A Rapid, Whole Genome Sequencing Assay for Detection and Characterization of Novel Coronavirus (SARS-CoV-2) Clinical Specimens Using Nanopore Sequencing.

A new human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged at the end of 2019 in Wuhan, China that caused a range of disease severities; including fever, shortness of breath, and coughing. This disease, now known as coronavirus disease 2019 (COVID-19), quickly spread throughout the world, and was declared a pandemic by the World Health Organization in March of 2020. As the disease continues to spread, providing rapid characterization has proven crucial to better inform the design and execution of control measures, such as decontamination methods, diagnostic tests, antiviral drugs, and prophylactic vaccines for long-term control. Our work at the United States Army's Combat Capabilities Development Command Chemical Biological Center (DEVCOM CBC) is focused on engineering workflows to efficiently identify, characterize, and evaluate the threat level of any potential biological threat in the field and more remote, lower resource settings, such as forward operating bases. While we have successfully established untargeted sequencing approaches for detection of pathogens for rapid identification, our current work entails a more in-depth sequencing analysis for use in evolutionary monitoring. We are developing and validating a SARS-CoV-2 nanopore sequencing assay, based on the ARTIC protocol. The standard ARTIC, Illumina, and nanopore sequencing protocols for SARS-CoV-2 are elaborate and time consuming. The new protocol integrates Oxford Nanopore Technology's Rapid Sequencing Kit following targeted RT-PCR of RNA extracted from human clinical specimens. This approach decreases sample manipulations and preparation times. Our current bioinformatics pipeline utilizes Centrifuge as the classifier for quick identification of SARS-CoV-2 and RAMPART software for verification and mapping of reads to the full SARS-CoV-2 genome. ARTIC rapid sequencing results, of previous RT-PCR confirmed patient samples, showed that the modified protocol produces high quality data, with up to 98.9% genome coverage at >1,000x depth for samples with presumably higher viral loads. Furthermore, whole genome assembly and subsequent mutational analysis of six of these sequences identified existing and unique mutations to this cluster, including three in the Spike protein: V308L, P521R, and D614G. This work suggests that an accessible, portable, and relatively fast sample-to-sequence process to characterize viral outbreaks is feasible and effective.

Evaluation of EPISEQ SARS-CoV-2 and a Fully Integrated Application to Identify SARS-CoV-2 Variants from Several Next-Generation Sequencing Approaches.

Whole-genome sequencing has become an essential tool for real-time genomic surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) worldwide. The handling of raw next-generation sequencing (NGS) data is a major challenge for sequencing laboratories. We developed an easy-to-use web-based application (EPISEQ SARS-CoV-2) to analyse SARS-CoV-2 NGS data generated on common sequencing platforms using a variety of commercially available reagents. This application performs in one click a quality check, a reference-based genome assembly, and the analysis of the generated consensus sequence as to coverage of the reference genome, mutation screening and variant identification according to the up-to-date Nextstrain clade and Pango lineage. In this study, we validated the EPISEQ SARS-CoV-2 pipeline against a reference pipeline and compared the performance of NGS data generated by different sequencing protocols using EPISEQ SARS-CoV-2. We showed a strong agreement in SARS-CoV-2 clade and lineage identification (>99%) and in spike mutation detection (>99%) between EPISEQ SARS-CoV-2 and the reference pipeline. The comparison of several sequencing approaches using EPISEQ SARS-CoV-2 revealed 100% concordance in clade and lineage classification. It also uncovered reagent-related sequencing issues with a potential impact on SARS-CoV-2 mutation reporting. Altogether, EPISEQ SARS-CoV-2 allows an easy, rapid and reliable analysis of raw NGS data to support the sequencing efforts of laboratories with limited bioinformatics capacity and those willing to accelerate genomic surveillance of SARS-CoV-2.

Genome Assembly and Analysis of the Flavonoid and Phenylpropanoid Biosynthetic Pathways in Fingerroot Ginger (Boesenbergia rotunda).

Boesenbergia rotunda (Zingiberaceae), is a high-value culinary and ethno-medicinal plant of Southeast Asia. The rhizomes of this herb have a high flavanone and chalcone content. Here we report the genome analysis of B. rotunda together with a complete genome sequence as a hybrid assembly. B. rotunda has an estimated genome size of 2.4 Gb which is assembled as 27,491 contigs with an N50 size of 12.386 Mb. The highly heterozygous genome encodes 71,072 protein-coding genes and has a 72% repeat content, with class I TEs occupying ~67% of the assembled genome. Fluorescence in situ hybridization of the 18 chromosome pairs at the metaphase showed six sites of 45S rDNA and two sites of 5S rDNA. An SSR analysis identified 238,441 gSSRs and 4604 EST-SSRs with 49 SSR markers common among related species. Genome-wide methylation percentages ranged from 73% CpG, 36% CHG and 34% CHH in the leaf to 53% CpG, 18% CHG and 25% CHH in the embryogenic callus. Panduratin A biosynthetic unigenes were most highly expressed in the watery callus. B rotunda has a relatively large genome with a high heterozygosity and TE content. This assembly and data (PRJNA71294) comprise a source for further research on the functional genomics of B. rotunda, the evolution of the ginger plant family and the potential genetic selection or improvement of gingers.

GAAP: A GUI-based Genome Assembly and Annotation Package.

Background: Next-generation sequencing (NGS) technologies are being continuously used for high-throughput sequencing data generation that requires easy-to-use GUI-based data analysis software. These kinds of software could be used in-parallel with sequencing for the automatic data analysis. At present, very few software are available for use and most of them are commercial, thus creating a gap between data generation and data analysis. Methods: GAAP is developed on the NodeJS platform that uses HTML, JavaScript as the front-end for communication with users. We have implemented FastQC and trimmomatic tool for quality checking and control. Velvet and Prodigal are integrated for genome assembly and gene prediction. The annotation will be done with the help of remote NCBI Blast and IPR-Scan. In the back- end, we have used PERL and JavaScript for the processing of data. To evaluate the performance of GAAP, we have assembled a viral (SRR11621811), bacterial (SRR17153353) and human genome (SRR16845439). Results: We have used GAAP software to assemble, and annotate a COVID-19 genome on a desktop computer that resulted in a single contig of 27994bp with 99.57% reference genome coverage. This assembly predicted 11 genes, of which 10 were annotated using annotation module of GAAP. We have also assembled a bacterial and human genome 138 and 194281 contigs with N50 value 100399 and 610, respectively. Conclusion: In this study, we have developed freely available, platform-independent genome assembly and annotation (GAAP) software (www.deepaklab.com/gaap). The software itself acts as a complete data analysis package with quality check, quality control, de-novo genome assembly, gene prediction and annotation (Blast, PFAM, GO-Term, pathway and enzyme mapping) modules.

De Novo-Whole Genome Assembly of the Roborovski Dwarf Hamster (Phodopus roborovskii) Genome: An Animal Model for Severe/Critical COVID-19.

The Roborovski dwarf hamster Phodopus roborovskii belongs to the Phodopus genus, one of the seven within Cricetinae subfamily. Like other rodents such as mice, rats, or ferrets, hamsters can be important animal models for a range of diseases. Whereas the Syrian hamster from the genus Mesocricetus is now widely used as a model for mild-to-moderate coronavirus disease 2019, Roborovski dwarf hamster shows a severe-to-lethal course of disease upon infection with the novel human coronavirus severe acute respiratory syndrome coronavirus 2.

The assembled and annotated genome of the masked palm civet (Paguma larvata).

BACKGROUND: The masked palm civet (Paguma larvata) acts as an intermediate host of severe acute respiratory syndrome coronavirus (SARS-CoV), which caused SARS, and transfered this virus from bats to humans. Additionally, P. larvata has the potential to carry a variety of zoonotic viruses that may threaten human health. However, genome resources for P. larvata have not been reported to date. FINDINGS: A chromosome-level genome assembly of P. larvata was generated using PacBio sequencing, Illumina sequencing, and Hi-C technology. The genome assembly was 2.44 Gb in size, of which 95.32% could be grouped into 22 pseudochromosomes, with contig N50 and scaffold N50 values of 12.97 Mb and 111.81 Mb, respectively. A total of 21,582 protein-coding genes were predicted, and 95.20% of the predicted genes were functionally annotated. Phylogenetic analysis of 19 animal species confirmed the close genetic relationship between P. larvata and species belonging to the Felidae family. Gene family clustering revealed 119 unique, 243 significantly expanded, and 58 significantly contracted genes in the P. larvata genome. We identified 971 positively selected genes in P. larvata, and one known human viral receptor gene PDGFRA is positively selected in P. larvata, which is required for human cytomegalovirus infection. CONCLUSIONS: This high-quality genome assembly provides a valuable genomic resource for exploring virus-host interactions. It will also provide a reliable reference for studying the genetic bases of the morphologic characteristics, adaptive evolution, and evolutionary history of this species.

Yeast goes viral: probing SARS-CoV-2 biology using S. cerevisiae.

The budding yeast Saccharomyces cerevisiae has long been an outstanding platform for understanding the biology of eukaryotic cells. Robust genetics, cell biology, molecular biology, and biochemistry complement deep and detailed genome annotation, a multitude of genome-scale strain collections for functional genomics, and substantial gene conservation with Metazoa to comprise a powerful model for modern biological research. Recently, the yeast model has demonstrated its utility in a perhaps unexpected area, that of eukaryotic virology. Here we discuss three innovative applications of the yeast model system to reveal functions and investigate variants of proteins encoded by the SARS-CoV-2 virus.

Chromosome-Scale Genome of Masked Palm Civet (Paguma larvata) Shows Genomic Signatures of Its Biological Characteristics and Evolution.

The masked palm civet (Paguma larvata) is a small carnivore with distinct biological characteristics, that likes an omnivorous diet and also serves as a vector of pathogens. Although this species is not an endangered animal, its population is reportedly declining. Since the severe acute respiratory syndrome (SARS) epidemic in 2003, the public has been particularly concerned about this species. Here, we present the first genome of the P. larvata, comprising 22 chromosomes assembled using single-tube long fragment read (stLFR) and Hi-C technologies. The genome length is 2.41 Gb with a scaffold N50 of 105.6 Mb. We identified the 107.13 Mb X chromosome and one 1.34 Mb Y-linked scaffold and validated them by resequencing 45 P. larvata individuals. We predicted 18,340 protein-coding genes, among which 18,333 genes were functionally annotated. Interestingly, several biological pathways related to immune defenses were found to be significantly expanded. Also, more than 40% of the enriched pathways on the positively selected genes (PSGs) were identified to be closely related to immunity and survival. These enriched gene families were inferred to be essential for the P. larvata for defense against the pathogens. However, we did not find a direct genomic basis for its adaptation to omnivorous diet despite multiple attempts of comparative genomic analysis. In addition, we evaluated the susceptibility of the P. larvata to the SARS-CoV-2 by screening the RNA expression of the ACE2 and TMPRSS2/TMPRSS4 genes in 16 organs. Finally, we explored the genome-wide heterozygosity and compared it with other animals to evaluate the population status of this species. Taken together, this chromosome-scale genome of the P. larvata provides a necessary resource and insights for understanding the genetic basis of its biological characteristics, evolution, and disease transmission control.

Rapid screening and identification of viral pathogens in metagenomic data.

BACKGROUND: Virus screening and viral genome reconstruction are urgent and crucial for the rapid identification of viral pathogens, i.e., tracing the source and understanding the pathogenesis when a viral outbreak occurs. Next-generation sequencing (NGS) provides an efficient and unbiased way to identify viral pathogens in host-associated and environmental samples without prior knowledge. Despite the availability of software, data analysis still requires human operations. A mature pipeline is urgently needed when thousands of viral pathogen and viral genome reconstruction samples need to be rapidly identified. RESULTS: In this paper, we present a rapid and accurate workflow to screen metagenomics sequencing data for viral pathogens and other compositions, as well as enable a reference-based assembler to reconstruct viral genomes. Moreover, we tested our workflow on several metagenomics datasets, including a SARS-CoV-2 patient sample with NGS data, pangolins tissues with NGS data, Middle East Respiratory Syndrome (MERS)-infected cells with NGS data, etc. Our workflow demonstrated high accuracy and efficiency when identifying target viruses from large scale NGS metagenomics data. Our workflow was flexible when working with a broad range of NGS datasets from small (kb) to large (100 Gb). This took from a few minutes to a few hours to complete each task. At the same time, our workflow automatically generates reports that incorporate visualized feedback (e.g., metagenomics data quality statistics, host and viral sequence compositions, details about each of the identified viral pathogens and their coverages, and reassembled viral pathogen sequences based on their closest references). CONCLUSIONS: Overall, our system enabled the rapid screening and identification of viral pathogens from metagenomics data, providing an important piece to support viral pathogen research during a pandemic. The visualized report contains information from raw sequence quality to a reconstructed viral sequence, which allows non-professional people to screen their samples for viruses by themselves (Additional file 1).

Nanopore Genome Sequencing and Variant Analysis of the Susceptible Candida auris Strain L1537/2020, Salvador, Brazil.

Candida auris has been reported worldwide, but only in December 2020, the first strain from a COVID-19 patient in Brazil was isolated. Here, we describe the genome sequence of this susceptible C. auris strain and performed variant analysis of the genetic relatedness with strains from other geographic localities.

Impacts of reference selection on the assembly of suspicious coronavirus genome.

The pandemic caused by SARS-CoV-2 has had a significant impact on the whole world. In a theory of the origin of SARS-CoV-2, pangolins are considered as a potential intermediate host. To assemble the genome of suspicious coronavirus (CoV) found in pangolins, SARS-CoV-2 was used as a reference in most of the previous studies, implicitly assuming the pangolin CoV and SARS-CoV-2 are the closest neighbors in evolution. However, this assumption may not be true. We investigated how the choice of reference genome affected the resulting CoV genome assembly. We explored various representative CoVs as the reference genome, and found significant differences in the resulting assemblies. The assembly obtained using RaTG13 as a reference showed better statistics in total length, N50, and pairwise distance reconstruction (PDR) scores than the assembly guided by SARS-CoV-2, indicating that RaTG13 may be a better reference. Therefore, RaTG13 should also be considered as a reference for assembling suspicious CoV found in pangolins and other potential intermediate hosts.

Sequencing of SARS-CoV-2 genome using different nanopore chemistries.

Nanopore sequencing has emerged as a rapid and cost-efficient tool for diagnostic and epidemiological surveillance of SARS-CoV-2 during the COVID-19 pandemic. This study compared the results from sequencing the SARS-CoV-2 genome using R9 vs R10 flow cells and a Rapid Barcoding Kit (RBK) vs a Ligation Sequencing Kit (LSK). The R9 chemistry provided a lower error rate (3.5%) than R10 chemistry (7%). The SARS-CoV-2 genome includes few homopolymeric regions. Longest homopolymers were composed of 7 (TTTTTTT) and 6 (AAAAAA) nucleotides. The R10 chemistry resulted in a lower rate of deletions in thymine and adenine homopolymeric regions than the R9, at the expenses of a larger rate (~10%) of mismatches in these regions. The LSK had a larger yield than the RBK, and provided longer reads than the RBK. It also resulted in a larger percentage of aligned reads (99 vs 93%) and also in a complete consensus genome. The results from this study suggest that the LSK preparation library provided longer DNA fragments which contributed to a better assembly of the SARS-CoV-2, despite an impaired detection of variants in a R10 flow cell. Nanopore sequencing could be used in epidemiological surveillance of SARS-CoV-2. KEY POINTS: • Sequencing SARS-CoV-2 genome is of great importance for the pandemic surveillance. • Nanopore offers a low cost and accurate method to sequence SARS-CoV-2 genome. • Ligation sequencing is preferred rather than the rapid kit using transposases.

Sequencing the Genome of Indian Flying Fox, Natural Reservoir of Nipah Virus, Using Hybrid Assembly and Conservative Secondary Scaffolding.

Indian fruit bats, flying fox Pteropus medius was identified as an asymptomatic natural host of recently emerged Nipah virus, which is known to induce a severe infectious disease in humans. The absence of P. medius genome sequence presents an important obstacle for further studies of virus-host interactions and better understanding of mechanisms of zoonotic viral emergence. Generation of the high-quality genome sequence is often linked to a considerable effort associated to elevated costs. Although secondary scaffolding methods have reduced sequencing expenses, they imply the development of new tools for the integration of different data sources to achieve more reliable sequencing results. We initially sequenced the P. medius genome using the combination of Illumina paired-end and Nanopore sequencing, with a depth of 57.4x and 6.1x, respectively. Then, we introduced the novel scaff2link software to integrate multiple sources of information for secondary scaffolding, allowing to remove the association with discordant information among two sources. Different quality metrics were next produced to validate the benefits from secondary scaffolding. The P. medius genome, assembled by this method, has a length of 1,985 Mb and consists of 33,613 contigs and 16,113 scaffolds with an NG50 of 19 Mb. At least 22.5% of the assembled sequences is covered by interspersed repeats already described in other species and 19,823 coding genes are annotated. Phylogenetic analysis demonstrated the clustering of P. medius genome with two other Pteropus bat species, P. alecto and P. vampyrus, for which genome sequences are currently available. SARS-CoV entry receptor ACE2 sequence of P. medius was 82.7% identical with ACE2 of Rhinolophus sinicus bats, thought to be the natural host of SARS-CoV. Altogether, our results confirm that a lower depth of sequencing is enough to obtain a valuable genome sequence, using secondary scaffolding approaches and demonstrate the benefits of the scaff2link application. The genome sequence is now available to the scientific community to (i) proceed with further genomic analysis of P. medius, (ii) to characterize the underlying mechanism allowing Nipah virus maintenance and perpetuation in its bat host, and (iii) to monitor their evolutionary pathways toward a better understanding of bats' ability to control viral infections.