Metabarcoding using multiplexed markers increases species detection in complex zooplankton communities

Abstract

Metabarcoding combines DNA barcoding with high‐throughput sequencing, often using one genetic marker to understand complex and taxonomically diverse samples. However, species‐level identification depends heavily on the choice of marker and the selected primer pair, often with a trade‐off between successful species amplification and taxonomic resolution. We present a versatile metabarcoding protocol for biomonitoring that involves the use of two barcode markers (COI and 18S) and four primer pairs in a single high‐throughput sequencing run, via sample multiplexing. We validate the protocol using a series of 24 mock zooplanktonic communities incorporating various levels of genetic variation. With the use of a single marker and single primer pair, the highest species recovery was 77%. With all three COI fragments, we detected 62%–83% of species across the mock communities, while the use of the 18S fragment alone resulted in the detection of 73%–75% of species. The species detection level was significantly improved to 89%–93% when both markers were used. Furthermore, multiplexing did not have a negative impact on the proportion of reads assigned to each species and the total number of species detected was similar to when markers were sequenced alone. Overall, our metabarcoding approach utilizing two barcode markers and multiple primer pairs per barcode improved species detection rates over a single marker/primer pair by 14% to 35%, making it an attractive and relatively cost‐effective method for biomonitoring natural zooplankton communities. We strongly recommend combining evolutionary independent markers and, when necessary, multiple primer pairs per marker to increase species detection (i.e., reduce false negatives) in metabarcoding studies.

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

“…Community diversity is estimated based on the number of recovered OTU sequences. In order to achieve a comprehensive taxon recovery in metabarcoding experiments, the use of more than one amplicon is advisable (Krehenwinkel et al 2018;Zhang et al 2018). Recent developments in clustering algorithms (Edgar 2018) also allow the inference of haplotypic information from bulk metabarcoding data.…”

Studying Ecosystems With DNA Metabarcoding: Lessons From Biomonitoring of Aquatic Macroinvertebrates

“…Refining primers for different taxonomic groups or species has taken considerable effort, but primers with broad coverage for invertebrates have now been established (e.g., Hajibabaei et al, 2012;Elbrecht and Leese, 2017). However, amplification bias due to variable affinity among sequence variants for amplification can distort the relationship between sample biomass and the number of sequence reads (Elbrecht and Leese, 2015;Zhang et al, 2018). Metabarcoding can therefore support a taxonomically broad universe of observation, but outputs should be treated as occurrences and do not support reliable estimation of organism biomass or abundance.…”

A practical guide to DNA metabarcoding for entomological ecologists

“…However, it is more challenging to identify NUMTs that do not have any in‐frame stop codons, insertions or deletions. The mitochondrial 16S and 12S ribosomal RNA genes have been used as alternative animal markers (Clarke et al ., ; Elbrecht et al ., ; Marquina et al ., ; Zhang et al ., ), but overall, we recommend using primers based on the COI barcode (Elbrecht et al ., ). The use of multiple or alternative markers does not necessarily improve species detection (Elbrecht et al ., ).…”