Amplification-Free cfDNA Library Prep for Preserving Fragment Profiles
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Solution Overview
Problem
Current methods for analyzing low amounts of DNA, particularly cell-free DNA, face challenges such as DNA degradation, loss of secondary information, and amplification biases, which complicate genetic profiling and disease diagnosis.
Innovation Solution
An amplification-free method involving repairing cfDNA to obtain single base overhang fragments and adding directional barcoded adapters for sequencing, enabling direct analysis of DNA without altering profiles or losing secondary information.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If amplification processes are used to analyze small amounts of DNA, then the quantity of DNA is increased for analysis, but amplification biases are introduced and secondary information is lost
Solution Approach 1:
The patent extracts only the necessary information (DNA fragment presence and characteristics) without performing amplification. By using direct sequencing approaches and avoiding whole genome amplification, the method retrieves genetic information while preserving the original DNA profile and secondary information such as fragment length and modifications.
Solution Approach 2:
The patent introduces intermediary steps such as adapter ligation and targeted enrichment that allow analysis of low-input DNA without amplification. These intermediaries enable direct sequencing or targeted analysis while maintaining the original DNA characteristics, serving as a bridge between limited DNA input and sufficient analytical information.
2Measurement precision
If advanced bioinformatics processes are used to correct amplification imbalances, then the accuracy of chromosome profile is improved, but the complexity of the analysis process increases
Solution Approach 1:
Instead of using amplification and then correcting its biases with complex bioinformatics, the patent inverts the approach by eliminating amplification entirely. This direct approach to DNA analysis avoids amplification biases from the outset, achieving accurate chromosome profiles without requiring complex corrective bioinformatics processes.
3Quantity of substance
If large volume of plasma is used as source of cell free DNA, then the amount of cfDNA is increased for analysis, but the complexity of purification and quantification increases
Solution Approach 1:
The patent uses partial action by working with the cfDNA that can be obtained from relatively small plasma volumes without requiring extensive purification. The method is designed to be effective with limited DNA input, performing only the necessary minimal purification steps rather than exhaustive purification protocols, thereby reducing process complexity while obtaining sufficient analytical material.
4Measurement precision
If chemical modification and sequencing are used to identify organ source of cfDNA, then the accuracy of tissue damage identification is improved, but the amount of starting DNA required increases
Solution Approach 1:
The patent performs preliminary action by using targeted enrichment approaches that pre-concentrate DNA from specific organs or tissues before sequencing. This preliminary enrichment step allows identification of organ sources with high accuracy while requiring minimal starting DNA, as the enrichment occurs before the sequencing step rather than requiring large amounts of DNA for direct analysis.
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 allows for accurate and efficient genetic profiling of low DNA amounts, preserving original fragment features and reducing amplification biases, applicable in IVF embryo testing, cancer detection, and chronic disease management.
Implementation Method 1
repairing the cfDNA to obtain single base overhang fragments
Implementation Method 2
adding a directional barcoded adapter to each of the ends of the repaired cfDNA, thereby generating a barcoded library for sequencing
Data Source
AI summary
Provided are an amplification-free method for the preimplantation genetic testing (PGT) of in-vitro fertilization (IVF) embryos, an amplification-free method of generating a library for the PGT, and a method of identifying the genetic background of IVF embryos. Provided are also a method of determining the degree of non-embryonic DNA contamination, and an amplification-free method of generating a library of cell-free DNAs (cfDNAs) from a biological material. Similarly provided are a method for enrichment of methylated fragments from the amplification free library, and a method for identifying methylation profiles and/or changes of same, in samples of different sources.