Papers by Awadhesh kumar Pandit
Optimized protocol for assay for transposase-accessible chromatin by sequencing (ATAC-seq) from Drosophila melanogaster brain tissue
STAR protocols, Jun 1, 2023
Phosphoinositides (PI) are key regulators of cellular organization in eukaryotes and genes that t... more Phosphoinositides (PI) are key regulators of cellular organization in eukaryotes and genes that tune PI signalling are implicated in human disease mechanisms. Biochemical analyses and studies in cultured cells have identified a large number of proteins that can mediate PI signalling. However, the role of such proteins in regulating cellular processes in vivo and development in metazoans remains to be understood. Here we describe a set of CRISPR based genome engineering tools that allow the manipulation of each of these proteins with spatial and temporal control during metazoan development. We demonstrate the use of these reagents to deplete a set of 103 proteins individually in the Drosophila eye and identify several new molecules that control eye development. Our work demonstrates the power of this resource in uncovering the molecular basis of tissue homeostasis during normal development and in human disease biology.
bioRxiv (Cold Spring Harbor Laboratory), Mar 14, 2023
Genomic surveillance in response to coronavirus disease (COVID-19) pandemic is crucial for tracki... more Genomic surveillance in response to coronavirus disease (COVID-19) pandemic is crucial for tracking spread, identify variants of concern (VoCs) and understand the evolution of its etiological agent, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). India has experienced three waves of COVID-19 cases, which includes a deadly wave of COVID-19 that was driven by the Delta lineages (second/Delta wave) followed by another wave driven by the Omicron lineages (third/Omicron wave). These waves were particularly dramatic in the metropolitan cities due to high population density. We evaluated the prevalence, and mutational spectrum of SARS-CoV-2 variants/lineages in one such megapolis, Bengaluru city, across these three waves between October 2020 and June 2022. 15,134 SARS-CoV-2 samples were subjected to whole genome sequencing (WGS). Phylogenetic analysis revealed, SARS-CoV-2 variants in Bengaluru city belonged to 18 clades and 196 distinct lineages. As expected, the Delta lineages were the most dominant lineages during the second wave of COVID-19. The Omicron lineage BA.2 and its sublineages accounted for most of the COVID-19 cases in the third wave. Most number of amino acid changes were observed in spike protein. Among the 18 clades, majority of the mutations and least similarity at nucleotide sequence level with the reference genome were observed in Omicron clades.
BMC Genomics, Mar 23, 2023
Background Illumina sequencing platform requires base diversity in the initial 11 cycles for effi... more Background Illumina sequencing platform requires base diversity in the initial 11 cycles for efficient cluster identification and colour matrix estimation. This limitation yields low-quality data for amplicon libraries having homogeneous base composition. Spike-in of PhiX library ensures base diversity but reduces the overall number of sequencing reads for data analysis. To overcome such low diversity issues during amplicon sequencing on illumina platforms, we developed a high throughput single amplicon sequencing method by introducing 'N' (0-10) spacers in target gene amplification primers that are pooled for simple handling. We evaluated the efficiency of 'N' (0-10) spacer-linked primers by targeting bacterial 16S V3-V4 region, demonstrating heterogeneous base library construction. The addition of 'N' (0-10) spacers causes sequencing frameshift at every base that leads to base diversity and produces heterogeneous high quality reads within a single amplicon library. We have written a python based command-line software, "MetReTrim", to trim the 'N' (0-10) spacers from the raw reads (). We further demonstrated the accuracy of this method by comparative mock community analysis with standard illumina V3-V4 primer method. The ZymoBIOMICS™ microbial community DNA standard was used as a control for this study. We performed data analysisusing the DADA2 pipeline where taxonomy was assigned using SILVA database as reference. We observed no difference between the communities represented by our method and standard illumina V3-V4 primer method. This method eliminates the need for PhiX spike-in for single amplicon sequencing on illumina MiSeq platform. This allows for sequencing of more number of samples in a run and a reduction in the overall cost. Given that Illumina sequencing works on SBS chemistry irrespective of the platform (such as HiSeq, MiSeq, NextSeq, NovaSeq, etc.) we propose that this strategy of using 'N' (0-10) spacer-linked primer design can be adopted for generating high-quality single locus amplicon sequencing in a high throughput manner across the illumina platform subject to further validation.
Journal of Cell Science, 2021
Meta-analysis of transcripts in colon adenocarcinoma patient tissues led to the identification of... more Meta-analysis of transcripts in colon adenocarcinoma patient tissues led to the identification of a DNA damage responsive miR signature called DNA damage sensitive miRs (DDSMs). DDSMs were experimentally validated in the cancerous colon tissues obtained from an independent cohort of colon cancer patients and in multiple cellular systems with high levels of endogenous DNA damage. All the tested DDSMs were transcriptionally upregulated by a common intestine-specific transcription factor, CDX2. Reciprocally, DDSMs were repressed via the recruitment of HDAC1/2-containing complexes onto the CDX2 promoter. These miRs downregulated multiple key targets in the DNA damage response (DDR) pathway, namely BRCA1, ATM, Chk1 (also known as CHEK1) and RNF8. CDX2 directly regulated the DDSMs, which led to increased tumor volume and metastasis in multiple preclinical models. In colon cancer patient tissues, the DDSMs negatively correlated with BRCA1 levels, were associated with decreased probability ...
Innovative Systems Design and Engineering, 2014
This paper has focused on the step-by-step design of a high frequency medium size (1-3 kW) air-co... more This paper has focused on the step-by-step design of a high frequency medium size (1-3 kW) air-cored resonance transformer commonly called tesla coil, that can be easily used for measurements and general research. Therefore, the task is to minimize the number of stochastic and unknown parameters influencing the device functionality and pointing out pros and cons of each solution. The physical dimension of the tesla transformer has been chosen due to cost effectiveness and availability of the regarding components. This design has mainly two units’ viz., a power supply and tesla coil. The power supply which fed the Tesla coil having voltage rating 12kV and current rating 120mA has been designed by using three microwave oven components. These components are assembled as dual MOTs voltage doubler circuit and necessary simulation has been carried out by using MicroSim Schematics for the verification of performance. Keywords: Microwave Oven , Transformer, Multi mini Capacitors, MicroSim S...
Immune profile and responses of a novel Dengue DNA vaccine encoding EDIII-NS1 consensus design based on Indo-African sequences
Following the recent clinical clearance of an Indian DNA COVID-19 vaccine, India and Africa are p... more Following the recent clinical clearance of an Indian DNA COVID-19 vaccine, India and Africa are potential regions where DNA vaccines may become a major delivery system subject to a range of immunological and regulatory scrutiny. The ongoing COVID pandemic highlights the need to tackle viral variants and expand the number of antigens and assess diverse delivery systems. To address some of these key issues, we have created a Dengue DNA vaccine candidate with the EDIII region as the key antigen given the promise of this segment in not causing ADE, a challenge with this disease. In addition, we have added the NS1 region to broaden the immune response. Following a large Dengue viral sequencing exercise in India, complemented with data from east Africa, our approach was to generate a consensus of four serotypes ED3-NS1 vaccine to explore tackling the issue of diversity. Our In silico structural analysis of EDIII consensus vaccine sequence revealed that epitopes are structurally conserved ...
BackgroundAfrica has one of the highest incidences of gonorrhoea, but not much information is ava... more BackgroundAfrica has one of the highest incidences of gonorrhoea, but not much information is available on the relatedness with strains from other geographical locations. Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a major public health threat, with the bacteria gaining resistance to most of the available antibiotics, compromising treatment across the world. Whole-genome sequencing is an efficient way of predicting AMR determinants and their spread in the human population. Previous studies on Kenyan gonococcal samples have focused on plasmid-mediated drug resistance and fluoroquinolone resistance using Illumina sequencing.Recent advances in next-generation sequencing technologies like Oxford Nanopore Technology (ONT) have helped in the generation of longer reads of DNA in a shorter duration with lower cost. However, long-reads are error-prone. The increasing accuracy ofbase-calling algorithms, high throughput, error-correction strategies, and ease of using the mobile ...
Journal of Experimental Botany, 2019
miRNA-mediated silencing of R2R3 MYB transcription factor promotes the production of specific sec... more miRNA-mediated silencing of R2R3 MYB transcription factor promotes the production of specific secondary metabolites in grapes.
Assessing diet variability between species, populations and individuals is of main importance to ... more Assessing diet variability between species, populations and individuals is of main importance to better understand the biology of bats and design conservation strategies. Although the advent of metabarcoding has facilitated such analyses, this approach does not come without challenges. Biases may occur throughout the whole experiment, from fieldwork to biostatistics, resulting in the detection of false negatives, false positives or low taxonomic resolution. We detail a rigorous metabarcoding approach based on a two-step PCR protocol enabling the "all at once" taxonomic identification of bats and their arthropod preys for several hundreds of samples. Our study includes faecal pellets collected in France from 357 bats representing 16 species, as well as insect mock communities that mimic bat meals of known composition, negative and positive controls. All samples were analysed in triplicate. We compare the efficiency of DNA extraction methods and we evaluate the effectiveness...
Single nucleotide polymorphism in sugar pathway and disease resistance genes in sugarcane
Plant cell reports, Jan 11, 2016
Single nucleotide polymorphism in sugar pathway and disease resistance genes showing genetic asso... more Single nucleotide polymorphism in sugar pathway and disease resistance genes showing genetic association with sugar content and red rot resistance would be useful in marker-assisted genetic improvement of sugarcane. Validation and genotyping of potential sequence variants in candidate genes are necessary to understand their functional significance and trait association potential. We discovered, characterized, validated and genotyped SNPs and InDels in sugar pathway and disease resistance genes of Saccharum complex and sugarcane varieties using amplicon sequencing and CAPS assays. The SNPs were abundant in the non-coding 3'UTRs than 5'UTRs and coding sequences depicting a strong bias toward C to T transition substitutions than transversions. Sequencing of cloned amplicons validated 61.6 and 45.2 % SNPs detected in silico in 21 sugar pathway and 16 disease resistance genes, respectively. Sixteen SNPs in four sugar pathway genes and 10 SNPs in nine disease resistance genes were...
Low Socioeconomic Status Is an Independent Risk Factor for Ischemic Stroke: A Case-Control Study in North Indian Population
Neuroepidemiology, Jan 21, 2015
Stroke is a multifactorial disease and is influenced by complex environmental interactions. The c... more Stroke is a multifactorial disease and is influenced by complex environmental interactions. The contribution of various risk factors to the burden of stroke worldwide is not well known, particularly in developing countries. The present case-control study is aimed at exploring the association between a low socioeconomic status and the risk of ischemic stroke among the North Indian population. The study design was a hospital-based, case-control study. Age- and sex-matched controls were included. The demographic characteristics and risk factor variables were documented by means of a personal interview through a standardized case record form. The household asset index for determining the socioeconomic status (HAISS) was used for the assessment of the socioeconomic status of the population. HAISS was validated with the widely used Kuppuswamy scale for measurement of socioeconomic status. The multivariable logistic regression model was used to estimate the odds ratio associated with strok...
Aging cell, Jan 26, 2015
Advanced glycation end products (AGEs) are formed when glucose reacts nonenzymatically with prote... more Advanced glycation end products (AGEs) are formed when glucose reacts nonenzymatically with proteins; these modifications are implicated in aging and pathogenesis of many age-related diseases including type II diabetes, atherosclerosis, and neurodegenerative disorders. Thus, pharmaceutical interventions that can reduce AGEs may delay age-onset diseases and extend lifespan. Using LC-MS(E) , we show that rifampicin (RIF) reduces glycation of important cellular proteins in vivo and consequently increases lifespan in Caenorhabditis elegans by up to 60%. RIF analog rifamycin SV (RSV) possesses similar properties, while rifaximin (RMN) lacks antiglycation activity and therefore fails to affect lifespan positively. The efficacy of RIF and RSV as potent antiglycating agents may be attributed to the presence of a p-dihydroxyl moiety that can potentially undergo spontaneous oxidation to yield highly reactive p-quinone structures, a feature absent in RMN. We also show that supplementing rifamp...
Plant Genome, 2009
The genome of tomato (Solanum lycopersicum L.) is being sequenced by an international consortium ... more The genome of tomato (Solanum lycopersicum L.) is being sequenced by an international consortium of 10 countries (Korea, China, the United Kingdom, India, the Netherlands, France, Japan, Spain, Italy, and the United States) as part of the larger "International Solanaceae Genome Project (SOL): Systems Approach to Diversity and Adaptation" initiative. The tomato genome sequencing project uses an ordered bacterial artifi cial chromosome (BAC) approach to generate a high-quality tomato euchromatic genome sequence for use as a reference genome for the Solanaceae and euasterids. Sequence is deposited at GenBank and at the SOL Genomics Network (SGN). Currently, there are around 1000 BACs fi nished or in progress, representing more than a third of the projected euchromatic portion of the genome. An annotation effort is also underway by the International Tomato Annotation Group. The expected number of genes in the euchromatin is ~40,000, based on an estimate from a preliminary annotation of 11% of fi nished sequence. Here, we present this fi rst snapshot of the emerging tomato genome and its annotation, a short comparison with potato (Solanum tuberosum L.) sequence data, and the tools available for the researchers to exploit this new resource are also presented. In the future, whole-genome shotgun techniques will be combined with the BAC-by-BAC approach to cover the entire tomato genome. The high-quality reference euchromatic tomato sequence is expected to be near completion by 2010.
Theoretical and Applied Genetics, 2008
Molecular Genetics and Genomics, 2005
Combining QTL mapping and transcriptome profiling of bulked RILs for identification of functional polymorphism for salt tolerance genes in rice (Oryza sativa L.)
Molecular Genetics and Genomics, 2010
Received: 3 March 2010 / Accepted: 11 June 2010 © Springer-Verlag 2010 ... Abstract Identificatio... more Received: 3 March 2010 / Accepted: 11 June 2010 © Springer-Verlag 2010 ... Abstract Identification of genes for quantitative traits is diYcult using any single approach due to complex inheri-tance of the traits and limited resolving power of the indi-vidual techniques. Here a combination ...
Isolation and molecular characterization of dTnp1, a mobile and defective transposable element of Nicotiana plumbaginifolia
Molecular and General Genetics MGG, 1994
By Northern blot analysis of nitrate reductase-deficient mutants of Nicotiana plumbaginifolia, we... more By Northern blot analysis of nitrate reductase-deficient mutants of Nicotiana plumbaginifolia, we identified a mutant (mutant D65), obtained after ?-ray irradiation of protoplasts, which contained an insertion sequence in the nitrate reductase (NR) mRNA. This insertion sequence was localized by polymerase chain reaction (PCR) in the first exon of NR and was also shown to be present in the NR gene. The mutant gene contained a 565 by insertion sequence that exhibits the sequence characteristics of a transposable element, which was thus named dTnp1. The dTnp1 element has 14 by terminal inverted repeats and is flanked by an 8-bp target site duplication generated upon transposition. These inverted repeats have significant sequence homology with those of other transposable elements. Judging by its size and the absence of a long open reading frame, dTnp1 appears to represent a defective, although mobile, transposable element. The octamer motif TTTAGGCC was found several times in direct orientation near the 5' and 3' ends of dTnp1 together with a perfect palindrome located after the 5' inverted repeat. Southern blot analysis using an internal probe of dTnp1 suggested that this element occurs as a single copy in the genome of N. plumbaginifolia. It is also present in N. tabacum, but absent in tomato or petunia. The dTnp1 element is therefore of potential use for gene tagging in Nicotiana species.
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Papers by Awadhesh kumar Pandit