`finaletoolkit coverage`¶

1. About¶

Calculates fragmentation coverage over intervals defined in a BED file.

Counts the fragments overlapping each interval and normalizes the counts by the sample's total coverage, so values are comparable across samples with different sequencing depths. In this pipeline the intervals are the TSS windows for the selected genome build, making the output a per-TSS measure of cfDNA abundance.

Coverage is the basis of the project-level merged workbook, so it is also the primary cross-sample comparison in a run.

2. As the pipeline runs it¶

finaletoolkit coverage bams/{sample}.sorted.bam <tss_interval> \
    -n \
    --scale 1e6 \
    -q <--mapscore> \
    -min <--fragment-minimum> \
    -max <--fragment-maximum> \
    -p any \
    -o coverage/{sample}_coverage.bed \
    -w <threads> \
    -v

2.1 Arguments¶

input_file

The analysis BAM. value: bams/{sample}.sorted.bam

interval_file

Intervals to quantify coverage over. value: the tss_interval file for the selected --genome build

Constant-length windows centered on transcription start sites. See Reference files.

-n, --normalize

Normalize by total coverage. set by the pipeline

Divides each interval's raw fragment count by the sample's genome-wide total, then multiplies by --scale-factor. Without this, values would track sequencing depth rather than relative fragment abundance and could not be compared between samples.

-s, --scale-factor

Scale factor applied to normalized values. value: 1e6 (passed as --scale)

Turns the normalized fraction into coverage per million fragments, which keeps values in a readable numeric range rather than very small decimals.

-q, --quality-threshold

Minimum mapping quality. value: --mapscore (default 20)

-min, --min-length / -max, --max-length

Fragment length window. value: --fragment-minimum / --fragment-maximum (defaults 50 / 500)

-p, --intersect-policy

How a fragment is assigned to an interval. value: any

A fragment counts toward an interval if it overlaps it anywhere. This is the appropriate choice for coverage — a fragment physically covers part of the interval whether or not its midpoint falls inside. Contrast with frag-length-bins, which uses midpoint.

-w, --workers

Worker processes. value: the step's threads from config/cluster.json (default 1)

3. Output¶

coverage/{sample}_coverage.bed — a headerless 5-column BED:

chr1    9873    11873   interval_name   14.2731
chr1    28084   30084   interval_name   9.8104

Column	Meaning
1	contig
2	interval start (0-based)
3	interval stop
4	interval name, from the interval BED
5	normalized, scaled coverage

These BEDs are merged into coverage/coverage_summary.xlsx by the project-level merge_coverage_excel step.

4. Controlling it¶

What	How
Fragment length window	`--fragment-minimum`, `--fragment-maximum`
Which intervals	the `tss_interval` entry of `config/genome.json` for the selected build
Threads / memory / walltime	the `coverage` entry of `config/cluster.json`

"coverage": {
    "threads": 1,
    "mem": "32000m",
    "time": "72:02:00",
    "gres": "lscratch:200",
    "partition": "norm"
}

Coverage is single-threaded by default and is one of the longer-running steps on large BAMs; raise threads in config/cluster.json to parallelize it.

5. Requires¶

The tss_interval file for the selected genome build. Unlike most analyses this step is not conditional on that entry being present — it always runs. That makes tss_interval the one reference key a build cannot leave out, so a --genome config file without it is rejected before the run starts rather than failing here. Both bundled builds provide it.

finaletoolkit coverage¶