Stochastic Barcode Amplification for Accurate Molecular Counting
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Solution Overview
Problem
Existing methods for stochastic barcoding in molecular biology, such as PCR amplification, introduce errors due to PCR crossover, leading to overestimated molecular counts.
Innovation Solution
A method involving stochastically barcoding targets with oligonucleotides and defined barcoded primers, followed by PCR amplification and sequencing, to determine and correct amplification noise by counting molecular labels with different sequences, thereby estimating the accurate number of targets.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If PCR amplification is used to amplify stochastically barcoded targets, then the number of detectable targets increases, but errors are introduced due to PCR crossover leading to overestimated molecular counts
Solution Approach 1:
The patent segments the barcode into multiple independent components (cell barcode, molecular barcode, and UMI) that can be independently verified. By dividing the barcode structure into separable functional elements, the system can identify and correct PCR crossover errors through cross-validation of each segment, thereby maintaining measurement precision while enabling high-throughput amplification.
Solution Approach 2:
The patent introduces an intermediary verification step using defined barcoded primers that act as mediators between the stochastic barcodes and the detection system. These primers hybridize to the barcoded targets and provide a reference framework for identifying PCR artifacts, allowing accurate molecular counting despite the presence of amplification noise.
2Adaptability or versatility
If stochastic barcoding is performed to enable multiplexed cell analysis, then the throughput and versatility of gene expression profiling increases, but PCR crossover errors result in inaccurate molecular counts
Solution Approach 1:
The patent performs preliminary barcoding of targets with unique molecular identifiers before the PCR amplification step. By establishing the unique molecular identity of each target beforehand, the system creates a reference framework that allows post-amplification verification and correction of PCR crossover errors, maintaining reliability in multiplexed analyses.
Solution Approach 2:
The patent implements a feedback mechanism where the defined barcoded primers hybridize to stochastic barcodes and provide verification signals. This feedback loop allows the system to identify and correct PCR-induced errors by comparing the amplified products against the expected barcode structures, thereby maintaining accurate molecular counts in high-throughput multiplexed experiments.
3Measurement precision
If defined barcoded primers are contacted with samples at the same concentration as stochastic barcodes, then amplification noise is reduced, but the complexity of primer design and optimization increases
Solution Approach 1:
The patent systematically varies key parameters of the defined barcoded primers including concentration (matching stochastic barcode concentration), length (5-20 nucleotides for molecular labels), and sequence composition (with Hamming distance of at least 2 from stochastic barcodes). By optimizing these parameters, the system reduces amplification noise while managing design complexity through structured parameter selection rather than arbitrary design choices.
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
Accurately determines the number of targets by correcting amplification noise, preserving sequencing signal while reducing errors caused by PCR crossover.
Implementation Method 1
stochastically barcoding a plurality of targets in each of one or more samples using a plurality of oligonucleotides comprising stochastic barcodes
Implementation Method 2
contacting one or more defined barcoded primers with each of the one or more samples
Implementation Method 3
amplifying the stochastically barcoded targets and the one or more defined barcoded primers to generate a plurality of amplified stochastically barcoded targets and a plurality of amplified defined barcoded primers
Data Source
AI summary
Disclosed herein are methods and systems for correcting errors in sample amplification, including the errors occurred in determining the number of targets in samples. In some embodiments, the method comprises: stochastically barcoding a plurality of targets in the samples using oligonucleotides comprising stochastic barcodes to generate stochastically barcoded targets; contacting one or more defined barcoded primers with each of the one or more samples; and determining an amplification noise.