Nucleic Acid Partitioning and Barcoding for Single-Cell Analysis
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Solution Overview
Problem
Current methods for processing nucleic acid samples, such as ATAC-seq, face limitations in single-cell analysis, including high variability, low read counts, and limited throughput, which hinder the generation of personal epigenomic profiles compatible with clinical decision-making.
Innovation Solution
A method involving partitioning nucleic acid molecules within a plurality of partitions, where DNA and RNA molecules are barcoded using specific barcode molecules and splint sequences, allowing for simultaneous processing and amplification, enabling the generation of barcoded nucleic acid products that can be recovered and subjected to PCR for sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional bulk processing methods are used for nucleic acid samples, then throughput is limited and cell-to-cell variation cannot be resolved, but partitioning into single-cell reactions increases complexity and reduces throughput
Solution Approach 1:
The patent partitions bulk nucleic acid samples into discrete droplets, with each droplet containing material from a single cell or nucleus. This segmentation enables individual processing of thousands of cells in parallel, resolving cell-to-cell variation while maintaining high throughput through parallelization of the segmentation process
Solution Approach 2:
The patent implements nested barcoding where unique molecular identifiers (UMIs) are embedded within droplet barcodes. This nested structure allows simultaneous tracking of individual molecules and their parent droplets (cells), enabling precise quantification at single-cell resolution without increasing processing complexity
2Productivity
If multiple nucleic acid types (DNA and RNA) are processed separately, then processing time increases and throughput decreases, but simultaneous processing requires complex partitioning schemes
Solution Approach 1:
The patent employs universal barcoding reagents and processing protocols that work for both DNA and RNA molecules. The same droplet partitioning and barcode attachment methodology is applied regardless of nucleic acid type, enabling simultaneous processing of multiple molecule types without increasing system complexity
Solution Approach 2:
The patent combines DNA and RNA processing into a single unified workflow within the same droplets. Both nucleic acid types are partitioned, barcoded, and amplified simultaneously in parallel reactions, merging previously separate processing streams into one integrated system that increases throughput without sacrificing specificity
3Reliability
If barcoding is performed after amplification, then read counts are sufficient, but variability in amplification efficiency dominates the data, but pre-amplification barcoding reduces read counts
Solution Approach 1:
The patent performs barcoding immediately after partitioning and before any amplification steps. By attaching unique molecular identifiers to nucleic acid molecules in the original single-cell partitions, the system captures the true distribution of molecules before amplification bias can occur, ensuring reliable quantitative data
Solution Approach 2:
The patent uses PCR amplification to generate multiple copies of the barcoded molecules for sequencing. The amplification occurs after barcoding, creating sufficient read counts while preserving the original barcode assignments, thus maintaining both data accuracy and adequate sequencing depth
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
This approach enables efficient analysis of both DNA and RNA molecules from single cells or cell nuclei, providing detailed insights into gene expression and chromatin accessibility with improved cell-to-cell variation resolution and increased throughput.
Implementation Method 1
the partition comprises a first nucleic acid barcode molecule, a second nucleic acid barcode molecule, and a splint sequence, wherein the first nucleic acid barcode molecule and the second nucleic acid barcode molecule comprise a common barcode sequence
Implementation Method 2
subjecting the barcoded nucleic acid products to one or more amplification processes (e.g., polymerase chain reaction (PCR))
Data Source
AI summary
The present disclosure provides methods, systems, and kits for processing nucleic acid molecules. A method may comprise providing a template nucleic acid fragment (e.g., within a cell, cell bead, or cell nucleus) within a partition (e.g., a droplet or well) and subjecting the template nucleic acid fragment to one or more processes including a barcoding process and a single primer extension or amplification process. The processed template nucleic acid fragment may then be recovered from the partition and subjected to further amplification to provide material for subsequent sequencing analysis. The methods provided herein may permit simultaneous processing and analysis of both DNA and RNA molecules originating from the same cell, cell bead, or cell nucleus.