Development of an amplicon-based sequencing approach in response to the global emergence of mpox.

The 2022 multicountry mpox outbreak concurrent with the ongoing Coronavirus Disease 2019 (COVID-19) pandemic further highlighted the need for genomic surveillance and rapid pathogen whole-genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early mpox infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there was an urgent need for a more sensitive and broadly applicable sequencing approach. Highly multiplexed amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented in public health laboratories during the COVID-19 pandemic. We sequenced clinical specimens that tested presumptively positive for human monkeypox virus with amplicon-based and metagenomic sequencing approaches. We found notably higher genome coverage across the virus genome, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach, particularly in higher PCR cycle threshold (Ct) (lower DNA titer) samples. Further testing demonstrated that Ct value correlated with the number of sequencing reads and influenced the percent genome coverage. To maximize genome coverage when resources are limited, we recommend selecting samples with a PCR Ct below 31 Ct and generating 1 million sequencing reads per sample. To support national and international public health genomic surveillance efforts, we sent out primer pool aliquots to 10 laboratories across the United States, United Kingdom, Brazil, and Portugal. These public health laboratories successfully implemented the human monkeypox virus primer scheme in various amplicon sequencing workflows and with different sample types across a range of Ct values. Thus, we show that amplicon-based sequencing can provide a rapidly deployable, cost-effective, and flexible approach to pathogen whole-genome sequencing in response to newly emerging pathogens. Importantly, through the implementation of our primer scheme into existing SARS-CoV-2 workflows and across a range of sample types and sequencing platforms, we further demonstrate the potential of this approach for rapid outbreak response.

Development of an amplicon-based sequencing approach in response to the global emergence of human monkeypox virus.

The 2022 multi-country monkeypox (mpox) outbreak concurrent with the ongoing COVID-19 pandemic has further highlighted the need for genomic surveillance and rapid pathogen whole genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early mpox infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there was an urgent need for a more sensitive and broadly applicable sequencing approach. Highly multiplexed amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for SARS-CoV-2. Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented in public health laboratories during the COVID-19 pandemic. We sequenced clinical samples that tested presumptive positive for human monkeypox virus with amplicon-based and metagenomic sequencing approaches. We found notably higher genome coverage across the virus genome, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach, particularly in higher PCR cycle threshold (lower DNA titer) samples. Further testing demonstrated that Ct value correlated with the number of sequencing reads and influenced the percent genome coverage. To maximize genome coverage when resources are limited, we recommend selecting samples with a PCR cycle threshold below 31 Ct and generating 1 million sequencing reads per sample. To support national and international public health genomic surveillance efforts, we sent out primer pool aliquots to 10 laboratories across the United States, United Kingdom, Brazil, and Portugal. These public health laboratories successfully implemented the human monkeypox virus primer scheme in various amplicon sequencing workflows and with different sample types across a range of Ct values. Thus, we show that amplicon based sequencing can provide a rapidly deployable, cost-effective, and flexible approach to pathogen whole genome sequencing in response to newly emerging pathogens. Importantly, through the implementation of our primer scheme into existing SARS-CoV-2 workflows and across a range of sample types and sequencing platforms, we further demonstrate the potential of this approach for rapid outbreak response.

Association of the BsmI, ApaI, TaqI, Tru9I and FokI Polymorphisms of the Vitamin D Receptor Gene with Nephrolithiasis in the Turkish Population.

PURPOSE: To analyze the relationship between nephrolithiasis and vitamin D receptor (VDR) gene BsmI (rs1544410), ApaI (rs7975232), TaqI (rs731236), Tru9I (rs757343) and FokI (rs2228570) polymorphisms in a study group from the Turkish population. MATERIALS AND METHODS: Ninety-eight patients with calcium oxalate kidney stones and 70 controls were enrolled in this study. Five polymorphisms of the VDR gene were studied using the Polymerase Chain Reaction Restriction Fragment Length Polymorphism (PCR-RFLP) method. RESULTS: For all polymorphisms, genotype frequencies were in line with Hardy-Weinberg equilibrium in the patients and controls. For the BsmI polymorphism, allele frequency distribution was found to differ significantly between the patients and the controls (P < .05). The "B" allele was found to increase the risk of nephrolithiasis by approximately 1.5-fold (odds ratio = 1.55, 95% confidence interval: 1.00-2.40; P = .048). However, we did not find any statistically significant differences in the allele and genotype frequencies for the ApaI, TaqI, Tru9I and FokI polymorphisms. Proportionally, the "BAt" and "baT" haplotypes were more common than other haplotypes in the cases and controls, respectively. For the haplotypes of the BsmI and TaqI polymorphisms, the "bT" haplotype frequency was found to be common in both the patients and the controls. However, we did not find statistically significant differences between the cases and the controls for either the BsmI / ApaI / TaqI or the BsmI/TaqI haplotypes. Moreover, no relationship was identified between family history and development of stone disease. CONCLUSION: The "B" allele of the BsmI polymorphism of the VDR gene may increase stone development risk. Further investigations are needed to improve our knowledge regarding the genetic factors affecting urinary stone development.