Reference Bead Indexing for Single-Cell Imaging–Sequencing Linkage
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Solution Overview
Problem
Current technologies face challenges in associating single-cell sequencing data with imaging data, particularly in determining gene expression and capturing phenotypic data of single cells, and there is a need for compositions and methods to link these data types effectively.
Innovation Solution
The use of reference particles with detectable moieties and particle indexing oligonucleotides, including unique particle identifiers, to associate sequencing data with imaging data by partitioning single cells and obtaining both data types, allowing for the identification and assignment of sequencing reads to specific partitions based on detectable signatures.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If single-cell sequencing and imaging are performed in parallel compartments, then both gene expression and phenotypic data can be captured, but the association between sequencing data and imaging data becomes difficult to establish
Solution Approach 1:
The system segments the identification process into two independent components: a detectable moiety attached to the bead that is captured by imaging, and a particle indexing oligonucleotide containing the unique identifier that is captured by sequencing. This segmentation allows each data type to be independently measured while maintaining their association through the dual-component identifier system.
Solution Approach 2:
The reference bead acts as an intermediary object that carries both the detectable moiety (for imaging capture) and the particle indexing oligonucleotide (for sequencing capture). This intermediary enables the linkage between imaging and sequencing data by providing a physical carrier that participates in both measurement modalities simultaneously.
2Reliability
If unique identifiers are assigned to each bead, then association between beads and data is enabled, but the complexity of the system increases
Solution Approach 1:
The system merges the unique identifier functionality into the existing bead structure by incorporating both the detectable moiety and the particle indexing oligonucleotide onto the same reference bead. This consolidation achieves reliable data association without requiring separate identification systems for imaging and sequencing.
Solution Approach 2:
The reference bead serves multiple functions simultaneously: it acts as a physical carrier for the cell, provides a detectable signal for imaging, and contains the unique identifier for sequencing. This multi-functionality eliminates the need for separate identification mechanisms and reduces overall system complexity.
3Difficulty of detecting and measuring
If detectable moieties are attached to reference beads, then imaging detection is enabled, but the beads become more complex and potentially less stable
Solution Approach 1:
The detectable moiety is attached locally to the surface of the reference bead at specific sites, rather than requiring modification of the entire bead structure. This localized attachment enables imaging detection while preserving the bulk stability and structural integrity of the bead.
Data Source
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AI summary
Disclosed herein include systems, methods, compositions, and kits for associating single cell sequencing data with imaging data (e.g., phenotypic data). In some embodiments, particle indexing oligonucleotides are associated with reference particles. Reference particles can comprise one or more detectable moieties. The particle indexing oligonucleotide can comprise a particle type identifier (PTI) and a unique particle identifier (UPI). The PTI and the UPI can be employed to identify of the imaged partition from which a sequenced nucleic acid target molecule originated.