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.

元条形码测序（Metabarcoding）将DNA条形码（DNA barcoding）与高通量测序（high-throughput sequencing）相结合，通常采用单个遗传标记（genetic marker）来解析复杂且分类学多样性丰富的样本。然而，物种水平鉴定（species-level identification）高度依赖遗传标记的选择与引物对（primer pair）的配置，且通常需在物种扩增成功率与分类学分辨率之间做出权衡。本研究开发了一种适用于生物监测（biomonitoring）的通用元条形码测序流程，该流程通过样品多重化（sample multiplexing）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）以及四组引物对。本研究利用一系列涵盖不同遗传变异水平的24个模拟浮游动物群落（mock zooplanktonic communities）对该流程进行验证。仅使用单个遗传标记与单组引物对时，最高物种回收率（species recovery）为77%。当使用全部三组COI扩增片段时，模拟群落中可检测到62%-83%的物种；而仅使用18S扩增片段时，物种检出率为73%-75%。当同时使用两种标记时，物种检出率可显著提升至89%-93%。此外，样品多重化并未对分配至各物种的测序读段（reads）比例产生负面影响，且总检出物种数与单独测序各标记时的结果相近。总体而言，本研究开发的元条形码测序方法通过同时使用两种条形码标记以及每个标记搭配多组引物对，将物种检出率相较于单标记/单引物对方案提升了14%至35%，使其成为一种极具应用前景且成本效益相对较高的自然浮游动物群落生物监测方法。本研究强烈建议，在元条形码测序研究中，结合进化独立标记（evolutionary independent markers），并在必要时为每个标记搭配多组引物对，以提升物种检出率（即降低假阴性false negatives）。