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Step 11: Runs of Homozygosity (ROH) Analysis

What This Does

Detects long stretches of homozygous genotypes (autozygous segments) in the genome. These arise when both copies of a chromosomal region are inherited from a common ancestor.

Why

ROH analysis screens for consanguinity and uniparental disomy (UPD). Long ROH segments increase the risk of autosomal recessive disease by unmasking deleterious variants. ROH patterns also provide population-level ancestry information.

Tool

  • bcftools roh (samtools/bcftools)

Docker Image

  • BCFTOOLS_IMAGE

Pinned in versions.env; Image versions lists the current tag.

Command

export GENOME_DIR=/path/to/your/data
./scripts/11-roh-analysis.sh your_sample

What the script runs. Only PASS records (and records with no filter, as chip VCFs have) go into bcftools roh: DeepVariant's RefCall and other filtered records are not genotypes to count on. The Nextflow ROH module reads the same records with the same flags.

source versions.env   # from the repository root
SAMPLE=your_sample
GENOME_DIR=/path/to/your/data

docker run --rm \
  -v ${GENOME_DIR}/${SAMPLE}/vcf:/data \
  "${BCFTOOLS_IMAGE}" \
  bash -euo pipefail -c "bcftools view -f PASS,. -Ou /data/${SAMPLE}.vcf.gz \
    | bcftools roh --AF-dflt 0.4 -o /data/${SAMPLE}_roh.txt -"

For chip data (no FORMAT/PL), the script adds -G30.

Output

  • ${SAMPLE}/vcf/${SAMPLE}_roh.txt — bcftools roh output: per-site states (ST lines) and segments (RG lines)
  • ${SAMPLE}/vcf/${SAMPLE}_roh_summary.txt — the autosomal segments of 5 Mb or more (chrom, start, end, length in bp and Mb). The script and the Nextflow module write the same file with the same threshold.

Interpretation

Total ROH and parental relationship

When parents are related, a child is autozygous (both copies from the same ancestor) over a fraction F of the genome, the inbreeding coefficient. The expected total of long ROH is F times the length of the autosomes, about 2,900 Mb. The table counts autosomal segments of 5 Mb or more (the ones the script prints), leaving out the centromeric artifacts listed below. Shorter segments come mostly from distant shared ancestry and population history, not from the parents' relationship.

Parents' relationship F Expected total of ROH segments of 5 Mb or more
Not related ~0 None or a few segments
Second cousins 1/64 ~45 Mb
First cousins once removed 1/32 ~90 Mb
First cousins 1/16 ~180 Mb
Half siblings, uncle and niece, double first cousins 1/8 ~360 Mb
Parent and child, full siblings 1/4 ~720 Mb

These are averages. Inheritance is random, so one person's total can be well above or below the value for their parents' relationship, and the ranges of neighbouring rows overlap. A total near one row is consistent with it; it does not prove it.

To compute the total from the output:

grep '^RG' "${GENOME_DIR}/${SAMPLE}/vcf/${SAMPLE}_roh.txt" \
  | awk '$3 ~ /^chr[0-9]+$/ && $6 >= 5000000 {sum += $6; n++} END {printf "%d segments, %.0f Mb\n", n, sum / 1e6}'

Subtract any segment that lies in one of the centromeric regions below.

Single segments

Individual ROH Segment Interpretation
<1 Mb Common, population-level background
1-5 Mb Distant shared ancestry; many of them is typical of population isolates
5-10 Mb Counted in the total above; a few can occur even when the parents are not related
>10 Mb Recent shared ancestry; possible uniparental disomy if confined to one chromosome

Important Notes

  • The script auto-detects chip data (no FORMAT/PL tag) and adds -G30 for genotype-only mode
  • --AF-dflt 0.4 sets a default allele frequency when population AF data is unavailable — suitable for single-sample WGS
  • Not done yet: a population allele-frequency file (--AF-file, for example from gnomAD) would replace that one default value with real frequencies per site and give better segment edges. Neither the script nor the module uses one today.
  • Known false-positive regions (centromeric/pericentromeric, always appear as ROH in WGS):
  • chr1: 125-143 MB
  • chr9: 42-60 MB
  • chr18: 15-20 MB
  • These centromeric artifacts should be excluded when calculating total ROH burden
  • A single large ROH (>20 MB) confined to one chromosome may indicate uniparental disomy — verify with SNP array or parental samples
  • ROH within known disease gene regions warrants checking for homozygous pathogenic variants