Benchmarking the quantitative performance of metabarcoding and shotgun sequencing using mock communities of marine nematodes

Abstract

High-throughput sequencing has transformed biodiversity assessment and ecological monitoring, yet its quantitative reliability remains unclear. Here, we assembled two experiments of nematode mock communities: one based on extracted DNA and one on individual specimens. Although DNA extraction was required in both experiments to assess the quantitative performance of the sequencing approaches, we essentially evaluated whether this performance was influenced by differences in the start material used to constructed the mock communities. Each community was analyzed using 18S and 28S metabarcoding and shotgun sequencing to evaluate their ability to resolve quantitative information. Across datasets, the number of observed taxa increased with sequencing depth despite controlled input, indicating that higher read numbers primarily revealed intragenomic variation in nematodes than true diversity. Community composition was more accurately recovered by 18S metabarcoding and shotgun sequencing than by 28S. Both sequencing approaches reflected DNA input reasonably well; however, shotgun sequencing provided more consistent abundance estimates relative to individual counts, particularly for nematodes with relatively large-bodied size. In contrast, all methods showed limited ability to accurately quantify taxa with low DNA input or small body size. Comparisons between mock community types showed strong correspondence between read abundance and DNA input, but weaker relationships with individual counts. Overall, both metabarcoding and shotgun sequencing effectively detected community-level patterns and within-taxon abundance, but shotgun sequencing was more reliable for cross-taxon quantitative comparisons. Our findings demonstrate how input material, primer choice, and sequencing approach influence the accuracy of nematode abundance estimates, and provide guidance for improving quantitative applications in nematode-based bioindication and, more broadly environmental DNA biomonitoring.