Selective cDNA Amplification for Depleting Uninformative RNA
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Solution Overview
Problem
Current RNA sequencing methods are hindered by the abundance of uninformative transcripts like rRNA and globin mRNA, which introduce 'noise' and require high RNA input and multiple time-consuming steps for depletion, especially in traditional and post-library construction techniques.
Innovation Solution
The use of sequence-specific primers during reverse transcription that target undesired RNA sequences, incorporating nucleotide analogs to prevent amplification, allowing selective amplification of desired RNA sequences, such as mRNA, through methods like SPIA, without subtractive hybridization or enzymatic degradation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If subtractive hybridization or enzymatic degradation methods are used to deplete rRNA prior to library construction, then rRNA depletion is achieved, but the process becomes time consuming and requires high RNA input
Solution Approach 1:
The patent incorporates depletion capability directly into the reverse transcription step by using sequence-specific primers that target both desired and undesired RNA sequences. This preliminary action integrates depletion and library construction into a single workflow, eliminating time-consuming separate depletion steps and reducing overall processing time while maintaining low RNA input requirements.
Solution Approach 2:
The invention merges the depletion function with the reverse transcription and library construction processes. By using sequence-specific primers during reverse transcription that incorporate nucleotide analogs into cDNA from undesired sequences, the method combines multiple functions (depletion, reverse transcription, and library preparation) into an integrated workflow, reducing both time and RNA input requirements.
2Quantity of substance
If multiple steps of enzymatic treatment and purification are used for post-library construction depletion, then non-desired sequences are removed, but the process becomes time consuming
Solution Approach 1:
The patent performs depletion action during the reverse transcription step itself, before library construction is complete. By incorporating nucleotide analogs into cDNA from undesired sequences during reverse transcription, the depletion is achieved preliminarily, eliminating the need for multiple post-library construction enzymatic treatment and purification steps.
Solution Approach 2:
The invention extracts or removes the need for multiple post-library construction depletion steps by integrating the depletion function into the reverse transcription process. The sequence-specific primers with nucleotide analogs selectively mark undesired sequences during reverse transcription, effectively extracting the depletion function from the later library construction workflow.
3Quantity of substance
If sequence-specific primers with nucleotide analogs are used during reverse transcription, then undesired sequences are selectively depleted, but the method requires development of new primer designs
Solution Approach 1:
The patent applies local quality by incorporating nucleotide analogs specifically at certain positions within the sequence-specific primers that target undesired sequences. This localized modification allows selective depletion of undesired sequences while maintaining standard primer design for desired sequences, reducing overall design complexity while achieving effective depletion.
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 enables efficient depletion of non-desired sequences, reducing the need for large RNA input and time-consuming steps, while maintaining accurate representation of desired sequences, thus lowering costs and improving sequencing efficiency.
Implementation Method 1
annealing a sequence-specific amplification-suppressing primer to a non-desired RNA fragment in a single-stranded nucleic acid library; annealing an amplification-supporting primer to a desired RNA fragment in the single-stranded nucleic acid library
Implementation Method 2
transcribing the non-desired RNA fragment and the desired RNA fragment to generate a non-desired cDNA molecule comprising an amplification-suppressing sequence and a desired cDNA molecule comprising a amplification-supporting sequence
Implementation Method 3
The inclusion of an enzymatic treatment step prior to PCR amplification of the transcribed cDNA library results in cleavage or other enzymatic processing of the nucleotide analogs and prevents their subsequent amplification
Data Source
AI summary
The use of different primer sets in reverse transcription incorporates tags allowing for the selective amplification of cDNA transcribed using the different primers. Primers targeting non-desired RNA sequences such as ribosomal RNA can be used to prevent subsequent amplification of cDNA transcribed from those non-coding fragments. Accordingly effective depletion of non-desired sequences after cDNA amplification can be achieved. Systems and methods of the invention have applications in whole-transcriptome analysis. Non-coding sequence targeting primers can include nucleotide analogs that, when enzymatically processed, prevent subsequent amplification. Library preparation can include single primer isothermal amplification (SPIA) techniques wherein an RNA sequence required for SPIA is included in random primers but is absent from primers targeting non-coding RNA.