{"title":"利用NanoString nCounter快速定量评估多种海洋环境中微生物氮循环关键功能基因的时空变化","authors":"Jennifer Tolman, Dhwani Desai, Julie LaRoche","doi":"10.1002/lom3.10594","DOIUrl":null,"url":null,"abstract":"<p>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 <i>nifH</i> 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, <i>nifH</i> amplicon sequencing, and shotgun metagenomic sequencing.</p>","PeriodicalId":18145,"journal":{"name":"Limnology and Oceanography: Methods","volume":"22 2","pages":"103-117"},"PeriodicalIF":2.1000,"publicationDate":"2023-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/lom3.10594","citationCount":"0","resultStr":"{\"title\":\"Rapid quantitative assessment of temporal and spatial variation in key functional genes of the microbial nitrogen cycle across multiple marine environments using the NanoString nCounter\",\"authors\":\"Jennifer Tolman, Dhwani Desai, Julie LaRoche\",\"doi\":\"10.1002/lom3.10594\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p>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 <i>nifH</i> 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, <i>nifH</i> amplicon sequencing, and shotgun metagenomic sequencing.</p>\",\"PeriodicalId\":18145,\"journal\":{\"name\":\"Limnology and Oceanography: Methods\",\"volume\":\"22 2\",\"pages\":\"103-117\"},\"PeriodicalIF\":2.1000,\"publicationDate\":\"2023-12-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://onlinelibrary.wiley.com/doi/epdf/10.1002/lom3.10594\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Limnology and Oceanography: Methods\",\"FirstCategoryId\":\"89\",\"ListUrlMain\":\"https://onlinelibrary.wiley.com/doi/10.1002/lom3.10594\",\"RegionNum\":3,\"RegionCategory\":\"地球科学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"LIMNOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Limnology and Oceanography: Methods","FirstCategoryId":"89","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/lom3.10594","RegionNum":3,"RegionCategory":"地球科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"LIMNOLOGY","Score":null,"Total":0}
Rapid quantitative assessment of temporal and spatial variation in key functional genes of the microbial nitrogen cycle across multiple marine environments using the NanoString nCounter
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.
期刊介绍:
Limnology and Oceanography: Methods (ISSN 1541-5856) is a companion to ASLO''s top-rated journal Limnology and Oceanography, and articles are held to the same high standards. In order to provide the most rapid publication consistent with high standards, Limnology and Oceanography: Methods appears in electronic format only, and the entire submission and review system is online. Articles are posted as soon as they are accepted and formatted for publication.
Limnology and Oceanography: Methods will consider manuscripts whose primary focus is methodological, and that deal with problems in the aquatic sciences. Manuscripts may present new measurement equipment, techniques for analyzing observations or samples, methods for understanding and interpreting information, analyses of metadata to examine the effectiveness of approaches, invited and contributed reviews and syntheses, and techniques for communicating and teaching in the aquatic sciences.