Multimeric Barcoding Reagents for Accurate Single-Cell Sequencing
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Solution Overview
Problem
Current DNA sequencing machines are limited by finite raw readlengths and raw accuracy, and experimental DNA samples like FFPE samples pose biophysical challenges due to DNA fragmentation and damage, limiting their scientific and medical applications.
Innovation Solution
The use of multimeric barcoding reagents to label nucleic acids of single cells, enabling high-throughput single-cell sequencing by appending unique barcode sequences to sub-sequences of target nucleic acids, which are then sequenced to produce sets of sequence reads corresponding to nucleic acid molecules of a single cell.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If DNA sequencing is performed on fragmented and damaged samples (e.g., FFPE samples), then the applicability to medical and scientific research is improved, but the sequencing accuracy and reliability deteriorate due to DNA fragmentation and damage
Solution Approach 1:
The patent segments the barcoding process into multiple independent components: (1) attachment of unique molecular barcodes to individual DNA fragments before sequencing, and (2) use of multimeric barcoding reagents that can simultaneously process multiple fragments. This segmentation allows each barcode to independently track its associated DNA fragment through the sequencing process, maintaining accuracy even when DNA is fragmented and damaged.
Solution Approach 2:
The patent applies preliminary barcoding to DNA fragments before they undergo sequencing. By attaching unique molecular barcodes to each DNA fragment in advance (before the sequencing process), the system establishes a reliable tracking mechanism that persists through subsequent fragmentation and damage, enabling accurate reconstruction of original sequences despite degraded sample conditions.
2Measurement precision
If unique molecular barcodes are attached to each DNA fragment through ligation or primer extension, then molecular counting and redundant sequencing are enabled, but the process complexity and time required increase
Solution Approach 1:
The patent merges multiple functions into the barcoding reagents: the barcoding oligonucleotides simultaneously serve as identification tags and as primers for subsequent amplification and sequencing. This consolidation eliminates separate steps for tagging and priming, reducing overall process time while maintaining the ability to perform accurate molecular counting and redundant sequencing.
Solution Approach 2:
The barcoding oligonucleotides are designed with multi-functionality: they provide unique molecular identification, serve as primers for PCR amplification, and enable both molecular counting and redundant sequencing. This universal design allows a single reagent to perform multiple critical functions, streamlining the workflow and reducing the time required compared to separate specialized reagents for each function.
3Productivity
If multimeric barcoding reagents are used to label nucleic acids of single cells, then high-throughput single-cell sequencing is enabled, but the device complexity and reagent design complexity increase
Solution Approach 1:
The patent employs a nested structure where barcoding oligonucleotides are integrated within multimeric barcoding reagents, which themselves are components of a larger single-cell sequencing system. The barcoding oligos are nested within the multimeric reagent structure, allowing them to be delivered and functionally integrated into single cells in a coordinated manner, thereby enabling high-throughput processing without proportionally increasing operational complexity.
Solution Approach 2:
The patent uses identical or highly similar barcoding oligonucleotide sequences across multiple multimeric reagents, allowing for standardized, reproducible barcoding across thousands of single cells. This copying approach enables high-throughput processing by using replicated, well-characterized reagent designs rather than customizing complex reagents for each individual cell, thereby increasing productivity without proportionally increasing design complexity.
Data Source
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AI summary
Reagents and methods for preparing nucleic acid samples for sequencing are provided. The reagents include multimeric barcoding reagents that comprise barcode regions linked together and a cell-binding moiety. The methods comprise contacting a nucleic acid sample comprising cells with a library of multimeric barcoding reagents, wherein each multimeric barcoding reagent comprises barcode regions linked together, and appending barcode sequences of a first multimeric barcoding reagent to sub-sequences of a target nucleic acid of a first cell, and appending barcode sequences of a second multimeric barcoding reagent to sub-sequences of a target nucleic acid of a second cell. Methods are also provided that comprise steps of internalising multimeric barcoding reagents into cells (e.g. by endocytosis) or exposing multimeric barcoding reagents to target nucleic acids by lysing cells or permeabilizing cell membranes.