Single-Cell Barcode Molecules With Abasic Sites to Minimize Exchange
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Solution Overview
Problem
Existing methods for sequencing nucleic acids from single cells face challenges in minimizing barcode exchange during processing, especially in partitioned environments like droplets or wells, which can lead to inaccurate identification and quantification of sequencing reads.
Innovation Solution
A method is introduced to generate partially double-stranded nucleic acid barcode molecules with a noncanonical base, such as uracil, which is treated with uracil-DNA-glycosylase to create an abasic site, allowing for barcoding of tagmented nucleic acid molecules and reducing barcode exchange by less than 10% through hybridization and amplification in partitions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If barcode molecules are processed in partitioned environments (droplets or wells), then single-cell sequencing accuracy is improved, but barcode exchange between partitions occurs leading to identification errors
Solution Approach 1:
The patent applies local quality by making the barcode molecule structurally asymmetric with a 5' overhang that is single-stranded while the rest of the molecule is double-stranded. This creates a localized region of different properties (single-stranded vs double-stranded) that enables selective hybridization. The overhang contains a noncanonical base (uracil) that creates a mismatch with canonical bases in other barcodes, preventing exchange while allowing hybridization to the intended adapter sequence.
Solution Approach 2:
The patent changes the chemical composition parameter of the barcode by incorporating a noncanonical base (uracil) at the 5' end of the overhang sequence. This parameter change creates a chemical mismatch that prevents hybridization with other barcodes containing only canonical bases (A, T, G, C), thereby preventing barcode exchange while maintaining the ability to hybridize to the complementary adapter sequence.
2Ease of manufacture
If conventional barcode molecules are used, then processing is simple, but barcode exchange occurs at rates exceeding 10%
Solution Approach 1:
The patent modifies the chemical composition of the barcode by incorporating a noncanonical base (uracil) at a specific position in the 5' overhang. This parameter change creates a chemical mismatch that prevents hybridization with other barcodes, reducing barcode exchange to less than 10%. The modification is integrated into the standard synthesis process without requiring complex additional steps.
Solution Approach 2:
The barcode molecule is constructed as a composite structure combining double-stranded DNA with a single-stranded 5' overhang containing a noncanonical base. This composite structure integrates the stability of double-stranded DNA with the hybridization capability of single-stranded overhangs, while the noncanonical base provides selective compatibility only with its complementary adapter sequence.
3Reliability
If abasic sites are generated at noncanonical bases, then barcode exchange is minimized, but additional processing steps are required
Solution Approach 1:
The patent performs preliminary action by incorporating the noncanonical base (uracil) into the barcode structure during synthesis, and then treating the barcode with uracil-DNA-glycosylase to generate an abasic site before the barcode is used in partitioning. This preliminary modification ensures that when barcodes are introduced to partitions, they cannot exchange with other barcodes, as the abasic site prevents proper hybridization. The processing step is performed once in advance rather than requiring continuous monitoring or correction.
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
The method effectively minimizes barcode exchange, ensuring accurate identification and quantification of sequencing reads by maintaining the integrity of barcodes in partitioned environments, thereby enhancing the reliability of nucleic acid sequencing from single cells.
Implementation Method 1
treating said partially double-stranded nucleic acid barcode molecule with an agent, thereby generating an abasic site at a position of said noncanonical base
Implementation Method 2
hybridizing said 5' overhang sequence to said adapter sequence
Data Source
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AI summary
Methods and compositions to minimize barcode exchange during the preparation of barcoded next-generation sequencing libraries prepared from a single cell. The methods utilize oligonucleotides containing a 3'-terminated blocking group or sequences that prevent amplification or extension.