RNA Template Tagging and Amplification for Low-Input cfRNA Sequencing
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Solution Overview
Problem
Current methods for preparing sequencing libraries from cell-free RNA (cfRNA) are inefficient due to its low levels in test samples, typically present at 10 ng or less, which hinders effective analysis using next-generation sequencing (NGS).
Innovation Solution
A method involving the purification of RNA sequences, synthesis of complementary DNA (cDNA) strands with C-tailing, annealing of complementary template switching oligonucleotides, and subsequent ligation using strand-displacement reverse transcriptase to produce tagged and amplified RNA templates, allowing for the generation of sequencing libraries from low-input RNA samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If current protocols are used to prepare sequencing libraries from cell-free RNA samples, then the standard library preparation process can be followed, but the low levels of cfRNA (10 ng or less) result in insufficient material for effective NGS analysis
Solution Approach 1:
The method performs preliminary tagging of RNA molecules with unique identifiers before amplification. This preliminary action allows subsequent tracking and quantification of original RNA molecules, enabling effective analysis even when starting with very low amounts (10 ng or less) of cfRNA
Solution Approach 2:
The method creates multiple copies of the original RNA templates through amplification after tagging. By first tagging then amplifying, the system generates sufficient quantities of library material from low-input samples while preserving the ability to trace back to original molecules for accurate quantification
2Manufacturing precision
If multiple steps are performed separately for tagging and amplifying RNA templates, then each step can be optimized independently, but the process requires multiple reaction tubes and increases complexity
Solution Approach 1:
The method merges template tagging and amplification into a single unified reaction system. The strand-displacement reverse transcriptase performs both functions sequentially in one tube: first synthesizing cDNA from RNA templates, then using the C-tail and template switching oligonucleotide to enable amplification of tagged products, eliminating the need for separate reaction tubes
Solution Approach 2:
The strand-displacement reverse transcriptase enzyme serves multiple functions: it performs reverse transcription to synthesize cDNA, enables template switching by incorporating the template switching oligonucleotide, and facilitates amplification. This multi-functional approach reduces the number of reagents and reaction vessels needed while maintaining process optimization
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 the effective preparation of sequencing libraries from low-input RNA samples, enhancing the analysis of cfRNA through next-generation sequencing by amplifying and tagging RNA templates, thereby overcoming the limitations of low RNA levels.
Implementation Method 1
synthesizing first complementary DNA (cDNA) strands based on the RNA sequences
Implementation Method 2
the reverse transcriptase comprises reverse transcription and terminal transferase activities, to generate a plurality of DNA sequences complementary to the one or more RNA templates, and wherein the complementary DNA (cDNA) sequences further comprise a plurality of non-templated bases at the 3'-end of the cDNA sequences
Implementation Method 3
annealing a complementary template switching oligonucleotide to the C-tail of the cDNA
Implementation Method 4
ligating the complementary template switching oligonucleotide to the 5'-ends of the RNA sequences
Implementation Method 5
synthesizing a plurality of cDNA strands from the RNA templates using a strand- displacement reverse transcriptase
Data Source
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AI summary
Methods for preparing sequencing libraries based on a plurality of RNA molecules which are tagged and amplified by tagging the molecule with an oligonucleotide hybridising to a polyC tail introduced by the terminal transferase activity of the reverse transcriptase, e.g. MMLV RT, and ligating the oligonucleotide to the RNA molecule using a ligase, e.g. T4 RNA ligase, and producing cDNA molecules based on mRNA and strand displacing reverse transcriptases, e.g.g MMLV RT, and producing a second cDNA strand in order to produce a dsDNA library for sequencing. In some embodiments, the subject methods comprise sequencing at least a portion of a sequencing library to obtain sequencing data or sequence reads from a test sample (e.g., a biological sample from a subject).