Protocol For Whole Genome Sequencing Research Articles

A novel isothermal whole genome sequencing approach for Monkeypox Virus

Monkeypox virus (MPXV) is the zoonotic agent responsible for mpox, an often-self-limiting pox-like disease. Since May 2022, an outbreak characterized by increased human-to-human transmission was detected outside the endemic regions. Whole genome sequencing (WGS) has been successfully used to keep track of viral evolution during outbreaks or for surveillance of multiple pathogens of public health significance. Current WGS protocols for MPXV are either based on metagenomic sequencing or tiled-PCR amplification. The latter allows multiplexing due to the efficient enrichment of the viral DNA, however, mutations or the presence of different clades can negatively influence genome coverage yield. Here, we present the establishment of a novel isothermal WGS method for MPXV based on Phi29 DNA polymerase-based multiple displacement amplification (MDA) properties making use of only 6 primers. This approach yielded from 88% up to 100% genome coverage using either alkaline denatured extracted DNA or clinical material as starting material, with the highest coverage generated by clinical material. We demonstrate that this novel isothermal WGS protocol is suitable for monitoring viral evolution during MPXV outbreaks and surveillance in any conventional laboratory setting.

Validated WGS and WES protocols proved saliva-derived gDNA as an equivalent to blood-derived gDNA for clinical and population genomic analyses

BackgroundWhole exome sequencing (WES) and whole genome sequencing (WGS) have become standard methods in human clinical diagnostics as well as in population genomics (POPGEN). Blood-derived genomic DNA (gDNA) is routinely used in the clinical environment. Conversely, many POPGEN studies and commercial tests benefit from easy saliva sampling. Here, we evaluated the quality of variant call sets and the level of genotype concordance of single nucleotide variants (SNVs) and small insertions and deletions (indels) for WES and WGS using paired blood- and saliva-derived gDNA isolates employing genomic reference-based validated protocols.MethodsThe genomic reference standard Coriell NA12878 was repeatedly analyzed using optimized WES and WGS protocols, and data calls were compared with the truth dataset published by the Genome in a Bottle Consortium. gDNA was extracted from the paired blood and saliva samples of 10 participants and processed using the same protocols. A comparison of paired blood–saliva call sets was performed in the context of WGS and WES genomic reference-based technical validation results.ResultsThe quality pattern of called variants obtained from genomic-reference-based technical replicates correlates with data calls of paired blood–saliva-derived samples in all levels of tested examinations despite a higher rate of non-human contamination found in the saliva samples. The F1 score of 10 blood-to-saliva-derived comparisons ranged between 0.8030–0.9998 for SNVs and between 0.8883–0.9991 for small-indels in the case of the WGS protocol, and between 0.8643–0.999 for SNVs and between 0.7781–1.000 for small-indels in the case of the WES protocol.ConclusionSaliva may be considered an equivalent material to blood for genetic analysis for both WGS and WES under strict protocol conditions. The accuracy of sequencing metrics and variant-detection accuracy is not affected by choosing saliva as the gDNA source instead of blood but much more significantly by the genomic context, variant types, and the sequencing technology used.

Identification of SARS-CoV-2 variants in indoor dust.

Environmental surveillance of pathogens underlying infectious disease is critical to ensure public health. Recent efforts to track SARS-CoV-2 have utilized wastewater sampling to infer community trends in viral abundance and variant composition. Indoor dust has also been used for building-level inferences, though to date no sequencing data providing variant-scale resolution have been reported from dust samples, and strategies to monitor circulating variants in dust are needed to help inform public health decisions. In this study, we demonstrate that SARS-CoV-2 lineages can be detected and sequenced from indoor bulk dust samples. We collected 93 vacuum bags from April 2021 to March 2022 from buildings on The Ohio State University's (OSU) Columbus campus, and the dust was used to develop and apply an amplicon-based whole-genome sequencing protocol to identify the variants present and estimate their relative abundances. Three variants of concern were detected in the dust: Alpha, Delta, and Omicron. Alpha was found in our earliest sample in April 2021 with an estimated frequency of 100%. Delta was the primary variant present from October of 2021 to January 2022, with an average estimated frequency of 91% (±1.3%). Omicron became the primary variant in January 2022 and was the dominant strain in circulation through March with an estimated frequency of 87% (±3.2%). The detection of these variants on OSU's campus correlates with the circulation of these variants in the surrounding population (Delta p<0.0001 and Omicron p = 0.02). Overall, these results support the hypothesis that dust can be used to track COVID-19 variants in buildings.

An amplicon-based protocol for whole-genome sequencing of human respiratory syncytial virus subgroup A.

It is convenient to study complete genome sequences of human respiratory syncytial virus (hRSV) for ongoing genomic characterization and identification of highly transmissible or pathogenic variants. Whole genome sequencing of hRSV has been challenging from respiratory tract specimens with low viral loads. Herein, we describe an amplicon-based protocol for whole genome sequencing of hRSV subgroup A validated with 24 isolates from nasopharyngeal swabs and infected cell cultures, which showed cycle threshold (Ct) values ranging from 10 to 31, as determined by quantitative reverse-transcription polymerase chain reaction. MinION nanopore generated 3200 to 5400 reads per sample to sequence over 93% of the hRSV-A genome. Coverage of each contig ranged from 130× to 200×. Samples with Ct values of 20.9, 25.2, 27.1, 27.7, 28.2, 28.8, and 29.6 led to the sequencing of over 99.0% of the virus genome, indicating high genome coverage even at high Ct values. This protocol enables the identification of hRSV subgroup A genotypes, as primers were designed to target highly conserved regions. Consequently, it holds potential for application in molecular epidemiology and surveillance of this hRSV subgroup.

Development and application of a whole genome amplicon sequencing method for infectious salmon anemia virus (ISAV).

Infectious salmon anemia (ISA) is an infectious disease primarily affecting farmed Atlantic salmon, Salmo salar, which is caused by the ISA virus (ISAV). ISAV belongs to the Orthomyxoviridae family. The disease is a serious condition resulting in reduced fish welfare and high mortality. In this study, we designed an amplicon-based sequencing protocol for whole genome sequencing of ISAV. The method consists of 80 ISAV-specific primers that cover 92% of the virus genome and was designed to be used on an Illumina MiSeq platform. The sequencing accuracy was investigated by comparing sequences with previously published Sanger sequences. The sequences obtained were nearly identical to those obtained by Sanger sequencing, thus demonstrating that sequences produced by this amplicon sequencing protocol had an acceptable accuracy. The amplicon-based sequencing method was used to obtain the whole genome sequence of 12 different ISAV isolates from a small local epidemic in the northern part of Norway. Analysis of the whole genome sequences revealed that segment reassortment took place between some of the isolates and could identify which segments that had been reassorted.

Feasibility and clinical utility of local rapid Nanopore influenza A virus whole genome sequencing for integrated outbreak management, genotypic resistance detection and timely surveillance.

Rapid respiratory viral whole genome sequencing (WGS) in a clinical setting can inform real-time outbreak and patient treatment decisions, but the feasibility and clinical utility of influenza A virus (IAV) WGS using Nanopore technology has not been demonstrated. A 24 h turnaround Nanopore IAV WGS protocol was performed on 128 reverse transcriptase PCR IAV-positive nasopharyngeal samples taken over seven weeks of the 2022-2023 winter influenza season, including 25 from patients with nosocomial IAV infections and 102 from patients attending the Emergency Department. WGS results were reviewed collectively alongside clinical details for interpretation and reported to clinical teams. All eight segments of the IAV genome were recovered for 97/128 samples (75.8 %) and the haemagglutinin gene for 117/128 samples (91.4 %). Infection prevention and control identified nosocomial IAV infections in 19 patients across five wards. IAV WGS revealed two separate clusters on one ward and excluded transmission across different wards with contemporaneous outbreaks. IAV WGS also identified neuraminidase inhibitor resistance in a persistently infected patient and excluded avian influenza in a sample taken from an immunosuppressed patient with a history of travel to Singapore which had failed PCR subtyping. Accurate IAV genomes can be generated in 24 h using a Nanopore protocol accessible to any laboratory with SARS-CoV-2 Nanopore sequencing capacity. In addition to replicating reference laboratory surveillance results, IAV WGS can identify antiviral resistance and exclude avian influenza. IAV WGS also informs management of nosocomial outbreaks, though molecular and clinical epidemiology were concordant in this study, limiting the impact on decision-making.

Metagenomic sequencing, molecular characterization, and Bayesian phylogenetics of imported type 2 vaccine-derived poliovirus, Spain, 2021.

In 2021, a type 2 vaccine-derived poliovirus (VDPV2) was isolated from the stool of a patient with acute flaccid paralysis (AFP) admitted to Spain from Senegal. A virological investigation was conducted to characterize and trace the origin of VDPV2. We used an unbiased metagenomic approach for the whole-genome sequencing of VDPV2 from the stool (pre-treated with chloroform) and from the poliovirus-positive supernatant. Phylogenetic analyses and molecular epidemiological analyses relying on the Bayesian Markov Chain Monte Carlo methodology were used to determine the geographical origin and estimate the date of the initiating dose of the oral poliovirus vaccine for the imported VDPV2. We obtained a high percentage of viral reads per total reads mapped to the poliovirus genome (69.5% for pre-treated stool and 75.8% for isolate) with a great depth of sequencing coverage (5,931 and 11,581, respectively) and complete genome coverage (100%). The two key attenuating mutations in the Sabin 2 strain had reverted (A481G in the 5'UTR and Ile143Thr in VP1). In addition, the genome had a recombinant structure between type-2 poliovirus and an unidentified non-polio enterovirus-C (NPEV-C) strain with a crossover point in the protease-2A genomic region. VP1 phylogenetic analysis revealed that this strain is closely related to VDPV2 strains circulating in Senegal in 2021. According to Bayesian phylogenetics, the most recent common ancestor of the imported VDPV2 could date back 2.6 years (95% HPD: 1.7-3.7) in Senegal. We suggest that all VDPV2s circulating in 2020-21 in Senegal, Guinea, Gambia, and Mauritania have an ancestral origin in Senegal estimated around 2015. All 50 stool samples from healthy case contacts collected in Spain (n = 25) and Senegal (n = 25) and four wastewater samples collected in Spain were poliovirus negative. By using a whole-genome sequencing protocol with unbiased metagenomics from the clinical sample and viral isolate with high sequence coverage, efficiency, and throughput, we confirmed the classification of VDPV as a circulating type. The close genomic linkage with strains from Senegal was consistent with their classification as imported. Given the scarce number of complete genome sequences for NPEV-C in public databases, this protocol could help expand poliovirus and NPEV-C sequencing capacity worldwide.

WHOLE-GENOME SINGLE MOLECULE REAL-TIME SEQUENCING OF SARS-CoV-2 OMICRON.

New variants and genetic mutations of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome can only be identified using accurate sequencing methods. Single molecule real-time (SMRT) sequencing has been used to characterize Alpha and Delta variants, but not Omicron variants harbouring numerous mutations in the SARS-CoV-2 genome. This study assesses the performance of a target capture SMRT sequencing protocol for whole genome sequencing (WGS) of SARS-CoV-2 Omicron variants and compared it to that of an amplicon SMRT sequencing protocol optimized for Omicron variants. The failure rate of the target capture protocol (6%) was lower than that of the amplicon protocol (34%, p<0.001) on our dataset, and the median genome coverage with the target capture protocol (98.6% [IQR: 86-99.4]) was greater than that with the amplicon protocol (76.6% [IQR: 66-89.6], (p<0.001)). The percentages of samples with >95% whole genome coverage were 64% with the target capture protocol and 19% with the amplicon protocol (p<0.05). The clades of 96 samples determined with both protocols were 93% concordant and the lineages of 59 samples were 100% concordant. Thus, target capture SMRT sequencing appears to be an efficient method for WGS, genotyping and detecting mutations of SARS-CoV-2 Omicron variants. This article is protected by copyright. All rights reserved.

WHOLE GENOME SEQUENCING AND GENOTYPING PROTOCOL OF RABIES VIRUS ISOLATES

This study describes the developed protocol for whole genome sequencing of clinical Rabies virus RNA samples on the Illumina MiSeq platform. Previously, genotyping of the rabies virus circulating in Kazakhstan was based on the nucleoprotein gene sequence (N gene). The protocol describes the methods of sample preparation, whole genome sequencing and genotyping currently used for epidemiological analysis not only of Rabies virus, but also for other viral pathogens such as SARS-CoV-2, Influenza and others. The aim of the work was to develop and test a protocol for whole genome sequencing and genetyping of rabies virus isolates for further use in epidemiological monitoring. As a result of the research, whole genome sequences of 31 isolates of Rabies virus isolated from various infected animals and regions of Kazakhstan were obtained for the first time. All the isolates belong to the major steppe-type clade – Cosmopolitan (30 isolates belong to the minor clade CA1, while 1 isolate belongs to an unassigned minor clade). 2 local outbreaks and 6 separated rabies virus lineages were identified. Estimated dates of divergence from a common ancestor were calculated for both the Cosmopolitan clade and the minor clade CA1.

Development of multiplex PCR based NGS protocol for whole genome sequencing of West Nile virus lineage 2 directly from biological samples using Oxford Nanopore platform

West Nile virus (WNV) can affect humans, birds, horses and another mammals, causing asymptomatic infection, mild febrile disease, neurological and systematic disease and death. In order to gain insight into the prevalence of WNV, a monitoring program has been established in the Republic of Serbia. Whole genome sequencing is essential for the molecular epizootiological analysis of virus entry and transmission routes, especially in high-risk regions. This paper describes the development of a multiplex PCR based NGS protocol for whole genome sequencing of WNV lineage 2 directly from biological samples using Oxford Nanopore (ONT) platform. The results obtained using this platform, confirmed by Sanger sequencing, indicate that this protocol can be applied to obtain whole sequences of the WNV genome, even when the virus concentration in the sample is medium, Ct value is approximately 30. The use of this protocol does not require prior virus isolation on cell culture nor the depletion of host nucleic acids.

DEVELOPMENT OF A PROTOCOL FOR WHOLE GENOME SEQUENCING OF THE SARS-COV-2 VIRUS

Whole-genome sequencing of the SARS-CoV-2 virus during the pandemic has become&nbsp;an essential&nbsp;part of the epidemiological control of the spread of coronavirus infection.&nbsp;This made it possible to&nbsp;get&nbsp;actual&nbsp;data&nbsp;of&nbsp;circulating genetic variants and mutational changes&nbsp;of&nbsp;SARS-CoV-2 during the pandemic. The obtained results provided an opportunity for researchers to&nbsp;collect&nbsp;additional data&nbsp;to evaluate the&nbsp;virulence, infectivity, and the likelihood of evading an immune response when using vaccines and therapeutic monoclonal antibodies. There are different technological approaches for whole-genome sequencing of SARS-CoV-2, but multiplex PCR amplification is used most often due to ease of implementation and cost-effectiveness. When performing research on viral genome sequencing, researchers need to optimize existing sequencing protocols&nbsp;with&nbsp;available reagents or develop new approaches. This study&nbsp;is&nbsp;devoted to the development of a protocol for whole-genome sequencing of the SARS-CoV-2 virus based on the use of RT-PCR amplification using 39 primer pairs in 3 reaction mixtures. The protocol made it possible to obtain 15 complete genome&nbsp;data of the SARS-CoV-2 and can&nbsp;probably&nbsp;be used for large-scale studies on viral genome sequencing.

From Clinical Specimen to Whole Genome Sequencing of A(H3N2) Influenza Viruses: A Fast and Reliable High-Throughput Protocol.

(1) Background: Over the last few years, there has been growing interest in the whole genome sequencing (WGS) of rapidly mutating pathogens, such as influenza viruses (IVs), which has led us to carry out in-depth studies on viral evolution in both research and diagnostic settings. We aimed at describing and determining the validity of a WGS protocol that can obtain the complete genome sequence of A(H3N2) IVs directly from clinical specimens. (2) Methods: RNA was extracted from 80 A(H3N2)-positive respiratory specimens. A one-step RT-PCR assay, based on the use of a single set of specific primers, was used to retro-transcribe and amplify the entire IV type A genome in a single reaction, thus avoiding additional enrichment approaches and host genome removal treatments. Purified DNA was quantified; genomic libraries were prepared and sequenced by using Illumina MiSeq platform. The obtained reads were evaluated for sequence quality and read-pair length. (3) Results: All of the study specimens were successfully amplified, and the purified DNA concentration proved to be suitable for NGS (at least 0.2 ng/µL). An acceptable coverage depth for all eight genes of influenza A(H3N2) virus was obtained for 90% (72/80) of the clinical samples with viral loads >105 genome copies/mL. The mean depth of sequencing ranged from 105 to 200 reads per position, with the majority of the mean depth values being above 103 reads per position. The total turnaround time per set of 20 samples was four working days, including sequence analysis. (4) Conclusions: This fast and reliable high-throughput sequencing protocol should be used for influenza surveillance and outbreak investigation.

The Brazilian Rare Genomes Project: Validation of Whole Genome Sequencing for Rare Diseases Diagnosis.

Rare diseases affect up to 13.2 million individuals in Brazil. The Brazilian Rare Genomes Project is envisioned to further the implementation of genomic medicine into the Brazilian public healthcare system. Here we report the validation results of a whole genome sequencing (WGS) procedure for implementation in clinical laboratories. In addition, we report data quality for the first 1,200 real-world patients sequenced. We sequenced a well-characterized group of 76 samples, including seven gold standard genomes, using a PCR-free WGS protocol on Illumina Novaseq 6,000 equipment. We compared the observed variant calls with their expected calls, observing good concordance for single nucleotide variants (SNVs; mean F-measure = 99.82%) and indels (mean F-measure = 99.57%). Copy number variants and structural variants events detection performances were as expected (F-measures 96.6% and 90.3%, respectively). Our WGS protocol presented excellent intra-assay reproducibility (coefficients of variation ranging between 0.03% and 0.20%) and inter-assay reproducibility (coefficients of variation ranging between 0.02% and 0.09%). Limitations of the WGS protocol include the inability to confidently detect variants such as uniparental disomy, balanced translocations, repeat expansion variants, and low-level mosaicism. In summary, the observed performance of the WGS protocol was in accordance with that seen in the best centers worldwide. The Rare Genomes Project is an important initiative to bring pivotal improvements to the quality of life of the affected individuals.

A robust, cost-effective and widely applicable whole-genome sequencing protocol for capripoxviruses

The diseases caused by capripoxviruses (CaPVs) are of major economic concern in sheep, goat and cattle as they are inexorably spreading into non-endemic regions. As CaPV strains are serologically indistinguishable and genetically highly homologous, typing closely related strains can only be achieved by whole genome sequencing. Unfortunately the number of publicly available genomes remains low as most sequencing methods rely on virus isolation. Therefore, we developed a robust, cost-effective and widely applicable method that allows to generate (nearly) complete CaPV genomes directly from clinical samples or commercial vaccine batches. A set of pan-CaPVs long-range PCRs spanning the entire genome was designed to generate PCR amplicons that can be sequenced on commonly used high-throughput sequencing platforms: MiSeq (Illumina), RSII (PacBio) and MinION (Oxford Nanopore Technologies). The robustness of the LR-PCR strategy was evaluated for all 3 members of CaPV directly from a variety of samples, including clinical samples (N = 7), vaccine batches (N = 6), and virus isolates (N = 2). The sequencing method described here allows to reconstruct (nearly) complete CaPV genomes in less than a week and will aid researchers studying closely-related CaPV strains worldwide.

Optimisation of methodology for whole genome sequencing of Measles Virus directly from patient specimens

In an era of decreasing genetic diversity of Measles Virus (MeV), effective surveillance requires a higher-resolution genotyping method or whole genome sequencing (WGS) to document elimination. Through optimization of MeV WGS protocol, we developed a MeV-specific probe enrichment method that allows next generation sequencing from clinical specimens. With the probe enrichment method, 70% of specimens can be sequenced at a read depth of greater than 10 reads with minimal off-target sequences.

A Case of Whole Genome Analysis of SARS-CoV-2 Using Oxford Nanopore MinION System

The application of whole genome sequencing on the SARS-CoV-2 viral genome is essential for our understanding of the molecular epidemiology and spread of viruses in the community. The portable whole genome sequencer MinION (Oxford Nanopore Technologies, ONT, UK) could be feasibly used in a clinical microbiology laboratory without the need for vast resources or stringent operating conditions. We used the MinION sequencer to analyze the viral genome sequence of one SARS-CoV-2 strain. In June 2020, a nasopharyngeal specimen from one patient was subjected to whole-genome analysis using the nCoV-2019 sequencing protocol ARTIC V2 using the MinION sequencer. The ONT MinKNOW software, RAMPART tool, and Genome Workbench were used. We identified 11 nucleotide variants using the Wuhan-Hu-1 isolate (NC_045512.2) as the reference sequence. There were six nucleotide variants (T265I,F924, Y3884L, P4715L, L5462, and Q6804L) in the ORF1ab region, one variant (D614G) in the S gene, one variant (Q57H) in ORF3a, one variant (P302) in the N gene, and two variants in each the 5’-UTR and 3’-UTR. In this prolonged coronavirus disease 2019 (COVID-19) pandemic season, the MinION system that operates an amplicon-based whole-genome sequencing protocol could be a rapid and reliable sequencer without the need for cumbersome viral cultivation.

Whole-genome sequencing of SARS-CoV-2 in Uganda: implementation of the low-cost ARTIC protocol in resource-limited settings

Background: In January 2020, a previously unknown coronavirus strain was identified as the cause of a severe acute respiratory syndrome (SARS-CoV-2). The first viral whole-genome was sequenced using high-throughput sequencing from a sample collected in Wuhan, China. Whole-genome sequencing (WGS) is imperative in investigating disease outbreak transmission dynamics and guiding decision-making in public health.Methods: We retrieved archived SARS-CoV-2 samples at the Integrated Biorepository of H3Africa Uganda, Makerere University (IBRH3AU). These samples were collected previously from individuals diagnosed with coronavirus disease 2019 (COVID-19) using real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR). 30 samples with cycle thresholds (Cts) values <25 were selected for WGS using SARS-CoV-2 ARTIC protocol at Makerere University Molecular Diagnostics Laboratory.Results: 28 out of 30 (93.3%) samples generated analyzable genomic sequence reads. We detected SARS-CoV-2 and lineages A (22/28) and B (6/28) from the samples. We further show phylogenetic relatedness of these isolates alongside other 328 Uganda (lineage A = 222, lineage B = 106) SARS-CoV-2 genomes available in GISAID by April 22, 2021 and submitted by the Uganda Virus Research Institute.Conclusions: Our study demonstrated adoption and optimization of the low-cost ARTIC SARS-CoV-2 WGS protocol in a resource limited laboratory setting. This work has set a foundation to enable rapid expansion of SARS-CoV-2 WGS in Uganda as part of the Presidential Scientific Initiative on Epidemics (PRESIDE) CoV-bank project and IBRH3AU.

A benchmarking study of SARS-CoV-2 whole-genome sequencing protocols using COVID-19 patient samples

SummarySevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging new type of coronavirus that is responsible for the COVID-19 pandemic and the unprecedented global health emergency. Whole-genome sequencing (WGS) of SARS-CoV-2 plays a critical role in understanding the disease. Performance variation exists across SARS-CoV-2 viral WGS technologies, but there is currently no benchmarking study comparing different WGS sequencing protocols. We compared seven different SARS-CoV-2 WGS library protocols using RNA from patient nasopharyngeal swab samples under two storage conditions with low and high viral inputs. We found large differences in mappability and genome coverage, and variations in sensitivity, reproducibility, and precision of single-nucleotide variant calling across different protocols. For certain amplicon-based protocols, an appropriate primer trimming step is critical for accurate single-nucleotide variant calling. We ranked the performance of protocols based on six different metrics. Our findings offer guidance in choosing appropriate WGS protocols to characterize SARS-CoV-2 and its evolution.

Whole-genome sequencing of SARS-CoV-2 in Uganda: implementation of the low-cost ARTIC protocol in resource-limited settings.

Background: In January 2020, a previously unknown coronavirus strain was identified as the cause of a severe acute respiratory syndrome (SARS-CoV-2). The first viral whole-genome was sequenced using high-throughput sequencing from a sample collected in Wuhan, China. Whole-genome sequencing (WGS) is imperative in investigating disease outbreak transmission dynamics and guiding decision-making in public health. Methods: We retrieved archived SARS-CoV-2 samples at the Integrated Biorepository of H3Africa Uganda, Makerere University (IBRH3AU). These samples were collected previously from individuals diagnosed with coronavirus disease 2019 (COVID-19) using real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR). 30 samples with cycle thresholds(Cts) values <25 were selected for WGS using SARS-CoV-2 ARTIC protocol at Makerere University Molecular Diagnostics Laboratory. Results: 28 out of 30 (93.3%) samples generated analyzable genomic sequence reads. We detected SARS-CoV-2 and lineages A (22/28) and B (6/28) from the samples. We further show phylogenetic relatedness of these isolates alongside other 328 Uganda (lineage A = 222, lineage B = 106) SARS-CoV-2 genomes available in GISAID by April 22, 2021 and submitted by the Uganda Virus Research Institute. Conclusions: Our study demonstrated adoption and optimization of the low-cost ARTIC SARS-CoV-2 WGS protocol in a resource limited laboratory setting. This work has set a foundation to enable rapid expansion of SARS-CoV-2 WGS in Uganda as part of the Presidential Scientific Initiative on Epidemics (PRESIDE) CoV-bank project and IBRH3AU.

A Half-Day Genome Sequencing Protocol for Middle East Respiratory Syndrome Coronavirus.

Recent coronavirus (CoV) outbreaks, including that of Middle East respiratory syndrome (MERS), have presented a threat to public health worldwide. A primary concern in these outbreaks is the extent of mutations in the CoV, and the content of viral variation that can be determined only by whole genome sequencing (WGS). We aimed to develop a time efficient WGS protocol, using universal primers spanning the entire MERS-CoV genome. MERS and synthetic Neoromicia capensis bat CoV genomes were successfully amplified using our developed PCR primer set and sequenced with MinION. All experimental and analytical processes took 6 h to complete and were also applied to synthetic animal serum samples, wherein the MERS-CoV genome sequence was completely recovered. Results showed that the complete genome of MERS-CoV and related variants could be directly obtained from clinical samples within half a day. Consequently, this method will contribute to rapid MERS diagnosis, particularly in future CoV epidemics.