Template-Switch Adapter Addition for Low-Input RNA Sequencing
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Solution Overview
Problem
Ligation-based methods for adding adapters to nucleic acids are cumbersome, lack directionality, and are unsuitable for small sample amounts due to inefficiencies and sensitivity issues, preventing efficient hybridization and amplification.
Innovation Solution
A template-switch polymerization reaction is used to combine template RNA, a template switch oligonucleotide, a polymerase, and dNTPs to create a hybrid nucleic acid with predetermined adapter sequences at both ends, facilitating efficient adapter addition and directionality.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If ligation-based methods are used to add adapters to nucleic acids, then adapter sequences can be added to nucleic acids, but the process becomes cumbersome with multiple enzymatic and wash steps
Solution Approach 1:
The patent combines multiple separate steps (adapter annealing, fill-in reaction, ligation) into a single one-pot reaction. The template switch oligonucleotide serves multiple functions: it provides the adapter sequence, acts as a primer for fill-in reaction, and enables directional adapter addition without requiring separate wash steps between operations.
Solution Approach 2:
The template switch oligonucleotide performs multiple functions simultaneously: it serves as a primer for DNA synthesis, provides the adapter sequence through its 5' overhang, and enables directional cloning through its specific binding to the 3' end of the nucleic acid. This multi-functionality eliminates the need for separate reagents and steps.
2Reliability
If ligation-based approaches are used, then adapters can be added to nucleic acids, but the sensitivity is low and unsuitable for small sample quantities
Solution Approach 1:
The template switch oligonucleotide self-primers at the 3' end of the nucleic acid through complementary base pairing, eliminating the need for external primers or complex preparation steps. This self-service mechanism reduces handling steps that could lead to sample loss and improves sensitivity for low-input samples.
3Adaptability or versatility
If ligation-based approaches are used, then adapters can be added to nucleic acids, but directionality is lost making it difficult to have different adapters at different ends
Solution Approach 1:
The template switch oligonucleotide creates asymmetric adapter addition by specifically binding to the 3' end of the nucleic acid through complementary base pairing. The 5' overhang of the template switch oligonucleotide provides a unique adapter sequence that is directional, allowing different adapters to be added to different ends of the nucleic acid in a controlled manner.
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 method simplifies the adapter addition process, enhances sensitivity, and allows for efficient amplification even with limited sample material, improving sequencing accuracy and efficiency.
Implementation Method 1
a polymerase, and dNTPs. The reaction mixture components are combined under conditions sufficient to produce a product nucleic acid that includes the template RNA and the template switch oligonucleotide each hybridized to adjacent regions of a single product nucleic acid that includes a region polymerized from the dNTPs by the polymerase
Implementation Method 2
a template switch oligonucleotide including a 3' hybridization domain and a sequencing platform adapter construct
Data Source
AI summary
Provided are methods of adding adapters to nucleic acids. The methods include combining in a reaction mixture a template ribonucleic acid (RNA), a template switch oligonucleotide including a 3′ hybridization domain and a sequencing platform adapter construct, a polymerase, and dNTPs. The reaction mixture components are combined under conditions sufficient to produce a product nucleic acid that includes the template RNA and the template switch oligonucleotide each hybridized to adjacent regions of a single product nucleic acid that includes a region polymerized from the dNTPs by the polymerase. Aspects of the invention further include compositions and kits.