Single-Cell Full-Length RNA Sequencing for Non-Polyadenylated RNA
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Solution Overview
Problem
Current single-cell sequencing methods fail to capture non-polyadenylated RNA species such as long non-coding RNA, tRNA, miRNA, snoRNA, and snRNA, and lack the ability to tag RNA molecules with barcodes and unique molecular identifiers (UMIs), limiting high-throughput sequencing and molecule counting.
Innovation Solution
A method for preparing a sequencing library involving RNA fragmentation, end-repair, polyadenylation, hybridization with a poly-T primer containing a barcode and UMI, reverse transcription, second strand synthesis, in vitro transcription, ribosomal-RNA depletion, and adapter ligation, enabling the sequencing of non-polyadenylated RNAs and providing strand-specific information.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If current single-cell sequencing methods (Cel-Seq, Smart-seq) are used to capture polyadenylated RNA, then the sequencing process is straightforward, but important RNA species such as non-polyadenylated long non-coding RNA, tRNA, miRNA, snoRNA and snRNA are missed
Solution Approach 1:
The RNA population is segmented into polyadenylated and non-polyadenylated fractions through selective polyadenylation treatment. Non-polyadenylated RNAs are enriched by preventing their polyadenylation while allowing polyadenylated RNAs to be polyadenylated, enabling separate processing and analysis of different RNA species with appropriate complexity levels for each fraction
Solution Approach 2:
Template-switching oligonucleotides serve as intermediaries to add universal adapter sequences to the 5' ends of cDNAs during reverse transcription. This intermediary mechanism enables subsequent PCR amplification and sequencing while preserving the original RNA's polyadenylation status information, allowing comprehensive RNA species coverage without excessive complexity
2Productivity
If high-throughput sequencing is performed without barcodes and UMIs, then the sequencing process is simpler, but molecule counting and high-throughput analysis become difficult
Solution Approach 1:
Barcodes and unique molecular identifiers (UMIs) are incorporated into the poly-T primers used during reverse transcription before the sequencing process. This preliminary tagging of individual RNA molecules enables subsequent high-throughput molecule counting and analysis by allowing differentiation of original molecules from amplification duplicates, achieving high productivity with manageable complexity
Solution Approach 2:
The poly-T primers serve multiple functions: they anneal to poly-A tails of mRNA for reverse transcription initiation, incorporate barcodes for sample identification, and include UMIs for molecule counting. This multi-functionality reduces the need for separate steps and reagents, maintaining library preparation complexity at acceptable levels while enabling high-throughput sequencing
3Loss of information
If full length RNA sequencing is performed on single cells, then comprehensive transcriptome information is obtained, but the ability to capture non-polyadenylated RNA species is lost
Solution Approach 1:
Different quality controls and processing conditions are applied to different RNA fractions based on their specific characteristics. Non-polyadenylated RNA fractions undergo different poly-A selection conditions compared to polyadenylated fractions, allowing each RNA type to be optimized for its specific properties while maintaining comprehensive transcriptome coverage
Solution Approach 2:
The polyadenylation status of RNA molecules is dynamically assessed and utilized throughout the workflow. The method adapts processing conditions based on whether RNAs are polyadenylated or not, with non-polyadenylated RNAs being selectively enriched and processed differently to preserve their full-length information while expanding detection range
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
The method allows for high-throughput, full-length RNA sequencing of single cells, capturing a broader range of RNA species with reduced technical noise and improved cell-to-cell heterogeneity analysis.
Implementation Method 1
hybridizing a poly-T primer to the polyadenylated RNA
Implementation Method 2
performing reverse transcription of the hybridized RNA thereby obtaining cDNA
Implementation Method 3
in vitro transcription of the cDNA obtained in step d) thereby obtaining amplified RNA (aRNA)
Data Source
Figure 1
Figure 2A~2C
Figure 2D~2E
AI summary
The invention relates to methods for processing an RNA sample and allows for single cell sequencing of full length total RNA. The method includes labeling the RNA sample with at least one of a barcode and a unique molecular identifier.