Affinity-Tagged Nucleic Acid Preparation for Accurate Multiplex Sequencing
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Solution Overview
Problem
Current next-generation sequencing platforms face challenges in accurately mapping and aligning regions of repetitive, homologous, and variable sequences, leading to bias and increased computational requirements for longer read lengths, necessitating improved methods for targeting specific genomic regions.
Innovation Solution
A method involving contacting nucleic acid samples with primers, strand displacing polymerase, and ddNTP coupled with an affinity conjugate to generate amplified nucleic acids, followed by hybridization and binding to a binding molecule, resulting in nucleic acids with random 3' ends and at least 200 bp length, allowing for multiplexed and accurate sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If alignment to a reference genome is performed for short read sequences, then sequence read alignment is achieved, but bias and computational steps are dramatically increased
Solution Approach 1:
The method performs preliminary enrichment of target genomic regions before sequencing by using hybridization capture with probes specific to regions of interest. This pre-concentration of target sequences eliminates the need for complex post-sequencing alignment to reference genomes, as the enriched samples can be directly analyzed with reduced computational requirements while maintaining high alignment accuracy.
Solution Approach 2:
The invention extracts and isolates specific genomic regions of interest from the entire genome using hybridization capture technology. By taking out only the relevant target sequences and removing non-target genomic DNA, the method eliminates the computational burden of aligning millions of short reads across the entire genome, thereby reducing computational complexity while preserving alignment precision for the extracted target regions.
2Productivity
If next-generation sequencing is used to amplify clonal errors, then sequencing throughput is increased, but clonal errors are amplified
Solution Approach 1:
The method combines hybridization capture enrichment with unique molecular identifiers (UMIs) and targeted amplification strategies. By merging the enrichment step with UMI tagging before amplification, the system maintains high sequencing throughput while enabling computational correction of clonal errors through UMI-based consensus building, thus preserving reliability despite the necessary amplification steps.
Solution Approach 2:
The invention uses unique molecular identifiers (UMIs) as digital copies or tags attached to each original DNA molecule before amplification. These UMI copies serve as fingerprints that allow computational distinction between true biological variants and clonal amplification errors, enabling error correction while maintaining the high throughput benefits of next-generation sequencing amplification.
3Quantity of substance
If regions of repetitive sequence, homologous sequence, and variable sequence are mapped, then genome coverage is improved, but mapping reliability decreases
Solution Approach 1:
The method applies local quality control by designing probes with varying degrees of specificity for different genomic regions. For repetitive and homologous regions, the invention uses probe pools with diverse specificities and employs local enrichment strategies that adapt to the complexity of each region, thereby improving mapping reliability in difficult-to-map areas while maintaining overall genome coverage through targeted approaches.
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 reliability and accuracy of targeted, multiplexed, high-throughput sequencing by enabling more precise alignment and assembly of longer sequences, reducing bias and computational complexity.
Implementation Method 1
generating a plurality of amplified nucleic acids from said nucleic acid sample using said strand displacing polymerase
Implementation Method 2
contacting said nucleic acid sample to a binding molecule to said affinity conjugate
Implementation Method 3
hybridizing said primers to said nucleic acid sample
Data Source
Figure 1A
Figure 1B
Figure 2A~2B
AI summary
The present disclosure provides methods, compositions, and kits for methods that can improve techniques nucleic acid analysis, and can allow for more reliable and accurate targeted, multiplexed, high throughput sequencing. The methods, compositions, and kits can be used for sequencing target loci of nucleic acid. The methods, compositions, and kits disclosed herein can be used for assisted de novo targeted sequencing. The methods, compositions, and kits disclosed herein can also be used for library labeling for de novo sequencing and phasing.