Full-Length Single Cell RNA Sequencing via Nested Barcoding
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Solution Overview
Problem
Current methods for molecular barcoding in single cell transcriptomics and proteomics face challenges in efficiently labeling and quantitatively analyzing nucleic acid targets, particularly in obtaining sequences from internal regions and determining copy numbers accurately.
Innovation Solution
The method involves contacting nucleic acid targets with oligonucleotide barcodes, extending them with reverse transcriptase and a template switch oligonucleotide, and using a transposome to generate barcoded DNA fragments, which are then anchored and further barcoded to yield double-stranded DNA products, allowing for the determination of copy numbers based on distinct molecular labels.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If molecular barcoding is used for single cell transcriptomics analysis, then gene expression profiles can be deciphered, but the efficiency of labeling and quantitative analysis is insufficient
Solution Approach 1:
The method segments the barcoding process into multiple stages: initial barcoding with first oligonucleotide barcodes, template switching with bait sequences, and secondary barcoding with second oligonucleotide barcodes. This segmentation allows parallel processing and amplification at different stages, significantly improving throughput and reducing overall analysis time while maintaining accurate copy number determination.
Solution Approach 2:
The template switch oligonucleotide with bait sequence is introduced in advance to enable preliminary capture and enrichment of barcoded molecules before final sequencing. This preliminary action concentrates target molecules and prepares them for efficient amplification, thereby improving labeling efficiency and reducing the time required for subsequent analysis steps.
2Loss of information
If current molecular barcoding methods are used, then nucleic acid targets can be labeled, but sequence information of internal regions cannot be obtained
Solution Approach 1:
The template switch oligonucleotide acts as an intermediary that bridges the initial barcode and the internal region of the nucleic acid target. It contains a bait sequence that hybridizes to the internal region, enabling capture and sequencing of internal sequences without requiring complex direct amplification methods, thus obtaining complete sequence information while managing process complexity.
Solution Approach 2:
The method employs nested barcoding where second oligonucleotide barcodes are added to molecules that already contain first barcodes and template switch sequences. This nesting allows multiple layers of information (cell identity, molecular identity, and internal sequence) to be captured in a hierarchical structure, enabling comprehensive sequence retrieval through systematic decoding.
3Measurement precision
If molecular barcoding is applied, then single cell analysis can be performed, but accurate copy number determination is challenging
Solution Approach 1:
The method uses multiple copies of barcoded molecules generated through PCR amplification, where each copy retains the original molecular barcode. By counting the number of unique molecular barcodes associated with a cell rather than counting amplified reads, the method achieves accurate copy number determination that is independent of amplification efficiency, thereby improving measurement precision while using standard amplification techniques.
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 enables efficient labeling and quantification of nucleic acid targets, providing sequence information of internal regions and accurate copy number determination, enhancing the precision of single cell analysis.
Implementation Method 1
extending the first plurality of oligonucleotide barcodes hybridized to the copies of the nucleic acid target in the presence of a reverse transcriptase
Implementation Method 2
contacting the barcoded nucleic acid molecules with a transposome to generate a first and a second plurality of barcoded DNA fragments
Implementation Method 3
each oligonucleotide barcode of the first plurality of oligonucleotide barcodes comprises a first universal sequence, a first molecular label, and a first target-binding region capable of hybridizing to the nucleic acid target
Data Source
AI summary
Disclosed herein include systems, methods, compositions, and kits for full-length whole transcriptome analysis (WTA). Some embodiments comprise 5′-based, 3′-based, and internal-based gene expression profiling. In some embodiments, nucleic acid targets (e.g., mRNAs) are initially barcoded on the 3′ end with the first plurality of oligonucleotide barcodes and subsequently barcoded on the 5′ end following a template switching reaction and intermolecular hybridization with a second plurality of oligonucleotide barcodes and extension. In some embodiments, extended barcoded nucleic acid molecules are contacted with a transposome and tagmentation products are barcoded with a third plurality of oligonucleotide barcodes. Immune repertoire profiling methods are also provided in some embodiments.