Trivedi Christopher B, Keuschnig Christoph, Larose Catherine, Rissi Daniel Vasconcelos, Mourot Rey, Bradley James A, Winkel Matthias, Benning Liane G
Interface Geochemistry, GFZ German Research Centre for Geosciences, Potsdam, Germany.
Environmental Microbial Genomics, Université de Lyon, Ecully Cedex, France.
Front Microbiol. 2022 May 23;13:894893. doi: 10.3389/fmicb.2022.894893. eCollection 2022.
The preservation of nucleic acids for high-throughput sequencing is an ongoing challenge for field scientists. In particular, samples that are low biomass, or that have to be collected and preserved in logistically challenging environments (such as remote sites or during long sampling campaigns) can pose exceptional difficulties. With this work, we compare and assess the effectiveness of three preservation methods for DNA and RNA extracted from microbial communities of glacial snow and ice samples. Snow and ice samples were melted and filtered upon collection in Iceland, and filters were preserved using: (i) liquid nitrogen flash freezing, (ii) storage in RNAlater, or (iii) storage in Zymo DNA/RNA Shield. Comparative statistics covering nucleic acid recovery, sequencing library preparation, genome assembly, and taxonomic diversity were used to determine best practices for the preservation of DNA and RNA samples from these environments. Our results reveal that microbial community composition based on DNA was comparable at the class level across preservation types. Based on extracted RNA, the taxonomic composition of the active community was primarily driven by the filtered sample volume (i.e., biomass content). In low biomass samples (where <200 ml of sample volume was filtered) the taxonomic and functional signatures trend toward the composition of the control samples, while in samples where a larger volume (more biomass) was filtered our data showed comparable results independent of preservation type. Based on all comparisons our data suggests that flash freezing of filters containing low biomass is the preferred method for preserving DNA and RNA (notwithstanding the difficulties of accessing liquid nitrogen in remote glacial field sites). Generally, RNAlater and Zymo DNA/RNA Shield solutions work comparably well, especially for DNA from high biomass samples, but Zymo DNA/RNA Shield is favored due to its higher yield of preserved RNA. Biomass quantity from snow and ice samples appears to be the most important factor in regards to the collection and preservation of samples from glacial environments.
对于野外科学家而言,保存用于高通量测序的核酸一直是一项挑战。特别是低生物量的样本,或者必须在后勤保障具有挑战性的环境(如偏远地区或长时间采样活动期间)收集和保存的样本,可能会带来特殊困难。在这项研究中,我们比较并评估了三种保存方法对从冰川雪冰样本微生物群落中提取的DNA和RNA的有效性。雪冰样本在冰岛采集后融化并过滤,滤膜采用以下方法保存:(i)液氮速冻,(ii)保存在RNAlater中,或(iii)保存在Zymo DNA/RNA Shield中。通过涵盖核酸回收率、测序文库制备、基因组组装和分类多样性的比较统计,来确定保存这些环境中DNA和RNA样本的最佳方法。我们的结果表明,基于DNA的微生物群落组成在不同保存类型的类水平上具有可比性。基于提取的RNA,活跃群落的分类组成主要由过滤后的样本体积(即生物量含量)决定。在低生物量样本(过滤样本体积<200 ml)中,分类和功能特征趋向于对照样本的组成,而在过滤体积较大(生物量更多)的样本中,我们的数据显示无论保存类型如何,结果都具有可比性。基于所有比较,我们的数据表明,对低生物量滤膜进行速冻是保存DNA和RNA的首选方法(尽管在偏远冰川野外地点获取液氮存在困难)。一般来说,RNAlater和Zymo DNA/RNA Shield溶液的效果相当,特别是对于高生物量样本中的DNA,但由于其保存RNA的产量更高,Zymo DNA/RNA Shield更受青睐。就冰川环境样本的采集和保存而言,雪冰样本的生物量数量似乎是最重要的因素。