5' End-Specific Capture Probes for Spatial Transcriptomics
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Solution Overview
Problem
Current spatial analysis methods for biological samples face challenges in providing comprehensive data on analyte location within tissues due to biases in analyte migration and capture protocols, particularly with poly(A) tail-based methods that lead to 3' bias in gene expression libraries.
Innovation Solution
The method involves generating a cDNA molecule complementary to the target nucleic acid using a reverse transcription primer with an adaptor sequence, ligating a second adaptor sequence, and releasing it to contact a capture probe with a spatial barcode and capture domain on an array, allowing for the identification of the target nucleic acid's location through sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If poly(A) tail-based capture methods are used, then analyte capture efficiency is improved, but 3' bias in gene expression libraries occurs
Solution Approach 1:
The patent inverts the conventional capture approach by using 5' end-specific capture probes instead of poly(A) tail-based 3' end capture. This inversion allows the capture to occur at the opposite end of the transcript, eliminating the 3' bias while maintaining capture efficiency through high-affinity probe-design at the 5' end
Solution Approach 2:
The patent applies preliminary action by performing in situ reverse transcription and cDNA synthesis within the tissue section before capture. This preliminary conversion of RNA to cDNA at the native location preserves spatial information and enables subsequent specific capture of the synthesized cDNA with 5' end probes
2Quantity of substance
If single-cell analyte data is provided, then comprehensive analyte information is achieved, but spatial position information is lost
Solution Approach 1:
The patent uses spatial barcodes as intermediaries that bridge the gap between single-cell analyte data and spatial position information. These barcodes are incorporated into the cDNA during in situ reverse transcription, serving as a mediator that carries spatial location data through subsequent processing steps to final sequencing analysis
Solution Approach 2:
The patent implements nesting by embedding spatial barcode sequences within the cDNA molecule structure. The spatial information is nested inside the genetic material itself, allowing both the analyte identity and spatial position to be read from the same molecular entity during sequencing
3Reliability
If in situ reverse transcription with adaptor sequences is used, then spatial context is retained, but process complexity increases
Solution Approach 1:
The patent applies universality by designing multi-functional adaptor sequences that perform multiple roles: they serve as priming sites for reverse transcription, contain spatial barcode information, and provide binding sites for subsequent capture probes. This multi-functionality reduces the need for separate components and simplifies the overall workflow despite the sophisticated chemistry involved
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 provides high spatial resolution analyte and expression data while retaining native spatial context, addressing biases in existing methods and enabling accurate localization of nucleic acids within biological samples.
Implementation Method 1
generating a cDNA molecule comprising a sequence that is substantially complementary to the target nucleic acid using a reverse transcription primer
Implementation Method 2
a capture domain that binds specifically to the second adaptor sequence ligated to the cDNA
Data Source
AI summary
Provided herein are methods of determining a location of a target nucleic acid in a biological sample.