DNA metabarcoding of insects and allies: An evaluation of primers and pipelines

Metabarcoding, the coupling of DNA-based species identification and high-throughput sequencing offers enormous promise for arthropod biodiversity studies but factors such as cost, speed, and ease-of-use of bioinformatic pipelines, crucial for making the leapt from demonstration studies to a real-world application, have not been adequately addressed. Here, four published and one newly designed primer set were tested across a diverse set of 80 arthropod species, representing 11 orders, to establish an optimal single primer set for Illumina-based metabarcoding of tropical Malaise trap samples. Two primer sets which showed the best amplification success with individual specimen PCR (98%) were used for bulk PCR and Illumina MiSeq sequencing. The sequencing outputs were subjected to both manual and simple metagenomics quality control and filtering pipelines. We obtained acceptable detection rates (80-90% of input species) after bulk PCR and high-throughput sequencing but analyses were complicated by potential heteroplasmic sequences and contamination. The manual pipeline produced similar or better outputs to the simple metagenomics pipelines (1.3 “real/error” sequences compared to 0.4). Our study suggests that metabarcoding is slowly becoming as cheap, fast and easy as conventional DNA barcoding, and that Malaise trap metabarcoding may soon fulfill its potential, providing a thermometer for biodiversity.