Nucleic Acid Fragment Tiling for Sequencing Library Preparation
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Solution Overview
Problem
Current methods for nucleic acid sequencing and analysis face challenges in efficiently labeling and preparing nucleic acids for sequencing, particularly in fragmenting, purifying, and amplifying DNA and RNA samples to achieve high multiplexing and parallel sequencing reactions.
Innovation Solution
The method involves fragmenting nucleic acids into short and long fragments, tailing and hybridizing the short fragments onto the long ones, followed by purification and exposure to barcoded primers for sequencing and amplification, utilizing microfluidic droplets for sequestration and analysis, and employing barcode and probe-type labels for identification and detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If nucleic acids are fragmented and processed through multiple purification steps, then sequencing quality is improved, but processing time and complexity increase
Solution Approach 1:
The nucleic acid sample is divided into multiple size fractions (short fragments 100-1000 bases and long fragments 5Kb-100Kb) that are processed differently. Short fragments are tiled onto long fragments, allowing parallel processing of different size classes to improve overall throughput while maintaining sequencing quality for each fraction
Solution Approach 2:
Adaptor sequences are pre-attached to the short fragments before hybridization to the long fragments. This preliminary tiling and adapter attachment simplifies subsequent purification steps and enables more efficient library preparation, reducing overall processing time while maintaining high sequencing quality
2Adaptability or versatility
If multiple short fragments are tiled onto each long fragment, then multiplexing capability is improved, but purification difficulty increases
Solution Approach 1:
Adaptor sequences serve as intermediary elements that are attached to short fragments and enable their specific hybridization to complementary sequences on long fragments. These adaptors act as mediators that facilitate the tiling process and provide handles for subsequent purification steps, making it easier to separate tiled fragments from unhybridized short fragments
Solution Approach 2:
Different regions of the nucleic acid library are treated with different properties: short fragments receive adaptors for tiling, while long fragments serve as scaffolds. This local differentiation in fragment processing creates distinct physical and chemical properties that simplify purification by allowing selective capture of tiled versus unhybridized fragments
3Productivity
If barcoded primers are used for sequencing, then sample tracking efficiency is improved, but reagent complexity increases
Solution Approach 1:
The barcode sequence and the sequencing primer are merged into a single oligonucleotide molecule. This combination allows the primer to simultaneously perform its function of initiating DNA synthesis and carry the barcode label for sample identification, reducing the number of separate reagents needed while maintaining efficient sample tracking
Solution Approach 2:
The barcoded primers are designed with universal features that allow them to function both as sequencing initiators and as sample identifiers. The same primer structure serves multiple purposes: hybridizing to the template, initiating synthesis, and providing the barcode for tracking, thereby simplifying the overall reagent set despite the increased functionality required
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 highly multiplexed and parallel sequencing reactions, improving the efficiency of nucleic acid analysis by allowing for the tracking of sequence reads through coded primers and enabling digital PCR assays, while maintaining the integrity and specificity of the nucleic acid samples.
Implementation Method 1
the short fragments are hybridized along the length of the large fragments (i.e., tiled)
Data Source
Figure 1A~1B
Figure 2A
Figure 2B
AI summary
The invention provides methods for sequencing and sample preparation for sequencing and amplification.