In silico evaluation of the impact of the Omicron variant on the sensitivity of RT-qPCR assays for SARS-CoV-2 detection using whole genome sequencing

Background The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant of concern (VoC) Omicron (B.1.1.529) has rapidly spread around the world presenting a new threat to global public human health. Due to the large number of mutations possessed by Omicron, concerns have emerged over potentially reduced diagnostic accuracy of reverse transcription polymerase chain reaction (RT-qPCR), the gold standard diagnostic test for SARS-CoV-2. Here, we aimed to assess the impact of Omicron on the integrity and sensitivity of RT-qPCR assays used for coronavirus disease-2019 (COVID-19) diagnosis via in silico analysis employing whole genome sequencing data and evaluated the potential for false negatives or test failure due to mismatches between primers/probes and viral genome. Methods In silico sensitivity of 12 RT-qPCR tests (containing 30 primers and probe sets) developed for detection of SARS-CoV-2 reported by the World Health Organization (WHO) or available in the literature, was assessed for use in detecting SARS-CoV-2 Omicron BA.1 and BA.2 sublineages, obtained after removing redundancy from publicly available genomes from National Center for Biotechnology Information (NCBI) and Global Initiative on Sharing Avian Influenza Data (GISAID) databases. The mismatches between the amplicon regions of the SARS-CoV-2 Omicron VoC and primers and probe sets were evaluated, and the clustering analysis of the corresponding amplicon sequences was carried out. Results From the 232 representative SARS-CoV-2 BA.1 Omicron sublineage genomes analyzed, 229 showed substitutions at the forward primer annealing site for assay China-CDC N, 226 showed mismatches in the reverse primer annealing site for assay Thai N, and all 232 had substitution at the 3’ end of the reverse primer annealing site for assay HKUniv RdRp/Hel. Therefore, the lowest sensitivity was observed for assay ChinaCDC N, Thai N and HKUniv RdRp/Hel for SARS-CoV-2 BA.1 sublineage genomes. For 5 SARS-CoV-2 BA.2 Omicron sublineage genomes, false negative results were observed for assays ChinaCDC N, Thai N, HKUniv RdRp/Hel, SigmAldr S5, SigmAldr S6 and HKUniv S. Conclusion In this study, we observed three (25%) assays (ChinaCDC N, Thai N, and HKUniv RdRp/Hel) demonstrated potential for false negatives for the SARS-CoV-2 Omicron BA.1 sublineage, while four (33.3%) assays (ChinaCDC N, Thai N, HKUniv RdRp/Hel, HKUniv S, SigmAldr S5 and SigmAldr S6) demonstrated potential false negative results for the for SARS-CoV-2 Omicron BA.2 sublineage, which also has the potential for Spike (S) gene dropout despite lacking 69-70 deletion in the S gene. Further, amplicon clustering and additional substitutions analysis along with the sensitivity analysis could be used for modification and development of RT-qPCR assays for detection of SARS-CoV-2 Omicron VoC lineages.

False negatives in GBA1 sequencing due to polymerase dependent allelic imbalance

A variant in the GBA1 gene is one of the most common genetic risk factors to develop Parkinson’s disease (PD). Here the serendipitous finding is reported of a polymerase dependent allelic imbalance when using next generation sequencing, potentially resulting in false-negative results when the allele frequency falls below the variant calling threshold (by default commonly at 30%). The full GBA1 gene was sequenced using next generation sequencing on saliva derived DNA from PD patients. Four polymerase chain reaction conditions were varied in twelve samples, to investigate the effect on allelic imbalance: (1) the primers (n = 4); (2) the polymerase enzymes (n = 2); (3) the primer annealing temperature (Ta) specified for the used polymerase; and (4) the amount of DNA input. Initially, 1295 samples were sequenced using Q5 High-Fidelity DNA Polymerase. 112 samples (8.6%) had an exonic variant and an additional 104 samples (8.0%) had an exonic variant that did not pass the variant frequency calling threshold of 30%. After changing the polymerase to TaKaRa LA Taq DNA Polymerase Hot-Start Version: RR042B, all samples had an allele frequency passing the calling threshold. Allele frequency was unaffected by a change in primer, annealing temperature or amount of DNA input. Sequencing of the GBA1 gene using next generation sequencing might be susceptible to a polymerase specific allelic imbalance, which can result in a large amount of flase-negative results. This was resolved in our case by changing the polymerase. Regions displaying low variant calling frequencies in GBA1 sequencing output in previous and future studies might warrant additional scrutiny.

Transferability and Polymorphism of SSR Markers Located in Flavonoid Pathway Genes in Fragaria and Rubus Species

Strawberry (Fragaria) and raspberry (Rubus) are very popular crops, and improving their nutritional quality and disease resistance are important tasks in their breeding programs that are becoming increasingly based on use of functional DNA markers. We identified 118 microsatellite (simple sequence repeat—SSR) loci in the nucleotide sequences of flavonoid biosynthesis and pathogenesis-related genes and developed 24 SSR markers representing some of these structural and regulatory genes. These markers were used to assess the genetic diversity of 48 Fragaria and Rubus specimens, including wild species and rare cultivars, which differ in berry color, ploidy, and origin. We have demonstrated that a high proportion of the developed markers are transferable within and between Fragaria and Rubus genera and are polymorphic. Transferability and polymorphism of the SSR markers depended on location of their polymerase chain reaction (PCR) primer annealing sites and microsatellite loci in genes, respectively. High polymorphism of the SSR markers in regulatory flavonoid biosynthesis genes suggests their allelic variability that can be potentially associated with differences in flavonoid accumulation and composition. This set of SSR markers may be a useful molecular tool in strawberry and raspberry breeding programs for improvement anthocyanin related traits.

Quantitating primer-template interactions using a deconstructed PCR methodology

When the polymerase chain reaction (PCR) is used to amplify simultaneously multiple templates, preferential amplification of certain templates (PCR bias) leads to a distorted representation of the original templates in the final amplicon pool. PCR selection, a type of PCR bias, is influenced by mismatches between primers and templates, the locations of mismatches, and the nucleotide pairing of mismatches. Direct measurement of primer-template interactions has not been possible, leading to uncertainty when attempting to optimize PCR reactions and degenerate primer pools. In this study, we developed an experimental system to systematically study primer-template interactions. We synthesized 10 double-stranded DNA templates with unique priming sites, as well as 64 primers with 0, 1, 2 or 3 mismatches with each of the 10 templates. By using a previously described deconstructed PCR (DePCR) methodology, we generated empirical data showing individual primer interactions with templates in complex template-primer amplification reactions. Standard PCR and DePCR amplification protocols were used to amplify templates in a series of 16 experiments in which templates, primers, and annealing temperature were varied. We observed that although perfect match primer-template interactions are important, the dominant type of interactions are mismatch amplifications, and that mismatched primer annealing and polymerase copying starts immediately during the first two cycle of PCR. In reactions with degenerate primer pools, multiple mismatches between primer and template are tolerated, and these do not have a strong effect on observed template ratios after amplification when employing the DePCR methodology. When employing the DePCR methodology, mismatched primer-template interactions were able to amplify source templates with significantly lower distortion relative to standard PCR. We establish here a quantitative experimental system for interrogating primer-template interactions and demonstrate the efficacy of the DePCR method for amplification of complex template mixtures with complex primer pools.

Quantitating primer-template interactions using a deconstructed PCR methodology

When the polymerase chain reaction (PCR) is used to amplify simultaneously multiple templates, preferential amplification of certain templates (PCR bias) leads to a distorted representation of the original templates in the final amplicon pool. PCR selection, a type of PCR bias, is influenced by mismatches between primers and templates, the locations of mismatches, and the nucleotide pairing of mismatches. Direct measurement of primer-template interactions has not been possible, leading to uncertainty when attempting to optimize PCR reactions and degenerate primer pools. In this study, we developed an experimental system to systematically study primer-template interactions. We synthesized 10 double-stranded DNA templates with unique priming sites, as well as 64 primers with 0, 1, 2 or 3 mismatches with each of the 10 templates. By using a previously described deconstructed PCR (DePCR) methodology, we generated empirical data showing individual primer interactions with templates in complex template-primer amplification reactions. Standard PCR and DePCR amplification protocols were used to amplify templates in a series of 16 experiments in which templates, primers, and annealing temperature were varied. We observed that although perfect match primer-template interactions are important, the dominant type of interactions are mismatch amplifications, and that mismatched primer annealing and polymerase copying starts immediately during the first two cycle of PCR. In reactions with degenerate primer pools, multiple mismatches between primer and template are tolerated, and these do not have a strong effect on observed template ratios after amplification when employing the DePCR methodology. When employing the DePCR methodology, mismatched primer-template interactions were able to amplify source templates with significantly lower distortion relative to standard PCR. We establish here a quantitative experimental system for interrogating primer-template interactions and demonstrate the efficacy of the DePCR method for amplification of complex template mixtures with complex primer pools.

Intrinsic conformational dynamics of the HIV-1 genomic RNA 5′UTR

The highly conserved 5′ untranslated region (5′UTR) of the HIV-1 RNA genome is central to the regulation of virus replication. NMR and biochemical experiments support a model in which the 5′UTR can transition between at least two conformational states. In one state the genome remains a monomer, as the palindromic dimerization initiation site (DIS) is sequestered via base pairing to upstream sequences. In the second state, the DIS is exposed, and the genome is competent for kissing loop dimerization and packaging into assembling virions where an extended dimer is formed. According to this model the conformation of the 5′UTR determines the fate of the genome. In this work, the dynamics of this proposed conformational switch and the factors that regulate it were probed using multiple single-molecule and in-gel ensemble FRET assays. Our results show that the HIV-1 5′UTR intrinsically samples conformations that are stabilized by both viral and host factor binding. Annealing of tRNALys3, the primer for initiation of reverse transcription, can promote the kissing dimer but not the extended dimer. In contrast, HIV-1 nucleocapsid (NC) promotes formation of the extended dimer in both the absence and presence of tRNALys3. Our data are consistent with an ordered series of events that involves primer annealing, genome dimerization, and virion assembly.

DNA metabarcode choice and design

Chapter “DNA metabarcode choice and design” develops the properties of the ideal metabarcode in a given context, including conservation of the primer annealing regions and resolution power across the target taxonomic group of interest. It also highlights the experimental constraints influencing the choice of a metabarcode in practice. A detailed tutorial illustrates how to design and test metabarcoding primers in silico with the programs ecoPrimers, ecoPCR, and the software suite OBITools. Command lines and example files are provided to design and test universal metabarcoding primers for Bacteria. Chapter 2 also gives statistics about the taxonomic resolution and primer conservation of more than 60 metabarcodes available for DNA metabarcoding analysis of a wide range of taxonomic groups.

PCR Multiplexes Discriminate Fusarium Symbionts of Invasive Euwallacea Ambrosia Beetles that Inflict Damage on Numerous Tree Species Throughout the United States

Asian Euwallacea ambrosia beetles vector Fusarium mutualists. The ambrosial fusaria are all members of the ambrosia Fusarium clade (AFC) within the Fusarium solani species complex (FSSC). Several Euwallacea–Fusarium mutualists have been introduced into nonnative regions and have caused varying degrees of damage to orchard, landscape, and forest trees. Knowledge of symbiont fidelity is limited by current identification methods, which typically requires analysis of DNA sequence data from beetles and the symbionts cultured from their oral mycangia. Here, polymerase chain reaction (PCR)-based diagnostic tools were developed to identify the six Fusarium symbionts of exotic Euwallacea spp. currently known within the United States. Whole-genome sequences were generated for representatives of six AFC species plus F. ambrosium and aligned to the annotated genome of F. euwallaceae. Taxon-specific primer-annealing sites were identified that rapidly distinguish the AFC species currently within the United States. PCR specificity, reliability, and sensitivity were validated using a panel of 72 Fusarium isolates, including 47 reference cultures. Culture-independent multiplex assays accurately identified two AFC fusaria using DNA isolated from heads of their respective beetle partners. The PCR assays were used to show that Euwallacea validus is exclusively associated with AF-4 throughout its sampled range within eastern North America. The rapid assay supports federal and state agency efforts to monitor spread of these invasive pests and mitigate further introductions.

Real-time polymerase chain reaction optimised for hepatitis C virus detection in dried blood spots from HIV-exposed infants, KwaZulu-Natal, South Africa

Background: There is a paucity of data on the prevalence of hepatitis C virus (HCV) in children, particularly in sub-Saharan Africa. A major obstacle in resource-limited settings for polymerase chain reaction (PCR) testing is the necessity for specimen transportation and storage at low temperatures. There are numerous recent studies of using real-time HCV PCR for diagnosis and screening of plasma and serum, but few have looked at using dried blood spot (DBS) specimens.Objectives: The aim of this study was to optimise a real-time HCV PCR method to detect HCV RNA from infant DBS specimens for use as a tool for HCV surveillance in KwaZulu-Natal, South Africa.Method: The LightCycler® 2.0 instrument was used for the HCV PCR using the LightCycler® RNA Master SYBR Green I kit. Template volume, primer concentration and primer annealing temperatures were optimised and the method was used on 179 DBS specimens from HIV-exposed infants in KwaZulu-Natal.Results: Primer concentrations adjusted to 0.25 µM and a template volume of 10 µL improved the PCR amplification. Primer annealing temperatures lowered from 65 °C to 58 °C resulted in higher quantities of amplified PCR product. The limit of detection of the optimised HCV PCR assay was between 1200 IU/mL and 3580 IU/mL of HCV RNA. HCV was not detected in any of the 179 DBS specimens.Conclusion: The optimised real-time HCV PCR on infant DBS specimens performed well, but HCV was not found in this surveillance study. HIV infection may have little impact on the vertical transmission of HCV in this region.