Biology subjects

Dixit, A.

Publications and source records attributed to Dixit, A..

17 recordsLinked to original sources

bioRxiv2026

A pathogen-associated odorant induces fear-like response regulated by an olfactory receptor STR-211 in Caenorhabditis elegans

Exposure to chemical cues released by predator or pathogen can evoke anxiety or fear responses in prey/host animals such as fight, flight or freeze both at behavioral and molecular levels. Freezing is a fundamental anxiety response when fighting or fleeing arent feasible. Despite the potential relevance of freezing as a stress-coping mechanism, its behavioral and molecular underpinnings are not understood yet. At molecular level danger cues are perceived by chemosensory receptors expressed in sensory neurons which may further regulate the animals behavioral responses(Ye et al., 2024){Citation}. 2-nonanone (2-NA) is one of the principal volatile organic compounds secreted by many pathogenic bacteria infecting Caenorhabditis elegans as well as humans and may signal danger to worms. Here, we show that olfactory exposure to threat-associated cue 2-NA induces a reversible fear-like freezing response characterized by immobility and halted feeding in C. elegans. With the application of in silico and behavioral approaches we showed that 2-NA is one of the ligands for an olfactory G-protein Coupled Receptor (GPCR) STR-211 and RNAi knockdown of the receptor leads to a defect in 2-NA induced avoidance behavior in worms. We next discovered that STR-211 is required for immediate behavioral changes in C. elegans during freezing response against 2-NA. The study proposes an environment relevant animal model to mimic human anxiety and fear-like behavior, along with the identification of one of the olfactory GPCRs mediating this behavior. The model may help in understanding the neuromolecular basis of freezing response in human anxiety, contributing towards treatment of mental health disorders.

neuroscience↗

bioRxiv2024

Effect of Emoji on Autonomic Nervous System: Evidence from EDA and RSA studies

BackgroundFacial expression, gesture, and posture play an important role in perceiving emotions during communication. In virtual communication platforms, users have devised and learned to use a variety of expressive emojis along with text messages to express certain emotions. The effect of emojis on human psychology and associated autonomic responses is still vague. MethodsA total of 100 healthy individuals (50 males and 50 females) aged between 18-40 years were recruited. Electrodermal activity (skin conductance level (SCL) and skin conductance response (SCR) amplitude) and Respiratory Sinus Arrhythmia (RSA) were assessed during the Emotional Stroop Task (EST). EST having expressive emojis superimposed with the congruent and incongruent words was used. ResultMean SCL and SCR amplitude was significantly increased during EST in incongruent and congruent blocks as compared to neutral block (respectively: 14.64 {+/-} 6.73, 12.99 {+/-} 6.26 vs. 7.75 {+/-} 4.93 S, p < 0.001 and 0.182 {+/-} 0.168, 0.158 {+/-} 0.134 vs. 0.021 {+/-} 0.015 S, p < 0.001). RSA was significantly decreased in incongruent and congruent blocks as compared to neutral blocks (respectively: 36.47 {+/-} 10.53, 39.40 {+/-} 10.15 vs. 48.66 {+/-} 10.27 msec2, p < 0.001). We found an increased sympathetic activity and parasympathetic withdrawal while performing the task. ConclusionThe results of this study suggested that emojis are adequate stimuli to elicit autonomic responses and change both sympathetic (EDA) as well as parasympathetic responses (RSA). Males and females showed similar autonomic arousal for emoji but the baseline emotional status was different for both genders.

physiology↗

bioRxiv2023

Exploring the Role of MMP9 and its Interaction with TGFβ Signalling in Patients with Mesial Temporal Lobe Epilepsy-Hippocampal Sclerosis

Inflammation and blood brain barrier (BBB) damage are associated with epileptogenesis in Mesial Temporal lobe epilepsy with Hippocampal sclerosis (MTLE-HS). Animal studies have predicted the role of Matrix metalloproteinase 9 (MMP9) in extracellular matrix (ECM) modulation, BBB leakage and neuro-inflammation, while Transforming growth factor beta (TGF{beta}) signalling in astrocytes potentiates hyper-excitability leading to seizure generation. We hypothesize whether changes in activity and expression of MMP9, and the ratio of MMP9 and its inhibitor, Tissue inhibitor of metalloproteinase 1 (TIMP1), have a role in epileptogenesis in the patients with MTLE-HS through zona occludens 1 (ZO1) modulation. We also proposed the role of astrocytic TGF{beta} signalling in these patients. mRNA expression of MMP9 and TIMP1 was significantly up-regulated. The ratio of MMP9 to its inhibitor TIMP1 was greater than one, suggesting activation of MMP9, further confirmed by gelatin zymography. MMP9 activity as well as immunoreactivity was higher in patients with MTLE-HS as compared to non-seizure controls, whereas the immunoreactivity of ZO1 was significantly lower in the patients. The downstream TGF{beta} signalling effector molecules, SMAD3 and pSMAD3 immunoreactivity were also significantly higher in MTLE-HS patients and both molecules showed co-localisation with astrocytes in the hippocampal region. Further, we showed preliminary data about interaction of MMP9 and TGF{beta}1 in these patients as evidenced by a co-immunoprecipitation assay. This study highlighted the MMP9 and astrocytic TGF{beta} signalling mediated potential mechanism of epileptogenesis in MTLE-HS patients.

neuroscience↗

bioRxiv2022

A novel multi-epitope peptide vaccine candidate targeting Hepatitis E virus: an in-silico approach

HEV is a foodborne virus transmitted through the fecal-oral route that causes viral hepatitis in humans worldwide. Ever since its discovery as a zoonotic agent, HEV was isolated from several species with an expanding range of hosts. HEV possesses several features of other RNA viruses but also has certain HEV-specific traits that make its viral-host interactions inimitable. HEV leads to severe morbidity and mortality in immunocompromised people and pregnant women across the world. The situation in underdeveloped countries is even more alarming. Even after creating a menace across the world, we still lack an effective vaccine against HEV. Till date, there is only one licensed vaccine for HEV available only in China. The development of an anti-HEV vaccine that can reduce HEV-induced morbidity and mortality is required. Live attenuated and killed vaccines against HEV are not accessible due to the lack of a tolerant cell culture system, slow viral replication kinetics and varying growth conditions. Thus, the main focus for anti-HEV vaccine development is now on the molecular approaches. In the current study, we have designed a multi-epitope vaccine against HEV through a reverse vaccinology approach (Figure 1). For the first time, we have used viral ORF3, capsid protein and polyprotein altogether for epitope prediction. These are crucial for viral replication and persistence and are major vaccine targets against HEV. The proposed in-silico vaccine construct comprises of highly immunogenic and antigenic T-cell and B-cell epitopes of HEV proteins and ORF3. The construct is capable of inducing an effective and long-lasting host immune response as evident from the simulations results. In addition, the construct is stable, non-allergic and antigenic for the host. Altogether, our findings suggest that the in-silico vaccine construct may be useful as a vaccine candidate for preventing HEV infections. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=164 SRC="FIGDIR/small/520355v1_fig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@354f48org.highwire.dtl.DTLVardef@1e80119org.highwire.dtl.DTLVardef@a04ed5org.highwire.dtl.DTLVardef@97bab0_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1:C_FLOATNO An overview of multi-epitope vaccine construct designing using different in-silico tools. C_FIG

immunology↗

bioRxiv2022

DNARecords: An extensible sparse format for petabyte scale genomics analysis

Recent growth in population scale sequencing initiatives involve both cohort scale and proportion of genome surveyed, with a transition from genotyping arrays to broader genome sequencing approaches. The resulting datasets can be challenging to analyze. Here we introduce DNARecords a novel sparse-compatible format for large scale genetic data. The structure enables integration of complex data types such as medical images and drug structures towards the development of machine learning methods to predict disease risk and drug response. We demonstrate its speed and memory advantages for various genetics analyses. These performance advantages will become more pronounced as it becomes feasible to analyze variants of lower population allele frequencies. Finally, we provide an open-source software plugin, built on top of Hail, to allow researchers to write and read such records as well as a set of examples for how to use them.

genomics↗

bioRxiv2022

The PREGCARE study: Personalized recurrence risk assessment following the birth of a child with a pathogenic de novo mutation

Next-generation sequencing has led to a dramatic improvement in molecular diagnoses of serious pediatric disorders caused by apparently de novo mutations (DNMs); by contrast, clinicians ability to counsel the parents about the risk of recurrence in a future child has lagged behind. Owing to the possibility that one of the parents could be mosaic in their germline, a recurrence risk of 1-2% is frequently quoted, but for any specific couple, this figure is usually incorrect. We present a systematic approach to providing individualized recurrence risk stratification, by combining deep-sequencing of multiple tissues in the mother-father-child trio with haplotyping to determine the parental origin of the DNM. In the first 58 couples analysed (total of 59 DNMs in 49 different genes), the risk for 35 (59%) DNMs was decreased below 0.1% but for 6 (10%) couples it was increased owing to parental mosaicism - that could be quantified in semen (recurrence risks of 5.6-12.1%) for the paternal cases. Deep-sequencing of the DNM efficiently identifies couples at greatest risk for recurrence and may qualify them for additional reproductive technologies. Haplotyping can further reassure many other couples that their recurrence risk is very low, but its implementation is more technically challenging and will require better understanding of how couples respond to information that reduces their risks.

genetics↗

bioRxiv2022

A metabolic labeling-based chemoproteomic platform unravels the physiological roles of choline metabolites

Choline is an essential nutrient for mammalian cells. Our understanding of the cellular functions of choline and its metabolites, independent of their roles as choline lipid metabolism intermediates, remains limited. In addition to fundamental cellular physiology, this knowledge has implications for cancer biology because elevated choline metabolite levels are a hallmark of cancer. Here, we establish the mammalian choline metabolite-interacting proteome by utilizing a photocrosslinkable choline probe. To design this probe, we performed metabolic labeling experiments with structurally diverse choline analogs that resulted in the serendipitous discovery of a choline lipid headgroup remodeling mechanism involving sequential dealkylation and methylation steps. We demonstrate that phosphocholine inhibits the binding of one of the proteins identified, the attractive anticancer target, p32, to its endogenous ligands and to the promising p32-targeting anticancer agent, Lyp-1. Our results reveal that choline metabolites play vital roles in cellular physiology by serving as modulators of protein function.

biochemistry↗

bioRxiv2022

Association of seminal polyaromatic hydrocarbons exposome with idiopathic male factor infertility: A proteomic insight into sperm function

Oxidative stress (OS) is implicated in 80% of idiopathic male infertility (IMI) patients where exposure to redox active environmental toxicants such as polyaromatic hydrocarbons (PAHs) may play. In the present study the seminal exposome of various PAH was analyzed in two separate cohorts including 43 fertile donors and 60 IMI patients by HPLC and receiver operator characteristic curve was applied to find out the cut-off limits. Furthermore, spermatozoa from both the groups were subjected to label free liquid chromatography mass spectroscopy (LC-MS/MS) followed by bioinformatics analysis to elucidate the molecular mechanism(s) involved and the key proteins from the affected pathways were validated by western blot along with oxidative modification of proteins. Of the 16 standard PAH 13 were detected in the semen. Receiver Operator Character (ROC) Curve analysis (AUCROC) revealed the PAHs having most significant effect on fertility are of the following order Anthracene<benzo(a)pyrene<benzo[b]fluoranthene<Fluoranthene<benzo(a)anthracene<indol (123CD)pyrene<pyrene<naphthalene<dibenzo(AH)anthracene<fluorene<2bromonaphthalene <chrysene<benzo(GH1)perylene. Benzo[a] pyrene is invariably present in all infertile patients while naphthalene is present in both fertile and IMI group. Of the total 773 detected proteins (Control: 631 and PAH: 717); 71 were differentially expressed (13 underexpressed, 58 overexpressed) in IMI patients resulting in impaired mitochondrial dysfunction and oxidative phosphorylation, DNA damage, Aryl hydrocarbon receptor (AHR) signalling, xenobiotic metabolism and induction of NRF-2 mediated oxidative stress response (increased in 4-hydroxynonenal and nitrosylated protein adduct formation, and declined antioxidant defence). The increased GSH/GSSG ratio in patients may be an adaptive response to metabolize the xenobiotics via conjugation as evidenced by overexpression of AHR and Heat shock protein 90 beta (HSP90{beta}) in patients. Seminal PAH concentrations, oxidative protein modification along with protein markers (e.g. AHR and HSP90{beta}) may help in better prediction and management of IMI. Contribution of environment borne PAH in semen should not undermined in infertility evaluation.

pharmacology and toxicology↗

bioRxiv2021

GenTB: A user-friendly genome-based predictor for tuberculosis resistance powered by machine learning

IntroductionMultidrug-resistant Mycobacterium tuberculosis (Mtb) is a significant global public health threat. Genotypic resistance prediction from Mtb DNA sequences offers an alternative to laboratory-based drug-susceptibility testing. User-friendly and accurate resistance prediction tools are needed to enable public health and clinical practitioners to rapidly diagnose resistance and inform treatment regimens. MethodsWe present Translational Genomics platform for Tuberculosis (GenTB), a web-based application to predict antibiotic resistance from next-generation sequence data. The user can choose between two potential predictors, a Random Forest (RF) classifier and a Wide and Deep Neural Network (WDNN) to predict phenotypic resistance to 13 and 10 anti-tuberculosis drugs, respectively. We benchmark GenTBs predictive performance along with leading TB resistance prediction tools (Mykrobe and TB-Profiler) using a ground truth dataset of 20,408 isolates with laboratory-based drug susceptibility data. ResultsAll four tools reliably predicted resistance to first-line tuberculosis drugs but had varying performance for second-line drugs. The mean sensitivities for GenTB-RF and GenTB-WDNN across the nine shared drugs was 77.6% (95% CI 76.6 - 78.5%) and 75.4% (95% CI 74.5 - 76.4%) respectively, and marginally higher than the sensitivities of TB-Profiler at 74.4% (95% CI 73.4 - 75.3%) and Mykrobe at 71.9% (95% CI 70.9 - 72.9%). The higher sensitivities were at an expense of [≤]1.5% lower specificity: Mykrobe 97.6% (95% CI 97.5 - 97.7%), TB-Profiler 96.9% (95% CI 96.7 to 97.0%), GenTB-WDNN 96.2% (95% CI 96.0 to 96.4%), and GenTB-RF 96.1% (95% CI 96.0 to 96.3%). Genotypic resistance sensitivity was 11% and 9% lower for isoniazid and rifampicin respectively, on isolates sequenced at low depth (<10x across 95% of the genome) emphasizing the need to quality control input sequence data before prediction. We discuss differences between tools in reporting results to the user including variants underlying the resistance calls and any novel or indeterminate variants ConclusionGenTB is an easy-to-use online tool to rapidly and accurately predict resistance to anti-tuberculosis drugs. GenTB can be accessed online at https://gentb.hms.harvard.edu, and the source code is available at https://github.com/farhat-lab/gentb-site.

bioinformatics↗

bioRxiv2020

SARS-CoV2 genome analysis of Indian isolates and molecular modelling of D614G mutated spike protein with TMPRSS2 depicted its enhanced interaction and virus infectivity

COVID-19 that emerged as a global pandemic is caused by SARS-CoV-2 virus. The virus genome analysis during disease spread reveals about its evolution and transmission. We did whole genome sequencing of 225 clinical strains from the state of Odisha in eastern India using ARTIC protocol-based amplicon sequencing. Phylogenetic analysis identified the presence of all five reported clades 19A, 19B, 20A, 20B and 20C in the population. The analyses revealed two major routes for the introduction of the disease in India i.e. Europe and South-east Asia followed by local transmission. Interestingly, 19B clade was found to be much more prevalent in our sequenced genomes (17%) as compared to other genomes reported so far from India. The haplogroup analysis for clades showed evolution of 19A and 19B in parallel whereas the 20B and 20C appeared to evolve from 20A. Majority of the 19A and 19B clades were present in cases that migrated from Gujarat state in India suggesting it to be one of the major initial points of disease transmission in India during month of March and April. We found that with the time 20A and 20B clades evolved drastically that originated from central Europe. At the same time, it has been observed that 20A and 20B clades depicted selection of four common mutations i.e. 241 C>T (5UTR), P323L in RdRP, F942F in NSP3 and D614G in the spike protein. We found an increase in the concordance of G614 mutation evolution with the viral load in clinical samples as evident from decreased Ct value of spike and Orf1ab gene in qPCR. Molecular modelling and docking analysis identified that D614G mutation enhanced interaction of spike with TMPRSS2 protease, which could impact the shedding of S1 domain and infectivity of the virus in host cells.

genomics↗

bioRxiv2020

Stagewise identification of significantly mutated gene clusters in major molecular classes of breast invasive carcinoma

Breast invasive carcinoma (BRCA) is most malignant and leading cause of death in women. The efforts are ongoing for improvement in early detection, prevention and treatment. Therefore, identification of biomarkers/candidate genes has become very important. The current work includes comprehensive analysis of RNA-sequencing data of 1097 BRCA samples and 114 normal adjacent tissues to identify dysregulated genes in major molecular classes of BRCA in various clinical stages. Huge number of dysregulated genes were found, some were stage-specific, and others were common. The pathways as interferon signaling, tryptophan degradation III, granulocyte adhesion & diapedesis and catecholamine biosynthesis were found in ER/PR+/HER-2- (p-value<0.010), pathways as RAR activation, adipogenesis, role of JAK1, JAK2 in interferon signaling, TGF-{beta} and STAT3 signaling (p-value<0.014) intricated in ER/PR-/HER-2+ and pathways as IL-1/IL-8 signaling, TNFR1/TNFR2 signaling, TWEAK and relaxin signaling (p-value<0.005) were found in triple negative breast cancer. The genes were clustered based on their mutation profile which revealed nine mutated clusters, some of which were known to be involved in well-known cancer signalling pathways while others were less characterized. Each cluster was analyzed in detail which led us to discover genes viz. NLGN3, MAML2, TTN, SYNE1, ANK2 as candidates in BRCA. The genes were found to be involved in important processes as chemotaxis, axon guidance, notch binding, cell-adhesion-molecule binding etc. They are central genes in the protein-protein-interaction network indicating they can have important regulatory roles. The qRT-PCR and western blot confirmed our findings in breast cancer cell-lines. Further, immunohistochemistry corroborated the results in ~100 tissue samples. The genes can be used as biomarker in BRCA.

bioinformatics↗

bioRxiv2019

Shuffle-Seq: En masse combinatorial encoding for n-way genetic interaction screens

Genetic interactions, defined as the non-additive phenotypic impact of combinations of genes, are a hallmark of the mapping from genotype to phenotype. However, genetic interactions remain challenging to systematically test given the massive number of possible combinations. In particular, while large-scale screening efforts in yeast have quantified pairwise interactions that affect cell viability, or synthetic lethality, between all pairs of genes as well as for a limited number of three-way interactions, it has previously been intractable to perform the large screens needed to comprehensively assess interactions in a mammalian genome. Here, we develop Shuffle-Seq, a scalable method to assay genetic interactions. Shuffle-Seq leverages the co-inheritance of genetically encoded barcodes in dividing cells and can scale in proportion to sequencing throughput. We demonstrate the technical validity of Shuffle-Seq and apply it to screening for mechanisms underlying drug resistance in a melanoma model. Shuffle-Seq should allow screens of hundreds of millions of combinatorial perturbations and facilitate the understanding of genetic dependencies and drug sensitivities.

genomics↗

bioRxiv2019

Tuberculosis resistance acquisition in space and time: an analysis of globally diverse M. tuberculosis whole genome sequences

BackgroundMycobacterium tuberculosis (MTB) whole genome sequencing data can provide insights into temporal and geographic trends in resistance acquisition and inform public health interventions.\n\nMethodsWe curated a set of clinical MTB isolates with high quality sequencing and culture-based drug susceptibility data spanning four lineages and more than 20 countries. We constructed geographic and lineage specific MTB phylogenies and used Bayesian molecular dating to infer the most-recent-common-susceptible-ancestor age for 4,869 instances of resistance to 10 drugs.\n\nFindingsOf 8,550 isolates curated, 6,099 from 15 countries met criteria for molecular dating. The number of independent resistance acquisition events was lower than the number of resistant isolates across all countries, suggesting ongoing transmission of drug resistance. Ancestral age distributions supported the presence of old resistance, [≥]20 years prior, in the majority of countries. A consistent order of resistance acquisition was observed globally starting with resistance to isoniazid, but resistance ancestral age varied by country. We found a direct correlation between country wealth and resistance age (R2= 0.47, P-value= 0.014). Amplification of fluoroquinolone and second-line injectable resistance among multidrug-resistant isolates is estimated to have occurred very recently (median ancestral age 4.7 years IQR 1.9-9.8 prior to sample collection). We found the sensitivity of commercial molecular diagnostics for second-line resistance to vary significantly by country (P-value <0.0003)\n\nInterpretationOur results highlight that both resistance transmission and amplification are contributing to disease burden globally but are variable by country. The observation that wealthier nations are more likely to have old resistance suggests that programmatic improvements can reduce resistance amplification, but that fit resistant strains can circulate for decades subsequently.\n\nFundingThis work was supported by the NIH BD2K grant K01 ES026835, a Harvard Institute of Global Health Burke Fellowship (MF), Boston Childrens Hospital OFD/BTREC/CTREC Faculty Career Development Fellowship and Bushrod H. Campbell and Adah F. Hall Charity Fund/Charles A. King Trust Postdoctoral Fellowship (AD).\n\nResearch in contextO_ST_ABSEvidence before this studyC_ST_ABSAcquisition and spread of drug-resistance by Mycobacterium tuberculosis (MTB) varies across countries. Local factors driving evolution of drug resistance in MTB are not well studied.\n\nAdded value of this studyWe applied molecular dating to 6,099 global MTB patient isolates and found the order of resistance acquisition to be consistent across the countries examined, i.e. acquisition of isoniazid resistance first followed by rifampicin and streptomycin followed by resistance to other drugs. In all countries with data available there was evidence for transmission of resistant strains from patient-to-patient and in the majority for extended periods of time (>20 years).\n\nCountries with lower gross wealth indices were found to have more recent resistance acquisition to the drug rifampicin. Based on the resistance patterns identified in our study we estimate that commercial diagnostic tests vary considerably in sensitivity for second-line resistance diagnosis by country.\n\nImplications of all available evidenceThe longevity of resistant MTB in many parts of the world emphasizes its fitness for transmission and its continued threat to public health. The association between country wealth and recent resistance acquisition emphasizes the need for continued investment in TB care delivery and surveillance programs. Geographically relevant diagnostics that take into account a countrys unique distribution of resistance are necessary.

bioinformatics↗

bioRxiv2019

Novel truncating mutations in CTNND1 cause a dominant craniofacial and cardiac syndrome

AbstractCTNND1 encodes the p120-catenin (p120) protein, which has a wide range of functions, including the maintenance of cell-cell junctions, regulation of the epithelial-mesenchymal transition and transcriptional signaling. Due to advances in next generation sequencing, CTNND1 has been implicated in human diseases including cleft palate and blepharocheilodontic syndrome (BCD) albeit only recently. In this study, we identify eight novel protein-truncating variants, six de novo, in thirteen participants presenting with craniofacial dysmorphisms including cleft palate and hypodontia, as well as congenital cardiac anomalies, limb dysmorphologies and neurodevelopmental disorders. Using conditional deletions in mice as well as CRISPR/Cas9 approaches to target CTNND1 in Xenopus, we identified a subset of phenotypes that can be linked to p120-catenin in epithelial integrity and turnover, and additional phenotypes that suggest mesenchymal roles of CTNND1. We propose that CTNND1 variants have a wider developmental role than previously described, and that variations in this gene underlie not only cleft palate and BCD but may be expanded to a broader velocardiofacial-like syndrome.

genetics↗

bioRxiv2019

Relocation of macrophages maintains the barrier function of the urothelium and protects against persistent infection

Macrophages perform essential functions during bacterial infections, such as phagocytosis of pathogens and elimination of neutrophils to reduce spreading of infection, inflammation and tissue damage. The spatial distribution of macrophages is critical to respond to tissue specific adaptations upon infections. Using a novel algorithm for correlative mass spectrometry imaging and state-of-the-art multiplex microscopy, we report here that macrophages within the urinary bladder are positioned in the connective tissue underneath the urothelium. Invading uropathogenic E.coli induced an IL-6-dependent CX3CL1 expression by urothelial cells, facilitating relocation of macrophages from the connective tissue into the urothelium. These cells phagocytosed UPECs and eliminated neutrophils to maintain barrier function of the urothelium, preventing persistent and recurrent urinary tract infection. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY GRAPHICAL ABSTRACT C_FIG_DISPLAY

immunology↗

bioRxiv2018

Genotypic clustering does not imply recent tuberculosis transmission in a high prevalence setting: A genomic epidemiology study in Lima, Peru

BackgroundWhole genome sequencing (WGS) can elucidate Mycobacterium tuberculosis (Mtb) transmission patterns but more data is needed to guide its use in high-burden settings. In a household-based transmissibility study of 4,000 TB patients in Lima, Peru, we identified a large MIRU-VNTR Mtb cluster with a range of resistance phenotypes and studied host and bacterial factors contributing to its spread.\n\nMethodsWGS was performed on 61 of 148 isolates in the cluster. We compared transmission link inference using epidemiological or genomic data with and without the inclusion of controversial variants, and estimated the dates of emergence of the cluster and antimicrobial drug resistance acquisition events by generating a time-calibrated phylogeny. We validated our findings in genomic data from an outbreak of 325 TB cases in London. Using a larger set of 12,032 public Mtb genomes, we determined bacterial factors characterizing this cluster and under positive selection in other Mtb lineages.\n\nFindingsFour isolates were distantly related and the remaining 57 isolates diverged ca. 1968 (95% HPD: 1945-1985). Isoniazid resistance arose once, whereas rifampicin resistance emerged subsequently at least three times. Amplification of other drug resistance occurred as recently as within the last year of sampling. High quality PE/PPE variants and indels added information for transmission inference. We identified five cluster-defining SNPs, including esxV S23L to be potentially contributing to transmissibility.\n\nInterpretationClusters defined by MIRU-VNTR typing, could be circulating for decades in a high-burden setting. WGS allows for an improved understanding of transmission, as well as bacterial resistance and fitness factors.\n\nFundingThe study was funded by the National Institutes of Health (Peru Epi study U19-AI076217 and K01-ES026835 to MRF). The funding sources had no role in any aspect of the study, manuscript or decision to submit it for publication.\n\nResearch in contextO_ST_ABSEvidence before this studyC_ST_ABSUse of whole genome sequencing (WGS) to study tuberculosis (TB) transmission has proven to have higher resolution that traditional typing methods in low-burden settings. The implications of its use in high-burden settings are not well understood.\n\nAdded value of this studyUsing WGS, we found that TB clusters defined by traditional typing methods may be circulating for several decades. Genomic regions typically excluded from WGS analysis contain large amount of genetic variation that may affect interpretation of transmission events. We also identified five bacterial mutations that may contribute to transmission fitness.\n\nImplications of all the available evidenceAdded value of WGS for understanding TB transmission may be even higher in high-burden vs. low-burden settings. Methods integrating variants found in polymorphic sites and insertions and deletions are likely to have higher resolution. Several host and bacterial factors may be responsible for higher transmissibility that can be targets of intervention to interrupt TB transmission in communities.

microbiology↗

bioRxiv2016

Correcting Chimeric Crosstalk in Single Cell RNA-seq Experiments

As part of the process of preparing sequencing libraries that include unique molecular identifiers (UMIs) such as many single cell RNA-seq (scRNA-seq) libraries, a diverse template must be amplified. During amplification, spurious chimeric molecules can be formed between molecules originating in different cells. While several computational and experimental strategies have been suggested to mitigate the impact of chimeric molecules, suitable approaches for scRNA-seq experiments do not exist. We demonstrate that chimeras become increasingly problematic as samples are sequenced deeply and propose both supervised and unsupervised computational solutions. These solutions are validated in the context of a deeply sequenced species mixing experiment, and, orthogonally, using replicate PCR amplifications of the same scRNA-seq library. Our code is publicly available at https://github.com/asncd/schimera.

bioinformatics↗