Analysis of unmapped reads was performed using 53,831 samples from TOPMed data freeze 5. From each sample, we extracted and filtered all read pairs with at least one unmapped mate and used them to discover human sequences that were absent from the reference. The pipeline included four steps: (1) per-sample de novo assembly of unmapped reads; (2) contig alignment to the Pan paniscus, Pan troglodytes, Gorilla gorilla and Pongo abelii genome references and subsequent hominid-reference-based merging and scaffolding of sequences pooled together from all samples; (3) reference placement and breakpoint calling; and (4) variant genotyping. The detailed description of each step is provided in Supplementary Information 1.7.
