Template Switching Nucleic Acid Library Preparation
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Solution Overview
Problem
Current methods for preparing nucleic acid libraries, particularly for next-generation sequencing (NGS), face challenges in efficiently generating high-quality libraries from single cells or RNA samples, especially for immune cell receptor repertoire analysis.
Innovation Solution
The method involves generating double-stranded complementary DNA (cDNA) through a template-switching reaction from a RNA sample, allowing for the production of libraries such as expression libraries and immune cell receptor repertoire libraries, which can be indexed at the single cell level.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional library preparation methods are used, then library preparation can be performed, but the efficiency and quality of libraries from single cells or RNA samples are insufficient
Solution Approach 1:
The library preparation process is divided into distinct modular steps: template switching to generate double-stranded cDNA, selective amplification of desired fragments, and indexing. This segmentation allows each step to be optimized independently, improving overall efficiency and reliability.
Solution Approach 2:
The template-switching reaction is performed as a preliminary step before amplification to generate double-stranded cDNA with known sequence boundaries. This preliminary action enables subsequent selective amplification and indexing, ensuring high-quality libraries from limited starting material.
2Adaptability or versatility
If multiple libraries are prepared from a single RNA sample, then analysis versatility is improved, but the complexity of the preparation process increases
Solution Approach 1:
A single template-switching reaction and amplification protocol can generate multiple library types (expression libraries, TCR repertoire libraries, etc.) from one RNA sample by using different primers or probes. This multi-functionality improves adaptability without proportionally increasing process complexity.
Solution Approach 2:
The process uses selective amplification steps that can be configured to target different nucleic acid sequences for different analysis types. By segmenting the amplification step, the same framework supports multiple library preparations without requiring separate complete protocols.
3Measurement precision
If single cell level indexing is implemented, then measurement precision is improved, but the difficulty of detecting and measuring increases
Solution Approach 1:
Unique molecular identifiers (UMIs) or barcodes are incorporated during the template-switching or amplification step as a preliminary action. This allows single cell identification to be encoded early in the process, simplifying downstream detection and measurement while maintaining high precision.
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 the simultaneous preparation of multiple libraries from a single RNA sample, including libraries from single cells, with high efficiency and accuracy, facilitating advanced analyses like differential expression and TCR profiling.
Implementation Method 1
double stranded complementary DNA (cDNA) generated through a template-switching reaction involving a RNA sample
Data Source
AI summary
Methods of preparing nucleic acid libraries are provided. Aspects of the methods include producing one or more libraries, including e.g., expression libraries and/or immune cell receptor repertoire libraries, from double stranded complementary DNA (cDNA) generated through a template-switching reaction involving a RNA sample. In some aspects, the methods include preparing a library from a single cell and/or a library indexed at the single cell level. Compositions and kits for use in performing the methods are also provided.