Nucleic Acid Sample Normalization by Depleting Abundant RNA Sequences
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Solution Overview
Problem
RNA sequencing is hindered by the dominance of highly expressed house-keeping genes, leading to inefficient sampling of genes of interest and excessive redundant data generation, which increases costs and storage requirements, and existing normalization methods like DSN and hydroxyapatite column methods introduce biases or PCR chimeras, making them unsuitable for many applications.
Innovation Solution
A method involving contacting nucleic acid samples with oligonucleotide arrays to separate abundant sequences, using oligo-dT molecules for RNA and cDNA, and extending these molecules to generate DNA arrays, allowing for the extraction of unannealed nucleic acids to achieve a more uniform distribution of sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If RNA sequencing is performed on typical biological samples, then sequencing data is generated, but the data is dominated by highly expressed house-keeping genes which reduces sampling efficiency for genes of interest
Solution Approach 1:
The patent extracts and removes highly abundant house-keeping gene sequences from the RNA sample before sequencing. This is achieved through selective depletion methods that identify and eliminate dominant sequences, thereby enriching the remaining pool for low-abundance genes of interest and improving sampling efficiency.
Solution Approach 2:
The patent applies normalization or depletion treatments to the RNA sample before sequencing to pre-adjust the distribution of sequences. This preliminary action ensures that highly expressed genes do not dominate the sequencing data, allowing for more efficient detection of low-abundance transcripts in the initial sequencing run.
2Measurement precision
If sequencing depth is increased to detect low abundance genes, then detection sensitivity improves, but cost and time requirements increase
Solution Approach 1:
By removing highly abundant house-keeping gene sequences from the sample prior to sequencing, the patent concentrates sequencing capacity on low-abundance genes of interest. This extraction approach achieves equivalent detection sensitivity at lower sequencing depths, thereby reducing the time and cost required for sequencing while maintaining the ability to detect rare transcripts.
3Stability of the object's composition
If DSN or hydroxyapatite column normalization methods are used, then sequence distribution is normalized, but PCR chimeras are formed or biases are introduced
Solution Approach 1:
The patent employs depletion probes that are designed to bind and remove specific high-abundance sequences. These probes are consumed in the process of selective depletion, preventing the formation of PCR chimeras and biases associated with reusable normalization methods like DSN or hydroxyapatite columns, thereby maintaining sequence accuracy while achieving uniform distribution.
4Measurement precision
If more sequencing data is generated to overcome sampling inefficiencies, then gene detection completeness improves, but data storage requirements increase
Solution Approach 1:
The patent extracts and removes redundant high-abundance house-keeping gene sequences before sequencing, thereby concentrating sequencing efforts on detecting low-abundance genes of interest. This approach achieves comparable gene detection completeness with reduced sequencing depth, resulting in smaller data volumes and lower storage requirements while maintaining detection effectiveness.
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
The method reduces variability and achieves a more uniform distribution of nucleic acid sequences, enhancing the analysis efficiency and reducing the need for excessive sequencing, thereby lowering costs and storage requirements.
Implementation Method 1
one or more nucleic acid molecules from the nucleic acid sample anneal to one or more oligonucleotides of the oligonucleotide array
Implementation Method 2
extending two or more of the oligonucleotides by reverse transcription using the annealed RNA molecules as templates to generate a DNA array
Data Source
AI summary
Methods and devices for preparing processed RNA and DNA samples are provided. A first nucleic acid sample is used to produce a probe set based on the intrinsic sequence abundances in the sample. Abundant sequences will produce more probes. When a second nucleic acid sample is applied to the probes more of the abundant sequences will bind to the probes enabling these sequences to be separated from the sample. Methods of extracting RNA and detecting target sequences using probes are also provided. Device for microfluidic processing of RNA in a flow-path is also claimed. Method for processing nucleic acid in which a surface comprising probes having a length of more than 100 nucleotides is used.