High dynamic range capillary electrophoresis method for sensitive detection of low-frequency driver mutations

High dynamic range capillary electrophoresis method for sensitive detection of low-frequency driver mutations

Abstract Cancer genomics aims to personalize treatments by identifying genetic abnormalities in cancer cells. However, current analytical techniques face limitations in simplicity and cost-effectiveness. To address these issues, we developed an enhanced capillary gel electrophoresis (CE) sequencer u...

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Main Authors: Nobue Tamamura, Yoshihiko Hagiwara, Hirokazu Kato, Yusuke Ono, Kenji Takahashi, Kazuya Koyama, Hiroki Sato, Tetsuhiro Okada, Hidemasa Kawabata, Yu Ohtaki, Chiho Maeda, Miyuki Mori, Shin-ichi Chiba, Mishie Tanino, Kenzui Taniue, Takashi Anazawa, Ryoji Inaba, Yusuke Mizukami
Format: Article
Language:English
Published: Nature Portfolio 2025-07-01
Series:Scientific Reports
Subjects:
HiDy
Capillary gel electrophoresis
Dynamic range
Low-frequency mutation
KRAS
Fragment analysis
Online Access:https://doi.org/10.1038/s41598-025-01884-5
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Summary:Abstract Cancer genomics aims to personalize treatments by identifying genetic abnormalities in cancer cells. However, current analytical techniques face limitations in simplicity and cost-effectiveness. To address these issues, we developed an enhanced capillary gel electrophoresis (CE) sequencer using a fluorescence-acquisition technique called “HiDy” (High Dynamic range) (HiDy-CE). The HiDy-CE reduces the hardware binning region size and increases the number of regions on a charge-coupled device image sensor, expanding the dynamic range and reducing saturation risk. By applying the multi-base primer extension method to the HiDy-CE with control DNA containing known mutations, we detected variant allele frequencies (VAFs) as low as 0.5% for major KRAS hotspot mutation at codon 12 and 13. With 10 ng of DNA from small tissues obtained via fine-needle biopsy from patients with suspected pancreaticoduodenal tumors, HiDy-CE produced equivalent VAFs in KRAS compared with targeted amplicon sequencing. This demonstrated the world’s first capability of detecting mutations below 1% on CE using pathological specimens, leveraging its wide dynamic range. With only 2 ng of input DNA, the HiDy-CE provided results highly concordant with digital PCR with minimal non-specific noise. These findings underscore the HiDy-CE’s potential for sensitive detection of oncogenes such as KRAS, facilitating pre-testing before comprehensive genome profiling.
ISSN:2045-2322

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