SARS-CoV-2 NCBI consensus submission protocol: GenBank v2

This is a SARS-CoV-2 specific protocol that covers the steps needed to submit SARS-CoV-2 consensus sequence to GenBank. If you need a pipeline for frequent or large volume submissions, follow Step 1 in the SARS-CoV-2 NCBI submission protocol: SRA, BioSample, and BioProject to get your NCBI submission environment established, then contact [email protected] to set up an account for submitting through the API. This protocol assumes (and requires) that the user has a BioProject and BioSamples(s) already registered. Complete in order:: 1. Populate your templates first. 2. SARS-CoV-2 NCBI submission protocol: SRA, BioSample, and BioProject Step-by-step instructions for establishing a new NCBI laboratory submission account and for creating and linking a new BioProject to an existing umbrella effort. SARS-CoV-2 raw data submission to SRA (Sequence Read Archive) and metadata to BioSample. Users can modify this protocol to just create a BioSample with no linked raw data. 3. SARS-CoV-2 NCBI consensus submission protocol: GenBank (included protocol) Required: established BioProject and BioSamples Submit SARS-CoV-2 assemblies to NCBI GenBank, linking to existing BioProject, BioSamples, and raw data. Version history: V3: Direct links provided to download metadata templates (instead of hosting duplicate files). minor edits throughout the protocol.

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

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.

Overview of NCBI's SARS-CoV-2 submission process and the metadata required v2

PURPOSE: This protocol explains the metadata requirements for the following two protocols: 1. SARS-CoV-2 NCBI submission protocol: SRA, BioSample, and BioProject Step-by-step instructions for establishing a new NCBI laboratory submission account and for creating and linking a new BioProject to an existing umbrella effort. SARS-CoV-2 raw data submission to SRA (Sequence Read Archive) and metadata to BioSample. Users can modify this protocol to just create a BioSample with no linked raw data. 2. SARS-CoV-2 NCBI consensus submission protocol: GenBank Required: established BioProject and BioSamples Submit SARS-CoV-2 assemblies to NCBI GenBank, linking to existing BioProject, BioSamples, and raw data. Version history: V4: Updated metadata templates to reflect updated PHA4GE templates (V3) plus minor text edits.

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

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

MaveDB v2: a curated community database with over three million variant effects from multiplexed functional assays

A central problem in genomics is understanding the effect of individual DNA variants. Multiplexed Assays of Variant Effect (MAVEs) can help address this challenge by measuring all possible single nucleotide variant effects in a gene or regulatory sequence simultaneously. Here we describe MaveDB v2, which has become the database of record for MAVEs. MaveDB now contains a large fraction of published studies, comprising over two hundred datasets and three million variant effect measurements. We created tools and APIs to streamline data submission and access, transforming MaveDB into a hub for the analysis and dissemination of these impactful datasets.

NCBI submission protocol for microbial pathogen surveillance v2

PURPOSE: Step-by-step instructions for submitting pathogen whole genome sequence data to NCBI and to the NCBI Pathogen Detection portal. This protocol covers the steps needed 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. SCOPE: for use by any laboratory submitting WGS data for species under active surveillance within NCBI’s Pathogen Detection. (This includes US laboratories in GenomeTrakr, NARMS, Vet-LIRN, PulseNet, and other non-US networks and submitters). 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 to get your NCBI submission environment established, then contact [email protected] to set up an account for submitting through the API. This protocol covers submission using NCBI's Submission Portal web-interface. Version history: V5: Linking directly to the metadata template guidance instead of including duplicate copies of the files in this protocol. Updated screenshot for choosing the pathogen template to reflect changes at NCBI. V4: updated screenshots to reflect NCBI submission portal changes. Updated custom BioSample template.

RNA Extraction from Wastewater Concentrates Using RNeasy and Zymo Kits v1

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 extraction of RNA from viral concentrates using the RNeasy and Zymo kits.

5N HCl v1

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.

2X PEG v1

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.

Primer TE (10mM Tris, 0.1mM EDTA, pH8.0) v1

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.