Mutations in emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineages can interfere with laboratory methods used to generate viral genome sequences for public health surveillance. We identified 20 mutations that are widespread in variant of concern lineages and affect widely used sequencing protocols by the ARTIC network and Freed et al. Three of these mutations disrupted sequencing of P.1 lineage specimens during a recent outbreak in British Columbia, Canada. We provide laboratory validation of protocol modifications that restored sequencing performance. The study findings indicate that genomic sequencing protocols require immediate updating to address emerging mutations. This work also suggests that routine monitoring and protocol updates will be necessary as SARS-CoV-2 continues to evolve. The bioinformatic and laboratory approaches used here provide guidance for this kind of assay maintenance.

Viral genome sequencing using the ARTIC protocol has been a vital tool for understanding the spread of epidemics including Ebola, Zika, COVID-19 and Mpox and has seen widespread adoption due to its low cost and high sensitivity. Here, we describe PrimalScheme, an open-source toolkit and website that allows users to easily design primer schemes for amplicon sequencing of viruses and has generated over 67,000 primer schemes for a global community since 2017. In January 2020, PrimalScheme was used to rapidly generate a primer scheme for SARS-CoV-2, with primer pools distributed to researchers from 44 countries to help scale-up genomic surveillance efforts. Overall, these primers were used to generate an estimated 18M genome sequences and the protocols were viewed online ~250K times. To complement PrimalScheme, we have built PrimalScheme Labs, a scheme repository which allows users to find and share primers schemes as well as establishing a set of data standards. Through improvements to the primer design process, including the use of discrete primer clouds, we have expanded the use of amplicon sequencing to include diverse virus species. We demonstrate the utility of this approach through a high diversity pan-genotype Measles virus (MeV) scheme. We also demonstrate its use on a high sensitivity, short amplicon Monkeypox virus (MPXV) scheme with over 1000 primers, showing high genome recovery on low-titre clinical samples. These developments have implications for sequencing from samples such as wastewater, for genomic surveillance of endemic pathogens and in preparing for future pandemics.

The ongoing COVID-19 pandemic necessitates cost-effective, high-throughput, and timely genomic sequencing of SARS-CoV-2 viruses for outbreak investigations, identifying variants of concern (VoC), characterizing vaccine breakthrough infections, and public health surveillance. Additionally, the enormous demand of genomic sequencing on supply chains and the resulting shortages of laboratory supplies necessitate the use of low-reagent and low-consumable methods. Here, we report an optimized library preparation method where the same protocol can be used in a STAT scenario, from sample to sequencer in as little as eight hours, and a high-throughput scenario, where one technologist can perform 576 library preparations over the course of one 8-hour shift. This new method uses Freed et al.’s 1200 bp primer sets (Biol Methods Protoc 5:bpaa014, 2020, https://doi.org/10.1093/biomethods/bpaa014 ) and a modified and truncated Illumina DNA Prep workflow (Illumina, CA, USA). Compared to the original, application of this new method in hundreds of clinical specimens demonstrated equivalent results to the full-length DNA Prep workflow at 45% the cost, 15% of consumables required (such as pipet tips), 25% of manual hands-on time, and 15% of on-instrument time if performing on a liquid handler, with no compromise in sequence quality. Results suggest that this new method is a rapid, simple, cost-effective, and high-quality SARS-CoV-2 whole genome sequencing protocol.

Mutations in emerging SARS-CoV-2 lineages can interfere with the laboratory methods used to generate high-quality genome sequences for COVID-19 surveillance. Here, we identify 46 mutations in current variant of concern lineages affecting the widely used laboratory protocols for SARS-CoV-2 genomic sequencing by Freed et al. and the ARTIC network. We provide laboratory data showing how three of these mutations disrupted sequencing of P.1 lineage specimens during a recent outbreak in British Columbia, Canada, and we also demonstrate how we modified the Freed et al. protocol to restore performance.

Background: Saline mouth rinse/gargle samples have recently been shown to be a suitable option for swab-independent self-collection for SARS-CoV-2 diagnosis. We sought to evaluate a simplified process for direct reverse transcriptase PCR (RT-qPCR) testing of this novel sample type and to compare performance with routine RT-qPCR using automated nucleic acid extraction. Methods: Clinical saline mouth rinse/gargle samples were subjected to automated nucleic acid extraction (“standard method”), followed by RT-qPCR using three assays including the FDA authorized US-CDC’s N1/N2 assay, which was the reference standard for determining sensitivity/specificity. For extraction-free workflow, an aliquot of each gargle sample underwent viral heat inactivation at 65 °C for 20 minutes followed by RT-qPCR testing, without an intermediate extraction step. An in-house validated RT-qPCR lab developed test (LDT), targeting the SARS-CoV-2’s S/ORF8 genes (SORP triplex assay) and the N1/N2 US-CDC assay was used to evaluate the extraction-free protocol. To improve the analytical sensitivity, we developed a single-tube hemi-nested (STHN) version of the SORP triplex assay. Results: A total of 38 SARS-CoV-2 positive and 75 negative saline mouth rinse/gargle samples were included in this evaluation. A 100% concordance in detection rate was obtained between the standard method and the extraction-free approach for the SORP assay. An average increase of +2.63 to +5.74 of the cycle threshold (C _T ) values was observed for both the SORP and N1/N2 assay when extraction-free was compared between the standard method. The average ΔC _T [ΔC _T =C _{T(Direct PCR)} -C _{T(Extracted RNA)} ], for each of the gene targets were: S (ΔC _T = +4.24), ORF8 (ΔC _T =+2.63), N1 (ΔC _T =+2.74) and N2 (ΔC _T =+5.74). The ΔC _T for the STHN SORP assay was +1.51 and −2.05 for the S and ORF8 targets respectively, when extracted method was compared to the standard method. Conclusion: Our Gargle-Direct SARS-CoV-2 method is operationally simple, minimizes pre-analytical sample processing and is potentially implementable by most molecular diagnostic laboratories. The empirical demonstration of single-tube hemi-nested RT-qPCR, to specifically address and alleviate the widely-acknowledged problem of reduced analytical sensitivity of detection of extraction-free templates, should help diagnostic laboratories in choosing Gargle-Direct protocol for high-throughput testing.

Treatment of Mycobacterium tuberculosis requires multi-drug regimens, and resistance to any individual antibiotic can compromise outcomes. For slow-growing organisms like M. tuberculosis, rapid detection of resistance-conferring mutations enables timely initiation of effective therapy. Conversely, confirming wild-type status in resistance-associated genes supports confidence in standard regimens. We developed an amplicon-based next generation sequencing (amplicon tNGS) assay on the Illumina platform targeting eight genes linked to resistance to isoniazid, rifampin, ethambutol, pyrazinamide, and fluoroquinolones. Sequencing results were analyzed using a custom bioinformatics pipeline. Forty-seven samples were used for assay development, and 37 additional samples underwent post-implementation clinical validation. Compared to whole genome sequencing (WGS), amplicon tNGS demonstrated 97.7% sensitivity, 98.9% specificity, and 98.7% overall accuracy for variant detection in targeted regions. Resistance prediction showed 79.3% concordance with WGS; discrepancies were primarily due to mutations outside of target regions. Among post-implementation samples, 27/37 passed quality metrics for all targets, with 95.7% concordance between amplicon tNGS results and final susceptibility results. This assay is now in use in our laboratory and offers significantly faster turnaround than both WGS and phenotypic methods on cultured isolates, enabling more rapid, informed treatment decisions for tuberculosis patients.