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

Despite the increasing use of different sequencing techniques in ecological applications, the mechanistic factors driving their quantitative performance remain poorly understood. Here, we assembled two types of mock communities: one using DNA extracted from pooled marine nematodes and one using the individual nematodes as input. The composition and relative abundances within those communities were then characterized using 18S and 28S rRNA metabarcoding and shotgun sequencing. A qualitatively similar {beta}-diversity was revealed by both methods. Shotgun read proportions generally tracked input DNA across well-represented genera, whereas metabarcoding performance depended on primer choice. Under the analytical frameworks applied, shotgun sequencing provided more consistent estimates of individual counts for nematode genera. However, although shotgun sequencing provided a more consistent estimation of taxon abundance than metabarcoding, particularly for nematodes represented by a high DNA input, neither method was able to accurately quantify nematodes with low input DNA or a small body size. The correlation analyses revealed that relative read abundances from both sequencing approaches were more strongly associated with DNA quantity than with individual counts. This suggests that variation in starting material can influence quantitative outcomes, and that differences in nematode body size across genera may significantly affect community composition assessment. Our findings show that metabarcoding and shotgun sequencing are equally effective in detecting structural changes at the community level as well as abundance shifts within individual taxa, but shotgun sequencing is more reliable for across-taxon comparisons. We provide a comprehensive assessment of how input material, primer choice, and sequencing approach influence the accuracy of nematode abundance quantification. Our study advances quantitative practices in the application of these methods for nematode-based bioindication and, more broadly environmental DNA biomonitoring.