Dataset Information

The room temperature preservation of filtered environmental DNA samples and assimilation into a phenol-chloroform-isoamyl alcohol DNA extraction.

ABSTRACT: Current research targeting filtered macrobial environmental DNA (eDNA) often relies upon cold ambient temperatures at various stages, including the transport of water samples from the field to the laboratory and the storage of water and/or filtered samples in the laboratory. This poses practical limitations for field collections in locations where refrigeration and frozen storage is difficult or where samples must be transported long distances for further processing and screening. This study demonstrates the successful preservation of eDNA at room temperature (20 °C) in two lysis buffers, CTAB and Longmire's, over a 2-week period of time. Moreover, the preserved eDNA samples were seamlessly integrated into a phenol-chloroform-isoamyl alcohol (PCI) DNA extraction protocol. The successful application of the eDNA extraction to multiple filter membrane types suggests the methods evaluated here may be broadly applied in future eDNA research. Our results also suggest that for many kinds of studies recently reported on macrobial eDNA, detection probabilities could have been increased, and at a lower cost, by utilizing the Longmire's preservation buffer with a PCI DNA extraction.

SUBMITTER: Renshaw MA

PROVIDER: S-EPMC4312482 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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The room temperature preservation of filtered environmental DNA samples and assimilation into a phenol-chloroform-isoamyl alcohol DNA extraction.

Renshaw Mark A MA Olds Brett P BP Jerde Christopher L CL McVeigh Margaret M MM Lodge David M DM

Molecular ecology resources 20140611 1

Current research targeting filtered macrobial environmental DNA (eDNA) often relies upon cold ambient temperatures at various stages, including the transport of water samples from the field to the laboratory and the storage of water and/or filtered samples in the laboratory. This poses practical limitations for field collections in locations where refrigeration and frozen storage is difficult or where samples must be transported long distances for further processing and screening. This study dem ...[more]

PMID: 24834966

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Project description: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.

Project description:The primary concentration and molecular process are critical to implement wastewater-based epidemiology for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the previously developed methods were optimized for nonenveloped viruses. Few studies evaluated if the methods are applicable to the efficient recovery of enveloped viruses from various types of raw sewage. This study aims (1) to compare the whole process recovery of Pseudomonas phage φ6, a surrogate for enveloped viruses, among combinations of primary concentration [ultrafiltration (UF), electronegative membrane vortex (EMV), and polyethylene glycol precipitation (PEG)] and RNA extraction methods (spin column-based method using QIAamp Viral RNA Mini Kit and acid guanidinium thiocyanate-phenol-chloroform extraction using TRIzol reagent) for three types of raw sewage and (2) to test the applicability of the method providing the highest φ6 recovery to the detection of SARS-CoV-2 RNA. Among the tested combinations, PEG+TRIzol provided the highest φ6 recovery ratio of 29.8% to 49.8% (geometric mean). UF + QIAamp Viral RNA Mini Kit provided the second highest φ6 recovery of 6.4% to 35.8%. The comparable φ6 recovery was observed for UF + TRIzol (13.8-30.0%). PEG + QIAamp Viral RNA Mini Kit provided only 1.4% to 3.0% of φ6 recovery, while coliphage MS2, a surrogate for nonenveloped viruses, was recovered comparably with PEG + TRIzol. This indicated that the nonenveloped surrogate (MS2) did not necessarily validate the efficient recovery for enveloped viruses. EMV + QIAamp Viral RNA Mini Kit provided significantly different φ6 recovery (1.6-21%) among the types of raw sewage. Then, the applicability of modified PEG + TRIzol was examined for the raw sewage collected in Tokyo, Japan. Of the 12 grab samples, 4 were positive for SARS-CoV-2 CDC N1 and N3 assay. Consequently, PEG + TRIzol provided the highest φ6 recovery and allowed for the detection of SARS-CoV-2 RNA from raw sewage.

Dataset Information

The room temperature preservation of filtered environmental DNA samples and assimilation into a phenol-chloroform-isoamyl alcohol DNA extraction.

Publications

The room temperature preservation of filtered environmental DNA samples and assimilation into a phenol-chloroform-isoamyl alcohol DNA extraction.

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