Abstract
Although DNA metabarcoding is an attractive approach for monitoring biodiversity, it is often difficult to detect all the species present in a bulk sample. In particular, sequence recovery for a given species depends on its biomass and mitome copy number as well as the primer set employed for PCR. To examine these variables, we constructed a mock community of terrestrial arthropods comprised of 374 species. 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 segments (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 amplicons 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, although the Miseq delivered the highest sequence quality. As expected, species recovery was most efficient from the Single Leg treatment because amplicon abundance varied little among taxa. Among the three treatments where PCR occurred after pooling, the Bulk Abdomen treatment produced a more uniform read abundance than the Bulk Leg or Composite Leg treatment. Primer choice also influenced species recovery and evenness. Our results reveal how variation in protocols can have substantial impacts on perceived diversity unless sequencing coverage is sufficient to reach an asymptote.
Highlights
It is generally accepted that we have entered a period of unprecedented global biodiversity loss (Pimm et al 2014, Vogel 2017)
Barcode reference library with the analytical capacity of high-throughput sequencers (HTS), DNA metabarcoding provides a path to rapid, low-cost assessments of species composition (Hajibabaei et al 2011; Yu et al 2012; Brandon-Mong et al 2015; Moriniere et al 2016)
The present study has examined the impacts of diverse factors including source DNA, PCR primers, sequencing platform, and sequencing depth on species recovery from a mock community of arthropods
Summary
It is generally accepted that we have entered a period of unprecedented global biodiversity loss (Pimm et al 2014, Vogel 2017). As arthropods account for the majority of terrestrial biodiversity (Medeiros et al 2013), they are an obvious target for bio-surveillance They are collected in large numbers (Russo et al 2011), the subsequent processing and identification of specimens has traditionally been a barrier to large-scale monitoring programs (Bassett et al.2012). By coupling a DNA barcode reference library with the analytical capacity of high-throughput sequencers (HTS), DNA metabarcoding provides a path to rapid, low-cost assessments of species composition (Hajibabaei et al 2011; Yu et al 2012; Brandon-Mong et al 2015; Moriniere et al 2016). It achieves this goal by the amplification and sequence characterization of the barcode region from bulk DNA extracts which can be assigned to operational taxonomic units (OTUs) that can be queried against reference sequences to ascertain their source species
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