Histone-Like Protein Tagmentation for Sequencing Insert Size Control

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Solution Overview

Problem

The existing methods for DNA fragmentation in library preparation for next-generation sequencing are sensitive to input DNA and transposition system concentrations, requiring precise quantification, and lack control over fragment size distribution, especially for applications needing longer fragments.

Innovation Solution

Incorporating histone-like proteins, particularly from thermophilic or hyperthermophilic Archaea such as Thermococcus or Pyrococcus, into the tagmentation process to mitigate the need for precise quantification and enhance control over DNA fragment size distribution.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Manufacturing precision

If traditional tagmentation is used without histone-like proteins, then the process is simple and fast, but precise control over fragment size is lost and sensitivity to input concentration increases

Engineering Contradiction:

Improvefragment size controlVSAvoidtagmentation process complexity

Core Design Contradiction:

Manufacturing precisionVSDevice complexity

Solution Approach 1:

Histone-like proteins act as intermediary components that mediate between the transposase and DNA, providing a buffering effect that stabilizes fragment size distribution. These proteins temporarily bind to DNA and modulate transposase accessibility, creating a more predictable fragmentation pattern without requiring precise input quantification.

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The invention changes the chemical and physical parameters of the tagmentation reaction by introducing histone-like proteins, which alter the reaction kinetics and DNA accessibility. This parameter change enables broader tolerance ranges for input DNA and transposase concentrations while maintaining consistent fragment size distributions.

Inventive Principle:

Principle #35Parameter changes

2Manufacturing precision

If precise quantification of input DNA and transposition system is performed, then fragment size control improves, but time and cost increase

Engineering Contradiction:

Improvefragment size distribution controlVSAvoidquantification and preparation time

Core Design Contradiction:

Manufacturing precisionVSLoss of time

Solution Approach 1:

The tagmentation system becomes self-regulating through the inclusion of histone-like proteins, which automatically buffer concentration variations. The system self-corrects for imprecise input quantification, eliminating the need for time-consuming precise measurement steps while maintaining fragment size control.

Inventive Principle:

Principle #25Self-service

Solution Approach 2:

Histone-like proteins provide beforehand cushioning by pre-binding to DNA and creating a protected complex that resists excessive fragmentation. This cushioning effect compensates for potential over-fragmentation that would occur with imprecise transposase dosing, allowing broader acceptable ranges for input concentrations.

Inventive Principle:

Principle #11Beforehand cushioning (Prior cushioning)

3Productivity

If higher transposition system concentration is used to ensure sufficient fragmentation, then fragmentation efficiency increases, but fragment size becomes harder to control and shorter fragments predominate

Engineering Contradiction:

Improvefragmentation efficiencyVSAvoidfragment size distribution

Core Design Contradiction:

ProductivityVSManufacturing precision

Solution Approach 1:

Histone-like proteins provide a counterbalancing effect that opposes the excessive fragmentation driven by high transposase concentrations. These proteins physically block transposase access to certain DNA regions and stabilize intermediate fragments, preventing the system from producing predominantly short fragments even when transposase is in excess.

Inventive Principle:

Principle #8Anti-weight (Counterweight)

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 allows for precise control over fragment size and distribution, facilitating efficient library preparation for sequencing, especially in applications requiring longer inserts, thereby simplifying and reducing the cost of the sequencing process.

Implementation Method 1

Incorporating histone-like proteins, particularly from thermophilic or hyperthermophilic Archaea such as Thermococcus or Pyrococcus, into the tagmentation process

Methodology Applied

Scientific EffectDNA-protein binding:

Implementation Method 2

A hyperactive variant of the bacterial Tn5 transposase that mediates the fragmentation of double-stranded DNA and ligates synthetic oligonucleotides

Methodology Applied

Scientific EffectTransposase enzymatic activity: Enzyme

Implementation Method 3

heating the fragmentation reaction mixture for a predetermined amount of time at a predetermined temperature

Methodology Applied

Scientific EffectThermal heating: Heating

Data Source

PatentUS20250368983A1Controlling for tagmentation sequencing library insert size using archaeal histone-like proteins

Publication Date: 2025.12.04 KAPA BIOSYSTEMS INC

AI summary

The present disclosure provides compositions and kits for the tagmentation of double stranded DNA. In some embodiments, the compositions and kits for the tagmentation of double stranded DNA include one or more histone-like proteins and/or one or more transposition systems. The present disclosure also provides methods for the tagmentation of double stranded DNA in the presence of one or more histone-like proteins.