In Situ Reverse Transcription for Spatial Copy Number Mapping
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Solution Overview
Problem
Existing methods struggle to accurately determine the spatial location and copy number of nucleic acid targets and cellular component targets within a sample, particularly when the number of molecules is small, leading to amplification bias and inaccurate gene expression measurements.
Innovation Solution
A method involving a substrate with spatially distinct oligonucleotide barcodes that hybridize to nucleic acid targets, allowing for sequencing data analysis to determine the spatial location and copy number of targets, combined with cellular component-binding reagents for specific binding and counting.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If amplification methods are used to detect nucleic acid targets, then detection sensitivity is improved, but amplification bias is introduced leading to inaccurate measurements
Solution Approach 1:
The patent performs reverse transcription in situ at the spatial location of RNA targets before any amplification occurs. By converting RNA to cDNA at the original location and incorporating spatial barcodes during this preliminary step, the method establishes accurate spatial and quantitative information before amplification bias can distort the measurements.
Solution Approach 2:
The patent introduces spatial barcodes and molecular labels as intermediary elements that capture spatial location and molecular identity information. These intermediaries are incorporated during reverse transcription and serve as reliable markers that are not affected by subsequent amplification processes, thereby resolving the contradiction between detection sensitivity and measurement accuracy.
2Measurement precision
If spatial barcoding is implemented to determine location, then spatial resolution is improved, but method complexity increases
Solution Approach 1:
The patent combines multiple functions into the reverse transcription step: RNA template switching, spatial barcode incorporation, molecular labeling, and cDNA synthesis all occur in a single in situ reaction. This merging of operations achieves high spatial resolution without proportionally increasing method complexity, as the spatial barcode is integrated during the essential reverse transcription process rather than as a separate step.
3Measurement precision
If in situ reverse transcription with template switching is used, then copy number accuracy is improved, but reaction conditions become more stringent
Solution Approach 1:
The patent utilizes template switching oligos with specific sequence parameters designed to facilitate efficient template switching during reverse transcription. By optimizing the sequence parameters of the TSO and incorporating it into the reaction, the method achieves accurate copy number determination through the template switching mechanism while managing reaction condition stringency through careful oligo design.
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 precise determination of nucleic acid and cellular component targets' spatial location and copy number, correcting for amplification bias and providing accurate gene expression analysis.
Implementation Method 1
a target-binding region capable of hybridizing to a nucleic acid target
Implementation Method 2
extending the plurality of oligonucleotide barcodes hybridized to the copies of a nucleic acid target
Implementation Method 3
obtaining sequencing data comprising a plurality of sequencing reads of the plurality of barcoded nucleic acid molecules
Data Source
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AI summary
Disclosed herein include systems, methods, compositions, and kits for determining the spatial location and copy number of targets (e.g., nucleic acid targets, cellular component targets) in a sample. There are provided, in some embodiments, substrates comprising a plurality of spatial regions. A plurality of oligonucleotide barcodes can be associated with each of the spatial regions and can comprise a predetermined spatial label. Oligonucleotide barcodes of the same spatial region can comprise the same spatial label, and oligonucleotide barcodes of the different spatial regions can comprise different spatial labels. The method can comprise contacting the substrate with a sample such that each distinct spatial region contacts a distinct spatial location of the sample. The method can comprise in situ extension (e.g., reverse transcription) of the oligonucleotide barcodes.