High Throughput RNA Extraction and PCR Inhibitor Removal of Settled Solids for Wastewater Surveillance of SARS-CoV-2 RNA v2

2021 ◽  
Author(s):  
Aaron Topol ◽  
marlene.wolfe not provided ◽  
Krista Wigginton ◽  
Bradley White ◽  
Alexandria B Boehm
Keyword(s):  
Nucleic Acids ◽  
High Throughput ◽  
Magnetic Beads ◽  
Rna Extraction ◽  
Poly Vinyl Alcohol ◽  
Enzymatic Reactions ◽  
Efficient Removal ◽  
Dna And Rna ◽  
Pcr Inhibitor ◽  
Wastewater Samples

Please note that while this protocol is for TNA extraction using the Perkin Elmer Chemagic 360, RNA extraction with resuspended solids from this protocol has been verified to perform well using the Kingfisher MagMax kit as another high throughput, automated option and two manual Qiagen kits - the All Prep Powerviral DNA/RNA Kit and the Qiamp Viral RNA Mini Kit. This process instruction describes the steps for purification of nucleic acids from wastewater solids and preparation for downstream quantitative analysis with Reverse Transcriptase droplet digital Polymerase Chain Reaction (RT-ddPCR). Due to the large quantities of substances that have inhibitory effects on PCR in wastewater samples, a subsequent PCR inhibitor removal step is required after nucleic acid purification. Both steps of the process are carried out in a 96-well plate format. This method uses the resuspended solids generated using this protocol: High Throughput pre-analytical processing of wastewater settled solids for SARS-CoV-2 RNA analyses. RNA purification is carried out using a kit optimized for the purification of viral on for the Perkin Elmer Chemagic 360. Although only RNA is used in the downstream applications from this protocol, DNA is also eluted in this process. A crucial component of the purification kit are the magnetic particles coated with poly vinyl alcohol (M-PVA Magnetic Beads) which have a hydrophilic surface giving them an affinity for nucleic acids but not many other biological molecules. The workflow involves binding nucleic acids in a sample to the beads which are then transferred through a series of wash buffers to remove debris with a robotic head with magnetic rods. The OneStep PCR Inhibitor Removal Kits are PCR inhibitor clean up kits that contain all the components needed for efficient removal of contaminants that can inhibit downstream enzymatic reactions (e.g. PCR and RT) from DNA and RNA preparations. The column matrices in these PCR inhibitor clean up kits have been specifically designed for the efficient removal of polyphenolic compounds, humic/fulvic acids, tannins, melanin, etc. from the most impure DNA and RNA preparations. This process instruction applies to extraction of RNA from wastewater samples using the Chemagic™ Viral DNA/RNA 300 Kit H96 for the Perkin Elmer Chemagic 360 followed by PCR Inhibitor Removal with the Zymo OneStep-96 PCR Inhibitor Removal Kit.

Quantification of SARS-CoV-2 variant mutations (HV69-70, E484K/N501Y, and del156-157/R158G) in settled solids using digital RT-PCR v2

2021 ◽  
Author(s):  
Bridgette Hughes ◽  
Bradley J. White ◽  
Marlene K. Wolfe ◽  
Krista Wigginton ◽  
Alexandria B Boehm
Keyword(s):  
Quantitative Analysis ◽  
Nucleic Acid ◽  
High Throughput ◽  
Rna Extraction ◽  
Population Level ◽  
Digital Pcr ◽  
N Gene ◽  
Rt Pcr ◽  
Pcr Inhibitor ◽  
Wastewater Samples

This process instruction describes the steps for quantitative analysis of nucleic acid from SARS-CoV-2 with a triplex Reverse Transcriptase droplet digital Polymerase Chain Reaction (RT-ddPCR) assay targeting the N Gene, S Gene and 3 mutation assays (one for HV69-70, one for E484K/N501Y, and one for del156-157/R158G) in extracted and purified RNA samples from solid wastewater samples for population level SARS-CoV-2 community surveillance. RT-ddPCR is a modified version of conventional RT-PCR workflows which involves separating the reaction mixture into many partitions (~20,000) before thermal cycling which allows for direct absolute quantification of the target RNA molecules. Future protocols will be published that are complementary to this one and describe assays targeting additional SARS-CoV-2 mutations. This protocol uses RNA extracted using this protocol: High Throughput RNA Extraction and PCR Inhibitor Removal of Settled Solids for Wastewater Surveillance of SARS-CoV-2 RNA. That RNA is generated from samples subjected to pre-analytical steps outlined in: High Throughput pre-analytical processing of wastewater settled solids for SARS-CoV-2 RNA analyses. It is recommended that these assays be run along assays for PMMoV and BCoV as controls as described in the companion protocol High Throughput SARS-COV-2, PMMOV, and BCoV quantification in settled solids using digital RT-PCR The readout of this assay is a concentration of each target in the extracted RNA samples (copies/µL). Scope This process instruction applies to quantitative analysis of nucleic acid from SARS-CoV-2 RNA from solid wastewater samples with ddPCR using a Bio-Rad AutoDG Droplet Digital PCR system consisting of the AutoDG Automated Droplet Generator and the QX200 droplet reader.

Quantification of SARS-CoV-2 variant mutations (HV69-70, E484K/N501Y,del156-157/R158G, and Del143-145) in settled solids using digital RT-PCR v2

2021 ◽  
Author(s):  
Bridgette Hughes ◽  
Bradley J. White ◽  
Marlene K. Wolfe ◽  
Krista Wigginton ◽  
Alexandria B Boehm
Keyword(s):  
Quantitative Analysis ◽  
Nucleic Acid ◽  
High Throughput ◽  
Rna Extraction ◽  
Population Level ◽  
Digital Pcr ◽  
N Gene ◽  
Rt Pcr ◽  
Pcr Inhibitor ◽  
Wastewater Samples

This process instruction describes the steps for quantitative analysis of nucleic acid from SARS-CoV-2 with a triplex Reverse Transcriptase droplet digital Polymerase Chain Reaction (RT-ddPCR) assay targeting the N Gene, S Gene and 3 mutation assays (one each for HV69-70, E484K/N501Y, del156-157/R158G, and Del143-145) in extracted and purified RNA samples from solid wastewater samples for population level SARS-CoV-2 community surveillance. RT-ddPCR is a modified version of conventional RT-PCR workflows which involves separating the reaction mixture into many partitions (~20,000) before thermal cycling which allows for direct absolute quantification of the target RNA molecules. Future protocols will be published that are complementary to this one and describe assays targeting additional SARS-CoV-2 mutations. This protocol uses RNA extracted using this protocol: High Throughput RNA Extraction and PCR Inhibitor Removal of Settled Solids for Wastewater Surveillance of SARS-CoV-2 RNA. That RNA is generated from samples subjected to pre-analytical steps outlined in: High Throughput pre-analytical processing of wastewater settled solids for SARS-CoV-2 RNA analyses. It is recommended that these assays be run along assays for PMMoV and BCoV as controls as described in the companion protocol High Throughput SARS-COV-2, PMMOV, and BCoV quantification in settled solids using digital RT-PCR The readout of this assay is a concentration of each target in the extracted RNA samples (copies/µL). Scope This process instruction applies to quantitative analysis of nucleic acid from SARS-CoV-2 RNA from solid wastewater samples with ddPCR using a Bio-Rad AutoDG Droplet Digital PCR system consisting of the AutoDG Automated Droplet Generator and the QX200 droplet reader.

High Throughput SARS-COV-2, PMMOV, and BCoV quantification in settled solids using digital RT-PCR v2

2021 ◽  
Author(s):  
Aaron Topol (Verily Life Sciences) ◽  
marlene.wolfe not provided ◽  
Brad White (Verily Life Sciences) ◽  
Krista Wigginton ◽  
Alexandria B Boehm
Keyword(s):  
Quantitative Analysis ◽  
Nucleic Acid ◽  
High Throughput ◽  
Rna Extraction ◽  
Digital Pcr ◽  
N Gene ◽  
Acid Extraction ◽  
Rt Pcr ◽  
Pcr Inhibitor ◽  
Wastewater Samples

This process instruction describes the steps for quantitative analysis of nucleic acid from SARS-CoV-2 with a triplex Reverse Transcriptase droplet digital Polymerase Chain Reaction (RT-ddPCR) assay targeting the N Gene, S Gene and ORF1a and a duplex assay targeting Bovine Coronavirus Vaccine (BCoV) and Pepper Mild mottle virus (PMMoV) in extracted and purified RNA samples from solid wastewater samples for population level SARS-CoV-2 community surveillance. RT-ddPCR is a modified version of conventional RT-PCR workflows which involves separating the reaction mixture into many partitions (~20,000) before thermal cycling which allows for direct absolute quantification of the target RNA molecules. This protocol uses RNA extracted using this protocol: High Throughput RNA Extraction and PCR Inhibitor Removal of Settled Solids for Wastewater Surveillance of SARS-CoV-2 RNA. That RNA is generated from samples subjected to pre-analytical steps outlined in: High Throughput pre-analytical processing of wastewater settled solids for SARS-CoV-2 RNA analyses. This protocol describes 2 separate PCR reactions, one containing primer/probe mixtures targeting the three SARS-CoV-2 targets and one containing primer/probe mixtures targeting BCoV and PMMoV. BCoV is spiked into samples before nucleic acid extraction and serves as a process control as well as an indicator of PCR inhibition. PMMoV is an enveloped virus which is abundant in human fecal waste and serves as an endogenous control for data normalization. PMMoV RNA is abundant at such high levels in wastewater samples that the samples must be diluted by a factor of 100 before quantification. The readout of this assay is a concentration of each target in the extracted RNA samples (copies/uL). Scope This process instruction applies to quantitative analysis of nucleic acid from SARS-CoV-2 RNA from solid wastewater samples with ddPCR using a Bio-Rad AutoDG Droplet Digital PCR system consisting of the AutoDG Automated Droplet Generator and the QX200 droplet reader.

scholarly journals A fast, flexible and inexpensive protocol for DNA and RNA extraction for forest trees

2020 ◽  
Vol 29 (2) ◽  
pp. e018
Author(s):  
Yusuf Kurt ◽  
Lilian Matallana-Ramirez ◽  
William Kohlway ◽  
Ross Whetten ◽  
John Frampton
Keyword(s):  
Next Generation Sequencing ◽  
Nucleic Acids ◽  
Tree Species ◽  
Rna Extraction ◽  
The United States ◽  
Forest Tree ◽  
Next Generation ◽  
Forest Tree Species ◽  
Dna And Rna ◽  
Generation Sequencing

Aim of the study: DNA and RNA extraction are still one of the most important and challenging steps of many molecular genetics applications such as Next-Generation Sequencing technologies. In this study, traditional laboratory preparation protocols and commercially available nucleic acids extraction kits’ features were combined into a procedure suitable for extraction of either DNA or RNA in 96-well plate format at high throughput.Area of study: The study covers forest tree species from the United States of America.Materials and methods: The DNA and RNA protocol were tested on 27 species, including especially recalcitrant forest tree species, from five angiosperm and three gymnosperm families. DNA was also extracted from stored (from 2 to 6 years) silica-dried samples of 11 species of Pinaceae.Main results: The spectrophotometric analysis of DNA and RNA showed that gymnosperms yielded lower quantity, but higher quality nucleic acids than angiosperms which have variable results among species. The quantity and quality of DNA from stored samples were generally lower than fresh silica-dried samples. The RNA results showed high-enough yield (6.6 to 8.8 RIN) for downstream analyses.Research highlights: It was demonstrated that high quality and high molecular weight nucleic acids for Next-Generation Sequencing applications can be isolated from hundreds of samples from a wide range of taxonomic groups. The new protocol has features similar to both traditional laboratory and commercial extraction kits; is easy to set up in any molecular research laboratory, can be applied to a large number of samples (hundreds) in a working day, uses inexpensive reagents and supplies, and is compatible with automation.Key words: Angiosperms; gymnosperms; isolation protocol; nucleic acids.

High Throughput RNA Extraction and PCR Inhibitor Removal of Settled Solids for Wastewater Surveillance of SARS-CoV-2 RNA v1 (protocols.io.btyrnpv6)

protocols.io ◽  
2021 ◽  
Author(s):  
Aaron Topol ◽  
marlene.wolfe not provided ◽  
Krista Wigginton ◽  
Bradley White ◽  
Alexandria B
Keyword(s):  
High Throughput ◽  
Rna Extraction ◽  
Pcr Inhibitor

scholarly journals A comparison of DNA/RNA extraction protocols for high-throughput sequencing of microbial communities

2020 ◽  
Author(s):  
Justin P. Shaffer ◽  
Clarisse Marotz ◽  
Pedro Belda-Ferre ◽  
Cameron Martino ◽  
Stephen Wandro ◽  
...  
Keyword(s):  
Microbial Community ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Limit Of Detection ◽  
Sequence Data ◽  
Rna Virus ◽  
Microbial Community Composition ◽  
Rna Extraction ◽  
Acid Extraction ◽  
Dna And Rna

AbstractOne goal among microbial ecology researchers is to capture the maximum amount of information from all organisms in a sample. The recent COVID-19 pandemic, caused by the RNA virus SARS-CoV-2, has highlighted a gap in traditional DNA-based protocols, including the high-throughput methods we previously established as field standards. To enable simultaneous SARS-CoV-2 and microbial community profiling, we compare the relative performance of two total nucleic acid extraction protocols and our previously benchmarked protocol. We included a diverse panel of environmental and host-associated sample types, including body sites commonly swabbed for COVID-19 testing. Here we present results comparing the cost, processing time, DNA and RNA yield, microbial community composition, limit of detection, and well-to-well contamination, between these protocols.Accession numbersRaw sequence data were deposited at the European Nucleotide Archive (accession#: ERP124610) and raw and processed data are available at Qiita (Study ID: 12201). All processing and analysis code is available on GitHub (github.com/justinshaffer/Extraction_test_MagMAX).Methods summaryTo allow for downstream applications involving RNA-based organisms such as SARS-CoV-2, we compared the two extraction protocols designed to extract DNA and RNA against our previously established protocol for extracting only DNA for microbial community analyses. Across 10 diverse sample types, one of the two protocols was equivalent or better than our established DNA-based protocol. Our conclusion is based on per-sample comparisons of DNA and RNA yield, the number of quality sequences generated, microbial community alpha- and beta-diversity and taxonomic composition, the limit of detection, and extent of well-to-well contamination.

scholarly journals Evaluation of commercial DNA and RNA extraction methods for high-throughput sequencing of FFPE samples

PLoS ONE ◽  
2018 ◽  
Vol 13 (5) ◽  
pp. e0197456 ◽  
Author(s):  
Stine H. Kresse ◽  
Heidi M. Namløs ◽  
Susanne Lorenz ◽  
Jeanne-Marie Berner ◽  
Ola Myklebost ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Rna Extraction ◽  
Extraction Methods ◽  
Dna And Rna ◽  
Ffpe Samples ◽  
Rna Extraction Methods

scholarly journals Bio-On-Magnetic-Beads (BOMB): Open platform for high-throughput nucleic acid extraction and manipulation

2018 ◽  
Author(s):  
Phil Oberacker ◽  
Peter Stepper ◽  
Donna M Bond ◽  
Sven Höhn ◽  
Jule Focken ◽  
...  
Keyword(s):  
Open Source ◽  
High Throughput ◽  
Magnetic Beads ◽  
Acid Extraction ◽  
Open Platform ◽  
Dna And Rna ◽  
Rna Fragments ◽  
Different Sources ◽  
High Throughput Manner ◽  
Selection Of

AbstractCurrent molecular biology laboratories rely heavily on the purification and manipulation of nucleic acids. Yet, commonly used centrifuge-and column-based protocols require specialised equipment, often use toxic reagents and are not economically scalable or practical to use in a high-throughput manner. Although it has been known for some time that magnetic beads can provide an elegant answer to these issues, the development of open-source protocols based on beads has been limited. In this article, we provide step-by-step instructions for an easy synthesis of functionalised magnetic beads, and detailed protocols for their use in the high-throughput purification of plasmids, genomic DNA and total RNA from different sources, as well as environmental TNA and PCR amplicons. We also provide a bead-based protocol for bisulfite conversion, and size selection of DNA and RNA fragments. Comparison to other methods highlights the capability, versatility and extreme cost-effectiveness of using magnetic beads. These open source protocols and the associated webpage (https://bomb.bio) can serve as a platform for further protocol customisation and community engagement.

scholarly journals A comparison of DNA/RNA extraction protocols for high-throughput sequencing of microbial communities

BioTechniques ◽  
2021 ◽  
Author(s):  
Justin P Shaffer ◽  
Clarisse Marotz ◽  
Pedro Belda-Ferre ◽  
Cameron Martino ◽  
Stephen Wandro ◽  
...  
Keyword(s):  
Microbial Community ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Limit Of Detection ◽  
Rna Virus ◽  
Microbial Community Composition ◽  
Rna Extraction ◽  
Acid Extraction ◽  
Dna And Rna ◽  
Community Profiling

One goal of microbial ecology researchers is to capture the maximum amount of information from all organisms in a sample. The recent COVID-19 pandemic, caused by the RNA virus SARS-CoV-2, has highlighted a gap in traditional DNA-based protocols, including the high-throughput methods the authors previously established as field standards. To enable simultaneous SARS-CoV-2 and microbial community profiling, the authors compared the relative performance of two total nucleic acid extraction protocols with the authors' previously benchmarked protocol. The authors included a diverse panel of environmental and host-associated sample types, including body sites commonly swabbed for COVID-19 testing. Here the authors present results comparing the cost, processing time, DNA and RNA yield, microbial community composition, limit of detection and well-to-well contamination between these protocols.

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