Genotyping

Rastair uses ML scores to determine which alternative alleles represent true genetic variants, then applies statistical methods to estimate zygosity. Rastair only support diploid genotyping (two chromosome copies) at this time.

Multi-Allelic Sites

When multiple alternate alleles are present at the same genomic position, they are combined into a single VCF record with comma-separated ALT values. For example, with REF=A and ALT=T,G, the genotype represents the complete set of observed alleles:

• A genotype of 1/2 means "on one chromosome there was a T (allele 1), on the other was a G (allele 2)"
• A genotype of 0/1 means "on one chromosome there was the reference A (allele 0), on the other was a T (allele 1)" (G alt did not pass filters)
• A genotype of 1/1 means "both chromosomes had a T (allele 1)" (only T alt passed filters)

This representation follows VCF specification standards where allele indices start at 0 for the reference, and 1, 2, 3... for the alternate alleles in order.

Genotype Calls

• Homozygous Reference (0/0): no alternate alleles passed filters
• Heterozygous (0/1): One alternate allele passed filters but read counts show a mix of reference and alternate reads
• Homozygous Alternate (1/1): an alternate allele passed filters and read counts show predominantly alternate reads, consistent with two copies of the variant
• Compound Heterozygous (1/2, 2/3, etc.): Multiple alternates passed filters and read counts support different variants on each chromosome copy

Confidence Scoring

Confidence values reflect call certainty:

• For 0/0 calls: based on the margin between the ML threshold and the highest-scoring alternate
• For variant calls: based on how well read count ratios match the expected distribution

Biological Interpretation

A genotype represents the complete set of observed alleles in an individual. All alternate alleles listed in a VCF record's ALT field are simultaneously present at that position in at least one chromosome copy. In most cases:

• If only one variant passes filters, the genotype indicates whether it's present on one chromosome copy (heterozygous 0/1) or both copies (homozygous alternate 1/1)
• If multiple variants pass filters, they represent compound heterozygosity where different variants are present on different chromosome copies (e.g., 1/2)

Strand-Specific Counting

For C→T and G→A variants, only one strand is used to avoid confounding with methylation. For all other variant types, both strands contribute to genotyping.