APEX Method for Multi-Omic Analysis on Tethered Nucleic Acid
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Solution Overview
Problem
Current single cell sequencing methods face challenges in maximizing biological information by measuring multiple omic features from the same cells and molecules, as they often require significant sample splits and consume precious primary samples, leading to depletion and destruction of sample material.
Innovation Solution
The attachment-based primer extension (APEX) method allows for the perpetual reuse of genetic material from a cell by conjugating genomic material to a solid phase support, enabling non-destructive analysis through multiple primer extension reactions and data integration from different assays.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If conventional sequencing methods are used to measure multiple omic features, then various assays can be performed, but significant sample splits are required which deplete and destroy precious primary sample material
Solution Approach 1:
The patent uses attachment-based primer extension to create multiple copies of the same nucleic acid template through sequential primer extension reactions. Each reaction produces a copy of the template without consuming it, enabling multiple omic feature measurements from a single original sample.
Solution Approach 2:
The method implements a universal platform where the same attached nucleic acid template can serve multiple functions across different assays. By sequentially performing primer extension reactions with different primers, the system can measure gene expression, isoform usage, and other omic features from the same original sample.
2Adaptability or versatility
If sample splits are performed to perform multiple experiments, then different omic features can be analyzed, but dropout complexities are introduced in the measured data
Solution Approach 1:
By generating multiple copies of the template through primer extension rather than splitting the original sample, the method maintains consistent data quality across all measurements. Each copy is an identical replica of the template, eliminating the dropout and variability issues that arise from splitting and re-amplifying different portions of the sample.
3Loss of information
If primary human samples are used for sequencing, then biological information can be obtained, but the samples are precious and limited, requiring careful conservation
Solution Approach 1:
The method creates multiple identical copies of the nucleic acid template through sequential primer extension reactions. This allows extraction of multiple different biological information types from a single original sample, maximizing information yield while consuming minimal sample material.
Solution Approach 2:
The template remains intact and reusable across all primer extension reactions. The same template molecule can be used for multiple different assays in sequence, continuously extracting useful biological information without depleting the original sample.
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
APEX enables multiple assays on the same genetic material without sample loss, integrating various omic features to maximize informativity and reduce noise, thereby conserving limited sample material and enhancing data integration across different sequencing platforms.
Implementation Method 1
incubating a nucleic acid sample with a terminal transferase and a cyclooctene-functionalized nucleotide to produced cyclooctene-functionalized nucleic acid molecules
Implementation Method 2
tethering the cyclooctene-functionalized nucleic acid molecules to a tetrazine-functionalized support via an Alder cycloaddition reaction
Implementation Method 3
performing at least two separate primer extension reactions using the tethered nucleic acid molecules as a template to produce multiple distinct sets of primer extension products
Data Source
AI summary
Provided herein is a method for sample analysis. In some embodiments, the method may involve: (a) incubating a nucleic acid sample with a terminal transferase and a cyclooctene-functionalized nucleotide to produced cyclooctene-functionalized nucleic acid molecules; (b) tethering the cyclooctene-functionalized nucleic acid molecules to a tetrazine-functionalized support via an Alder cycloaddition reaction; (c) performing at least two separate primer extension reactions using the tethered nucleic acid molecules as a template to produce multiple distinct sets of primer extension products; (d) separately analyzing the sets of primer extension products using different methods to produce multiple data sets; and (e) integrating the data sets.