Abstract
Metabarcoding can rapidly determine the species composition of bulk samples and thus aids biodiversity and ecosystem assessment. However, it is essential to use primer sets that minimize amplification bias among taxa to maximize species recovery. Despite this fact, the performance of primer sets employed for metabarcoding terrestrial arthropods has not been sufficiently evaluated. This study tests the performance of 36 primer sets on a mock community containing 374 insect species. Amplification success was assessed with gradient PCRs and the 21 most promising primer sets selected for metabarcoding. These 21 primer sets were also tested by metabarcoding a Malaise trap sample. We identified eight primer sets, mainly those including inosine and/or high degeneracy, that recovered more than 95% of the species in the mock community. Results from the Malaise trap sample were congruent with the mock community, but primer sets generating short amplicons produced potential false positives. Taxon recovery from both mock community and Malaise trap sample metabarcoding were used to select four primer sets for additional evaluation at different annealing temperatures (40–60 °C) using the mock community. The effect of temperature varied by primer pair but overall it only had a minor effect on taxon recovery. This study reveals the weak performance of some primer sets employed in past studies. It also demonstrates that certain primer sets can recover most taxa in a diverse species assemblage. Thus, based our experimental set up, there is no need to employ several primer sets targeting the same gene region. We identify several suitable primer sets for arthropod metabarcoding, and specifically recommend BF3 + BR2, as it is not affected by primer slippage and provides maximal taxonomic resolution. The fwhF2 + fwhR2n primer set amplifies a shorter fragment and is therefore ideal when targeting degraded DNA (e.g., from gut contents).
Highlights
IntroductionThe coupling of high-throughput sequencers (HTS) with DNA barcoding, commonly known as metabarcoding, allows for characterization of biodiversity at unprecedented scales (Creer et al 2016) as shown by studies on terrestrial (Gibson et al 2014; Beng et al 2016), freshwater (Hajibabaei et al 2011; Carew et al 2013; Andújar et al 2017), and marine (Leray & Knowlton 2015) ecosystems
Over the past decade, two methodological and technological advances have made it possible to address the urgent need for the capacity to undertake large-scale surveys of biodiversity (Vörösmarty et al 2010; Dirzo et al 2014; Steffen et al 2015)
We used two samples to test a range of primer sets for metabarcoding: a mock community of species (Braukmann et al 2019) and a sample collected with a Malaise trap (Figure 1)
Summary
The coupling of HTS with DNA barcoding, commonly known as metabarcoding, allows for characterization of biodiversity at unprecedented scales (Creer et al 2016) as shown by studies on terrestrial (Gibson et al 2014; Beng et al 2016), freshwater (Hajibabaei et al 2011; Carew et al 2013; Andújar et al 2017), and marine (Leray & Knowlton 2015) ecosystems. Metabarcoding studies on bulk collections of animals usually targets a 658 bp region of the cytochrome c oxidase subunit I (COI) (Folmer et al 1994; Andújar et al 2018). This gene region has gained broad adoption because of the rapidly expanding reference database
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
