Sequencing Calibration Cycles Using Embedded Reference Sequences
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Solution Overview
Problem
Existing sequencing systems face errors and inefficiencies due to predetermined sequencing parameters that may not adapt to changes in sequencing machine environments, random failures in early genomic sequencing cycles, and resource consumption for calibration, leading to inaccurate nucleobase calls and reduced sequencing efficiency.
Innovation Solution
A calibration-sequencing system that incorporates short calibration sequences into library fragments or a sample-nucleotide slide to run calibration cycles, determining sequencing parameters through direct detection and adjustment based on known nucleobase calls, allowing for real-time adaptation to the sequencing device's current state.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If predetermined sequencing parameters are used for a sequencing machine, then initial sequencing can be performed, but sequencing accuracy deteriorates due to environmental changes and hardware drift over time
Solution Approach 1:
The system performs preliminary calibration by incorporating known calibration sequences (such as Phi-X) into the library fragments before actual sequencing. This preliminary action establishes baseline parameters that account for the specific sequencing machine's environmental conditions and hardware characteristics, thereby improving subsequent sequencing accuracy without sacrificing throughput
Solution Approach 2:
The system implements feedback mechanisms by continuously monitoring sequencing quality metrics and adjusting parameters based on the known calibration sequences. This feedback loop allows the system to compensate for hardware drift and environmental changes over time, maintaining high accuracy while preserving sequencing productivity
2Measurement precision
If genomic sequencing cycles are used for calibration, then sequencing parameters can be determined, but computing resources are consumed and sequencing efficiency is reduced
Solution Approach 1:
The system segments the sequencing process by dedicating specific library fragments (containing known calibration sequences) to calibration purposes while other fragments are used for actual sample sequencing. This segmentation allows calibration to occur in parallel with sample sequencing, eliminating the need to sacrifice entire sequencing runs for calibration and thereby reducing time loss while maintaining parameter accuracy
Solution Approach 2:
The system uses a partial approach by incorporating calibration sequences into only a subset of library fragments rather than using all fragments for calibration. This partial calibration action is sufficient to determine sequencing parameters accurately while preserving the majority of sequencing capacity for actual sample analysis, thus minimizing time loss
3Adaptability or versatility
If early genomic sequencing cycles are used to configure parameters, then initial calibration is achieved, but random failures introduce errors into subsequent cycles
Solution Approach 1:
The system performs preliminary calibration using known calibration sequences before actual sample sequencing begins. This preliminary action allows the system to configure parameters under controlled conditions with known outcomes, avoiding the random failures that occur during early sample sequencing cycles. The calibration parameters are then used as a stable foundation for subsequent reliable sequencing
Solution Approach 2:
The system prepares for potential failures by using robust calibration sequences with known properties that can tolerate variations in early sequencing conditions. This beforehand cushioning ensures that even if early cycles experience random failures (such as bubble formation or focus issues), the calibration parameters remain reliable and do not propagate errors into subsequent sequencing cycles
4Measurement precision
If calibration sequences are incorporated into library fragments, then device-specific parameter customization is achieved, but library preparation complexity increases
Solution Approach 1:
The system uses universal calibration sequences (such as Phi-X) that can be incorporated into library fragments using standard library preparation protocols. These calibration sequences serve multiple functions: they enable device-specific parameter customization, provide quality control benchmarks, and can be processed alongside sample DNA without requiring separate preparation workflows. This universality achieves device-specific accuracy while minimizing the increase in library preparation complexity
Data Source
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AI summary
This disclosure describes methods, non-transitory computer readable media, and systems that can introduce short calibration sequences into a sequencing device and run calibration cycles to adjust or otherwise determine a sequencing parameter corresponding to the sequencing device. For instance, the disclosed systems can detect a flow cell (or other sample-nucleotide slide) with calibration sequences incorporated into samples' library fragments or into a surface of the sample-nucleotide slide. By running one or more calibration cycles to incorporate nucleobases on oligonucleotides corresponding to calibration sequences and capture corresponding images for calibration sequences—separate from genomic sequencing cycles for sample genomic sequences—the disclosed systems can determine a sequencing parameter corresponding to the sequencing device.