In Situ Copy Number Detection with Shared Genomic Barcodes
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Solution Overview
Problem
Current methods for in situ detection of copy number variations (CNVs) in biological samples face limitations in multiplexing RNA detection, leading to challenges in accurately analyzing copy number variations due to practical constraints on image resolution and time requirements, particularly in single-cell or spatial-array-based transcriptomic analyses.
Innovation Solution
The use of a common barcode sequence to encode multiple genes within a genomic region, combined with different barcodes for different analytes, allows for improved detection of copy number variations and gene expression signatures by averaging signals across multiple genes, enabling more accurate analysis of CNVs in biological samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If multiple different barcode sequences are used to detect different genes in situ, then the number of detectable genes increases, but the image resolution and optical crowding worsen, limiting the number of barcodes that can be detected in the same imaging round
Solution Approach 1:
A single barcode sequence is designed to represent multiple genes within the same genomic region, allowing the barcode to serve multiple detection functions simultaneously. This multi-functionality approach enables the system to detect copy number variations across multiple genes without requiring proportionally more distinct barcodes, thereby maintaining image resolution while increasing the effective detection capacity.
Solution Approach 2:
Multiple gene detection capabilities are merged into a single barcode sequence. Instead of assigning unique barcodes to each gene, the invention combines the detection function for multiple genes into one shared barcode, reducing optical crowding and enabling detection of more genes within the same imaging round without sacrificing resolution.
2Quantity of substance
If multiple imaging rounds are used to detect different subsets of RNA transcripts, then the number of different barcodes detected increases, but the time required for the assay increases
Solution Approach 1:
Each barcode sequence is designed to function as a universal identifier for multiple genes within a genomic region, allowing a single imaging round to simultaneously capture information about copy number variations across multiple genes. This eliminates the need for multiple sequential imaging rounds, thereby maintaining high detection capacity while significantly reducing total assay time.
3Device complexity
If a lower degree of multiplexing is used, then the complexity of the experiment decreases, but the ability to sufficiently average over many genes to achieve good representation of large scale genomic events is reduced
Solution Approach 1:
The invention merges the detection of multiple genes into shared barcode sequences, allowing experiments with lower multiplexing (fewer distinct barcodes) to still achieve robust statistical representation. By combining signals from multiple genes that share the same barcode, the system maintains reliable detection of large-scale genomic events even when the total number of distinct barcodes is limited.
Solution Approach 2:
The genomic region is segmented into multiple subregions, each with its own gene set that shares a common barcode. This segmentation allows the experiment to cover many genes across multiple subregions using a limited number of barcodes, achieving both reduced complexity and reliable representation through the aggregation of signals across segmented regions.
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 enhances the ability to detect CNVs with greater reliability by reducing the confounding effect of natural fluctuations in gene expression levels, allowing for more precise identification of copy number variations, such as gains or losses of chromosomes or chromosomal segments, through the use of common barcode sequences and rolling circle amplification.
Implementation Method 1
the plurality of probes comprises at least two different probes capable of hybridizing to at least two different RNA molecules expressed from different subregions within the genomic region
Implementation Method 2
the use of common barcode sequences and rolling circle amplification
Data Source
AI summary
The present disclosure relates in some aspects to methods, systems, and kits for analyzing copy number of a genomic region, including at spatial regions in a biological sample. The methods can comprise contacting the biological sample with a plurality of probes for detecting RNA expression from a plurality of subregions within a genomic region, wherein the plurality of probes comprises at least two different probes capable of hybridizing to at least two different RNA molecules expressed from different subregions within the genomic region, and wherein the plurality of probes individually comprise a common barcode sequence that identifies the genomic region. Detection of the common barcode sequence for the genomic region can be used to infer copy number.