Pathogen Sequencing Protocols

The Pathogen Genomics Centers of Excellence (PGCoE) Network is currently conducting a viral respiratory benchmarking study, which aims to compare the results of pathogen whole genome sequencing protocols used across the United States. When complete, the protocols listed below will be annotated according to their applicability for different sequencing platforms and effectiveness for specific pathogens.

Further information on this benchmarking study, including details about the tool we are using to evaluate sequencing results, are available in our benchmarking repository.

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Measles resources

Many of the viral sequencing protocols below can be used or adapted for sequencing measles virus. For protocols that have been specifically used and tested by PGCoE sites and collaborators for sequencing measles virus, please see our measles resources page.

Sample Collection Protocols

Sample Collection for Genomic Endpoints

The PGCoE network has developed a document that contains guidance for sample collection and storage with genomic endpoints in mind. This sample collection protocol is available in the PGCoE resources repository.

Sequencing Primers and Probes

Primal Scheme Amplicon Sequencing Primers

Primer sets for amplicon-based sequencing of many different pathogens (including RSV, SARS-CoV-2, measles, mpox, and others) are available on the labs.primalscheme site, to which NE PGCoE partners regularly contribute.

Viral Sequencing Protocols

Broad Institute Metagenomic Sequencing Protocol

Pooled metagenomics sequencing protocol (paired with hybrid capture using the Twist Comprehensive Viral Research Panel) that greatly decreases library preparation costs by allowing 24–96 samples to be processed through a single shared downstream reaction after reverse transcription.

Washington State Hybrid-Capture Measles Sequencing Protocol

Hybrid-capture sequencing protocol using the Illumina Viral Surveillance Panel v2 for over 200 viral species, including the measles virus.

H5N1-specific Molecular Inversion Probes

Protocol for sequencing H5N1 viral genomes from animal specimens at Washington Animal Disease Diagnostic Laboratory using hybrid capture with Molecular Inversion Probes. This protocol will be linked here when available.

Automated Viral Amplicon Sequencing on Liquid Handlers

This repository contains protocols for automated viral sequencing on Hamilton Microlab STAR liquid handlers. These protocols have been validated by the DAMP Lab at Boston University.

SARS-CoV-2 Wastewater Sequencing

A wet lab protocol for the preparation of purified SARS-CoV-2 RNA from raw wastewater samples for short-read sequencing.

iNextEra Library Preparation

Protocol for the preparation of multiplexed short-read DNA libraries for WGS.

Metagenomic Sequencing for Respiratory Virus Detection

Metagenomic sequencing protocol for RNA metagenomic sequencing to identify and characterize viruses from nasopharyngeal swab samples.

PathMIP Co-Seq Method

A multiplexed sequencing protocol for three viral respiratory pathogens simultaneously using molecular inversion probes on the Oxford Nanopore. Link coming soon!