Concatenated DNA Templates Boost Single-Molecule Sequencing Throughput
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Solution Overview
Problem
Current single-molecule sequencing platforms suffer from low throughput, particularly when dealing with short nucleic acid fragments like cell-free DNA or circulating tumor DNA, which are present in trace amounts, limiting their effectiveness in applications such as oncology and prenatal testing.
Innovation Solution
A method is developed to create libraries of concatenated target nucleic acid molecules by attaching adaptors with specific regions to DNA molecules, using exonucleases and polymerases to join them, and adding barcodes and priming sites for enhanced sequencing, thereby increasing the length and versatility of DNA templates for long-read sequencing technologies.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Length of moving object
If single-molecule sequencing platforms are used to sequence short nucleic acid fragments, then long read lengths are achieved, but throughput remains low
Solution Approach 1:
The patent merges multiple short DNA fragments into concatenated templates by ligating them together with adaptors. This combining approach allows short fragments to be processed as longer molecules, increasing the number of fragments that can be sequenced simultaneously and thereby improving throughput while maintaining compatibility with long-read sequencing platforms
Solution Approach 2:
The patent segments the sequencing process into distinct phases: fragment preparation with adaptors, concatenation of multiple fragments into templates, and sequencing. This segmentation allows optimization of each step independently, particularly enabling the concatenation step to increase the effective number of sequencable molecules without compromising the long-read capability of the platform
2Adaptability or versatility
If short nucleic acid fragments are sequenced directly, then applications like oncology and prenatal testing are enabled, but sequencing throughput is limited
Solution Approach 1:
The patent introduces adaptors as intermediary molecules that bridge short nucleic acid fragments and the sequencing platform. These adaptors contain necessary sequencing motifs and enable the fragments to be ligated into concatemers, serving as a mediator that preserves the ability to sequence short fragments from trace samples while dramatically increasing throughput by processing them as extended templates
3Productivity
If multiple short DNA fragments are concatenated into longer templates, then sequencing throughput increases, but library preparation complexity increases
Solution Approach 1:
The patent performs preliminary actions by pre-attaching adaptors to short DNA fragments before concatenation. This preliminary adaptor ligation simplifies the subsequent concatenation step and standardizes the fragments for efficient joining, reducing overall library preparation complexity while enabling high-throughput sequencing of the resulting concatemers
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 sequencing throughput by generating longer DNA templates, allowing for more efficient analysis of short DNA fragments and improving the ability to sequence both long and short DNA molecules, thereby enhancing the versatility and cost-effectiveness of single-molecule sequencing platforms.
Implementation Method 1
contacting the sample with an exonuclease to generate partially single-stranded adaptor regions at the ends of the target molecule
Implementation Method 2
The joining of the target molecules may comprise a polymerase fill-in, wherein the polymerase may lack the 3′-5′ exonuclease activity
Implementation Method 3
The joining of the target molecules may comprise a ligation step
Data Source
AI summary
The invention comprises a method and compositions for sequencing library preparation, which increases the throughput of single-molecule sequencing (SMS) platforms by generating long concatenated templates from pools of short DNA molecules.