Single-cell sequencing with mapped cellular observations
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Solution Overview
Problem
Current single-cell sequencing methods have limited throughput and scope in mapping genomic diversity to cellular observations, making it difficult to construct large-scale cellular databases for applications in biology, personalized drug discovery, and machine learning.
Innovation Solution
A novel method combining an aligned sequencing element array with cellular well arrays and optical or magnetic observation techniques, using solid phase PCR probes with molecular barcodes and UMIs to map cellular observations to individual cell sequences in a massively parallel manner.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional bulk sequencing methods are used, then sequencing cost and time are reduced, but the ability to sequence individual cells and detect cellular heterogeneity is lost
Solution Approach 1:
The invention segments the sequencing process into individual cell-level operations using sub-nanoliter cellular wells that physically isolate single cells. Each well contains a bead with molecular barcodes that are unique to that specific well, enabling parallel sequencing of thousands of individual cells while maintaining the ability to map observations back to specific cells through the barcode-well correspondence
Solution Approach 2:
The invention introduces molecular barcodes as an intermediary element that bridges the connection between physical cellular observations and sequencing data. The barcodes are attached to beads in specific wells, and when cells are sequenced, the barcode sequences serve as identifiers that map the genomic data back to the specific cellular observations made in each well
2Measurement precision
If droplet-based methods like Chromium 10× are used, then single-cell sequencing capability is improved, but the cost and complexity increase
Solution Approach 1:
The invention uses disposable sub-nanoliter cellular wells that are simple in structure compared to complex droplet-based systems. Each well is a straightforward microstructured compartment that can be manufactured at low cost using standard microfabrication techniques, eliminating the need for complex microfluidic droplet generation and manipulation systems
Solution Approach 2:
The cellular well array serves multiple functions: it physically isolates individual cells, provides a platform for optical or magnetic observation, contains the sequencing reagents, and through the barcode system, enables data mapping. This multi-functionality reduces the need for separate specialized components that would increase overall system complexity
3Ease of manufacture
If Seq-Well method is used, then cost effectiveness is improved, but the ability to map cellular observations to sequences is lost
Solution Approach 1:
The invention reintroduces the molecular barcode intermediary that Seq-Well lacks. Barcodes are attached to beads in each well, creating a unique identifier system. When cellular observations are made in each well and sequencing is performed, the barcode sequences serve as the linking key that reconstructs the mapping between physical observations and genomic data
Solution Approach 2:
The invention applies local quality by making each well unique through its specific barcode assignment. While the overall system structure remains simple and cost-effective like Seq-Well, each individual well has a distinctive molecular barcode that enables traceability. This localized differentiation preserves mapping capability without requiring complex global system changes
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
Enables the creation of high-throughput databases for diverse proteins and transcriptomes, allowing for the collection of binding and affinity data from hundreds of thousands of cells, and facilitating personalized treatments for cancer, autoimmune diseases, and allergies.
Implementation Method 1
solid phase PCR probes that are added onto a substrate that align with cellular wells. The probes comprise a spacer, to avoid steric hindrance of the polymerase used in the reaction
Implementation Method 2
optical observation, fluorescence, or interaction with magnetic fields
Data Source
AI summary
This invention pertains to a novel method of single cell sequencing where cellular observations are paired directly with sequences. This method consists of a sequencing element with barcoded solid-phase PCR probes, cellular wells, and an optional cellular trap. Cells are loaded with reagents directly into wells or captured with cellular traps where the cellular wells are then lowered over the trapped cells. The sequencing element is then added. Cellular observations such as binding and affinity are then recorded. Cells are lysed and RT-PCR is performed to create a cDNA library that is then sequenced. Barcodes in the library allow sequences to be mapped back to individual wells. This method has applications from basic biology to personalized drug discovery to creating large databases for machine learning training sets.