Transformable Tagging Moieties for Oligonucleotide Library Simplification
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Solution Overview
Problem
Current barcoding strategies in life sciences require large, diverse libraries of oligonucleotide barcodes, which are challenging to prepare and manage, especially for large numbers of samples, leading to inefficiencies and increased costs.
Innovation Solution
The use of transformable tagging moieties that can be transformed in situ into a collection of unique tagging or 'barcode' moieties, allowing for differential tagging of molecular species with a smaller number of original tags, such as oligonucleotides with degenerate nucleotides that can randomly or semi-randomly replicate to generate diverse sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If large, diverse libraries of oligonucleotide barcodes are used for tagging molecular species, then the ability to distinguish between large numbers of different samples is improved, but the complexity of preparing and managing these libraries increases significantly
Solution Approach 1:
The barcode library is segmented into multiple smaller sub-libraries, each containing a subset of the total barcode sequences. These sub-libraries can be independently prepared, managed, and pooled, reducing the complexity of handling the complete library while maintaining the ability to distinguish between samples through combinatorial pooling strategies.
Solution Approach 2:
The patent introduces a temporal dimension to barcode identification by using barcode sequences that can be read at different stages of the workflow. Additionally, spatial dimensionality is utilized through combinatorial pooling where barcodes are distributed across multiple pools and samples, allowing identification through the pattern of presence/absence across pools rather than requiring direct sequencing of all barcodes.
2Quantity of substance
If large, diverse libraries of oligonucleotide barcodes are prepared and allocated across large numbers of samples, then comprehensive tagging coverage is achieved, but the time and resources required for library preparation and management increase
Solution Approach 1:
Barcode libraries are pre-prepared and stored in a standardized format before sample processing begins. The libraries are organized into predetermined pools with known compositions, allowing rapid allocation to samples without requiring de novo library preparation for each experimental run. This preliminary organization significantly reduces the time required for library preparation and allocation.
Solution Approach 2:
The patent utilizes variable parameters in barcode design, such as degenerate nucleotides that can adopt multiple base identities, allowing a single barcode sequence to represent multiple specific sequences. This parameter variation enables comprehensive tagging coverage with fewer physical barcode sequences, reducing the time and resources needed for library preparation while maintaining the ability to distinguish between large numbers of samples.
3Measurement precision
If diverse barcode libraries are used for tagging, then accurate identification of molecular species is enabled, but the cost of preparing and maintaining these libraries increases
Solution Approach 1:
Instead of synthesizing and maintaining large numbers of unique barcode sequences, the patent uses a smaller set of barcode sequences that are replicated and distributed across multiple pools and samples. The identification accuracy is maintained through the combinatorial pooling strategy and degenerate nucleotide design, which allow a single barcode sequence to serve multiple identification purposes, thereby reducing the cost of library preparation and maintenance.
Solution Approach 2:
The barcode library is designed with universal features that allow the same set of barcodes to be used across multiple experiments, samples, and applications. The degenerate nucleotide sequences and standardized pool structures enable the library to serve multiple functions simultaneously, reducing the need for separate specialized libraries and thereby lowering the overall cost of library preparation and maintenance while maintaining accurate identification capabilities.
Data Source
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AI summary
The present disclosure provides methods, systems and compositions that provide transformable tagging moieties for use in analytical operations, and particularly in analysis biological systems, such as in the analysis of gene expression in cell based systems.