Single-Stranded Adaptor Genotyping Reduces Sequencing Costs
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Solution Overview
Problem
Conventional genotyping-by-sequencing (GBS) methods require stringent control of template:adaptor concentration ratios and high-quality input DNA, and they need numerous sequencing reads to achieve adequate coverage, making them inefficient and costly for genotyping large numbers of individuals.
Innovation Solution
The use of single-stranded oligonucleotides instead of double-stranded adaptors for ligation reactions, allowing for barcode indexing of nucleic acids, enabling the multiplexing of samples while reducing the criticality of template:adaptor ratios and the number of sequencing reads required, and the selection of restriction enzymes and barcode sequences to tune the analysis for specific genetic markers.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If double-stranded adaptors are used for ligation reactions, then the ligation can proceed with conventional methods, but stringent control of template:adaptor concentration ratio and high-quality input DNA are required
Solution Approach 1:
The patent changes the fundamental parameter of adaptor structure from double-stranded to single-stranded. This parameter change fundamentally alters the ligation kinetics and reduces the sensitivity to template:adaptor concentration ratios, thereby resolving the contradiction between ease of manufacture and manufacturing precision requirements
Solution Approach 2:
The patent introduces barcode sequences at specific locations within the single-stranded adaptor structure. This local differentiation allows for sample multiplexing while the rest of the adaptor structure maintains simplicity, enabling easy ligation without stringent concentration controls
2Measurement precision
If conventional GBS methods survey hundreds of thousands or more sites, then comprehensive genomic coverage is achieved, but numerous sequencing reads are required generating high costs
Solution Approach 1:
The patent extracts and focuses sequencing efforts on specific genomic regions by using restriction enzyme cutting sites as anchors. Instead of uniformly sequencing the entire genome, the method targets only regions adjacent to restriction sites, thereby achieving sufficient genotyping coverage with far fewer sequencing reads
Solution Approach 2:
The patent segments the genome into regions defined by restriction enzyme cutting sites. By using these predetermined segments as targets for sequencing, the method reduces the total sequencing burden while maintaining adequate coverage for genotyping purposes
3Ease of operation
If single-stranded oligonucleotides are used instead of double-stranded adaptors, then template:adaptor ratio control becomes less critical, but the method requires development of new ligation protocols
Solution Approach 1:
The patent employs single-stranded oligonucleotides that are chemically synthesized and can be easily prepared in large quantities with precise sequences. These disposable oligonucleotides eliminate the need for complex adaptor preparation and concentration control, simplifying the overall operation despite requiring initial protocol development
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 allows for efficient genotyping of hundreds to thousands of individuals with reduced sequencing needs, lowering costs and enabling flexible application in various fields such as breeding and forensics, while maintaining accurate data deconvolution and genotype assignment.
Implementation Method 1
a single-stranded oligonucleotide is less subject to self-ligation as a double-stranded adaptor is and thus the template:adaptor ratio is much less critical than in conventional approaches
Implementation Method 2
two restriction enzymes are used to generate two sites with different overhangs at each end of the digested fragments
Implementation Method 3
amplifying the template to produce an amplicon
Data Source
AI summary
Provided herein is technology relating to genotyping and particularly, but not exclusively, to methods for genotyping one or more organisms by genome sequencing.