Role of NGS in SARS-CoV-2 surveillance and COVID-19 research
NGS methods for SARS-CoV-2 Sequencing
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.
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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.
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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.
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Individual components, rather than complete kits, are often used in this method:
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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).
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.