Trans-Splicing Barcoding for High-Throughput Transcriptome Profiling
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Solution Overview
Problem
Current methods for transcriptome profiling are limited by low throughput, high cost, and labor intensity, as they often require generating RNA libraries in compartmentalized wells or droplets, which restrict the ability to analyze multiple cell populations simultaneously.
Innovation Solution
A nucleic acid construct with a barcoding construct linked to an antisense promoter and a perturbation element, using trans-splicing to attach barcodes to endogenous RNA molecules, allowing for the generation of barcoded libraries in a single volume, enabling high-throughput analysis of cell populations and whole-organism RNA barcoding.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If RNA libraries are generated in compartmentalized wells or droplets, then library generation is achieved, but throughput is limited and cost increases
Solution Approach 1:
The patent extracts the barcode attachment process from compartmentalized structures (wells/droplets) and performs it in a single-volume homogeneous mixture. Cells are lysed in one volume and barcodes are attached to RNA molecules throughout the entire volume simultaneously, eliminating the need for physical compartmentalization and dramatically increasing throughput.
Solution Approach 2:
The patent merges multiple functions into a single volume: cell lysis, barcode delivery, RNA release, and barcode attachment all occur simultaneously in one homogeneous mixture. This consolidation eliminates the need for separate compartmentalized steps and enables high-throughput processing of multiple cell populations together.
2Productivity
If compartmentalized well or droplet methods are used, then RNA library generation is possible, but labor intensity increases
Solution Approach 1:
The patent removes the complex compartmentalization steps (well plate handling, droplet generation and manipulation) and replaces them with a simple single-volume lysis and attachment process. This extraction of the problematic steps dramatically reduces labor intensity while maintaining library generation capability.
Solution Approach 2:
The system uses endogenous RNA molecules themselves as the target for barcode attachment, eliminating the need for external priming or complex preparation steps. The RNA molecules naturally present in the lysed cells serve as the substrate for barcode attachment, simplifying the overall process.
3Productivity
If multiple cell populations are analyzed simultaneously, then throughput increases, but specificity maintenance becomes challenging
Solution Approach 1:
The patent attaches unique barcodes specific to each cell population's RNA molecules through trans-splicing. Each population's RNA acquires a distinctive barcode signature that maintains local quality and identity even though all populations are processed together in the same volume. This enables precise identification and differentiation of populations during sequencing.
Solution Approach 2:
The patent creates barcode copies that are attached to each RNA molecule through trans-splicing. These barcode copies serve as molecular tags that replicate population identity information onto the RNA molecules, enabling accurate tracking and identification of cells of origin even after mixed population processing.
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 increases the throughput of transcriptome profiling by allowing multiple cell populations to be analyzed simultaneously, reducing costs and labor, and enabling the identification of perturbation effects on RNA molecules through sequencing, while maintaining specificity and accuracy.
Implementation Method 1
splicing the barcoding sequence onto endogenous RNA molecules in the cell, thereby generating a barcoded library
Data Source
AI summary
Method of generating a barcoded library, comprising delivering a polynucleotide into a cell, each polynucleotide comprising: (i) a sequence encoding a barcoding construct operably linked to a first promoter that is an antisense promoter, wherein the barcoding construct comprises a trans-splicing element and a barcode sequence; and a sequence encoding a perturbation element operably linked to a second promoter; generating RNA transcripts of the polynucleotide delivered into the cell, wherein the RNA transcripts comprise the barcoding construct and the perturbation element; and splicing the barcoding sequence onto endogenous RNA molecules in the cell, thereby generating a barcoded library, each member of the barcoded library comprising the barcode sequence and the endogenous RNA molecule attached with the barcode sequence.