Single Cell Sequencing Multiplexing via Sample Barcodes
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Solution Overview
Problem
Current genomic research faces challenges in achieving a quantitative and predictive understanding of cellular circuits due to non-linear interactions among biological components, making it difficult to reconstruct cellular responses and predict effects of genetic perturbations on a genomic scale.
Innovation Solution
The development of methods for multiplexing single cell sequencing using sample barcode oligonucleotides to construct multiplexed libraries, allowing for simultaneous perturbation and analysis of multiple components, and the use of computational tools like demuxEM for demultiplexing and identifying singlets and doublets, enabling a combinatorial approach to study genetic interactions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If multiple tissue samples are analyzed separately using single cell sequencing, then each sample can be analyzed with high precision, but the experimental cost and time increase significantly
Solution Approach 1:
The patent combines multiple tissue samples into a single sequencing run by labeling cells from different samples with unique sample-specific barcodes. This allows simultaneous analysis of multiple samples in one experiment, dramatically increasing throughput while maintaining single-cell resolution. The barcoded cells are pooled and processed together through the sequencing workflow.
Solution Approach 2:
The patent introduces sample barcodes as intermediary molecular tags that carry sample identity information through the sequencing process. These barcodes serve as mediators that allow the sequencing system to distinguish and track cells from different original samples without requiring separate sequencing runs for each sample.
2Adaptability or versatility
If sample barcodes are added to single cell sequencing, then multiple samples can be multiplexed, but the library construction complexity increases
Solution Approach 1:
The patent segments the library construction process into distinct modular steps: sample-specific barcode labeling, pooling, and sequencer read assignment. This segmentation allows each step to be optimized independently and simplifies the overall complex process by breaking it into manageable, standardized operations that can be performed in sequence.
3Measurement precision
If computational demultiplexing is performed using demuxEM, then accurate sample assignment is achieved, but computational resources and processing time increase
Solution Approach 1:
The patent replaces manual or simple computational demultiplexing methods with the demuxEM algorithm, which uses probabilistic modeling and statistical inference to accurately assign reads to samples. This computational approach substitutes simpler but less accurate methods, achieving high precision in sample assignment through sophisticated algorithms that analyze barcode patterns and sequencing data characteristics.
Data Source
AI summary
The present invention provides methods and tools for analyzing genetic interactions. The subject matter disclosed herein is generally directed to single cell genomics and proteomics. In one embodiment provided is a method of cell and nuclei hashing using sample barcodes. In another embodiment are method of performing genomewide CRISPR perturbation screens.