Modified NEBNext® VarSkip Short SARS-CoV-2 Library Prep Kit for Illumina Platforms - adapted for wastewater samples v1

2021 ◽  
Author(s):  
Padmini Ramachandran ◽  
Tamara Walsky ◽  
Amanda Windsor ◽  
maria.hoffmann not provided ◽  
Chris Grim
Keyword(s):  
Rna Extraction ◽  
Library Preparation ◽  
Cdna Synthesis ◽  
Optimal Variant ◽  
Decision Points ◽  
Sequencing Quality ◽  
Project Monitoring ◽  
Variant Detection ◽  
Pandemic Response ◽  
Wastewater Samples

PURPOSE: This method was developed at the FDA’s Center for Food Safety and Applied Nutrition for GenomeTrakr’s pandemic response project, monitoring SARS-CoV-2 variants in wastewater. Protocols developed for this project cover wastewater collection, concentration, RNA extraction, RT-qPCR, library prep, genome sequencing, quality control checks, and data submission to NCBI. This modified protocol details methods for cDNA synthesis and library preparation for sequencing of wastewater samples containing SARS-CoV-2. The protocol is based primarily on the NEBNext® ARTIC SARS-CoV-2 Library Prep Kit (Illumina®), NEB #E7650S/L 24/96 reactions, with a few modifications. Primarily, VarSkip Short primers are used in place of the ARTIC V3 primers. These primers are available in the NEBNext®ARTIC SARS-CoV-2 FS Library Prep Kit (Illumina®); however, for optimal variant detection from wastewater, sequenced fragments should be as large as possible, so we discourage fragmentation prior to end prep. There are a couple of decision points in this protocol. Examining cDNA amplicon samples on an Agilent TapeStation system or similar fragment analyzer is extremely helpful in making these decisions.

Collection from Wastewater Treatment Plant, Transportation, and Storage of Raw Wastewater v1

2021 ◽  
Author(s):  
Kevin Calci ◽  
Kevin Calci
Keyword(s):  
Wastewater Treatment ◽  
Wastewater Treatment Plant ◽  
Rna Extraction ◽  
Treatment Plant ◽  
Sequencing Quality ◽  
Project Monitoring ◽  
Pandemic Response ◽  
Wastewater Samples ◽  
And Storage ◽  
Raw Wastewater

This method was developed at the FDA’s Center for Food Safety and Applied Nutrition for GenomeTrakr’s pandemic response project, monitoring SARS-CoV-2 variants in wastewater. Protocols developed for this project cover wastewater collection, concentration, RNA extraction, RT-qPCR detection, library prep, genome sequencing, quality control checks, and data submission to NCBI. This method describes the collection and transport of raw wastewater samples from a wastewater treatment plant.

6M GITC v1

2021 ◽  
Author(s):  
Jacquelina.Woods not provided
Keyword(s):  
Quality Control ◽  
Food Safety ◽  
Genome Sequencing ◽  
Rna Extraction ◽  
Data Submission ◽  
Sequencing Quality ◽  
Project Monitoring ◽  
Pandemic Response ◽  
Wastewater Collection

This method was developed at the FDA’s Center for Food Safety and Applied Nutrition for GenomeTrakr’s pandemic response project, monitoring SARS-CoV-2 variants in wastewater​​. Protocols developed for this project cover wastewater collection, concentration, RNA extraction, RT-qPCR detection, library prep, genome sequencing, quality control checks, and data submission to NCBI. This method provides a reagent formula required in the extraction of RNA from viral concentrates using the RNeasy and Zymo kits.

RT-qPCR Detection of Process Controls (Murine noroviurs and crAssphage) from Wastewater using AB 7500 v1

2021 ◽  
Author(s):  
Jacquelina.Woods not provided ◽  
rachel.rodriguez not provided
Keyword(s):  
Rna Extraction ◽  
Murine Norovirus ◽  
False Negatives ◽  
Multiplexed Detection ◽  
Detection Assay ◽  
Sequencing Quality ◽  
Project Monitoring ◽  
Version 2.0 ◽  
Pandemic Response ◽  
Process Controls

This method was developed at the FDA’s Center for Food Safety and Applied Nutrition for GenomeTrakr’s pandemic response project, monitoring SARS-CoV-2 variants in wastewater​​. Protocols developed for this project cover wastewater collection, concentration, RNA extraction, RT-qPCR detection, library prep, genome sequencing, quality control checks, and data submission to NCBI. This protocol describes the murine norovirus (MNV; extraction control) and crAssphage (human indicator) RT-qPCR assay developed for use on the AB 7500 platform using software version 2.0 or 2.3. The assay incorporates an internal amplification control (IC) to prevent the reporting of false negatives due to inhibition or failure of the RT-qPCR. This multiplexed detection assay was developed for the determination crAssphage extracted from wastewater, as an endogenous control, and MNV as an extraction control. The assay is designed to be used in conjunction with the SARS-CoV-2 RT-qPCR detection assay. Valid sample results for SARS-CoV-2 detection are contingent upon the detection of the MNV extraction control from the sample being tested.

2X PEG v1

2021 ◽  
Author(s):  
Jacquelina.Woods not provided
Keyword(s):  
Quality Control ◽  
Food Safety ◽  
Genome Sequencing ◽  
Rna Extraction ◽  
Data Submission ◽  
Sequencing Quality ◽  
Project Monitoring ◽  
Pandemic Response ◽  
Wastewater Collection

This method was developed at the FDA’s Center for Food Safety and Applied Nutrition for GenomeTrakr’s pandemic response project, monitoring SARS-CoV-2 variants in wastewater​​. Protocols developed for this project cover wastewater collection, concentration, RNA extraction, RT-qPCR detection, library prep, genome sequencing, quality control checks, and data submission to NCBI. This method provides a reagent formula required in the rapid concentration of intact viruses from wastewater using a combination of PEG precipitation and ultracentrifugation.

0.05 M Glycine v1

2021 ◽  
Author(s):  
Jacquelina.Woods not provided
Keyword(s):  
Quality Control ◽  
Food Safety ◽  
Genome Sequencing ◽  
Rna Extraction ◽  
Data Submission ◽  
Sequencing Quality ◽  
Project Monitoring ◽  
Pandemic Response ◽  
Wastewater Collection

This method was developed at the FDA’s Center for Food Safety and Applied Nutrition for GenomeTrakr’s pandemic response project, monitoring SARS-CoV-2 variants in wastewater​​. Protocols developed for this project cover wastewater collection, concentration, RNA extraction, RT-qPCR detection, library prep, genome sequencing, quality control checks, and data submission to NCBI. This method provides a reagent formula required in the rapid concentration of intact viruses from wastewater using a combination of PEG precipitation and ultracentrifugation.

NCBI submission protocol for SARS-CoV-2 wastewater data: SRA, BioSample, and BioProject v2

2021 ◽  
Author(s):  
Ruth E Timme ◽  
Maria Balkey
Keyword(s):  
Quality Control ◽  
Food Safety ◽  
Genome Sequencing ◽  
Sequence Data ◽  
Rna Extraction ◽  
Data Submission ◽  
Sequencing Quality ◽  
Project Monitoring ◽  
Pandemic Response ◽  
Set Up

PURPOSE: This method was developed at the FDA’s Center for Food Safety and Applied Nutrition for GenomeTrakr’s pandemic response project, monitoring SARS-CoV-2 variants in wastewater; however, this protocol was written to be broadly applicable for all wastewater sequence data submissions to NCBI. Protocols developed for this project cover wastewater collection, concentration, RNA extraction, RT-qPCR, library prep, genome sequencing, quality control checks, and data submission to NCBI. This protocol covers the last step of making your data public at NCBI. Specifically, it provides the steps to establish a new NCBI submission environment for your laboratory, including the creation of new BioProject(s) and submission groups. Once these are step up, the protocol then walks through the process for submitting raw reads to SRA and sample metadata to BioSample through the Submission portal. For new submitters, there's quite a bit of groundwork that needs to be established before a laboratory can start its first data submission. We recommend that one person in the laboratory take a few days to get everything set up in advance of when you expect to do your first data submission. If you need a pipeline for frequent or large volume submissions, follow Step 1 in this protocol to get your NCBI submission environment established, then contact [email protected] to set up an account for submitting through the API. Version updates: V2: minor edits to the BioSample and SRA templates V3: Many minor edits were made to make the protocol more broadly applicable to submitters outside of FDA's wastewater project. Updates were also made to both metadata templates, including a new attribute to the SRA metadata template, called "enrichment_kit". There are now three custom attributes we recommend populating to more specifically capture relevant methods for ww sequencing: enrichment approach, primer set, and library prep kit.

Tissue Culture (tc) PBS v1

2021 ◽  
Author(s):  
Jacquelina.Woods not provided
Keyword(s):  
Tissue Culture ◽  
Quality Control ◽  
Food Safety ◽  
Genome Sequencing ◽  
Rna Extraction ◽  
Data Submission ◽  
Sequencing Quality ◽  
Project Monitoring ◽  
Pandemic Response ◽  
Wastewater Collection

This method was developed at the FDA’s Center for Food Safety and Applied Nutrition for GenomeTrakr’s pandemic response project, monitoring SARS-CoV-2 variants in wastewater​​. Protocols developed for this project cover wastewater collection, concentration, RNA extraction, RT-qPCR detection, library prep, genome sequencing, quality control checks, and data submission to NCBI. This method provides a reagent formula required in the rapid concentration of intact viruses from wastewater using a combination of PEG precipitation and ultracentrifugation.

RT-qPCR Detection of SARS-CoV-2 from Wastewater Using the AB 7500 v1

2021 ◽  
Author(s):  
Jacquelina.Woods not provided ◽  
rachel.rodriguez not provided
Keyword(s):  
Rna Extraction ◽  
Murine Norovirus ◽  
False Negatives ◽  
Nucleocapsid Gene ◽  
Sequencing Quality ◽  
Project Monitoring ◽  
Version 2.0 ◽  
Pandemic Response ◽  
Process Controls ◽  
Qualitative Determination

This method was developed at the FDA’s Center for Food Safety and Applied Nutrition for GenomeTrakr’s pandemic response project, monitoring SARS-CoV-2 variants in wastewater​​. Protocols developed for this project cover wastewater collection, concentration, RNA extraction, RT-qPCR detection, library prep, genome sequencing, quality control checks, and data submission to NCBI. This protocol describes triplex and duplex assays for the RT-qPCR detection of the nucleocapsid region of the SARS-CoV-2 genome. These assays, along with the murine norovirus (MNV; extraction control) and crAssphage (human indicator) RT-qPCR assay (RT-qPCR Detection of Process Controls (Murine noroviurs and crAssphage) from Wastewater (protocols.io)), were developed for use on the AB 7500 platform using software version 2.0 or 2.3. All assays incorporate an internal amplification control (IC) to prevent the reporting of false negatives due to inhibition or failure of the RT-qPCR. These multiplexed detection assays were developed for the qualitative determination SARS-CoV-2 nucleocapsid gene extracted from wastewater. Valid sample results are contingent upon the detection of the MNV extraction control from the sample being tested.

Virus Concentration from Wastewater Using PEG Precipitation and Ultracentrifugation v1

2021 ◽  
Author(s):  
Jacquelina.Woods not provided ◽  
rachel.rodriguez not provided
Keyword(s):  
Quality Control ◽  
Food Safety ◽  
Genome Sequencing ◽  
Rna Extraction ◽  
Virus Concentration ◽  
Data Submission ◽  
Sequencing Quality ◽  
Project Monitoring ◽  
Pandemic Response ◽  
Wastewater Collection

This method was developed at the FDA’s Center for Food Safety and Applied Nutrition for GenomeTrakr’s pandemic response project, monitoring SARS-CoV-2 variants in wastewater​​. Protocols developed for this project cover wastewater collection, concentration, RNA extraction, RT-qPCR detection, library prep, genome sequencing, quality control checks, and data submission to NCBI. This method describes the rapid concentration of intact viruses from wastewater using a combination of PEG precipitation and ultracentrifugation.

NCBI submission protocol for SARS-CoV-2 wastewater data: SRA, BioSample, and BioProject v2

2021 ◽  
Author(s):  
Ruth E Timme ◽  
Maria Balkey
Keyword(s):  
Quality Control ◽  
Food Safety ◽  
Genome Sequencing ◽  
Large Volume ◽  
Rna Extraction ◽  
Data Submission ◽  
Sequencing Quality ◽  
Project Monitoring ◽  
Pandemic Response ◽  
Set Up

PURPOSE: This method was developed at the FDA’s Center for Food Safety and Applied Nutrition for GenomeTrakr’s pandemic response project, monitoring SARS-CoV-2 variants in wastewater​​. Protocols developed for this project cover wastewater collection, concentration, RNA extraction, RT-qPCR, library prep, genome sequencing, quality control checks, and data submission to NCBI. This protocol covers the last step of making your data public at NCBI. Specifically, it provides the steps to establish a new NCBI submission environment for your laboratory, including the creation of new BioProject(s) and submission groups. Once these are step up, the protocol then walks through the process for submitting raw reads to SRA and sample metadata to BioSample through the Submission portal. For new submitters, there's quite a bit of groundwork that needs to be established before a laboratory can start its first data submission. We recommend that one person in the laboratory take a few days to get everything set up in advance of when you expect to do your first data submission. If you need a pipeline for frequent or large volume submissions, follow Step 1 in this protocol to get your NCBI submission environment established, then contact [email protected] to set up an account for submitting through the API. Version updates: V2: minor edits to the BioSample and SRA templates

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