Oxford Nanopore Third Generation Sequencing for Analysis of FMR1 5'UTR CGG Repeat Expansions

Xu Yang; Bowei Han; Jie Huang; Min Zhang; Shi Weng; Guojun Ouyang; Wanqing Han; Wenyu Wang; Li Zhang; Juanjuan Chen; Juan Du; Yingsong Wu; Xuexi Yang

doi:10.1016/j.ab.2025.115931

Oxford Nanopore Third Generation Sequencing for Analysis of FMR1 5'UTR CGG Repeat Expansions

Anal Biochem. 2025 Jul 11:115931. doi: 10.1016/j.ab.2025.115931. Online ahead of print.

Authors

Xu Yang¹, Bowei Han², Jie Huang³, Min Zhang², Shi Weng², Guojun Ouyang⁴, Wanqing Han⁴, Wenyu Wang⁴, Li Zhang⁴, Juanjuan Chen⁵, Juan Du⁶, Yingsong Wu⁷, Xuexi Yang⁸

Affiliations

¹ Institute of Antibody Engineering, School of Laboratory Medicine and Biotechnology, Southern Medical University, Guangzhou, China; Guangzhou Darui Biotechnology Co., Ltd., Guangzhou, China.
² Institute of Antibody Engineering, School of Laboratory Medicine and Biotechnology, Southern Medical University, Guangzhou, China.
³ Department of in vitro Diagnostic Reagent, National Institutes for Food and Drug Control, Beijing, China.
⁴ Guangzhou Darui Biotechnology Co., Ltd., Guangzhou, China.
⁵ School of Laboratory Medicine and Biotechnology, Southern Medical University, Guangzhou, China.
⁶ Institute of Reproductive and Stem Cell Engineering, School of Basic Medical Science, Central South University, Changsha, China; Reproductive and Genetic Hospital of CITIC-Xiangya, Changsha, China; National Engineering and Research Center of Human Stem Cells, Changsha, China. Electronic address: tandujuan@csu.edu.cn.
⁷ Institute of Antibody Engineering, School of Laboratory Medicine and Biotechnology, Southern Medical University, Guangzhou, China. Electronic address: wg@smu.edu.cn.
⁸ Institute of Antibody Engineering, School of Laboratory Medicine and Biotechnology, Southern Medical University, Guangzhou, China. Electronic address: yangxuexi2017@126.com.

PMID: 40653294
DOI: 10.1016/j.ab.2025.115931

Abstract

Objective: Fragile X syndrome is mainly caused by the expansion of GC-rich cytosine-guanine-guanine (CGG) repeat in FMR1 5'UTR region, as well as rare gene point mutations or deletions in its open reading frame. Currently, third-generation long-read sequencing is a potential technology for simultaneously detecting CGG repeat expansions, point mutations, and deletions. However, a major challenge remains in obtaining the target long-fragment CGG repeat region with ultra-high GC content for sequencing.

Methods: We developed a novel approach combining long-fragment ultra-high GC polymerase chain reaction (PCR) amplification with Oxford Nanopore sequencing to detect the full spectrum of FMR1 5'UTR CGG repeat mutations. The method was validated using 10 standard cell line samples (males: n_normal=1, n_intermediate=1, n_pre-mutation=1, and n_{full mutation}=2; females: n_normal=1, n_intermediate=1, n_pre-mutation=2, and n_{full mutation}=1) and 53 retrospective clinical blood samples (males: n_normal=7, n_pre-mutation=3, n_{full mutation}=15, and n_{mosaic mutaion}=2; females: n_normal=9, n_pre-mutation=13, and n_{full mutation}=4).

Results: Our method demonstrated that the 100% concordance with the triplet repeat-primed PCR and Southern blot analysis in genotyping 10 cell line samples and 53 clinical samples. Additionally, CGG repeat numbers showed strong correlation with reference mehods (male cell lines, n=5, R²=0.9996; female cell lines, n=5, R²=0.9972; clinical male samples, n=26, R²=1.0000; clinical female samples, n=25, R²=0.9854).

Conclusion: This study presents a simple and cost-effective strategy for preparing FMR1 5'UTR CGG repeat regions with long-fragment ultra-high GC content for third-generation sequencing. The approach could serve as a model for detecting other challenging disorders caused by short tandem repeat expansions, such as myotonic dystrophy and Huntington' s disease.

Keywords: CGG repeat expansion; FMR1; Long read; Oxford Nanopore Sequencing; Ultra-high GC.