Metatranscriptomics of environmental samples enables the identification of community activities without a priori knowledge of taxonomic or functional composition. However, several technical challenges associated with the RNA preparation protocols can affect the relative representation of transcripts and data interpretation. Here, seven replicate metatranscriptomes from planktonic freshwater samples (Lake Lanier, USA) were sequenced to evaluate technical and biological reproducibility of different RNA extraction protocols. Organic versus bead‐beating extraction showed significant enrichment for low versus high G + C% mRNA populations respectively. The sequencing data were best modelled by a negative binomial distribution to account for the large technical and biological variation observed. Despite the variation, the transcriptional activities of populations that persisted in year‐round metagenomes from the same site consistently showed distinct expression patterns, reflecting different ecologic strategies and allowing us to test prevailing models on the contribution of both rare biosphere and abundant members to community activity. For instance, abundant members of the Verrucomicrobia phylum systematically showed low transcriptional activity compared with other abundant taxa. Our results provide a practical guide to the analysis of metatranscriptomes and advance understanding of the activity and ecology of abundant and rare members of temperate freshwater microbial communities.

中文翻译：

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转录组学方案的评价及其在淡水微生物群落研究中的应用。

环境样品的超转录组学无需先验即可鉴定社区活动分类或功能组成方面的知识。但是，与RNA制备方案相关的一些技术挑战可能会影响转录本和数据解释的相对表示。在这里，对来自浮游淡水样品（美国Lake Lanier）的七个重复的转录组进行测序，以评估不同RNA提取方案的技术和生物学可重复性。有机提取法与珠子提取法分别显示出低G + C％和高C％mRNA群体的显着富集。测序数据最好通过负二项分布来建模，以说明观察到的较大的技术和生物学差异。尽管有变化，来自同一位点的全年元基因组中持续存在的种群的转录活动始终显示出不同的表达方式，反映了不同的生态策略，并使我们能够测试稀有生物圈和丰富成员对社区活动的贡献的主流模型。例如，V errucomicrobia与其他丰富的类群相比，门系统呈低转录活性。我们的结果提供了实用的指导，以分析超转录组，并进一步了解温带淡水微生物群落的丰富和稀有成员的活性和生态。