Spatial RNA Location Identification via Enzymatic Adaptor Ligation
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Solution Overview
Problem
Current methods for generating sequencing libraries from spatial arrays are inefficient, leading to a loss of sensitivity and specificity in spatial gene expression analysis, as they fail to effectively incorporate sequencing adaptors, resulting in unamplified analytes and incomplete gene expression data.
Innovation Solution
The method involves using RNAse H to create nicks in RNA:DNA hybrids, followed by end repair, A-tailing, and ligation of a double-stranded sequencing adaptor to a blunt-ended DNA product, significantly improving the efficiency of adaptor incorporation and sensitivity of the sequencing process.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional adaptor incorporation methods are used, then the process is simpler, but the efficiency and sensitivity of sequencing are reduced
Solution Approach 1:
The method performs preliminary actions by creating nicks in the RNA strand before second-strand synthesis, then performing end repair to create blunt-ended DNA products. This preliminary processing enables highly efficient adaptor ligation in subsequent steps, resolving the contradiction between efficiency and complexity by preparing the substrate in advance for optimal adaptor incorporation.
Solution Approach 2:
The method changes the physical and chemical parameters of the DNA product through a series of transformations: from RNA:DNA hybrid to nicked RNA, then to blunt-ended double-stranded DNA, and finally to A-tailed product. These parameter changes (chemical composition, structural form) enable highly efficient adaptor ligation, achieving high productivity despite increased method complexity.
2Loss of time
If adaptor incorporation is performed using conventional methods, then the protocol is shorter, but the hands-on time and processing capacity are limited
Solution Approach 1:
The method enables continuous processing of multiple analytes through standardized blunt-ended DNA product generation and A-tailing. The continuous workflow allows parallel processing of numerous samples without extensive manual intervention between steps, reducing hands-on time while increasing the number of analytes that can be processed simultaneously.
Solution Approach 2:
The method employs self-service mechanisms through enzymatic reactions that automatically perform end repair, A-tailing, and adaptor ligation without extensive manual manipulation. The biochemical systems self-correct and self-assemble the sequencing libraries, reducing hands-on time while maintaining high processing capacity for multiple analytes.
3Reliability
If sequencing adaptors are not effectively incorporated, then the protocol is simpler, but the sensitivity and specificity of spatial gene expression analysis are lost
Solution Approach 1:
The method replaces mechanical or manual adaptor incorporation with biochemical enzymatic reactions. RNAse H creates nicks, DNA polymerase performs end repair and A-tailing, and ligase incorporates adaptors through specific biochemical recognition. This substitution of mechanical processes with enzyme-driven chemistry ensures high sensitivity and specificity while managing the complexity through standardized biochemical protocols.
Solution Approach 2:
The method introduces intermediary molecules and enzymes (RNAse H, DNA polymerase, ligase) that mediate between the cDNA and sequencing adaptors. These intermediaries ensure specific and sensitive recognition and incorporation of adaptors through biochemical complementarity, achieving high reliability despite the increased complexity of the multi-step process.
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 reduces hands-on time for adding sequencing oligonucleotides to cDNA generated on spatial arrays, allowing for more accurate spatial gene expression analysis by increasing the number of analytes processed and sequenced, thereby enhancing sensitivity and specificity.
Implementation Method 1
RNAse H or a functional equivalent to produce nicks in the RNA of a RNA:DNA hybrid
Implementation Method 2
performing random-primed DNA synthesis, thereby replacing the nicked RNA with a second strand DNA molecule
Implementation Method 3
performing end repair on the second strand DNA molecule hybridized to the extended capture probe to generate a blunt-ended double-stranded DNA product
Implementation Method 4
adding a single adenosine (A) nucleotide to the 3′ end of the extended capture probe in the blunt-ended double-stranded DNA product
Implementation Method 5
ligating a double-stranded sequencing adaptor to the double-stranded DNA product
Data Source
AI summary
Provided herein are methods of identifying a location of an RNA in a sample that include: (a) contacting the sample with an array comprising capture probes, where a capture probe comprises a capture domain and a spatial barcode; (b) releasing the RNA from the sample; (c) extending a 3′ end of the capture probe using the capture domain-bound RNA as a template; (d) generating nick(s) in the extended capture probe-hybridized RNA and performing random-primed DNA synthesis; (e) performing end repair on the second strand DNA molecule; (f) adding a single adenosine nucleotide to the 3′ end of the extended capture probe; (g) ligating a double-stranded sequencing adaptor to the double-stranded DNA product; and (h) determining all or a part of the sequence of the RNA, and the sequence of the spatial barcode, or complements thereof, and using the determined sequences to identify the location of the RNA in the sample.