Revealing the Complexities of Metabarcoding with a Diverse Arthropod Mock Community

Abstract

DNA metabarcoding 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, mitome copy number, and primer set employed for PCR. 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 generated by these four treatments were then sequenced on three platforms (Illumina MiSeq, Ion Torrent PGM and Ion Torrent S5). 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 choice also influenced species recovery. Our results reveal how variation in protocols can have substantive impacts on perceived diversity unless sequencing coverage is sufficient to reach an asymptote. Although metabarcoding is a powerful approach, further optimization of analytical protocols is crucial to obtain reproducible results and increase its cost-effectiveness.

A new primer for metabarcoding of spider gut contents

“…In order to assess the arthropod detection efficiency of the primer pair, it was also used to metabarcode an insect mock sample (Braukmann et al, ) and a malaise trap sample from Ontario, Canada, both previously tested with 21 primer sets (Elbrecht et al, ). A two‐step PCR was used to amplify and tag the DNA fragments.…”

Scaling up DNA metabarcoding for freshwater macrozoobenthos monitoring

“…Failed extractions or polymerase chain reactions (PCRs) can be excluded from the sequencing run, and repeated on a new plate. With the newly designed BF2 + BR2 fusion primers developed in this publication up to three 96-well plates can be multiplexed for a single run (Braukmann et al 2018). To ensure complete homogenisation, it is recommended that dried bulk samples are ground using e.g.…”

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“…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). The mock community is comprised of 374 species (Figure 1A), each specimen represented by a individual BIN (taxonomic breakdown shown in Figure S1A, (Ratnasingham & Hebert 2013)).…”

“…Well performing primers showed no bias against specific orders, while less suitable primers did struggle with detection of Hymenoptera and Hemiptera. The decreased detection of Hymenoptera in the mock community can likely be attributed to the lysis protocol used for DNA extraction from the insect abdomens (Braukmann et al 2019). This was not the case for the malaise sample, where the bulk sample was ground to a fine powder, making the tissue more accessible to the lysis buffer.…”

“…While primer choice is critical for metabarcoding projects, PCR is also biased by the polymerase used (Nichols et al 2018), cycle number (Vierna et al 2017;Krehenwinkel et al 2017), GC content (Braukmann et al 2019), inhibitors (Demeke & Jenkins 2009;Sellers et al 2018), and annealing temperature (Aylagas et al 2016;Clarke et al 2017;Krehenwinkel et al 2018). It is generally assumed that primers bind better at lower annealing temperatures as potential mismatches between template and primer have less influence.…”