1. Roche Sequencing
  2. Covid-19 Research & SARS-CoV-2 Surveillance
  3. NGS Workflow

COVID-19 Research and SARS-CoV-2 Surveillance

Next-generation sequencing solutions for SARS-CoV-2 sequencing and surveillance

As the SARS-CoV-2 virus that causes COVID-19 has mutated, there has been a growing need to sequence the viral genome from many more COVID-19-positive samples to better understand the virus and its transmission.

Surveillance by sequencing enables labs to track known variants, such as the B.1.1.7 variant first identified in the UK, the P.1 variant first discovered in Brazil, and the B.1.351 variant originally identified in South Africa. Surveillance sequencing also enables the Identification of new and emerging variants that could have an impact on the utility of diagnostic tests, vaccines, and therapeutic agents. Many other labs leverage the power of NGS to research COVID-19 or the virus itself.

Role of NGS in SARS-CoV-2 surveillance and COVID-19 research

  • Track the transmission trends for known variants
  • Identify novel SARS-CoV-2 variants, and monitor viral evolution
  • Track the spread of the pandemic
  • Study host immune response
  • Find the intermediate host of SARS-CoV-2 

NGS methods for SARS-CoV-2 Sequencing

SARS-CoV-2 NGS Workflows

Hybridization-based target enrichment plus library prep

SARS-CoV-2 genome enrichment methods based on hybrid capture of the viral sequences by oligonucleotide probes offer an alternative to the more common, amplicon-based methods, such as the ARTIC protocol.

The target-enriched RNA-seq workflow starts with whole-transcriptome library preparation using an RNA library preparation workflow. The resulting libraries are then enriched for target sequences by hybridization-based target enrichment using SARS-CoV-2 specific probes.

Workflow steps with icons and text for Hybrid Capture
When to use Pros Cons Roche Reagents
  • For accurate, complete coverage of the entire viral genome
  • When RNA samples are degraded (e.g., in waste water and environmental samples)
  • When the sequence of subgenomic RNAs is needed  (e.g., for research applications)
  • Most tolerant of viral mutation, yielding reliable data even if the virus mutates by as much as 10-20%
  • Complete workflow reagents  are available from a single vendor, ensuring uniform support for all steps of the workflow
  • Longer assay time
  • Higher cost
  • More hands-on steps

PrimalSeq workflow with ARTIC amplicons (ARTIC Protocol)

The method most broadly used in the early phases of the pandemic has been the “ARTIC protocol.” Based on a PrimalSeq technique first developed for Zika virus, the ARTIC protocol was created by the UK-based ARTIC Network, which designed and published primer sets that would amplify the 30 kb SARS-CoV-2 genome in 1200-bp amplicons for NGS. Roche has built upon the ARTIC protocol by combining the ARTIC primer sets (available from oligonucleotide suppliers) with high-performing Roche NGS reagents in a PrimalSeq workflow that mimics the ARTIC protocol. Although the ARTIC protocol was originally developed to generate long amplicons for sequencing on Oxford Nanopore sequencers, labs have subsequently adapted this method for short-read sequencing on Illumina platforms by adding an enzymatic fragmentation step prior to library prep.

The PrimalSeq workflow utilizes virus-specific PCR primer sets designed by the ARTIC Network to amplify viral genome sequence from cDNA. These amplicons are then used as input into a DNA library preparation workflow, during which they are converted to barcoded libraries.

Workflow steps with icons and text for PrimalSeq workflow
When to use Pros Cons Roche Reagents
  • When wanting to mimic the broadly published ARTIC Protocol method
  • When fast turnaround time is desired
  • Most widely adopted/published method
  • Fast
  • Fairly complete workflow from a single vendor ensures single-partner support

 
  • Doesn't cover the end regions of the viral genome
  • Primer-binding sites can be disrupted by mutations, preventing efficient amplification
  • Coverage uniformity can be highly variable, especially with low viral-load/copy number samples

Tailed amplicon method

The Tailed Amplicon method developed at the University of Minnesota offers another attractive amplicon-based option for labs generating SARS-CoV2 viral libraries for sequencing on Illumina, Oxford Nanopore, and Pacific Bioscience sequencers.

This method utilizes tailed primers specific for viral sequences to amplify viral genome sequence from cDNA; the core primer sequences are available from the ARTIC network. A second round of PCR is then used to add the index sequences, yielding the barcoded libraries.  

Workflow steps with icons and text for tailed amplicon workflow
When to use Pros Cons Roche Reagents
  • For samples with a high viral load (have low Ct values)
  • When fast turnaround time is the top priority
  • When cost is a driving factor
  • Fastest of the three workflows
  • Inexpensive
  • Fewest hands-on steps (after pooling the 98 primers)

 
  • Less tolerant method for samples with low viral load
  • Doesn't cover the end regions of the viral genome
  • Primer-binding sites can be disrupted by mutations, preventing efficient amplification
Individual components, rather than complete kits, are often used in this method:

Featured Products

KAPA SARS-CoV-2 Target Enrichment Panel

Monitor the sequence of the SARS-CoV-2 virus and the emergence of new variants with the KAPA SARS-CoV-2 Target Enrichment Panel. Prepare libraries from input RNA using the robust KAPA RNA HyperPrep Kit and then enrich for viral sequences; this panel targets 100% of the reference SARS-CoV-2 genome (NC_045512) and >99.7% of another 183 publicly available SARS-CoV-2 genomes (GenBank).

  • Identify multiple variants of SARS-CoV-2 in a single reaction
  • Achieve 1X coverage of >97% of the SARS-CoV-2 genome down to 1000 viral copies and obtain genomic sequence from as few as 10 viral copies (in a background of 20 ng or 100 ng RNA with 1 million 2x75 bp reads)
  • Save valuable time with hybridization as short as 1 hour

 

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KAPA RNA HyperPrep Kits

Start the RNA-seq process off the right way by making high-quality libraries through the streamlined, single-day workflow of the KAPA RNA HyperPrep Kits. Combine these flexible kits with KAPA Dual-Indexed Adapters in an automation-friendly workflow that is compatible with mRNA capture, ribosomal depletion, and globin depletion.

  • Construct libraries in a single day, inclusive of RNA enrichment
  • Save steps and reduce hands-on time with streamlined protocols
  • Achieve robust performance across different sample types and low-input amounts, including degraded samples
  • Rely on Roche Support throughout the entire workflow, including custom depletion

     

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Let us help you start or improve SARS-Cov-2 sequencing

Our dedicated team of scientific support specialists is available to discuss your workflow for SARS-CoV-2 genome sequencing.

US Customers only:

Email: Support.SeqLS@roche.com 

Phone: 800.262.4911 (Monday - Friday, 9 am - 5 pm EST)

Research Use Only. Not for use in diagnostic procedures.
HYPERCAP and KAPA are trademarks of Roche.