Rapid and cost-effective generation of single specimen multilocus barcoding data from whole arthropod communities by multiple levels of multiplexing

Abstract

In light of the current biodiversity crisis, molecular barcoding has developed into an irreplaceable tool. Barcoding has been considerably simplified by developments in high throughput sequencing technology, but still can be prohibitively expensive and laborious when community samples of thousands of specimens need to be processed. Here, we outline an Illumina amplicon sequencing approach to generate multilocus data from large collections of arthropods. We reduce cost and effort up to 50-fold, by combining multiplex PCRs and DNA extractions from pools of presorted and morphotyped specimens and using two levels of sample indexing. We test our protocol by generating a comprehensive, community wide dataset of barcode sequences for several thousand Hawaiian arthropods from 14 orders, which were collected across the archipelago using various trapping methods. We explore patterns of diversity across the Archipelago and compare the utility of different arthropod trapping methods for biodiversity explorations on Hawaii, highlighting undergrowth beating as highly efficient method. Moreover, we show the effects of barcode marker, taxonomy and relative biomass of the targeted specimens and sequencing coverage on taxon recovery. Our protocol enables rapid and inexpensive explorations of diversity patterns and the generation of multilocus barcode reference libraries across whole ecosystems.

High-throughput sequencing for community analysis: the promise of DNA barcoding to uncover diversity, relatedness, abundances and interactions in spider communities

“…Further reductions in processing cost can be achieved by limiting the number of DNA extractions. This can be done by pooling specimens of divergent lineages, then performing bulk extractions (de Kerdrel et al 2020). PCR and library preparation are then performed on the pooled extract, and the resultant sequences are assigned back to their specimens using a reference database.…”

“…By limiting the number of DNA extractions, using multiplex PCRs, and using multiple levels of sample indexing, barcodes can now be generated at a cost of $ 0.2-1 each and with a considerable reduction of workload (de Kerdrel et al 2020;Meier et al 2016;Srivathsan et al 2019). This enables researchers to generate barcode sequences for thousands of specimens, thereby allowing estimates of both abundance and taxonomic richness within a community (see below).…”

“…A major drawback of this approach, however, is that it only yields a list of OTU sequences, which cannot be linked back to individual specimens because the DNA is bulk-extracted from mixed samples. As sequences cannot be assigned back to specimens, individual sequences from multilocus barcoding cannot be linked together, limiting the phylogenetic resolution of this approach unless the specimens can be linked to an extensive reference library (de Kerdrel et al 2020). Metabarcoding can also lead to inflated diversity estimates, as spurious sequences coamplify with the targeted specimen's DNA barcodes.…”

Methodological considerations for monitoring soil/litter arthropods in tropical rainforests using DNA metabarcoding, with a special emphasis on ants, springtails and termites

“…These authors suggested that samples with a considerable amount of soil and debris may include compounds that may inhibit PCR. Water in the soil may also increase DNA degradation (de Kerdrel et al 2020).…”

“…This may be related to lower extraction rate and sequence recovery. For instance, out of 14 arthropod orders screened by de Kerdrel et al (2020), Hymenoptera (including ants) showed the lowest sequence recovery rate. Implementing our three collecting methods with an ideal sample size of 100 soil, 40 Malaise and 80 light trap samples annually, would generate reliable time-series for the 24 ant species that we labeled as being common and amenable to statistical analysis of long-term trends.…”