Chromatin Analysis via Template Switching for Single-Cell Sensitivity
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Solution Overview
Problem
Current methods for analyzing chromatin information, such as chromatin accessibility and nucleosome positioning, suffer from low sensitivity when dealing with low input sources, like single cells, and often result in the loss of valuable information.
Innovation Solution
A method involving a mixture of native, intact chromatin DNA, transposase molecules, and sequencing adaptors is used to generate DNA molecules with sequencing adaptors, allowing for the identification of nucleosome positions through sequencing, which includes a template switching reaction to preserve nucleosome positioning information.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional chromatin analysis methods are used, then chromatin accessibility and nucleosome positioning can be analyzed, but sensitivity is low when dealing with low input sources like single cells
Solution Approach 1:
The method segments the chromatin analysis process into distinct molecular steps: transposase-mediated tagmentation of chromatin followed by template switching PCR. This segmentation allows each step to be optimized independently, with the template switching step specifically designed to amplify low-input samples while preserving nucleosome positioning information through full-length fragment amplification.
Solution Approach 2:
The invention introduces an intermediary template switching reaction between the initial tagmentation and final sequencing. This intermediary step uses template switching oligonucleotides that bind to the 3' end of amplified fragments, enabling full-length amplification of nucleosome-positioning information that would otherwise be lost in conventional methods.
2Measurement precision
If methods amenable to low input are used, then sensitivity is improved, but valuable information such as nucleosome positioning is lost
Solution Approach 1:
The method performs preliminary tagmentation of intact chromatin before amplification, creating fragment ends that serve as templates for subsequent PCR. This preliminary action preserves the spatial information of nucleosome positions within the fragment lengths, which are then fully amplified in the template switching step to recover the positioning information that would otherwise be lost.
Solution Approach 2:
The invention changes the amplification parameters by using template switching PCR instead of conventional PCR. This parameter change allows for full-length amplification of tagmented chromatin fragments, preserving the size information that corresponds to nucleosome positioning, while still maintaining the sensitivity needed for low-input samples through efficient amplification.
3Productivity
If conventional PCR and sequencing are used, then nucleic acid can be amplified and detected, but nucleosome positioning information is lost
Solution Approach 1:
The method ensures continuity of useful action by designing the template switching oligonucleotides to bind to the extreme 3' ends of amplified fragments. This continuous action from tagmentation through template switching PCR preserves the full length of chromatin fragments, maintaining the nucleosome positioning information throughout the amplification process rather than losing it at any stage.
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 method enhances the sensitivity of chromatin analysis from low input sources, providing accurate nucleosome positioning information while maintaining valuable data, even from a single cell.
Implementation Method 1
subjecting the mixture to conditions sufficient to cause transposition of at least some of the plurality of first nucleic acid molecules into the template DNA molecules with the aid of a transposon-nucleic acid complex generated from at least a subset of the plurality of transposase molecules and the plurality of first nucleic acid molecules
Implementation Method 2
using the promoter region to generate a plurality of ribonucleic acid (RNA) molecules comprising sequences that correspond to the first sequencing adaptor region
Implementation Method 3
generating from (i) a plurality of second nucleic acid molecules each comprising a second sequencing adaptor region, and (ii) the plurality of RNA molecules, a plurality of DNA molecules comprising the first sequencing adaptor region and the second sequencing adaptor region. In some embodiments, generating the plurality of double-stranded DNA molecules comprises a template switching reaction
Data Source
AI summary
The present disclosure provides methods and systems for nucleic acid preparation and/or analysis. Nucleic acids may be derived from one or more cells. Nucleic acid preparation may comprise generating nucleic acid molecules of varying lengths. Nucleic acid analysis may comprise identifying nucleic acid sequence information with nucleosome position information.