Partition Barcoding with Barcode Bead Colocalization in Sequencing
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Solution Overview
Problem
Existing methods for sequencing sample preparation in partitions result in low utilization of partitions due to low bead concentrations, leading to sample loss and increased reagent use, and higher bead concentrations cause multiple barcodes per partition, resulting in data sensitivity loss and overrepresentation of data points.
Innovation Solution
A method involving partitioning a sample into partitions with multiple particles per partition, each with distinct barcode sequences, and using a transposase to introduce heterologous end adaptor sequences, followed by pairwise comparison of sequencing read overlaps to determine shared DNA fragments, allowing for accurate nucleotide sequence generation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If bead concentrations are adjusted so that only about 1 out of 10 partitions are occupied by a bead, then partitions have only one bead and are uniquely labeled by the barcode, but this results in low utilization of partitions, sample loss and increases the amount of sample and reagents that are needed
Solution Approach 1:
The patent segments the barcode identification problem into two independent components: (1) particle-level barcodes for partition identification, and (2) template-level barcodes for sample identification. This allows multiple particles per partition without compromising the uniqueness of sample labeling, as the template barcode remains singular and traceable even when multiple particle barcodes are present.
Solution Approach 2:
The patent introduces template barcodes as an intermediary layer between the sample and the detection system. These template barcodes are incorporated into the amplified DNA products and serve as the primary identifier for sample origin, while particle barcodes become secondary markers that can be used for additional validation or multiplexing without interfering with the primary identification function.
2Productivity
If higher bead concentrations are used, then partition occupancy and utilization of partitions increase, but this leads to a greater number of partitions having more than one bead, resulting in sample split between multiple barcodes and loss of sensitivity
Solution Approach 1:
The patent segments the barcode system into particle barcodes and template barcodes, allowing multiple particles (each with its own barcode) to coexist in a single partition without causing sample splitting. The template barcode remains unique to the sample, ensuring that sensitivity is maintained because all reads from that template can be confidently attributed to a single source.
Solution Approach 2:
The patent changes the critical parameter for identification from particle barcode uniqueness to template barcode uniqueness. This parameter change allows the system to tolerate multiple particles per partition (higher bead concentration) while maintaining measurement precision, because the template barcode serves as the definitive identifier for sample origin rather than the particle barcode.
3Productivity
If higher bead concentrations are used, then partition occupancy increases, but this causes overrepresentation of certain data points by having more than one data point generated from a single partition
Solution Approach 1:
The patent segments the identification hierarchy so that template barcodes (unique to each sample) take precedence over particle barcodes (shared among particles). This segmentation allows multiple particles with different particle barcodes to contribute data to the same template, and the system correctly attributes all such reads to a single sample, preventing overrepresentation while maintaining high partition occupancy.
Solution Approach 2:
The patent implements a feedback mechanism where the system detects multiple particle barcodes within a single partition through the shared template barcode, and uses this information to correctly aggregate reads from multiple particles into a single sample profile. This feedback loop prevents data overrepresentation by recognizing that multiple particle barcodes can legitimately contribute to one template.
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
Improves sequencing data quality by increasing partition utilization and reducing sample loss, while maintaining sensitivity and accuracy by distinguishing between barcodes from the same partition.
Implementation Method 1
the providing comprises randomly cleaving template DNA. In some embodiments, the randomly cleaving comprises contacting the template DNA with a transposase that introduces heterologous end adaptor sequences into the template DNA
Implementation Method 2
linking oligonucleotide primers from the solid support to at least a fragment of DNA template, thereby forming barcoded DNA template fragments
Data Source
AI summary
Comparison of common sequencing reads from sequencing based on partition-based barcoding can be used to improve sequencing results. Increased loading of barcodes per partition can also improve sequencing results.