Libraries were sequenced on an Illumina MiSeq and HiSeq 2500 platforms in 76-cycle paired-end runs (28). For a detailed description of the read processing, see note S3. When analyzing the relationship of HST and Scladina to Vindija and the Altai Neandertal, further processing was necessary to avoid a reference bias of the alignments. First, we aligned DNA sequences to both the human reference genome (GRCh37/hg19) and a modified (“Neandertalized”) version of the reference genome that includes the alternative alleles seen in Vindija and/or the Altai Neandertal. If there was more than one alternative base at a given site (i.e., a triallelic site), then a random base was chosen. We then merged sequences that aligned to either reference genome and removed one duplicate of the sequences that mapped to both. If a sequence aligned to the two references at different positions, then both alignments were excluded (representing 522 and 332 such sequences for HST and Scladina, respectively). We developed an algorithm called bam-mergeRef to perform these merging steps, wrote it in C++, and made it available on GitHub (https://github.com/StephanePeyregne/bam-mergeRef). For a description of the reference bias and the effects of this processing, see note S9. Sequences from libraries enriched for mtDNA fragments were aligned to the revised Cambridge reference sequence (30) or the Altai Neandertal mtDNA with the same parameters as those applied to nuclear sequences (note S3).

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