Data from: Metabarcoding using multiplexed markers increases species detection in complex zooplankton communities

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.

元条形码（Metabarcoding）将DNA条形码（DNA barcoding）与高通量测序（high-throughput sequencing）相结合，通常借助单遗传标记解析复杂且分类学多样性丰富的样本。然而，物种级鉴定高度依赖标记基因与所选引物对的选择，往往需要在物种成功扩增与分类分辨率之间做出权衡。本研究提出一种适用于生物监测的通用元条形码实验方案：通过样本多重化技术，在单次高通量测序中同时使用两种条形码标记（COI与18S）以及四对引物。本研究利用24组包含不同遗传变异水平的模拟浮游动物群落对该方案进行验证。仅使用单标记与单引物对时，物种回收率最高可达77%。当使用全部3段COI序列片段时，模拟群落的物种检出率为62%~83%；而仅使用18S序列片段时，物种检出率为73%~75%。若同时使用两种标记，物种检出率可显著提升至89%~93%。此外，样本多重化并未对各物种的测序读段（reads）分配比例产生负面影响，且总检出物种数与单独测序各标记时相近。总体而言，我们提出的元条形码方法同时依托两种条形码标记，并为每个条形码标记配置多对引物，相较于单标记/单引物对方案，物种检出率提升了14%~35%，成为一种兼具应用价值与成本效益的天然浮游动物群落生物监测方法。我们强烈建议在元条形码研究中结合进化独立的标记，必要时为每个标记配置多对引物，以提升物种检出率（即减少假阴性结果）。