Nucleic Acid Sequencing Phasing Error Reduction via Periodic Cleavage
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Solution Overview
Problem
Nucleic acid sequencing methods, particularly sequencing-by-synthesis, face challenges in reducing phasing errors that arise from loss of phase synchrony, leading to inaccuracies in base calling and difficulties in handling large sequencing data volumes.
Innovation Solution
A method for nucleic acid sequencing involves disposing template nucleic acid molecules on a sensor array, advancing nucleotide species with sequencing primers and polymerase bound, measuring signals from nucleotide incorporations, and exposing the templates to a cleaving reagent to remove labeling reagents. This process is repeated for different orders of nucleotide species to minimize phase errors.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If sequencing-by-synthesis is used to determine nucleotide order, then high throughput sequencing data can be generated, but phasing errors occur due to loss of phase synchrony leading to inaccurate base calls
Solution Approach 1:
The patent implements periodic cleavage cycles where a cleavage agent is applied at regular intervals to remove labels from incorporated nucleotides. This periodic action resets the phase of all template molecules, ensuring they remain synchronized throughout the sequencing process. The cycle repeats: incorporate labeled nucleotides, detect signals, apply cleavage agent to remove labels, then repeat with next nucleotide type. This periodic intervention prevents cumulative phase drift and maintains measurement precision across many sequencing cycles.
Solution Approach 2:
The patent discards the labels from incorporated nucleotides by applying a cleavage agent that removes the labeling reagents. This discarding of labels is essential because they interfere with subsequent detection cycles. By removing (discarding) the labels after their detection purpose is fulfilled, the system recovers the template molecules in a clean state, ready for the next round of nucleotide incorporation without phase interference from residual labels.
2Loss of information
If multiple nucleotide species are advanced over template molecules repeatedly, then complete sequencing data can be obtained, but phase synchrony is lost leading to phasing errors
Solution Approach 1:
The patent applies the cleavage agent periodically after each complete cycle of advancing all nucleotide species (A, T, C, G). This periodic reset ensures that even after multiple rounds of complete sequencing cycles, all template molecules remain in phase. The periodic intervention counteracts the natural drift that would occur from repeated incorporation and detection cycles, maintaining reliability throughout the complete sequencing process.
Solution Approach 2:
The system uses signal detection as feedback to monitor incorporation events. After detecting signals from incorporated labeled nucleotides, the system triggers the cleavage step. This feedback loop ensures that cleavage occurs at the optimal moment to maintain phase synchrony. The feedback mechanism allows the system to adapt to actual incorporation patterns while maintaining reliable phase coordination across all template molecules.
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
The method effectively reduces phasing errors, enhances the accuracy of nucleic acid sequencing, and improves the handling of large sequencing data volumes, particularly for long sequences and homopolymers.
Implementation Method 1
advancing one or more nucleotide species over the plurality of template nucleic acid molecules with the sequencing primer and the polymerase operably bound therewith
Implementation Method 2
measuring a signal generated by nucleotide incorporations resulting from advancing the one or more nucleotide species
Implementation Method 3
exposing the plurality of template nucleic acid molecules to a cleaving reagent subsequent to the advancing and measuring. The cleaving reagent removes labeling reagents attached to the one or more nucleotide species
Data Source
AI summary
A method for nucleic acid sequencing may include disposing a plurality of template nucleic acid molecules in a plurality of defined spaces disposed on a sensor array, at least some of the plurality of template nucleic acid molecules having a sequencing primer and a polymerase operably bound therewith; advancing one or more nucleotide species over the plurality of template nucleic acid molecules with the sequencing primer and the polymerase operably bound therewith; measuring a signal generated by nucleotide incorporations resulting from advancing the one or more nucleotide species; and exposing the plurality of template nucleic acid molecules to a cleaving reagent subsequent to the advancing and measuring. The cleaving reagent can remove labeling reagents attached to the one or more nucleotide species. The advancing and measuring steps can be performed for different orders of the one or more nucleotide species prior to a subsequent exposing of the plurality of template nucleic acid molecules to the cleaving reagent.