In Situ ATAC Sequencing for Spatial Epigenomic Mapping
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for measuring epigenomes, such as ATAC-Seq and ChIP-Seq, fail to resolve the three-dimensional structure or nuclear regulatory complexes that drive cellular variation in situ, limiting our understanding of cellular regulation.
Innovation Solution
A method involving an insertional enzyme complex that fragments and tags genomic DNA in situ, allowing for the construction of a genomic library, circularization, and amplification, followed by sequencing to produce an epigenetic map, which includes chromatin accessibility, nucleosome positioning, and DNA binding protein occupancy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If ATAC-Seq and ChIP-Seq methods are used to measure epigenomes, then transcription factors and cis-regulatory elements can be identified, but the three-dimensional structure and nuclear regulatory complexes in situ cannot be resolved
Solution Approach 1:
The biological sample is fixed before epigenomic analysis to preserve the three-dimensional structure and spatial relationships of chromatin and nuclear regulatory complexes. This preliminary fixation step allows subsequent ATAC-Seq or ChIP-Seq procedures to be performed while maintaining in situ structural context, thereby preventing loss of spatial information while retaining epigenomic measurement capability
Solution Approach 2:
The invention integrates spatial dimension information into traditional epigenomic analysis by performing sequencing reactions within the fixed three-dimensional tissue architecture. This adds the spatial dimension to the conventional two-dimensional epigenomic data, enabling simultaneous measurement of epigenomic features and their spatial organization
2Loss of information
If genomic DNA is fragmented and tagged using insertional enzyme complex in situ, then spatially resolved epigenomic maps can be created, but the complexity of the procedure increases
Solution Approach 1:
The invention combines multiple steps into a single in situ procedure: genomic DNA fragmentation, adapter tagging, and spatial preservation are performed simultaneously within the fixed tissue sample using an insertional enzyme complex. This merging of steps reduces the number of separate operations needed compared to traditional methods that require DNA extraction, separate tagging, and spatial reconstruction
Solution Approach 2:
The insertional enzyme complex acts as an intermediary that performs both fragmentation and tagging functions within the fixed tissue. This single enzymatic complex mediates the conversion of genomic DNA into spatially resolved tagged fragments without requiring separate enzymatic steps or complex sample processing intermediaries
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
Enables the creation of spatially resolved epigenomic maps that provide insights into gene expression and cellular regulation, allowing for diagnostic and prognostic applications by analyzing chromatin in its native context.
Implementation Method 1
treating a fixed biological sample with an insertional enzyme complex to produce tagged fragments of genomic DNA
Implementation Method 2
circularizing the tagged fragments of genomic DNA
Implementation Method 3
amplifying the tagged fragments of genomic DNA
Implementation Method 4
sequencing all or a portion of the tagged fragments to produce a plurality of sequence reads
Data Source
AI summary
The present invention provides methods for analyzing polynucleotides such as genomic DNA. In some embodiments, the disclosure provides a method for preparing and amplifying a genomic DNA library in situ in a fixed biological sample. The method comprises treating a fixed biological sample with an insertional enzyme complex to produce tagged fragments of genomic DNA. The method further comprises circularizing the tagged fragments of genomic DNA. The method further comprises amplifying the tagged fragments of genomic DNA.