Revealing the Complexities of Metabarcoding with a Diverse Arthropod Mock Community

Abstract:Because DNA metabarcoding can reveal the species composition of bulk samples, it is an attractive approach for monitoring biodiversity. However, it is subject to biases that often impede detection of all species in a sample. In particular, the proportion of sequences recovered from each species depends on its biomass and mitome copy number as well as the primer set employed for PCR. The choice of sequencing platform may also influence results. To examine these variables, we constructed a mock community of terrestrial arthropods comprised of 374 BINs, a species proxy. We used this community to examine how species recovery was impacted when amplicon pools were constructed in four ways. The first two protocols involved the construction of bulk DNA extracts from different body partitions (Bulk Abdomen, Bulk Leg). The other protocols involved the production of DNA extracts from single legs which were then merged prior to PCR (Composite Leg) or PCR-amplified separately (Single Leg) and then pooled. The amplicon arrays generated by these four treatments were then sequenced on three platforms (Illumina MiSeq, Ion Torrent PGM and S5). Although the choice of sequencing platform did not substantially influence species recovery, other variables did. As expected, the best recovery was obtained from the Single Leg treatment, but the Bulk Abdomen produced a more uniform read abundance than the Bulk Leg or Composite Leg samples. Primer sets also influenced species recovery. The overall results reveal how variation in protocols can have substantive impacts on perceived diversity unless sequencing coverage is sufficient to achieve an asymptote. Although metabarcoding is a powerful approach, further optimization of analytical protocols is crucial to increase its cost-effectiveness and the interpretability of results.