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Coronavirus Sequencing

Coronavirus

Benefits of NGS for COVID-19

Next-generation sequencing (NGS) provides an effective, unbiased way to identify coronavirus strains and other pathogens without prior knowledge of the organisms.1 Sequencing was used to identify the novel coronavirus causing COVID-19 (SARS-CoV-2) early in the outbreak2. NGS continues to provide public health officials, vaccine and drug developers, and researchers with critical evidence, and allows labs to:

  • Determine the source of infection and route of transmission
  • Identify and characterize co-infections and the role of complex disease
  • Provide information on coronavirus strain typing to monitor viral spread
  • Screen targets for possible COVID-19 therapeutics
  • Comprehensively detect respiratory pathogens and antimicrobial resistance

Compare Coronavirus NGS Methods

  Detect Novel Virus Detect SARS-COV-2 and Diagnose COVID-19 Detect and Monitor Respiratory Infections Detect Patient Immunity
Testing Needs Metagenomics PCR/qPCR Amplicon Illumina COVIDSeq Test Targeted Enrichment Serology
Speed and TAT          
Scalable and Cost Effective          
Identification of Novel Pathogen          
Track Transmissions          
Unaffected by Mutational Changes          
Identify Co-Infection and Complex Disease          
Detection of Antimicrobial Resistance          

Adequately meets laboratory testing needs

Partially meets laboratory testing needs

More About Coronavirus Sequencing Methods

Shotgun Metagenomics

Comprehensively sequence all organisms in a given sample and identify novel pathogens such as coronaviruses. This NGS method can help accelerate outbreak investigations and support development of new laboratory tests.

Target Enrichment

Detect and characterize respiratory pathogens and antimicrobial resistance. Universal detection of pathogenic respiratory tract organisms and antimicrobial resistance markers can help researchers detect and monitor respiratory infections like SARS-CoV-2 and optimize infection control strategies.

Amplicon Sequencing

Detect the presence of SARS-CoV-2 by identifying specific regions of the pathogen genome. This method involves analyzing genomic regions of interest with ultra-deep sequencing of PCR amplicons.

Coronavirus Software Tools
These tools accelerate coronavirus detection and identification, simplify sample tracking, facilitate host response studies, and make it easier for scientists to contribute to public databases.

COVID-19 Application Notes

Detect Respiratory Pathogens & Associated Resistance Genes

Rapid target enrichment workflow for broad detection of respiratory pathogens (including SARS-CoV-2 and flu viruses) and antimicrobial resistance genes.

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Find and Characterize Respiratory Viruses with Illumina RNA Prep with Enrichment

A rapid target enrichment sequencing workflow for highly sensitive detection and characterization of respiratory viruses, including COVID-19 strains.

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Rapid Detection of Respiratory Infections on Illumina MiniSeq System

Detection of respiratory viruses including coronavirus in <24 hours.

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Clinical Metagenomics Analysis to Address SARS-CoV-2 Detection

The COVID-19 pandemic has underscored the need for new tools to detect, diagnose and monitor emerging pathogens like SARS-CoV-2. NGS has proven advantageous as it led to the initial detection of the coronavirus itself, and accelerated test as well as vaccine development.

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FAQs

At a basic level, diagnostic testing helps clinicians manage patients and surveillance is required to manage populations.

Diagnostic testing provides important yes/no answers for individual patients so that appropriate management can be provided.

Surveillance helps public health officials track the path of the epidemic, understand transmission routes, perform contact tracing, determine the rate of viral evolution, and understand if the virus is changing in ways that could impact diagnostic or therapeutic effectiveness.

NGS can provide unbiased detection of a novel pathogen in patient samples without prior knowledge of the organism.

A key challenge in infectious disease detection is that many of the microbes, including viruses, that cause respiratory, digestive, and other diseases in humans, have not been researched and characterized and thus are not known or detected by targeted approaches, such as PCR, the development of which require knowledge of the pathogen genome. NGS plays a critical role in discovering these unknown, novel pathogens and the resulting genome sequence can then be used to develop routine tests such as PCR to help clinicians manage patients.

NGS can be used to track the evolution of the pathogen genome to help public health officials monitor the spread of infection and determine the best isolation plan at a population level. Sequencing the virus from different patients over time can determine the rate of viral evolution and understanding if the virus is changing in ways that could impact pathogenicity as well as diagnostic or therapeutic effectiveness. PCR is designed to detect the presence of specific regions of the pathogen genome and will not identify new mutations across these rapidly evolving pathogen genomes. Furthermore, PCR performance can suffer if mutations occur in the primer or probe binding regions.

Epidemiologists study the mutations of the viral genome from patient samples across the globe and can use this information to build a genetic tree (or map) that can indicate the path of transmission between patients. Clusters due to genetic similarities in the pathogen belong to patients within the same transmission chains. These transmission chains enable public health officials to quickly determine the pathogen origin, track the path of the epidemic, understand transmission routes and inform appropriate containment measures.

A shotgun metagenomic workflow enables detection of both novel and known species. When faced with an unknown outbreak, multiple molecular diagnostic tests are often used which may lead to unnecessary cost and delays in identifying the pathogen. Shotgun Metagenomics can be used as a single comprehensive screening test for identifying and characterizing pathogens. This research workflow can help accelerate outbreak investigations and support development of new laboratory tests for large scale screening efforts.

Once a pathogen, such as SARS-CoV-2 is identified, a target enrichment workflow can provide the high sensitivity needed to detect the virus and provide information about its epidemiology and evolution to help researchers optimize infection control strategies including monitoring when its acceptable to de-escalate isolation mechanisms and resume normal activities, and aid in the development of vaccines.

These complementary workflows using Illumina sequencing can be performed alongside traditional testing methods and integrated into a comprehensive outbreak response model.

There are 7,800 probes to detect common respiratory viruses, recent flu strains, and SARS-CoV-2 as well as human probes to act as positive controls. These probes are 80-mer oligos, spaced very close together providing full genome coverage of all viruses in the panel. Table of viruses in the panel:

  • Human coronavirus 229E
  • Human coronavirus NL63
  • Human coronavirus OC43
  • Human coronavirus HKU1
  • SARS-CoV-2
  • Human adenovirus B1
  • Human adenovirus C2
  • Human adenovirus E4
  • Human bocavirus 1 (Primate bocaparvovirus 1 isolate st2)
  • Human bocavirus 2c PK isolate PK-5510
  • Human bocavirus 3
  • Human parainfluenza virus 1
  • Human parainfluenza virus 2
  • Human parainfluenza virus 3
  • Human parainfluenza virus 4a
  • Human metapneumovirus (CAN97-83)
  • Respiratory syncytial virus (type A)
  • Human Respiratory syncytial virus 9320 (type B)
  • Influenza A virus (A/Puerto Rico/8/1934(H1N1))
  • Influenza A virus (A/Korea/426/1968(H2N2))
  • Influenza A virus (A/New York/392/2004(H3N2))
  • Influenza A virus (A/goose/Guangdong/1/1996(H5N1))
  • Human bocavirus 4 NI strain HBoV4-NI-385
  • KI polyomavirus Stockholm 60
  • WU Polyomavirus
  • Human parechovirus type 1 PicoBank/HPeV1/a
  • Human parechovirus 6
  • Human rhinovirus A89
  • Human rhinovirus C (strain 024)
  • Human rhinovirus B14
  • Human enterovirus C104 strain: AK11
  • Human enterovirus C109 isolate NICA08-4327
  • Influenza A virus (A/Zhejiang/DTID-ZJU01/2013(H7N9))
  • Influenza A virus (A/Hong Kong/1073/99(H9N2))
  • Influenza A virus (A/Texas/50/2012(H3N2))
  • Influenza A virus (A/Michigan/45/2015(H1N1))
  • Influenza B virus (B/Lee/1940)
  • Influenza B virus (B/Wisconsin/01/2010)
  • Influenza B virus (B/Brisbane/60/2008)
  • Influenza B virus (B/Colorado/06/2017)
  • Influenza B virus (B/Washington/02/2019)
  • Human control genes

Target enrichment is a resequencing method that captures genomic regions of interest by hybridization to target-specific biotinylated probes. Target enrichment through hybrid–capture methods allows for highly sensitive detection and therefore does not require high read depth. Additionally, the target enrichment NGS workflow allows for near-complete sequence data of targets and opens up applications such as variant analysis for viral evolution or viral surveillance.

Alternatively, amplicon sequencing is designed to detect the presence of the target pathogen in a sample by identifying specific regions of the pathogen genome. This method does not enable identification of new mutations across these rapidly evolving pathogen genomes which is required for viral evolution or viral surveillance studies.

The target enrichment NGS workflow allows for near-complete sequence data of targets and opens up applications such as variant analysis for viral evolution or viral surveillance. Compared to other targeted resequencing methods, such as amplicon sequencing, enrichment through hybrid capture allows for dramatically larger probe panels with more comprehensive profiling of the target regions. Additionally, the oligo probes used for hybrid–capture protocols remain effective, even within highly mutagenic regions, which can be difficult for amplicon-based assays such as qPCR, allowing targeting of rapidly evolving viruses, such as RNA viruses.

Once the pathogen has been identified, amplicon can provide cost effective, rapid, and scalable detection of SARS-CoV-2. When used as a general whole genome sequencing diagnostic approach, it allows for broader target coverage, making it less susceptible to mutational effects. For research, whole genome sequencing can be used to monitor viral mutations and allows phylogenetic analysis.

Once the pathogen has been identified, the viral enrichment panel provides high sensitivity detection coupled with epidemiology information by detecting the full SARS-CoV-2 genome and the genomic mutations found across different samples. This information helps define the epidemiology of transmission and public officials can optimize infection control strategies.

When used with Illumina’s Respiratory Virus Oligo Panel, detection is expanded to ~30 families of respiratory viruses and allows researchers to study and identify co-infections with other viruses in the panel.

This amplicon-based NGS test includes 2019-nCoV primers designed to detect RNA from the SARS-CoV-2 virus.

Learn More

Visit our Sequencing Platforms page to explore our portfolio. The choice of sequencer depends on which method(s) you use most frequently. See the workflows above for recommendations on which sequencer is optimal for which method.

llumina and IDbyDNA Partner to Deliver NGS Infectious Disease Solutions

The partnership aims to expand NGS adoption for clinical infectious disease testing. It has been accelerated to provide improved support to customers globally performing NGS for surveillance purposes during the COVID-19 pandemic.

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llumina and IDbyDNA

Technical Tips

Decontaminating Illumina Sequencers for Novel Coronavirus

In keeping with recommendations from the United States CDC and World Health Organization, Illumina recommends this procedure for decontaminating NGS instruments suspected or known to have come in contact with the novel coronavirus SARS-CoV-2 (2019-nCoV).

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Impact of Ammonium-Based Cleaning Products on Sequencing Run Performance

Use of ammonia-based cleaners or sanitizing products in proximity to sequencing run setup can result in decreased sequencing run performance metrics. View tips on how to avoid these issues.

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Shotgun Metagenomics Supporting Products

Illumina Stranded Total RNA Prep with Ribo-Zero Plus

Rapid library preparation from a broad range of sample types for studying the coding and non-coding transcriptome with unparalleled study flexibility.

rRNA Depletion Kit

The stand-alone Illumina Ribo-Zero Plus kit allows for ribosomal RNA removal in human, mouse, rat, and bacterial samples.

NextSeq 2000

Groundbreaking benchtop sequencers allow you to explore new science across a variety of current and emerging applications, with higher efficiency and fewer restraints.

Enrichment Supporting Products

iSeq 100

The iSeq 100 system leverages the speed and affordability of complementary metal-oxide-semiconductor (CMOS) technology and the accuracy of sequencing by synthesis (SBS) chemistry.

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MiSeq

The MiSeq benchtop sequencer enables targeted and microbial genome applications, with high-quality sequencing, simple data analysis, and cloud storage.

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Illumina RNA Prep with Enrichment

Illumina RNA Prep with Enrichment is the fastest and most flexible targeted sequencing solution for RNA in the Illumina library prep portfolio.

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References
  1. Bulcha B. Review on viral metagenomics and its future perspective in zoonotic and arboviral disease surveillance. J Biol Agr Healthc. 2017; 7(21): 35–41.
  2. A Novel Coronavirus from Patients with Pneumonia in China, 2019. N Engl J Med. 2020; 382(8): 727–733.