Efficient High-throughput Techniques for the Analysis of Disease-Resistant Plant Varieties and Detection of Food Adulteration

2021 ◽  
Vol 23 ◽  
Author(s):  
Romesh Kumar Salgotra ◽  
Rafiq Ahmad Bhat ◽  
Deyue Yu ◽  
Javaid Akhter Bhat
Keyword(s):  
Multiplex Pcr ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Low Cost ◽  
Crop Improvement ◽  
Small Sample ◽  
Food Crops ◽  
Genomic Resources ◽  
Breeding Programmes ◽  
Next Generation Sequencing Ngs

Abstract: Over the past two decades, the advances in the next generation sequencing (NGS) platforms have led to the identification of numerous genes/QTLs at high-resolution for their potential use in crop improvement. The genomic resources generated through these high-throughput sequencing techniques have been efficiently used in screening of particular gene of interest particularly for numerous types of plant stresses and quality traits. Subsequently, the identified-markers linked to a particular trait have been used in marker-assisted backcross breeding (MABB) activities. Besides, these markers are also being used to catalogue the food crops for detection of adulteration to improve the quality of food. With the advancement of technologies, the genomic resources are originating with new markers; however, to use these markers efficiently in crop breeding, high-throughput techniques (HTT) such as multiplex PCR and capillary electrophoresis (CE) can be exploited. Robustness, ease of operation, good reproducibility and low cost are the main advantages of multiplex PCR and CE. The CE is capable of separating and characterizing proteins with simplicity, speed and small sample requirements. Keeping in view the availability of vast data generated through NGS techniques and development of numerous markers, there is a need to use these resources efficiently in crop improvement programmes. In summary, this review describes the use of molecular markers in the screening of resistance genes in breeding programmes and detection of adulterations in food crops using high-throughput techniques.

scholarly journals Application of High-Throughput Sequencing in the Diagnosis of Inherited Thrombocytopenia

2018 ◽  
Vol 24 (9_suppl) ◽  
pp. 94S-103S ◽  
Author(s):  
Qi Wang ◽  
Lijuan Cao ◽  
Guangying Sheng ◽  
Hongjie Shen ◽  
Jing Ling ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Gene Sequencing ◽  
Hereditary Diseases ◽  
Sequencing Platform ◽  
Pathogenic Variants ◽  
Diagnosis Method ◽  
Inherited Thrombocytopenia ◽  
Thrombotic Diseases ◽  
Next Generation Sequencing Ngs

Inherited thrombocytopenia is a group of hereditary diseases with a reduction in platelet count as the main clinical manifestation. Clinically, there is an urgent need for a convenient and rapid diagnosis method. We introduced a high-throughput, next-generation sequencing (NGS) platform into the routine diagnosis of patients with unexplained thrombocytopenia and analyzed the gene sequencing results to evaluate the value of NGS technology in the screening and diagnosis of inherited thrombocytopenia. From a cohort of 112 patients with thrombocytopenia, we screened 43 patients with hereditary features. For the blood samples of these 43 patients, a gene sequencing platform for hemorrhagic and thrombotic diseases comprising 89 genes was used to perform gene detection using NGS technology. When we combined the screening results with clinical features and other findings, 15 (34.9%) of 43patients were diagnosed with inherited thrombocytopenia. In addition, 19 pathogenic variants, including 8 previously unreported variants, were identified in these patients. Through the use of this detection platform, we expect to establish a more effective diagnostic approach to such disorders.

scholarly journals Recent Advances in Phalaenopsis Orchid Improvement using Omics Approaches

2019 ◽  
Vol 29 (1) ◽  
pp. 133-149
Author(s):  
Khosro Balilashaki ◽  
Hedayat Zakizadeh ◽  
Jamal Ali Olfati ◽  
Maryam Vahedi ◽  
Anuj Kumar ◽  
...  
Keyword(s):  
High Throughput ◽  
Plant Tissue ◽  
Industrial Production ◽  
High Throughput Sequencing ◽  
Genomic Resources ◽  
Recent Advances ◽  
Recent Developments

With recent advances in high-throughput sequencing (HTS) technologies to improve plants, there is a need to release orchid specific genomic resources and platforms that are crucial for managing omics elements in systematic manner. Authors provide details about the recent developments in biotechnological techniques, genomics, transcriptomics, proteomics, metabolomics and their applications for the industrial production, propagation, conservation and manipulation of Phalaenopsis orchid. Plant Tissue Cult. & Biotech. 29(1): 133-149, 2019 (June)

scholarly journals Hi-Plex2: a simple and robust approach to targeted sequencing-based genetic screening

BioTechniques ◽  
2019 ◽  
Vol 67 (3) ◽  
pp. 118-122 ◽  
Author(s):  
Fleur Hammet ◽  
Khalid Mahmood ◽  
Thomas R Green ◽  
Tu Nguyen-Dumont ◽  
Melissa C Southey ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Dna Sequencing ◽  
Multiplex Pcr ◽  
High Throughput ◽  
Genetic Screening ◽  
Low Cost ◽  
Substantial Reduction ◽  
Targeted Sequencing ◽  
Robust Approach ◽  
Panel Size

We have previously reported Hi-Plex, a multiplex PCR methodology for building targeted DNA sequencing libraries that offers a low-cost protocol compatible with high-throughput processing. Here, we detail an improved protocol, Hi-Plex2, that more effectively enables the robust construction of small-to-medium panel-size libraries while maintaining low cost, simplicity and accuracy benefits of the Hi-Plex platform. Hi-Plex2 was applied to three panels, comprising 291, 740 and 1193 amplicons, targeting genes associated with risk for breast and/or colon cancer. We show substantial reduction of off-target amplification to enable library construction for small-to-medium-sized design panels not possible using the previous Hi-Plex chemistry.

scholarly journals Broad-Range Virus Detection and Discovery Using Microfluidic PCR Coupled with High-throughput Sequencing

2020 ◽  
Author(s):  
Ying Tao ◽  
Clinton R. Paden ◽  
Krista Queen ◽  
Jing Zhang ◽  
Eishita Tyagi ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Virus Detection ◽  
Viral Pathogens ◽  
Large Numbers ◽  
Straightforward Method ◽  
Sequence Identification ◽  
Pcr Assays ◽  
Taxonomic Groups ◽  
Next Generation Sequencing Ngs

AbstractThere is a need for a comprehensive and sensitive method to test for a broad range of viral pathogens in samples without any identifiable pathogen detected. Real-time PCR assays are sensitive and rapid, but their specificity limits their utility in detecting divergent agents. Shotgun high-throughput sequencing methods provide unbiased sequence identification, however, they have limited sensitivity and require complex analyses. In order to meet the need for a sensitive, high-throughput virus detection and discovery platform with good sensitivity, we combine two existing technologies, broadly-reactive consensus-degenerate pan-viral group PCR and the MiSeq sequencer (Illumina), using the Access Array (Fluidigm), a commercially-available microfluidic PCR system. Pan-viral group primers target conserved regions of virus taxonomic groups and can amplify known and potentially novel species. The Access Array employs dozens of these assays in parallel, which are then sequenced all at once on the MiSeq. In this study, we run a respiratory panel of pan-viral group PCR assays using AA-PCR-Seq. We validate the panel on a collection of representative human and animal samples, comparing it to qPCR and shotgun next-generation sequencing (NGS). AA-PCR-Seq provides a robust, straightforward method for screening large numbers of samples for virus detection and discovery.

scholarly journals MitoSuite: a graphical tool for human mitochondrial genome profiling in massive parallel sequencing

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3406 ◽  
Author(s):  
Koji Ishiya ◽  
Shintaroh Ueda
Keyword(s):  
Mitochondrial Genome ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Sequencing Data ◽  
High Coverage ◽  
Graphical Tool ◽  
Genome Variations ◽  
Next Generation Sequencing Ngs ◽  
Ngs Data ◽  
Human Mitochondrial Genome

Recent rapid advances in high-throughput, next-generation sequencing (NGS) technologies have promoted mitochondrial genome studies in the fields of human evolution, medical genetics, and forensic casework. However, scientists unfamiliar with computer programming often find it difficult to handle the massive volumes of data that are generated by NGS. To address this limitation, we developed MitoSuite, a user-friendly graphical tool for analysis of data from high-throughput sequencing of the human mitochondrial genome. MitoSuite generates a visual report on NGS data with simple mouse operations. Moreover, it analyzes high-coverage sequencing data but runs on a stand-alone computer, without the need for file upload. Therefore, MitoSuite offers outstanding usability for handling massive NGS data, and is ideal for evolutionary, clinical, and forensic studies on the human mitochondrial genome variations. It is freely available for download from the website https://mitosuite.com.

scholarly journals High-throughput profiling and analysis of plant responses over time to abiotic stress

2017 ◽  
Author(s):  
Kira M. Veley ◽  
Jeffrey C. Berry ◽  
Sarah J. Fentress ◽  
Daniel P. Schachtman ◽  
Ivan Baxter ◽  
...  
Keyword(s):  
Abiotic Stress ◽  
High Throughput ◽  
Stress Responses ◽  
Low Cost ◽  
Abiotic Stress Tolerance ◽  
Crop Improvement ◽  
Plant Responses ◽  
Nitrogen Deprivation ◽  
Plant Nitrogen ◽  
Over Time

ABSTRACTSorghum (Sorghum bicolor (L.) Moench) is a rapidly growing, high-biomass crop prized for abiotic stress tolerance. However, measuring genotype-by-environment (G × E) interactions remains a progress bottleneck. Here we describe strategies for identifying shape, color and ionomic indicators of plant nitrogen use efficiency. We subjected a panel of 30 genetically diverse sorghum genotypes to a spectrum of nitrogen deprivation and measured responses using high-throughput phenotyping technology followed by ionomic profiling. Responses were quantified using shape (16 measurable outputs), color (hue and intensity) and ionome (18 elements). We measured the speed at which specific genotypes respond to environmental conditions, both in terms of biomass and color changes, and identified individual genotypes that perform most favorably. With this analysis we present a novel approach to quantifying color-based stress indicators over time. Additionally, ionomic profiling was conducted as an independent, low cost and high throughput option for characterizing G × E, identifying the elements most affected by either genotype or treatment and suggesting signaling that occurs in response to the environment. This entire dataset and associated scripts are made available through an open access, user-friendly, web-based interface. In summary, this work provides analysis tools for visualizing and quantifying plant abiotic stress responses over time. These methods can be deployed as a time-efficient method of dissecting the genetic mechanisms used by sorghum to respond to the environment to accelerate crop improvement.

scholarly journals McQ – An open-source multiplexed SARS-CoV-2 quantification platform

2020 ◽  
Author(s):  
Sibylle C Vonesch ◽  
Danila Bredikhin ◽  
Nikolay Dobrev ◽  
Laura Villacorta ◽  
Rozemarijn Kleinendorst ◽  
...  
Keyword(s):  
Supply Chain ◽  
High Throughput ◽  
Reverse Transcription ◽  
High Throughput Sequencing ◽  
Early Stage ◽  
Low Cost ◽  
Negative Control ◽  
Clinical Samples ◽  
Sample Pooling ◽  
Cost Efficient

McQ is a SARS-CoV-2 quantification assay that couples early-stage barcoding with high-throughput sequencing to enable multiplexed processing of thousands of samples. McQ is based on homemade enzymes to enable low-cost testing of large sample pools, circumventing supply chain shortages.Implementation of cost-efficient high-throughput methods for detection of RNA viruses such as SARS-CoV-2 is a potent strategy to curb ongoing and future pandemics. Here we describe Multiplexed SARS-CoV-2 Quantification platform (McQ), an in-expensive scalable framework for SARS-CoV-2 quantification in saliva samples. McQ is based on the parallel sequencing of barcoded amplicons generated from SARS- CoV-2 genomic RNA. McQ uses indexed, target-specific reverse transcription (RT) to generate barcoded cDNA for amplifying viral- and human-specific regions. The barcoding system enables early sample pooling to reduce hands-on time and makes the ap-proach scalable to thousands of samples per sequencing run. Robust and accurate quantification of viral load is achieved by measuring the abundance of Unique Molecular Identifiers (UMIs) introduced during reverse transcription. The use of homemade reverse transcriptase and polymerase enzymes and non-proprietary buffers reduces RNA to library reagent costs to 92 cents/sample and circumvents potential supply chain short-ages. We demonstrate the ability of McQ to robustly quantify various levels of viral RNA in 838 clinical samples and accu-rately diagnose positive and negative control samples in a test-ing workflow entailing self-sampling and automated RNA ex-traction from saliva. The implementation of McQ is modular, scalable and could be extended to other pathogenic targets in future.

scholarly journals Towards high–throughput analyses of fecal samples from wildlife

2020 ◽  
pp. 171-183
Author(s):  
C. Sarabia ◽  
I. Salado ◽  
A. Cornellas ◽  
A. Fernández-Gil ◽  
C. Vilà ◽  
...  
Keyword(s):  
Dna Extraction ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Low Cost ◽  
Molecular Ecology ◽  
Extraction Methods ◽  
Success Rates ◽  
Sample Collection ◽  
Field Sampling ◽  
Fecal Samples

High–throughput sequencing offers new possibilities in molecular ecology and conservation studies. However, its potential has not yet become fully exploited for noninvasive studies of free–ranging animals, such as those based on feces. High–throughput sequencing allows sequencing of short DNA fragments and could allow simultaneous genotyping of a very large number of samples and markers at a low cost. The application of high throughput genotyping to fecal samples from wildlife has been hindered by several labor–intensive steps. We evaluate alternative protocols which could allow higher throughput for two of these steps: sample collection and DNA extraction. Two different field sampling and seven different DNA extraction methods are tested here on grey wolf (Canis lupus) feces. There was high variation in genotyping success rates. The field sampling method based on surface swabbing performed much worse than the extraction from a fecal fragment. In addition, there is a lot of room for improvement in the DNA extraction step. Optimization of protocols can lead to very much more efficient, cheaper and higher throughput noninvasive monitoring. Selection of appropriate markers is still of paramount importance to increase genotyping success.

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