Bi-directional In Situ Nucleic Acid Sequencing
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for analyzing biological samples fail to provide comprehensive spatial information about analytes and their positions within tissues, lacking the ability to effectively analyze nucleic acids in situ, especially in the context of intact tissues or single cells.
Innovation Solution
A method involving nucleic acid sequencing by synthesis and ligation using detection probes with detectable labels, where probes hybridize to specific regions of interest flanking an adaptor region, allowing for the generation and detection of ligation products to determine nucleotide sequences, enabling in situ analysis of nucleic acids in samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of information
If sequencing by synthesis or ligation is performed on extracted nucleic acids, then nucleotide sequence information can be obtained, but spatial information about the analytes in the original tissue context is lost
Solution Approach 1:
The method performs preliminary actions by hybridizing anchors to the adaptor region and extending them with nucleotides before sequencing, while the tissue structure is preserved. This allows spatial information to be retained and later associated with the sequenced nucleotide sequences through the spatial coordinates of the detected signals.
Solution Approach 2:
The invention creates copies of the nucleic acid regions of interest through in situ synthesis and ligation reactions, generating multiple copies at the same spatial location. This amplification enables sufficient signal detection while maintaining the original spatial context within the tissue sample.
2Loss of information
If traditional sequencing methods are used on isolated nucleic acids, then comprehensive nucleotide sequence data can be obtained, but the positional context and morphology of the source cells are lost
Solution Approach 1:
The method segments the sequencing process into two independent components: (1) performing synthesis and ligation reactions in situ within the tissue to preserve spatial context, and (2) detecting signals at specific spatial coordinates. This segmentation allows both morphological context and sequence data to be obtained and subsequently correlated.
Solution Approach 2:
The invention adds a spatial dimension to traditional sequencing by performing reactions in three-dimensional tissue context and detecting signals with spatial coordinates. This transforms the sequencing from a one-dimensional nucleotide sequence determination to a three-dimensional spatially-resolved sequencing process.
3Measurement precision
If in situ analysis is performed to preserve spatial information, then positional context is maintained, but the ability to obtain detailed nucleotide sequence information is limited
Solution Approach 1:
The method changes parameters by using detectable labels with high signal amplification capability, allowing detailed nucleotide sequence information to be obtained through multiple cycles of synthesis and ligation while maintaining the same spatial location. The detectable labels enable sensitive detection that preserves spatial precision.
4Adaptability or versatility
If multiple analytes are analyzed simultaneously in intact tissue, then spatial heterogeneity can be studied, but the technical complexity of the analysis increases
Solution Approach 1:
The invention creates a universal platform that can analyze multiple different analytes (different nucleic acid sequences) using the same in situ synthesis and ligation methodology. The universal anchor-adaptor-region structure and detection protocol enable multiplexed analysis of various analytes while preserving their spatial context in the tissue.
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 enables detailed in situ analysis of nucleic acids, providing spatial information about analytes within tissues, improving upon existing techniques by allowing for the determination of nucleotide sequences and positioning of analytes, thereby enhancing the understanding of cellular morphology and function.
Implementation Method 1
each detection probe is configured to hybridize to the nucleic acid adjacent to an end of the anchor
Implementation Method 2
ligating a detection probe complementary to the first region of interest to the end of the anchor to generate a ligation product
Implementation Method 3
incorporating a nucleotide and/or analog thereof into the anchor by the polymerase using the second region of interest or a probe bound thereto as a template
Implementation Method 4
each detection probe comprises an interrogatory region and a detectable label
Implementation Method 5
detecting a signal associated with the detectable label of the ligation product
Data Source
AI summary
In some aspects, provided herein are methods for analyzing a nucleic acid comprising first and second regions of interest flanking an adaptor region, comprising hybridizing an anchor to the adaptor region, analyzing the first region of interest from one end of the anchor using probe ligation (e.g., sequencing-by-ligation), and binding a polymerase to the other end of the anchor and optionally incorporating a nucleotide and/or analog thereof into the anchor by the polymerase using the second region of interest or a probe bound thereto as a template. In some embodiments, the second region of interest is used as a template for sequencing-by-synthesis. In some embodiments, spatially resolved detections of analytes are performed at a cellular or subcellular resolution which involve correlating signals associated with analytes with specific spatial locations in a biological sample.