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Related Concept Videos

Next-generation Sequencing03:00

Next-generation Sequencing

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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.
Next-Generation Sequencing Methods
Although all next-generation methods use different technologies, they all share a set of standard features....
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Related Experiment Video

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An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing
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High-Throughput Microdissection for Next-Generation Sequencing.

Avi Z Rosenberg1, Michael D Armani2, Patricia A Fetsch3

  • 1Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America.

Plos One
|March 22, 2016
PubMed
Summary
This summary is machine-generated.

Expression microdissection (xMD) efficiently isolates specific cells from complex tissues. This tool enhances molecular pathology by enriching target cells, significantly improving mutation detection in next-generation sequencing (NGS).

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Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling |
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Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling |
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Area of Science:

  • Molecular Pathology
  • Surgical Pathology
  • Precision Medicine

Background:

  • Precision medicine relies on molecular technologies (genomics, transcriptomics, proteomics) for enhanced patient treatment.
  • Current surgical pathology tools struggle to isolate specific cell populations from complex tissues, potentially confounding molecular analysis.
  • Efficient isolation of target cells is crucial for accurate molecular profiling in diagnostics and research.

Purpose of the Study:

  • To evaluate the accuracy and precision of expression microdissection (xMD) for isolating specific cellular targets.
  • To assess the impact of xMD-based target enrichment on molecular data, particularly for next-generation sequencing (NGS).
  • To explore the utility of xMD in clinical workflows requiring tumor cell enrichment or analysis of samples with scarce target cells.

Main Methods:

  • Expression microdissection (xMD), an immuno-based technique, was used to isolate immunostained targets from tissue sections.
  • Procured targets included cytokeratin AE1/AE3, p53, estrogen receptor (ER) positive cells/nuclei, GFP-expressing fibroblasts, Epstein-Barr virus (EBV) positive nuclei, and silver-stained fungi.
  • xMD was employed to isolate specific targets constituting only 5% of a mixed cell population before NGS analysis.

Main Results:

  • xMD demonstrated accurate and precise isolation of various immunostained targets and nucleic acid targets from tissue specimens.
  • Target enrichment using xMD from a mixed cell population (5% target cells) resulted in a minimum 13-fold increase in mutation allele frequency detection via NGS.
  • The technique successfully isolated diverse targets, including protein markers, reporter proteins, viral nucleic acids, and microbial elements.

Conclusions:

  • Expression microdissection (xMD) is a valuable tool for isolating specific cell populations and subcellular targets in pathology.
  • xMD significantly enhances the sensitivity of molecular analyses, such as NGS, by enriching for low-frequency targets.
  • xMD shows promise for applications in molecular pathology research and clinical settings, particularly for samples requiring target cell enrichment.