Biology subjects

Shah, M.

Publications and source records attributed to Shah, M..

62 records · Page 4Linked to original sources

bioRxiv2019

Deep sequencing of 3 cancer cell lines on 2 sequencing platforms

To test the performance of a new sequencing platform, develop an updated somatic calling pipeline and establish a reference for future benchmarking experiments, we sequenced 3 common cancer cell lines along with their matched normal cell lines to great sequencing depths (up to 278X coverage) on both Illumina HiSeqX and NovaSeq sequencing instruments. Somatic calling was generally consistent between the two platforms despite minor differences at the read level. We designed and implemented a novel pipeline for the analysis of tumor-normal samples, using multiple variant callers. We show that coupled with a high-confidence filtering strategy, it improves the accuracy of somatic calls. We also demonstrate the utility of the dataset by creating an artificial purity ladder to evaluate the somatic pipeline and benchmark methods for estimating purity and ploidy from tumor-normal pairs. The data and results of the pipeline are made accessible to the cancer genomics community.

genomics↗

bioRxiv2019

Single cell q-PCR derived expression profiles of identified sensory neurons

Sensory neurons are chemically and functionally heterogeneous and this heterogeneity has been examined extensively over the last several decades. These studies have employed a variety of different methodologies, including anatomical, electrophysiological and molecular approaches. Recent studies using next generation sequencing techniques have examined the transcriptome of single sensory neurons. Although, these reports have provided a wealth of exciting new information on the heterogeneity of sensory neurons, correlation with functional types is lacking. Here, we employed retrograde tracing of cutaneous and muscle afferents to examine the variety of mRNA expression profiles of individual, target-specific sensory neurons. In addition, we used an ex vivo skin/nerve/DRG/ spinal cord preparation to record and characterize the functional response properties of individual cutaneous sensory neurons that were then intracellularly labeled with fluorescent dyes, recovered from dissociated cultures and analyzed for gene expression. We found that by using single cell qPCR techniques and a limited set of genes, we can identify transcriptionally distinct groups. We have also used calcium imaging and single cell qPCR to determine the correlation between levels of mRNA expression and functional protein expression and how functional properties correlated with the different transcriptional groups. These studies show that although transcriptomics does map to functional types, within any one functional subgroup, there are highly variable patterns of gene expression. Thus, studies that rely on the expression pattern of one or a few genes as a stand in for physiological experiments, runs a high risk of data misinterpretation with respect to function. Significance statementExpression profiles of unidentified sensory neurons have been recently studied using RNASeq techniques. Here, we utilize a multifactorial approach to target identified cutaneous and muscle afferents to examine expression and functional levels of specific high priority candidate genes using ex vivo electrophysiology, Ca2+ imaging, and single cell qPCR. Using this methodology, we were able to identify specific groups of neurons with distinct functional properties that corresponded to unique transcriptional profiles. This represents the first attempt to relate neuronal phenotype with levels of gene expression in single identified afferents and highlights the importance of combining functional analysis with transcriptomics.

neuroscience↗

bioRxiv2018

Genetic mechanisms of primary chemotherapy resistance in pediatric acute myeloid leukemia: A report from the TARGET initiative

Acute myeloid leukemias (AML) are characterized by mutations of tumor suppressor and oncogenes, involving distinct genes in adults and children. While certain mutations have been associated with the increased risk of AML relapse, the genomic landscape of primary chemotherapy resistant AML is not well defined. As part of the TARGET initiative, we performed whole-genome DNA and transcriptome (RNA and miRNA) sequencing analysis of pediatric AML with failure of induction chemotherapy. We identified at least three genetic groups of patients with induction failure, including those with NUP98 rearrangements, somatic mutations of WT1 in the absence of NUP98 mutations, and additional recurrent variants including those in KMT2C and MLLT10. Comparison of specimens before and after chemotherapy revealed distinct and invariant gene expression programs. While exhibiting overt therapy resistance, these leukemias nonetheless showed diverse forms of clonal evolution upon chemotherapy exposure. This included selection for mutant alleles of FRMD8, DHX32, PIK3R1, SHANK3, MKLN1, as well as persistence of WT1 and TP53 mutant clones, and elimination or contraction of FLT3, PTPN11, and NRAS mutant clones. These findings delineate genetic mechanisms of primary chemotherapy resistance in pediatric AML, which should inform improved approaches for its diagnosis and therapy.

cancer biology↗

bioRxiv2018

Streamlined, recombinase-free genome editing with CRISPR-Cas9 in Lactobacillus plantarum reveals barriers to efficient editing

Lactic-acid bacteria such as Lactobacillus plantarum are commonly used for fermenting foods and as probiotics, where increasingly sophisticated genome-editing tools are currently being employed to elucidate and enhance these microbes beneficial properties. The most advanced tools to-date require heterologous single-stranded DNA recombinases to integrate short oligonucleotides followed by using CRISPR-Cas9 to eliminate cells harboring unedited sequences. Here, we show that encoding the recombineering template on a replicating plasmid allowed efficient genome editing with CRISPR-Cas9 in multiple L. plantarum strains without a recombinase. This strategy accelerated the genome-editing pipeline and could efficiently introduce a stop codon in ribB, silent mutations in ackA, and a complete deletion of lacM. In contrast, oligo-mediated recombineering with CRISPR-Cas9 proved far less efficient in at least one instance. We also observed unexpected outcomes of our recombinase-free method, including an ~1.3-kb genomic deletion when targeting ribB in one strain, and reversion of a point mutation in the recombineering template in another strain. Our method therefore can streamline targeted genome editing in different strains of L. plantarum, although the best means of achieving efficient editing may vary based on the selected sequence modification, gene, and strain.

synthetic biology↗

bioRxiv2018

Recombinant expression of Proteorhodopsin and biofilm regulators in Escherichia coli for nanoparticle binding and removal in a wastewater treatment model

The small size of nanoparticles is both an advantage and a problem. Their high surface-area-to-volume ratio enables novel medical, industrial, and commercial applications. However, their small size also allows them to evade conventional filtration during water treatment, posing health risks to humans, plants, and aquatic life. This project aims to remove nanoparticles during wastewater treatment using genetically modified Escherichia coli in two ways: 1) binding citrate-capped nanoparticles with the membrane protein Proteorhodopsin, and 2) trapping nanoparticles using Escherichia coli biofilm produced by overexpressing two regulators: OmpR234 and CsgD. We demonstrate experimentally that Escherichia coli expressing Proteorhodopsin binds to 60 nm citrate-capped silver nanoparticles. We also successfully upregulate biofilm production and show that Escherichia coli biofilms are able to trap 30 nm gold particles. Finally, both Proteorhodopsin and biofilm approaches are able to bind and remove nanoparticles in simulated wastewater treatment tanks. We envision integrating our trapping system in both rural and urban wastewater treatment plants to efficiently capture all nanoparticles before treated water is released into the environment.\n\nFinancial DisclosureThis work was funded by the Taipei American School. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\nCompeting InterestsThe authors have declared that no competing interests exist.\n\nEthics StatementN/A\n\nData AvailabilityYes - all data are fully available without restriction. Sequences for the plasmids used in this study are available through the Registry of Standard Biological Parts. Links to raw data are included in Supplementary Information.

synthetic biology↗

bioRxiv2018

Pleiotropic and Epistatic Network-Based Discovery: Integrated Networks for Target Gene Discovery

Biological organisms are complex systems that are composed of functional networks of interacting molecules and macromolecules. Complex phenotypes are the result of orchestrated, hierarchical, heterogeneous collections of expressed genomic variants. However, the effects of these variants are the result of historic selective pressure and current environmental and epigenetic signals, and, as such, their co-occurrence can be seen as genome-wide correlations in a number of different manners. Biomass recalcitrance (i.e., the resistance of plants to degradation or deconstruction, which ultimately enables access to a plants sugars) is a complex polygenic phenotype of high importance to biofuels initiatives. This study makes use of data derived from the re-sequenced genomes from over 800 different Populus trichocarpa genotypes in combination with metabolomic and pyMBMS data across this population, as well as co-expression and co-methylation networks in order to better understand the molecular interactions involved in recalcitrance, and identify target genes involved in lignin biosynthesis/degradation. A Lines Of Evidence (LOE) scoring system is developed to integrate the information in the different layers and quantify the number of lines of evidence linking genes to lignin-related lignin-phenotypes across the network layers. The resulting Genome Wide Association Study networks, integrated with Single Nucleotide Polymorphism (SNP) correlation, co-methylation and co-expression networks through the LOE scores are proving to be a powerful approach to determine the pleiotropic and epistatic relationships underlying cellular functions and, as such, the molecular basis for complex phenotypes, such as recalcitrance.

bioinformatics↗

bioRxiv2017

Lancet: genome-wide somatic variant calling using localized colored DeBruijn graphs

Reliable detection of somatic variations is of critical importance in cancer research. Lancet is an accurate and sensitive somatic variant caller which detects SNVs and indels by jointly analyzing reads from tumor and matched normal samples using colored DeBruijn graphs. Extensive experimental comparison on synthetic and real whole-genome sequencing datasets demonstrates that Lancet has better accuracy, especially for indel detection, than widely used somatic callers, such as MuTect, MuTect2, LoFreq, Strelka, and Strelka2. Lancet features a reliable variant scoring system which is essential for variant prioritization and detects low frequency mutations without sacrificing the sensitivity to call longer insertions and deletions empowered by the local assembly engine. In addition to genome-wide analysis, Lancet allows inspection of somatic variants in graph space, which augments the traditional read alignment visualization to help confirm a variant of interest. Lancet is available as an open-source program at https://github.com/nygenome/lancet.

bioinformatics↗

bioRxiv2017

Human PGBD5 DNA transposase promotes site-specific oncogenic mutations in rhabdoid tumors

Genomic rearrangements are a hallmark of childhood solid tumors, but their mutational causes remain poorly understood. Here, we identify the piggyBac transposable element derived 5 (PGBD5) gene as an enzymatically active human DNA transposase expressed in the majority of rhabdoid tumors, a lethal childhood cancer. Using assembly-based whole-genome DNA sequencing, we observed previously unknown somatic genomic rearrangements in primary human rhabdoid tumors. These rearrangements were characterized by deletions and inversions involving PGBD5-specific signal (PSS) sequences at their breakpoints, with some recurrently targeting tumor suppressor genes, leading to their inactivation. PGBD5 was found to be physically associated with human genomic PSS sequences that were also sufficient to mediate PGBD5-induced DNA rearrangements in rhabdoid tumor cells. We found that ectopic expression of PGBD5 in primary immortalized human cells was sufficient to promote penetrant cell transformation in vitro and in immunodeficient mice in vivo. This activity required specific catalytic residues in the PGBD5 transposase domain, as well as end-joining DNA repair, and induced distinct structural rearrangements, involving PSS-associated breakpoints, similar to those found in primary human rhabdoid tumors. This defines PGBD5 as an oncogenic mutator and provides a plausible mechanism for site-specific DNA rearrangements in childhood and adult solid tumors.

cancer biology↗