Polynucleotide Quantification Accuracy via Unique Molecular Identifiers
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Solution Overview
Problem
Current methods for quantifying polynucleotides in mixed samples face challenges in accuracy and sensitivity, particularly with low copy numbers and high diversity, as they are prone to errors in PCR and sequencing, leading to over-estimation or under-estimation of target counts due to sequencing artifacts and assumptions of error-free processes.
Innovation Solution
The method involves attaching a unique molecular identifier (UMI) and a universal adapter sequence to target sequences during reverse transcription or primer extension, followed by amplification with universal primers, and using statistical filtering to correct for sequencing errors, ensuring accurate counting of polynucleotides by distinguishing between random errors and authentic sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional PCR and sequencing methods are used to quantify polynucleotides, then the process is simple and widely applicable, but accuracy deteriorates due to sequencing errors and PCR artifacts leading to over- or under-estimation of target counts
Solution Approach 1:
A unique molecular identifier (UMI) is introduced as an intermediary element that is attached to each target polynucleotide before amplification. This UMI serves as a mediator that allows tracking of individual target molecules through the PCR and sequencing process, enabling differentiation between true biological variation and artifacts introduced during amplification and sequencing.
Solution Approach 2:
The method creates multiple copies of each target polynucleotide while preserving the original UMI tag. By sequencing these amplified copies and grouping them by their shared UMI, the true count of original targets can be determined despite PCR amplification variations and sequencing errors in the copied regions.
2Measurement precision
If unique molecular identifiers (UMI) are used to improve quantification accuracy, then measurement precision improves, but device complexity increases due to additional labeling and processing steps
Solution Approach 1:
The UMI labeling step is merged with the adapter ligation step, where a single oligonucleotide contains both the UMI sequence and the adapter sequence required for subsequent PCR amplification. This combining of functions reduces the number of separate steps and reagents needed compared to adding UMI as a separate operation.
Solution Approach 2:
The adapter oligonucleotide serves multiple functions: it provides the UMI for tracking, contains the binding site for universal primers in PCR amplification, and enables subsequent sequencing. This multi-functionality reduces the need for separate specialized reagents and steps.
3Measurement precision
If sample dilution is performed to achieve accurate spectrophotometry measurement, then measurement precision improves for RNA quantitation, but sensitivity deteriorates for low copy number targets
Solution Approach 1:
The method replaces the mechanical/dilution-based approach of spectrophotometry with a molecular tagging approach using UMI. Instead of diluting samples to achieve accurate absorbance measurements, each molecule is individually tagged and counted through sequencing, enabling accurate quantification without dilution and maintaining sensitivity for low copy number targets.
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 enhances the accuracy and sensitivity of polynucleotide detection by reducing sequencing artifacts and correcting for errors, providing a more reliable quantification of diverse and low-copy number targets in complex genetic samples.
Implementation Method 1
attaching the UMI/universal adapter sequence to the target in a reverse transcription (RT) reaction at 50-60 degree Celsius for RNA targets
Implementation Method 2
amplifying, with universal primer, products with the universal primer binding site attached at both ends
Data Source
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AI summary
Disclosed is a method for improving the sensitivity and accuracy of quantitative detection of polynucleotides in a sample, such a clinical specimen, by a method that utilizes a two- or three-step process of tagging/labeling target molecules and adding an adapter sequence for adding a universal primer for efficient amplification of targets while decreasing target amplification bias. When combined with the step of statistically correcting for sequencing errors, the method can significantly increase the accuracy of quantitative detection of polynucleotides in a sample.