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pp 1-14 | Cite as

Targeted, Amplicon-Based, Next-Generation Sequencing to Detect Age-Related Clonal Hematopoiesis

  • Brooke Snetsinger
  • Christina K. Ferrone
  • Michael J. RauhEmail author
Protocol
Part of the Methods in Molecular Biology book series

Abstract

Aging hematopoietic stem cells acquire mutations that sometimes impart a selective advantage. Next-generation DNA sequencing (NGS) can be used to detect expanded peripheral blood progeny of a mutant clone, usually carrying just one cancer-driver mutation, most often in the epigenetic regulator genes, DNMT3A or TET2. This phenomenon is known as clonal hematopoiesis (CH), age-related CH (ARCH) when considering its association with age, and CH of indeterminate potential (CHIP) when the variant allele fraction (VAF) is at least 2% in peripheral leukocytes. CHIP is present in at least 10–15% of adults older than 65 years and is a risk factor for hematological neoplasms and diseases exacerbated by mutant, hyper-inflammatory, monocytes/macrophages, such as atherosclerotic cardiovascular disease. Therefore, the detection of CHIP has important clinical consequences. Herein, we present a protocol for the generation of targeted, amplicon-based, NGS libraries for ion semiconductor sequencing and CHIP detection, using Ion Torrent platforms.

Keywords

Clonal hematopoiesis (CH) Age-related clonal hematopoiesis (ARCH) Clonal hematopoiesis of indeterminate potential (CHIP) Next-generation DNA sequencing (NGS) Amplicon Library Ion semiconductor Ion Torrent Peripheral blood DNMT3A TET2 

Notes

Acknowledgments

The authors thank Dr. Harriet Feilloter, Dr. Xudong Liu, Dr. Amy McNaughton, Dr. Xiao Zhang, and Dr. Paul Park for initial assistance with Ion Torrent Sequencing. Funding was provided by the Southeastern Ontario Academic Medical Organization (SEAMO) Innovation Fund, the University Hospitals Kingston Fund (UHKF)/Women’s Giving Circle, and the Ontario Institute for Cancer Research (OICR)/Ontario Molecular Pathology Research Network (OMPRN).

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Copyright information

© Springer Science+Business Media New York 2019

Authors and Affiliations

  • Brooke Snetsinger
    • 1
  • Christina K. Ferrone
    • 1
  • Michael J. Rauh
    • 1
    Email author
  1. 1.Department of Pathology and Molecular MedicineQueen’s UniversityKingstonCanada

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