cDNA Library Preparation via RNA Normalization and Barcoding
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current high-throughput sequencing methods for bulk RNA samples are hindered by high costs and complexity due to inefficient RNA extraction, normalization, and barcoding processes, which lead to uneven sequencing read distribution and increased experimental effort.
Innovation Solution
Integrating RNA normalization, barcoding, and reverse transcription into a single reaction step using biotinylated and barcoded oligo-dT sequences with streptavidin magnetic beads to normalize and pool RNA samples, ensuring uniform contribution of each sample in the sequencing library.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If standard RNA library preparation methods are used, then sequencing can be performed, but the cost and complexity are high due to separate processing of each sample
Solution Approach 1:
The patent combines multiple separate processing steps (RNA extraction, normalization, barcoding, and reverse transcription) into a single integrated reaction. Multiple samples are processed simultaneously in one tube with pooled RNA, eliminating the need for separate handling of each sample and reducing both time and operational complexity.
Solution Approach 2:
The patent employs universal adapters and a single barcoding strategy that works across all samples simultaneously. The barcoded oligo-dT primers serve multiple functions: they capture poly-A tails, incorporate sample-specific barcodes, and enable subsequent PCR amplification and sequencing, replacing multiple specialized reagents and steps.
2Measurement precision
If separate processing of each sample is performed, then individual sample quality can be controlled, but the time and cost increase significantly
Solution Approach 1:
The patent pools RNA from multiple samples into a single reaction tube for simultaneous processing. This maintains measurement accuracy through uniform reverse transcription conditions while reducing total processing time by eliminating sequential handling of individual samples.
Solution Approach 2:
The patent uses sample-specific barcodes that act as molecular copies or tags for each original sample within the pooled mixture. These barcodes allow computational separation and quantification of each sample's contribution to the pooled sequencing data, preserving individual sample information while enabling bulk processing.
3Productivity
If early multiplexing with barcoding is implemented, then cost and processing time are reduced, but uniform normalization of samples becomes challenging
Solution Approach 1:
The patent creates equipotential conditions for all samples by pooling equal amounts of RNA from each sample before processing. This ensures that each sample starts with equal representation in the reaction, and the use of universal adapters and barcoded primers maintains this equality throughout the amplification and sequencing process.
Solution Approach 2:
The patent incorporates sequencing depth information as feedback to normalize sample contributions. By sequencing the barcodes themselves, the method quantifies how much each sample contributed to the pool, allowing computational normalization to account for any variations in input amounts or amplification efficiency.
4Speed
If bulk RNA sequencing is performed without normalization, then processing is faster, but sequencing read distribution becomes uneven
Solution Approach 1:
The patent performs normalization as a preliminary action before sequencing by pooling equal amounts of RNA from each sample. This upfront equalization ensures that subsequent amplification and sequencing produce relatively uniform read distribution across samples, avoiding the need for corrective normalization later.
Solution Approach 2:
The patent uses barcode sequencing to provide feedback on each sample's contribution to the pool. This information is used to computationally normalize the data, adjusting for any unevenness in read distribution that arises during the rapid bulk processing, thereby maintaining precision despite speed optimizations.
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 significantly reduces experimental complexity and costs, achieving uniform sequencing read distribution and efficient high-throughput RNA sequencing by normalizing RNA samples before pooling, thereby enhancing the cost-effectiveness and accuracy of bulk RNA sequencing.
Implementation Method 1
Contacting separately for each sample, the obtained mixture with streptavidin magnetic beads at a pre-defined concentration
Implementation Method 2
Incubating separately each sample with reverse transcription enzyme (RT) under reverse transcription reaction conditions
Data Source
AI summary
The invention relates to methods for the preparation of method of preparation of cDNA library based on one or many RNA samples useful for efficient RNA sequencing and uses thereof. The invention further relates to related tools and kits useful in said method.