Single-Flow-Cell Sequencing With Phi X 174 Complexity Control
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Whole genome sequencing remains costly and inefficient, with biased sequencing applications being too expensive for single runs on high-output sequencers and unbiased sequencing runs being cost-effective but lacking complexity, leading to sequencing challenges and increased costs.
Innovation Solution
A method involving a single flow cell for sequencing a pooled nucleic acid sample comprising both biased and unbiased libraries, using a well-characterized control genome like Phi X 174 bacteriophage to maintain complexity and recover lost sequencing capacity, allowing parallel assays and efficient use of high-output instruments.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If biased sequencing is performed to enhance confidence in specific genomic regions, then sequencing accuracy for disease assessment is improved, but sample complexity is reduced making base calling difficult
Solution Approach 1:
The patent introduces Phi X 174 bacteriophage control genome as an intermediary substance that provides necessary sequence diversity without interfering with the biased sequencing targets. This control sample acts as a mediator that maintains overall library complexity while allowing biased sequencing to proceed, enabling the sequencer to properly call bases by having diverse sequences present in the run.
2Measurement precision
If Phi X 174 bacteriophage control genome is added to maintain sequencing complexity, then base calling accuracy is improved, but sequencing capacity is reduced due to reads consumed by control
Solution Approach 1:
The patent combines multiple sequencing objectives into a single integrated run by pooling biased sequencing libraries with unbiased control libraries and Phi X 174 control genomes. This merging allows the system to achieve both biased sequencing accuracy and sufficient complexity without requiring separate dedicated control runs, thereby maximizing the utilization of available sequencing capacity.
Solution Approach 2:
The control libraries serve multiple functions simultaneously: they maintain sequence diversity for accurate base calling, provide complexity for the sequencer algorithm, and enable quality control metrics. This multi-functionality eliminates the need for separate control runs, thereby recovering sequencing capacity that would otherwise be lost.
3Productivity
If high-output sequencers are used for biased sequencing applications, then throughput is improved, but cost per run increases requiring multiplexing of large numbers of specimens
Solution Approach 1:
The patent changes the compositional parameters of the sequencing library by incorporating control genomes and control libraries at specific percentage ranges (e.g., 5-20% control material). This parameter optimization allows high-output sequencers to operate efficiently with smaller numbers of biased sequencing specimens while maintaining necessary complexity and accuracy, thereby reducing the multiplexing burden.
4Productivity
If unbiased sequencing is performed to maximize sequencing capacity utilization, then cost per base is reduced, but complexity is insufficient leading to sequencing challenges
Solution Approach 1:
The patent creates a composite sequencing library that combines different types of nucleic acid materials: biased sequencing targets, unbiased control libraries, and Phi X 174 control genomes. This composite structure provides both the high capacity utilization of unbiased sequencing and the necessary sequence diversity for accurate base calling, resolving the contradiction between efficiency and complexity.
Data Source
AI summary
The present disclosure provides methods and systems for nucleic acid sequencing. Such systems and methods may use a single flow cell to perform unbiased and/or biased sequencing to generate libraries of nucleic acid molecules. An aspect of the present disclosure provides a method for increasing complexity of a sample for sequencing, the method comprising: providing a first nucleic acid sample having a first degree of complexity that differs from a desired degree of complexity; providing a second nucleic acid sample having a second degree of complexity that differs from the first degree of complexity and that differs from the desired degree of complexity; pooling at least a portion of the first nucleic acid sample and at least a portion of the second nucleic acid sample, thereby generating a pooled nucleic acid sample having the desired degree of complexity; and sequencing at least a portion of the pooled nucleic acid sample.