Biology subjects

Mahajan, S.

Publications and source records attributed to Mahajan, S..

50 records · Page 3Linked to original sources

bioRxiv2021

Genome sequencing and assembly of Tinospora cordifolia (Giloy) plant

During the ongoing COVID-19 pandemic Tinospora cordifolia also known as Giloy gained immense popularity and use due to its immunity-boosting function and anti-viral properties. T. cordifolia is among the most important medicinal plants that has numerous therapeutic applications in health due to the production of a diverse array of secondary metabolites. Therefore, to gain genomic insights into the medicinal properties of T. cordifolia, the first genome sequencing was carried out using 10x Genomics linked read technology and the draft genome assembly comprised of 1.01 Gbp. This is also the first genome sequenced from the plant family Menispermaceae. We also performed the first genome size estimation for T. cordifolia and was found to be 1.13 Gbp. The deep sequencing of transcriptome from the leaf tissue was also performed followed by transcriptomic analysis to gain insights into the gene expression and functions. The genome and transcriptome assemblies were used to construct the gene set in T. cordifolia that resulted in 19,474 coding gene sequences. Further, the phylogenetic position of T. cordifolia was also determined through the construction of a genome-wide phylogenetic tree using 35 other dicot species and one monocot species as an outgroup species.

genomics↗

bioRxiv2021

Meeting experiments at the diffraction barrier: an in-silico widefield fluorescence microscopy

Fluorescence microscopy allows the visualization of live cells and their components, but even with advances in super- resolution microscopy, atomic resolution remains unattainable. On the other hand, molecular simulations (MS) can easily access atomic resolution, but comparison with experimental microscopy images has not been possible. In this work, a novel in-silico widefield fluorescence microscopy is proposed, which reduces the resolution of MS to generate images comparable to experiments. This technique will allow cross-validation and compound the knowledge gained from experiments and MS. We demonstrate that in-silico images can be produced with different optical axis, object focal planes, exposure time, color combinations, resolution, brightness and amount of out-of-focus fluorescence. This allows the generation of images that resemble those obtained from widefield, confocal, light-sheet, two-photon and super-resolution microscopy. This technique not only can be used as a standalone visualization tool for MS, but also lays the foundation for other in-silico microscopy methods.

biophysics↗

bioRxiv2021

Evolutionary jumps in bacterial GC content

Genomic GC content is a fundamental molecular trait linked with many key genomic features such as codon and amino acid use. Across bacteria, GC content is surprisingly diverse and has been studied for many decades; yet its evolution remains incompletely understood. Since it is difficult to observe GC content evolve on laboratory time-scales, phylogenetic comparative approaches are instrumental; but this dimension is rarely studied systematically in the case of bacterial GC content. We applied phylogenetic comparative models to analyse GC content evolution in multiple bacterial groups across two major bacterial phyla. We find that GC content diversifies via a combination of gradual evolution and evolutionary "jumps". Surprisingly, unlike prior reports that solely focused on reductions in GC, we found a comparable number of jumps with both increased and decreased GC content. Overall, many of the identified jumps occur in lineages beyond the well-studied peculiar examples of endosymbiotic and AT-rich marine bacteria, and do not support the predicted role of oxygen dependence. Our analysis of rapid and large shifts in GC content thus identifies new clades and novel contexts to further understand the ecological and evolutionary drivers of this important genomic trait.

evolutionary biology↗

bioRxiv2020

TCRMatch: Predicting T-cell receptor specificity based on sequence similarity to previously characterized receptors

The adaptive immune system in vertebrates has evolved to recognize non-self-antigens, such as proteins expressed by infectious agents and mutated cancer cells. T cells play an important role in antigen recognition by expressing a diverse repertoire of antigen-specific receptors, which bind epitopes to mount targeted immune responses. Recent advances in high-throughput sequencing have enabled the routine generation of T-cell receptor (TCR) repertoire data. Identifying the specific epitopes targeted by different TCRs in these data would be valuable. To accomplish that, we took advantage of the ever-increasing number of TCRs with known epitope specificity curated in the Immune Epitope Database (IEDB) since 2004. We compared six metrics of sequence similarity to determine their power to predict if two TCRs have the same epitope specificity. We found that a comprehensive k-mer matching approach produced the best results, which we have implemented into TCRMatch, an openly accessible tool (http://tools.iedb.org/tcrmatch/) that takes TCR {beta}-chain CDR3 sequences as an input, identifies TCRs with a match in the IEDB, and reports the specificity of each match. We anticipate that this tool will provide new insights into T cell responses captured in receptor repertoire and single cell sequencing experiments and will facilitate the development of new strategies for monitoring and treatment of infectious, allergic, and autoimmune diseases, as well as cancer.

immunology↗

bioRxiv2020

Immunodominant T-cell epitopes from the SARS-CoV-2 spike antigen reveal robust pre-existing T-cell immunity in unexposed individuals

The COVID-19 pandemic has revealed a range of disease phenotypes in infected patients with asymptomatic, mild or severe clinical outcomes, but the mechanisms that determine such variable outcomes remain unresolved. In this study, we identified immunodominant CD8 T-cell epitopes in the RBD and the non-RBD domain of the spike antigen using a novel TCR-binding algorithm. A selected pool of 11 predicted epitopes induced robust T-cell activation in unexposed donors demonstrating pre-existing CD4 and CD8 T-cell immunity to SARS-CoV-2 antigen. The T-cell reactivity to the predicted epitopes was higher than the Spike-S1 and S2 peptide pools containing 157 and 158 peptides both in unexposed donors and in convalescent patients suggesting that strong T-cell epitopes are likely to be missed when larger peptide pools are used in assays. A key finding of our study is that pre-existing T-cell immunity to SARS-CoV-2 is contributed by TCRs that recognize common viral antigens such as Influenza and CMV, even though the viral epitopes lack sequence identity to the SARS-CoV-2 epitopes. This finding is in contrast to multiple published studies in which pre-existing T-cell immunity is suggested to arise from shared epitopes between SARS-CoV-2 and other common cold-causing coronaviruses. Whether the presence of pre-existing T-cell immunity provides protection against COVID-19 or contributes to severe disease phenotype remains to be determined in a larger cohort. However, our findings raise the expectation that a significant majority of the global population is likely to have SARS-CoV-2 reactive T-cells because of prior exposure to flu and CMV viruses, in addition to common cold-causing coronaviruses.

immunology↗

bioRxiv2020

Genome sequencing of turmeric provides evolutionary insights into its medicinal properties

Curcuma longa, or turmeric, is traditionally known for its immense medicinal properties and has diverse therapeutic applications. However, the absence of a reference genome sequence is a limiting factor in understanding the genomic basis of the origin of its medicinal properties. In this study, we present the draft genome sequence of Curcuma longa, the first species sequenced from Zingiberaceae plant family, constructed using 10x Genomics linked reads. For comprehensive gene set prediction and for insights into its gene expression, the transcriptome sequencing of leaf tissue was also performed. The draft genome assembly had a size of 1.24 Gbp with ~74% repetitive sequences, and contained 56,036 coding gene sequences. The phylogenetic position of Curcuma longa was resolved through a comprehensive genome-wide phylogenetic analysis with 16 other plant species. Using 5,294 orthogroups, the comparative evolutionary analysis performed across 17 species including Curcuma longa revealed evolution in genes associated with secondary metabolism, plant phytohormones signaling, and various biotic and abiotic stress tolerance responses. These mechanisms are crucial for perennial and rhizomatous plants such as Curcuma longa for defense and environmental stress tolerance via production of secondary metabolites, which are associated with the wide range of medicinal properties in Curcuma longa.

genomics↗

bioRxiv2020

Label-free and Multimodal Second Harmonic Generation Light Sheet Microscopy

Light sheet microscopy (LSM) has emerged as one of most profound three dimensional (3D) imaging tools in the life sciences over the last decade. However, LSM is currently performed with fluorescence detection on one- or multi-photon excitation. Label-free LSM imaging approaches have been rather limited. Second Harmonic Generation (SHG) imaging is a label-free technique that has enabled detailed investigation of collagenous structures, including its distribution and remodelling in cancers and respiratory tissue, and how these link to disease. SHG is generally regarded as having only forward- and back-scattering components, apparently precluding the orthogonal detection geometry used in Light Sheet Microscopy. In this work we demonstrate SHG imaging on a light sheet microscope (SHG-LSM) using a rotated Airy beam configuration that demonstrates a powerful new approach to direct, without any further processing or deconvolution, 3D imaging of harmonophores such as collagen in biological samples. We provide unambiguous identification of SHG signals on the LSM through its wavelength and polarisation sensitivity. In a multimodal LSM setup we demonstrate that SHG and two-photon signals can be acquired on multiple types of different biological samples. We further show that SHG-LSM is sensitive to changes in collagen synthesis within lung fibroblast 3D cell cultures. This work expands on the existing optical methods available for use with light sheet microscopy, adding a further label-free imaging technique which can be combined with other detection modalities to realise a powerful multi-modal microscope for 3D bioimaging.

biophysics↗

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↗

bioRxiv2020

Analysis of the potential impact of genomic variants in SARS-CoV-2 genomes from India on molecular diagnostic assays

An isolated epidemic of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causing Coronavirus Diseases (COVID-19) originating in Wuhan, China has now rapidly emerged into a global pandemic affecting millions of people worldwide. Molecular detection of SARS-CoV-2 using reverse transcription polymerase chain reaction (RT-PCR) forms the mainstay in screening, diagnosis and epidemiology of disease. The virus has been evolving through base substitutions. The recent availability of genomes of SARS-CoV-2 isolates from different countries including India motivated us to assess the presence and potential impact of variations in target sites for the oligonucleotide primers and probes used in molecular diagnosis. We catalogued a total of 132 primers or probes sequences from the literature and the public domain. Our analysis revealed a total of 125 unique genetic variants in 80 either primers or probes binding sites. A total of 13 unique variants had allele frequency of [≥] 1% in Indian SARS-CoV-2 genomes mapped to the primers or probes binding sites. A total of 15 primers or probes binding sites had cumulative variant frequency of [≥] 1% in the SARS-CoV-2 genomes. These included primers or probes sites which are widely used in India and across the world for molecular diagnosis as well as approved by national and international agencies. This highlights the need for sequencing genomes of emerging pathogens to make evidence based policies for development and approval of diagnostics. To the best of our knowledge, ours is the most comprehensive analysis of genomic variants in genomes of SARS-CoV-2 isolates from India and their potential impact on efficacy of molecular diagnostics.

genomics↗

bioRxiv2020

The genome sequence of Aloe vera reveals adaptive evolution of drought tolerance mechanisms

Aloe vera is a species from Asphodelaceae plant family having unique characteristics such as drought resistance and also possesses numerous medicinal properties. However, the genetic basis of these phenotypes is yet unknown, primarily due to the unavailability of its genome sequence. In this study, we report the first Aloe vera draft genome sequence comprising of 13.83 Gbp and harboring 86,177 coding genes. It is also the first genome from the Asphodelaceae plant family and is the largest angiosperm genome sequenced and assembled till date. Further, we report the first genome-wide phylogeny of monocots with Aloe vera using 1,440 one-to-one orthologs that resolves the genome-wide phylogenetic position of Aloe vera with respect to the other monocots. The comprehensive comparative analysis of Aloe vera genome with the other available high-quality monocot genomes revealed adaptive evolution in several genes of the drought stress response, CAM pathway, and circadian rhythm in Aloe vera. Further, genes involved in DNA damage response, a key pathway in several biotic and abiotic stress response mechanisms, were found to be positively selected. This provides the genetic basis of the evolution of drought stress tolerance capabilities of Aloe vera. This also substantiates the previously suggested notion that the evolution of unique characters in this species is perhaps due to selection and adaptive evolution rather than the phylogenetic divergence or isolation.

genomics↗

bioRxiv2020

Super-resolved polarisation-enhanced second harmonic generation for direct imaging of nanoscale changes in collagen architecture

Super-resolution (SR) optical microscopy has allowed the investigation of many biological structures below the diffraction limit, however, most of the techniques are hampered by the need for fluorescent labels. Non-linear label-free techniques such as Second Harmonic Generation (SHG) provide structurally specific contrast without the addition of exogenous labels, allowing observation of unperturbed biological systems. Here we achieve super-resolution SHG (SR-SHG) for the first time. We use the photonic nanojet (PNJ) phenomena to achieve a resolution of ~{lambda}/6 with respect to the fundamental wavelength, a ~2.7-fold improvement over diffraction-limited SHG under the same imaging conditions. Crucially we find that the polarisation properties of excitation are maintained in a PNJ allowing the resolution to be further enhanced by detection of polarisation-resolved SHG (p-SHG) by observing anisotropy in signals. These new findings allowed us to visualise biological SHG-active structures such as collagen at an unprecedented and previously unresolvable spatial scale. Moreover, we demonstrate that the use of an array of self-assembled high-index spheres overcomes the issue of a limited field of view for such a method, allowing PNJ-assisted SR-SHG to be used over a large area. Dysregulation of collagen at the nanoscale occurs in many diseases and is an underlying cause in diseases such as lung fibrosis. Here we are able to demonstrate that pSR-SHG allows unprecedented observation of changes at the nanoscale that are invisible by conventional diffraction-limited SHG imaging. The ability to non-destructively image SHG-active biological structures without labels at the nanoscale with a relatively simple optical method heralds the promise of a new tool to understand biological phenomena and drive drug discovery.

biophysics↗

bioRxiv2018

Massive gene amplification on a recently formed Drosophila Y chromosome

Widespread loss of genes on the Y is considered a hallmark of sex chromosome differentiation. Here we show that the initial stages of Y evolution are driven by massive amplification of distinct classes of genes. The neo-Y chromosome of Drosophila miranda initially contained about 3000 protein-coding genes, but has gained over 3200 genes since its formation about 1.5 MY ago, primarily by tandem amplification of protein-coding genes ancestrally present on this chromosome. We show that distinct evolutionary processes may account for this drastic increase in gene number on the Y. Testis-specific and dosage sensitive genes appear to have amplified on the Y to increase male fitness. A distinct class of meiosis-related multi-copy Y genes independently co-amplified on the X, and their expansion is likely driven by conflicts over segregation. Co-amplified X/Y genes are highly expressed in testis, enriched for meiosis and RNAi functions, and are frequently targeted by small RNAs in testis. This suggests that their amplification is driven by X vs. Y antagonism for increased transmission, where sex chromosome drive suppression is likely mediated by sequence homology between the suppressor and distorter, through RNAi mechanism. Thus, our analysis suggests that newly emerged sex chromosomes are a battleground for sexual and meiotic conflict.

evolutionary biology↗

bioRxiv2018

De novo assembly of a young Drosophila Y chromosome using Single-Molecule sequencing and Chromatin Conformation capture

While short-read sequencing technology has resulted in a sharp increase in the number of species with genome assemblies, these assemblies are typically highly fragmented. Repeats pose the largest challenge for reference genome assembly, and pericentromeric regions and the repeat-rich Y chromosome are typically ignored from sequencing projects. Here, we assemble the genome of Drosophila miranda using long reads for contig formation, chromatin interaction maps for scaffolding and short reads, optical mapping and BAC clone sequencing for consensus validation. Our assembly recovers entire chromosomes and contains large fractions of repetitive DNA, including ~41.5 Mb of pericentromeric and telomeric regions, and >100Mb of the recently formed highly repetitive neo-Y chromosome. While Y chromosome evolution is typically characterized by global sequence loss and shrinkage, the neo-Y increased in size by almost 3-fold, due to the accumulation of repetitive sequences. Our high-quality assembly allows us to reconstruct the chromosomal events that have led to the unusual sex chromosome karyotype in D. miranda, including the independent de novo formation of a pair of sex chromosomes at two distinct time points, or the reversion of a former Y chromosome to an autosome.

genomics↗

bioRxiv2017

PB-kPRED: Knowledge-Based Prediction Of Protein Backbone Conformation Using A Structural Alphabet

Libraries of structural prototypes that abstract protein local structures are known as structural alphabets and have proven to be very useful in various aspects of protein structure analyses and predictions. One such library, Protein Blocks (PBs), is composed of 16 standard 5-residues long structural prototypes. This form of analyzing proteins involves drafting its structure as a string of PBs. Thus, predicting the local structure of a protein in terms of protein blocks is a step towards the objective of predicting its 3-D structure. Here a new approach, kPred, is proposed towards this aim that is independent of the evolutionary information available. It involves (i) organizing the structural knowledge in the form of a database of pentapeptide fragments extracted from all protein structures in the PDB and (ii) apply a purely knowledge-based algorithm, not relying on secondary structure predictions or sequence alignment profiles, to scan this database and predict most probable backbone conformations for the protein local structures.\n\nBased on the strategy used for scanning the database, the method was able to achieve efficient mean Q16 accuracies between 40.8% and 66.3% for a non-redundant subset of the PDB filtered at 30% sequence identity cut-off. The impact of these scanning strategies on the prediction was evaluated and is discussed. A scoring function that gives a good estimate of the accuracy of prediction was further developed. This score estimates very well the accuracy of the algorithm (R2 of 0.82). An online version of the tool is provided freely for non-commercial usage at http://www.bo-protscience.fr/kpred/.

bioinformatics↗