RNA Barcode Imaging for High-Throughput Spatial Sequencing
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Solution Overview
Problem
Existing transcriptomics assays face challenges in achieving high-throughput analysis of mRNA sequences with subcellular resolution, as array-based sequencing struggles with subcellular resolution, FISH assays are limited to known sequences and low throughput, and in situ sequencing has low throughput.
Innovation Solution
A method involving forming randomized barcodes on RNA molecules using permanent nucleic acid adapters, followed by imaging and sequencing to determine both spatial location and sequence information, utilizing detection motifs and transient adapters for reversible binding.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If array-based sequencing is used, then cellular scale mRNA sequencing is achieved, but subcellular resolution is lost
Solution Approach 1:
The patent segments the mRNA analysis process into two distinct phases: (1) in situ imaging phase where spatial information is captured at subcellular resolution using imaging techniques, and (2) sequencing phase where high-throughput sequence data is obtained. This segmentation allows each phase to optimize for its specific requirement - spatial precision for imaging and throughput for sequencing - thereby resolving the contradiction between the two.
Solution Approach 2:
The patent introduces barcodes as an intermediary element that bridges the gap between imaging and sequencing. These barcodes are attached to mRNA molecules in situ, capturing spatial information through their location in the image, and then serve as targets for high-throughput sequencing. The barcode acts as a mediator that translates spatial position into sequenceable data, enabling both subcellular resolution and high throughput to be achieved simultaneously.
2Measurement precision
If FISH assays are used, then single molecule resolution and spatial information are achieved, but throughput is limited
Solution Approach 1:
The patent makes the FISH assay multi-functional by combining it with barcode attachment and high-throughput sequencing capabilities. The same in situ hybridization process that provides single-molecule resolution also serves as the platform for attaching barcodes that can be sequenced en masse. This universality allows the assay to simultaneously achieve spatial precision and high throughput by serving multiple functions: localization, barcoding, and sequencing target preparation.
Solution Approach 2:
The patent adds a temporal dimension to the traditional FISH assay by introducing a time-separated two-step process: first capturing spatial information through imaging, then extracting and sequencing the barcodes. This dimensional transformation allows the assay to overcome the throughput limitation of traditional FISH by separating the low-throughput imaging step from the high-throughput sequencing step, which can process many samples in parallel.
3Measurement precision
If in situ sequencing is used, then single molecule resolution is achieved, but throughput remains low
Solution Approach 1:
The patent extracts the sequencing target (barcode) from its original mRNA context after in situ imaging has captured the spatial information. By detaching and isolating the barcodes from the complex in situ environment, the patent enables these barcodes to be processed through high-throughput sequencing pipelines designed for bulk DNA/RNA samples. This extraction separates the spatial information capture (done in situ with single-molecule resolution) from the sequencing operation (done in a high-throughput format), thereby resolving the throughput limitation.
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 high-throughput identification of RNA molecules with single molecule resolution and spatial information, overcoming limitations of existing assays by providing both sequence and spatial data in a large-scale manner.
Implementation Method 1
attaching one by one in a stepwise manner, a plurality of permanent nucleic acid adapters to each of the plurality of RNA molecules, thereby forming a linear array of permanent nucleic acid adapters on each of the plurality of RNA molecules
Implementation Method 2
each permanent nucleic acid adapter from the pool of different permanent nucleic acid adapters is associated with and identifiable by a detection motif
Implementation Method 3
The detection motifs are introduced into the sample together with a pool of transient nucleic acid adapters, each nucleic acid probe of the pool of transient nucleic acid adapters hybridizes with a corresponding transient nucleic acid adapter, and each transient nucleic acid adapter hybridizes with the readout region of a corresponding permanent nucleic acid adapter in a reversible manner
Implementation Method 4
acquiring imaging signals of the detection motifs in the sample
Data Source
AI summary
Described herein is an imaging-based method for identifying RNA molecules in a sample, which is able to determine both the location and sequences of the RNA molecules. The method comprises: forming randomized barcode attached to the RNA molecules in the sample by sequential stepwise addition of permanent nucleic acid adapters randomly selected from a pool; acquiring imaging signals from the sequence-specific staining of the newly added permanent nucleic acid adapters after each addition of a permanent nucleic acid adapter, which links the location information of the RNA molecules with the barcoding; sequencing the barcoded RNA molecules, which links the sequences of the RNA molecules with the barcoding; and matching the location of the RNA molecules to the sequences thereof by matching the barcoding. Also described are kits for performing the method.