A novel high-throughput molecular counting method with single base-pair resolution enables accurate single-gene NIPT

Area of Science:

• Molecular Biology
• Genetics
• Bioinformatics

Background:

• Next-generation DNA sequencing faces limitations in accurately quantifying input DNA molecules.
• Accurate molecular counting is essential for sensitive diagnostic applications like single-gene non-invasive prenatal testing (sgNIPT) and liquid biopsy.
• Current methods lack the precision needed for reliable genetic disease detection from low-input samples.

Purpose of the Study:

• To develop a novel molecular counting method for accurate reconstruction of input DNA molecules from sequencing data.
• To apply this method for the development of highly sensitive and specific single-gene non-invasive prenatal tests (sgNIPTs).
• To validate the performance of the developed sgNIPTs for common monogenic disorders.

Main Methods:

• Development of Quantitative Counting Template (QCT) molecular counting technique.
• Application of QCT for reconstructing the number of input DNA molecules using sequencing data.
• Design and implementation of sgNIPTs for sickle cell disease, cystic fibrosis, spinal muscular atrophy, alpha-thalassemia, and beta-thalassemia.

Main Results:

• The Quantitative Counting Template (QCT) molecular counting method accurately reconstructs input DNA molecule counts.
• Developed sgNIPTs demonstrated high analytical sensitivity (>98%) and specificity (>99%).
• Clinical validation with maternal blood samples showed 100% concordance with newborn follow-up results.

Conclusions:

• QCT molecular counting provides a robust solution for accurate DNA quantification in sequencing data.
• The developed sgNIPTs are highly accurate and reliable for prenatal screening of multiple genetic disorders.
• This technology has significant potential to improve diagnostic accuracy in prenatal and liquid biopsy applications.