Biology subjects

Tallapaka, K. B.

Publications and source records attributed to Tallapaka, K. B..

7 recordsLinked to original sources

bioRxiv2024

Comprehensive benchmarking of tools for nanopore-based detection of DNA methylation

Long read sequencing technologies such as Oxford Nanopore (ONT) offer direct, simultaneous detection of DNA base modifications. The recent migration of ONT to the upgraded R10 chemistry has spurred the development of diverse methylation detection models. However, their performance and accuracy remain unclear. Here, leveraging diverse bacterial, plant, and mammalian datasets, we systematically evaluate the current landscape of tools and models for studying DNA methylation using nanopore sequencing. Our results demonstrate that the older models remain the best choice for studying CpG methylation. We note substantial improvement of newer tools in identifying 5-methylcytosine in non-CG contexts, 6-methyladenine, and 4-methylcytosine. We highlight the sensitivity of various tools to confounding methylation nearby. We also assess the computational performance of various tools, and effects of sequencing depth, methylation abundance, read quality, and basecalling mode. Our reusable pipelines and fully open access datasets provide a framework of resources to empower future benchmarking efforts. Our work thus details the strengths and limitations of the state-of-the-art methylation models and outlines practical guidelines for researchers using nanopore sequencing to study DNA modifications.

genomics↗

bioRxiv2024

NEMO: Improved and accurate models for identification of 6mA using Nanopore sequencing

DNA methylation plays a key role in epigenetic regulation across lifeforms. Nanopore sequencing enables direct detection of base modifications. While multiple tools are currently available for studying 5-methylcytosine (5mC), there is a paucity of models that can detect 6-methyladenine (6mA) from raw nanopore data. Leveraging the motif-driven nature of bacterial methylation systems, we generated 6mA identification models that vastly surpass the accuracy of the current best model. Our work enables the study of 6mA at a single-base resolution in new as well as existing nanopore datasets.

genomics↗

bioRxiv2023

Composition of nasopharyngeal microbiota in individuals with SARS-COV-2 infection across three COVID-19 waves in India

Multiple variants of the SARS-CoV-2 virus have been plaguing the world through successive waves of infection over the past three years. Studies by independent research groups across geographies have shown that the microbiome composition in COVID-19 patients (CP) differ from that of healthy individuals (CN). However, such observations were based on limited-sized sample-sets collected primarily from the early days of the pandemic. Here, we study the nasopharyngeal microbiota in COVID-19 patients, wherein the samples have been collected across the three COVID-19 waves witnessed in India, which were driven by different variants of concern. We also present the variations in microbiota of symptomatic vs asymptomatic COVID-19 patients. The nasopharyngeal swabs were collected from 589 subjects providing samples for diagnostics purposes at Centre for Cellular and Molecular Biology (CSIR-CCMB), Hyderabad, India. CP showed a marked shift in the microbial diversity and composition compared to CN, in a wave-dependent manner. Rickettsiaceae was the only family that was noted to be consistently depleted in CP samples across the waves. The genera Staphylococcus, Anhydrobacter, Thermus, and Aerococcus were observed to be highly abundant in the symptomatic CP patients when compared to the asymptomatic group. In general, we observed a decrease in the burden of opportunistic pathogens in the host microbiota during the later waves of infection. To our knowledge, this is the first longitudinal study which was designed to understand the relation between the evolving nature of the virus and the changes in the human nasopharyngeal microbiota. Such studies not only pave way for better understanding of the disease pathophysiology but also help gather preliminary evidence on whether interventions to the host microbiota can help in better protection or faster recovery.

microbiology↗

bioRxiv2021

Host transcriptional response to SARS-CoV-2 infection in COVID-19 patients

BackgroundOne of the most perplexing aspects of infection with the SARS-CoV-2 virus has been the variable response elicited in its human hosts. Investigating the transcriptional changes in individuals affected by COVID-19 can help understand and predict the degree of illness and guide clinical outcomes in diverse backgrounds. MethodsAnalysis of host transcriptome variations via RNA sequencing from naso/oropharyngeal swabs of COVID-19 patients. ResultsWe report strong upregulation of the innate immune response, especially type I interferon pathway, upon SARS-CoV-2 infection. Upregulated genes were subjected to a comparative meta-analysis using global datasets to identify a common network of interferon stimulated and viral response genes that mediate the host response and resolution of infection. A large proportion of mis-regulated genes showed a reduction in expression level, suggesting an overall decrease in host mRNA production. Significantly downregulated genes included those encoding olfactory, taste and neuro-sensory receptors. Many pro-inflammatory markers and cytokines were also downregulated or remained unchanged in the COVID-19 patients. Finally, a large number of non-coding RNAs were identified as down-regulated, with a few of the lncRNAs associated with functional roles in directing the response to viral infection. ConclusionsSARS-CoV-2 infection results in the robust activation of the bodys innate immunity. Reduction of gene expression is well correlated with the clinical manifestations and symptoms of COVID-19 such as the loss of smell and taste, and myocardial and neurological complications. This study provides a critical dataset of genes that will enhance our understanding of the nature and prognosis of COVID-19.

bioinformatics↗

bioRxiv2021

Dry Swab Method of sample collection for SARS-CoV2 testing can be used for culturing virus

Back groundEarlier studies suggested the use of dry swab method for SARS-CoV-2 detection as it does not need VTM and subsequent RNA extraction step making the process cheaper, safer and faster. In this study we explore whether the virus in the dry swab is viable and can be cultured and propagated. MethodSwabs were spiked with SARS-CoV-2 and stored in three different conditions: a) as dry swab (SD, eluted in 1 mL DMEM), b) in 1 mL of Viral Transport Medium (SVTM), and c) in 1 mL of Tris-EDTA buffer (STE). The sample groups were stored either at room temperature (RT, 25{degrees}C{+/-}1{degrees}C) or at 4{degrees}C for 1, 4, 8, 12, 24, 48 and 72 hours before being used as viral inoculums for the propagation studies in Vero cells. ResultsThe RT-qPCR data suggests that SD incubated both at RT and 4{degrees}C harbors viral particles that are viable and culturable at par with SVTM and STE. ConclusionThe dry swab method, in addition to its advantages in detection of the virus, also renders viable viral particles that can be cultured and propagated.

microbiology↗

bioRxiv2020

An Inexpensive RT-PCR Endpoint Diagnostic Assay for SARS-CoV-2 Using Nested PCR: Direct Assessment of Detection Efficiency of RT-qPCR Tests and Suitability for Surveillance

With a view to extending testing capabilities for the ongoing SARS-CoV-2 pandemic we have developed a test that lowers cost and does not require real time quantitative reverse transcription polymerase chain reaction (RT-qPCR). We developed a reverse transcription nested PCR endpoint assay (RT-nPCR) and showed that RT-nPCR has comparable performance to the standard RT-qPCR test. In the course of comparing the results of both tests, we found that the standard RT-qPCR test can have low detection efficiency (less than 50%) in a real testing scenario which may be only partly explained by low viral representation in many samples. This finding points to the importance of directly monitoring detection efficiency in test environments. We also suggest measures that would improve detection efficiency.

molecular biology↗

bioRxiv2020

Improved and Simplified Diagnosis of Covid-19 using TE Extraction from Dry Swabs

Rigorous testing is the way forward to fight the Covid-19 pandemic. Here we show that the currently used and most reliable RT-PCR based SARS-CoV-2 procedure can be further simplified to make it faster, safer and economical by bypassing the RNA isolation step. The modified method is not only fast and convenient but also at par with the traditional method in terms of accuracy, and therefore, can be used for mass screening. Our method takes about half the time and is cheaper by about 40% compared to current most widely used method. We also provide a variant of the new method that increases the efficiency of detection by about 20% compared to the currently used method. Taken together, we demonstrate a more effective and reliable method of SARS-CoV-2 detection.

molecular biology↗