A DNA pool of FLT3-ITD positive DNA samples can be used efficiently for analytical evaluation of NGS-based FLT3-ITD quantitation - Testing several different ITD sequences and rates, simultaneously

Zoltán A. Mezei, Dávid Tornai, Róza Földesi, László Madar, Andrea Sümegi, M. Papp, P. Antal-Szalmás

Research output: Article

Abstract

Internal tandem duplication (ITD) in the fms-like tyrosine kinase 3 (FLT3) gene is one of the most frequent genetic alteration in acute myeloid leukemia (AML), and it is associated with worse clinical outcome. Not only the presence but also the size, localization and the rate of this variant or the presence of multiple ITDs has prognostic information. The traditional PCR based diagnostic methods cannot provide information about all of these parameters in one assay, however the application of next generation sequencing (NGS) technique can be a reliable solution for this diagnostic problem. In order to evaluate the analytical properties of an NGS-based FLT3-ITD detection assay a quality control sample was prepared from DNA of AML patients containing 19 different FLT3-ITD variants identified by NGS. The higher the total read count was in a certain sample of the NGS run, the more ITD variant types could be detected. The maximal sensitivity of FLT3-ITD detection by NGS technique was as low as 0.007% FLT3-ITD/total allele rate, however, below 0.1% rate, the reproducibility of the quantitation was poor (CV > 25%). DNA pools with several FLT3-ITDs can be used efficiently for analytical evaluation of NGS-based FLT3-ITD quantitation testing several different ITD sequences and rates, simultaneously.

Original languageEnglish
Pages (from-to)25-29
Number of pages5
JournalJournal of Biotechnology
Volume303
DOIs
Publication statusPublished - szept. 10 2019

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fms-Like Tyrosine Kinase 3
Assays
DNA
Testing
Quality control
Genes
Acute Myeloid Leukemia
Quality Control
Alleles
Polymerase Chain Reaction

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

Cite this

@article{80719f6f11a84637b53e65f99088591b,
title = "A DNA pool of FLT3-ITD positive DNA samples can be used efficiently for analytical evaluation of NGS-based FLT3-ITD quantitation - Testing several different ITD sequences and rates, simultaneously",
abstract = "Internal tandem duplication (ITD) in the fms-like tyrosine kinase 3 (FLT3) gene is one of the most frequent genetic alteration in acute myeloid leukemia (AML), and it is associated with worse clinical outcome. Not only the presence but also the size, localization and the rate of this variant or the presence of multiple ITDs has prognostic information. The traditional PCR based diagnostic methods cannot provide information about all of these parameters in one assay, however the application of next generation sequencing (NGS) technique can be a reliable solution for this diagnostic problem. In order to evaluate the analytical properties of an NGS-based FLT3-ITD detection assay a quality control sample was prepared from DNA of AML patients containing 19 different FLT3-ITD variants identified by NGS. The higher the total read count was in a certain sample of the NGS run, the more ITD variant types could be detected. The maximal sensitivity of FLT3-ITD detection by NGS technique was as low as 0.007{\%} FLT3-ITD/total allele rate, however, below 0.1{\%} rate, the reproducibility of the quantitation was poor (CV > 25{\%}). DNA pools with several FLT3-ITDs can be used efficiently for analytical evaluation of NGS-based FLT3-ITD quantitation testing several different ITD sequences and rates, simultaneously.",
keywords = "Analytical validation, Deep next generation sequencing (NGS), Fms-like tyrosine kinase 3 (FLT3), Internal tandem duplication (ITD)",
author = "Mezei, {Zolt{\'a}n A.} and D{\'a}vid Tornai and R{\'o}za F{\"o}ldesi and L{\'a}szl{\'o} Madar and Andrea S{\"u}megi and M. Papp and P. Antal-Szalm{\'a}s",
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T1 - A DNA pool of FLT3-ITD positive DNA samples can be used efficiently for analytical evaluation of NGS-based FLT3-ITD quantitation - Testing several different ITD sequences and rates, simultaneously

AU - Mezei, Zoltán A.

AU - Tornai, Dávid

AU - Földesi, Róza

AU - Madar, László

AU - Sümegi, Andrea

AU - Papp, M.

AU - Antal-Szalmás, P.

PY - 2019/9/10

Y1 - 2019/9/10

N2 - Internal tandem duplication (ITD) in the fms-like tyrosine kinase 3 (FLT3) gene is one of the most frequent genetic alteration in acute myeloid leukemia (AML), and it is associated with worse clinical outcome. Not only the presence but also the size, localization and the rate of this variant or the presence of multiple ITDs has prognostic information. The traditional PCR based diagnostic methods cannot provide information about all of these parameters in one assay, however the application of next generation sequencing (NGS) technique can be a reliable solution for this diagnostic problem. In order to evaluate the analytical properties of an NGS-based FLT3-ITD detection assay a quality control sample was prepared from DNA of AML patients containing 19 different FLT3-ITD variants identified by NGS. The higher the total read count was in a certain sample of the NGS run, the more ITD variant types could be detected. The maximal sensitivity of FLT3-ITD detection by NGS technique was as low as 0.007% FLT3-ITD/total allele rate, however, below 0.1% rate, the reproducibility of the quantitation was poor (CV > 25%). DNA pools with several FLT3-ITDs can be used efficiently for analytical evaluation of NGS-based FLT3-ITD quantitation testing several different ITD sequences and rates, simultaneously.

AB - Internal tandem duplication (ITD) in the fms-like tyrosine kinase 3 (FLT3) gene is one of the most frequent genetic alteration in acute myeloid leukemia (AML), and it is associated with worse clinical outcome. Not only the presence but also the size, localization and the rate of this variant or the presence of multiple ITDs has prognostic information. The traditional PCR based diagnostic methods cannot provide information about all of these parameters in one assay, however the application of next generation sequencing (NGS) technique can be a reliable solution for this diagnostic problem. In order to evaluate the analytical properties of an NGS-based FLT3-ITD detection assay a quality control sample was prepared from DNA of AML patients containing 19 different FLT3-ITD variants identified by NGS. The higher the total read count was in a certain sample of the NGS run, the more ITD variant types could be detected. The maximal sensitivity of FLT3-ITD detection by NGS technique was as low as 0.007% FLT3-ITD/total allele rate, however, below 0.1% rate, the reproducibility of the quantitation was poor (CV > 25%). DNA pools with several FLT3-ITDs can be used efficiently for analytical evaluation of NGS-based FLT3-ITD quantitation testing several different ITD sequences and rates, simultaneously.

KW - Analytical validation

KW - Deep next generation sequencing (NGS)

KW - Fms-like tyrosine kinase 3 (FLT3)

KW - Internal tandem duplication (ITD)

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