Dual-Barcoded Beads for Accurate 5' Gene Expression Profiling
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Solution Overview
Problem
Existing methods for molecular barcoding in single cell transcriptomics and proteomics analysis face challenges in efficiently quantifying gene expression and correcting for amplification bias, particularly in low-abundance nucleic acid targets.
Innovation Solution
A method involving the use of dual sets of oligonucleotide barcodes with universal sequences and molecular labels, combined with reverse transcriptase and template switch oligonucleotides, to generate barcoded nucleic acid molecules, allowing for accurate determination of nucleic acid copy numbers through molecular label counting.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional molecular barcoding methods are used for single cell transcriptomics analysis, then gene expression profiles can be obtained, but amplification bias occurs leading to inaccurate quantification of nucleic acid targets
Solution Approach 1:
The patent attaches molecular labels to oligonucleotide barcodes before the amplification process. This preliminary labeling allows the original molecule count to be recorded before amplification occurs, enabling accurate quantification that is not affected by subsequent amplification bias. The molecular labels serve as a permanent record of the initial nucleic acid target copy numbers.
Solution Approach 2:
The patent uses molecular labels as copies or markers that represent the original nucleic acid targets. Instead of directly measuring the amplified products, the system counts the molecular labels that were attached to the original targets, providing an accurate representation of initial copy numbers without being influenced by amplification variations.
2Measurement precision
If existing barcoding techniques are applied to low-abundance nucleic acid targets, then detection is possible, but measurement precision deteriorates due to amplification bias and low molecule counts
Solution Approach 1:
By attaching molecular labels to oligonucleotide barcodes before amplification, the system captures the exact number of low-abundance targets present initially. This preliminary recording of molecule counts allows accurate quantification even when starting with very few target molecules, as the molecular labels preserve the original copy number information.
Solution Approach 2:
The patent replaces direct measurement of amplified DNA products with counting of molecular labels that were attached earlier in the process. This substitution allows accurate counting of low-abundance targets by measuring the stable molecular labels rather than the variable amplified products, improving precision for low-copy number targets.
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
Enables precise quantification of nucleic acid targets by correcting amplification bias and providing accurate copy number determination, even in samples with low molecule counts, enhancing the reliability of gene expression analysis.
Implementation Method 1
extending the first plurality of oligonucleotide barcodes hybridized to the copies of the nucleic acid target in the presence of a reverse transcriptase
Implementation Method 2
a target-binding region capable of hybridizing to the nucleic acid target
Data Source
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AI summary
Disclosed herein include systems, methods, compositions, and kits for 5'-based gene expression profiling. Some embodiments provide synthetic particles (e.g., beads) associated with a first plurality of oligonucleotide barcodes and a second plurality of oligonucleotide barcodes. In some embodiments, nucleic acid targets (e.g., mRNAs) are initially barcoded on the 3' end with the first plurality of oligonucleotide barcodes and subsequently barcoded on the 5' end following a template switching reaction and intermolecular hybridization with the first plurality of oligonucleotide barcodes and extension. Immune repertoire profiling methods are also provided.