Molecular Tagging for Low-Abundance Variant Detection
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Solution Overview
Problem
Current methods for detecting genetic variants in nucleic acid samples face challenges in accurately identifying low-abundance variants amidst a mixture of target and non-target sequences, often due to high error rates in sequencing data.
Innovation Solution
The method employs a molecular tagging procedure where polynucleotides are appended with oligonucleotide tags, allowing for the formation of tagged amplicons which are then sequenced. This approach enables the detection of target polynucleotides at low abundance levels by reducing sequencing errors through error-corrected sequencing data generation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional sequencing methods are used to detect genetic variants, then the process is simple and fast, but the error rate is high and low-abundance variants cannot be accurately identified
Solution Approach 1:
The patent segments the sequencing process by introducing molecular tags that divide the population of polynucleotides into distinct groups. Each tag identifies a specific subset of molecules, allowing error correction through comparison of multiple tagged sequences. This segmentation enables accurate detection of low-abundance variants by tracking them through their unique tags throughout the sequencing process.
Solution Approach 2:
The patent implements feedback mechanisms through the molecular tagging system where sequencing reads are compared against reference tags and each other. The system uses the information from tagged sequences to correct errors in individual reads, providing feedback that improves overall sequencing accuracy. This feedback loop is particularly important for identifying low-abundance variants that may be obscured by sequencing errors.
2Measurement precision
If molecular tagging with multiple steps is employed, then sequencing error rate is reduced and low-abundance variants are detected accurately, but the process complexity increases
Solution Approach 1:
The patent merges multiple functions into the molecular tagging step. The tags simultaneously serve as identifiers for molecular families, primers for amplification, and references for error correction. By combining these functions into a single tagging operation, the patent reduces the need for separate steps while maintaining high accuracy for detecting low-abundance variants.
Solution Approach 2:
The molecular tags designed in the patent serve multiple purposes: they act as sequence identifiers, amplification primers, and error correction references. This multi-functionality reduces the overall process complexity by eliminating the need for separate operations for each function, while still enabling accurate detection of low-abundance genetic variants.
3Reliability
If conventional sequencing is used without molecular tags, then the method is simpler, but sequencing errors cannot be corrected and low-abundance DNA molecules are missed
Solution Approach 1:
The patent applies preliminary action by attaching molecular tags to polynucleotides before sequencing occurs. This pre-tagging step establishes the error correction references and family identifiers in advance, enabling error correction and accurate detection of low-abundance variants during the sequencing process. The preliminary tagging action pays off by providing built-in correction mechanisms that simplify the overall workflow.
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 method effectively detects and identifies genetic variants present at low abundance (0.05-5%) in nucleic acid samples, achieving high sensitivity and specificity by reducing sequencing errors and accurately confirming the presence of low-abundance DNA and RNA molecules.
Implementation Method 1
The tags are appended to the polynucleotides by primer extension or ligation
Implementation Method 2
The tags are appended to the polynucleotides by primer extension or ligation
Implementation Method 3
The tagged polynucleotides are amplified to generate tagged amplicons
Implementation Method 4
Sequencing reads are generated by sequencing the tagged amplicons
Implementation Method 5
The molecular tags enable error correction by allowing identification and removal of erroneous sequencing reads
Data Source
AI summary
In some embodiments, the disclosure relates generally to methods, as well as related systems, compositions, kits, apparatuses and computer-readable media, comprising a multiplex molecular tagging procedure that employs a plurality of tags that are appended to a plurality of polynucleotides. The tags have characteristics, including a sequence, length and/or detectable moiety, or any other characteristic, that uniquely identifies the polynucleotide molecule to which it is appended, and permits tracking individual tagged molecules in a mixture of tagged molecules. For example, the tag having a unique tag sequence, can uniquely identify an individual polynucleotide to which it is appended, and distinguish the individual polynucleotide from other tagged polynucleotides in a mixture. In some embodiments, the multiplex molecular tagging procedure can be used for generating error-corrected sequencing data and for detecting a target polynucleotide which is present at low abundance in a nucleic acid sample.