Navigating Past Oceans: Comparing Metabarcoding and Metagenomics of Marine Ancient Sediment Environmental <scp>DNA</scp>

The condition of ancient marine ecosystems provides context for contemporary biodiversity changes in human-impacted oceans. Sequencing sedimentary ancient DNA (sedaDNA) is an emerging method for generating high-resolution biodiversity time-series data, offering insights into past ecosystems. However, few studies directly compare the two predominant sedaDNA sequencing approaches: metabarcoding and shotgun-metagenomics, and it remains unclear if these methodological differences affect diversity metrics. We compared these methods using sedaDNA from an archived marine sediment record sampled in the Skagerrak, North Sea, spanning almost 8000 years. We performed metabarcoding of a eukaryotic 18S rRNA region (V9) and sequenced 153-229 million metagenomic reads per sample. Our results show limited overlap between metabarcoding and metagenomics, with only three metazoan genera detected by both methods. For overlapping taxa, metabarcoding detections became inconsistent for samples older than 2000 years, while metagenomics detected taxa throughout the time series. We observed divergent patterns of alpha diversity, with metagenomics indicating decreased richness towards the present and metabarcoding showing an increase. However, beta diversity patterns were similar between methods, with discrepancies only in metazoan data comparisons. Our findings demonstrate that the choice of sequencing method significantly impacts detected biodiversity in an ancient marine sediment record. While we stress that studies with limited variation in DNA degradation among samples may not be strongly affected, researchers should exonerate methodological explanations for observed biodiversity changes in marine sediment cores, particularly when considering alpha diversity, before making ecological interpretations.