RNA Tail Addition for Unified Short and Long RNA Sequencing
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Solution Overview
Problem
Current sequencing technologies, particularly those using Oxford Nanopore Technologies, do not allow for the simultaneous sequencing of short and long RNAs due to biochemical incompatibilities, necessitating separate library construction and sequencing, which is inefficient and costly.
Innovation Solution
A method involving the attachment of a polymeric nucleic acid tail to both coding and non-coding RNAs, enabling simultaneous sequencing of short and long RNAs in a single library using nanopore sequencing, with accompanying bioinformatics workflows to differentiate and map sequence information.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If separate library construction protocols are used for coding and non-coding RNAs, then sequencing accuracy is maintained, but device complexity and operational cost increase
Solution Approach 1:
The patent merges the sequencing of coding and non-coding RNAs into a single library construction protocol. The method uses a universal polyadenylation step that works for both mRNA and non-coding RNAs, followed by a single round of PCR amplification and sequencing, eliminating the need for separate library preparations while maintaining sequencing accuracy through optimized reaction conditions
Solution Approach 2:
The patent creates a universal library construction protocol that handles multiple RNA types (coding and non-coding) simultaneously. The polyadenylation step and subsequent sequencing protocol are designed to be multi-functional, accommodating RNAs of different lengths and types without requiring protocol modifications, thereby reducing operational complexity
2Measurement precision
If separate sequencing runs are performed for short and long RNAs, then sequencing quality is maintained, but productivity decreases
Solution Approach 1:
The patent combines short and long RNA sequencing into a single experimental run using a unified library construction approach. The method processes RNAs across a broad size range (from 20 nt to several kb) in the same reaction mixture and sequencing lane, maximizing throughput while maintaining quality through optimized polymerase and adapter designs
Solution Approach 2:
The patent optimizes reaction parameters (polymerase selection, extension conditions, adapter concentrations) to accommodate a wide range of RNA lengths simultaneously. By adjusting these parameters, the protocol achieves high-quality sequencing for both short non-coding RNAs and long coding RNAs in the same run, eliminating the need for separate size-based sequencing experiments
3Length of stationary object
If conventional nanopore sequencing protocols are used, then long RNA sequencing is achieved, but short RNA detection is lost
Solution Approach 1:
The patent applies preliminary polyadenylation to all RNAs in the sample before library construction. This preliminary step adds uniform poly-A tails to both short and long RNAs, enabling them to be captured by poly-T primers during reverse transcription. This pre-treatment ensures that short RNAs are properly prepared for nanopore sequencing without losing detection sensitivity
Solution Approach 2:
The patent uses poly-A tails as an intermediary element that bridges short and long RNAs for simultaneous processing. The poly-A tail serves as a universal handle that allows both short non-coding RNAs and long coding RNAs to be captured, amplified, and sequenced by the same nanopore protocol, effectively mediating their co-detection despite size differences
Data Source
AI summary
Provided herein are methods of simultaneously detecting coding and non-coding ribonucleic acids (RNAs) in a sample that include attaching a polymeric nucleic acid tail to a plurality of the non-coding linear RNAs in the sample to produce a population of RNA molecules that each comprise polymeric nucleic acid tails. The methods also include obtaining sequence information from the population of RNA molecules that each comprise polymeric nucleic acid tails and/or from derivative nucleic acid molecules thereof irrespective of lengths of the RNA molecules or the derivative nucleic acid molecules thereof using a long-read sequencing technique. Related methods, systems, and computer readable media are also provided.