A short plus long-amplicon based sequencing approach improves genomic coverage and variant detection in the SARS-CoV-2 genome

High viral transmission in the COVID-19 pandemic has enabled SARS‐CoV‐2 to acquire new mutations that may impact genome sequencing methods. The ARTIC.v3 primer pool that amplifies short amplicons in a multiplex-PCR reaction is one of the most widely used methods for sequencing the SARS-CoV-2 genome. We observed that some genomic intervals are poorly captured with ARTIC primers. To improve the genomic coverage and variant detection across these intervals, we designed long amplicon primers and evaluated the performance of a short (ARTIC) plus long amplicon (MRL) sequencing approach. Sequencing assays were optimized on VR-1986D-ATCC RNA followed by sequencing of nasopharyngeal swab specimens from fifteen COVID-19 positive patients. ARTIC data covered 94.47% of the virus genome fraction in the positive control and patient samples. Variant analysis in the ARTIC data detected 217 mutations, including 209 single nucleotide variants (SNVs) and eight insertions & deletions. On the other hand, long-amplicon data detected 156 mutations, of which 80% were concordant with ARTIC data. Combined analysis of ARTIC + MRL data improved the genomic coverage to 97.03% and identified 214 high confidence mutations. The combined final set of 214 mutations included 203 SNVs, 8 deletions and 3 insertions. Analysis showed 26 SARS-CoV-2 lineage defining mutations including 4 known variants of concern K417N, E484K, N501Y, P618H in spike gene. Hybrid analysis identified 7 nonsynonymous and 5 synonymous mutations across the genome that were either ambiguous or not called in ARTIC data. For example, G172V mutation in the ORF3a protein and A2A mutation in Membrane protein were missed by the ARTIC assay. Thus, we show that while the short amplicon (ARTIC) assay provides good genomic coverage with high throughput, complementation of poorly captured intervals with long amplicon data can significantly improve SARS-CoV-2 genomic coverage and variant detection.

Rapid and Accurate Identification of SARS-CoV-2 Omicron Variants Using Droplet Digital PCR (RT-ddPCR)

Background Mutations in the receptor binding domain of the SARS-CoV-2 Spike protein are associated with increased transmission or substantial reductions in vaccine efficacy, including in the recently described Omicron variant. The changing frequencies of these mutations combined with their differing susceptibility to available therapies have posed significant problems for clinicians and public health professionals. Objective To develop an assay capable of rapidly and accurately identifying variants including Omicron in clinical specimens to enable case tracking and/or selection of appropriate clinical treatment. Study Design Using three duplex RT-ddPCR reactions targeting four amino acids, we tested 419 positive clinical specimens from February to December 2021 during a period of rapidly shifting variant prevalences and compared genotyping results to genome sequences for each sample, determining the sensitivity and specificity of the assay for each variant. Results Mutation determinations for 99.7% of detected samples agree with NGS data for those samples, and are accurate despite wide variation in RNA concentration and potential confounding factors like transport medium, presence of additional respiratory viruses, and additional mutations in primer and probe sequences. The assay accurately identified the first 15 Omicron variants in our laboratory including the first Omicron in Washington State and discriminated against S-gene dropout Delta specimen. Conclusion We describe an accurate, precise, and specific RT-ddPCR assay for variant detection that remains robust despite being designed prior the emergence of Delta and Omicron variants. The assay can quickly identify mutations in current and past SARS-CoV-2 variants, and can be adapted to future mutations.

A robust benchmark for evaluating and improving mosaic variant calling strategies

The rapid advances in sequencing and analysis technologies have enabled the accurate detection of diverse forms of genomic variants, including germline, somatic, and mosaic mutations. However, unlike for the former two mutations, the best practices for mosaic variant calling still remain chaotic due to the technical and conceptual difficulties faced in evaluation. Here, we present our benchmark of nine feasible strategies for mosaic variant detection based on a systematically designed reference standard that mimics mosaic samples, with 390,153 control positive and 35,208,888 negative single-nucleotide variants and insertion–deletion mutations. We identified the condition-dependent strengths and weaknesses of the current strategies, instead of a single winner, regarding variant allele frequencies, variant sharing, and the usage of control samples. Moreover, feature-level investigation directs the way for immediate to prolonged improvements in mosaic variant calling. Our results will guide researchers in selecting suitable calling algorithms and suggest future strategies for developers.

The Rapid Antigen Detection Test for SARS-CoV-2 Underestimates the Identification of COVID-19 Positive Cases and Compromises the Diagnosis of the SARS-CoV-2 (K417N/T, E484K, and N501Y) Variants

Timely detection of severe acute respiratory syndrome due to coronavirus 2 (SARS-CoV-2) by reverse transcription quantitative polymerase chain reaction (RT-qPCR) has been the gold- strategy for identifying positive cases during the current pandemic. However, faster and less expensive methodologies are also applied for the massive diagnosis of COVID-19. In this way, the rapid antigen test (RAT) is widely used. However, it is necessary to evaluate its detection efficiency considering the current pandemic context with the circulation of new viral variants. In this study, we evaluated the sensitivity and specificity of RAT (SD BIOSENSOR, South Korea), widely used for testing and SARS-CoV-2 diagnosis in Santiago of Chile. The RAT showed a 90% (amplification range of 20 ≤ Cq <25) and 10% (amplification range of 25 ≤ Cq <30) of positive SARS-CoV-2 cases identified previously by RT-qPCR. Importantly, a 0% detection was obtained for samples within a Cq value>30. In SARS-CoV-2 variant detection, RAT had a 42.8% detection sensitivity in samples with RT-qPCR amplification range 20 ≤ Cq <25 containing the single nucleotide polymorphisms (SNP) K417N/T, N501Y and E484K, associated with beta or gamma SARS-CoV-2 variants. This study alerts for the special attention that must be paid for the use of RAT at a massive diagnosis level, especially in the current scenario of appearance of several new SARS-CoV-2 variants which could generate false negatives and the compromise of possible viral outbreaks.

RT-PCR Detection of SARS-CoV-2 Among Individuals from the Upper Silesian Region—Analysis of 108 516 Tests.

Background: The COVID-19 pandemic triggered by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has left a huge mark on everyday lives, introducing restrictions and plunging the global economy. This study aimed to analyze the available epidemiological data from the register of one of the largest laboratories testing for SARS-CoV-2 in the Silesian voivodship of Poland. Methods: This analysis is based upon the epidemiological records collected between 30 March 2020, and 30 April 2021, by the Silesian Park of Medical Technology Kardio-Med Silesia (Zabrze, Poland). In addition, we performed SARS-CoV-2 variant detection in samples from patients reinfected with SARS-CoV-2. Results: Our results confirm that SARS-CoV-2 infections are more common in urban areas. Laboratoryconfirmed COVID19 cases represent 13.21% of all RT-PCR test results during the 13 months of our laboratory diagnostics for SARS-CoV-2 infections. Detection of SARS-CoV-2 variants in samples of potentially reinfected patients showed discrepancies in the results. Conclusions: Due to the higher risk of SARS-CoV-2 infection among the Upper Silesian population, the region is at greater risk of deteriorating economic situation and healthcare as compared to other areas of Poland. RT-PCR methods are inexpensive and suitable for large-scale screening, but they can be untrustworthy so detection of SARS-CoV-2 variants in samples should be confirmed by sequencing.

Modified NEBNext® VarSkip Short SARS-CoV-2 Library Prep Kit for Illumina Platforms - adapted for wastewater samples v1

PURPOSE: This method was developed at the FDA’s Center for Food Safety and Applied Nutrition for GenomeTrakr’s pandemic response project, monitoring SARS-CoV-2 variants in wastewater. Protocols developed for this project cover wastewater collection, concentration, RNA extraction, RT-qPCR, library prep, genome sequencing, quality control checks, and data submission to NCBI. This modified protocol details methods for cDNA synthesis and library preparation for sequencing of wastewater samples containing SARS-CoV-2. The protocol is based primarily on the NEBNext® ARTIC SARS-CoV-2 Library Prep Kit (Illumina®), NEB #E7650S/L 24/96 reactions, with a few modifications. Primarily, VarSkip Short primers are used in place of the ARTIC V3 primers. These primers are available in the NEBNext®ARTIC SARS-CoV-2 FS Library Prep Kit (Illumina®); however, for optimal variant detection from wastewater, sequenced fragments should be as large as possible, so we discourage fragmentation prior to end prep. There are a couple of decision points in this protocol. Examining cDNA amplicon samples on an Agilent TapeStation system or similar fragment analyzer is extremely helpful in making these decisions.

Multiplexed CRISPR-based microfluidic platform for clinical testing of respiratory viruses and SARS-CoV-2 variants

The COVID-19 pandemic has demonstrated a clear need for high-throughput, multiplexed, and sensitive assays for detecting SARS-CoV-2 and other respiratory viruses as well as their emerging variants. Here, we present microfluidic CARMEN (mCARMEN), a cost-effective virus and variant detection platform that combines CRISPR-based diagnostics and microfluidics with a streamlined workflow for clinical use. We developed the mCARMEN respiratory virus panel (RVP) and demonstrated its diagnostic-grade performance on 533 patient specimens in an academic setting and then 166 specimens in a clinical setting. We further developed a panel to distinguish 6 SARS-CoV-2 variant lineages, including Delta and Omicron, and evaluated it on 106 patient specimens, with near-perfect concordance to sequencing-based variant classification. Lastly, we implemented a combined Cas13 and Cas12 approach that enables quantitative measurement of viral copies in samples. mCARMEN enables high-throughput surveillance of multiple viruses and variants simultaneously.

COVID-19 Variant Detection with a High-Fidelity CRISPR-Cas12 Enzyme

Laboratory tests for the accurate and rapid identification of SARS-CoV-2 variants have the potential to guide the treatment of COVID-19 patients and inform infection control and public health surveillance efforts. Here we present the development and validation of a COVID-19 variant DETECTR assay incorporating loop-mediated isothermal amplification (LAMP) followed by CRISPR-Cas12 based identification of single nucleotide polymorphism (SNP) mutations in the SARS-CoV-2 spike (S) gene. This assay targets the L452R, E484K, and N501Y mutations associated with nearly all circulating viral lineages. In a comparison of three different Cas12 enzymes, only the newly identified enzyme CasDx1 was able to accurately identify all three targeted SNP mutations. We developed a data analysis pipeline for CRISPR-based SNP identification using the assay from 91 clinical samples (Ct < 30), yielding an overall SNP concordance and agreement with SARS-CoV-2 lineage classification of 100% compared to viral whole-genome sequencing. These findings highlight the potential utility of CRISPR-based mutation detection for clinical and public health diagnostics.