Sarah Trusiak1, Jonathan Nowacki1, Ranjit Kumar1, Spencer Debenport1
1Roche Sequencing & Life. Science, Wilmington, MA
Since the emergence of the SARS-CoV-2 and its resulting disease, COVID-19, over 30 million people have been infected worldwide and over 900,000 people have died. Many research programs have pivoted to studying the virus and understanding its health impacts and disease complications to develop treatments. RNA sequencing (RNA-seq) is a high-throughput method that enables research into the host’s transcriptional response to viral infection and the RNA genome of the virus itself; this is critical for understanding the impact of new viral mutations and emerging strains. Samples from infected hosts typically contain a single SARS-CoV-2 strain, while environmental samples like soil and sewage often contain a mixture of strains. When studying the RNA genome of viruses in mixed-RNA samples, such as total RNA from a human host, it is necessary to enrich for the viral RNA molecules above the much-more-abundant background RNA. Hybridization-based target enrichment (TE) using well-designed probes complementary to viral sequences has the potential to isolate and greatly enrich for viral reads of interest, and enable the capture of diverse viral strain mutations with a single TE panel, providing insights into the distribution and evolution of the virus. To enable hybridization capture RNA-seq for SARS-CoV-2 genome sequencing, we have developed a KAPA COVID-19 TE panel and workflow. Our probe panel covers >99.7% of 184 publicly available SARS-CoV-2 sequences (NCBI). Using this panel, we have developed a new target-enriched RNA-seq workflow that incorporates the KAPA COVID-19 TE panel into a modified version of the HyperCap Workflow v3 and includes the KAPA RNA HyperPrep Kit. In order to determine the lowest viral load that yields full coverage of the SARS-CoV-2 genome using the KAPA COVID-19 TE workflow, we tested varying levels of viral genome copies in different amounts of human total RNA. We then compared the performance of the KAPA COVID-19 TE panel workflow to panels from three different suppliers to identify differences in minimum viral load required for full genome coverage, as well as the ability to detect strain mutations. We show here that the KAPA COVID-19 TE panel detects mutations from six different strains of SARS-CoV-2 within a single target enrichment reaction. We conclude that this panel, when used with the modified HyperCap Workflow v3 adapted for RNA-seq, is a powerful tool for studying the SARS-CoV-2 genome in both infected host samples and samples containing mixed viral strains, and for distinguishing between multiple strains in a single sample.
Sandra Theall1, Sarah Trusiak1, Ranjit Kumar1, Jonathan Nowacki1, Spencer Debenport1, Rachel Kasinskas1.
1Roche Sequencing & Life. Science, Wilmington, MA
Scientists around the globe have pivoted their research to focus on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for the rampant and fatal COVID-19 pandemic. Amplicon sequencing is a critical tool that uses ultra-deep sequencing of PCR products (amplicons) which allows efficient variant identification and characterization in specific genomic regions. Valuable information such as this is used to monitor the evolution and transmission of the virus. Better understanding of the virus’s genetic composition could ultimately save lives by shaping strategies for public health and clinical care, as well as facilitating the production of therapies and treatments to combat the virus.
The increasing number and accessibility of SARS-CoV-2 sequencing protocols that are compatible across different products gives researchers the flexibility to use resources that are trusted and convenient for them, which is critical as the virus continues to quickly spread. The ARTIC network has responded to the pressing need to understand the human coronavirus by making a set of materials widely available to help researchers with PrimalSeq amplicon sequencing for SARS-CoV-2.
The Roche PrimalSeq workflow is an adaptation of the previously published PrimalSeq-Nextera XT workflow and demonstrates the benefits of using PCR and NGS reagents from a single vendor, including trusted KAPA polymerases and library prep kits-- reagents designed to work together seamlessly to simplify NGS workflows. This protocol integrates reverse transcription of RNA, multiplexed PCR, DNA library preparation, deep sequencing, and data analysis, enabling accurate reproducible sequencing readouts. The Roche PrimalSeq protocol will be compared to the PrimalSeq protocol as written using Nextera XT products and performance metrics will be reported.
Mariana Fitarelli-Kiehl1, Brian Sogoloff1, Jonathan Nowacki1, Ranjit Kumar1, Spencer Debenport1, Rachel Kasinskas1
1Roche Sequencing, Wilmington, MA
The rapid growth of targeted next-generation sequencing (NGS) applications has increased the demand for workflows with short turnaround times. However, most hybridization-based target enrichment workflows include an overnight hybridization step, presenting a major hurdle in the development of single-day protocols for whole-exome sequencing (WES) and other targeted sequencing applications. The KAPA HyperCap Workflow v3 is a high-performance, streamlined target enrichment solution that combines high-efficiency KAPA DNA library preparation kits with high-performance KAPA Target Enrichment probes. This portfolio includes the new comprehensive-yet-compact ~43 Mb KAPA HyperExome panel, which covers exonic regions defined by the CCDS, RefSeq, Ensembl, GENCODE, and ClinVar databases− including medically-relevant variants. Towards the development of a single-day WES workflow, we have tested the effectiveness of the KAPA HyperCap Workflow v3 with hybridization times as short as 15 minutes using KAPA HyperExome, and compared the results to data produced using the recommended overnight hybridization step. We have also compared the results to WES workflows from two other suppliers using the same hybridization times (15 minutes, 1.5 hours, 4 hours or 16 hours). Library preparation and target enrichment protocols were followed as instructed by the manufacturers. All workflows utilized in this study share several steps, such as enzymatic fragmentation of genomic DNA targeting 200 bp inserts, ligation to universal adapters followed by indexing via PCR amplification with Unique Dual-Indexed (UDI) primers, and probe panels designed for whole exome capture. For all workflows, libraries were prepared in triplicate for each condition and hybridization was performed using respective exome panels for 15 minutes, 1.5 hours, 4 hours or 16 hours. Enriched libraries were sequenced on an Illumina® NextSeq 500 sequencer (paired-end 2x76 bp). Library performance was evaluated using key sequencing metrics: mean coverage, percent of on-target reads, fold-80 base penalty, percent of duplicate reads, and GC coverage uniformity. Preliminary data generated with the KAPA HyperCap Workflow v3 and KAPA HyperExome showed very promising results with hybridization times as short as 1 hour, and data were similar for libraries produced with either the KAPA HyperPlus Kit (with enzymatic fragmentation) or the KAPA HyperPrep Kit (which uses mechanically-fragmented DNA). Overall, we have demonstrated that the entire HyperCap Workflow v3 may be readily adapted to a single-day target-enrichment workflow.