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Related Concept Videos

RNA-seq03:21

RNA-seq

RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases. 
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Next-generation Sequencing03:00

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The first human genome sequencing project cost $2.7 billion and was declared complete in 2003, after 15 years of international cooperation and collaboration between several research teams and funding agencies. Today, with the advent of next-generation sequencing technologies, the cost and time of sequencing a human genome have dropped over 100 fold.
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Sanger Sequencing

DNA sequencing is a fundamental technique that is routinely used in the biological sciences. This method can be applied to a range of questions at different scales - from the sequencing of a cloned DNA fragment or the study of a mutation in a gene up to whole-genome sequencing. However, despite the widespread use of sequencing today, it was not until 1977 that Fredrick Sanger and his collaborators developed the chain-termination method to decode DNA sequences. It relies on the separation of a...
PCR01:32

PCR

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Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry
05:53

Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry

Published on: June 21, 2018

A high-plex PCR approach for massively parallel sequencing.

Tú Nguyen-Dumont1, Bernard J Pope, Fleur Hammet

  • 1Genetic Epidemiology Laboratory, Department of Pathology, The University of Melbourne, Victoria, Australia.

Biotechniques
|August 13, 2013
PubMed
Summary
This summary is machine-generated.

We developed Hi-Plex, a cost-effective PCR-based massively parallel sequencing (MPS) strategy for high-throughput screening of multiple genomic regions. This method simplifies primer design and reduces costs, making it ideal for broad genomic analysis.

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Area of Science:

  • Genomics
  • Molecular Biology
  • Biotechnology

Background:

  • Current targeted massively parallel sequencing (MPS) methods face limitations in design flexibility, cost, and complexity.
  • These drawbacks restrict MPS applications to smaller target sizes and high-throughput, low-cost scenarios.

Purpose of the Study:

  • To introduce Hi-Plex, a novel PCR-MPS strategy designed for high-throughput screening of multiple genomic target regions.
  • To address the limitations of existing MPS methods by offering a simpler, more cost-effective, and flexible approach.

Main Methods:

  • Developed Hi-Plex, a PCR-MPS strategy incorporating automated primer design software for precise control over product size.
  • Utilized permissive thermocycling conditions and clamp bias reduction for a simplified, cost- and time-effective protocol.
  • Applied Hi-Plex to a 60-plex assay targeting breast cancer predisposition genes (PALB2, XRCC2) using various DNA inputs (LCL and FFPE).

Main Results:

  • Achieved high on-target read percentages (≥86.94%) with excellent amplicon representation (within 25-fold of mean) across different DNA types.
  • Demonstrated superior performance with FFPE DNA (25 ng input), reaching 95.14% on-target reads and a narrow relative representation range (10.1-fold lower to 5.8-fold higher than mean).
  • Results were obtained using only automatically designed primers at equal concentrations, highlighting protocol efficiency.

Conclusions:

  • Hi-Plex offers a powerful and efficient new approach for screening multiple genomic target regions.
  • The method is cost-effective, time-efficient, and requires minimal optimization, making it suitable for high-throughput genomic analysis.
  • Hi-Plex overcomes limitations of current MPS techniques, broadening its applicability in genetic research and diagnostics.