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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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A Semiautomated ChIP-Seq Procedure for Large-scale Epigenetic Studies
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A technical application of quantitative next generation sequencing for chimerism evaluation.

Michelangelo Aloisio1, Danilo Licastro2, Luciana Caenazzo3

  • 1Department of Life Sciences, University of Trieste, I‑34127 Trieste, Italy.

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|August 9, 2016
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Summary

This study introduces a new next-generation sequencing (NGS) method for precise chimerism quantification after hematopoietic stem cell transplantation. The developed NGS protocol demonstrates a 1% limit of detection for evaluating chimerism stability over time.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Transplantation Science

Background:

  • Current chimerism evaluation post-hematopoietic stem cell transplantation relies on microsatellite analysis via capillary electrophoresis.
  • Monitoring chimerism is crucial for assessing transplant success and detecting graft rejection or relapse.
  • Next-generation sequencing (NGS) offers potential for enhanced genetic diagnostic capabilities.

Purpose of the Study:

  • To evaluate a novel next-generation sequencing (NGS) protocol for accurate chimerism quantification.
  • To establish the stability of mixed chimerism over time using the developed NGS method.
  • To assess the feasibility of applying NGS in routine clinical diagnostics for chimerism analysis.

Main Methods:

  • Development of a 44-amplicon custom chimerism panel using Ion AmpliSeq technology.
  • Creation of a custom bioinformatics pipeline for genotyping and quantifying NGS data.
  • Application of the NGS protocol following standard Ion AmpliSeq library preparation and Ion Torrent guidelines.

Main Results:

  • The custom NGS panel identified an average of 16 informative recipient alleles per sample.
  • The developed protocol achieved a limit of detection of 1% for chimerism quantification.
  • NGS background noise was determined to be less than 1%.

Conclusions:

  • The study successfully established a novel NGS protocol for chimerism quantification.
  • The developed method provides technical details for potential future clinical application of NGS in transplantation.
  • NGS offers a promising alternative for precise and sensitive chimerism monitoring.