Benchmarking Metagenomic Classifiers on Simulated Ancient and Modern Metagenomic Data.

Taxonomic profiling of ancient metagenomic samples is challenging due to the accumulation of specific damage patterns on DNA over time. Although a number of methods for metagenome profiling have been developed, most of them have been assessed on modern metagenomes or simulated metagenomes mimicking modern metagenomes. Further, a comparative assessment of metagenome profilers on simulated metagenomes representing a spectrum of degradation depth, from the extremity of ancient (most degraded) to current or modern (not degraded) metagenomes, has not yet been performed. To understand the strengths and weaknesses of different metagenome profilers, we performed their comprehensive evaluation on simulated metagenomes representing human dental calculus microbiome, with the level of DNA damage successively raised to mimic modern to ancient metagenomes. All classes of profilers, namely, DNA-to-DNA, DNA-to-protein, and DNA-to-marker comparison-based profilers were evaluated on metagenomes with varying levels of damage simulating deamination, fragmentation, and contamination. Our results revealed that, compared to deamination and fragmentation, human and environmental contamination of ancient DNA (with modern DNA) has the most pronounced effect on the performance of each profiler. Further, the DNA-to-DNA (e.g., Kraken2, Bracken) and DNA-to-marker (e.g., MetaPhlAn4) based profiling approaches showed complementary strengths, which can be leveraged to elevate the state-of-the-art of ancient metagenome profiling.