Duplex Sequencing Cell-Free DNA Error Filtering
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Solution Overview
Problem
Current methods for liquid biopsy-based molecular testing face challenges in capturing and sequencing circulating tumor DNA (ctDNA) due to DNA fragmentation and dilution in plasma, leading to false positives and difficulties in distinguishing true variants from background errors, especially at low allelic frequencies.
Innovation Solution
A method involving end-repair, A-tailing, stem-loop adaptor ligation, and hybridization capture with RNA baits to enrich and sequence ctDNA, using dual molecular barcodes for error elimination and bioinformatics strategies to correct sequencing artifacts and identify low-frequency variants.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional sequencing methods are used on cfDNA, then sequencing can be performed, but background errors occur predominantly from DNA-damaging events leading to false positive variants
Solution Approach 1:
The method segments the sequencing process into two independent library preparations from the same cfDNA template. Each library is sequenced separately and results are integrated computationally. This segmentation allows identification of true variants that appear in both libraries while filtering out background errors that occur in only one library.
Solution Approach 2:
The method implements feedback through iterative computational analysis where sequencing results from both libraries are compared and integrated. Variants are validated by checking their presence and consistency across both independent sequencing runs, with background errors identified and filtered based on their inconsistent appearance.
2Quantity of substance
If cfDNA is captured and sequenced, then molecular profiling is obtained, but circulating tumor DNA is diluted into abundant cell-free DNA making it challenging to capture and retain
Solution Approach 1:
The method performs preliminary action by preparing two independent libraries from the same cfDNA sample before sequencing. This duplicate preparation ensures that true ctDNA variants are captured in both libraries while statistical analysis can later distinguish true signals from background noise, improving both capture efficiency and reliability.
Solution Approach 2:
The method changes parameters by using different library preparation protocols or conditions for the two independent libraries. This allows optimization for different aspects of detection and provides complementary data that improves overall sensitivity and specificity for capturing rare ctDNA molecules.
3Measurement precision
If sequencing is performed on cfDNA, then variants can be detected, but it is difficult to distinguish true variants from background errors at low allelic frequencies
Solution Approach 1:
The method segments variant validation into independent observation events across two libraries. True variants at low frequency will appear consistently in both libraries, while background errors will appear randomly in only one. This segmentation enables statistical discrimination between true signals and noise.
Solution Approach 2:
The computational integration process provides feedback by comparing variant calls from both libraries. Variants that meet consistency criteria across both independent sequencing runs are validated as true positives, while inconsistent variants are flagged as potential background errors and filtered out.
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 effectively maintains variant allele frequencies and improves the accuracy of ctDNA sequencing, enabling the monitoring of cancer progression and therapeutic responses by distinguishing true variants from false positives, with high concordance with traditional imaging methods.
Implementation Method 1
contacting the amplified adaptor-ligated cfDNA with RNA baits that hybridize to selected molecules of the plurality of cfDNA
Implementation Method 2
contacting between about 2.5 ng and about 15 ng of the plurality of cfDNA with a population of stem-loop adaptors and a ligase in a second reaction volume, wherein the stem-loop adaptors each comprise an inverted repeat and a loop, wherein the loop comprises at least one cleavable base, thereby ligating a stem-loop adaptor to each end of the plurality of cfDNA
Implementation Method 3
amplifying the linearized adaptor-ligated cfDNA to produce amplified adaptor-ligated cfDNA
Data Source
AI summary
Provided herein are methods of preparing cell-free DNA (cfDNA) for sequencing such that variant allele frequencies are maintained. Also provided are sequencing libraries prepared according to such methods. In addition, methods are provided for analyzing sequencing reads to determine variant allele frequencies. These methods may be used for diagnosing and/or evaluating cancer patients.