A quantitative protocol for DNA metabarcoding of springtails (Collembola)

Abstract

Abstract:We developed a novel protocol with superior quantitative analysis results for DNA metabarcoding of Collembola, a major soil microarthropod order. Degenerate PCR primers were designed for conserved regions in the mitochondrial cytochrome c oxidase subunit I (mtCOI) and 16S ribosomal RNA (mt16S) genes based on published collembolan mitogenomes. The best primer pair was selected based on its ability to amplify each gene, irrespective of the species. DNA was extracted from 10 natural communities sampled in a temperate forest (with typically 25-30 collembolan species per 10 soil samples) and 10 mock communities (with seven cultured collembolan species). The two gene regions were then amplified using the selected primers, ligated with adapters for 454 technology, and sequenced. Examination of the natural community samples showed that 32 and 36 operational taxonomic units (defined at a 90% sequence similarity threshold) were recovered from the mtCOI and mt16S data, respectively, which were comparable to the results of the microscopic identification of 25 morphospecies. Further, sequence abundances for each collembolan species from the mtCOI and mt16S data of the mock communities, after normalization by using a species as the internal control, showed good correlation with the number of individuals in the samples (R = 0.91-0.99), although relative species abundances within a mock community sample estimated from sequences were skewed from community composition in terms of the number of individuals or biomass of the species. Thus, this protocol enables the comparison of collembolan communities in a quantitative manner by metabarcoding.Key words: metabarcoding, Collembola, 16S, COX1, quantification. Résumé :Les auteurs ont mis au point un protocole pour le métacodage à barres de l'ADN chez les collemboles, un ordre important parmi les micro-arthropodes du sol. Des amorces PCR dégénérées ont été conçues pour les gènes codant pour la sous-unité I de la cytochrome c oxydase mitochondriale (mtCOI) et pour l'ARN ribosomique 16S mitochondrial (mt16S) sur la base des génomes mitochondriaux déjà séquencés chez les collemboles. La meilleure paire d'amorces a été choisie sur la base de sa capacité à amplifier chaque gène, sans égard à l'espèce.

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

“…Instead, position and type of mismatch can have widely different effects on amplification efficiency (Kwok et al 1990). Nevertheless, the accurate quantification of relative abundances of taxa is critical for many biodiversity analyses, and much effort is therefore being made to optimize methods for quantitative metabarcoding (Krehenwinkel et al 2017a;Piñol et al 2019;Saitoh et al 2016).…”

Barcoding the food chain: from Sanger to high-throughput sequencing

“…For example, it is generally difficult to determine the geographic provenance of a sample with DNA barcoding, which could be useful for uncovering illegal fishing or protected designation of origin substitutions, although some populations do contain distinct COI sequences (Bogdanowicz et al 2000). Inferring data on biomass from copy numbers of sequences is still contentious (Deagle et al 2013) and the subject of system-specific research (Saitoh et al 2016), so frauds such as product dilution will be difficult to detect.…”

“…Some studies have shown that eDNA extracted from water is a good indicator of relative species abundance in aquatic systems (Lodge et al 2012;Thomsen et al 2012;Pilliod et al 2014), and while this provides a good starting point, it remains a thorny issue for terrestrial systems (Saitoh et al 2016), for invertebrate species (due to primer bias), or when degraded DNA is used (King et al 2008). One solution is to model occupancy (species-level) or spatial mark-recapture (individual-level) data, although careful experimental designs are needed before data collection can begin (Schnell et al 2015).…”

From Barcodes to Biomes: Special Issues from the 6th International Barcode of Life Conference

“…However, quantification of organismal abundance via metabarcoding and HTS has remained an underdeveloped area of methodological study. In this special issue, Saitoh et al (2016) present a novel protocol employing HTS for the quantification of springtail (Collembola) communities, which form an important component of the soil biota. Using natural and artificially constructed test communities, the authors find that reliable quantification is possible under standardized molecular conditions involving the inclusion of a known species as an internal control.…”

“…The current open-access special issues feature 24 full articles. Part 1 includes two reviews that expand upon conference presentations and themes and 10 additional research articles associated with conference presentations, four of which are first authored by winners of a postdoctoral or graduate student prize at the 6th Conference (Beet et al 2016;Mark et al 2016;Saitoh et al 2016;Sing et al 2016). Part 2 includes 12 additional articles-three reviews, eight research articles, and one opinion piece-that cover topics ranging from a review of the field of fungal DNA barcoding (Xu 2016) to applications of DNA barcoding for biosecurity (Hodgetts et al 2016) to multiple novel methods of species detection (discussed below).…”