Biology subjects

Maheshwari, S.

Publications and source records attributed to Maheshwari, S..

10 recordsLinked to original sources

bioRxiv2025

Comprehensive benchmarking of somatic structural variant detection at ultra-low allele fractions

Postzygotic mosaicism gives rise to somatic structural variants (SVs) at ultra-low variant allele fractions (VAFs), which pose challenges for detection due to the high-coverage sequencing required and noise introduced by sequencing artifacts. Although somatic SV detection has been extensively studied in cancer, these studies are not directly applicable to the study of tissue mosaicism, as they rely on matched normals, target higher VAF ranges, and are enriched for different types of SVs. We present comprehensive benchmark data and best practices for non-cancer somatic SV detection. We created a synthetic mosaic sample by combining six HapMap individuals at varying proportions, generating allele fractions as low as 0.25%. This sample was sequenced to [~]2,300x total coverage using Illumina, PacBio, and Nanopore technologies across multiple sequencing centers. A high-confidence benchmark SV set containing over 21,000 pseudo-somatic insertions and deletions [≥]50bp was derived from haplotype-resolved assemblies. We evaluated 12 SV discovery pipelines and identified caller-specific strengths and sequencing platform-specific shortcomings. We find that short read-based approaches show reduced recall for insertions and repeat-associated SVs, whereas long-read sequencing achieves high accuracy throughout the genome, increasing linearly with coverage. The best algorithms sensitivity exceeded 80% for VAFs [≥]4% and 15% for VAFs of 0.5-1% with 60x coverage. The publicly available benchmarking data and comparative analysis of current methods provide a foundation for robust discovery of SV mosaicism in non-cancer tissues..

bioinformatics↗

bioRxiv2020

Leveraging a WGS compression and indexing format with dynamic graph references to call structural variants.

We introduce a novel structural variant calling pipeline (BioGraph) that leverages a read compression and indexing format to discover alleles, assess their supporting coverage, and assign useful quality scores. To evaluate BioGraphs performance, we run five sequencing replicates of the individual HG002 through the BioGraph variant calling pipeline, as well as two other short-read sv-calling pipelines. BioGraph detects the GIAB benchmark SVs at a peak sensitivity of {approx}59% compared to {approx}42% sensitivity from the other pipelines. The overall precision of BioGraph is lower than other pipelines ({approx}81% and {approx}90%, respectively), however, adjusting for quality score, BioGraph calls were sensitive to a greater number of SV calls given the same false discovery rate compared to the other pipelines. Cumulatively {approx}77% of GIAB benchmark SVs are discovered by BioGraph in at least one replicate. After merging discovered calls and running BioGraph Coverage to create a squared-off project-level VCF, we find {approx}90% percent of discovered true positive alleles have at least 5x coverage in all replicates, thus increasing per-replicate recall of alleles having at least 1x coverage to {approx}76.9%.

bioinformatics↗

bioRxiv2020

Salinomycin inhibits epigenetic modulator EZH2 to enhance Death Receptors in Colon Cancer Stem Cells

Drug resistance is one of the trademark features of Cancer Stem Cells (CSCs). We and others have recently shown that paucity of functional death receptors (DR4/5) on the cell surface of tumor cells is one of the major reasons for drug resistance, but their involvement in the context of in CSCs is poorly understood. By harnessing CSC specific cytotoxic function of salinomycin, we discovered a critical role of epigenetic modulator EZH2 in regulating the expression of DRs in colon CSCs. Our unbiased proteome profiler array approach followed by ChIP analysis of salinomycin treated cells indicated that the expression of DRs, especially DR4 is epigenetically repressed in colon CSCs. Concurrently, EZH2 knockdown demonstrated increased expression of DR4/DR5, significant reduction of CSC phenotype such as spheroid formation in-vitro and tumorigenic potential in-vivo in colon cancer. TCGA data analysis of human colon cancer clinical samples shows strong inverse correlation between EZH2 and DR4. Taken together, this study provides an insight about epigenetic regulation of DR4 in colon CSCs and advocates that drug resistant colon cancer can be therapeutically targeted by combining TRAIL and small molecule EZH2 inhibitors.

cancer biology↗

bioRxiv2020

Unequal contribution of two paralogous centromeric histones to function the cowpea centromere

The legume cowpea (Vigna unguiculata, 2n=2x=22) has significant tolerance to drought and heat stress. Here we analysed and manipulated cowpea centromere-specific histone H3 (CENH3) genes, aiming to establish a centromere-based doubled-haploid method for use in genetic improvement of this dryland crop in future. Cowpea encodes two functional CENH3 variants (CENH3.1 and CENH3.2) and two CENH3 pseudogenes. Phylogenetic analysis suggests that gene duplication of CENH3 occurred independently during the speciation of V. unguiculata and the related V. mungo without a genome duplication event. Both functional cowpea CENH3 variants are transcribed, and the corresponding proteins are intermingled in subdomains of different types of centromere sequences in a tissue-specific manner together with the outer kinetochore protein CENPC. CENH3.2 is removed from the generative cell of mature pollen, while CENH3.1 persists. Differences between both CENH3 paralogs are restricted to the N-terminus. The complete CRISPR/Cas9-based inactivation of CENH3.1 resulted in delayed vegetative growth and sterility, indicating that this variant is needed for plant development and reproduction. By contrast, CENH3.2 knockout individuals did not show obvious defects during vegetative and reproductive development, suggesting that the gene is an early stage of subfunctionalization or pseudogenization. One-sentence summaryThe two paralogous centromeric histones (CENH3) of cowpea contribute unequal to the function of the centromere.

genetics↗

bioRxiv2019

CXCR4 intracellular protein regulates drug resistance and tumorigenic potential via modulating the expression of Death Receptor 5

CXCR4 overexpression in solid tumors has been strongly associated with poor prognosis and adverse clinical outcome. However, CXCR4 signaling inhibitor drug Plerixafor has shown limited clinical success in cancer treatment. Therefore, CXCR4 signaling may not be the exclusive contributor to its pro-tumorigenic functions. In our continuous effort to understand the chemokine receptor signaling inhibition as cancer therapy, here we unexpectedly discovered that instead of its signaling, intracellular CXCR4 protein augments therapy resistance and pro-tumorigenic functions. Unbiased proteome profiler apoptosis array followed by immunoblot, FACS, real-time PCR and ChIP analyses demonstrate that CXCR4 promotes DR5 downregulation via modulating differential recruitment of transcription factors p53 and YY1 to its promoter. Surprisingly, inhibiting CXCR4 mediated signals failed to block the above phenotype. Irrespective of CXCR4 surface expression, its loss compromised colon tumor growth in vivo. Finally, TCGA data mining and human patient sample data analysis showed CXCR4 and DR5 are inversely regulated in human cancers. Together, we showed evidence for the first time that targeting CXCR4 intracellular protein may be critical to dampen the pro-tumorigenic functions of CXCR4.

cancer biology↗

bioRxiv2019

Resolving sub-clonal heterogeneity within cell-line growths by single cell sequencing genomic DNA

We performed shallow single-cell sequencing of genomic DNA across 1,475 cells from a well-studied cell-line, COLO829, to resolve overall tumor complexity and clonality. This melanoma tumor-line has been previously characterized by multiple technologies and provides a benchmark for evaluating somatic alterations, though has exhibited conflicting and indeterminate copy number states. We identified at least four major sub-clones by discriminant analysis of principal components (DAPC) of single cell copy number data. Break-point and loss of heterozygosity (LOH) analysis of aggregated data from sub-clones revealed a complex rearrangement of chromosomes 1, 10 and 18 that was maintained in all but two sub-clones. Likewise, two of the sub-clones were distinguished by loss of 1 copy of chromosome 8. Re-analysis of previous spectral karyotyping data and bulk sequencing data recapitulated these sub-clone hallmark features and explains why the original bulk sequencing experiments generated conflicting copy number results. Overall, our results demonstrate how shallow copy number profiling together with clustering analysis of single cell sequencing can uncover significant hidden insights even in well studied cell-lines.

genomics↗

bioRxiv2018

Joint single cell DNA-Seq and RNA-Seq of gastric cancer reveals subclonal signatures of genomic instability and gene expression

Sequencing the genomes of individual cancer cells provides the highest resolution of intratumoral heterogeneity. To enable high throughput single cell DNA-Seq across thousands of individual cells per sample, we developed a droplet-based, automated partitioning technology for whole genome sequencing. We applied this approach on a set of gastric cancer cell lines and a primary gastric tumor. In parallel, we conducted a separate single cell RNA-Seq analysis on these same cancers and used copy number to compare results. This joint study, covering thousands of single cell genomes and transcriptomes, revealed extensive cellular diversity based on distinct copy number changes, numerous subclonal populations and in the case of the primary tumor, subclonal gene expression signatures. We found genomic evidence of positive selection - where the percentage of replicating cells per clone is higher than expected - indicating ongoing tumor evolution. Our study demonstrates that joining single cell genomic DNA and transcriptomic features provides novel insights into cancer heterogeneity and biology. SIGNIFICANCEWe conducted a massively parallel DNA sequencing analysis on a set of gastric cancer cell lines and a primary gastric tumor in combination with a joint single cell RNA-Seq analysis. This joint study, covering thousands of single cell genomes and transcriptomes, revealed extensive cellular diversity based on distinct copy number changes, numerous subclonal populations and in the case of the primary tumor, subclonal gene expression signatures. We found genomic evidence of positive selection where the percentage of replicating cells per clone is higher than expected indicating ongoing tumor evolution. Our study demonstrates that combining single cell genomic DNA and transcriptomic features provides novel insights into cancer heterogeneity and biology.

genomics↗

bioRxiv2017

Resolving the Full Spectrum of Human Genome Variation using Linked-Reads

Large-scale population based analyses coupled with advances in technology have demonstrated that the human genome is more diverse than originally thought. To date, this diversity has largely been uncovered using short read whole genome sequencing. However, standard short-read approaches, used primarily due to accuracy, throughput and costs, fail to give a complete picture of a genome. They struggle to identify large, balanced structural events, cannot access repetitive regions of the genome and fail to resolve the human genome into its two haplotypes. Here we describe an approach that retains long range information while harnessing the advantages of short reads. Starting from only [~]1ng of DNA, we produce barcoded short read libraries. The use of novel informatic approaches allows for the barcoded short reads to be associated with the long molecules of origin producing a novel datatype known as Linked-Reads. This approach allows for simultaneous detection of small and large variants from a single Linked-Read library. We have previously demonstrated the utility of whole genome Linked-Reads (lrWGS) for performing diploid, de novo assembly of individual genomes (Weisenfeld et al. 2017). In this manuscript, we show the advantages of Linked-Reads over standard short read approaches for reference based analysis. We demonstrate the ability of Linked-Reads to reconstruct megabase scale haplotypes and to recover parts of the genome that are typically inaccessible to short reads, including phenotypically important genes such as STRC, SMN1 and SMN2. We demonstrate the ability of both lrWGS and Linked-Read Whole Exome Sequencing (lrWES) to identify complex structural variations, including balanced events, single exon deletions, and single exon duplications. The data presented here show that Linked-Reads provide a scalable approach for comprehensive genome analysis that is not possible using short reads alone.

genomics↗

bioRxiv2017

Mechanics of Hydra Detachment from Subrates: The Role of Substrate Rigidity and Starvation

Hydra is a fresh water hydrozoan living as a solitary polyp with a sedentary feeder lifestyle attached to a substrate. In times of food shortage they are reported to detach from their substrate and move either by drifting or somer-saulting. The attachment to the substrate is usually by the basal-body which secretes a mucosal adhesive. The mechanical strength of the adhesion of Hydra has not been quantified so far. Here, we measure the force required to detach Hydra vulgaris and Hydra magnipapillata from a surface and the role of physical and physiological factors. In order to do this, we have developed a flow chamber with a calibrated jet of water. We find H. vulgaris adhering to a hard substrate - a glass cover slip- requires more force to detach it as compared to a soft substrate- polyacrylamide gel. While H. vulgaris after one week of starvation detaches with very similar values of stress, H. magnipapillata detaches more readily when starved. These results suggest that the strength of adhesion is strongly affected by the stiffness of the substrate, while nutritional status dependence of detachment force appears to be species dependent. Given that Hydra detachment is required during locomotion, our measurements on the one hand suggest the magnitude of forces the animal must exert to detach itself. Additionally, our results suggest active detachment of the base might be required for Hydra to achieve movement, and only a small contribution coming from weakening adhesion.

biophysics↗

bioRxiv2017

Reference Quality Assembly of the 3.5 Gb genome of Capsicum annuum from a Single Linked-Read Library

BackgroundLinked-Read sequencing technology has recently been employed successfully for de novo assembly of multiple human genomes, however the utility of this technology for complex plant genomes is unproven. We evaluated the technology for this purpose by sequencing the 3.5 gigabase (Gb) diploid pepper (Capsicum annuum) genome with a single Linked-Read library. Plant genomes, including pepper, are characterized by long, highly similar repetitive sequences. Accordingly, significant effort is used to ensure the sequenced plant is highly homozygous and the resulting assembly is a haploid consensus. With a phased assembly approach, we targeted a heterozygous F1 derived from a wide cross to assess the ability to derive both haplotypes for a pungency gene characterized by a large insertion/deletion.\n\nResultsThe Supernova software generated a highly ordered, more contiguous sequence assembly than all currently available C. annuum reference genomes. Eighty-four percent of the final assembly was anchored and oriented using four de novo linkage maps. A comparison of the annotation of conserved eukaryotic genes indicated the completeness of assembly. The validity of the phased assembly is further demonstrated with the complete recovery of both 2.5 kb insertion/deletion haplotypes of the PUN1 locus in the F1 sample that represents pungent and non-pungent peppers.\n\nConclusionsThe most contiguous pepper genome assembly to date has been generated through this work which demonstrates that Linked-Read library technology provides a rapid tool to assemble de novo complex highly repetitive heterozygous plant genomes. This technology can provide an opportunity to cost-effectively develop high-quality reference genome assemblies for other complex plants and compare structural and gene differences through accurate haplotype reconstruction.

genomics↗