Multiplexed Chromatin Modification Quantification via Barcoded ChIP-Seq
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Solution Overview
Problem
Current methods for assessing chromatin modifications are limited by their qualitative or semi-quantitative nature, high costs, and inability to perform high-throughput epigenomic profiling, particularly in drug characterization and toxicity studies, where there is a need for unifying readouts to detect epigenetic side effects and filter out drug candidates early in the development process.
Innovation Solution
A method involving chromatin immunoprecipitation and sequencing with unique molecular identifiers (UMIs) and barcoding, allowing for the multiplexed, quantitative assessment of chromatin modifications by sequencing a small subset of DNA fragments, enabling accurate global and locus-specific quantification of chromatin modifications across multiple samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional ChIP-Seq or WGBS methods are used to profile chromatin modifications, then quantitative accuracy can be achieved, but the cost and time required increase linearly with the number of samples and antibodies analyzed
Solution Approach 1:
The patent combines multiple samples and antibodies into a single pooled workflow using molecular barcodes. Chromatin fragments from different samples are tagged with unique barcodes, allowing parallel processing of up to 96 samples across multiple antibodies in one sequencing run, thereby achieving high throughput without sacrificing quantitative accuracy
Solution Approach 2:
The patent creates a universal platform that can simultaneously analyze multiple chromatin modifications across multiple samples using a single standardized workflow. The barcoded ChIP-Seq method serves multiple functions: quantification, multiplexing, and normalization, eliminating the need for separate workflows for each sample-antibody combination
2Productivity
If molecular barcodes are added to chromatin fragments for multiplexing, then throughput increases, but technical challenges arise that hamper accurate quantification
Solution Approach 1:
The patent incorporates feedback mechanisms through unique molecular identifiers (UMIs) that track individual chromatin fragments throughout the workflow. By counting unique UMIs rather than raw reads, the method corrects for PCR amplification biases and sequencing errors, providing accurate quantification despite the complexity of multiplexed barcoded samples
Solution Approach 2:
The patent uses molecular barcodes as intermediaries that link chromatin fragments to their original samples and antibodies. These barcodes enable the pooling and demultiplexing of samples while preserving quantitative information, serving as a bridge between the physical chromatin and the digital sequencing data
3Measurement precision
If extensive sequencing is performed to achieve accurate quantification, then measurement precision improves, but cost and time consumption increase
Solution Approach 1:
The patent applies partial action by sequencing only a small subset of barcoded chromatin fragments rather than performing extensive whole-genome sequencing. The barcoded approach allows accurate quantification to be achieved with minimal sequencing depth because the barcodes provide the necessary multiplexing and normalization information
Solution Approach 2:
The patent performs preliminary actions by adding molecular barcodes and UMIs to chromatin fragments before sequencing. This preprocessing step enables subsequent accurate quantification with reduced sequencing requirements, as the barcodes carry the identification and normalization information that would otherwise require extensive sequencing to obtain
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 enables accurate and cost-effective, high-throughput quantification of chromatin modifications, reducing the need for extensive sequencing and allowing for the determination of drug effects on epigenetic alterations, thereby improving drug characterization and reducing animal testing.
Implementation Method 1
a plurality of different antibodies, each specifically binding a chromatin modification
Implementation Method 2
obtaining chromatin fragments binding each antibody, thereby obtaining a sub-pool comprising tagged gDNA fragments
Data Source
AI summary
The invention provides methods for assessing the global levels of a plurality of different chromatin modifications in parallel in a plurality of samples. The methods disclosed herein also relate to assessing the levels of a plurality of different chromatin modifications, in a plurality of locations of interest within genome, in a plurality of samples. The methods are highly multiplexed, quantitative and involve chromatin immunoprecipitation and sequencing technology.