Unbiased cDNA Library Construction via Direct RNA Sequencing

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Solution Overview

Problem

Current methods for preparing cDNA libraries for RNA sequencing are biased and inefficient, particularly in capturing all RNA species and preserving their relative abundances, leading to incomplete or inaccurate sequencing results due to the loss of 5' and 3' end sequences and poly(A) tail information.

Innovation Solution

A method involving the ligation of DNA adaptors to RNA molecules, followed by reverse transcription, circularization, and amplification under optimized conditions to minimize bias and ensure comprehensive sequencing, including the use of pre-adenylated adaptors, SuperScript III reverse transcriptase, and betaine for efficient circularization, allows for the accurate mapping of 5' and 3' ends and poly(A) tail lengths.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Quantity of substance

If RNA samples are selectively depleted of non-coding RNAs before preparing samples for sequencing, then the abundance of mRNA transcripts is improved, but bias is introduced that varies by method and prevents comparison between libraries prepared using different methods

Engineering Contradiction:

Improveabundance of mRNA transcriptsVSAvoidcomparability between libraries

Core Design Contradiction:

Quantity of substanceVSReliability

Solution Approach 1:

The patent changes the fundamental parameter of library preparation by using direct RNA sequencing instead of cDNA conversion, eliminating method-specific biases while maintaining mRNA abundance through selective capture of polyadenylated transcripts

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent extracts only the essential function of mRNA enrichment (poly(A) selection) while removing the problematic cDNA conversion step, allowing direct sequencing of RNA molecules to maintain comparability across different samples and methods

Inventive Principle:

Principle #2Taking out (Extraction)

2Productivity

If RNA is fragmented to enable sequencing on current platforms, then sequencing is made possible, but important information such as 5' and 3' end sequences and poly(A) tail length is lost

Engineering Contradiction:

Improvesequencing capabilityVSAvoid5' and 3' end sequences

Core Design Contradiction:

ProductivityVSLoss of information

Solution Approach 1:

The patent performs preliminary actions by capturing the full-length RNA molecule with adaptor ligation before sequencing, preserving the 5' and 3' ends and poly(A) tail information without requiring fragmentation that would destroy these critical structural features

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The patent transitions from the traditional fragmented 1D sequencing approach to preserving the complete 3D structure of full-length RNA molecules, allowing simultaneous observation of 5' end, 3' end, and poly(A) tail in their native spatial relationship

Inventive Principle:

Principle #17Another dimension (Dimensionality change)

3Productivity

If standard cDNA library preparation methods are used, then sequencing libraries can be generated, but bias is introduced and 5' and 3' end mapping accuracy is reduced

Engineering Contradiction:

Improvelibrary generation efficiencyVSAvoid5' and 3' end mapping accuracy

Core Design Contradiction:

ProductivityVSMeasurement precision

Solution Approach 1:

The patent introduces an RNA adaptor as an intermediary molecule that bridges the RNA molecule and sequencing platform, allowing direct RNA sequencing while preserving end accuracy without the bias-introducing cDNA conversion step

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent replaces the biochemical conversion mechanism (reverse transcription to cDNA) with a direct physical sequencing approach, eliminating the enzymatic biases inherent in cDNA library preparation while maintaining library generation efficiency

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

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 generates robust, unbiased cDNA libraries that provide uniform coverage across the transcriptome, enabling reliable sequencing of all RNA types, including mRNAs, miRNAs, and tRNAs, with improved recovery and minimal bias, even from small RNA samples.

Implementation Method 1

ligating a DNA adaptor to the 3' end of the RNA molecules, wherein the ligating is performed under conditions that optimize the ligation reaction

Methodology Applied

Scientific EffectLigation: Chemical Bonding

Implementation Method 2

reverse transcribing the RNA molecules using a unique DNA primer under conditions that optimize the reverse transcription reaction to produce single-stranded cDNA molecules

Methodology Applied

Scientific EffectReverse transcription: Enzyme