SC3-seq mRNA Amplification for Single-Cell Quantitative Transcriptomics
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Solution Overview
Problem
Existing nucleic acid sequence amplification methods for mRNA analysis using next-generation sequencers face challenges with low quantitativity and high unit costs, particularly when analyzing small numbers of cells, leading to poor reproducibility and precision in quantitative transcriptome analysis.
Innovation Solution
A method called SC3-seq, which involves amplifying cDNA by targeting the 3' terminal side of mRNA using a poly A sequence, fragmenting it, and selectively adding primers, allowing for the simultaneous analysis of multiple samples with enhanced quantitativity and reduced costs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If PCR amplification is used for cDNA amplification, then amplification efficiency is high and the method is convenient, but reproducibility of copy number after amplification is poor when small number of copies are used
Solution Approach 1:
The invention segments the cDNA amplification process into two distinct phases: (1) multiple displacement amplification (MDA) phase that generates full-length cDNA copies with high reproducibility, and (2) PCR phase that amplifies specific regions for sequencing. This segmentation allows each method to perform its optimal function - MDA for quantitative fidelity and PCR for amplification efficiency.
Solution Approach 2:
The invention performs preliminary full-length cDNA synthesis using MDA before PCR amplification. By first creating accurate full-length copies that maintain the original copy number relationship, the subsequent PCR step operates on already-amplified templates, reducing the impact of PCR stochastic effects on quantitative accuracy.
2Adaptability or versatility
If RNA-seq is used for transcriptome analysis, then comprehensive analysis is achieved, but unit cost of analysis per cell is high and many samples are required
Solution Approach 1:
The invention extracts and sequences only the 3' terminal region of cDNA rather than the full-length sequence. This extraction approach maintains the ability to identify and quantify transcripts while dramatically reducing the sequencing depth and number of samples needed, as the 3' end contains sufficient information for gene identification and quantification.
3Loss of information
If full-length cDNA is targeted for sequencing, then complete transcript information is obtained, but quantitativity of mRNA analysis becomes low when cDNA is long
Solution Approach 1:
The invention extracts the 3' terminal region of cDNA for sequencing while discarding the 5' region. This extraction provides sufficient transcript identification information from the 3' end (including poly-A tail region) while improving quantitativity by reducing amplification biases associated with long cDNA molecules and enabling more efficient sequencing coverage.
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 SC3-seq method provides a reliable and cost-effective technique for quantitative mRNA analysis, significantly improving reproducibility and accuracy at the single-cell level by focusing on the 3' ends of mRNA sequences, enabling efficient gene expression profiling with improved precision and reduced errors.
Implementation Method 1
amplifying cDNA by utilizing a poly A sequence of mRNA
Data Source
AI summary
The invention provides a method of preparing a nucleic acid population suitable for RNA sequencing. The method involves amplifying a double-stranded DNA and a poly T sequence by using the DNA constituted of any additional nucleic acid sequence X, poly T sequence, mRNA sequence isolated from a biological sample, poly A sequence and any additional nucleic acid sequence Y in this order as a template, a first primer containing any additional nucleic acid sequence X having amine added to the 5′-terminal (and a poly T sequence), and a second primer containing any additional nucleic acid sequence Y (and a poly T sequence), followed by fractionalizing the DNA, phosphorylating the DNA, preparing cDNA by using the DNA as a template and a third primer, adding adenine (A) to the cDNA, linking a DNA, and amplifying the DNA by using the DNA as a template, a fourth primer, and a fifth primer.