Nucleic Acid Barcoding for Single-Cell Haplotype Phasing
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Solution Overview
Problem
Current nucleic acid sequencing methods struggle to provide complete haplotype phase information due to limited sequencing read length and are unable to perform genotyping at the single cell level, making it difficult to distinguish tumor cells from normal cells.
Innovation Solution
The method involves sequestering cells or nuclei into compartments with barcode templates, amplifying cellular content, fragmenting it, and attaching barcodes to the fragments, allowing for single cell nucleic acid sequencing and detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of information
If conventional sequencing methods are used, then sequencing can be performed, but complete haplotype phase information cannot be obtained due to limited read length
Solution Approach 1:
The method segments the haplotype information capture problem by using multiple shorter sequencing reads, each tagged with barcodes indicating their parental origin (maternal or paternal chromosome). These segmented reads are then computationally assembled to reconstruct complete haplotype phases, effectively overcoming the limited read length constraint while preserving full haplotype information.
Solution Approach 2:
The patent introduces barcodes as an intermediary element that bridges the gap between short sequencing reads and long haplotype reconstruction. Each barcode serves as a mediator that carries parental origin information, allowing reads from different chromosomes to be correctly paired and assembled into complete haplotypes despite the short read lengths.
2Productivity
If bulk cell sequencing is performed, then sequencing efficiency is high, but single cell genotyping cannot be achieved
Solution Approach 1:
The method segments the bulk sample into individual single-cell units, each processed separately with unique barcodes. This segmentation enables single-cell resolution while maintaining high throughput by processing many cells in parallel, thus achieving both single-cell precision and high productivity.
Solution Approach 2:
The patent changes the organizational parameter from bulk mixing to individual cell compartmentalization with unique barcode assignment. By assigning distinct barcode combinations to each cell, the method transforms the measurement parameter from population-averaged signals to cell-specific identifiers, enabling single-cell genotyping while maintaining scalability.
3Device complexity
If tumor cells are mixed with normal cells, then sample complexity is reduced, but tumor cell identification becomes difficult
Solution Approach 1:
The patent uses barcode tags as intermediaries that carry cell origin information. By incorporating cell-type-specific barcode combinations during single-cell processing, the method enables differentiation between tumor and normal cells even when mixed in the same sample, maintaining low sample complexity while achieving high detection accuracy.
Data Source
AI summary
Described and featured herein are methods to barcode nucleic acids for detection and sequencing, particularly at a single cell level. The methods involve application of a barcode template in a compartment with various targets, including nucleic acid fragments, nuclei and/or cells. After clonal amplification within the compartment, the barcode sequence integrates into its target before the compartment is broken so that it will effectively barcode nucleic acid fragments originated from a nucleic acid fragment, a nucleus or a cell clonally. The barcode information can be used for tracking the origin of the fragment, nucleus or cell and be used for haplotype phasing and a variety of single cell-based applications including whole genome sequencing, metagenome sequencing, targeted sequencing, RNA sequencing and immune repertoire sequencing.