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Updated: Feb 19, 2026
Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors
Published on: September 20, 2016
Single-cell whole exome and targeted sequencing in NPM1/FLT3 positive pediatric acute myeloid leukemia
Christiane Walter1, Christian Pozzorini2, Katarina Reinhardt1
1Department of Pediatrics III, University Children's Hospital Essen, University of Duisburg-Essen, Essen, Germany.
Background:
The small portion of leukemic stem cells (LSCs) in acute myeloid leukemia (AML) present in children and adolescents is often masked by the high background of AML blasts and normal hematopoietic cells. The aim of the current study was to establish a simple workflow for reliable genetic analysis of single LSC-enriched blasts from pediatric patients.
Procedure:
For three AMLs with mutations in nucleophosmin 1 and/or fms-like tyrosine kinase 3, we performed whole genome amplification on sorted single-cell DNA followed by whole exome sequencing (WES). The corresponding bulk bone marrow DNAs were also analyzed by WES and by targeted sequencing (TS) that included 54 genes associated with myeloid malignancies.
Results:
Analysis revealed that read coverage statistics were comparable between single-cell and bulk WES data, indicating high-quality whole genome amplification. From 102 single-cell variants, 72 single nucleotide variants and insertions or deletions (70%) were consistently found in the two bulk DNA analyses. Variants reliably detected in single cells were also present in TS. However, initial screening by WES with read counts between 50-72× failed to detect rare AML subclones in the bulk DNAs.
Conclusions:
In summary, our study demonstrated that single-cell WES combined with bulk DNA TS is a promising tool set for detecting AML subclones and possibly LSCs.
