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pISSN 2950-9114 eISSN 2950-9122
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Original Article

Lab Med Qual Assur 2022; 44(2): 76-81

Published online June 30, 2022


Copyright © Korean Association of External Quality Assessment Service.

Detection of ASXL1 Codon 646 Variant Using Amplicon-Based Next-Generation Sequencing

Miyoung Kim1 , Nan Young Kim2 , Sangkyoon Hong2 , Jiwon Lee3 , Yonggeun Cho4 , Han-Sung Kim4 , Hee Jung Kang4 , and Young Kyung Lee4

1Department of Laboratory Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul; 2Hallym Institute of Translational Genomics and Bioinformatics, Hallym University Medical Center, Anyang; 3Department of Laboratory Medicine, Green Cross Laboratories, Yongin; 4Department of Laboratory Medicine, Hallym University Sacred Heart Hospital, Hallym University College of Medicine, Anyang, Korea

Correspondence to:Miyoung Kim
Department of Laboratory Medicine, Asan Medical Center, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Korea
Tel +82-2-3010-4498
E-mail miyoungkim@amc.seoul.kr

Received: December 16, 2021; Revised: April 20, 2022; Accepted: April 25, 2022

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.


Background: The ASXL1 codon 646 variant is the most common ASXL1 variant that negatively impacts the prognoses of patients with myeloid malignancies, particularly those with myelodysplastic syndromes and acute myeloid leukemia. However, it has been suggested that this mutation is not somatic but rather an artifact of next-generation sequencing (NGS) owing to its location in an 8 bp guanine mononucleotide repeat. In this study, we evaluated the performance of amplicon-based NGS in discriminating the ASXL1 codon 646 variant.
Methods: Amplicon-based NGS was performed on the Myeloid DNA Reference Standard HD829 in varying reference material dilution ratios using the TruSight Myeloid panel and a MiSeqDx system.
Results: The expected and measured variant allele frequencies (VAFs) of the ASXL1 codon 646 mutation in the reference material were 40.00% and 18.65%, respectively. The measured VAFs in reference materials serially diluted at 1:1, 1:2, 1:4, and 1:8 were 9.09%, 5.82%, 1.92%, and 2.87%, respectively (y=0.4391x+0.8642; r2=0.9846). Most of the other variants showed VAFs comparable to expected VAFs.
Conclusions: The measured allele frequencies of the ASXL1 codon 646 variant in the serially diluted reference materials were approximately half their expected values, suggesting difficulties in the correct detection of the variant using amplicon-based NGS.

Keywords: ASXL1, Amplicon, Variant allele frequency, Next-generation sequencing

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