Sequencing Protocol Research Articles

Invasive group A streptococcal disease surveillance in Canada, 2021-2022.

Invasive group A streptococcal (iGAS, Streptococcus pyogenes) disease has been a nationally notifiable disease in Canada since 2000. This report summarizes the demographics, emm types, and antimicrobial resistance of iGAS isolates collected in Canada in 2021 and 2022. The Public Health Agency of Canada's National Microbiology Laboratory collaborates with provincial and territorial public health laboratories to conduct national surveillance of invasive S. pyogenes. Emm typing was performed using the Centers for Disease Control and Prevention emm sequencing protocol or extracted from whole-genome sequencing data. Antimicrobial susceptibilities were determined using Kirby-Bauer disk diffusion according to Clinical and Laboratory Standards Institute guidelines or predicted from whole-genome sequencing data based on the presence of resistance determinants. Overall, the incidence of iGAS disease in Canada was 5.56 cases per 100,000 population in 2021, decreasing from the peak of 8.6 cases per 100,000 population in 2018. A total of 2,630 iGAS isolates were collected during 2022, representing an increase from 2021 (n=2,179). In particular, there was a large increase in isolates collected from October to December 2022. The most predominant emm type overall in 2021 and 2022 was emm49, at 21.5% (n=468) and 16.9% (n=444), respectively, representing a significant increase in prevalence since 2018 (p<0.0001). The former most prevalent type, emm1, increased from 0.5% (n=10) in 2021 to 4.8% (n=125) in 2022; similarly, emm12 increased from 1.0% (n=22) in 2021 to 5.8% (n=151) in 2022. These two types together accounted for almost 25% of isolates collected in late 2022 (October to December). Antimicrobial resistance rates in 2021 and 2022 included: 14.9%/14.1% erythromycin resistance, 4.8%/3.0% clindamycin resistance, and <1% chloramphenicol resistance. The increase of iGAS isolates collected in Canada is an important public health concern. Continued surveillance of iGAS is critical to monitor expanding emm types and antimicrobial resistance patterns.

#2696 The effect of laxatives on reducing serum phosphate levels of probiotics in hemodialysis patients: a subanalysis of RCT

Abstract Background and Aims Dysbiosis of the intestinal microbiota in chronic kidney disease increases uremic toxin, which damage the epithelial tight junctions and increase the permeability of the intestinal wall via endotoxemia and systemic inflammation. Some articles reported that probiotics reduced the uremic toxin and decreased inflammation biomarkers in CKD patients. Meanwhile, we have already reported that oral administration of Bifidobacterium longum in a gastro-resistant seamless capsule decreases serum phosphate levels in patients receiving hemodialysis (Clin Kidney J, 5:373-374, 2012). In addition, in this congress, we reported that three-combination probiotics decreased serum phosphate levels. Meanwhile, some articles reported the relationship hypophosphatemia and laxative abuse. The aim of this subanalysis study is to evaluate the efficacy of laxatives on reducing serum phosphate levels of three-combination probiotic in patients receiving hemodialysis. Method A 6-month prospective, randomized, double-blind, placebo-controlled study was conducted at three hemodialysis centers. As probiotic therapy, of three-combination probiotics (Bio-Three) tablets, each containing 2 mg of lactomin (Streptococcus faecalis T-110), 10 mg of Clostridium butyricum TO-A, and 10 mg of Bacillus mesentericus TO-A, were used. The patients receiving hemodialysis were randomly assigned to the Bio-Three group (n = 37) and the placebo group (n = 36). In both the Bio-Three group and the placebo group, patients orally received 6 tablets 3 times daily. We evaluated the effect of probiotic on serum phosphate level and gut microbiome before and 6 months after the start of treatment. The gut microbiome composition was analyzed by a 16s rRNA gene-based sequencing protocol. In addition, in patients with laxative (n = 24), we evaluate the effect of probiotic on serum phosphate levels and gut microbiome. Results The serum phosphate levels in the Bio-Three group significantly decreased after 6-month treatment, while did not change in the placebo group. Furthermore, the serum phosphate level in patients with Bio-Three and laxative additionally decreased. In a 16s rRNA gene-based sequencing protocol analysis, serum phosphate levels were positively correlated with the occupancy ratio of Erysipelotrichaceae, Lachnospiraceae, Oscillospiraceae, Ruminococcaceae, and Sutterellaceae and were negatively correlated with the occupancy ratio of Leuconostocaceae and Lachnospiraceae in patients with Bio-Three and laxatives. Conclusion In this subanalysis study, the effect of reducing serum phosphate levels on three-combination probiotic might be augmented by laxatives and the specific bacteria in the gut microbiome might contribute to the decrease of serum phosphate levels.

#181 Dysbiosis of gut microbiota in patients with chronic kidney disease

Abstract Background and Aims The human intestinal tract contains 100 trillion microorganisms, and 500–1000 microbial strains exist in each individual, classified into two main phyla: Firmicutes and Bacteroidetes. The gut bacterial microbiota is altered in patients with chronic kidney disease (CKD). However, the bacterial composition at each stage of CKD is unclear in these patients, including those receiving renal replacement therapy. In this report, we present the changes in the gut microbiota among patients with CKD. Method A total of 93 individuals were recruited for the study. Seventy-three patients had stage 3–5 CKD, including those receiving renal replacement therapy (CKD group), and 20 were age- and sex-matched controls (CKD stage 1–2). Gut microbiome composition was analyzed using a 16S ribosomal RNA gene-based sequencing protocol. Results The beta diversity of the samples was evaluated via principal coordinates analysis (PCoA). The unweighted and weighted UniFrac distances revealed a tendency toward separation of microbiota in the samples according to the CKD stage 1–2 and CKD stage 3–5, 5D groups. At the genus level, the butyrate-producing bacteria Lachnospira, Blautia, Coprococcus, Anaerostipes, and Roseburia were more abundant in the control group (Linear Discriminant Analysis score of &amp;gt;3) than in the CKD group. Lachnospira was more abundant in the control group than in patients with CKD stage 3a. Compared to the control group, multiplex butyrate-producing bacteria were deficient in patients with CKD stage 3b–5D, including in patients receiving renal replacement therapy. Conclusion Our findings highlight that the gut bacterial composition, including butyrate-producing bacteria, deteriorates from CKD stage 3b. Even after renal replacement therapy, the bacterial composition did not change.

Deep Batch Integration and Denoise of Single-Cell RNA-Seq Data.

Numerous single-cell transcriptomic datasets from identical tissues or cell lines are generated from different laboratories or single-cell RNA sequencing (scRNA-seq) protocols. The denoising of these datasets to eliminate batch effects is crucial for data integration, ensuring accurate interpretation and comprehensive analysis of biological questions. Although many scRNA-seq data integration methods exist, most are inefficient and/or not conducive to downstream analysis. Here, DeepBID, a novel deep learning-based method for batch effect correction, non-linear dimensionality reduction, embedding, and cell clustering concurrently, is introduced. DeepBID utilizes a negative binomial-based autoencoder with dual Kullback-Leibler divergence loss functions, aligning cell points from different batches within a consistent low-dimensional latent space and progressively mitigating batch effects through iterative clustering. Extensive validation on multiple-batch scRNA-seq datasets demonstrates that DeepBID surpasses existing tools in removing batch effects and achieving superior clustering accuracy. When integrating multiple scRNA-seq datasets from patients with Alzheimer's disease, DeepBID significantly improves cell clustering, effectively annotating unidentified cells, and detecting cell-specific differentially expressed genes.

Multiplexed in situ RNA imaging by combFISH.

Multiplexed in situ RNA imaging offers new opportunities for gene expression profiling by providing high-throughput spatial information. In this work, we present a cyclic combinatorial fluorescent in situ hybridization (combFISH) assay to achieve multiplexed detection of RNA in cell cultures and tissues. Specifically, multiplexing is achieved through cyclic interrogation of barcode sequences on the rolling circle amplicons generated from the padlock probe assay by using sets of combinatorial detection probes. Theoretically, combFISH can detect 64 genes in three hybridization cycles by combinatorial barcoding using 12 fluorescently labeled detection probes. Our method eliminates sequencing-by-ligation (SBL) chemistry in the in situ sequencing protocol and directly uses RNA as targets for ligation, making it more straightforward. We showed that our method works in fresh-frozen and formalin-fixed paraffin-embedded tissue sections. With its straightforward protocols, we expect our method to be adopted by the scientific community and extended to clinical settings.

Identification and in vitro characterization of UDP-GlcNAc-RNA cap-modifying and decapping enzymes.

In recent years, several noncanonical RNA caps derived from cofactors and metabolites have been identified. Purine-containing RNA caps have been extensively studied, with multiple decapping enzymes identified and efficient capture and sequencing protocols developed for nicotinamide adenine dinucleotide (NAD)-RNA, which allowed for a stepwise elucidation of capping functions. Despite being identified as an abundant noncanonical RNA-cap, UDP-sugar-capped RNA remains poorly understood, which is partly due to its complex in vitro preparation. Here, we describe a scalable synthesis of sugar-capped uridine-guanosine dinucleotides from readily available protected building blocks and their enzymatic conversion into several cell wall precursor-capped dinucleotides. We employed these capped dinucleotides in T7 RNA polymerase-catalyzed in vitro transcription reactions to efficiently generate RNAs capped with uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), its N-azidoacetyl derivative UDP-GlcNAz, and various cell wall precursors. We furthermore identified four enzymes capable of processing UDP-GlcNAc-capped RNA in vitro: MurA, MurBand MurC from Escherichia coli can sequentially modify the sugar-cap structure and were used to introduce a bioorthogonal, clickable moiety, and the human Nudix hydrolase Nudt5 was shown to efficiently decap UDP-GlcNAc-RNA. Our findings underscore the importance of efficient synthetic methods for capped model RNAs. Additionally, we provide useful enzymatic tools that could be utilized in the development and application of UDP-GlcNAc capture and sequencing protocols. Such protocols are essential for deepening our understanding of the widespread yet enigmatic GlcNAc modification of RNA and its physiological significance.

A new long-read mitochondrial-genome protocol (PacBio HiFi) for haemosporidian parasites: a tool for population and biodiversity studies

BackgroundStudies on haemosporidian diversity, including origin of human malaria parasites, malaria's zoonotic dynamic, and regional biodiversity patterns, have used target gene approaches. However, current methods have a trade-off between scalability and data quality. Here, a long-read Next-Generation Sequencing protocol using PacBio HiFi is presented. The data processing is supported by a pipeline that uses machine-learning for analysing the reads.MethodsA set of primers was designed to target approximately 6 kb, almost the entire length of the haemosporidian mitochondrial genome. Amplicons from different samples were multiplexed in an SMRTbell® library preparation. A pipeline (HmtG-PacBio Pipeline) to process the reads is also provided; it integrates multiple sequence alignments, a machine-learning algorithm that uses modified variational autoencoders, and a clustering method to identify the mitochondrial haplotypes/species in a sample. Although 192 specimens could be studied simultaneously, a pilot experiment with 15 specimens is presented, including in silico experiments where multiple data combinations were tested.ResultsThe primers amplified various haemosporidian parasite genomes and yielded high-quality mt genome sequences. This new protocol allowed the detection and characterization of mixed infections and co-infections in the samples. The machine-learning approach converged into reproducible haplotypes with a low error rate, averaging 0.2% per read (minimum of 0.03% and maximum of 0.46%). The minimum recommended coverage per haplotype is 30X based on the detected error rates. The pipeline facilitates inspecting the data, including a local blast against a file of provided mitochondrial sequences that the researcher can customize.ConclusionsThis is not a diagnostic approach but a high-throughput method to study haemosporidian sequence assemblages and perform genotyping by targeting the mitochondrial genome. Accordingly, the methodology allowed for examining specimens with multiple infections and co-infections of different haemosporidian parasites. The pipeline enables data quality assessment and comparison of the haplotypes obtained to those from previous studies. Although a single locus approach, whole mitochondrial data provide high-quality information to characterize species pools of haemosporidian parasites.

Long-Read Nanopore-Based Sequencing of Anelloviruses.

Routinely used metagenomic next-generation sequencing (mNGS) techniques often fail to detect low-level viremia (<104 copies/mL) and appear biased towards viruses with linear genomes. These limitations hinder the capacity to comprehensively characterize viral infections, such as those attributed to the Anelloviridae family. These near ubiquitous non-pathogenic components of the human virome have circular single-stranded DNA genomes that vary in size from 2.0 to 3.9 kb and exhibit high genetic diversity. Hence, species identification using short reads can be challenging. Here, we introduce a rolling circle amplification (RCA)-based metagenomic sequencing protocol tailored for circular single-stranded DNA genomes, utilizing the long-read Oxford Nanopore platform. The approach was assessed by sequencing anelloviruses in plasma drawn from people who inject drugs (PWID) in two geographically distinct cohorts. We detail the methodological adjustments implemented to overcome difficulties inherent in sequencing circular genomes and describe a computational pipeline focused on anellovirus detection. We assessed our protocol across various sample dilutions and successfully differentiated anellovirus sequences in conditions simulating mixed infections. This method provides a robust framework for the comprehensive characterization of circular viruses within the human virome using the Oxford Nanopore.

Changes in intrahost genetic diversity according to lesion severity in longitudinal HPV16 samples.

Human papillomavirus type 16 (HPV16) is the most common cause of cervical cancer, but most infections are transient with lesions not progressing to cancer. There is a lack of specific biomarkers for early cancer risk stratification. This study aimed to explore the intrahost HPV16 genomic variation in longitudinal samples from HPV16-infected women with different cervical lesion severity (normal, low-grade, and high-grade). The TaME-seq deep sequencing protocol was used to generate whole genome HPV16 sequences of 102 samples collected over time from 40 individuals. Single nucleotide variants (SNVs) and intrahost SNVs (iSNVs) were identified in the viral genomes. A majority of individuals had a unique set of SNVs and these SNVs were stable over time. Overall, the number of iSNVs and APOBEC3-induced iSNVs were significantly lower in high-grade relative to normal and low-grade samples. A significant increase in the number of APOBEC3-induced iSNVs over time was observed for normal samples when compared to high-grade. Our results indicates that the lower incidence of iSNVs and APOBEC3-induced iSNVs in high-grade lesions may have implications for novel biomarkers discoveries, potentially aiding early stratification of HPV-induced cervical precancerous lesions.

DengueSeq: a pan-serotype whole genome amplicon sequencing protocol for dengue virus

BackgroundThe increasing burden of dengue virus on public health due to more explosive and frequent outbreaks highlights the need for improved surveillance and control. Genomic surveillance of dengue virus not only provides important insights into the emergence and spread of genetically diverse serotypes and genotypes, but it is also critical to monitor the effectiveness of newly implemented control strategies. Here, we present DengueSeq, an amplicon sequencing protocol, which enables whole-genome sequencing of all four dengue virus serotypes.ResultsWe developed primer schemes for the four dengue virus serotypes, which can be combined into a pan-serotype approach. We validated both approaches using genetically diverse virus stocks and clinical specimens that contained a range of virus copies. High genome coverage (>95%) was achieved for all genotypes, except DENV2 (genotype VI) and DENV 4 (genotype IV) sylvatics, with similar performance of the serotype-specific and pan-serotype approaches. The limit of detection to reach 70% coverage was 10-100 RNA copies/μL for all four serotypes, which is similar to other commonly used primer schemes. DengueSeq facilitates the sequencing of samples without known serotypes, allows the detection of multiple serotypes in the same sample, and can be used with a variety of library prep kits and sequencing instruments.ConclusionsDengueSeq was systematically evaluated with virus stocks and clinical specimens spanning the genetic diversity within each of the four dengue virus serotypes. The primer schemes can be plugged into existing amplicon sequencing workflows to facilitate the global need for expanded dengue virus genomic surveillance.

A simple sequencing protocol for genotyping the &lt;i&gt;HLA-C&lt;/i&gt;locus by the Sanger method

The HLA-C gene, which belongs to the HLA superfamily (Human Leukocyte Antigen), codes for the Major Histocompatibility Complex (MHC), which plays crucial roles in the human immune system. This study aimed to develop a simple sequencing protocol by the Sanger method using fewer primers and reactions for genotyping the HLA-C gene than published protocols. The simple protocol with three primers includes one PCR reaction and two sequencing reactions. The primer set comprising SEQ ID1 and SEQ ID2 was used for the PCR reaction to specifically amplify the exon 2 – exon 3 region of the HLA-C locus, which contains the typical SNPs of each HLA-C allele. The PCR product was purified and used as a template for the sequencing reactions. Two forward primers, SEQ ID1 and SEQ ID3 were used for sequencing, in which, the SEQ ID1 forward primer is located in the intron 1 region and the SEQ ID3 forward primer is located in the intron 2 region of the HLA-C gene. Testing the simple sequencing protocol on four samples of known HLA-C genotypes showed 100% accurate results. The established Sanger sequencing protocol is simple to implement, and reduces cost and time. Thus, this protocol can be used for HLA-C sequencing for pharmacogenetic studies and applications.

Combination of topical silymarin 0.7%, dual toning and chemical peels (SeQuenCe protocol) for lichen planus pigmentosus in Indian skin

Combination of topical silymarin 0.7%, dual toning and chemical peels (SeQuenCe protocol) for lichen planus pigmentosus in Indian skin

Lessons learned: overcoming common challenges in reconstructing the SARS-CoV-2 genome from short-read sequencing data via CoVpipe2

Background Accurate genome sequences form the basis for genomic surveillance programs, the added value of which was impressively demonstrated during the COVID-19 pandemic by tracing transmission chains, discovering new viral lineages and mutations, and assessing them for infectiousness and resistance to available treatments. Amplicon strategies employing Illumina sequencing have become widely established for variant detection and reference-based reconstruction of SARS-CoV-2 genomes, and are routine bioinformatics tasks. Yet, specific challenges arise when analyzing amplicon data, for example, when crucial and even lineage-determining mutations occur near primer sites. Methods We present CoVpipe2, a bioinformatics workflow developed at the Public Health Institute of Germany to reconstruct SARS-CoV-2 genomes based on short-read sequencing data accurately. The decisive factor here is the reliable, accurate, and rapid reconstruction of genomes, considering the specifics of the used sequencing protocol. Besides fundamental tasks like quality control, mapping, variant calling, and consensus generation, we also implemented additional features to ease the detection of mixed samples and recombinants. Results We highlight common pitfalls in primer clipping, detecting heterozygote variants, and dealing with low-coverage regions and deletions. We introduce CoVpipe2 to address the above challenges and have compared and successfully validated the pipeline against selected publicly available benchmark datasets. CoVpipe2 features high usability, reproducibility, and a modular design that specifically addresses the characteristics of short-read amplicon protocols but can also be used for whole-genome short-read sequencing data. Conclusions CoVpipe2 has seen multiple improvement cycles and is continuously maintained alongside frequently updated primer schemes and new developments in the scientific community. Our pipeline is easy to set up and use and can serve as a blueprint for other pathogens in the future due to its flexibility and modularity, providing a long-term perspective for continuous support. CoVpipe2 is written in Nextflow and is freely accessible from \href{https://github.com/rki-mf1/CoVpipe2}{github.com/rki-mf1/CoVpipe2} under the GPL3 license.

Antimalarial drug resistance profiling of Plasmodium falciparum infections in India using Ion Torrent deep sequencing

IntroductionTracking the emergence and spread of antimalarial drug resistance is critical for supporting progress towards the control and eventual elimination of malaria in South Asia, especially India. Plasmodium falciparum has evolved resistance to virtually every antimalarial drug, and significant progress has been made to identify the molecular genetic mechanisms involved in the most common types of resistance.MethodsAn amplicon sequencing protocol was used for molecular surveillance of antimalarial drug resistance in a total of 158 patient isolates collected from December 2012 to September 2015 from three sites in south, west and east India: Tamil Nadu, Gujarat, and Odisha respectively. Five full length Plasmodium falciparum genes whose mutant proteins are implicated in antimalarial drug resistance were investigated: Pfcrt for chloroquine, Pfdhfr for pyrimethamine, Pfdhps for sulfadoxine, Pfk13 for artemisinin and Pfmdr1 for resistance to multiple antimalarial drugs.ResultsWe observed a high proportion of wild-type Pfcrt and Pfdhfr haplotypes from the P. falciparum-dominant site Rourkela, while mutant Pfcrt and Pfdhfr haplotypes were fixed at the P. vivax dominant sites Chennai and Nadiad. The wild-type Pfdhps haplotype was predominant across all study sites. We identified mutations in the propeller domain of Pfk13, although they are not associated with resistance to artemisinin. Finally, using samples taken from the same patient on day 2, day 7, and day 14 after artemisinin combination treatment, we were able to observe changes in allele frequency of drug resistance genes during the course of an infection.DiscussionThis is the first high-throughput deep sequencing study of five full-length P. falciparum genes in clinical isolates from three different study sites in India with varying transmission ecologies. Amplicon sequencing based on ion-torrent has the potential to track changes in the frequency of drug resistant alleles as a patient is undergoing drug therapy, and to identify new resistance mutations as they increase in frequency in the patient. This study showed possibility of whole gene sequencing, leads to in-depth molecular surveillance of multiple antimalarial resistant candidates and furthermore suggests investigations on reversal of resistant genotypes due to usage of artemisinin combination therapy in P. falciparum prevalent areas of the country.

Measurement of Implant Insertion Torque in Intentionally Undersized Osteotomy Site Preparation in the Posterior Maxilla: A Prospective Clinical Study.

Achieving adequate primary stability for dental implants in the posterior maxilla is challenging due to the area's low bone density and fine trabecular bone. Low primary implant stability can compromise the long-term success of the implant. This study aims to evaluate the effect of intentionally undersized osteotomy sites on the insertion torque of implants placed in type 4 bone density in the posterior maxilla, regardless of the surgeon's tactile sense of the bone density. The conventional implant placement technique was performed in the posterior maxilla by intentionally omitting the last drill of the recommended drilling sequence protocol. The insertion torque was measured using an integrated surgical motor software. The study included 72 implants in patients of both genders and different age groups. The retrieved samples were divided into four groups, with insertion torque ranging from 11 Ncm to 48 Ncm. The mean insertion torque was 26.51 ± 0.18 Ncm for all cases (P = .001). No significant difference was found in the mean insertion torque between different age groups (26.51 ± 2.845, P = .84). However, a significant difference was observed between male and female cases (P = .84). Most implant insertion torque measurements vary significantly between cases in the intentionally underprepared posterior maxillary osteotomy site. The last drill should be omitted for all preparations of the posterior maxillary implant sites. Intentionally minimizing the osteotomy site is recommended for all posterior maxillary implant cases.

Procrustes is a machine-learning approach that removes cross-platform batch effects from clinical RNA sequencing data

With the increased use of gene expression profiling for personalized oncology, optimized RNA sequencing (RNA-seq) protocols and algorithms are necessary to provide comparable expression measurements between exome capture (EC)-based and poly-A RNA-seq. Here, we developed and optimized an EC-based protocol for processing formalin-fixed, paraffin-embedded samples and a machine-learning algorithm, Procrustes, to overcome batch effects across RNA-seq data obtained using different sample preparation protocols like EC-based or poly-A RNA-seq protocols. Applying Procrustes to samples processed using EC and poly-A RNA-seq protocols showed the expression of 61% of genes (N = 20,062) to correlate across both protocols (concordance correlation coefficient > 0.8, versus 26% before transformation by Procrustes), including 84% of cancer-specific and cancer microenvironment-related genes (versus 36% before applying Procrustes; N = 1,438). Benchmarking analyses also showed Procrustes to outperform other batch correction methods. Finally, we showed that Procrustes can project RNA-seq data for a single sample to a larger cohort of RNA-seq data. Future application of Procrustes will enable direct gene expression analysis for single tumor samples to support gene expression-based treatment decisions.

Time-Saving 3D MR Imaging Protocols with Millimeter and Submillimeter Isotropic Spatial Resolution for Face and Neck Imaging as Implemented at a Single-Site Major Referral Center.

MR imaging has become the routine technique for staging nasopharyngeal carcinoma, evaluating perineural tumor spread, and detecting cartilage invasion in laryngeal carcinoma. However, these protocols traditionally require in the range of 25 to 35 minutes of acquisition time. 3D sequences offer the potential advantage of time savings through the acquisition of 1-mm or submillimeter resolution isotropic data followed by multiplanar reformats that require no further imaging time. We have iteratively optimized vendor product 3D T1-weighted MR imaging sequences for morphologic face and neck imaging, reducing the average acquisition time of our 3T protocols by 9 minutes 57 seconds (40.9%) and of our 1.5T protocols by 9 minutes 5 seconds (37.0%), while simultaneously maintaining or improving spatial resolution. This clinical report describes our experience optimizing and implementing commercially available 3D T1-weighted MR imaging pulse sequence protocols for clinical face and neck MR imaging examinations using illustrative cases. We provide protocol details to allow others to replicate our implementations, and we report challenges we faced along with our solutions.

Temporal analysis and comparative genomic study of SARS-CoV-2 variants across Uttar Pradesh, India: Insights from the first and second COVID-19 waves

The rapid emergence of genetic variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) after late 2020, lead to origin of the term “variant of concern” and became the prime issue for the health of the human populations across the globe. Genome surveillance provides the information about the functional consequences of the variants and their divergence. Presently, we report and compare the nucleotide sequences of the isolated SARS-CoV-2 genomes at two different time-frame i.e. first wave (June- September, 2020) and second wave (April-May, 2021) from Lucknow, Uttar Pradesh, India. We employed the Oxford Nanopore MINION system and ARTIC protocol for sequencing, achieving >90x genome coverage in approximately 90% of samples. Increased mutation density in viral spike proteins were the cause of higher transmission and infectivity of second wave. The second wave exhibited a higher mutation density in viral spike proteins, likely contributing to increased transmission and infectivity. Notable spike mutations included receptor-binding domain (RBD) mutations (L452R, T478K, D614G), N-terminal domain (NTD) mutations (T17R, G142D, Δ156–157, A222V), and furin cleavage site (FCS) mutations (P681R, D950N). The functional consequences of common and unique variants among the first and second wave were evaluated by Sorting Intolerant from Tolerant (SIFT) and Protein Variant Effect Analyser (PROVEAN). Furthermore, we extensively investigated phylogenetic clustering of the genome sequences by Nextstrain and identified the contrasting divergence between first and second wave. Different sub division of delta clade (21I, 21 A, 21 J) and 21 B (kappa) with mutations primarily in spike proteins and nucleocapsid proteins, were exclusive to the second wave. Our study identified changes in spike proteins and nucleocapsid potentially contributing to increased transmission and infectivity during the second wave. Loci specific variant map for different countries across the world suggested the appearance of large number of variants associated with ORF1a during the second wave in India. Overall, the data underscores the dynamic nature of viral evolution during different phases of an outbreak, highlighting the importance of continuous genomic surveillance to track and understand viral mutations and their implications for public health interventions.

Random Amplified Polymorphic DNA (RAPD) Markers Protocol of Bacterial Isolates from Two selected General Hospitals Wastewater (HWW)

In this research work, Random amplified polymorphic DNA (RAPD) markers Protocol was used to characterize bacterial isolates from two selected General Hospitals Waste Water (HWW). The hospital environment and its wastes accumulate diseases from both inward and outward patients. It is pertinent to investigate the wastewater and study its microbial community. Wastewater samples were collected from two major General Hospitals in Akoko area, namely, Ikare and Akungba-Akoko General Hospitals. Samples were microbiologically examined for the presence of bacterial colonies. Isolated bacterial species were preliminarily identified through conventional biochemical tests and were further characterized using 16s rRNA sequencing protocol. The bacterial gene was amplified using selected primers. DNA was isolated, purified, and amplified using 16s rRNA gene sequence protocol, and the results were analyzed and assessed with the standard bacteria sequence in the NCBI database. Molecular evolutionary analyses and Phylogenetic trees were mapped out from the results obtained. it was observed that the Bacterial counts in Cfu/ml range between 10.0 x 103Cfu/ml to 24.0 x 103Cfu/ml. The highest bacterial count was observed from the test sample from Ikare followed by Akungba Akoko General Hospitals with the Cfu/ml value of 24.0 x 103 and 18.0 x103 Cfu/ml respectively. Molecular data sequence, when compared with NCBI database using BLAST showed 99.60 –99.87% bactaria phylogenic identity and E-value equal to 0 for all closely related taxa. The following bacteria were characterized using Random amplified polymorphic DNA (RAPD) markers protocol namely: Streptococcus pneumoniae, Staphylococcus aureus, Bacillus cereus, and Bacillus subtilis. this study has revealed the presence of different bacterial species in hospital waste water which pose threat to public health. Hence, proper monitoring and sewage treatment should be encouraged in different hospital settings before the disposal of these wastewaters to the public water runways

Validation of the Meditech ABPM-06 24-hour blood pressure monitoring system in a pediatric population according to International Organization for Standardization Protocol 81060-2:2018.

Ambulatory blood pressure monitoring (ABPM) devices play a crucial role in diagnosing hypertension, not only in adults but also in pediatric patients. ABPM-06, the latest oscillometric device from Meditech Ltd. (Budapest, Hungary), is the focus of this study. The objective was to validate the ABPM-06 device using the International Organization for Standardization (ISO) 81060-2 : 2018 standard. A total of 86 healthy patients, consisting of 34 males and 52 females, aged between 3 and 17 years, were enrolled in this study. During the ambulatory phase, a total of 50 patients were enrolled, with 35 patients falling within the 3- to 12-year-old age range and 15 patients aged between 12 and 17 years. Additionally, for the dynamic test, 36 patients were selected, comprising of 10 individuals aged 3-12 years and 26 patients aged 12-17 years. These patients were recruited from the outpatient clinics of the Department of Pediatrics at Albert Szent-Györgyi University in Szeged, Hungary. The validation process involved utilizing the same-arm sequence protocol, both in resting positions and during stress testing. The ABPM-06 performed well in both clinical and ambulatory validations. In terms of validation criterion 1, the mean ± SD of the differences between the test device and reference blood pressure readings was -1.3 ± 3.5 mmHg for systolic and -0.1 ± 2.3 mmHg for diastolic, in children under the age of 12 years. For those over the age of 12 years, the mean ± SD of the differences was -2.8 ± 4.6 mmHg for systolic and -0.5 ± 2.7 mmHg for diastolic. Regarding the ambulatory validation, for children under 12 years old, the mean ± SD of the differences was -1.3 ± 3.5 mmHg for systolic and -0.1 ± 2.3 mmHg for diastolic. In the age group above 12 years, the mean ± SD of the differences was -2.8 ± 4.6 mmHg for systolic and -0.5 ± 2.7 mmHg for diastolic. Both tests successfully met the established criteria regarding the mean and SD values of the differences between the device readings and the observed SBP and DBP measurements. The ABPM-06 oscillometric device fully adheres to the ISO 81060-2 : 2018 standard requirements for ABPM determination in the pediatric population (ages 3-17 years). Consequently, this ABPM device proves to be suitable for effectively managing hypertension in children and adolescents.