Evaluation of commercial RNA extraction kits for long-read metatranscriptomics in soil.

IF 4 2区 生物学 Q1 GENETICS & HEREDITY
Daniel G Barber, Christian A Davies, Iain P Hartley, Richard K Tennant
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引用次数: 0

Abstract

Metatranscriptomic analysis of the soil microbiome has the potential to reveal molecular mechanisms that drive soil processes regulated by the microbial community. Therefore, RNA samples must be of sufficient yield and quality to robustly quantify differential gene expression. While short-read sequencing technology is often favoured for metatranscriptomics, long-read sequencing has the potential to provide several benefits over short-read technologies. The ability to resolve complete transcripts on a portable sequencing platform for a relatively low capital expenditure makes Oxford Nanopore Technology an attractive prospect for addressing many of the challenges of soil metatranscriptomics. To fully enable long-read metatranscriptomic analysis of the functional molecular pathways expressed in these diverse habitats, RNA purification methods from soil must be optimised for long-read sequencing. Here we compare RNA samples purified using five commercially available extraction kits designed for use with soil. We found that the Qiagen RNeasy PowerSoil Total RNA Kit performed the best across RNA yield, quality and purity and was robust across different soil types. We found that sufficient sequencing depth can be achieved to characterise the active community for total RNA samples using Oxford Nanopore Technology, and discuss its current limitations for differential gene expression analysis in soil studies.

评估用于土壤长读元转录组学的商用 RNA 提取试剂盒。
对土壤微生物组进行元转录组学分析,有可能揭示驱动由微生物群落调控的土壤过程的分子机制。因此,RNA 样本必须有足够的产量和质量,才能稳健地量化不同基因的表达。虽然短线程测序技术通常是元转录组学的首选,但与短线程技术相比,长线程测序技术有可能提供多种优势。牛津纳米孔技术能够以相对较低的资本支出在便携式测序平台上解析完整的转录本,这使其在应对土壤元转录组学的许多挑战方面具有极具吸引力的前景。要想完全实现对这些不同生境中表达的功能分子通路的长读数元转录组学分析,必须优化土壤中的 RNA 纯化方法,以实现长读数测序。在此,我们比较了使用五种市售土壤提取试剂盒纯化的 RNA 样品。我们发现,Qiagen RNeasy PowerSoil 总 RNA 试剂盒在 RNA 产量、质量和纯度方面表现最佳,而且在不同土壤类型中都很稳定。我们发现,使用牛津纳米孔技术可以达到足够的测序深度,以描述总 RNA 样品的活性群落特征,并讨论了其目前在土壤研究中进行差异基因表达分析的局限性。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Microbial Genomics
Microbial Genomics Medicine-Epidemiology
CiteScore
6.60
自引率
2.60%
发文量
153
审稿时长
12 weeks
期刊介绍: Microbial Genomics (MGen) is a fully open access, mandatory open data and peer-reviewed journal publishing high-profile original research on archaea, bacteria, microbial eukaryotes and viruses.
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