Partition ID Tag Oligonucleotides for Barcode Deconvolution
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
In microfluidic detection applications, such as high-throughput sequencing, beads conjugated to oligonucleotides often result in low partition utilization due to adjusted bead concentrations to ensure unique labeling. Higher bead concentrations lead to increased partition occupancy, but also result in some partitions having more than one bead, causing barcode overlap and loss of sensitivity.
Innovation Solution
The method involves forming partitions with forward primers and partition ID tag oligonucleotides, where different partitions contain different forward primers and ID tag sequences. These primers and ID tags are designed to hybridize and form amplicons, allowing for the detection of multiple barcodes in a partition by sequencing the amplicons and identifying shared ID tag sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If bead concentrations are adjusted to ensure unique labeling (1 out of 10 partitions occupied), then partition labeling accuracy is improved, but partition utilization decreases and sample/reagent consumption increases
Solution Approach 1:
The patent introduces partition ID tag oligonucleotides as intermediary molecules that mediate between the barcode oligonucleotides and the detection system. These ID tags are distributed throughout the partition and serve as unique identifiers for the partition, allowing multiple barcode-conjugated beads to be present without causing labeling errors, as long as they share the same ID tag. This mediator enables higher bead concentrations while maintaining labeling accuracy.
2Productivity
If bead concentrations are increased to improve partition occupancy, then partition utilization is improved, but the number of partitions with multiple beads increases causing barcode overlap and sensitivity loss
Solution Approach 1:
The partition ID tag oligonucleotide acts as a mediator that allows multiple barcode-conjugated beads to coexist in the same partition without causing barcode overlap issues. The ID tag is shared by all beads in a partition and serves as a partition-specific identifier, enabling the system to distinguish between different partitions even when multiple beads are present. This resolves the contradiction by allowing high bead concentrations while maintaining barcode uniqueness through the shared ID tag mechanism.
Solution Approach 2:
The patent segments the identification function into two separate components: the barcode sequence (which may be duplicated across multiple beads) and the partition ID tag (which is shared across all beads in a partition). This segmentation allows the barcode to serve its original purpose of identifying the bead while the ID tag provides partition-level identification, eliminating the conflict between having multiple beads and maintaining unique labeling.
3Quantity of substance
If higher bead concentrations are used to decrease sample and reagent consumption, then cost efficiency is improved, but barcode overlap increases resulting in sensitivity loss
Solution Approach 1:
The partition ID tag oligonucleotide serves as a mediator that enables the use of higher bead concentrations without sacrificing detection sensitivity. By providing a shared partition-specific identifier, the ID tag allows the system to tolerate multiple beads per partition while maintaining the ability to accurately detect and quantify target molecules. This mediator thus enables cost efficiency through reduced sample/reagent consumption while preserving sensitivity.
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 detection of multiple barcodes in a partition, allowing for improved data accuracy and sensitivity by distinguishing between barcodes that share the same partition ID tag sequence, thus addressing the issue of barcode overlap and enhancing partition utilization.
Implementation Method 1
hybridizing at least one forward primer to the partition ID tag oligonucleotide to form a hybridized product
Implementation Method 2
extending the forward primer with a polymerase using the partition ID tag oligonucleotide as a template to form an amplicon
Data Source
AI summary
Methods of assessing whether multiple differently-barcodes primers are in partitions are provided.