RNA Sequencing via Dual Barcode Segmentation
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Solution Overview
Problem
Current methods for single-cell RNA sequencing, such as sub-nanoliter well plates and microfluidic droplet generators, face limitations in throughput due to the need for cells to be loaded at limiting dilution to avoid cell doublets, leading to inefficient use of reaction compartments and high costs with labor-intensive parallelization, and combinatorial indexing methods suffer from material loss, synthesis errors, and labor-intensive processes.
Innovation Solution
A method for sequencing RNA oligonucleotides involving permeabilized cells or nuclei pre-indexed with a first barcode, followed by microfluidic droplet-based sequencing with a second barcode, allowing multiple cells per droplet without creating indistinguishable labeled readouts, thereby increasing throughput and reducing labor and reagent wastage.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If cells are loaded at limiting dilution to avoid cell doublets, then cell identification accuracy is improved, but throughput and productivity deteriorate due to inefficient use of reaction compartments
Solution Approach 1:
The patent divides the barcoding process into two separate stages: (1) combinatorial indexing in microwells that assigns first barcodes to cells, and (2) microfluidic droplet sequencing that assigns second barcodes. This segmentation allows multiple cells to be processed per droplet while maintaining identification accuracy through the combination of both barcode types.
Solution Approach 2:
The patent performs combinatorial indexing with first barcodes as a preliminary step before microfluidic droplet sequencing. Cells are pre-barcoded in microwells with first barcodes, then pooled and processed through microfluidics. This preliminary action enables higher throughput in the microfluidic stage without sacrificing cell identification accuracy.
2Productivity
If parallelization is used to increase throughput, then productivity is improved, but device complexity and labor intensity worsen
Solution Approach 1:
The patent merges two different barcoding approaches (combinatorial indexing and microfluidic droplet sequencing) into a unified workflow. The first barcodes from combinatorial indexing and second barcodes from microfluidics are combined to create unique cell identifiers, achieving high throughput without requiring complex parallelized systems.
3Productivity
If combinatorial indexing is used to increase throughput, then productivity is improved, but material loss and synthesis errors worsen
Solution Approach 1:
The patent performs combinatorial indexing as a preliminary step with controlled material usage, then uses the pre-barcoded cells in microfluidic droplets. This preliminary action with first barcodes reduces the need for excessive material consumption in subsequent high-throughput microfluidic processing, minimizing overall material loss while maintaining throughput.
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, named scifi-RNA-seq, achieves a significant increase in throughput by enabling loading of multiple cells per droplet, reducing material loss, and improving library preparation efficiency, resulting in cleaner data with high single-cell purity and the ability to handle large-scale sequencing projects.
Implementation Method 1
combining said cells and/or nuclei of (a) with a second oligonucleotide comprising DNA in a first reaction compartment, wherein the second oligonucleotide comprises at least a first sequence at least partially complementary to a sequence of the first oligonucleotide, under conditions to allow annealing of the first sequence of the second oligonucleotide to the first oligonucleotide
Implementation Method 2
reversely transcribing the first oligonucleotide in said cells/nuclei to obtain an elongated second oligonucleotide
Implementation Method 3
wherein for (i), the method further comprises a step of second strand DNA synthesis subsequent to step (c) and prior to step (d)
Implementation Method 4
wherein for (ii) the method further comprises a step of DNA ligation
Data Source
AI summary
The invention relates to a method for sequencing oligonucleotides comprising RNA, wherein two indexing sequences are introduced in RNA oligonucleotides. The invention furthermore relates to uses of such methods and devices used for such methods. Further provided are kits comprising one or more components used in the methods of the invention.