Barcoded DNA Library Construction from Damaged Samples
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Solution Overview
Problem
Current sequencing technologies face challenges in constructing high-quality nucleic acid libraries from low-quality and low-quantity samples, particularly in clinical applications where rare mutations are difficult to detect due to DNA damage and the presence of single-stranded DNA segments.
Innovation Solution
A method and kit for constructing a nucleic acid library involving dephosphorylation and dissociation of DNA samples to create single-stranded molecules, followed by ligation of adaptors with barcode sequences and subsequent synthesis of complementary strands to form barcoded double-stranded DNA molecules, which can be immobilized and amplified for sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If current sequencing library construction methods are used, then standard DNA sequencing can be performed, but single-stranded DNA segments from damaged samples cause ligation failures and sequencing errors
Solution Approach 1:
The patent applies preliminary action by performing dephosphorylation of the 5' ends of DNA fragments before ligation. This pre-treatment removes phosphate groups that would otherwise prevent efficient ligation of single-stranded DNA segments, enabling subsequent adaptor ligation to proceed successfully even on damaged DNA molecules
Solution Approach 2:
The patent changes the chemical state of the DNA 5' ends by removing phosphate groups through dephosphorylation. This parameter change transforms the DNA ends from a state that resists ligation to a state that readily accepts adaptor ligation, thereby resolving the issue of single-stranded segment ligation failures
2Manufacturing precision
If standard library construction is applied to ultra-low amount DNA samples, then sequencing can be attempted, but the low quantity results in insufficient library complexity and poor sequencing quality
Solution Approach 1:
The patent applies preliminary action by dephosphorylating DNA fragments before ligation to prevent concatemer formation. This ensures that each DNA fragment is ligated individually to a unique adaptor molecule, maximizing library complexity from limited starting material and improving sequencing quality
Solution Approach 2:
The patent segments the library construction process into distinct steps: dephosphorylation, adaptor ligation, and barcode assignment. This segmentation allows each step to be optimized independently, ensuring high efficiency even when working with ultra-low DNA quantities
3Productivity
If conventional ligation methods are used, then adaptors can be attached to DNA fragments, but single-stranded segments lead to poor ligation efficiency and loss of rare mutations
Solution Approach 1:
The patent applies preliminary action by dephosphorylating DNA fragments before ligation. This pre-treatment creates chemically reactive 5' ends that efficiently accept adaptor ligation, thereby preventing loss of rare mutations due to ligation failure and improving overall ligation efficiency
Solution Approach 2:
The patent introduces adaptors as intermediary molecules that bridge the damaged DNA fragments and the sequencing platform. These adaptors contain barcodes and sequencing priming sites, enabling even heavily damaged fragments to be successfully sequenced and analyzed for rare mutations
4Measurement precision
If standard library construction is used on clinical samples with mixed normal and diseased tissue, then bulk sequencing can be performed, but rare disease mutations are diluted and difficult to detect
Solution Approach 1:
The patent introduces unique molecular barcodes as intermediaries that tag each original DNA fragment. During sequencing, reads with matching barcodes are grouped together (consolidated), allowing rare disease mutations to be detected by comparing multiple reads from the same original molecule, thereby overcoming the dilution effect in mixed tissue samples
Solution Approach 2:
The patent implements feedback through barcode-based read consolidation. The barcode information provides feedback about the origin of each read, allowing computational methods to identify and prioritize reads that contain rare mutations by comparing them against the consensus sequence of their barcode family
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 enables the construction of high-quality nucleic acid libraries from ultra-low amounts of DNA, improving the detection of rare mutations and enhancing the sensitivity of sequencing, particularly in clinical samples with damaged DNA.
Implementation Method 1
preparing a DNA sample from the biological sample, wherein the DNA sample comprises a plurality of single-stranded DNA molecules, each having a dephosphorylated 5' end
Implementation Method 2
ligating a first strand of a first adaptor to a 3' end of each of the plurality of single-stranded DNA molecules, wherein the first strand of the first adaptor comprises a phosphate group
Implementation Method 3
synthesizing a complementary strand for each of the plurality of single-stranded DNA molecules ligated to the first strand of the first adaptor to obtain a uniquely barcoded double-stranded DNA molecule
Data Source
AI summary
A method and kit for constructing a barcoded single-stranded DNA library are disclosed. The method includes preparing single-stranded DNA molecules each having a dephosphorylated 5′ end, ligating a first adaptor to a 3′ end of each single-stranded DNA molecule, and synthesizing a complementary strand of each single-stranded DNA molecule ligated to the first strand of the first adaptor. The kit includes the first adaptor having a first strand, which includes, from a 5′ end to a 3′ end, a phosphate group, a barcode sequence, and a first primer recognition sequence. The kit also includes a DNA ligase for a ligation between the 5′ end of the first strand of the first adaptor to each single-stranded DNA molecule, and a first primer for the synthesis of the complementary strand. The method allows for analysis of rare mutations and from nucleic acid samples of low quality and quantity.