Clinical chemistry · 2026
Genetic Investigation of X-Linked Defects with Optimized Targeted Long-Read Sequencing.
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Zheng Jia, Li Keying, Han Jin, Chan Chi Chun, Wang Huilin, Law Jasmine Yan Tung, Chen Lin, Jiang Fan, Zhang Rui, Liang Hailei, Chen Xiaofang, Shi Mengmeng, Chau Matthew Hoi Kin, Cao Ye, Kwok Yvonne K, Zhu Yuanfang, Leung Tak Yeung, Choy Kwong Wai, Dong Zirui
Аннотация
BACKGROUND: X-linked genetic diseases affect males and females through different inheritance patterns influenced by sex, where differential methylation caused by X-chromosome inactivation can present with a range of asymptomatic, mild, and severe symptoms in females. Targeted long-read sequencing (LRS) through adaptive sampling (AS) shows robust performance in characterizing genomic composition and methylation status within the X chromosome, but it is not optimal for nonhigh-molecular-weight (non-HMW) DNA. Herein, we optimized a targeted LRS for non-HMW DNA and validated its diagnostic utility in families with X-linked genomic variants reported by routine methods.
METHODS: Twenty families with X-linked genetic defects reported by previous chromosomal microarray analysis and/or low-pass genome sequencing were recruited and underwent optimized targeted LRS. Genomic variants were detected and classified along with haplotype analysis for assessing allele-specific methylation changes.
RESULTS: The AS-based protocol was optimized for DNA fragmentation with size selection, library construction, and sequencing, and demonstrated a minimum 15-fold enrichment of targets against nontargets compared with the standard AS-based protocol showing 8.1-fold enrichment. In addition, our optimized LRS detected all of the X-linked variants reported previously and revealed additional findings in 7/20 families (35%). This led to variant reclassification in 3/20 cases (15%) achieved through breakpoint resolution for structural variants and determination of gene methylation status.
CONCLUSIONS: Our study demonstrated an AS-based protocol optimized for non-HMW DNAs that enabled the comprehensive characterization of genomic and methylation changes, highlighting its robust performance in the genomic evaluation of X-linked genetic defects with promising broader application to other disease states.
METHODS: Twenty families with X-linked genetic defects reported by previous chromosomal microarray analysis and/or low-pass genome sequencing were recruited and underwent optimized targeted LRS. Genomic variants were detected and classified along with haplotype analysis for assessing allele-specific methylation changes.
RESULTS: The AS-based protocol was optimized for DNA fragmentation with size selection, library construction, and sequencing, and demonstrated a minimum 15-fold enrichment of targets against nontargets compared with the standard AS-based protocol showing 8.1-fold enrichment. In addition, our optimized LRS detected all of the X-linked variants reported previously and revealed additional findings in 7/20 families (35%). This led to variant reclassification in 3/20 cases (15%) achieved through breakpoint resolution for structural variants and determination of gene methylation status.
CONCLUSIONS: Our study demonstrated an AS-based protocol optimized for non-HMW DNAs that enabled the comprehensive characterization of genomic and methylation changes, highlighting its robust performance in the genomic evaluation of X-linked genetic defects with promising broader application to other disease states.