Encoded Microcarriers for Genomic Sequencing Positioning
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Solution Overview
Problem
Current nucleic acid sequencing methods face limitations in individual read length, leading to reduced information content and difficulty in accurately positioning sequences within a reference genome, particularly in applications like cancer sequencing and clinical genetics, where speed and accuracy are crucial.
Innovation Solution
A method involving encoded microcarriers with capture oligonucleotide probes that hybridize with nucleic acid molecules, allowing for the determination of sequence information by generating sequence reads and decoding the microcarrier's code to identify the probe's sequence and location on the genome, thereby increasing information content and improving sequencing accuracy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of information
If current sequencing methods are used, then sequencing can be performed, but individual read length is short leading to reduced information content
Solution Approach 1:
The invention segments the sequencing process into multiple independent reactions performed in parallel on different microcarriers. Each microcarrier carries a capture probe that hybridizes to a specific genomic region, allowing multiple sequence reads to be obtained from different locations simultaneously. This segmentation enables longer effective read lengths by combining information from multiple shorter reads across different microcarriers.
Solution Approach 2:
The invention implements a nested structure where capture probes are attached to microcarriers, which are then hybridized to DNA fragments. The microcarriers serve as containers that hold the capture probes, creating a hierarchical organization that allows for multiplexing of multiple sequencing reactions within a single reaction volume while maintaining individual read length information through the microcarrier encoding system.
2Measurement precision
If current sequencing methods are used, then sequencing can be performed, but it is difficult to position sequences in reference genome
Solution Approach 1:
The invention introduces capture probes as intermediary molecules that bridge the connection between the sequenced DNA fragments and the reference genome. These capture probes have known sequences that hybridize to specific genomic locations, serving as anchors that provide positional information. By using these intermediaries, the system can accurately map short sequence reads to their corresponding locations in the reference genome even when the reads themselves are short.
Solution Approach 2:
The invention uses encoded microcarriers with different colors or fluorescent markers to identify and track specific capture probes throughout the sequencing process. This color-coding system allows for the identification of which genomic region each microcarrier represents, enabling accurate positioning of sequenced fragments in the reference genome by matching the color code to the corresponding genomic location.
3Adaptability or versatility
If bisulfate sequencing is performed, then methylation analysis can be done, but information for alignment is reduced making assembly difficult
Solution Approach 1:
The invention performs the hybridization of capture probes to DNA fragments before the bisulfate conversion step. By establishing the initial positioning and identity of DNA fragments through capture probe hybridization prior to chemical modification, the system preserves the positional information needed for alignment. The capture probes serve as reference points that remain unchanged during bisulfate conversion, allowing accurate mapping even after the DNA sequence is chemically modified for methylation analysis.
4Productivity
If multiple separate sequencing reactions are performed, then complete sequencing can be achieved, but time and sample loss increase
Solution Approach 1:
The invention merges multiple separate sequencing reactions into a single pooled reaction by attaching different capture probes to different microcarriers and hybridizing them all to the DNA sample simultaneously. This consolidation allows parallel processing of multiple genomic regions in one reaction vessel, dramatically reducing the total time required compared to performing separate reactions sequentially, while also minimizing sample loss by keeping all reactions in the same volume throughout the process.
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 information content of sequenced DNA fragments, reduces assay time and sample loss, and improves diagnostic sequencing test specificity by integrating genome enrichment and sequencing steps, facilitating faster and more accurate determination of sequence alterations.
Implementation Method 1
hybridizing said capture oligonucleotide probe with a sample comprising a nucleic acid molecule
Data Source
AI summary
The present invention relates to a method for determining the sequence of a nucleic molecule. Herein a capture oligonucleotide probe is attached to an encoded microcarrier, wherein the code of said microcarrier identifies the sequence of said oligonucleotide probe. The capture oligonucleotide probe is hybridized with a sample comprising nucleic acids molecules, wherein said DNA fragment comprises a sequence which is complementary to the sequence of the capture oligonucleotide probe. The sequence of the DNA molecule is determined, wherein the capture oligonucleotide probe serves as a primer for a DNA polymerase, in the case of single molecule sequencing this is a sequencing primer. After the sequence determination, the nucleotide sequence of the capture oligonucleotide probe is identified by determining the code on the microcarrier, which corresponds with the capture oligonucleotide probe. This sequence information directly identifies the location of the sequenced DNA fragment on the genome, allowing direct comparison.