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.

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.

Controllable and reversible DNA translocation through a single-layer molybdenum disulfide nanopore

Nanoscale ◽  
2018 ◽  
Vol 10 (41) ◽  
pp. 19450-19458 ◽  
Author(s):  
Wei Si ◽  
Yin Zhang ◽  
Jingjie Sha ◽  
Yunfei Chen
Keyword(s):  
Dna Sequencing ◽  
Molybdenum Disulfide ◽  
High Throughput ◽  
Low Cost ◽  
Single Layer ◽  
Dna Translocation ◽  
Nanopore Dna Sequencing

A challenge that remains to be solved in the high-throughput and low-cost nanopore DNA sequencing is that DNA translocates through the nanopore too quickly to be sequenced with enough accuracy.

scholarly journals Multiplex SNaPshot Genotyping for Detecting Loss of Heterozygosity in the Mismatch-Repair Genes MLH1 and MSH2 in Microsatellite-Unstable Tumors

2008 ◽  
Vol 54 (11) ◽  
pp. 1844-1854 ◽  
Author(s):  
Maria Bujalkova ◽  
Katarina Zavodna ◽  
Tomas Krivulcik ◽  
Denisa Ilencikova ◽  
Brigitte Wolf ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Dna Sequencing ◽  
Mismatch Repair ◽  
Loss Of Heterozygosity ◽  
Germline Mutation ◽  
Low Cost ◽  
Clinical Genetics ◽  
E Coli ◽  
Fresh Frozen ◽  
Mutl Homolog 1

Abstract Background: In the workup of patients with suspected hereditary nonpolyposis colorectal cancer (HNPCC), detection of loss of heterozygosity (LOH) could help pinpoint the mismatch-repair (MMR) gene carrying the germline mutation, but analysis of microsatellite markers has proved unreliable for this purpose. We developed a simple, low-cost method based on single-nucleotide polymorphism (SNP) genotyping and capillary electrophoresis for the assessment of LOH at 2 MMR loci simultaneously. Methods: We used the Applied Biosystems SNaPshot® Multiplex Kit with meticulously selected primers to assess 14 common SNPs in MLH1 [mutL homolog 1, colon cancer, nonpolyposis type 2 (E. coli)] and MSH2 [mutS homolog 2, colon cancer, nonpolyposis type 1 (E. coli)] and optimized the protocol for DNA isolated from peripheral blood and fresh/frozen or archival microsatellite-unstable tumors from patients with confirmed (n = 42) or suspected (n = 25) HNPCC. The 42 tumors from patients with confirmed MLH1 or MSH2 germline mutations were used to validate the method’s diagnostic accuracy against results obtained with DNA sequencing or multiplex ligation-dependent probe amplification. Results: The SNaPshot assay provided better detection of certain SNPs than DNA sequencing. The MLH1 and MSH2 SNP marker sets were informative in 82% and 76% of the 67 cases analyzed, respectively. The new assay displayed 100% specificity for detecting LOH and predicted the location of the germline mutation in 40% of the cases (54% of those involving MLH1, 22% in MSH2). Conclusions: Our SNP-based method for detecting LOH in MLH1 and MSH2 is simple to perform with instruments available in most clinical genetics laboratories. It can be a valuable addition to protocols now used to guide mutational screening of patients with suspected HNPCC.

scholarly journals Design and characterization of a nanopore-coupled polymerase for single-molecule DNA sequencing by synthesis on an electrode array

2016 ◽  
Vol 113 (44) ◽  
pp. E6749-E6756 ◽  
Author(s):  
P. Benjamin Stranges ◽  
Mirkó Palla ◽  
Sergey Kalachikov ◽  
Jeff Nivala ◽  
Michael Dorwart ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Dna Synthesis ◽  
Lipid Bilayers ◽  
High Throughput ◽  
Single Molecule ◽  
Low Cost ◽  
Electrode Array ◽  
Oxide Semiconductor ◽  
Sequencing Platform ◽  
Sequencing By Synthesis

Scalable, high-throughput DNA sequencing is a prerequisite for precision medicine and biomedical research. Recently, we presented a nanopore-based sequencing-by-synthesis (Nanopore-SBS) approach, which used a set of nucleotides with polymer tags that allow discrimination of the nucleotides in a biological nanopore. Here, we designed and covalently coupled a DNA polymerase to an α-hemolysin (αHL) heptamer using the SpyCatcher/SpyTag conjugation approach. These porin–polymerase conjugates were inserted into lipid bilayers on a complementary metal oxide semiconductor (CMOS)-based electrode array for high-throughput electrical recording of DNA synthesis. The designed nanopore construct successfully detected the capture of tagged nucleotides complementary to a DNA base on a provided template. We measured over 200 tagged-nucleotide signals for each of the four bases and developed a classification method to uniquely distinguish them from each other and background signals. The probability of falsely identifying a background event as a true capture event was less than 1.2%. In the presence of all four tagged nucleotides, we observed sequential additions in real time during polymerase-catalyzed DNA synthesis. Single-polymerase coupling to a nanopore, in combination with the Nanopore-SBS approach, can provide the foundation for a low-cost, single-molecule, electronic DNA-sequencing platform.

scholarly journals An Automated Sample Preparation System for Large-Scale DNA Sequencing

1999 ◽  
Vol 9 (5) ◽  
pp. 457-462 ◽  
Author(s):  
Andre Marziali ◽  
Thomas D. Willis ◽  
Nancy A. Federspiel ◽  
Ronald W. Davis
Keyword(s):  
Sample Preparation ◽  
Dna Sequencing ◽  
High Throughput ◽  
Large Scale ◽  
Low Cost ◽  
High Quality ◽  
Dna Templates ◽  
Sequencing Technologies ◽  
Front End ◽  
Automated Sample Preparation

Recent advances in DNA sequencing technologies, both in the form of high lane-density gels and automated capillary systems, will lead to an increased requirement for sample preparation systems that operate at low cost and high throughput. As part of the development of a fully automated sequencing system, we have developed an automated subsystem capable of producing 10,000 sequence-ready ssDNA templates per day from libraries of M13 plaques at a cost of $0.29 per sample. This Front End has been in high throughput operation since June, 1997 and has produced > 400,000 high-quality DNA templates.

scholarly journals Organ-specific, multimodal, wireless optoelectronics for high-throughput phenotyping of peripheral neural pathways

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Woo Seok Kim ◽  
Sungcheol Hong ◽  
Milenka Gamero ◽  
Vivekanand Jeevakumar ◽  
Clay M. Smithhart ◽  
...  
Keyword(s):  
High Throughput ◽  
Low Cost ◽  
Optoelectronic Device ◽  
Antenna System ◽  
Neural Pathways ◽  
Organ Specific ◽  
Coil Antenna ◽  
Sensory Fibers

AbstractThe vagus nerve supports diverse autonomic functions and behaviors important for health and survival. To understand how specific components of the vagus contribute to behaviors and long-term physiological effects, it is critical to modulate their activity with anatomical specificity in awake, freely behaving conditions using reliable methods. Here, we introduce an organ-specific scalable, multimodal, wireless optoelectronic device for precise and chronic optogenetic manipulations in vivo. When combined with an advanced, coil-antenna system and a multiplexing strategy for powering 8 individual homecages using a single RF transmitter, the proposed wireless telemetry enables low cost, high-throughput, and precise functional mapping of peripheral neural circuits, including long-term behavioral and physiological measurements. Deployment of these technologies reveals an unexpected role for stomach, non-stretch vagal sensory fibers in suppressing appetite and demonstrates the durability of the miniature wireless device inside harsh gastric conditions.

scholarly journals Design of Low-Cost and High-Strength Titanium Alloys Using Pseudo-Spinodal Mechanism through Diffusion Couple Technology and CALPHAD

Materials ◽  
2021 ◽  
Vol 14 (11) ◽  
pp. 2910
Author(s):  
Chaoyi Ding ◽  
Chun Liu ◽  
Ligang Zhang ◽  
Di Wu ◽  
Libin Liu
Keyword(s):  
Diffusion Couple ◽  
Titanium Alloys ◽  
High Throughput ◽  
Volume Fraction ◽  
Raw Materials ◽  
Low Cost ◽  
High Strength ◽  
High Yield ◽  
Α Phase ◽  
Cr System

The high cost of development and raw materials have been obstacles to the widespread use of titanium alloys. In the present study, the high-throughput experimental method of diffusion couple combined with CALPHAD calculation was used to design and prepare the low-cost and high-strength Ti-Al-Cr system titanium alloy. The results showed that ultra-fine α phase was obtained in Ti-6Al-10.9Cr alloy designed through the pseudo-spinodal mechanism, and it has a high yield strength of 1437 ± 7 MPa. Furthermore, application of the 3D strength model of Ti-6Al-xCr alloy showed that the strength of the alloy depended on the volume fraction and thickness of the α phase. The large number of α/β interfaces produced by ultra-fine α phase greatly improved the strength of the alloy but limited its ductility. Thus, we have demonstrated that the pseudo-spinodal mechanism combined with high-throughput diffusion couple technology and CALPHAD was an efficient method to design low-cost and high-strength titanium alloys.

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