Data from: Effect of marker choice and thermal cycling protocol on zooplankton DNA metabarcoding studies

DNA metabarcoding is a promising approach for rapidly surveying biodiversity and is likely to become an important tool for measuring ecosystem responses to environmental change. Metabarcoding markers need sufficient taxonomic coverage to detect groups of interest, sufficient sequence divergence to resolve species, and will ideally indicate relative abundance of taxa present. We characterized zooplankton assemblages with three different metabarcoding markers (nuclear 18S rDNA, mitochondrial COI, and mitochondrial 16S rDNA) to compare their performance in terms of taxonomic coverage, taxonomic resolution, and correspondence between morphology- and DNA-based identification. COI amplicons sequenced on separate runs showed that operational taxonomic units representing >0.1% of reads per sample were highly reproducible, although slightly more taxa were detected using a lower annealing temperature. Mitochondrial COI and nuclear 18S showed similar taxonomic coverage across zooplankton phyla. However, mitochondrial COI resolved up to threefold more taxa to species compared to 18S. All markers revealed similar patterns of beta-diversity, although different taxa were identified as the greatest contributors to these patterns for 18S. For calanoid copepod families, all markers displayed a positive relationship between biomass and sequence reads, although the relationship was typically strongest for 18S. The use of COI for metabarcoding has been questioned due to lack of conserved primer-binding sites. However, our results show the taxonomic coverage and resolution provided by degenerate COI primers, combined with a comparatively well-developed reference sequence database, make them valuable metabarcoding markers for biodiversity assessment.DNA宏条形码技术（DNA metabarcoding）是一种极具应用前景的生物多样性快速调查手段，有望成为衡量生态系统对环境变化响应的重要工具。宏条形码标记需具备足够的分类覆盖度（taxonomic coverage）以检出目标类群、足够的序列差异度（sequence divergence）以区分物种，且理想情况下可反映目标类群的相对丰度（relative abundance）。 本研究采用三类不同的宏条形码标记（核18S rDNA、线粒体COI、线粒体16S rDNA）对浮游动物群落（zooplankton assemblages）进行表征，以比较三者在分类覆盖度、物种分辨能力，以及形态学鉴定与DNA分子鉴定结果一致性方面的表现。 对不同测序批次的COI扩增子（COI amplicons）测序结果显示，占单样本reads数0.1%以上的操作分类单元（operational taxonomic units）具有极高的可重复性；尽管采用较低退火温度可检测到略多的类群。 线粒体COI与核18S rDNA在浮游动物各门的分类覆盖度上表现相近，但线粒体COI对物种的分辨能力可达核18S rDNA的三倍。 三类标记均呈现出相似的β多样性（beta-diversity）分布模式，但18S所鉴定的对该模式贡献最大的类群与另外两者存在差异。 针对哲水蚤科类群（calanoid copepod families），所有标记均显示生物量与序列reads数呈正相关关系，其中核18S rDNA的相关性通常最强。 此前由于保守引物结合位点的缺失，COI用于宏条形码研究的可行性曾受到质疑。但本研究结果表明，简并COI引物（degenerate COI primers）所提供的分类覆盖度与分辨能力，结合相对完善的参考序列数据库（reference sequence database），使其成为生物多样性评估中极具价值的宏条形码标记。