Random-Primed cDNA Barcoding for Uniform Full-Length RNA Sequencing
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Solution Overview
Problem
Current single cell RNA sequencing methods suffer from 5′ and 3′ coverage bias due to the use of cell-specific barcodes at either the 3′ or 5′ end, limiting full-length RNA sequencing and increasing costs per cell, which is a challenge for applications requiring comprehensive RNA analysis like splicing isoform and allele-specific expression analyses.
Innovation Solution
A method involving random priming oligonucleotides with a cell barcode (cID) and unique molecular index (UMI) is used, along with a random sequence region and template switching, to generate cDNA samples that provide uniform coverage from the 5′ to 3′ end, reducing primer dimer formation and enhancing cDNA synthesis efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If cell-specific barcodes are added at the 3′ or 5′ end of RNA, then high-throughput sequencing is enabled, but 5′ and 3′ coverage bias occurs
Solution Approach 1:
The patent divides the barcode insertion process into multiple segments by using multiple random priming sites distributed throughout the RNA sequence. Instead of inserting barcodes at a single location (3′ or 5′ end), the method uses multiple random priming oligonucleotides that bind at different positions along the RNA, with each primer carrying a cell barcode. This segmentation of the barcode placement process eliminates the coverage bias that occurs when all barcodes are concentrated at one end, while still enabling high-throughput sequencing by maintaining the ability to pool and sequence multiple cells simultaneously.
2Measurement precision
If traditional single cell barcoding methods are used, then cell-specific identification is achieved, but full-length RNA sequencing is not possible
Solution Approach 1:
The patent makes the random priming oligonucleotides multi-functional by incorporating multiple elements into a single primer structure: (1) a cell barcode for cell-specific identification, (2) a unique molecular index (UMI) for tracking individual molecules, and (3) a random sequence region that can bind at multiple positions along the RNA. This universal design allows the same primer to serve both as a cell barcode carrier and as a binding site distributed throughout the RNA sequence, enabling full-length sequencing while maintaining cell identification accuracy.
3Manufacturing precision
If random priming with long sequence tags is used, then full-length RNA coverage is achieved, but primer dimer formation increases
Solution Approach 1:
The patent applies local quality by making the random sequence region of the primers locally optimized for binding characteristics. The random sequence is designed to be 5-8 nucleotides long, which provides sufficient binding specificity to the RNA template while being short enough to minimize self-complementarity and primer-dimer formation. This local optimization of the random sequence length and composition allows the primers to effectively bind at multiple positions along the RNA without generating harmful primer dimers, thus achieving full-length coverage while reducing adverse effects.
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 allows for high-throughput, cost-effective full-length RNA sequencing with reduced bias, enabling better detection of low-expression transcripts and non-polyadenylated RNAs, and facilitating pooling of multiple cells for transcriptome analysis.
Implementation Method 1
a plurality of random priming oligonucleotides, each random priming oligonucleotide comprising a cell barcode (cID), a unique molecular index (UMI), and a random sequence region of 5 to 8 nucleotides
Implementation Method 2
performing a reverse transcription reaction (RT)
Implementation Method 3
adding a template switching oligonucleotide (TSO) and performing a template switching reaction (TS) to generate a cDNA sample comprising universal PCR handles on the 5′ and 3′ ends
Implementation Method 4
adding a homopolymer oligonucleotide tail to a first strand cDNA by adding a terminal transferase (TdT) and performing a primer extension reaction to generate a second strand cDNA
Data Source
AI summary
The invention relates to methods for preparing cDNA samples for RNA sequencing using random priming oligonucleotides comprising a cell barcode (cID), a unique molecular index (UMI), and a random sequence region, and performing a reverse transcription reaction (RT). The invention also relates to cDNA samples prepared by the methods and uses thereof.