Adapters were trimmed, sequences were merged, and reads shorter than 28 bp were discarded. Unmerged reads were kept apart and processed in parallel. All reads were then aligned to the Denisova mitochondrial genome sequence (1) using BWA aln (42), followed by either samse (merged reads) or sampe (nonmerged reads), and duplicate reads were removed. The two resulting bam files were then merged into a single file, and a consensus sequence was constructed.

The level of contaminating modern human DNA was measured by aligning the reads to a multi-fasta reference sequence containing both the Denisova and modern human rCRS mitochondrial sequences, removing PCR duplicates, and counting reads and bases overlapping any of the 182 sites where the Denisovan mitochondrial sequence is different from a panel of modern humans. This resulted in 12.1% bases and 13.9% reads originating from modern human mitochondria. A consensus modern human contaminating sequence covering 89% of bases was generated from reads mapping to the human rCRS rather than to the Denisovan sequence, and haplogroup assignment was assessed both by using the K-mer–based haplogroup assignment software Phy-Mer (43) and by its position on a maximum likelihood tree of reference haplogroups. A more detailed description is available in the Supplementary Materials and Methods.

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