Composite Barcode Partitioning for High-Occupancy Sequencing
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Solution Overview
Problem
In microfluidic detection applications like high-throughput sequencing, bead concentrations need to be adjusted to ensure unique labeling of partitions, resulting in significant dead volume and increased sample and reagent requirements.
Innovation Solution
The method involves partitioning a sample into partitions with particles conjugated to oligonucleotide primers, providing a substrate with barcode sequences, and associating the particles with the barcode sequences to generate a unique nucleic acid signature for each partition.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If bead concentrations are adjusted to ensure only one bead per partition, then unique labeling of partitions is achieved, but particle occupancy decreases to about 10% and dead volume increases
Solution Approach 1:
The invention segments the unique identification function into two parts: a common barcode shared by multiple beads and a unique identifier specific to each bead. This allows multiple beads per partition while maintaining unique identification capability, resolving the contradiction between unique labeling and particle occupancy.
Solution Approach 2:
The invention merges the common barcode sequence (present on all beads) with the unique identifier sequence (specific to each bead) into a composite barcode structure. This combination enables both high particle occupancy (multiple beads per partition) and accurate unique identification, directly resolving the technical contradiction.
2Measurement precision
If bead concentrations are adjusted to ensure only one bead per partition, then unique labeling is achieved, but the amount of sample and reagents needed increases
Solution Approach 1:
By segmenting the identification system into common and unique components, the invention enables high particle occupancy (85-95%) while maintaining unique partition identification. This reduces the total number of partitions needed, thereby decreasing the overall volume of sample and reagents required.
Solution Approach 2:
The invention changes the concentration parameter from low (1 bead per 10 partitions) to high (multiple beads per partition) while compensating through the composite barcode design. This parameter change reduces dead volume and minimizes sample and reagent requirements while preserving identification accuracy.
3Productivity
If multiple beads are allowed per partition, then particle occupancy increases to 85% or 95%, but unique identification of partitions becomes difficult
Solution Approach 1:
The composite barcode is segmented into a common portion (shared by all beads in a partition) and a unique portion (specific to each bead). During sequencing, the unique portion allows computational deconvolution to identify which beads are present in each partition, maintaining identification accuracy even with high particle occupancy.
Solution Approach 2:
The invention uses excessive action by allowing multiple beads (more than the traditional one bead per partition) to occupy each partition. The composite barcode system handles this excess by providing unique identifiers for each bead, enabling computational resolution of partition contents and maintaining identification precision despite high particle occupancy.
Data Source
AI summary
Methods of generating a nucleic acid signature for identifying particles associated in a partition are provided. In one aspect, the method comprises: partitioning a sample into a plurality of partitions comprising a particle comprising a solid support surface, the solid support surface having a plurality of oligonucleotide primers conjugated thereon, wherein the oligonucleotide primers comprise a barcode sequence, and wherein the partitions have 0, 1, or more than 1 particles per partition; providing in a partition a substrate comprising a barcode sequence or repeating clonal barcode sequences; and in the partition, associating a first particle conjugated to oligonucleotide primers comprising a first barcode sequence and a second particle conjugated to oligonucleotide primers comprising a second barcode sequence to a barcode sequence from the substrate, thereby generating a nucleic acid signature for the particles in the partition.