Sequencing Read Distance Determination via Accelerated Primer Extension
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Solution Overview
Problem
Traditional paired-end sequencing methods fail to provide significant information for the region between the 3′ and 5′ ends of a polynucleotide, limiting the detection of variants in unsequenced regions and being prone to sequencing errors and slow processing.
Innovation Solution
The method involves accelerated primer extension using labeled nucleotides in a flow order, measuring signals to determine distance information indicative of the length of unsequenced regions, and coupling sequencing data from 3′ and 5′ ends to derive additional sequencing information, allowing for the detection of structural variants and characterization of polynucleotides.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of information
If traditional paired-end sequencing methods are used, then sequencing data for the 3' and 5' ends can be obtained, but no significant information is derived for the region between the ends and the method is prone to sequencing errors
Solution Approach 1:
The polynucleotide is divided into multiple regions (first region, second region, third region) that are sequenced separately. The first and third regions are sequenced using traditional paired-end methods, while the second region is skipped during sequencing but its distance information is measured, allowing comprehensive coverage without sequencing the entire molecule in one continuous process.
Solution Approach 2:
Distance information measurement serves as an intermediary mechanism to bridge the gap between the sequenced first and third regions. By measuring the physical distance or number of bases in the unsequenced second region through labeled nucleotide incorporation, the system recovers information that would otherwise be lost while maintaining sequencing accuracy in the measured regions.
2Loss of information
If long-range sequencing techniques are used to sequence the region between ends, then information from the unsequenced region can be obtained, but the method becomes slow and prone to substantial sequencing errors
Solution Approach 1:
Instead of completely sequencing the second region (which would be slow and error-prone), the method performs partial action by only measuring distance information through labeled nucleotide incorporation. This partial measurement approach recovers the necessary information about the unsequenced region while maintaining fast sequencing speeds and avoiding the errors associated with complete long-range sequencing.
Solution Approach 2:
The sequencing process is segmented into three distinct regions where only the first and third regions undergo full sequencing, while the second region undergoes rapid distance measurement. This segmentation allows the system to obtain information from all regions without subjecting the entire polynucleotide to slow long-range sequencing procedures.
3Productivity
If traditional paired-end sequencing is used, then processing can be completed quickly, but the method cannot detect variants in the unsequenced region
Solution Approach 1:
The sequencing system performs multiple functions within a single workflow: it sequences the first and third regions using traditional paired-end methods (maintaining speed) while simultaneously measuring distance information in the second region (enabling variant detection). This multi-functionality allows the system to achieve both fast processing and comprehensive variant detection without requiring separate experiments.
4Productivity
If distance information measurement is performed through accelerated primer extension with labeled nucleotides, then speed is improved, but the system must manage complex flow orders and signal measurement protocols
Solution Approach 1:
The system applies different quality protocols to different regions: the first and third regions use standard sequencing protocols with detection after each flow step, while the second region uses accelerated measurement with detection only after multiple flow steps. This local differentiation optimizes speed where appropriate (second region) while maintaining accuracy where needed (first and third regions), managing complexity through regional specialization rather than uniform complexity throughout.
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
This approach enhances the detection of variants and characterizes polynucleotides by providing distance information and coupling sequencing data, overcoming the limitations of traditional paired-end sequencing, including improved accuracy and speed.
Implementation Method 1
extending the primer through the first region of the polynucleotide using labeled nucleotides
Implementation Method 2
detecting the presence or absence of an incorporated labeled nucleotide
Implementation Method 3
measuring a signal from the labeled nucleotides incorporated into the primer
Data Source
AI summary
Described herein are methods of sequencing a polynucleotide and methods of analyzing sequencing data obtained from such sequencing methods. The sequencing methods can include accelerated primer extension through a region of the polynucleotide using labeled nucleotides provided according to a flow order, measuring a signal from labeled nucleotides incorporated into the primer, and determining distance information that indicates the length of the region using the measured signal.