SAFARI: pangenome alignment of ancient DNA using purine/pyrimidine encodings

Aligning DNA sequences retrieved from fossils or other paleontological artifacts, referred to as ancient DNA (aDNA), is particularly challenging due to the short sequence length and chemical damage which creates a specific pattern of substitution (C[Formula: see text]T and G[Formula: see text]A) in addition to the heightened divergence between the sample and the reference genome thus exacerbating reference bias. This bias can be mitigated by aligning to pangenome graphs to incorporate documented organismic variation, but this approach still suffers from substitution patterns due to chemical damage. We introduce a novel methodology introducing the RYmer index, a variant of the commonly used minimizer index which represents purines (A,G) and pyrimidines (C,T) as R and Y, respectively. This creates an indexing scheme robust to the aforementioned chemical damage. We implemented SAFARI (Sensitive Alignments From A RYmer Index), an aDNA damage-aware version of the pangenome aligner vg giraffe, which uses RYmers to rescue alignments containing deaminated seeds. For highly damaged samples, the recovery rate could be upwards of 10%, an amount which could well affect downstream results. We show that our approach produces more correct alignments from aDNA sequences than current approaches while maintaining a tolerable rate of spurious alignments. In addition, we demonstrate that our algorithm improves the estimate of the rate of aDNA damage, especially for highly damaged samples. Crucially, we show that this improved alignment can directly translate into better insights gained from the data by showcasing its integration with a number of extant pangenome tools.