Amplicon Source Identification via Pool-Specific Identifiers
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Solution Overview
Problem
Current methods for generating physical maps of genomes are limited by indirect fragment-pattern matching, variation in DNA migration, and the inefficiency of sequencing technology, which hinders accurate and cost-effective genome mapping and mutation detection, especially in complex genomes and large populations.
Innovation Solution
A method involving the creation of artificial chromosome clone banks, pooling strategies, restriction endonuclease digestion, adaptor ligation, and high-throughput sequencing to generate sequence-based physical maps, allowing for direct contig building and accurate genome mapping, and the detection of mutations in mutagenized populations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If fingerprinting technology is used for physical map construction, then genome mapping can be performed, but the method is indirect and based on fragment-pattern matching which reduces accuracy
Solution Approach 1:
The patent replaces the mechanical/indirect fragment-pattern matching approach of fingerprinting with direct DNA sequencing technology. Instead of using restriction enzymes to create fragments and matching patterns, the invention uses sequencing to directly read the DNA sequences, thereby substituting an indirect mechanical process with a direct molecular reading process that provides higher accuracy.
Solution Approach 2:
The patent introduces pool-specific identifiers as intermediaries that link pooled DNA samples to their original sources. These identifiers act as mediators between the pooled samples and the sequencing results, enabling accurate traceability and source identification without requiring direct analysis of each individual sample, thus simplifying the process while maintaining accuracy.
2Productivity
If high-throughput sequencing is used for mutation detection, then large populations can be screened efficiently, but the cost and complexity of processing increase
Solution Approach 1:
The patent combines multiple individual DNA samples into pooled groups, where each pool contains DNA from multiple individuals. By sequencing the pooled samples rather than individual samples, the method achieves high-throughput screening of large populations while reducing the number of sequencing reactions required, thereby improving productivity without proportionally increasing complexity.
Solution Approach 2:
The patent uses pool-specific identifiers as intermediaries to track which individuals are contained in each pool. These identifiers enable the system to manage and trace large numbers of samples through the sequencing process without requiring complex individual sample management, thus facilitating high-throughput processing while controlling complexity.
3Loss of information
If pool-specific identifiers are added to adaptors, then amplicon source identification is enabled, but the adaptor design and processing become more complex
Solution Approach 1:
The patent incorporates pool-specific identifiers into the adaptor sequences before the DNA samples are pooled and processed. By pre-loading the identifier information into the adaptors, the system ensures that traceability information is already embedded in the DNA fragments before pooling occurs, eliminating the need for complex post-processing tracking and simplifying the overall workflow despite the added identifier sequences.
4Measurement precision
If direct sequence-based physical mapping is used instead of fingerprinting, then mapping accuracy is improved, but the cost of sequencing the entire restriction fragment increases
Solution Approach 1:
The patent combines multiple DNA samples into pooled groups and sequences the pools rather than individual samples. This merging approach allows direct sequence-based mapping to be performed on pooled material, reducing the total number of sequencing reactions needed while maintaining mapping accuracy through the use of pool-specific identifiers for traceability.
Solution Approach 2:
The patent sequences only the necessary portions of the pooled DNA samples required to achieve accurate physical mapping, rather than sequencing every single base pair of every individual sample. By focusing sequencing efforts on the pooled material with sufficient depth to resolve individual contributions, the method achieves high accuracy without the prohibitive cost of complete individual sequencing.
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 accuracy and efficiency of genome mapping and mutation detection, providing a more direct and cost-effective method for constructing physical maps and identifying mutations in complex genomes and large populations.
Implementation Method 1
digesting the DNA of one or more pools with one or more restriction endonucleases to provide for a set of restriction fragments for each pool
Implementation Method 2
ligating adaptors to one or both sides of the restriction fragments, wherein at least one adaptor contains a pool-specific identifier
Implementation Method 3
amplifying the adaptor-ligated restriction fragments of step (e) with at least one primer, which primer contains a pool-specific section corresponding to the pool-specific identifier section in the adaptor
Implementation Method 4
determining the sequence of at least the pool-specific identifier and part of the restriction fragment of the amplicons or set of combined amplicons
Data Source
AI summary
The present invention relates to a method for identifying the source of an amplicon, comprising: providing a plurality of pools of amplicons from different sources, wherein the amplicons from different sources are present in more than one pool, and wherein the amplicons in each pool are tagged with a unique pool-specific identifier; sequencing at least part of the amplicons that comprise the pool-specific identifiers; and assigning one or more of the amplicons to corresponding pools and/or sources using the pool-specific identifiers.