cfDNA Library Prep from Whole Blood
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Solution Overview
Problem
Conventional methods for genetic screening using cell-free DNA (cfDNA) are invasive, costly, and complex due to the need for plasma isolation and purification, which limits their sensitivity and reliability in clinical settings.
Innovation Solution
A method involving freezing and thawing whole blood samples to separate solids, followed by a single centrifugation step to obtain a liquid fraction for sequencing library preparation without prior cfDNA purification, using fixatives to stabilize blood cells and mild detergents or heating to reduce cfDNA binding to nucleosomal proteins.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional plasma isolation and purification steps are performed to obtain cfDNA, then cfDNA can be obtained for sequencing, but the process becomes complex, costly, and time-consuming
Solution Approach 1:
The invention extracts only the essential step of plasma separation from the complex conventional purification process. By using a single centrifugation step to separate plasma from whole blood and directly using this plasma for library preparation, the method eliminates multiple purification steps while maintaining sufficient cfDNA quality for sequencing analysis
Solution Approach 2:
The simplified protocol makes cfDNA sequencing accessible for routine clinical use by reducing specialized equipment and complex procedures. The method can be performed in standard clinical laboratories without requiring sophisticated purification systems, enabling widespread adoption for prenatal and cancer diagnostics
2Manufacturing precision
If plasma is separated from whole blood by centrifugation to avoid cellular DNA contamination, then cfDNA purity is improved, but the process requires additional steps and time
Solution Approach 1:
The invention performs plasma separation as a preliminary action before library preparation, but uses a single centrifugation step rather than multiple sequential separations. This preliminary plasma isolation removes the majority of cellular components that would contaminate cfDNA, while the subsequent library preparation steps are designed to work efficiently with this partially purified plasma
Solution Approach 2:
The method skips intermediate purification steps that would otherwise be required between plasma separation and library preparation. By rushing through the process with direct library preparation from centrifuged plasma, the invention maintains sufficient purity while dramatically reducing processing time
3Quantity of substance
If multiple purification steps are performed to isolate cfDNA, then cfDNA recovery is maximized, but the cost and complexity of the diagnostic procedure increase
Solution Approach 1:
The invention performs a single centrifugation step that recovers sufficient cfDNA for clinical applications without attempting to maximize recovery through multiple sequential purifications. The library preparation protocol is designed to be efficient with the cfDNA obtained from this partial purification, achieving adequate sensitivity for detecting chromosomal abnormalities without requiring exhaustive purification
Solution Approach 2:
The invention changes the parameters of the centrifugation step (single step with specific g-force and duration) to optimize the balance between cfDNA recovery and procedural simplicity. By adjusting these parameters, the method achieves sufficient cfDNA yield for clinical diagnostics while maintaining ease of execution in routine laboratory settings
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach simplifies the process, increases cfDNA recovery, and reduces costs by eliminating the need for multi-step purification, enabling reliable and sensitive detection of chromosomal abnormalities without contaminating DNA from white blood cells.
Implementation Method 1
fixing blood cells in the whole blood sample using a fixative
Implementation Method 2
freezing the whole blood sample; thawing the frozen whole blood sample
Implementation Method 3
thawing the frozen whole blood sample
Implementation Method 4
separating solids from the thawed whole blood sample comprises centrifuging the thawed whole blood sample
Implementation Method 5
The reduction of binding may be achieved by treating with a detergent or heating
Data Source
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AI summary
The disclosure provides methods and kits for preparing sequencing library to detect chromosomal abnormality using cell-free DNA (cfDNA) without the need of first isolating the cfDNA from a liquid fraction of a test sample. In some embodiments, the method involves reducing the binding between the cfDNA and nucleosomal proteins without unwinding the cfDNA from the nucleosomal proteins. In some embodiments, the reduction of binding may be achieved by treating with a detergent or heating. In some embodiments, the method further involves freezing and thawing the test sample before reducing the binding between the cfDNA and the nucleosomal proteins. In some embodiments, the test sample is a peripheral blood sample from a pregnant woman including cfDNA of both a mother and a fetus, wherein the methods may be used to detect fetal chromosomal abnormality such as copy number variation. In other embodiments, the test sample is a peripheral blood sample from a patient known or suspected to have cancer, wherein the methods can be used to detect chromosomal abnormalities in the cfDNA of the patient. Kits for detection of copy number variation of the fetus using the disclosed methods are also provided.