fragment-length-estimation-cross-correlation

fragment-length-estimation-cross-correlation

Summary

Estimate the DNA fragment length d in ChIP-Seq experiments by cross-correlation analysis of read pileups, a critical parameter that determines downstream peak calling accuracy and local background normalization. This skill uses MACS3's predictd subcommand to infer the typical distance between forward and reverse strand reads.

When to use

When beginning ChIP-Seq analysis with single-end BED/SAM input and no prior knowledge of the library's fragment length. The fragment length is essential before generating coverage tracks and building local bias backgrounds, as it directly controls peak calling resolution (minimum peak length) and background normalization scales. Apply this skill immediately after duplicate filtering but before pileup track generation.

When NOT to use

• Input is paired-end BED (BEDPE format) — use fragment length from BEDPE read pairs directly instead.
• Fragment length is already known from prior QC (e.g., Bioanalyzer, library prep documentation) — skip prediction and use known value to save compute time.
• Control-only or Input-only samples — predictd requires ChIP sample; cannot infer fragment length from background alone.

Inputs

• Deduplicated ChIP-Seq BED file (single-end, post-filterdup)
• Genome size (integer, e.g., 'hs' for human haploid ~2.7 billion bp)
• mfold range (integers, default 5 50; defines fold-enrichment window for cross-correlation peak detection)

Outputs

• Predicted fragment length d (integer, in base pairs)
• Cross-correlation plot (diagnostic, optional PDF output)
• Shift distance metrics (diagnostic output from predictd analysis)

How to apply

Run macs3 predictd on deduplicated ChIP sample using default parameters: macs3 predictd -i <filtered_chip.bed> -g <genome_size> -m 5 50 (mfold range). The tool performs cross-correlation analysis by shifting reads across strands and identifying the lag distance with maximum correlation, yielding fragment length d (e.g., 254 bp for CTCF data). The predicted d is then used as --extsize parameter in macs3 pileup (ChIP coverage extension), as the extension size in control background tracks (d/2 for d-background, typically 127 bp), and as minimum peak length constraint in macs3 bdgpeakcall. Validate by checking that d is biologically plausible for your protocol (typically 100–500 bp for sonication-based ChIP).