scholarly journals A Short-Read Multiplex Sequencing Method for Reliable, Cost-Effective and High-Throughput Genotyping in Large-Scale Studies

2013 ◽  
Vol 35 (2) ◽  
pp. 270-270 ◽  
Author(s):  
Hongzhi Cao ◽  
Yu Wang ◽  
Wei Zhang ◽  
Xianghua Chai ◽  
Xiandong Zhang ◽  
...  
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Cost Effective ◽  
Short Read ◽  
Sequencing Method ◽  
Multiplex Sequencing

A Short-Read Multiplex Sequencing Method for Reliable, Cost-Effective and High-Throughput Genotyping in Large-Scale Studies

2013 ◽  
Vol 34 (12) ◽  
pp. 1715-1720 ◽  
Author(s):  
Hongzhi Cao ◽  
Yu Wang ◽  
Wei Zhang ◽  
Xianghua Chai ◽  
Xiandong Zhang ◽  
...  
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Cost Effective ◽  
Short Read ◽  
Sequencing Method ◽  
Multiplex Sequencing

scholarly journals STAMP: a multiplex sequencing method for simultaneous evaluation of mitochondrial DNA heteroplasmies and content

2020 ◽  
Author(s):  
Xiaoxian Guo ◽  
Yiqin Wang ◽  
Ruoyu Zhang ◽  
Zhenglong Gu
Keyword(s):  
Large Scale ◽  
Nuclear Dna ◽  
Large Population ◽  
High Sensitivity ◽  
Cost Effective ◽  
Cost Effective Method ◽  
Sequencing Method ◽  
Scale Population ◽  
Mtdna Variants ◽  
Multiplex Sequencing

ABSTRACTHuman mitochondrial genome (mtDNA) variations, such as mtDNA heteroplasmies (the co-existence of mutated and wild-type mtDNA), have received increasing attention in recent years for their clinical relevance to numerous diseases. But large-scale population studies of mtDNA heteroplasmies have been lagging due to the lack of a labor- and cost-effective method. Here, we present a novel human mtDNA sequencing method called STAMP (sequencing by targeted amplification of multiplex probes) for measuring mtDNA heteroplasmies and content in a streamlined workflow. We show that STAMP has high mapping rates to mtDNA, deep coverage of unique reads, and high tolerance to sequencing and PCR errors when applied to human samples. STAMP also has high sensitivity and low false positive rates in identifying artificial mtDNA variants at fractions as low as 0.5% in genomic DNA samples. We further extend STAMP, by including nuclear DNA-targeting probes, to enable assessment of relative mtDNA content in the same assay. The high cost-effectiveness of STAMP, along with the flexibility of using it for measuring various aspects of mtDNA variations, will accelerate the research of mtDNA heteroplasmies and content in large population cohorts, and in the context of human diseases and aging.

scholarly journals STAMP: a multiplex sequencing method for simultaneous evaluation of mitochondrial DNA heteroplasmies and content

2020 ◽  
Vol 2 (4) ◽  
Author(s):  
Xiaoxian Guo ◽  
Yiqin Wang ◽  
Ruoyu Zhang ◽  
Zhenglong Gu
Keyword(s):  
Large Scale ◽  
Nuclear Dna ◽  
Large Population ◽  
High Sensitivity ◽  
Cost Effective ◽  
Cost Effective Method ◽  
Sequencing Method ◽  
Scale Population ◽  
Mtdna Variants ◽  
Multiplex Sequencing

Abstract Human mitochondrial genome (mtDNA) variations, such as mtDNA heteroplasmies (the co-existence of mutated and wild-type mtDNA), have received increasing attention in recent years for their clinical relevance to numerous diseases. But large-scale population studies of mtDNA heteroplasmies have been lagging due to the lack of a labor- and cost-effective method. Here, we present a novel human mtDNA sequencing method called STAMP (sequencing by targeted amplification of multiplex probes) for measuring mtDNA heteroplasmies and content in a streamlined workflow. We show that STAMP has high-mapping rates to mtDNA, deep coverage of unique reads and high tolerance to sequencing and polymerase chain reaction errors when applied to human samples. STAMP also has high sensitivity and low false positive rates in identifying artificial mtDNA variants at fractions as low as 0.5% in genomic DNA samples. We further extend STAMP, by including nuclear DNA-targeting probes, to enable assessment of relative mtDNA content in the same assay. The high cost-effectiveness of STAMP, along with the flexibility of using it for measuring various aspects of mtDNA variations, will accelerate the research of mtDNA heteroplasmies and content in large population cohorts, and in the context of human diseases and aging.

scholarly journals Development of High-Throughput Multiplex Serology to Detect Serum Antibodies against Coxiella burnetii

2021 ◽  
Vol 9 (11) ◽  
pp. 2373
Author(s):  
Rima Jeske ◽  
Larissa Dangel ◽  
Leander Sauerbrey ◽  
Dimitrios Frangoulidis ◽  
Lauren R. Teras ◽  
...  
Keyword(s):  
Coxiella Burnetii ◽  
High Throughput ◽  
Large Scale ◽  
Q Fever ◽  
Reference Group ◽  
Clinical Manifestations ◽  
Cost Effective ◽  
Population Monitoring ◽  
Increased Risk ◽  
Multiplex Serology

The causative agent of Q fever, the bacterium Coxiella burnetii (C. burnetii), has gained increasing interest due to outbreak events and reports about it being a potential risk factor for the development of lymphomas. In order to conduct large-scale studies for population monitoring and to investigate possible associations more closely, accurate and cost-effective high-throughput assays are highly desired. To address this need, nine C. burnetii proteins were expressed as recombinant antigens for multiplex serology. This technique enables the quantitative high-throughput detection of antibodies to multiple antigens simultaneously in a single reaction. Based on a reference group of 76 seropositive and 91 seronegative sera, three antigens were able to detect C. burnetii infections. Com1, GroEL, and DnaK achieved specificities of 93%, 69%, and 77% and sensitivities of 64%, 72%, and 47%, respectively. Double positivity to Com1 and GroEL led to a combined specificity of 90% and a sensitivity of 71%. In a subgroup of seropositives with an increased risk for chronic Q fever, the double positivity to these markers reached a specificity of 90% and a sensitivity of 86%. Multiplex serology enables the detection of antibodies against C. burnetii and appears well-suited to investigate associations between C. burnetii infections and the clinical manifestations in large-scale studies.

Rapid screening of SNPs in metastatic colorectal cancer (mCRC) utilizing multiplex sequencing technology (Sequenom).

2012 ◽  
Vol 30 (4_suppl) ◽  
pp. 418-418
Author(s):  
Radhashree Maitra ◽  
Jay B. Nayak ◽  
Atrayee Basu-Mallick ◽  
Arjun Sood ◽  
Titto A Augustine ◽  
...  
Keyword(s):  
High Throughput ◽  
Direct Sequencing ◽  
Cost Effective ◽  
Rapid Screening ◽  
Accurate Identification ◽  
Multiple Snps ◽  
Fast Screening ◽  
Effective Manner ◽  
Multiplex Sequencing ◽  
Multiple Samples

418 Background: Accurate and fast screening of mutations is essential for designing individualized therapy necessary and critical for efficient disease management and better patient outcome in mCRC. Detection of hotspots by gold standard direct sequencing (DS) is time consuming and cost ineffective. Pyrosequencing (PS) technique is rapid and precisely committed towards SNP detection. Recent introduction of high throughput multiplex PCR based extension on microarray (Sequenom, SEQ) offers a robust platform capable of detecting multiple SNPs simultaneously in a rapid and cost effective manner. The current study analyzes the concordance and efficacy of the cutting edge SEQ technique to the well established DS and PS methods. Methods: DNA isolated from 122 specimens from 76 mCRC patients were sequenced by all three methods. DS and PS were performed on 4 genes at 10 hotspots. SEQ multiplexing was performed on 31 hotspots in 19 genes by 4 multiplex reactions. Results: We were able to make "calls" for all samples by DS and PS. With the multiplex system, the “calls” rate was 97.8% of successful reactions. Using PS data as our standard in the assay we calculated the percent concordance of DS and SEQ. Futhermore SEQ offered a more accurate identification of the substituted nucleotide in Kras codon 12 as compared to PS. Conclusions: The multiplexing of PCR reactions offers an excellent advantage of high throughput with strong feasibility of analyzing several samples for multiple SNPs simultaneously. The concordance rate of > 90% when compared to PS along with the ability to analyze multiple samples/ hotspots plexed together in a time effective rapid mode provided a trifold advantage of the sequenom technology. It is therefore the next generation technology for rapid genetic evaluation of cancer patients. [Table: see text]

Cell-based models for mycotoxin screening and toxicity evaluation: an update

2014 ◽  
Vol 7 (2) ◽  
pp. 153-166 ◽  
Author(s):  
F. Cheli ◽  
E. Fusi ◽  
A. Baldi
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Mycotoxin Research ◽  
Cost Effective ◽  
Screening Tests ◽  
Test Model ◽  
Related Factors ◽  
Technical Problems ◽  
Third Dimension

This review presents the applications of cell-based models in mycotoxin research, with a focus on models for mycotoxin screening and cytotoxicity evaluation. Various cell-based models, cell and cell culture condition related factors, toxicity endpoints and culture systems as well as predictive value of cell-based bioassays are reviewed. Advantages, drawbacks and technical problems regarding set up and validation of consistent, robust, reproducible and high-throughput cell-based models are discussed. Various cell-based models have been developed and used as screening tests for mycotoxins but the data obtained are difficult to compare. However, the results highlight the potential of cell-based models as promising in vitro platforms for the initial screening and cytotoxicity evaluation of mycotoxins and as a significant analytical approach in mycotoxin research before any animal or human clinical studies. To develop cell-based models as powerful high-throughput laboratory platforms for the analysis of large numbers of samples, there are mainly two fundamental requirements that should be met, i.e. the availability of easy-to-use and, if possible, automated cell platforms and the possibility to obtain reproducible results that are comparable between laboratories. The transition from a research model to a test model still needs optimisation, standardisation, and validation of analytical protocols. The validation of a cell-based bioassay is a complex process, as several critical points, such as the choice of the cellular model, the assay procedures, and the appropriate use and interpretation of the results, must be strictly defined to ensure more consistency in the results. The development of cell-based models exploring the third dimension together with automation and miniaturisation will bring cellular platforms to a level appropriate for cost-effective and large-scale analysis in the field of mycotoxin research.

scholarly journals A 454 multiplex sequencing method for rapid and reliable genotyping of highly polymorphic genes in large-scale studies

BMC Genomics ◽  
2010 ◽  
Vol 11 (1) ◽  
pp. 296 ◽  
Author(s):  
Maxime Galan ◽  
Emmanuel Guivier ◽  
Gilles Caraux ◽  
Nathalie Charbonnel ◽  
Jean-Francois Cosson
Keyword(s):  
Large Scale ◽  
Polymorphic Genes ◽  
Sequencing Method ◽  
Multiplex Sequencing

scholarly journals MultiFRAGing: Rapid and Simultaneous Genotyping of Multiple Alleles in a Single Reaction

2019 ◽  
Author(s):  
Cassidy Petree ◽  
Gaurav K Varshney
Keyword(s):  
Genome Editing ◽  
High Throughput ◽  
Large Scale ◽  
Cost Effective ◽  
Fold Increase ◽  
Targeted Mutagenesis ◽  
Model Organisms ◽  
Multiple Alleles ◽  
Cost Effective Method ◽  
Single Reaction

AbstractThe powerful and simple RNA-guided CRISPR/Cas9 technology is a versatile genome editing tool that has revolutionized targeted mutagenesis. CRISPR-based genome editing has enabled large-scale functional genetic studies through the generation of gene knockouts in a variety of model organisms including zebrafish. CRISPR/Cas9 can also be used to target multiple genes simultaneously. One of the challenges associated with applying this technique to zebrafish in a high-throughput manner is the absence of a cost-effective method by which to identify mutants. To address this, we optimized the high-throughput, high-resolution fluorescent PCR-based fragment analysis method to develop MultiFRAGing, a robust and cost-effective method for genotyping of multiple targets in a single reaction. Our approach can identify indels in 4 targets from a single reaction, which represents a four-fold increase in genotyping throughput. This method can be used by any laboratory with access to capillary electrophoresis based sequencing equipment.

scholarly journals A Robust and Cost-Effective Luminescent-Based High-Throughput Assay for Fructose-1,6-Bisphosphate Aldolase A

2020 ◽  
Vol 25 (9) ◽  
pp. 1038-1046
Author(s):  
Eun Jeong Cho ◽  
Ashwini K. Devkota ◽  
Gabriel Stancu ◽  
Ramakrishna Edupunganti ◽  
Ginamarie Debevec ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Large Scale ◽  
Cancer Survival ◽  
Hypoxia Inducible Factor ◽  
Cost Effective ◽  
High Rate ◽  
Statistical Parameters ◽  
Fructose 1,6 Bisphosphate ◽  
Aldolase A

Hypoxic solid tumors induce the stabilization of hypoxia-inducible factor 1 alpha (HIF1α), which stimulates the expression of many glycolytic enzymes and hypoxia-responsive genes. A high rate of glycolysis supports the energetic and material needs for tumors to grow. Fructose-1,6-bisphosphate aldolase A (ALDOA) is an enzyme in the glycolytic pathway that promotes the expression of HIF1α. Therefore, inhibition of ALDOA activity represents a potential therapeutic approach for a range of cancers by blocking two critical cancer survival mechanisms. Here, we present a luminescence-based strategy to determine ALDOA activity. The assay platform was developed by integrating a previously established ALDOA activity assay with a commercial NAD/NADH detection kit, resulting in a significant (>12-fold) improvement in signal/background (S/B) compared with previous assay platforms. A screening campaign using a mixture-based compound library exhibited excellent statistical parameters of Z′ (>0.8) and S/B (~20), confirming its robustness and readiness for high-throughput screening (HTS) application. This assay platform provides a cost-effective method for identifying ALDOA inhibitors using a large-scale HTS campaign.

scholarly journals Cost-effective epigenetic age profiling in shallow methylation sequencing data

2021 ◽  
Author(s):  
Alexandre Trapp ◽  
Vadim N Gladyshev
Keyword(s):  
High Throughput ◽  
Aging Process ◽  
Large Scale ◽  
Cost Effective ◽  
Cellular Reprogramming ◽  
Biological Aging ◽  
Sequencing Data ◽  
Epigenetic Aging ◽  
Epigenetic Age ◽  
Fold Reduction

There is a critical need for robust, high-throughput assays of biological aging trajectories. Among various approaches, epigenetic aging clocks emerged as reliable molecular trackers of the aging process. However, current methods for epigenetic age profiling are inherently costly and lack throughput. Here, we leverage the scAge framework for accurate prediction of biological age from very few bisulfite sequencing reads in bulk samples, thereby enabling drastic (100-1,000-fold) reduction in sequencing costs per sample. Our approach permits age assessment based on distinct assortments of CpG sites in different samples, without the need for targeted site enrichment or specialized reagents. We demonstrate the efficacy of this method to quantify the age of mouse blood samples across independent cohorts, identify the effect of calorie restriction as an attenuator of the aging process, and discern rejuvenation upon cellular reprogramming. We propose that this framework may be used for epigenetic age prediction in extremely high-throughput applications, enabling robust, large-scale and inexpensive interventions testing and age profiling.

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