Prenatal cfDNA Sequencing Library Prep for Reliable Aneuploidy Detection
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Solution Overview
Problem
Current prenatal diagnostic methods for fetal aneuploidies face challenges due to insufficient sensitivity and sequencing bias, leading to the need for noninvasive methods that provide specificity, sensitivity, and applicability in clinical settings.
Innovation Solution
A novel protocol for preparing sequencing libraries from a mixture of fetal and maternal nucleic acids using end-repairing, dA-tailing, and adaptor ligation, followed by sequencing and comparing chromosome doses to threshold values to identify aneuploidies, utilizing next-generation sequencing technologies.
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 genetic material (cell-free DNA) from maternal blood plasma, separating the diagnostic target from the invasive sampling requirement. This allows noninvasive acquisition of fetal genetic information while maintaining diagnostic capability for aneuploidies
Solution Approach 2:
The patent uses maternal blood plasma as an intermediary medium to access fetal genetic material. Instead of directly sampling fetal tissue through invasive procedures, the method mediates the diagnostic process by detecting fetal cfDNA that circulates in the maternal circulation
2Measurement precision
If next-generation sequencing is used to sequence entire genomes, then measurement precision is improved, but sequencing bias and insufficient sensitivity worsen the diagnostic reliability
Solution Approach 1:
The patent applies local quality by focusing sequencing efforts on specific chromosomal regions or targets rather than attempting to sequence entire genomes. This targeted approach improves sensitivity for detecting aneuploidies while reducing the sequencing bias inherent in whole-genome sequencing of low-abundance fetal cfDNA
Solution Approach 2:
The patent changes key parameters including the target concentration (enriching fetal cfDNA), sequencing depth, and computational thresholds for aneuploidy detection. These parameter adjustments optimize the balance between sensitivity and bias reduction for clinical diagnostic reliability
3Manufacturing precision
If conventional library preparation methods are used, then manufacturing precision is maintained, but the process is time-consuming and material loss increases
Solution Approach 1:
The patent merges multiple library preparation steps into streamlined protocols that reduce the number of separate operations. This consolidation maintains manufacturing precision for library quality while significantly reducing processing time and minimizing material loss during transfers between steps
Solution Approach 2:
The patent performs preliminary actions such as pre-enrichment of fetal cfDNA and pre-preparation of sequencing libraries before actual sequencing. This advance preparation optimizes the subsequent sequencing process, improving both speed and material utilization efficiency
4Object-affected harmful factors
If noninvasive methods using fetal nucleic acids from maternal blood are used, then harmful factors are reduced, but sensitivity is insufficient due to limited levels of cfDNA
Solution Approach 1:
The patent uses copying by amplifying fetal cfDNA sequences through targeted enrichment methods before sequencing. This creates sufficient copies of the limited fetal genetic material present in maternal blood, thereby improving detection sensitivity while maintaining the noninvasive advantage
Solution Approach 2:
The patent employs composite approaches combining multiple enrichment techniques and sequencing strategies to overcome the limitation of low cfDNA levels. By integrating biochemical enrichment with optimized sequencing protocols, the method achieves high sensitivity without requiring invasive sampling
Data Source
AI summary
The invention provides methods for determining aneuploidy and/or fetal fraction in maternal samples comprising fetal and maternal cfDNA 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.