Prenatal Sequencing Library Preparation for Reliable Aneuploidy Detection
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Solution Overview
Problem
Current prenatal diagnostic methods for fetal aneuploidies face limitations in sensitivity and sequencing bias, leading to the need for noninvasive methods that provide specificity, sensitivity, and applicability for reliable diagnosis.
Innovation Solution
A method involving massively parallel sequencing of fetal and maternal nucleic acids, including steps of end-repairing, dA-tailing, and adaptor ligation, followed by sequencing and comparison to threshold values, to determine fetal aneuploidy and fetal fraction in maternal samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If invasive testing methods (amniocentesis or CVS) are used for prenatal diagnosis, then diagnostic reliability is improved, but the risk of miscarriage increases
Solution Approach 1:
The patent extracts fetal nucleic acids from maternal peripheral blood plasma, separating the diagnostic target (fetal DNA) from the invasive sampling process. This allows obtaining fetal genetic material without directly accessing the fetus through invasive procedures, thereby maintaining diagnostic reliability while eliminating miscarriage risk
Solution Approach 2:
The patent uses maternal peripheral blood plasma as an intermediary medium to access fetal genetic material. Instead of directly sampling fetal tissue, the method captures fetal nucleic acids that have circulated into the maternal bloodstream, serving as a safe intermediary that provides diagnostic information without invasive risk
2Productivity
If next generation sequencing is used to sequence entire genomes, then sequencing speed is improved, but sensitivity is insufficient due to limited cDNA levels
Solution Approach 1:
The patent performs preliminary enrichment of fetal nucleic acids from maternal plasma before sequencing. By pre-concentrating the fetal DNA targets and removing maternal background DNA, the method ensures sufficient signal strength and sensitivity for accurate detection, enabling NGS to achieve both high speed and high sensitivity
Solution Approach 2:
The patent changes the concentration parameter of fetal nucleic acids in the sample through enrichment procedures. By increasing the relative concentration of fetal DNA from trace levels to detectable levels, the method overcomes the sensitivity limitation of NGS while maintaining its high sequencing speed capability
3Reliability
If conventional sequencing methods are used, then sequencing bias is reduced, but sensitivity and applicability are insufficient for reliable fetal aneuploidy diagnosis
Solution Approach 1:
The patent segments the diagnostic process into distinct modules: fetal nucleic acid enrichment, library preparation, NGS sequencing, and bioinformatic analysis. This segmentation allows optimization of each step independently, achieving high diagnostic reliability through systematic control while managing complexity through modular design
Solution Approach 2:
The patent develops a universal enrichment and sequencing platform that can detect various fetal aneuploidies (trisomy 21, 18, 13, sex chromosome abnormalities) using the same methodology. This multi-functional approach achieves broad diagnostic applicability and high reliability across different conditions without requiring separate specialized methods for each anomaly type
Data Source
AI summary
The invention provides methods for determining aneuploidy and/or fetal fraction in maternal samples comprising fetal and maternal ctDNA by massively parallel sequencing. The method comprises a novel protocol for preparing sequencing libraries that unexpectedly improves the quality of library DNA while expediting the process of analysis of samples for prenatal diagnoses.