Feasibility of High-Throughput Genome-Wide Genotyping using DNA from Stored Buccal Cell Samples

It is unclear if buccal cell samples contain sufficient human DNA with adequately sized fragments for high throughput genetic bioassays. Yet buccal cell sample collection is an attractive alternative to gathering blood samples for genetic epidemiologists engaged in large-scale genetic biomarker studies. We assessed the genotyping efficiency (GE) and genotyping concordance (GC) of buccal cell DNA samples compared to corresponding blood DNA samples, from 32 Nurses' Health Study (NHS) participants using the Illumina Infinium 660W-Quad platform. We also assessed how GE and GC accuracy varied as a function of DNA concentration using serial dilutions of buccal DNA samples. Finally we determined the nature and genomic distribution of discordant genotypes in buccal DNA samples. The mean GE of undiluted buccal cell DNA samples was high (99.32%), as was the GC between the paired buccal and blood samples (99.29%). GC between the dilutions versus the undiluted buccal DNA was also very high (>97%), though both GE and GC notably declined at DNA concentrations less than 5 ng/μl. Most (>95%) genotype determinations in buccal cell samples were of the “missing call” variety (as opposed to the “alternative genotype call” variety) across the spectrum of buccal DNA concentrations studied. Finally, for buccal DNA concentration above 1.7 ng/ul, discordant genotyping calls did not cluster in any particular chromosome. Buccal cell-derived DNA represents a viable alternative to blood DNA for genotyping on a high-density platform.

Download Full-text

A high-throughput microfluidic nano-immunoassay for detecting anti-SARS-CoV-2 antibodies in serum or ultra-low volume dried blood samples

10.1101/2020.10.07.20208280 ◽

2020 ◽

Author(s):

Zoe Swank ◽

Grégoire Michielin ◽

Hon Ming Yip ◽

Patrick Cohen ◽

Diego O. Andrey ◽

...

Keyword(s):

High Throughput ◽

Whole Blood ◽

Large Scale ◽

Test Strip ◽

Sample Collection ◽

Serum Samples ◽

Blood Samples ◽

Novel Technologies ◽

Glucose Test ◽

Low Volume

AbstractNovel technologies are needed to facilitate large-scale detection and quantification of SARS-CoV-2 specific antibodies in human blood samples. Such technologies are essential to support seroprevalence studies, vaccine clinical trials, and to monitor quality and duration of immunity. We developed a microfluidic nano-immunnoassay for the detection of anti-SARS-CoV-2 IgG antibodies in 1024 samples per device. The method achieved a specificity of 100% and a sensitivity of 98% based on the analysis of 289 human serum samples. To eliminate the need for venipuncture, we developed low-cost, ultra-low volume whole blood sampling methods based on two commercial devices and repurposed a blood glucose test strip. The glucose test strip permits the collection, shipment, and analysis of 0.6 µL whole blood easily obtainable from a simple fingerprick. The nano-immunoassay platform achieves high-throughput, high sensitivity and specificity, negligible reagent consumption, and a decentralized and simple approach to blood sample collection. We expect this technology to be immediately applicable to current and future SARS-CoV-2 related serological studies and to protein biomarker diagnostics in general.

Download Full-text

Factors Enhancing Serum Syndecan-1 Concentrations: A Large-Scale Comprehensive Medical Examination

Journal of Clinical Medicine ◽

10.3390/jcm8091320 ◽

2019 ◽

Vol 8 (9) ◽

pp. 1320

Author(s):

Kazumasa Oda ◽

Hideshi Okada ◽

Akio Suzuki ◽

Hiroyuki Tomita ◽

Ryo Kobayashi ◽

...

Keyword(s):

Large Scale ◽

Prospective Observational Study ◽

Nitrogen Concentration ◽

Laboratory Data ◽

University Hospital ◽

Endothelial Glycocalyx ◽

Sample Collection ◽

Blood Samples ◽

Triglyceride Concentration ◽

Medical Examinations

Endothelial disorders are related to various diseases. An initial endothelial injury is characterized by endothelial glycocalyx injury. We aimed to evaluate endothelial glycocalyx injury by measuring serum syndecan-1 concentrations in patients during comprehensive medical examinations. A single-center, prospective, observational study was conducted at Asahi University Hospital. The participants enrolled in this study were 1313 patients who underwent comprehensive medical examinations at Asahi University Hospital from January 2018 to June 2018. One patient undergoing hemodialysis was excluded from the study. At enrollment, blood samples were obtained, and study personnel collected demographic and clinical data. No treatments or exposures were conducted except for standard medical examinations and blood sample collection. Laboratory data were obtained by the collection of blood samples at the time of study enrolment. According to nonlinear regression, the concentrations of serum syndecan-1 were significantly related to age (p = 0.016), aspartic aminotransferase concentration (AST, p = 0.020), blood urea nitrogen concentration (BUN, p = 0.013), triglyceride concentration (p < 0.001), and hematocrit (p = 0.006). These relationships were independent associations. Endothelial glycocalyx injury, which is reflected by serum syndecan-1 concentrations, is related to age, hematocrit, AST concentration, BUN concentration, and triglyceride concentration.

Download Full-text

A comprehensive RNA handling and transcriptomics guide for high-throughput processing of Plasmodium blood-stage samples

Malaria Journal ◽

10.1186/s12936-020-03436-w ◽

2020 ◽

Vol 19 (1) ◽

Author(s):

Michal Kucharski ◽

Jaishree Tripathi ◽

Sourav Nayak ◽

Lei Zhu ◽

Grennady Wirjanata ◽

...

Keyword(s):

High Throughput ◽

Large Scale ◽

Rna Extraction ◽

Transcriptome Data ◽

High Quality ◽

Diverse Range ◽

Human Haemoglobin ◽

Infected Blood ◽

Blood Samples ◽

Total Rna

Abstract Background Sequencing technology advancements opened new opportunities to use transcriptomics for studying malaria pathology and epidemiology. Even though in recent years the study of whole parasite transcriptome proved to be essential in understanding parasite biology there is no compiled up-to-date reference protocol for the efficient generation of transcriptome data from growing number of samples. Here, a comprehensive methodology on how to preserve, extract, amplify, and sequence full-length mRNA transcripts from Plasmodium-infected blood samples is presented that can be fully streamlined for high-throughput studies. Results The utility of various commercially available RNA-preserving reagents in a range of storage conditions was evaluated. Similarly, several RNA extraction protocols were compared and the one most suitable method for the extraction of high-quality total RNA from low-parasitaemia and low-volume blood samples was established. Furthermore, the criteria needed to evaluate the quality and integrity of Plasmodium RNA in the presence of human RNA was updated. Optimization of SMART-seq2 amplification method to better suit AT-rich Plasmodium falciparum RNA samples allowed us to generate high-quality transcriptomes from as little as 10 ng of total RNA and a lower parasitaemia limit of 0.05%. Finally, a modified method for depletion of unwanted human haemoglobin transcripts using in vitro CRISPR-Cas9 treatment was designed, thus improving parasite transcriptome coverage in low parasitaemia samples. To prove the functionality of the pipeline for both laboratory and field strains, the highest 2-hour resolution RNA-seq transcriptome for P. falciparum 3D7 intraerythrocytic life cycle available to date was generated, and the entire protocol was applied to create the largest transcriptome data from Southeast Asian field isolates. Conclusions Overall, the presented methodology is an inclusive pipeline for generation of good quality transcriptomic data from a diverse range of Plasmodium-infected blood samples with varying parasitaemia and RNA inputs. The flexibility of this pipeline to be adapted to robotic handling will facilitate both small and large-scale future transcriptomic studies in the field of malaria.

Download Full-text

Mutant resources for functional genomics in Dictyostelium discoideum using REMI-seq technology

BMC Biology ◽

10.1186/s12915-021-01108-y ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Nicole Gruenheit ◽

Amy Baldwin ◽

Balint Stewart ◽

Sarah Jaques ◽

Thomas Keller ◽

...

Keyword(s):

Dictyostelium Discoideum ◽

High Throughput ◽

Gene Function ◽

Evolutionary Biology ◽

Large Scale ◽

Function Analysis ◽

Model Systems ◽

Genome Wide ◽

The Social ◽

Drug Resistance Marker

Abstract Background Genomes can be sequenced with relative ease, but ascribing gene function remains a major challenge. Genetically tractable model systems are crucial to meet this challenge. One powerful model is the social amoeba Dictyostelium discoideum, a eukaryotic microbe widely used to study diverse questions in the cell, developmental and evolutionary biology. Results We describe REMI-seq, an adaptation of Tn-seq, which allows high throughput, en masse, and quantitative identification of the genomic site of insertion of a drug resistance marker after restriction enzyme-mediated integration. We use REMI-seq to develop tools which greatly enhance the efficiency with which the sequence, transcriptome or proteome variation can be linked to phenotype in D. discoideum. These comprise (1) a near genome-wide resource of individual mutants and (2) a defined pool of ‘barcoded’ mutants to allow large-scale parallel phenotypic analyses. These resources are freely available and easily accessible through the REMI-seq website that also provides comprehensive guidance and pipelines for data analysis. We demonstrate that integrating these resources allows novel regulators of cell migration, phagocytosis and macropinocytosis to be rapidly identified. Conclusions We present methods and resources, generated using REMI-seq, for high throughput gene function analysis in a key model system.

Download Full-text

Genome-wide association study of body fat distribution traits in Hispanics/Latinos from the HCHS/SOL Study

10.1101/2021.02.23.21251958 ◽

2021 ◽

Author(s):

Anne E. Justice ◽

Kristin Young ◽

Stephanie M. Gogarten ◽

Tamar Sofer ◽

Misa Graff ◽

...

Keyword(s):

Central Obesity ◽

Large Scale ◽

Mixed Model ◽

Linear Mixed Model ◽

Genome Wide Association Study ◽

Health Concern ◽

Health Study ◽

Central Adiposity ◽

Combined Analysis ◽

Genome Wide

AbstractCentral obesity is a leading health concern with a great burden carried by ethnic minority populations, and especially Hispanics/Latinos. Genetic factors contribute to the obesity burden overall and to inter-population differences. We aim to: 1) identify novel loci associated with central adiposity measured as waist-to-hip ratio (WHR), waist circumference (WC), and hip circumference (HIP), all adjusted for body mass index (adjBMI), using the Hispanic Community Health Study/Study of Latinos (HCHS/SOL); 2) determine if differences in genetic associations differ by background group within HCHS/SOL; 3) determine whether previously reported association regions generalize to HCHS/SOL. Our analyses included 7,472 women and 5,200 men of mainland (Mexican, Central and South American) and Caribbean (Puerto Rican, Cuban, and Dominican) background residing in the US, with genome-wide array data imputed to the 1000 genomes Phase I multiethnic reference panel. We analyzed associations stratified by sex in addition to sexes combined using linear mixed-model regression. We identified 16 variants for WHRadjBMI, 22 for WCadjBMI, and 28 for HIPadjBMI that reached suggestive significance (P<1×10−6). Many of the loci exhibited differences in strength of associations by ethnic background and sex. We brought a total of 66 variants forward for validation in nine cohort studies (N=34,161) with participants of Hispanic/Latino, African and European descent. We confirmed four novel loci (ancestry-specific P<0.05 in replication, consistent direction of effect with HCHS/SOL, and P<5×10−8 after meta-analysis with HCHS/SOL), including rs13301996 in the sexes-combined analysis, and rs79478137 for women-only for WHRadjBMI; rs28692724 in women-only for HIPadjBMI; and rs3168072 in the sexes combined analysis for WCadjBMI. Also, a total of eight previously reported WHRadjBMI association regions, 12 for HIPadjBMI, and 10 for WCadjBMI generalized to HCHS/SOL. Our study findings highlight the importance of large-scale genomic studies in ancestrally diverse Hispanic/Latino populations for identifying and characterizing central obesity-susceptibility that may be ancestry-specific.

Download Full-text

Genome-wide approaches for identification of nuclear receptor target genes

Nuclear Receptor Signaling ◽

10.1621/nrs.04018 ◽

2006 ◽

Vol 4 (1) ◽

pp. nrs.04018 ◽

Cited By ~ 6

Author(s):

Luz E. Tavera-Mendoza ◽

Sylvie Mader ◽

John H. White

Keyword(s):

Nuclear Receptor ◽

High Throughput ◽

Large Scale ◽

Target Genes ◽

Receptor Signaling ◽

Receptor Field ◽

Hormone Response Element ◽

Genome Wide ◽

Gene Switches ◽

On Chip

Large-scale genomics analyses have grown by leaps and bounds with the rapid advances in high throughput DNA sequencing and synthesis techniques. Nuclear receptor signaling is ideally suited to genomics studies because receptors function as ligand-regulated gene switches. This review will survey the strengths and limitations of three major classes of high throughput techniques widely used in the nuclear receptor field to characterize ligand-dependent gene regulation: expression profiling studies (microarrays, SAGE and related techniques), chromatin immunoprecipitation followed by microarray (ChIP-on-chip), and genome-wide in silico hormone response element screens. We will discuss each technique, and how each has contributed to our understanding of nuclear receptor signaling.

Download Full-text

Dub-seq: dual-barcoded shotgun expression library sequencing for high-throughput characterization of functional traits

10.1101/387399 ◽

2018 ◽

Author(s):

Vivek K. Mutalik ◽

Pavel S. Novichkov ◽

Morgan N. Price ◽

Trenton K. Owens ◽

Mark Callaghan ◽

...

Keyword(s):

High Throughput ◽

Large Scale ◽

Dna Barcode ◽

Gene Overexpression ◽

Expression Library ◽

Gain Of Function ◽

Experimental Conditions ◽

Genome Wide ◽

Barcode Sequencing

AbstractA major challenge in genomics is the knowledge gap between sequence and its encoded function. Gain-of-function methods based on gene overexpression are attractive avenues for phenotype-based functional screens, but are not easily applied in high-throughput across many experimental conditions. Here, we present Dual Barcoded Shotgun Expression Library Sequencing (Dub-seq), a method that greatly increases the throughput of genome-wide overexpression assays. In Dub-seq, a shotgun expression library is cloned between dual random DNA barcodes and the precise breakpoints of DNA fragments are associated to the barcode sequences prior to performing assays. To assess the fitness of individual strains carrying these plasmids, we use DNA barcode sequencing (BarSeq), which is amenable to large-scale sample multiplexing. As a demonstration of this approach, we constructed a Dub-seq library with total Escherichia coli genomic DNA, performed 155 genome-wide fitness assays in 52 experimental conditions, and identified 813 genes with high-confidence overexpression phenotypes across 4,151 genes assayed. We show that Dub-seq data is reproducible, accurately recapitulates known biology, and identifies hundreds of novel gain-of-function phenotypes for E. coli genes, a subset of which we verified with assays of individual strains. Dub-seq provides complementary information to loss-of-function approaches such as transposon site sequencing or CRISPRi and will facilitate rapid and systematic functional characterization of microbial genomes.ImportanceMeasuring the phenotypic consequences of overexpressing genes is a classic genetic approach for understanding protein function; for identifying drug targets, antibiotic and metal resistance mechanisms; and for optimizing strains for metabolic engineering. In microorganisms, these gain-of-function assays are typically done using laborious protocols with individually archived strains or in low-throughput following qualitative selection for a phenotype of interest, such as antibiotic resistance. However, many microbial genes are poorly characterized and the importance of a given gene may only be apparent under certain conditions. Therefore, more scalable approaches for gain-of-function assays are needed. Here, we present Dual Barcoded Shotgun Expression Library Sequencing (Dub-seq), a strategy that couples systematic gene overexpression with DNA barcode sequencing for large-scale interrogation of gene fitness under many experimental conditions at low cost. Dub-seq can be applied to many microorganisms and is a valuable new tool for large-scale gene function characterization.

Download Full-text

Characterisation of a Novel Benzopyran Library Using High-Throughput Microscopy

10.26686/wgtn.17151011.v1 ◽

2021 ◽

Author(s):

◽

Jeffrey Sheridan

Keyword(s):

Functional Groups ◽

High Throughput ◽

Large Scale ◽

Structural Diversity ◽

Ergosterol Biosynthesis ◽

Biological Interactions ◽

Diverse Range ◽

Genome Wide ◽

Wide Range ◽

Disciplinary Field

<p>Drug discovery is a multi-disciplinary field incorporating both chemistry and biology to create novel pharmaceuticals. Nature synthesizes a diverse range of chemical entities that can demonstrate a wide range of biological interactions, though often produces these compounds in small amounts. Using natures structural diversity as a template, organic synthetic chemistry can tap into the structures of natural products and provide novel structures as well as overcome supply issues through large-scale synthetic chemical processes. A novel benzopyran library was synthesised by Sandile Simelane by reacting 3,4,6,-tri-O-acetyl-D-galactal with various phenols to create a novel focused library of bridged benzopyrans. Each molecule has unique functional groups at defined points in the structure due to varying the functional groups on the phenol, allowing for variation within the library whilst retaining the core scaffold. In this thesis, the bioactivity of this novel benzopyran library was explored using a phenotypic screen measuring growth inhibition. A compound, S13, was determined to be the most potent in the library, therefore genome-wide screening was performed using S13. High-throughput microscopy of 4,100 strains, each with a different GFP-tagged protein, was utilized to determine proteins that increased in abundance or changed localization in response to perturbation with S13. Following treatment with S13, the yeast vacuole increased in size due to an aggregation of proteins in the vacuolar lumen. The increase in vacuole size was coincident with a decrease in vacuolar acidity, potentially disrupted autophagy and the upregulation of several proteins involved in ergosterol biosynthesis. Together, these results reveal a novel bridged benzopyran that increases vacuolar size and pH through an epistatic mechanism involving ergosterol biosynthesis.</p>

Download Full-text

FRET-Based Calcium Imaging

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057113502672 ◽

2013 ◽

Vol 18 (10) ◽

pp. 1309-1320 ◽

Cited By ~ 12

Author(s):

Kamran Honarnejad ◽

Achim K. Kirsch ◽

Alexander Daschner ◽

Aleksandra Szybinska ◽

Jacek Kuznicki ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

High Throughput ◽

Calcium Homeostasis ◽

Calcium Imaging ◽

Large Scale ◽

Calcium Release ◽

Amyloid Β ◽

Hek293 Cells ◽

Attractive Alternative ◽

Endoplasmic Reticulum Calcium

Perturbed intracellular store calcium homeostasis is suggested to play a major role in the pathophysiology of Alzheimer disease (AD). A number of mechanisms have been suggested to underlie the impairment of endoplasmic reticulum calcium homeostasis associated with familial AD-linked presenilin 1 mutations (FAD-PS1). Without aiming at specifically targeting any of those pathophysiological mechanisms in particular, we rather performed a high-throughput phenotypic screen to identify compounds that can reverse the exaggerated agonist-evoked endoplasmic reticulum calcium release phenotype in HEK293 cells expressing FAD-PS1. For that purpose, we developed a fully automated high-throughput calcium imaging assay using a fluorescence resonance energy transfer–based calcium indicator at single-cell resolution. This novel robust assay offers a number of advantages compared with the conventional calcium measurement screening technologies. The assay was employed in a large-scale screen with a library of diverse compounds comprising 20,000 low-molecular-weight molecules, which resulted in the identification of 52 primary hits and 4 lead structures. In a secondary assay, several hits were found to alter the amyloid β (Aβ) production. In view of the recent failure of AD drug candidates identified by target-based approaches, such a phenotypic drug discovery paradigm may present an attractive alternative for the identification of novel AD therapeutics.

Download Full-text