Full-Length cDNA Sequencing With Rolling Circle Nanopore Consensus
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Solution Overview
Problem
Current nucleic acid sequencing technologies, such as PacBio and nanopore-based methods, face challenges with low throughput, incomplete reads, or high error rates, making them inadequate for high-throughput full-length cDNA sequencing.
Innovation Solution
A method involving circularization of full-length cDNA with a known heterologous sequence, followed by rolling circle amplification to create a concatemer, and subsequent nanopore sequencing to generate consensus sequences through identifying repeating segments.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If nanopore sequencing is used to sequence full-length cDNA, then throughput is improved, but base accuracy deteriorates
Solution Approach 1:
The patent segments the cDNA sequencing process into multiple cycles of rolling circle amplification and sequencing. Each cycle produces a set of reads that are then used to generate a consensus sequence, dividing the overall task into manageable segments that can be processed iteratively to improve accuracy while maintaining throughput.
Solution Approach 2:
The patent performs preliminary circularization of cDNA molecules with known heterologous sequences before sequencing. This preliminary action creates a template structure that enables subsequent rolling circle amplification, allowing the same template to be sequenced multiple times to generate consensus sequences that correct errors while maintaining high throughput.
2Measurement precision
If PacBio sequencing is used to sequence full-length cDNA, then base accuracy is improved, but throughput deteriorates
Solution Approach 1:
The patent uses rolling circle amplification to create multiple copies of the circularized cDNA template before sequencing. This copying process generates numerous identical templates that can be sequenced in parallel, dramatically increasing throughput while maintaining the accuracy benefits of long-read sequencing by allowing consensus building from multiple copies.
3Measurement precision
If circular consensus sequencing is applied to increase base accuracy, then measurement precision is improved, but throughput deteriorates
Solution Approach 1:
The patent implements continuous rolling circle amplification where the circularized template is continuously copied and sequenced in an uninterrupted manner. This continuous action allows for high throughput while the repetitive nature of the process enables consensus sequence generation, maintaining both accuracy and productivity simultaneously.
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 method produces a large number of accurate full-length cDNA consensus sequences, enabling high-throughput analysis of isoform-level transcriptomes in both bulk and single-cell samples.
Implementation Method 1
performing rolling circle amplification using the circularized DNA as template to produce a concatemer including repeating segments including the full-length cDNA and the known heterologous sequence
Implementation Method 2
obtaining a raw sequencing read of the concatemer using a nanopore
Data Source
AI summary
Provided are methods of nucleic acid sequencing. The methods include producing a circularized DNA including a full-length cDNA and a known heterologous sequence, and performing rolling circle amplification using the circularized DNA as template to produce a concatemer including repeating segments including the full-length cDNA and the known heterologous sequence. The methods further include obtaining a raw sequencing read of the concatemer using a nanopore, identifying the repeating segments in the raw sequencing read, and producing a consensus sequence of the full-length cDNA based on the sequences of the repeating segments. Computer-readable media, computing devices, and systems that find use, e.g., in practicing the methods of the present disclosure are also provided.