DOI: 10.5061/dryad.m83jc20
title: Data from: Metabarcoding using multiplexed markers increases species detection in complex zooplankton communities

description: 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.
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