Control Sequence Monitoring for Sequencing Phasing Errors
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Solution Overview
Problem
Nucleic acid sequencing technologies face challenges in accurately detecting sequencing errors and noise accumulation due to incomplete cleavage of fluorophores or reversible terminators, leading to phasing issues in sequencing reactions.
Innovation Solution
Incorporation of a control sequence with a known sequence and a labeled control sequencing primer allows for real-time monitoring of nucleic acid sequencing reactions by comparing detected signals to expected signals, enabling error detection and correction through cycle-by-cycle phasing corrections and algorithm adjustments.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If sequencing reactions are performed without control sequences, then sequencing throughput is maintained, but noise accumulation and sequencing errors increase due to incomplete cleavage of fluorophores or reversible terminators
Solution Approach 1:
A control sequence with known sequence is introduced as an intermediary element alongside the target sequence. This control sequence serves as a reference standard that enables real-time monitoring and correction of sequencing reactions, allowing detection of noise accumulation and phasing errors without interfering with the primary sequencing function
Solution Approach 2:
The control sequence enables feedback mechanisms by providing expected signal patterns that can be compared against actual sequencing signals. This allows cycle-by-cycle monitoring of reaction efficiency and cleavage completeness, with the ability to adjust base-calling algorithms dynamically to maintain accuracy
2Measurement precision
If control sequences are incorporated for real-time monitoring, then sequencing error detection improves, but the complexity of the sequencing reaction increases
Solution Approach 1:
The control sequence acts as a copied reference version of the target sequence with known characteristics. By sequencing this known copy alongside the unknown target, the system can compare expected versus actual signals to detect errors, effectively using duplication for quality control purposes
3Reliability
If cycle-by-cycle phasing corrections are implemented, then phasing errors are reduced, but computational requirements and processing time increase
Solution Approach 1:
The control sequence provides pre-established expected signal patterns that can be used for immediate comparison during the sequencing reaction. This preliminary reference framework enables real-time phasing corrections without requiring extensive post-sequencing computational analysis
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
Enhances the accuracy of nucleic acid sequencing by identifying and correcting errors and noise, improving the fidelity of sequencing results and reducing chromatic blur and phasing errors.
Implementation Method 1
a control sequencing primer labeled with a first fluorescent dye, wherein the control sequencing primer binds adjacent to the control sequence
Data Source
AI summary
The present disclosure relates in some aspects to methods, systems, and kits for nucleic acid sequencing using a control sequencing primer to perform a plurality of cycles of a nucleic acid sequencing reaction of a control sequence comprising a known sequence. In some cases, the control sequencing primer is detected to identify the location of a plurality of copies of the control sequence. In certain embodiments, for a cycle of the plurality of cycles at the identified location, the method comprises comparing a signal from the nucleic acid sequencing reaction to an expected signal from the known sequence, thereby determining noise in the signal and/or identifying an error in the nucleic acid sequencing reaction.