On-Surface Sequencing Library Prep Using Transposomes
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Solution Overview
Problem
Next generation sequencing technologies require labor-intensive and inefficient library preparation methods involving multiple steps and transfers of reaction volumes, which can compromise haplotype information and sequencing efficiency.
Innovation Solution
A method involving a single reaction volume where transposomes are used to fragment and prepare sequencing templates directly on a surface, such as a flow cell, allowing for efficient sequencing and maintaining haplotype information by retaining physical proximity of template nucleic acids.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If traditional library preparation methods are used involving multiple steps and transfers of reaction volumes, then adaptors can be attached to DNA fragments, but the process becomes labor intensive and requires several hands-on steps at different stages
Solution Approach 1:
The patent combines multiple library preparation steps (DNA fragmentation, adaptor ligation, and amplification) into a single reaction volume performed on the flow cell surface. This merging eliminates the need for multiple transfers between tubes and reduces hands-on steps, directly resolving the contradiction between ease of manufacture and device complexity
Solution Approach 2:
The patent performs DNA fragmentation and adaptor ligation simultaneously using transposomes before the sequencing reaction begins. This preliminary action prepares the library in advance on the flow cell surface, eliminating subsequent transfer steps and reducing operational complexity
2Reliability
If traditional library preparation methods with multiple transfers are used, then adaptors can be ligated to fragments, but haplotype information may be compromised
Solution Approach 1:
The patent performs library preparation directly on the flow cell surface before sequencing begins, maintaining continuous physical presence of DNA fragments. This preliminary action on-surface prevents loss of spatial information and haplotype data while enabling immediate sequencing, thus preserving reliability and maintaining productivity
3Ease of manufacture
If DNA fragmentation and adaptor ligation are performed separately, then each step can be optimized, but the overall process becomes more labor intensive
Solution Approach 1:
The patent merges DNA fragmentation and adaptor ligation into a single simultaneous reaction using transposomes on the flow cell surface. This combination eliminates sequential processing time and reduces hands-on operations, directly addressing both ease of manufacture and loss of time
Solution Approach 2:
The transposome-mediated reaction continuously performs both fragmentation and ligation in one uninterrupted process on the flow cell surface. This continuous action eliminates idle time between steps and reduces total preparation time while maintaining operational simplicity
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 enhances sequencing efficiency and reduces preparation time while preserving haplotype information by immobilizing nucleic acids on a surface, facilitating direct sequencing and reducing the need for amplification steps.
Implementation Method 1
contacting the surface with a reaction volume comprising a plurality of template nucleic acids and transposomes, wherein each transposome comprising a transposon sequence and the transposase, wherein the template nucleic acids are prepared by contacting a target nucleic acid with a plurality of transposomes
Implementation Method 2
the capture moiety of the surface is a first affinity moiety and the template nucleic acids comprises a second affinity moiety having affinity for the first affinity moiety
Implementation Method 3
the amplifying comprises bridge amplification
Implementation Method 4
the capture moiety of the surface is a capture probe immobilized to the surface
Data Source
AI summary
Embodiments provided herein relate to methods and compositions for next generation sequencing. Some embodiments include the preparation of a template library from a target nucleic acid in contact with a surface, and sequencing the library on the surface.