Chimeric Artefact Detection Using Dual-End Identifier Sequences
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Solution Overview
Problem
Current methods for detecting chimeric artefact polynucleotides during amplification of mixed samples, particularly in long-read sequencing, are inadequate due to high error rates and the formation of chimeric molecules, which complicates the identification of true Unique Molecular Identifiers (UMIs).
Innovation Solution
A method involving the use of capture polynucleotides with identifier sequences and template switch oligonucleotides (TSOs) to generate libraries flanked by unique identifier sequences, allowing for the detection of chimeric artefacts by identifying underrepresented or mismatched pairings of these sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Length of moving object
If long-read sequencing technologies are used to sequence native RNA, then sequencing resolution and read length are improved, but error rates increase making it difficult to identify true UMI sequences
Solution Approach 1:
The UMI sequence is divided into multiple discrete nucleotide blocks (e.g., 3-5 blocks of 2-4 nucleotides each). Each block is independently sequenced and can be individually error-corrected, allowing the true UMI to be reconstructed even when individual blocks contain sequencing errors. This segmentation enables reliable UMI identification despite the high error rates of long-read sequencing technologies.
Solution Approach 2:
The method performs preliminary error correction by comparing each discrete nucleotide block against a pool of known UMI sequences before final UMI assignment. This preliminary action of blocking and comparing allows the system to anticipate and correct sequencing errors, distinguishing true UMIs from artefactual ones before downstream analysis.
2Quantity of substance
If PCR-based library methods are used to overcome sample size limitations, then sensitivity is improved, but PCR amplification bias and chimeric molecule formation increase
Solution Approach 1:
By segmenting the UMI into discrete nucleotide blocks that are flanking the cDNA molecule, the method creates unique molecular fingerprints that can track individual original RNA molecules through PCR amplification. This enables computational identification and removal of chimeric molecules that result from PCR artefacts, as they will have inconsistent UMI block pairings compared to true amplification products.
Solution Approach 2:
The method uses the paired discrete UMI blocks as feedback signals to verify the authenticity of amplified molecules. During data analysis, the system checks whether the UMI block pairings are consistent with the original capture polynucleotide and TSO pairings, providing feedback that allows differentiation between true PCR amplification products and chimeric artefacts.
3Measurement precision
If discrete nucleotide blocks with at least two nucleotide substitutions are used in identifier sequences, then sequencing error detection is improved, but library complexity and synthesis requirements increase
Solution Approach 1:
The method applies local quality control by ensuring that each discrete nucleotide block within the UMI has sufficient sequence diversity (at least two nucleotide substitutions from other blocks). This local differentiation within blocks provides error-detecting capability without requiring the entire library to be overly complex, as each block independently contributes to error correction.
Solution Approach 2:
The patent specifies particular parameters for the nucleotide blocks (2-4 nucleotides per block, 3-5 blocks per UMI, minimum two nucleotide substitutions between blocks) to optimize the balance between error detection capability and library complexity. These parameter changes create a UMI structure that is sufficiently complex to detect errors but manageable for synthesis and sequencing.
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
Effectively identifies and removes chimeric artefacts, improving the accuracy of sequencing by reducing PCR amplification bias and enhancing the reliability of UMIs, especially in long-read sequencing technologies.
Implementation Method 1
capturing RNA molecules of the sample on a set of capture polynucleotides
Implementation Method 2
performing reverse transcription of captured sample RNA molecules using a template switch reverse polymerase
Implementation Method 3
annealing a set of template switch oligonucleotides (TSOs) to the 3' end non-templated nucleotides
Implementation Method 4
amplifying the cDNA using the capture polynucleotide PCR handle sequences and TSO PCR handle sequences
Data Source
AI summary
The invention relates to methods for detecting chimeric artefact polynucleotides produced during amplification of a mixed sample of polynucleotide. The methods comprise adding identifier sequences to both ends of a sample polynucleotide. Also provided are arrays of annealed oligonucleotide strand pairs for providing a mixed pool of identifier sequences; kits and methods for producing a library of polynucleotides, or libraries of polynucleotides having identifier sequences at both ends of the polynucleotides; and arrays of template switch oligonucleotides.