Oligonucleotide Barcoding for Simultaneous Protein and Gene Expression Profiling
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Solution Overview
Problem
Current methods for determining protein and gene expressions in cells face challenges in accurately quantifying both highly and lowly expressed proteins simultaneously, particularly in single cells, due to limitations in measuring protein-protein interactions and gene expression correlations.
Innovation Solution
The method involves using oligonucleotide barcodes to specifically bind to cellular components, generating barcoded reagents and nucleic acid molecules, and creating sequencing libraries for simultaneous measurement of protein and gene expressions by attaching sequencing adaptors and obtaining sequencing data.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If current methods are used to determine protein and gene expressions, then measurement can be performed, but accurate quantification of both highly and lowly expressed proteins simultaneously is not achieved
Solution Approach 1:
The method segments the measurement process into distinct phases: (1) barcoding phase where oligonucleotide barcodes are attached to cellular components, (2) amplification phase where barcoded molecules are amplified, and (3) sequencing phase where expressions are quantified. This segmentation allows different measurement strategies to be applied to highly and lowly expressed proteins separately, then integrated for simultaneous quantification.
Solution Approach 2:
Oligonucleotide barcodes serve as intermediaries between cellular components (proteins and genes) and the sequencing detection system. Each barcode uniquely identifies a cellular component and is attached to corresponding molecules, enabling traceability and accurate quantification through sequencing read counts while distinguishing between highly and lowly expressed targets.
2Reliability
If amplification is performed to increase signal, then detection sensitivity improves, but amplification biases are introduced
Solution Approach 1:
The method incorporates feedback through unique molecular identifiers (UMIs) that are embedded in the oligonucleotide barcodes. These UMIs allow the system to track and correct for amplification biases by comparing the number of original barcoded molecules to the number of amplified copies, enabling accurate quantification despite variable amplification efficiency across different targets.
Solution Approach 2:
The method uses digital copying where each original cellular component is represented by a unique barcode copy. During amplification, these barcode copies are replicated, but the unique identifier allows reconstruction of the original molecule count by counting distinct barcodes rather than total amplified products, thereby eliminating amplification bias from quantification.
3Productivity
If high-throughput measurement is implemented, then productivity increases, but complexity of the method increases
Solution Approach 1:
The oligonucleotide barcodes are designed with universal features including: (1) a common sequencing adapter region that enables all barcoded molecules to be processed through the same sequencing pipeline, (2) UMI regions that provide universal error correction capability, and (3) target-specific regions that can adapt to different cellular components. This universal design allows simultaneous measurement of multiple targets through a unified protocol, increasing throughput while managing complexity.
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 the accurate and quantitative analysis of protein and gene expressions in cells, correcting for amplification biases and allowing for the determination of protein and gene expression profiles in a high-throughput manner.
Implementation Method 1
each of the plurality of first cellular component-binding reagents comprises a first cellular component-binding reagent specific oligonucleotide
Data Source
AI summary
Disclosed herein include systems, methods, compositions, and kits for determining the expression of highly expressed proteins and lowly expressed proteins. In some embodiments, primers allowing generation of separate libraries for abundant AbSeq protein profiling oligonucleotides and scarce AbSeq protein profiling oligonucleotides are provided.