Integrated DNA and RNA Library Preparation Without Physical Separation
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Solution Overview
Problem
Traditional methods for paired DNA and RNA profiling require separate workflows for DNA and RNA samples, leading to less correlative data due to sample heterogeneity and increased time and effort, with existing integrated methods primarily designed for single-cell applications.
Innovation Solution
A method for preparing DNA and cDNA libraries that integrates DNA and RNA profiling without physical separation, using unique molecular identifiers (UMIs) and template switching oligonucleotides (TSOs) to tag DNA and RNA molecules, followed by targeted enrichment and differential PCR amplification to generate separate libraries.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If separate workflows are used for DNA and RNA profiling, then sample preparation can be performed independently, but data correlation is reduced due to sample heterogeneity and time consumption increases
Solution Approach 1:
The patent merges DNA and RNA profiling workflows into a single integrated process where both nucleic acid types are processed simultaneously from the same cell population. The method uses universal primers and a unified library preparation protocol that handles both DNA and RNA molecules together, eliminating the need for separate parallel workflows and ensuring data from the same biological sample.
Solution Approach 2:
The patent employs universal primers and reagents that can bind to and amplify both DNA and RNA molecules. The library preparation kit contains components that function for both nucleic acid types, allowing a single workflow to serve multiple purposes and process different molecule types without requiring separate specialized protocols.
2Ease of manufacture
If separate workflows are used for DNA and RNA profiling, then each can be optimized independently, but overall process complexity and effort increase due to doubled workload
Solution Approach 1:
The patent combines multiple workflow steps into a unified process where DNA and RNA library preparation share common reagents, enzymes, and protocol steps. The kit provides a single set of instructions and materials that handle both nucleic acid types simultaneously, reducing the overall number of separate operations required.
Solution Approach 2:
The patent uses universal adapters, primers, and enzymes that work for both DNA and RNA processing. This multi-functional approach allows a single library preparation protocol to generate both DNA and RNA libraries, reducing the need for multiple specialized workflows and decreasing overall process complexity.
3Quantity of substance
If integrated DNA and RNA profiling is performed, then sample consumption is reduced and workflow is simplified, but sequencing capacity allocation becomes more challenging
Solution Approach 1:
The patent uses unique molecular identifiers (UMIs) and sample-specific barcodes that are incorporated into both DNA and RNA libraries during the unified workflow. These identifiers allow computational separation and independent analysis of DNA and RNA data after sequencing, enabling flexible allocation of sequencing capacity while maintaining the ability to analyze each nucleic acid type separately.
Solution Approach 2:
The patent employs universal adapters and linkers as intermediary elements that connect both DNA and RNA molecules to the sequencing platform. These adapters contain built-in identifiers that allow the sequencing data to be sorted and analyzed by nucleic acid type, providing flexibility in sequencing capacity allocation while maintaining integrated sample processing.
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 reduces sample consumption, simplifies the experimental process, and enhances the accuracy and flexibility of sequencing analysis, allowing for flexible allocation of sequencing capacity based on specific needs.
Implementation Method 1
ligating a DNA tag to an end of a DNA molecule in a sample, wherein the DNA tag comprises a unique molecular identifier (UMI) and a DNA identifier; and performing reverse transcription of a RNA molecule in the sample in the presence of a RNA tag, wherein the RNA tag comprises a RNA identifier, a UMI
Implementation Method 2
performing reverse transcription of a RNA molecule in the sample in the presence of a RNA tag
Implementation Method 3
amplifying the tagged DNA and tagged cDNA for enrichment with a set of gene specific primers
Implementation Method 4
ligating a DNA tag to an end of a DNA molecule in a sample
Data Source
AI summary
The invention relates to integrated DNA and RNA library preparations and methods of making and uses thereof. The methods do not require physical separation of DNA and RNA. The methods output two separate libraries from DNA and RNA, respectively, which helps flexible manipulation on downstream sequencing platform.