Dual-Barcode Bead Detection in Partitioned Sequencing Droplets

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Solution Overview

Problem

The presence of multiple barcoded beads in a partition leads to fractionated data points, causing issues with data splitting and reduced sequencing efficiency.

Innovation Solution

A method is described for detecting multiple barcoded solid supports in partitions by synthesizing complementary sequences of oligonucleotides from two solid support oligonucleotides, forming double-stranded polynucleotides, and sequencing these to identify partitions with multiple supports, using techniques such as R2 retrotransposase, reverse transcriptase, and DNA polymerase to generate sequencing reads with barcodes.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If barcode bead overloading is used to increase the number of barcoding competent partitions, then the substrate to sequencing data conversion rate is improved, but fractionated data points are created when multiple barcodes occur in discrete partitions

Engineering Contradiction:

Improvesubstrate to sequencing data conversion rateVSAvoidfractionated data points

Core Design Contradiction:

ProductivityVSLoss of information

Solution Approach 1:

The patent applies preliminary action by performing PCR amplification and sequencing of barcode oligos before the actual barcoding process. This allows the system to pre-determine which partitions contain multiple beads, enabling subsequent steps to handle these partitions appropriately and prevent data fractionation while maintaining high conversion rates through bead overloading

Inventive Principle:

Principle #10Preliminary action

2Quantity of substance

If multiple barcoded beads are present in a partition, then the number of barcoding competent partitions increases, but data splitting and reduced sequencing efficiency occur

Engineering Contradiction:

Improvenumber of barcoding competent partitionsVSAvoidsequencing efficiency

Core Design Contradiction:

Quantity of substanceVSProductivity

Solution Approach 1:

The patent applies segmentation by separating the detection of multiple barcodes from the actual barcoding process. Through PCR amplification and sequencing of barcode oligos, the system identifies partitions with multiple beads as distinct segments, allowing these to be processed differently to maintain sequencing efficiency while increasing the number of competent partitions

Inventive Principle:

Principle #1Segmentation

3Productivity

If barcode bead overloading is implemented, then more partitions become barcoding competent, but the complexity of detecting and measuring multiple barcodes per partition increases

Engineering Contradiction:

Improvepercentage of barcoding competent partitionsVSAvoiddetection of multiple barcodes per partition

Core Design Contradiction:

ProductivityVSDifficulty of detecting and measuring

Solution Approach 1:

The patent applies copying by creating multiple copies of barcode oligos through PCR amplification. This generates sufficient signal from the barcode sequences attached to beads in each partition, making it feasible to detect and measure multiple barcodes per partition without excessive complexity, thereby increasing the percentage of barcoding competent partitions

Inventive Principle:

Principle #26Copying

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 enhances data conversion rates by accurately identifying partitions with multiple barcoded beads, improving sequencing data quality and reducing fractionated data points.

Implementation Method 1

step b) comprises contacting the solid support oligonucleotides with a R2 retrotransposase

Methodology Applied

Scientific EffectRetrotransposition:

Implementation Method 2

step b) comprises contacting the solid support oligonucleotides with a reverse transcriptase

Methodology Applied

Scientific EffectReverse transcription:

Implementation Method 3

step b) comprises contacting the solid support oligonucleotides with a DNA polymerase

Methodology Applied

Scientific EffectDNA replication:

Implementation Method 4

synthesizing a complementary sequence of the oligonucleotides to produce a double-stranded polynucleotide

Methodology Applied

Scientific EffectHybridization:

Data Source

PatentUS12559788B2Multiple beads per droplet resolution

Publication Date: 2026.02.24 BIO RAD LABORATORIES INC

AI summary

Provided are methods for detecting the presence of multiple barcoded solid supports in partitions, the method comprising providing a plurality of partitions wherein at least some partitions comprises multiple solid supports, where each solid support is linked to different solid support oligonucleotides, the solid support oligonucleotides comprising a barcode unique for the solid support; synthesizing a complementary sequence of the oligonucleotides to produce a double-stranded polynucleotide having barcodes from two of the solid support oligonucleotides; and sequencing one or both strands of the double-stranded polynucleotides, wherein sequences comprising two different barcodes indicates the presence of two solid supports in the same partition. Also provided are compositions and methods for producing the oligonucleotides.