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.
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.
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.