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Updated: Feb 16, 2026
Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER
Published on: June 23, 2012
MLDP-AS: an optimized next-generation sequencing assay for enhanced detection of technically challenging variants in
Zhenhua Zhao1, Ganye Zhao1, Shaojun Li2
1Department of Obstetrics and Gynecology, Genetic and Prenatal Diagnosis Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China.
Background:
Next-generation sequencing (NGS) facilitates simultaneous carrier screening for multiple single-gene disorders. However, conventional NGS methods struggle to detect complex variants, such as F8 inversions, CYP21A2 variations, and single-exon copy number variations (CNVs), resulting in residual risk.
Methods:
We developed and validated MLDP-AS (Multiplex Long-Distance PCR followed by Amplicon Sequencing), a novel NGS assay, to screen for pathogenic variants in ten prevalent single-gene disorders in the Chinese population, including alpha- and beta-thalassemia, non-syndromic hearing loss, spinal muscular atrophy, Duchenne muscular dystrophy, phenylketonuria, 21-hydroxylase deficiency, Wilson disease, methylmalonic acidemia, and Hemophilia A. MLDP-AS detects routine variants and technically challenging types, such as F8 intron 22 inversions, CYP21A2 variations, and single-exon CNVs, in a single test. The assay was optimized using positive clinical samples, with sensitivity validated against gold-standard methods. Clinical applicability was evaluated through a prospective study of couples planning or undergoing pregnancy, with positive results confirmed by gold-standard techniques.
Results:
MLDP-AS achieved 100% sensitivity, identifying all 255 pathogenic variants in known positive samples, including 36 technically challenging variants. In a prospective trial involving 5,209 individuals, carriers for all targeted disorders were detected, with an overall carrier rate of 22.15%. Thirty-four couples (1.09%) were identified as at-risk, spanning seven of the ten diseases. A total of 289 pathogenic variants were detected 1,290 times, including 169 technically challenging variants. The assay demonstrated a positive predictive value of 99.7% compared to gold-standard methods. MLDP-AS is rapid and cost-effective, completing testing within three days at a cost under $25.
Conclusions:
MLDP-AS integrates detection of multiple complex variants into a single, comprehensive assay, overcoming limitations of conventional NGS. It significantly enhances variant detection and provides an efficient, cost-effective tool for carrier screening in the Chinese population.
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