The marine nitrogen cycle controls oceanic productivity through enzymatic processes mediated by microbes. Here, we report the construction, evaluation, and application of the OceansN CodeSet for the NanoString nCounter, which quantifies a suite of protein-coding genes that are central to microbially mediated nitrogen cycle processes in the ocean. We also placed emphasis on quantifying a diverse set of marine diazotrophs within known nifH phylogenetic clades. The OceansN CodeSet provided direct hybridization-based quantitation of 48 probes in a single sample, presenting advantages in terms of reduced sample handling, elimination of amplification bias, minimal DNA sample requirements, and the ability to assess targets ranging from relatively rare to abundant, with a reliable quantitation limit of ~ 1000 gene copies per target per sample. As such, our approach fills a unique methodological niche between the scale of high-throughput amplicon sequencing (a compositional method) and quantitative polymerase chain reaction (qPCR) (a targeted method with generally lower throughput). When applied to North Atlantic environmental DNA samples, the OceansN CodeSet revealed temporal and spatial patterns in nitrogen assimilation, nitrification, and denitrification, as well as the abundance and distribution of various nitrogen-fixing microorganisms (diazotrophs). Data from the nCounter was validated via internal and external controls, and by comparison to qPCR, nifH amplicon sequencing, and shotgun metagenomic sequencing.

中文翻译：

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使用 NanoString nCounter 快速定量评估多个海洋环境中微生物氮循环关键功能基因的时间和空间变化

海洋氮循环通过微生物介导的酶促过程控制海洋生产力。在这里，我们报告了 NanoString nCounter 的 OceansN CodeSet 的构建、评估和应用，该代码集量化了一套蛋白质编码基因，这些基因对于海洋中微生物介导的氮循环过程至关重要。我们还重点量化已知nifH系统发育进化枝内的一组不同的海洋固氮生物。OceansN CodeSet 提供了对单个样品中 48 个探针进行基于杂交的直接定量，在减少样品处理、消除扩增偏差、最小化 DNA 样品要求以及评估从相对稀有到丰富的靶标的能力方面具有优势，每个样本每个目标的可靠定量限制约为 1000 个基因拷贝。因此，我们的方法填补了高通量扩增子测序（一种组合方法）和定量聚合酶链式反应（qPCR）（一种吞吐量通常较低的靶向方法）之间的独特方法论利基。当应用于北大西洋环境 DNA 样本时，OceansN CodeSet 揭示了氮同化、硝化和反硝化的时间和空间模式，以及各种固氮微生物（固氮微生物）的丰度和分布。来自 nCounter 的数据通过内部和外部对照进行验证，并与 qPCR、nifH扩增子测序和鸟枪法宏基因组测序进行比较。