Nucleic Acid Library Normalization via Transposome Capture
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Solution Overview
Problem
Current high throughput library preparation methods for genome and transcriptome analysis are hindered by the complexity of sequence information, particularly in higher eukaryotes like plants, where repetitive DNA and abundant transcripts dominate, masking information from low copy genes and transcripts.
Innovation Solution
The method employs DNA reassociation kinetics and transposome-based normalization to selectively identify and analyze both abundant and less abundant nucleic acid sequences by using a biotinylated transposome to tag and capture abundant DNA molecules, reducing library complexity and enriching rare sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of information
If high throughput library preparation methods are used to preserve natural abundance of genes and transcripts, then sequence information is preserved, but library complexity increases and masks information from low copy genes
Solution Approach 1:
The patent segments the nucleic acid library into different complexity fractions based on reassociation kinetics. By treating abundant and rare sequences differently through selective capture and removal, the method divides the original complex library into manageable components that can be analyzed separately, resolving the contradiction between preserving sequence information and reducing library complexity.
Solution Approach 2:
The patent extracts abundant sequences from the library through selective capture using biotinylated transposomes. By removing these dominant sequences that mask rare genes, the method isolates the problematic component (abundant sequences) and separates it from the rare sequences of interest, thereby reducing library complexity while preserving information from low copy genes.
2Loss of information
If abundant sequences are removed to reduce library complexity, then rare sequences become accessible, but processing time and steps increase
Solution Approach 1:
The patent performs preliminary normalization of the library before sequencing by removing abundant sequences in advance. This preliminary action prevents the dominance of abundant sequences during sequencing, ensuring that rare sequences are adequately represented without requiring extended processing time during the actual sequencing run.
Solution Approach 2:
The patent changes the physical state and composition of the library by controlling reassociation kinetics through temperature and time parameters. By adjusting these parameters, abundant sequences are selectively captured while rare sequences remain in solution, enabling efficient separation and reducing overall processing time compared to alternative methods.
3Reliability
If repetitive DNA and abundant transcripts are present in the library, then natural abundance is preserved, but analysis of low copy genes is hindered
Solution Approach 1:
The patent introduces biotinylated transposomes as intermediary molecules that selectively bind to abundant sequences. These intermediaries act as mediators between the abundant sequences and the magnetic beads, enabling specific capture and removal of dominant sequences while leaving rare sequences untouched, thus resolving the detection difficulty without compromising natural abundance information.
Solution Approach 2:
The patent replaces traditional mechanical separation methods with a biochemical approach using reassociation kinetics and affinity capture. Instead of physically separating sequences by size or other mechanical properties, the method uses temperature-controlled reassociation and biotin-streptavidin binding to selectively isolate abundant sequences, enabling more precise detection of low copy genes.
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 effectively reduces the impact of abundant sequences, allowing for the analysis of low copy genes and transcripts by separating and processing less abundant DNA molecules for sequencing, thereby enhancing the discovery and analysis of rare nucleic acid species.
Implementation Method 1
The rate at which a particular sequence will reassociate is proportional to the number of copies of that sequence in the DNA sample. For example, highly-repetitive (abundant) sequence will reassociate rapidly, while complex sequences (less abundant) will reassociate more slowly and remain single-stranded for a longer period of time.
Implementation Method 2
a biotinylated transposome is used to selectively tag and capture the more abundant DNA molecules in a DNA library
Implementation Method 3
a biotinylated transposome is used to selectively tag and capture the more abundant DNA molecules in a DNA library
Data Source
AI summary
Presented herein are methods and compositions for analyzing rare nucleic acid species. Some methods presented herein use DNA reassociation kinetics following thermal denaturation to define populations of nucleic acid sequences, e.g., highly abundant (e.g., cDNA from rRNA), moderately abundant, and less abundant or rare sequences (e.g., cDNA from mRNA).