| name | bio-genome-intervals-coverage-analysis |
| description | Computes and interprets sequencing read depth and coverage over a genome, windows, or target regions with mosdepth (windowed depth, cumulative distribution, --quantize callable BEDs), bedtools genomecov/coverage (bedGraph tracks, per-target stats), samtools depth/coverage (per-base depth, per-contig depth+breadth). Covers the breadth-vs-mean distinction, the cumulative-coverage curve, evenness (CV/Fano/fold-80/Gini), what each tool silently counts (duplicates, secondary/supplementary, MAPQ, read span vs fragment, mate-overlap), the samtools-depth 8000-cap version trap, and the bedtools coverage -a/-b orientation flip. Use when assessing sequencing adequacy, building coverage tracks, computing breadth at a depth threshold, defining callable regions, or QCing target-capture uniformity. |
| tool_type | mixed |
| primary_tool | bedtools |
Version Compatibility
Reference examples tested with: bedtools 2.31+, mosdepth 0.3+, samtools 1.19+, pybedtools 0.10+, numpy 1.26+.
Before using code patterns, verify installed versions match. If versions differ:
- CLI:
<tool> --version then <tool> --help to confirm flags
- Python:
pip show <package> then help(module.function) to check signatures
samtools depth behaviour changed across versions: pre-1.13 capped depth at 8000 and truncated silently (-d 0 = unlimited); 1.13+ rewrote the subcommand with NO cap and -d/-m deprecated/ignored. Always check samtools --version before trusting a max-depth number. If code throws an error, introspect the installed tool and adapt rather than retrying.
Coverage Analysis
"Is my sequencing deep enough to answer the question?" -> Measure depth as a distribution over positions, then report median, breadth at a depth threshold, and an evenness number -- never the mean alone.
- CLI:
mosdepth --by 500 prefix in.bam (windowed depth + cumulative dist), samtools coverage in.bam (per-contig depth+breadth), bedtools genomecov -ibam in.bam -bga (bedGraph track)
- Python:
pybedtools.BedTool('in.bam').genome_coverage(bga=True) (pybedtools); parse prefix.mosdepth.global.dist.txt for the breadth curve
The Single Most Important Modern Insight -- Mean Depth Is a Budget, Not a Result; Report Breadth Off a Cumulative Curve
"30x WGS" describes what was paid for, not what was achieved. Coverage is a distribution over positions, and the mean is its worst summary: it is dragged up by a fat right tail (repeats, rDNA, mitochondria, PCR pileups, segmental dups) while staying blind to a hard left wall of zeros and near-zeros (GC-extreme exons, poorly-mappable regions, capture dropout). Two libraries with identical mean 30x can differ completely -- one even and callable everywhere, one spiky with 20% of the target uncallable. The mean hides both failures. Four load-bearing moves:
- Report MEDIAN, not mean. The median is robust to the right tail. When mean/median exceeds ~1.1-1.2 the distribution is skewed and the mean is overstating typical depth -- that gap is a free evenness diagnostic.
- Report a BREADTH / cumulative-coverage curve. "% of target >= 1x, >= 10x, >= 20x, >= 30x" is the honest summary, because adequacy is a breadth statement: a base that was not covered deeply enough is uncallable no matter how deep the rest of the genome is. mosdepth's
*.mosdepth.global.dist.txt IS this curve. The killer question for any "mean = 30x" claim is "breadth at 20x?".
- Quantify EVENNESS (CV, Fano factor, Picard fold-80, or Gini) -- an even 30x and a spiky 30x are different experiments, and a spiky library cannot be rescued by sequencing deeper (extra reads follow the same biased distribution; the holes stay holes). Fix the library (PCR-free, better capture, UMIs), not the lane count.
- Say WHAT WAS COUNTED. A depth number is meaningless until the recipe is stated: duplicates dropped (only if MARKED first)? secondary/supplementary included? MAPQ filter? read span or fragment? mate-overlap corrected? per-base or per-region? The tools disagree on every one of these by default.
Tool Taxonomy
| Tool | Counts what (defaults) | Per-base or region | When |
|---|
| mosdepth | corrects mate-overlap by default (off under --fast-mode/-x); -Q MAPQ filter; emits cumulative dist + summary | windowed (--by), per-region, or callable bins (--quantize) | the modern fast default for WGS/WES/targeted; gives the breadth curve directly |
| samtools coverage | per-reference summary (added 1.10); coverage column = breadth %, meandepth = depth | per-contig | quick "is this contig actually covered?" -- spots high-mean/low-breadth pileups |
| samtools depth | drops UNMAP/SECONDARY/QCFAIL/DUP by default; -Q/-q filters; -s de-double-counts overlap; CRAM needs --reference | per-base | exact per-base depth over small regions; watch the 8000-cap version trap |
| bedtools genomecov | counts READ coverage by default (double-counts mate overlap); -pc = fragment; -split for spliced | per-base / bedGraph / histogram | bedGraph tracks, genome-wide depth histogram |
| bedtools coverage | per-A-interval stats from B reads; -a/-b flipped at v2.24.0 | per-region (or -d per-base) | per-target counts/breadth/mean over a BED |
| Picard CollectHsMetrics | capture-kit QC | per-target panel | exome/panel uniformity: on-target %, fold-80, PCT_TARGET_BASES_20X |
Decision Tree by Scenario
| Scenario | Recommended | Why |
|---|
| WGS / WES breadth + adequacy | mosdepth --by then parse *.global.dist.txt | emits the cumulative curve + median directly; fast |
| Quick per-contig depth & breadth glance | samtools coverage | one line/contig; coverage col = breadth, meandepth = depth |
| Exact per-base depth, small region | samtools depth -a -r chr:from-to | per-base; add -s for short-insert; check version for 8000 cap |
| bedGraph coverage TRACK for a browser | bedtools genomecov -ibam -bga (or -bg) | -bga marks zero-coverage gaps; convert to bigWig -> bigwig-tracks |
| Per-target counts/breadth/mean over a BED | bedtools coverage -a targets.bed -b in.bam | A = targets, B = reads (post-v2.24.0); -mean for mean depth |
| Callable-region BED (NO/LOW/CALLABLE/HIGH) | mosdepth --quantize 0:1:4:150: | lightweight CallableLoci replacement at scale |
| Target-capture uniformity QC | -> Picard CollectHsMetrics (fold-80, on-target %) | the capture-QC standard; off-target loss + bait unevenness |
| Spliced/RNA-seq depth | add -split (genomecov/coverage) | without it an intron (N CIGAR) is counted as covered |
| Short-insert VAF (amplicon/cfDNA) | correct mate-overlap: samtools depth -s / genomecov -pc / mosdepth default | naive per-base double-counts the overlap, corrupting VAFs |
| Normalized cross-sample track | -> chip-seq/chipseq-visualization (deepTools bamCoverage) | library-size correction (RPGC/CPM/BPM) for comparison |
| Pileup/variant evidence from BAM | -> alignment-files/pileup-generation | depth is upstream of per-call DP/AD |
mosdepth -- The Modern Default
Goal: Get the median depth and the full breadth curve for a BAM in one fast pass.
Approach: Run mosdepth windowed (or whole-genome), then read the cumulative distribution file -- it already holds breadth at every depth threshold; no histogram integration needed.
mosdepth --by 500 -Q 20 sample in.bam
The *.global.dist.txt rows are chrom depth proportion_of_bases_at_least_this_depth -- the breadth curve directly. Read median as the depth where proportion crosses 0.5. --fast-mode/-x is ~2x faster but SILENTLY disables mate-overlap correction -- fine for a rough WGS glance, wrong for VAF-sensitive short-insert data.
Goal: Emit a callable-region BED (NO_COVERAGE / LOW / CALLABLE / HIGH) without GATK3.
Approach: Use --quantize to bin depth and merge adjacent equal-bin runs into a compact BED.
mosdepth --quantize 0:1:4:150: callable in.bam
zcat callable.quantized.bed.gz | head
bedtools genomecov -- Tracks and the Histogram Default
bedtools genomecov -ibam in.bam -bga > cov.bedGraph
bedtools genomecov -ibam in.bam -pc -bg > frag.bedGraph
bedtools genomecov -ibam in.bam -split -bg > rna.bedGraph
bedtools genomecov -ibam in.bam > hist.txt
The bare default is a histogram, not a bedGraph -- 5 columns: chrom depth bases_at_that_depth chrom_size fraction_of_chrom, with a final genome block for the whole genome. Breadth/mean must be integrated from it yourself (sum fraction over depth >= threshold) -- which is exactly why mosdepth's ready-made dist file is preferred.
bedtools coverage -- Per-Target Stats (mind the orientation)
bedtools coverage -a targets.bed -b in.bam > per_target.bed
bedtools coverage -a targets.bed -b in.bam -mean > mean.bed
As of bedtools v2.24.0 coverage is computed for the -a file (it was -b before) -- A = the regions stats are wanted for (targets), B = the reads. The default appends 4 columns to each A interval: (1) count of B features overlapping, (2) bases in A covered >=1x, (3) length of A, (4) fraction of A covered (col2/col3 = per-interval breadth). -d = per-base depth within each interval; -hist = depth histogram per interval plus an all summary; -counts = just the overlap count (faster).
samtools depth / coverage
samtools coverage in.bam
samtools depth -a -Q 20 -r chr1:1-100000 in.bam
samtools depth -s in.bam
samtools depth -a --reference ref.fa in.cram
In samtools coverage the column literally named coverage is breadth (% bases >=1x), and meandepth is depth -- a contig with coverage=9.7 and meandepth=3.5 is 3.5x over only 9.7% of the contig (a localized pileup), NOT "9.7x coverage". samtools depth drops UNMAP/SECONDARY/QCFAIL/DUP by default (so duplicates are excluded -- but only if they were MARKED first). Without -a/-aa, zero-depth positions are omitted, so a naive sum/lines mean over-counts by dropping the zeros.
Per-Method Failure Modes
Reporting mean depth as the result
Trigger: citing "mean = 30x" as adequacy. Mechanism: mean is inflated by the repeat/rDNA tail and blind to GC/mappability holes. Symptom: a genome with large uncallable gaps looks fine. Fix: report median + breadth at the caller's threshold (mosdepth dist).
samtools depth silent 8000 cap
Trigger: pre-1.13 samtools on high-depth loci (rDNA, mito, amplicon, ctDNA). Mechanism: old default -d/-m capped depth at 8000 and truncated with no warning. Symptom: depth plateaus near 8000. Fix: samtools --version; on old builds add -d 0; 1.13+ has no cap (flag ignored). Note mpileup has its own separate 8000 default.
Mate-overlap double-counting
Trigger: naive per-base depth on short-insert libraries (amplicon, cfDNA, FFPE). Mechanism: the two mates of a short fragment both cover the overlap, counted twice but not independent. Symptom: locally doubled depth, corrupted/inflated VAFs. Fix: samtools depth -s, genomecov -pc (fragment), or mosdepth default -- and do NOT use mosdepth --fast-mode/-x, which turns the correction off.
Coverage off an un-deduped BAM
Trigger: depth on a BAM whose duplicates were never marked. Mechanism: dedup-aware tools drop the DUP flag, but nothing was flagged. Symptom: inflated depth at amplified (often GC-extreme) loci, fatter right tail. Fix: Picard MarkDuplicates / samtools markdup FIRST, then measure.
MAPQ filtering and repeat coverage
Trigger: choosing a MAPQ threshold without considering repeats. Mechanism: repeats give low MAPQ; -Q 20+ makes repeat coverage vanish, MAPQ 0 lets multimappers pile up or smear. Symptom: repeats read as either empty or noisy -- no neutral choice. Fix: mask repeats (ENCODE blacklist / mappability) and report breadth over the MAPPABLE genome, not the whole genome.
bedtools coverage -a/-b backwards
Trigger: pre-v2.24.0 muscle memory / old tutorials. Mechanism: semantics flipped to report stats for -a at v2.24.0. Symptom: well-formed output describing per-read instead of per-target stats. Fix: A = targets, B = reads; sanity-check the row count equals the target count.
genomecov default misread / no -split on RNA-seq
Trigger: expecting a bedGraph from bare genomecov, or omitting -split on spliced reads. Mechanism: bare default is a histogram; without -split an N-CIGAR intron is counted as covered. Symptom: misparsed histogram, or every spliced gene appears fully covered across introns. Fix: add -bg/-bga for a track; always -split for spliced data.
Quantitative Thresholds
| Threshold | Source | Rationale |
|---|
| WGS germline ~30x mean -> ~95% of genome >= 20x | field convention (approx) | het-SNP sensitivity plateaus ~30x; frame as breadth, not mean |
| WES germline ~100x on-target -> ~90-95% target >= 10-20x | field convention (approx; ACMG-style, lab-dependent) | capture unevenness + off-target loss eat the raw mean |
| Somatic bulk tumor ~60-100x+ | field convention (approx) | low-VAF subclones need depth ~ 1/VAF; impure tumors need more |
| ctDNA/UMI panels 1000s-50000x raw | field convention (approx) | raw depth != usable depth after UMI collapse; report effective depth |
| Long-read WGS ~20-30x (HiFi ~30x, ONT SV ~20x+) | moving convention (approx) | flatter GC bias + better repeat mappability reach more genome per x |
| mean/median > ~1.1-1.2 = skewed | distribution diagnostic | the tail is inflating the mean; investigate dups/repeats/rDNA |
| Fano factor = 1 (Poisson ideal); real >> 1 | Lander & Waterman 1988 | overdispersion = evenness problem; deeper sequencing won't fill holes |
| Picard fold-80 ~1.3-2 good, >3 poor | practitioner heuristic (Picard defines only the metric) | fold extra sequencing to lift 80% of targets to the mean |
Common Errors
| Error / symptom | Cause | Solution |
|---|
| Depth plateaus at ~8000 | pre-1.13 samtools default cap | samtools --version; add -d 0; upgrade to 1.13+ |
| Inflated VAFs in amplicon/cfDNA | mate-overlap double-counting | samtools depth -s / genomecov -pc / mosdepth (not --fast-mode) |
| "30x" but variants missing in some genes | GC-shallow / uncallable holes hidden by mean | report breadth at threshold; mask blacklist; check fold-80 |
samtools coverage "coverage" looks tiny | it is breadth %, not depth | read meandepth for depth; coverage = % bases >=1x |
| genomecov gives a histogram not a track | no -bg/-bga flag | add -bga (with zeros) or -bg |
| Every spliced gene fully covered | missing -split on RNA-seq | add -split to genomecov/coverage |
| bedtools coverage stats look per-read | -a/-b backwards (pre-2.24 habit) | A = targets, B = reads |
| CRAM depth errors / empty | missing reference | samtools depth --reference ref.fa |
References
- Quinlan AR, Hall IM. 2010. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26:841-842.
- Pedersen BS, Quinlan AR. 2018. Mosdepth: quick coverage calculation for genomes and exomes. Bioinformatics 34:867-868.
- Danecek P, Bonfield JK, Liddle J, et al. 2021. Twelve years of SAMtools and BCFtools. GigaScience 10:giab008.
- Aird D, Ross MG, Chen WS, et al. 2011. Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries. Genome Biol 12:R18.
- Benjamini Y, Speed TP. 2012. Summarizing and correcting the GC content bias in high-throughput sequencing. Nucleic Acids Res 40:e72.
- Amemiya HM, Kundaje A, Boyle AP. 2019. The ENCODE blacklist: identification of problematic regions of the genome. Sci Rep 9:9354.
- Lander ES, Waterman MS. 1988. Genomic mapping by fingerprinting random clones: a mathematical analysis. Genomics 2:231-239.
Related Skills
- bedgraph-handling - bedGraph tracks this skill emits, and their normalization
- bigwig-tracks - Convert the coverage bedGraph to an indexed bigWig for browsers
- interval-arithmetic - Intersect coverage/callable BEDs with target regions
- alignment-files/pileup-generation - Per-base pileup upstream of depth and per-call DP
- alignment-files/bam-statistics - flagstat/idxstats and dup rate that explain coverage confounders
- chip-seq/chipseq-visualization - deepTools normalized coverage tracks for cross-sample comparison
- data-visualization/genome-tracks - Render the coverage tracks built here
