CATS RNA Sequencing Method for Degraded Samples
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Solution Overview
Problem
Current RNA sequencing methods, particularly next-generation sequencing technologies, face challenges in efficiently sequencing sensitive, degraded, and low-input RNA samples, including cell-free and non-coding RNA sequences, which are crucial for disease marker detection, due to limitations in library creation and sensitivity.
Innovation Solution
The method combines Capture and Amplification by Tailing and Switching (CATS) technology with Nanoball sequencing, involving steps like fragmentation, end-repairing, template switching, rolling circle replication, and immobilization of DNA nanoballs on a patterned array flow cell, enabling high-throughput sequencing of RNA sequences with improved sensitivity and reproducibility.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional RNA sequencing library creation methods are used, then the sequencing can be performed with standard protocols, but the sensitivity and efficiency for degraded, low-input RNA samples are insufficient
Solution Approach 1:
The patent combines capture and amplification steps into a single integrated reaction tube, eliminating the need for separate library preparation steps. This merging of operations simplifies the overall process while improving sensitivity for low-input and degraded RNA samples, directly addressing the contradiction between reliability and device complexity
Solution Approach 2:
The CATS protocol uses universal adaptors that can bind to various types of RNA sequences including degraded, cell-free, and non-coding RNAs. This multi-functional approach allows the same protocol to handle diverse sample types effectively, improving sequencing reliability across different sample conditions without requiring complex sample-specific procedures
2Productivity
If ligase-based adapter incorporation is used during cDNA synthesis, then the protocol is well-established, but the efficiency for incorporating adaptors is lower compared to ligase-free methods
Solution Approach 1:
The patent extracts the ligation step from the adaptor incorporation process, using a ligase-free method instead. By removing the ligation enzyme and its associated complex procedures, the protocol achieves higher adaptor incorporation efficiency while maintaining simplicity, resolving the contradiction between productivity and ease of manufacture
Solution Approach 2:
The patent introduces a template switching oligonucleotide as an intermediary that facilitates adaptor incorporation during reverse transcription without requiring ligation. This mediator enables efficient adaptor attachment to cDNA fragments while keeping the protocol simple and compatible with standard reverse transcription reagents
3Reliability
If manual library preparation procedures are used, then the process can be customized, but the hands-on time and technical variability are high
Solution Approach 1:
The patent implements continuous automated processing where DNA nanoballs are generated, immobilized, and sequenced in an uninterrupted workflow on the DNBSEQ platform. This continuous operation eliminates manual intervention steps, reducing both hands-on time and technical variability while maintaining high reproducibility across samples
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 efficient sequencing of sensitive and degraded RNA samples, including non-coding RNAs, with high reproducibility and sensitivity, reducing hands-on time and technical variability, and producing high-quality reads comparable to state-of-the-art methods like Illumina sequencing.
Implementation Method 1
obtaining DNA nanoballs (DNBs) by performing a rolling circle replication of the synthesized stranded nucleic acid sequence
Implementation Method 2
that can be immobilized on a substrate at a specific location and that remain separated from each other, because of their negatively charges upon the patterned substrate
Data Source
AI summary
A high throughput sequencing method and kit The present invention is in the field of diagnostic and sequencing technologies and is related to a high throughput sequencing method and a kit comprising tools for performing this method, that combine a capture and amplification by switching detection step, preferably the so-called “Capture and Amplification by Tailing and Switching” (CATS) and sequencing technology, preferably the so-called “Nanoballs sequencing” technologies.