Preservation of nucleic acids by freeze-drying for next generation sequencing analyses of soil microbial communities

Weißbecker, Christina; Buscot, François; Wubet, Tesfaye

doi:10.1093/jpe/rtw042

Cited by 38 publications

(31 citation statements)

References 45 publications

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“…Two important sample processing choices that may alter experimental results are (a) the type of soil storage method and (b) DNA extract thaw time, the length of time for which extracted DNA is transported and left to thaw. To date, only a handful of studies have examined consequences of soil storage methods on study results (i.e., temperature, absolute ethanol, freeze‐drying, RNAlater, PLFA) on targets such as DNA, RNA, bacteria, fungi, and arbuscular mycorrhizal fungi (Brandt, Breidenbach, Brenzinger, & Conrad, 2014; Cui et al, 2014; Harry, Gambier, & Garnier‐Sillam, 2000; Klammer, Mondini, & Insam, 2005; Lauber, Zhou, Gordon, Knight, & Fierer, 2010; Rissanen, Kurhela, Aho, Oittinen, & Tiirola, 2010; Rubin et al, 2013; Tzeneva et al, 2009; Weißbecker, Buscot, & Wubet, 2017). These studies have broadly found little impact of storage method, but they do not thoroughly explore common storage practices used in the field and focus overwhelmingly on bacteria.…”

Section: Introductionmentioning

confidence: 99%

“…This leaves researchers with an unclear understanding of how their choice of soil storage will impact study conclusions and interpretations. Most studies find no impact of soil sample storage over short periods of time under 1 month at temperatures from 4 to −80°C (Brandt et al, 2014; Harry et al, 2000; Klammer et al, 2005; Lauber et al, 2010; Schnecker, Wild, Fuchslueger, & Richter, 2012; Tatangelo, Franzetti, Gandolfi, Bestetti, & Ambrosini, 2014; Weißbecker et al, 2017). Some studies compare freeze‐drying in addition to storage at different temperatures (Cui et al, 2014; Klammer et al, 2005; Tatangelo et al, 2014; Weißbecker et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

“…Most studies find no impact of soil sample storage over short periods of time under 1 month at temperatures from 4 to −80°C (Brandt et al, 2014; Harry et al, 2000; Klammer et al, 2005; Lauber et al, 2010; Schnecker, Wild, Fuchslueger, & Richter, 2012; Tatangelo, Franzetti, Gandolfi, Bestetti, & Ambrosini, 2014; Weißbecker et al, 2017). Some studies compare freeze‐drying in addition to storage at different temperatures (Cui et al, 2014; Klammer et al, 2005; Tatangelo et al, 2014; Weißbecker et al, 2017). Nonetheless, most of these studies fail to investigate several practical and commonly used storage methods besides different temperatures (Brandt et al, 2014; Lauber et al, 2010; Rubin et al, 2013; Tatangelo et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

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Keeping it cool: Soil sample cold pack storage and DNA shipment up to 1 month does not impact metabarcoding results

Delavaux

Bever

Karppinen

et al. 2020

Ecology and Evolution

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With the advances of sequencing tools, the fields of environmental microbiology and soil ecology have been transformed. Today, the unculturable majority of soil microbes can be sequenced. Although these tools give us tremendous power and open many doors to answer important questions, we must understand how sample processing may impact our results and interpretations. Here, we test the impacts of four soil storage methods on downstream amplicon metabarcoding and qPCR analyses for fungi and bacteria. We further investigate the impact of thaw time on extracted DNA to determine a safe length of time during which this can occur with minimal impact on study results. Overall, we find that storage using standard cold packs with subsequent storage at −20°C is little different than immediate storage in liquid nitrogen, suggesting that the historical and current method is adequate. We further find evidence that storage at room temperature or with aid of RNAlater can lead to changes in community composition and in the case of RNAlater, lower gene copies. We therefore advise against these storage methods for metabarcoding analyses. Finally, we show that over 1 month, DNA extract thaw time does not impact diversity or qPCR metrics. We hope that this work will help researchers working with soil bacteria and fungi make informed decisions about soil storage and transport to ensure repeatability and accuracy of results and interpretations.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Keeping it cool: Soil sample cold pack storage and DNA shipment up to 1 month does not impact metabarcoding results

Delavaux

Bever

Karppinen

et al. 2020

Ecology and Evolution

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“…Although the advance in NGS and the possibility to analyze a large number of samples have led to large‐scale and integrated biodiversity studies at the global scale (Shoemaker, Locey, & Lennon, ), standardized soil sampling, storage, and transportation across continents still are a challenge. Accordingly, we developed a soil sampling, freeze‐drying, and preservation protocol that guarantees transportation of soil samples without nucleic acid degradation between laboratories across continents (Weißbecker, Buscot, & Wubet, ). The soil microbial nucleic acid extraction protocols have been optimized to a high‐throughput protocol, and the classical PCR‐based microbial diversity analysis protocols using microbial rDNA‐based barcodes (e.g., 16S for bacteria and ITS for fungi) have been adapted to meta‐barcoding protocols using NGS platforms (Lentendu et al., ; Wu et al., ).…”

Section: Bef‐china As a Case Study Of A Large Tree Diversity Experimentsmentioning

confidence: 99%

“…Recent advances in next-generation sequencing (NGS) techniques coupled with meta-barcoding approaches and the associated bioinformatics and statistical analysis tools enabled microbial ecologists to work in large-scale tree diversity experiments to shed light on the poorly understood role of microbial diversity on BEF relationships in forest ecosystems.Although the advance in NGS and the possibility to analyze a large number of samples have led to large-scale and integrated biodiversity studies at the global scale(Shoemaker, Locey, & Lennon, 2017), standardized soil sampling, storage, and transportation across continents still are a challenge. Accordingly, we developed a soil sampling, freeze-drying, and preservation protocol that guarantees transportation of soil samples without nucleic acid degradation between laboratories across continents(Weißbecker, Buscot, & Wubet, 2017). The soil microbial nucleic acid extraction protocols have been optimized to a high-throughput protocol, and the classical PCR-based microbial diversity analysis protocols using microbial rDNA-based barcodes (e.g., 16S for bacteria and ITS for fungi) have been adapted to metabarcoding protocols using NGS platforms(Lentendu et al, 2014;Wu et al, 2013).Another crucial point is the sampling strategy.…”

mentioning

confidence: 99%

Toward a methodical framework for comprehensively assessing forest multifunctionality

Trogisch

Schuldt

Bauhus

et al. 2017

Ecology and Evolution

Self Cite

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Biodiversity–ecosystem functioning (BEF) research has extended its scope from communities that are short‐lived or reshape their structure annually to structurally complex forest ecosystems. The establishment of tree diversity experiments poses specific methodological challenges for assessing the multiple functions provided by forest ecosystems. In particular, methodological inconsistencies and nonstandardized protocols impede the analysis of multifunctionality within, and comparability across the increasing number of tree diversity experiments. By providing an overview on key methods currently applied in one of the largest forest biodiversity experiments, we show how methods differing in scale and simplicity can be combined to retrieve consistent data allowing novel insights into forest ecosystem functioning. Furthermore, we discuss and develop recommendations for the integration and transferability of diverse methodical approaches to present and future forest biodiversity experiments. We identified four principles that should guide basic decisions concerning method selection for tree diversity experiments and forest BEF research: (1) method selection should be directed toward maximizing data density to increase the number of measured variables in each plot. (2) Methods should cover all relevant scales of the experiment to consider scale dependencies of biodiversity effects. (3) The same variable should be evaluated with the same method across space and time for adequate larger‐scale and longer‐time data analysis and to reduce errors due to changing measurement protocols. (4) Standardized, practical and rapid methods for assessing biodiversity and ecosystem functions should be promoted to increase comparability among forest BEF experiments. We demonstrate that currently available methods provide us with a sophisticated toolbox to improve a synergistic understanding of forest multifunctionality. However, these methods require further adjustment to the specific requirements of structurally complex and long‐lived forest ecosystems. By applying methods connecting relevant scales, trophic levels, and above‐ and belowground ecosystem compartments, knowledge gain from large tree diversity experiments can be optimized.

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Sample Preservation Solution Increases Nucleic Acid Yield and Environmental RNA Quality in Sediments Across an Estuarine Salinity Gradient

Keneally,

Gaget,

Kidd

et al. 2024

Environmental DNA

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Environmental nucleic acid‐based assessments are powerful tools for understanding microbial ecology, and environmental degradation in aquatic environments. This approach is particularly useful in guiding restoration in estuaries, some of the most degraded ecosystems in the world. The recent popularity of this approach has been accompanied by a parallel increase in the diversity of applied methods. A range of best practice methods exist across the field that can be employed and are selected based on environmental considerations such as physicochemical gradients, maximizing yield, and quality of nucleic acids sampled across sites within a study area. A consistent approach to intra‐study nucleic acid sampling also ensures accurate comparison between those sites. This study evaluates environmental nucleic acid (eNA) sampling methods across salinity gradients in aquatic ecosystems, focusing on the impact of preservation techniques on environmental DNA (eDNA) yield and environmental RNA (eRNA) yield and quality. Fieldwork was conducted at three sites within the Coorong estuary system in South Australia, representing low salinity, marine, and hypersaline conditions. Snap freezing and LifeGuard preservation solution treatments were applied in situ to compare their effects on nucleic acid yields and eRNA integrity. Snap freezing enhanced eDNA yield in low salinity sediments but negatively impacted eRNA integrity in marine and hypersaline conditions. Conversely, treatment with preservation solution consistently improved both eDNA and eRNA recovery across all salinity levels, which makes this approach a good candidate for preserving eNA molecules across environmental gradients. The study underscores the necessity of tailoring sample preservation methods to specific environmental conditions for accurate eNA‐based microbial community assessments in coastal ecosystems. These findings contribute to the development of robust eNA sampling protocols for benthic communities under varying salinity conditions.

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Preservation of nucleic acids by freeze-drying for next generation sequencing analyses of soil microbial communities

Cited by 38 publications

References 45 publications

Keeping it cool: Soil sample cold pack storage and DNA shipment up to 1 month does not impact metabarcoding results

Keeping it cool: Soil sample cold pack storage and DNA shipment up to 1 month does not impact metabarcoding results

Toward a methodical framework for comprehensively assessing forest multifunctionality

Sample Preservation Solution Increases Nucleic Acid Yield and Environmental RNA Quality in Sediments Across an Estuarine Salinity Gradient

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