AFLP Marker Detection via High-Throughput Sequencing
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Solution Overview
Problem
Current methods for detecting and identifying molecular markers, such as AFLP, are laborious and time-consuming, especially when dealing with large samples, and are limited by the resolving power of electrophoresis, which restricts throughput and cannot distinguish between similar mobility fragments, and high-throughput sequencing technologies struggle to provide sequencing reads that encompass entire AFLP fragments economically.
Innovation Solution
Incorporating a sample-specific identifier into adaptor-ligated restriction fragments and sequencing only part of the restriction fragment allows for efficient and reliable identification, enabling multiple samples to be sequenced in a single run and providing adequate identification of restriction fragments through high-throughput sequencing technology.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If electrophoresis is used to detect AFLP fragments, then the detection method is simple and widely applicable, but the resolving power is limited and throughput is restricted
Solution Approach 1:
The patent replaces the mechanical electrophoresis system with a sequencing-based detection system. Instead of separating fragments by mobility through electrophoresis, the invention sequences the fragments directly, substituting a biochemical process (sequencing) for a physical separation process. This enables high-throughput detection while maintaining the ability to identify AFLP fragments.
Solution Approach 2:
The patent introduces sample-specific identifiers as intermediary elements that are incorporated into the AFLP fragments before sequencing. These identifiers serve as mediators that allow multiple samples to be distinguished and processed simultaneously in a sequencing run, thereby increasing throughput while maintaining sample traceability.
2Adaptability or versatility
If electrophoresis is used to separate fragments, then the method can handle large genome sizes, but fragments of similar mobility cannot be distinguished
Solution Approach 1:
The patent replaces mobility-based separation with sequence-based identification. Instead of relying on electrophoretic mobility differences that fail for fragments of similar size, the invention directly sequences the fragments, using their nucleotide sequences as unique identifiers. This provides precise distinction between fragments regardless of their mobility similarities.
Solution Approach 2:
The patent changes the detection parameter from physical mobility (electrophoresis) to chemical sequence composition (sequencing). By measuring the nucleotide sequence rather than migration distance, the system can precisely distinguish between fragments that would co-migrate in electrophoresis, thereby improving measurement precision while maintaining adaptability to large genomes.
3Productivity
If high throughput sequencing is used to sequence entire AFLP fragments, then all fragments can be captured, but the cost is prohibitively high
Solution Approach 1:
The patent extracts and sequences only the essential portions of AFLP fragments - specifically the regions containing sample-specific identifiers and selective nucleotide positions - rather than sequencing entire fragments. This extraction approach captures the necessary information for fragment identification and polymorphism detection while significantly reducing sequencing costs and increasing throughput.
Solution Approach 2:
The patent applies partial sequencing action by sequencing only specific regions of AFLP fragments that contain discriminatory information (identifiers and selective positions) rather than performing complete fragment sequencing. This partial action achieves the necessary detection completeness for marker identification while reducing the overall sequencing burden and cost.
4Productivity
If multiple samples are processed simultaneously, then throughput increases, but sampling variation increases
Solution Approach 1:
The patent uses sample-specific identifiers as intermediary markers that are incorporated into each sample's fragments before pooling and sequencing. These identifiers allow computational differentiation and individual tracking of fragments from each sample within the pooled sequencing data, enabling accurate genotype calling despite the mixed sample environment and reducing sampling variation effects.
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 significantly increases the number of markers identified, reduces sampling variation, and improves accuracy, enabling the simultaneous processing of many samples while maintaining the versatility of AFLP technology, making it suitable for high-throughput applications across various organisms.
Implementation Method 1
digesting the sample nucleic acid with at least one restriction endonuclease to obtain a set of restriction fragments
Implementation Method 2
amplification of the set of adaptor-ligated restriction fragments, with one or more primers that are at least complementary to: the sample-specific identifier section, the section that is complementary to the remains of the recognition sequence of the restriction endonuclease
Data Source
Figure 1
Figure 2A~2B
AI summary
The present invention relates to a high throughput method for the identification and detection of molecular markers wherein restriction fragments are generated and suitable adaptors comprising (sample-specific) identifiers are ligated. The adapter-ligated restriction fragments may be selectively amplified with adaptor compatible primers carrying selective nucleotides at their 3' end. The amplified adapter-ligated restriction fragments are, at least partly, sequenced using high throughput sequencing methods and the sequence parts of the restriction fragments together with the sample-specific identifiers serve as molecular markers.