Dual Barcode Sequencing for Rare Variant Detection
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Solution Overview
Problem
Current methods for detecting rare genomic variants are costly and inefficient, requiring large amounts of sequencing data to identify rare mutants, with existing barcoding techniques only being sensitive to 5E-5 and unable to effectively utilize the information in a single blood draw, leading to high costs and limited sensitivity in cancer prognosis and screening applications.
Innovation Solution
The method involves digesting nucleic acids, performing linear amplification with primary and secondary barcoded adapters, removing unused barcodes, and using mutant enrichment techniques to generate a library for sequencing, which includes secondary barcoding to preserve error correction and increase sensitivity, allowing for the detection of rare mutants with reduced sequencing space and costs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If barcoding techniques are used to detect rare genomic variants, then sensitivity increases, but sequencing costs and the amount of sequencing space required increase significantly
Solution Approach 1:
The patent divides the barcode into two separate parts: a primary barcode that identifies the original genomic molecule and a secondary barcode that identifies the amplified copy. This segmentation allows the system to track the lineage of each molecule through amplification while maintaining the ability to detect rare variants, thereby improving detection sensitivity without proportionally increasing sequencing space requirements.
Solution Approach 2:
The patent introduces an intermediary structure - the secondary barcode - that mediates between the primary barcode and the amplified DNA copies. This intermediary allows the system to preserve error correction information from the primary barcode while enabling efficient sequencing of amplified copies, thus resolving the contradiction between detection sensitivity and sequencing space requirements.
2Measurement precision
If large amounts of sequencing are performed to identify rare mutants, then detection sensitivity improves, but costs increase by over 100-fold
Solution Approach 1:
The patent performs preliminary enrichment of rare mutants before sequencing by using the dual barcode system to identify and isolate molecules of interest. The primary barcode captures the original molecule's identity, and the secondary barcode tracks amplification copies, allowing selective enrichment of rare variants. This preliminary action reduces the amount of sequencing required, thereby improving detection sensitivity while controlling costs.
Solution Approach 2:
The patent changes the parameter of barcode structure from a single unified barcode to a two-part system with primary and secondary barcodes. This parameter change enables the system to differentiate between original molecules and amplified copies, allowing for more efficient sequencing strategies that reduce costs while maintaining or improving rare mutant detection sensitivity.
3Reliability
If current barcoding techniques are used, then error correction is achieved, but sensitivity is limited to 5E-5 and only 5% of information in a blood draw is utilized
Solution Approach 1:
The patent implements a nested structure where the secondary barcode is embedded within the context of the primary barcode. The primary barcode (identifying the original molecule) contains or is associated with the secondary barcode (identifying the amplified copy). This nesting allows the system to preserve error correction from the primary barcode while adding the functionality of tracking amplification copies, thereby improving sensitivity beyond the 5E-5 limitation without sacrificing error correction capability.
Solution Approach 2:
The patent adds another dimension to the barcode system by introducing a second barcode layer. Instead of relying on a single-dimensional barcode, the system uses a two-dimensional barcode structure where the primary barcode provides error correction and the secondary barcode provides amplification tracking. This dimensional expansion enables the system to achieve both error correction and higher sensitivity, utilizing more than 5% of the information in a blood draw.
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 significantly increases the sensitivity and efficiency of rare mutant detection, reducing costs by over 100-fold, enabling affordable screening of thousands of regions of interest from a single blood draw while maintaining high accuracy, thus improving cancer prognosis and screening capabilities.
Implementation Method 1
removing unused primary barcodes by digestion with a single-stranded DNA exonuclease
Data Source
AI summary
Exemplary embodiments of methods, kits, systems and computer-accessible medium can be provided for facilitating maximum depth enrichment sequencing for identifying rare genomic variants.