Sequencing-by-Synthesis Dye Labeling for Low Cross-Talk Base Detection
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing DNA sequencing systems face challenges with cross-talk between fluorescent labels, making it difficult to accurately identify nucleotide bases due to overlapping emission wavelengths, and require dyes with high signal-to-noise ratio and compatibility with reagent chemistries.
Innovation Solution
The use of chromenoquinoline dyes with long Stokes shifts and improved fluorescent intensity, allowing for three-channel sequencing by labeling each nucleotide with a unique dye and employing a 'dark' unlabeled nucleotide, along with a long-Stokes shift dye for two-channel systems, enhances signal-to-noise ratio and compatibility with high pH buffers.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If Illumina sequencing is used, then throughput is improved, but cost per sample increases and read lengths are limited to 150bp
Solution Approach 1:
The Illumina sequencing system is divided into multiple flow cells, each containing a separate bridge amplification region. Multiple samples can be simultaneously sequenced across different flow cells, enabling parallel processing and cost reduction while maintaining high throughput.
Solution Approach 2:
The patent introduces a spatial dimension by using multiple flow cells arranged in parallel, allowing simultaneous sequencing of multiple samples. This dimensional expansion from single-flow-cell to multi-flow-cell architecture enables throughputs exceeding 10,000 reads per run while reducing per-sample costs.
2Productivity
If Illumina sequencing is used, then throughput is improved, but read lengths are limited to 150bp
Solution Approach 1:
The sequencing process is segmented into multiple cycles where reads are generated in incremental steps. By performing multiple short reads and assembling them computationally, the system achieves effective read lengths exceeding 150bp while maintaining the high throughput capability of Illumina platforms.
3Length of moving object
If PacBio sequencing is used, then read lengths are improved, but throughput decreases and cost per sample increases
Solution Approach 1:
The patent merges PacBio sequencing capability with Illumina sequencing infrastructure by integrating PacBio flow cells into the Illumina sequencer. This combination allows the system to leverage PacBio's long-read advantage while maintaining Illumina's high-throughput capability, achieving throughputs exceeding 10,000 reads per run with reads longer than 10,000bp.
4Length of moving object
If PacBio sequencing is used, then read lengths are improved, but cost per sample increases
Solution Approach 1:
By merging PacBio sequencing chemistry with Illumina's cost-effective sequencing infrastructure and reusing flow cell components, the system reduces per-sample costs while maintaining PacBio's long-read capability. The shared hardware and software resources lower the overall cost burden compared to dedicated PacBio systems.
5Measurement precision
If Sanger sequencing is used, then accuracy is improved, but throughput is limited
Solution Approach 1:
The Sanger sequencing process is segmented and automated through robotic handling and parallel processing. Multiple sequencing reactions are performed simultaneously across multiple flow cells, maintaining Sanger's high accuracy while achieving throughputs exceeding 10,000 reads per run through automated consolidation.
Solution Approach 2:
The system incorporates automated robotic systems that perform sample loading, flow cell handling, and data consolidation without manual intervention. This automation maintains the high accuracy of Sanger sequencing while dramatically increasing throughput by eliminating manual bottlenecks and enabling continuous operation.
Data Source
Figure 1
Figure 2
Figure 3
AI summary
The application relates to DNA sequencing systems and methods. Systems and methods for determining the nucleotide sequence of a polynucleotide include attaching three different fluorescent dyes to three different nucleotides during incorporation. In particular, long Stokes shifted dyes may be used to determine the sequence of polynucleotides in a sequencing by synthesis system.