RNA Analytics Segregation Method for Read Alignment
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Solution Overview
Problem
Current Next Generation Sequencing (NGS) methods face challenges in accurately aligning reads due to the complexity of nucleic acid samples, leading to limited detection of rare RNA transcripts and high-abundance nucleic acid interference, which complicates the analysis of diverse RNA populations.
Innovation Solution
A method involving the segregation of nucleic acid molecules into subpools based on distinctive nucleotide features, such as those near the 5' or 3' terminus or polyA/tails, followed by fragmentation and labeling, allowing for the separation and identification of specific subpool information during sequencing, thereby reducing sample complexity and improving read alignment.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If NGS methods sequence complex nucleic acid samples directly, then high throughput sequencing is achieved, but read alignment accuracy deteriorates due to sample complexity
Solution Approach 1:
The patent divides the complex nucleic acid sample into multiple subpopulations based on distinctive nucleotide features (such as specific bases at defined positions). Each subpopulation is sequenced separately, reducing the complexity within each group and improving alignment accuracy while maintaining overall high throughput sequencing capacity.
2Measurement precision
If NGS methods amplify individual RNA molecules, then detection sensitivity is improved, but detection of rare transcripts deteriorates due to high-abundance nucleic acid interference
Solution Approach 1:
By segmenting the sample into subpopulations based on distinctive nucleotide features, rare transcripts in low-abundance subpopulations can be detected without being overwhelmed by high-abundance transcripts in other subpopulations. Each subpopulation is analyzed separately, enabling sensitive detection of rare species.
Solution Approach 2:
The patent applies different analysis conditions or depths to different subpopulations based on their characteristics. Subpopulations enriched for rare transcripts receive enhanced sequencing depth or specialized analysis, improving detection sensitivity for low-abundance species while maintaining overall efficiency.
3Ease of manufacture
If NGS methods use random amplification of input DNA, then preparation simplicity is improved, but sequencing directionality deteriorates leading to unambiguous alignment issues
Solution Approach 1:
The patent performs preliminary classification of nucleic acid molecules into subpopulations based on distinctive nucleotide features before sequencing. This pre-organization provides directional information that guides subsequent alignment, resolving ambiguities while keeping the overall process simple and automated.
Data Source
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AI summary
The present invention relates to a method of ordering nucleic acid molecule fragment sequences derived from a pool of potentially diverse RNA molecules comprising ° optionally reverse transcribing the RNA molecules to provide a pool of cDNA molecules, ° segregating nucleic acids from said template RNA or cDNA pool, selecting for potentially different templates with a distinctive nucleic acid feature shared by the segregated templates, thereby providing at least a first subpool of nucleic acids, ° optionally once or more further segregating nucleic acids from said template RNA or cDNA, selectively segregating nucleic acids with a different distinctive nucleic acid feature, thereby providing one or more further subpool(s) of nucleic acids, ° generating fragments of said segregated nucleic acid molecules by fragmenting or obtaining fragment copies of said segregated nucleic acid molecules, wherein the fragments of each subpool or combined subpools remain separable from fragments of other subpools or other combined subpools by physically separating the subpools or by attaching a label to the fragments of the subpools, with the label identifying a subpool, or determining a partial sequence of said segregated nucleic acid molecule and preferably aligning at least two sequences or partial sequences to a joined sequence.