Barcoded Particle Spatial Mapping by Serial Primer Extension

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Solution Overview

Problem

Existing genomics workflows face challenges in efficiently sequencing and analyzing samples due to the limitations of sample indexing methods, which struggle to provide spatial information and effectively eliminate PCR/sequencing errors.

Innovation Solution

A probe system utilizing barcoded particles with unique identifier sequences to extend nucleic acid molecules, allowing for the production of primer extension products that contain complementary sequences, enabling the mapping of binding events on a cellular sample.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If sample indexing methods are used to enable multiplex sequencing, then sequencing throughput is improved, but spatial information is lost

Engineering Contradiction:

Improvesequencing throughputVSAvoidspatial information

Core Design Contradiction:

ProductivityVSLoss of information

Solution Approach 1:

The invention segments the indexing function into two distinct components: sample indexes for multiplex identification and molecular indexes for spatial positioning. Each nucleic acid molecule receives both a sample-specific barcode and a unique molecular barcode, allowing simultaneous multiplex sequencing and spatial mapping without information loss

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The molecular index sequence is nested within the sequencing library structure alongside the sample index. The unique molecular barcode is incorporated into the adapter sequences flanking the insert, creating a nested structure where both indexing layers coexist within the same sequencing read

Inventive Principle:

Principle #7Nested doll (Nesting)

2Reliability

If molecular indexing is used to tag each molecule with a unique sequence, then PCR/sequencing errors are eliminated, but device complexity increases

Engineering Contradiction:

Improveerror eliminationVSAvoidindexing system complexity

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The indexing system is segmented into standardized components: sample index regions and molecular index regions with defined boundaries. This segmentation allows independent optimization and simplifies the implementation of error correction algorithms for each component

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The invention employs parameter changes in the form of variable-length molecular index sequences and configurable barcode structures. This flexibility allows tuning of the system to balance between error elimination capability and computational complexity based on specific application requirements

Inventive Principle:

Principle #35Parameter changes

3Measurement precision

If barcoded particles are used for spatial mapping, then measurement precision is improved, but manufacturing complexity increases

Engineering Contradiction:

Improvespatial mapping precisionVSAvoidparticle production complexity

Core Design Contradiction:

Measurement precisionVSEase of manufacture

Solution Approach 1:

Instead of manufacturing complex physical barcoded particles with precise spatial arrangements, the invention uses in silico copying of barcode sequences into the nucleic acid library. The spatial mapping information is copied into the molecular index sequences during library preparation, eliminating the need for complex particle manufacturing while maintaining measurement precision

Inventive Principle:

Principle #26Copying

Solution Approach 2:

The invention replaces the mechanical approach of physical barcoded particles with a biochemical system using nucleic acid sequences. The spatial information is encoded in the molecular index sequences rather than being physically embodied in particle structures, simplifying manufacturing while maintaining precision

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

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

The method provides spatial information and enhances the accuracy of sequencing by filtering out duplicates and errors, improving the analysis of molecular data.

Implementation Method 1

hybridizing the first set of barcoded particles with the first population of primer molecules

Methodology Applied

Scientific EffectHybridization:

Implementation Method 2

extending the hybridized primer molecules using the nucleotide sequence of the first set of barcoded particles as a template

Methodology Applied

Scientific EffectPrimer extension:

Data Source

PatentUS12416040B2Spatial mapping by serial primer extension

Publication Date: 2025.09.16 PIXELGEN TECH AB

AI summary

Provided herein is probe system comprising: a population of nucleic acid molecules that have an extendible end, a first set of barcoded particles that each have a nucleotide sequence comprising: (i) a binding sequence that is complementary to the extendible end of the nucleic acid molecules, (ii) a unique particle identifier sequence, and (iii) a first template sequence, and a second set of barcoded particles that each have a nucleotide sequence comprising: (i) the first template sequence and (ii) a unique particle identifier sequence. In use, extension of the nucleic acid molecules using the first set of barcoded particles of as a template produces extensions products that contain the complement of a unique particle identifier sequence of a particle and the complement of the first template sequence. Methods of using the probe system to map binding events in or on a cellular sample are also provided.