Pooled Duplex Sequencing for Rare Variant Genotyping
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Solution Overview
Problem
Current next-generation sequencing technologies face challenges in cost-effectively multiplexing small sequencing needs across large numbers of samples, particularly for applications involving rare genetic variants, due to high costs and inefficiencies in sample preparation and indexing for pooled sequencing.
Innovation Solution
The method employs Duplex Sequencing to generate error-corrected sequence reads by ligating adapter molecules to double-stranded DNA molecules, sequencing both strands, and comparing them to identify variant alleles within pooled samples, allowing for efficient and accurate genotyping of multiple samples using unique sub-pools.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If individual indexing and library preparation is performed for each sample, then sample identification accuracy is improved, but preparation cost and time increase significantly
Solution Approach 1:
The method segments samples into pooled groups and uses combinatorial indexing where each sample is represented by a unique combination of pool identifiers rather than individual sample identifiers. This segmentation allows multiplexed processing while maintaining traceability through the pooling matrix.
Solution Approach 2:
The patent introduces pooled samples as an intermediary layer between individual samples and sequencing. Each pooled sample contains multiple individual samples with unique combinations of pool identifiers, acting as a mediator that enables batch processing while preserving individual sample identification capability.
2Productivity
If pooled sequencing is used to increase throughput, then productivity is improved, but measurement precision of variant detection decreases
Solution Approach 1:
The method performs preliminary error correction by comparing forward and reverse strand sequences before final variant calling. This preliminary action removes sequencing errors and artifacts that would otherwise compromise variant detection accuracy in pooled samples.
Solution Approach 2:
The patent implements feedback through iterative error correction where sequence reads are compared against reference genomes and error patterns are identified and corrected. The system uses the information from both strands to feedback and refine the accuracy of variant detection.
3Measurement precision
If extensive library preparation is performed for high accuracy sequencing, then measurement precision is improved, but loss of time increases
Solution Approach 1:
The method merges multiple samples into pooled groups that share common library preparation steps. By combining samples in pools and using combinatorial indexing, the protocol performs library preparation once per pool rather than once per sample, significantly reducing total preparation time while maintaining accuracy through subsequent error correction.
Solution Approach 2:
Error correction and quality control steps are performed as preliminary actions during the library preparation and sequencing process itself, rather than requiring extensive post-sequencing analysis. This preliminary error correction reduces the need for time-consuming follow-up validation.
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 enables cost-effective and time-efficient genotyping of large numbers of samples with high sensitivity, maintaining original source identification and allowing for the detection of rare variant alleles with high confidence, suitable for pre-clinical and clinical disease assessment and screening.
Implementation Method 1
ligating adapter molecules to the plurality of target double-stranded DNA molecules to generate a plurality of adapter-DNA molecules
Implementation Method 2
sequencing one or more copies of the original first and second strands to provide a first strand sequence and a second strand sequence
Implementation Method 3
comparing the first strand sequence and the second strand sequence to identify one or more correspondences between the first and second strand sequences
Data Source
Figure 1A
Figure 1B~1C
Figure 2
AI summary
Methods and associated reagents for efficient genotyping of large numbers of samples via pooling are disclosed herein. Some of the embodiments of the technology are directed utilizing Duplex Sequencing for efficient genotyping of large numbers of samples (e.g., nucleic acid samples, patient samples, tissue samples, blood samples, etc.) and associated applications. Various aspects of the present technology have many applications in both pre-clinical and clinical disease assessment, screening large sample numbers where relatively infrequent variants are being sought, and others.