Direct RNA Nanopore Sequencing for Full-Length Native Transcripts
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Solution Overview
Problem
Existing RNA sequencing methods, such as poly-T priming and PCR-based library preparations, introduce bias and lose epigenetic information, and short-read sequencing fails to capture alternative splicing and splice variants.
Innovation Solution
A method involving annealing and ligating a polynucleotide with a 3′ terminal random multimer segment and stem-loop form, followed by reverse transcription, cleavage to yield a 3′ A overhang, and connecting an adaptor polynucleotide complex with an RNA translocase enzyme for sequencing through a transmembrane pore.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If poly-T priming or ribonucleic acid fragmentation followed by random hexamer initiation of synthetically complementary DNA is used, then sequencing can be performed, but bias is introduced and epigenetic information is lost
Solution Approach 1:
The invention extracts and preserves the native RNA strand for sequencing instead of converting it to synthetic cDNA copies. By using direct RNA sequencing on the Helicos platform, the original RNA molecule is sequenced while maintaining its native state, thus preserving epigenetic information that would be lost in PCR-based methods. The method takes out the RNA-to-cDNA conversion step that causes information loss.
Solution Approach 2:
Instead of the conventional approach of converting RNA to cDNA for sequencing, the invention inverts the process by directly sequencing the native RNA strand. This inversion eliminates the need for reverse transcription and PCR amplification, thereby preserving the original epigenetic information present in the RNA molecule.
2Quantity of substance
If PCR amplification is used in library preparation, then sufficient material for sequencing is obtained, but bias is introduced and certain RNA species are over-amplified or lost
Solution Approach 1:
The invention removes PCR amplification from the library preparation workflow entirely. By using direct RNA sequencing, sufficient native RNA material can be loaded directly onto the sequencing platform without requiring amplification, thus eliminating the bias introduced by differential amplification efficiencies of different RNA species.
Solution Approach 2:
The invention performs preliminary enrichment of the RNA sample through selective capture methods before sequencing, ensuring sufficient material is available for direct sequencing without requiring PCR amplification. This preliminary action maintains the native state of RNA molecules while obtaining adequate quantities for sequencing.
3Productivity
If short-read sequencing is used, then sequencing throughput is high, but alternative splicing and splice variants cannot be captured
Solution Approach 1:
The invention segments the transcriptome analysis into full-length individual RNA molecule sequencing events. Each RNA molecule is sequenced in its entirety rather than being fragmented into short reads, allowing complete capture of splice variants and alternative splicing patterns while maintaining high throughput through parallel processing of multiple molecules.
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
Enables direct RNA sequencing of all RNA species, including those without poly-A tails, preserving epigenetic information and capturing full-length transcripts.
Implementation Method 1
modifying the RNA polynucleotide by annealing and ligating a polynucleotide comprising a 3′ terminal random multimer segment and having a stem-loop form
Implementation Method 2
contacting the modified RNA polynucleotide obtained in step (v) with a transmembrane pore such that the RNA translocase controls the movement of the RNA polynucleotide through the transmembrane pore
Data Source
AI summary
A method includes (i) providing an RNA polynucleotide; (ii) modifying the RNA polynucleotide by annealing and ligating a polynucleotide comprising a 3′ terminal random multimer segment and having a stem-loop form; (iii) optionally performing a reverse transcription of the RNA polynucleotide; (iv) cleaving the stem-loop segment of the annealed polynucleotide to yield a 3′ A overhang; (v) connecting an adaptor polynucleotide complex associated with an RNA translocase enzyme and at least one cholesterol tether segment to the polynucleotide obtained in step (iv); (vi) contacting the modified RNA polynucleotide obtained in step (v) with a transmembrane pore such that the RNA translocase controls the movement of the RNA polynucleotide through the transmembrane pore and the cholesterol tether anchors the RNA polynucleotide in the vicinity of the transmembrane pore; and (vii) taking one or more measurements during the movement of the RNA polynucleotide through the transmembrane pore. Other features are also disclosed.