Modified Transposon End Sequences for Flexible NGS Library Preparation
Solution Overview
Problem
Current methods for DNA fragmentation in Next-Generation Sequencing (NGS) are limited by the need for expensive capital equipment in mechanical approaches, variable performance in enzymatic strategies, and limitations on library adapter structure in tagmentation-based methods, particularly due to the requirement for a defined 19-bp mosaic end sequence adjacent to library inserts.
Innovation Solution
The use of modified transposon end sequences with mutations such as uracil, inosine, ribose, 8-oxoguanine, thymine glycol, modified purines, and modified pyrimidines allows for decoupling of enzymatic fragmentation and adapter tagging steps, enabling hybrid Tn5-ligation library preparation approaches that eliminate the constraint of the 19-bp mosaic end sequence requirement, facilitating the incorporation of features like forked adapters and unique molecular identifiers while maintaining compatibility with standard sequencing methods.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If tagmentation-based approaches are used for DNA fragmentation, then enzymatic fragmentation is achieved without specialized equipment, but library adapter structure is limited due to the requirement for a defined 19-bp mosaic end sequence adjacent to library inserts
Solution Approach 1:
The patent extracts the mosaic end sequence requirement from the library preparation process by using fragmentase enzymes that generate fragments without requiring the 19-bp mosaic end sequence. This separates the fragmentation function from the adapter tagging function, allowing independent optimization of each step and enabling greater adapter structure flexibility.
Solution Approach 2:
The patent segments the library preparation workflow into distinct enzymatic steps: fragmentation by fragmentase, end repair, and adapter ligation. This segmentation allows each step to be independently optimized and performed with different enzymes, removing the constraint that coupled tagmentation imposed on adapter structure.
2Productivity
If transposase-mediated fragmentation is used, then adapter sequences are introduced during fragmentation, but the process is limited to one turnover and requires introduction of the mosaic end sequence into DNA fragments
Solution Approach 1:
The patent uses fragmentase enzymes that act once on the DNA substrate and are then discarded, rather than using transposases that require complex preloading with transposon substrates and are limited to one turnover. This simplifies the reaction system while maintaining high fragmentation efficiency.
3Ease of manufacture
If fragmentase enzymes are used for enzymatic fragmentation, then no specialized equipment is required, but the enzymes can turn-over several times making fragmentation time- and concentration-dependent requiring optimization
Solution Approach 1:
The patent performs preliminary optimization of fragmentase enzyme concentration and reaction time to achieve the desired fragment size distribution before proceeding with library preparation. This preliminary action establishes a standardized protocol that reduces the need for further optimization by the user.
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 the flexibility and efficiency of library preparation by allowing the inclusion of additional features in NGS libraries without the need for specialized equipment, improving fragment size distribution and sequencing performance, and enabling PCR-free and UMI library preparation workflows.
Implementation Method 1
transposition by bead-linked transposomes (BLTs)
Implementation Method 2
combining the sample with an endonuclease or an enzyme mixture and cleaving the modified mosaic end sequence at the uracil, inosine, ribose, 8-oxoguanine, thymine glycol, modified purine, and/or modified pyrimidine
Data Source
AI summary
Disclosed herein is a modified transposon end sequence comprising a mosaic end sequence, wherein the mosaic end sequence comprises one or more mutation as compared to a wild-type mosaic end sequence, wherein the mutation comprises a substitution with a uracil, an inosine, a ribose, an 8-oxoguanine, a thymine glycol, a modified purine, or a modified pyrimidine. Also disclosed are transposome complexes comprising these modified transposon end sequences and methods of library preparation using these modified transposon end sequences.