Biology subjects

Gupta, V.

Publications and source records attributed to Gupta, V..

At least 55 records · Page 3Linked to original sources

bioRxiv2022

A chromosome-scale assembly of Brassica carinata (BBCC) accession HC20 containing resistance to multiple pathogens and an early generation assessment of introgressions into B. juncea (AABB)

Brassica carinata (BBCC) commonly referred to as Ethiopian mustard is a natural allotetraploid containing the genomes of Brassica nigra (BB) and Brassica oleracea (CC). It is an oilseed crop endemic to the Northeastern regions of Africa. Although it is grown in a limited manner, B. carinata is of value as it is resistant/highly tolerant to most of the pathogens affecting cultivated Brassica species of the Us triangle that are grown worldwide as oilseed and vegetable crops. We report a chromosome-scale genome assembly of B. carinata accession HC20 using long-read Oxford Nanopore and Illumina sequencing and BioNano optical maps. The assembly has a scaffold N50 of ~39.8 Mb and covers ~1.11 Gb of the genome. We compared the available long-read genome assemblies of the six species of the Us triangle and found a highly conserved gene number and collinearity suggesting that B. juncea (AABB), B. napus (AACC), and B. carinata are strict allopolyploids. We cataloged the nucleotide-binding and leucine-rich repeat immune receptor (NLR) repertoire of B. carinata resulting in the identification of 465 NLRs. We investigated the extent and nature of early generation genomic interactions between the subgenomes of B. carinata and B. juncea in interspecific crosses between the two species. We found that C chromosome additions are well tolerated, with homoeologous exchanges occurring between the A and C genomes. Based on the genomic interactions, we propose strategies to utilize the interspecific crosses for transferring disease resistance from B. carinata to B. juncea and other Brassica species.

plant biology↗

bioRxiv2022

Mucosal gene expression in response to SARS-CoV-2 is associated with early viral load.

Little is known about the relationships between symptomatic early-time SARS-CoV-2 viral load and upper airway mucosal gene expression and immune response. To examine the association of symptomatic SARS-CoV-2 early viral load with upper airway mucosal gene expression, we profiled the host mucosal transcriptome from nasopharyngeal swab samples from 68 adults with symptomatic, mild-to-moderate COVID-19. We measured SARS-CoV-2 viral load using qRT-PCR. We then examined the association of SARS-CoV-2 viral load with upper airway mucosal immune response. We detected SARS-CoV-2 in all samples and recovered >80% of the genome from 85% of the samples from symptomatic COVID-19 adults. The respiratory virome was dominated by SARS-CoV-2, with limited co-detection of common respiratory viruses i.e., only the human Rhinovirus (HRV) being identified in 6% of the samples. We observed a significant positive correlation between SARS-CoV-2 viral load and interferon signaling (OAS2, OAS3, IFIT1, UPS18, ISG15, ISG20, IFITM1, and OASL), chemokine signaling (CXCL10 and CXCL11), and adaptive immune system (IFITM1, CD300E, and SIGLEC1) genes in symptomatic, mild-to-moderate COVID-19 adults, when adjusted for age, sex and race. Interestingly, the expression levels of most of these genes plateaued at a CT value of ~25. Overall, our data shows that early nasal mucosal immune response to SARS-CoV-2 infection is viral load dependent, which potentially could modify COVID-19 outcomes. AUTHOR SUMMARYSeveral prior studies have shown that SARS-CoV-2 viral load can predict the likelihood of disease spread and severity. A higher detectable SARS-CoV-2 plasma viral load was associated with worse respiratory disease severity. However, the relationship between SARS-CoV-2 viral load and airway mucosal gene expression and immune response remains elusive. We profiled the nasal mucosal transcriptome from nasal samples collected from adults infected with SARS-CoV-2 during Spring 2020 with mild-to-moderate symptoms using a comprehensive metatranscriptomics method. We observed a positive correlation between SARS-CoV-2 viral load with interferon signaling, chemokine signaling, and adaptive immune system in adults with COVID-19. Our data suggest that early nasal mucosal immune response to SARS-CoV-2 infection was viral load-dependent and may modify COVID-19 outcomes.

genomics↗

bioRxiv2022

GMHI-webtool: a user-friendly browser application for assessing health through metagenomic gut microbiome profiling

SummaryWe recently introduced the Gut Microbiome Health Index (GMHI), a stool-based indicator for monitoring health given the state of ones gut microbiome. GMHI depends on health-prevalent and health-scarce species determined and validated using a pooled dataset of 5,026 stool shotgun metagenomic samples from 43 independent studies. Encouragingly, GMHI has already been utilized in various studies focusing on identifying differences in the gut microbiome between cases and controls. However, current computational barriers and logistical issues prevent researchers from computing, interpreting, and contextualizing GMHI, thereby limiting its further widespread utilization. Herein, we introduce the GMHI-webtool, a user-friendly browser application that computes GMHI, health-prevalent/scarce species, -diversities, and taxonomic distributions of the gut microbiome from stool samples. Users of our interactive online tool can visualize their results and compare side-by-side with those from our pooled reference dataset, as well as export data in .csv format and high-resolution figures. Availability and implementationGMHI-webtool is freely available here: https://gmhi-webtool.github.io/. Source code: https://github.com/danielchang2002/GMHI-webtool.

bioinformatics↗

bioRxiv2022

The restriction factor pastrel is associated with host vigor, viral titer, and variation in disease tolerance during Drosophila C Virus infection

Genetic variation for both resistance and disease tolerance has been described in a range of species infected with bacterial, viral and fungal pathogens. In Drosophila melanogaster, genetic variation in mortality following systemic Drosophila C Virus (DCV) infection has been shown to be driven by large effect polymorphisms in the viral restriction factor pastrel (pst). However, it is unclear if pst impacts variation in DCV titres (i.e. resistance), or if it also contributes to disease tolerance. We investigated systemic infection across a range of DCV challenge doses spanning nine orders of magnitude, in males and females of ten Drosophila Genetic Reference Panel (DGRP) lines carrying either a susceptible (S) or resistant (R) pst allele. Our results uncover among-line variation in fly survival, viral titers, and disease tolerance measured both as the ability to maintain survival (mortality tolerance) and reproduction (fecundity tolerance). We confirm the role of pst in resistance, as fly lines with the resistant (R) pst allele experienced lower viral titers, and we uncover novel effects of pst on host vigor, as flies carrying the R allele exhibited higher survival and fecundity even in the absence of infection. Finally, we found significant variation in the expression of the JAK-STAT ligand upd3 and the epigenetic regulator of JAK-STAT G9a. While G9a has been previously shown to mediate tolerance of DCV infection, we found no correlation between the expression of either upd3 or G9a on fly tolerance or resistance. Our work highlights the importance of both resistance and tolerance in viral defence.

immunology↗

bioRxiv2022

Bimodal pilot study on inner speech decoding reveals the potential of combining EEG and fMRI

The recognition of inner speech, which could give a voice to patients that have no ability to speak or move, is a challenge for brain-computer interfaces (BCIs). A shortcoming of the available datasets is that they do not combine modalities to increase the performance of inner speech recognition. Multimodal datasets of brain data enable the fusion of neuroimaging modalities with complimentary properties, such as the high spatial resolution of functional magnetic resonance imaging (fMRI) and the temporal resolution of electroencephalography (EEG), and therefore are promising for decoding inner speech. This paper presents the first publicly available bimodal dataset containing EEG and fMRI data acquired nonsimultaneously during inner-speech production. Data were obtained from four healthy, right-handed participants during an inner-speech task with words in either a social or numerical category. Each of the 8-word stimuli were assessed with 40 trials, resulting in 320 trials in each modality for each participant. The aim of this work is to provide a publicly available bimodal dataset on inner speech, contributing towards speech prostheses.

neuroscience↗

bioRxiv2022

Pathogen dependence and inter-individual variability of post-infection reproductive fitness in Drosophila melanogaster

In the experiments reported in this manuscript, we explore the effect of bacterial infections on the reproductive output of Drosophila melanogaster females. Canonical view of host-pathogen interactions supposes two possible outcomes. Because of immune defence being an energy/resource intensive function, an infected female reallocates resources away from reproductive processes and towards immune defence, therefore compromising its reproductive output. Alternatively, faced with impending mortality, an infected female increases its reproductive output to compensate for lost opportunities of future reproduction. We tested if pathogen identity, infection outcome (survival vs. death), and/or time of death determines the reproductive output of females infected with three bacterial pathogens. Our results show that pathogen identity is a reliable predictor of population level response of infected females but does not reliably predict the behaviour of individual females. Additionally, females succumbing to infection exhibit greater variability in reproductive output, compared to both survivors and controls, but this variability is not explained by either the time of death or the identity of the infecting pathogen. Furthermore, survivors of infection have reproductive output similar to control females.

evolutionary biology↗

bioRxiv2022

Context-dependent triggering of STING-interferon signaling by CD11b agonists supports anti-tumor immunity in mouse models and human cancer patients

Chronic activation of inflammatory pathways and suppressed interferon signaling are hallmarks of myeloid cells in immunosuppressive tumors that drive poor responsiveness to conventional and immune therapies. Previous studies have identified agonistic activation of the CD11b integrin as a potential strategy to enhance anti-tumor immunity. However, the mechanisms by which CD11b-agonism reprogram tumor immunity are poorly understood, and this may impair patient selection and identification of effective treatment combinations. Herein we used a combination of in vitro systems, animal models, and samples from first in human clinical trials of the CD11b-agonist GB1275 to identify the mechanism of action of this approach and identify combinations for further testing. We found that CD11b agonism altered tumor-associated macrophage (TAM) phenotypes by simultaneously repressing NF{kappa}B/IL1-signaling and activating interferon (IFN) gene expression. Repression of NF{kappa}B/IL-1 signaling was due to rapid degradation of p65 protein by the proteosome and was not context dependent. In contrast, CD11b agonism triggered mitochondrial dysfunction to stimulate STING-induced, STAT1-mediated interferon signaling. The magnitude of CD11b agonist induction of STING/IFN signaling was dependent on the tumor microenvironment and was significantly amplified by cytotoxic therapies. Using tissues from phase I clinical trials, we demonstrated that GB1275 treatment activated STING and STAT1 signaling in TAMs in human tumors. Together, these mechanisms allowed macrophages to augment anti-tumor T cell immunity. These studies identified potential mechanism-based therapeutic strategies for CD11b agonist use and identified potential patient populations more likely to benefit from them. Statement of significanceCD11b agonists are a novel approach to reprogram myeloid cells in solid tumors. We show that GB1275, a CD11b-agonist, amplified STING/IFN signaling in TAMs to support anti-tumor immunity and this signaling is amplified further by cytotoxic therapy. These studies support new treatment strategies for advanced solid tumors with myeloid immunosuppression.

cancer biology↗

bioRxiv2022

Rethinking the Methods and Algorithms for Inner Speech Decoding - and Making them Reproducible

This study focuses on the automatic decoding of inner speech using noninvasive methods, such as electroencephalography (EEG)). While inner speech has been a research topic in philosophy and psychology for half a century, recent attempts have been made to decode nonvoiced spoken words by using various brain-computer interfaces. The main shortcomings of existing work are reproducibility and the availability of data and code. In this work, we investigate various methods (using Convolutional Neural Network (CNN), Gated Recurrent Unit (GRU), Long Short-Term Memory Networks (LSTM)) for the detection task of 5 vowels and 6 words on a publicly available EEG dataset. The main contributions of this work are (1) subject dependent vs. subject-independent approaches, (2) the effect of different preprocessing steps (Independent Component Analysis (ICA), down-sampling and filtering), and (3) word classification (where we achieve state-of-the-art performance on a publicly available dataset). Overall we achieve a performance accuracy of 35.20% and 29.21% when classifying 5 vowels and 6 words respectively in a publicly available dataset, using our tuned iSpeech-CNN architecture. All of our code and processed data are publicly available to ensure reproducibility. As such, this work contributes to a deeper understanding and reproducibility of experiments in the area of inner speech detection.

neuroscience↗

bioRxiv2021

Transcriptomic and proteomic retinal pigment epithelium signatures of age-related macular degeneration.

Induced pluripotent stem cells generated from patients with geographic atrophy as well as healthy individuals were differentiated to retinal pigment epithelium (RPE) cells. By integrating transcriptional profiles of 127,659 RPE cells generated from 43 individuals with geographic atrophy and 36 controls with genotype data, we identified 439 expression Quantitative Trait (eQTL) loci in cis that were associated with disease status and specific to subpopulations of RPE cells. We identified loci linked to two genes with known associations with geographic atrophy - PILRB and PRPH2, in addition to 43 genes with significant genotype x disease interactions that are candidates for novel genetic associations for geographic atrophy. On a transcriptome-only level, we identified molecular pathways significantly upregulated in geographic atrophy-RPE including in extracellular cellular matrix reorganisation, neurodegeneration, and mitochondrial functions. We subsequently implemented a large-scale proteomics analysis, confirming modification in proteins associated with these pathways. We also identified six significant protein (p) QTL that regulate protein expression in the RPE cells and in geographic atrophy - two of which share variants with cis-eQTL. Transcriptome-wide association analysis identified genes at loci previously associated with age-related macular degeneration. Further analysis conditional on disease status, implicated statistically significant RPE-specific eQTL. This study uncovers important differences in RPE homeostasis associated with geographic atrophy.

neuroscience↗

bioRxiv2021

Type I PRMTs and PRMT5 Inversely Regulate Post-Transcriptional Intron Detention

Protein arginine methyltransferases (PRMTs) are required for the regulation of RNA processing factors. Type I enzymes catalyze mono- and asymmetric dimethylation; Type II enzymes catalyze mono- and symmetric dimethylation. To understand the specific mechanisms of PRMT activity in splicing regulation, we inhibited Type I and II PRMTs and probed their transcriptomic consequences. Using the newly developed SKaTER-seq method, analysis of co-transcriptional splicing revealed that PRMT inhibition resulted in slower splicing rates. Surprisingly, altered co-transcriptional splicing kinetics correlated poorly with ultimate changes in alternative splicing of polyadenylated RNA--particularly intron retention (RI). Investigation of RI following inhibition of nascent transcription demonstrated that PRMTs inversely regulate RI post-transcriptionally. Subsequent proteomic analysis of chromatin-associated polyadenylated RNA identified aberrant binding of the Type I substrate, CHTOP, and the Type II substrate, SmB. Targeted mutagenesis of all methylarginine sites in SmD3, SmB, and SmD1 recapitulated splicing changes seen with Type II PRMT inhibition. Conversely, mutagenesis of all methylarginine sites in CHTOP recapitulated the splicing changes seen with Type I PRMT inhibition. Closer examination of subcellular fractions indicated that RI were isolated to the nucleoplasm and chromatin. Together, these data demonstrate that PRMTs regulate the post-transcriptional processing of nuclear, detained introns through Sm and CHTOP arginine methylation.

cancer biology↗

bioRxiv2021

Nopp140-chaperoned 2'-O-methylation of small nuclear RNAs in Cajal bodies ensures splicing fidelity

Spliceosomal small nuclear RNAs (snRNAs) are modified by small Cajal body (CB) specific ribonucleoproteins (scaRNPs) to ensure snRNP biogenesis and pre-mRNA splicing. However, the function and subcellular site of snRNA modification are largely unknown. We show that CB localization of the protein Nopp140 is essential for concentration of scaRNPs in that nuclear condensate; and that phosphorylation by casein kinase 2 (CK2) at some 80 serines targets Nopp140 to CBs. Transiting through CBs, snRNAs are apparently modified by scaRNPs. Indeed, Nopp140 knockdown-mediated release of scaRNPs from CBs severely compromises 2-O-methylation of spliceosomal snRNAs, identifying CBs as the site of scaRNP catalysis. Additionally, alternative splicing patterns change indicating that these modifications in U1, U2, U5, and U12 snRNAs safeguard splicing fidelity. Given the importance of CK2 in this pathway, compromised splicing could underlie the mode of action of small molecule CK2 inhibitors currently considered for therapy in cholangiocarcinoma, hematological malignancies, and COVID-19.

cell biology↗

bioRxiv2021

Closed-Loop Control between Two GTPase Switches makes the Secretory Functions of the Golgi Responsive to Growth Factors

Cancers represent complex autonomous systems, displaying self-sufficiency in growth signaling. Autonomous growth is fueled by a cancer cells ability to secrete-and-sense growth factors: a poorly understood phenomenon. Using an integrated systems and experimental approach, here we dissect the impact of a feedback-coupled GTPase circuit within the secretory pathway that imparts secretion-coupled autonomy. The circuit is assembled when the Ras-superfamily monomeric GTPase Arf1, and the heterotrimeric GTPase Gi{beta}{gamma} and their corresponding GAPs and GEFs are coupled by GIV/Girdin, a protein that is known to fuel aggressive traits in diverse cancers. One forward and two key negative feedback loops within the circuit create closed-loop control (CLC), allow the two GTPases to coregulate each other, and convert the expected switch-like behavior of Arf1-dependent secretion into an unexpected dose response alignment behavior of sensing and secretion. Such behavior translates into cell survival that is self-sustained by stimulus-proportionate secretion. Proteomic studies and protein-protein interaction network analyses pinpoint growth factors (e.g., the epidermal growth factor; EGF) as a key stimuli for such self-sustenance. Findings highlight how enhanced coupling of two biological switches in cancer cells is critical for multiscale feedback control to achieve secretion-coupled autonomy of growth factors. SYNOPSIS IMAGE O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY STANDFIRST TEXTThis work defines the inner workings of a Golgi-localized molecular circuitry comprised of coupled GTPases, which empowers cells to achieve self-sufficiency in growth factor signaling by creating a secrete-and-sense autocrine loop. HIGHLIGHTS/MAIN FINDINGSO_LIModeling and experimental approaches were used to dissect a coupled GTPase circuit. C_LIO_LICoupling enables closed loop feedback and mutual control of GTPases. C_LIO_LICoupling generates dose response alignment behavior of sensing and secretion of growth factors. C_LIO_LICoupling is critical for multiscale feedback control to achieve secretion-coupled autonomy. C_LI

systems biology↗

bioRxiv2021

Reliance on polyfunctional tissue leads to a reproduction-immunity tradeoff due to inherent constraint

The use of one tissue for multiple purposes can result in constraints, impaired function, and tradeoffs. The insect fat body performs remarkably diverse functions including metabolic control, reproductive provisioning, and systemic immune responses. Immunity and reproduction are observed to trade off in many organisms, although the mechanistic basis for the tradeoff is generally unknown. More generally, how do polyfunctional tissues simultaneously execute multiple distinct physiological functions? Using single-nucleus sequencing, we determined the Drosophila melanogaster fat body executes diverse basal functions with heterogenous cellular subpopulations. However, as an emergency function, the immune response engages the entire tissue. We found that reproductively active females exhibit impaired capacity to produce new protein in response to infection, resulting in the reproduction-immunity tradeoff. We suggest that such inherent internal limitations may provide a general explanation for the wide prevalence of physiological and evolutionary tradeoffs.

evolutionary biology↗

bioRxiv2021

A robust method to isolate Drosophila fat body nuclei for transcriptomic analysis

Gene expression profiles are typically described at the level of the tissue or, often in Drosophila, at the level of the whole organism. Collapsing the gene expression of entire tissues into single measures averages over potentially important heterogeneity among the cells that make up that tissue. The advent of single-cell RNA-sequencing technology (sc-RNAseq) allows transcriptomic evaluation of the individual cells that make up a tissue. However, sc-RNAseq requires a high-quality suspension of viable cells or nuclei, and cell dissociation methods that yield healthy cells and nuclei are still lacking for many important tissues. The insect fat body is a polyfunctional tissue responsible for diverse physiological processes and therefore is an important target for sc-RNAseq. The Drosophila adult fat body consists of fragile cells that are difficult to dissociate while maintaining cell viability. As an alternative, we developed a method to isolate single fat body nuclei for RNA-seq. Our isolation method is largely free of mitochondrial contamination and yields higher capture of transcripts per nucleus compared to other nuclei preparation methods. Our method works well for single cell nuclei sequencing and potentially can be implemented for bulk RNA-seq.

molecular biology↗

bioRxiv2020

Quantum Approximated Graph Cutting: A Rapid Replacement for T-REMD?

Determining an optimal protein configuration for the employment of protein binding analysis as completed by Temperature based Replica Exchange Molecular Dynamics (T-REMD) is an important process in the accurate depiction of a proteins behavior in different solvent environments, especially when determining a proteins top binding sites for use in protein-ligand and protein-protein docking studies. However, the completion of this analysis, which pushes out top binding sites through configurational changes, is an polynomial-state computational problem that can take multiple hours to compute, even on the fastest supercomputers. In this study, we aim to determine if graph cutting provide approximated solutions the MaxCut problem can be used as a method to provide similar results to T-REMD in the determination of top binding sites of Surfactant Protein A (SP-A) for binding analysis. Additionally, we utilize a quantum-hybrid algorithm within Iff Technologys Polar+ package using an actual quantum processor unit (QPU), an implementation of Polar+ using an emulated QPU, or Quantum Abstract Machine (QAM), on a large scale classical computing device, and an implementation of a classical MaxCut algorithm on a supercomputer in order to determine the types of advantages that can be gained through using a quantum computing device for this problem, or even using quantum algorithms on a classical device. This study found that Polar+ provides a dramatic speedup over a classical implementation of a MaxCut approximation algorithm or the use of GROMACS T-REMD, and produces viable results, in both its QPU and QAM implementations. However, the lack of direct configurational changes carried out onto the structure of SP-A after the use of graph cutting methods produces different final binding results than those produced by GROMACS T-REMD. Thus, further work needs to be completed into translating quantum-based probabilities into configurational changes based on a variety of noise conditions to better determine the accuracy advantage that quantum algorithms and quantum devices can provide in the near future.

bioinformatics↗

bioRxiv2020

Type I PRMTs and PRMT5 Independently Regulate Both snRNP Arginine Methylation and Post-Transcriptional Splicing

Protein arginine methyltransferases (PRMTs) methylate histones, splicing factors, and many other nuclear proteins. Type I enzymes (PRMT1-4,6,8) catalyze mono- (Rme1/MMA) and asymmetric (Rme2a/ADMA) dimethylation; Type II enzymes (PRMT5,9) catalyze mono- and symmetric (Rme2s/SDMA) dimethylation. Misregulation of PRMTs in multiple types of cancers is associated with aberrant gene expression and RNA splicing. To understand the specific mechanisms of PRMT activity in splicing regulation, we treated cells with the PRMT5 inhibitor GSK591 and the Type I inhibitor MS023 and probed their transcriptomic consequences. We discovered that Type I PRMTs and PRMT5 inversely regulate core spliceosomal Sm protein Rme2s and intron retention. Loss of Sm Rme2s is associated with the accumulation of polyadenylated RNA containing retained introns and snRNPs on chromatin. Conversely, increased Sm Rme2s correlates with decreased intron retention and chromatin-association of intron-containing polyadenylated RNA. Using the newly developed SKaTER-seq model, comprehensive and quantitative analysis of co-transcriptional splicing revealed that either Type I PRMT or PRMT5 inhibition resulted in slower splicing rates. Surprisingly, altered co-transcriptional splicing kinetics correlated poorly with ultimate changes in alternatively spliced mRNA. Quantitation of retained intron decay following inhibition of nascent transcription revealed that Type I PRMTs and PRMT5 reciprocally regulate post-transcriptional splicing efficiency.

cell biology↗

bioRxiv2020

SeqStain using fluorescent-DNA conjugated antibodies allows efficient, multiplexed, spatialomic profiling of human and murine tissues

Spatial organization of molecules and cells in complex tissue microenvironments provides essential cues during healthy growth and in disease. Novel techniques are needed for elucidation of their spatial relationships and architecture. Although a few multiplex immunofluorescence based techniques have been developed for visualization of the spatial relationships of various molecules in cells and tissues, there remains a significant need for newer methods that are rapid, easy to adapt and are gentle during the cyclic steps of fluorescence staining and de-staining. Here, we describe a novel, multiplex immunofluorescence imaging method, termed SeqStain, that uses fluorescent-DNA labelled antibodies for immunofluorescence staining of cells and tissues, and nuclease treatment for de-staining that allows selective enzymatic removal of the fluorescent signal. SeqStain can be used with primary antibodies, secondary antibodies and antibody fragments, such as Fabs, to efficiently analyse complex cells and tissues in multiple rounds of staining and de-staining. Additionally, incorporation of specific endonuclease restriction sites in antibody labels allows for selective removal of fluorescent signals, while retaining other signals that can serve as marks for subsequent analyses. The application of SeqStain on human kidney tissue provided spatialomic profile of the organization of >25 markers in the kidney, highlighting it as a versatile, easy to use and gentle new technique for spatialomic analyses of complex microenvironments.

molecular biology↗

bioRxiv2020

High throughput detection and genetic epidemiology of SARS-CoV-2 using COVIDSeq next generation sequencing

The rapid emergence of coronavirus disease 2019 (COVID-19) as a global pandemic affecting millions of individuals globally has necessitated sensitive and high-throughput approaches for the diagnosis, surveillance and for determining the genetic epidemiology of SARS-CoV-2. In the present study, we used the COVIDSeq protocol, which involves multiplex-PCR, barcoding and sequencing of samples for high-throughput detection and deciphering the genetic epidemiology of SARS-CoV-2. We used the approach on 752 clinical samples in duplicates, amounting to a total of 1536 samples which could be sequenced on a single S4 sequencing flow cell on NovaSeq 6000. Our analysis suggests a high concordance between technical duplicates and a high concordance of detection of SARS-CoV-2 between the COVIDSeq as well as RT-PCR approaches. An in-depth analysis revealed a total of six samples in which COVIDSeq detected SARS-CoV-2 in high confidence which were negative in RT-PCR. Additionally, the assay could detect SARS-CoV-2 in 21 samples and 16 samples which were classified inconclusive and pan-sarbeco positive respectively suggesting that COVIDSeq could be used as a confirmatory test. The sequencing approach also enabled insights into the evolution and genetic epidemiology of the SARS-CoV-2 samples. The samples were classified into a total of 3 clades. This study reports two lineages B.1.112 and B.1.99 for the first time in India. This study also revealed 1,143 unique single nucleotide variants and added a total of 73 novel variants identified for the first time. To the best of our knowledge, this is the first report of the COVIDSeq approach for detection and genetic epidemiology of SARS-CoV-2. Our analysis suggests that COVIDSeq could be a potential high sensitivity assay for detection of SARS-CoV-2, with an additional advantage of enabling genetic epidemiology of SARS-CoV-2.

genomics↗