Transposase Tagmentation on Isolated Chromatin for Low-Input Sequencing
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for preparing sequencing libraries from nucleic acids, such as ChIP-seq and Chem-seq, are laborious, require high input amounts, and are sensitive to DNA concentrations, making it difficult to study low-abundance molecular interactions, especially in low-input samples like primary patient cells or rare cell types.
Innovation Solution
A method involving the addition of an agent binding to chromatin, followed by isolation and subsequent addition of transposase to the chromatin, allowing for direct tagmentation on chromatin-bound nucleic acids, which reduces the need for extensive purification steps and is robust across varying DNA concentrations, enabling efficient sequencing library preparation from low-input samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If classical ChIP-seq library preparation methods are used, then sequencing libraries can be prepared, but the process is laborious and requires high input amounts (5-10 ng)
Solution Approach 1:
The patent combines chromatin immunoprecipitation (ChIP) with transposase-mediated tagmentation into a single integrated workflow. The transposase is added directly to the isolated chromatin without intermediate purification steps, merging the enrichment and library preparation processes. This reduces manual handling and minimizes sample loss, enabling successful library preparation from as little as 100-1000 cells while simplifying the overall protocol.
Solution Approach 2:
The patent performs transposase tagmentation directly on the isolated chromatin before nucleic acid extraction and library construction. By introducing adaptors and fragmenting the DNA in advance during the chromatin state, the method eliminates subsequent end-repair, A-tailing, and adapter ligation steps. This preliminary action significantly reduces the number of required input molecules and simplifies the workflow.
2Reliability
If extensive purification steps are performed, then library quality is improved, but the process becomes more laborious and time-consuming
Solution Approach 1:
The transposase-mediated tagmentation is performed preliminarily during the chromatin immunoprecipitation step, before nucleic acid extraction and library construction. The adaptors are introduced and DNA is fragmented in advance while the chromatin is still in a controlled state, eliminating the need for subsequent end-repair, A-tailing, and adapter ligation steps. This preliminary action maintains library quality while dramatically reducing preparation time and manual handling.
Solution Approach 2:
The patent extracts and removes unnecessary intermediate purification and processing steps from the classical ChIP-seq workflow. By performing tagmentation directly on isolated chromatin and eliminating end-repair, A-tailing, and separate adapter ligation steps, the method removes sources of sample loss and time consumption while maintaining or improving library quality through reduced manual handling.
3Productivity
If adapter ligation is used, then DNA fragments can be amplified, but adapters can self-ligate and require size-selection
Solution Approach 1:
The patent uses transposase as an intermediary enzyme that simultaneously fragments DNA and integrates adaptors in a single reaction step. The transposase mediates the insertion of sequencing adaptors directly into the fragmented chromatin DNA during tagmentation, eliminating the need for separate adapter ligation. This prevents adapter self-ligation and eliminates the requirement for size-selection steps to remove adapter-dimers, while maintaining high amplification efficiency.
4Productivity
If transposase is added to purified DNA, then library preparation is faster, but the method is sensitive to varying DNA concentrations
Solution Approach 1:
The patent performs transposase tagmentation preliminarily on isolated chromatin before DNA purification and library construction. By conducting the tagmentation reaction in the chromatin precipitation step rather than on purified DNA, the method takes advantage of the concentrated chromatin state and eliminates sensitivity to DNA concentration variations that would occur during later purification steps. This preliminary action ensures robust performance across varying input conditions.
Solution Approach 2:
The patent changes the physical and chemical state of the substrate from purified DNA to isolated chromatin for the tagmentation reaction. By performing tagmentation on chromatin that has been precipitated and isolated but not yet purified, the method creates optimal conditions for transposase activity that are less sensitive to concentration variations. The chromatin state provides a more stable substrate that maintains consistent tagmentation efficiency across different input amounts.
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 results in a faster, cheaper, and more robust method for preparing sequencing libraries, compatible with low-input samples, preserving local chromatin structure and reducing the need for optimized ratios of transposase to chromatin, thus enhancing the feasibility of studying rare cell populations and molecular interactions.
Implementation Method 1
addition of transposase to the isolated chromatin; isolating nucleic acid from chromatin
Data Source
AI summary
The present invention provides a novel method for preparing a sequencing library and studying molecular interactions involving a nucleic acid. In particular, the invention relates to a method for preparing a sequencing library, the method comprising the addition of an agent binding to chromatin to a sample comprising a nucleic acid; isolating chromatin bound by said agent; addition of transposase to the isolated chromatin; isolating nucleic acid from chromatin; and obtaining a sequencing library. Moreover, the present invention relates to a method for mapping of molecular interactions involving a nucleic acid, the method comprising the addition of an agent binding to chromatin to a sample comprising a nucleic acid; isolating chromatin bound by said agent; addition of transposase to the isolated chromatin; isolating nucleic acid from chromatin; amplification of nucleic acid; sequencing of amplified nucleic acid; and identifying molecular interactions.