly on aligned BAM instead.21- Working with raw, uncorrected ATAC-seq cutsite data for motif discovery without bias correction; apply ATACorrect first.2223## Inputs2425- Bias-corrected ATAC-seq signal data (as .bw output from TOBIAS ATACorrect, or intermediate cutsite BAM/bedGraph)26- Reference genome index or chrom.sizes file (for bedGraph-to-bigWig conversion if needed)2728## Outputs2930- BigWig (.bw) file containing bias-corrected signal track31- IGV/genome-browser compatible signal visualization3233## How to apply3435Following TOBIAS ATACorrect bias correction, the corrected signal output (stored initially as a bigWig file by ATACorrect itself) is ready for visualization and downstream analysis. ATACorrect natively outputs bias-corrected signal as a .bw file alongside uncorrected, bias model, and expected signal tracks. No additional conversion step is strictly required—ATACorrect produces the BigWig directly. However, if working with intermediate cutsite signals or custom corrected BAM files, conversion to BigWig can be achieved through standard genomic tools (e.g., bedtools, deeptools, or UCSC tools) that bin aligned reads into fixed-width windows and store normalized signal values. The resulting BigWig tracks preserve positional cutsite resolution while enabling rapid querying by genome browsers and footprinting downstream tools.3637## Related tools3839- **TOBIAS ATACorrect** (Produces bias-corrected signal as primary bigWig output; generates _corrected.bw file from aligned BAM and reference genome) — https://github.com/loosolab/TOBIAS40- **TOBIAS ScoreBigwig** (Consumes corrected bigWig tracks to calculate footprint scores within regulatory regions) — https://github.com/loosolab/TOBIAS41- **bedtools genomecov / bedGraphToBigWig** (Alternative tools for converting intermediate bedGraph or BAM-derived signal to bigWig if not using TOBIAS ATACorrect directly)4243## Examples4445```46TOBIAS ATACorrect --bam reads.bam --genome genome.fa --peaks peaks.bed --outdir ./corrected_tracks47```4849## Evaluation signals5051- Output file exists and is valid bigWig format (check header and chrom extent with `bigWigInfo` or similar utility).52- BigWig signal values align with corrected cutsite positions from the source BAM or intermediate track—spot-check a known footprint region.53- BigWig can be loaded into a genome browser (IGV, UCSC) without errors and displays expected ATAC-seq signal distribution.54- Downstream footprinting tools (TOBIAS ScoreBigwig, PlotAggregate, PlotHeatmap) successfully ingest the BigWig without format errors.55- Signal magnitude and range are consistent with the bias-corrected BAM read depth—no anomalous scaling artifacts.5657## Limitations5859- BigWig is a lossy format for some use cases; exact per-base read counts may be lost if binned at low resolution. For precise analysis, retain the original BAM.60- Conversion does not add new biological information—it is purely a format transformation for visualization and downstream tool compatibility.61- TOBIAS ATACorrect outputs multiple bigWig files (_uncorrected.bw, _bias.bw, _expected.bw, _corrected.bw); ensure you use the _corrected.bw for downstream footprinting, not the uncorrected or bias tracks.62- Single-cell ATAC-seq analysis requires pseudobulk aggregation (via SC-Framework) before conversion, as individual cell BAMs lack sufficient coverage for meaningful footprinting.6364## Evidence6566- [readme] Output files: - <outdir>/<prefix>_uncorrected.bw - <outdir>/<prefix>_bias.bw - <outdir>/<prefix>_expected.bw - <outdir>/<prefix>_corrected.bw: "Output files:67- <outdir>/<prefix>_uncorrected.bw68- <outdir>/<prefix>_bias.bw69- <outdir>/<prefix>_expected.bw70- <outdir>/<prefix>_corrected.bw"71- [other] Convert corrected signal to bigWig format for visualization and downstream footprinting analysis.: "Convert corrected signal to bigWig format for visualization and downstream footprinting analysis."72- [readme] ATACorrect corrects the cutsite-signal from ATAC-seq with regard to the underlying sequence preference of Tn5 transposase.: "ATACorrect corrects the cutsite-signal from ATAC-seq with regard to the underlying sequence preference of Tn5 transposase."73- [intro] the local distribution of Tn5 insertions contains information about transcription factor binding due to the visible depletion of insertions around sites bound by protein - known as _footprints_: "the local distribution of Tn5 insertions contains information about transcription factor binding due to the visible depletion of insertions around sites bound by protein - known as _footprints_"
HolobiomicsLab 