Dual 3′/5′ Circularized Barcoded Assay for Single-Cell Sequencing
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Solution Overview
Problem
Existing nucleic acid sequencing methods struggle to obtain sequence information from both the 3' and 5' ends of transcripts in a single assay, particularly for spatial or single-cell applications.
Innovation Solution
A method involving the use of oligonucleotides with barcode sequences and self-complementarity regions to generate circularized barcoded nucleic acid molecules, followed by amplification and sequencing, allowing for the generation of amplicons with both 3' and 5' end sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of information
If reverse transcription-based assays are used to obtain sequence information from either 3' end or 5' end of transcripts, then sequence information can be obtained, but it is not possible to obtain barcode-resolved sequence information from both 3' end and 5' end in a single assay
Solution Approach 1:
The assay is divided into two separate reverse transcription reactions: one using a first oligonucleotide that binds to the 3' end of the transcript, and another using a second oligonucleotide that binds to the 5' end. Each reaction generates a separate extended molecule with barcode information. This segmentation allows both ends to be processed simultaneously in parallel, achieving complete sequence information without excessive complexity.
Solution Approach 2:
The patent merges the two separate extension reactions into a single assay workflow by combining the first and second oligonucleotides in the same reaction mixture. The extended molecules from both reactions are then circularized together through ligation of self-complementary regions, creating a unified library preparation process that simultaneously captures both 3' and 5' end sequence information with barcodes.
2Loss of information
If oligonucleotides with self-complementarity regions are used to circularize extended molecules, then barcode-resolved sequence information from both ends can be obtained, but the assay procedure becomes more complex
Solution Approach 1:
The first and second oligonucleotides each contain self-complementary regions that enable them to anneal to each other through complementary base pairing without requiring external reagents. The 3' end of the first oligonucleotide is complementary to the 5' end of the second oligonucleotide, allowing automatic alignment and circularization of the extended molecules. This self-service mechanism simplifies the ligation step and reduces procedural complexity.
Solution Approach 2:
The patent employs circularization of the extended molecules to create circular DNA structures. By ligating the self-complementary ends together, the linear extended molecules are transformed into circular molecules that contain barcode information from both ends. This circular topology facilitates efficient amplification and sequencing while maintaining compact information storage.
3Productivity
If extension reactions are performed to generate extended molecules with barcode sequences, then simultaneous sequencing of both ends is enabled, but additional reaction steps are required
Solution Approach 1:
The first and second oligonucleotides are designed with self-complementary regions and barcode sequences incorporated in advance. During the extension reactions, these pre-designed features automatically align and circularize the extended molecules without requiring additional ligation steps. This preliminary design enables simultaneous sequencing capability while minimizing the number of reaction steps.
Solution Approach 2:
The first and second oligonucleotides serve multiple functions: they act as primers for reverse transcription, carry barcode sequences for identification, and provide self-complementary regions for circularization. This multi-functionality reduces the need for separate reagents and steps, increasing productivity while controlling assay complexity.
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 the simultaneous sequencing of both ends of nucleic acid analytes, providing comprehensive sequence information for spatial and single-cell analysis.
Implementation Method 1
a first oligonucleotide comprising at least one barcode sequence, a region that hybridizes to a first portion of the nucleic acid analyte
Implementation Method 2
annealing the first region of self-complementarity to the second region of self-complementarity
Implementation Method 3
ligating a 5' terminus and a 3' terminus of the extended molecule to generate a circularized barcoded nucleic acid molecule
Data Source
AI summary
Provided are methods, systems, and kits for circularization-based dual 3′/5′ assays for sequence analysis of barcoded nucleic acids. The circularization-based dual 3′/5′ assays included single cell sequencing assays and spatial sequencing assays.