Multimeric Molecular Barcoding for Accurate FFPE 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 barcode nucleic acids of single cells and microparticles, allowing for high-throughput sequencing by appending unique barcode sequences to sub-sequences of target nucleic acids, followed by freezing and thawing steps to enhance lysis and annealing efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If standard DNA sequencing machines are used, then sequencing throughput is achieved, but raw accuracy and readlength are limited
Solution Approach 1:
The patent segments the sequencing process by attaching unique barcodes to individual DNA molecules before sequencing. This allows multiple sequenced molecules to be grouped and analyzed together, effectively increasing throughput while maintaining accuracy through computational grouping rather than relying solely on the physical sequencing machine's raw capabilities.
Solution Approach 2:
The patent introduces molecular barcodes as intermediary elements that mediate between the DNA molecules and the sequencing process. These barcodes serve as identifiers that allow accurate tracking and grouping of molecules throughout the sequencing process, enabling improved accuracy without sacrificing throughput.
2Adaptability or versatility
If FFPE samples are analyzed, then historical clinical samples can be studied, but DNA fragmentation and damage limit analysis quality
Solution Approach 1:
The patent performs preliminary barcoding of DNA molecules before the DNA undergoes fragmentation and damage from FFPE processing. By attaching barcodes to intact molecules prior to degradation, the method preserves molecular identity information even when the DNA structure is compromised, enabling accurate analysis of degraded FFPE samples.
Solution Approach 2:
The patent converts the harmful effect of DNA fragmentation into a beneficial filtering mechanism. By using barcodes to track original molecules, the method can distinguish between true biological variants and artifacts introduced by fragmentation, actually improving the ability to detect real signals in noisy FFPE data.
3Productivity
If multiple cells are analyzed in parallel, then throughput is improved, but complexity of sample preparation increases
Solution Approach 1:
The patent creates universal barcoded reagents that can simultaneously perform multiple functions: cell identification, DNA molecule tracking, and sequencing group assignment. This multi-functionality simplifies the overall process by consolidating what would otherwise require separate operations into a single barcoding step, reducing complexity while enabling parallel analysis of multiple cells.
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 improves sequencing accuracy and throughput, enabling efficient analysis of fragmented and damaged DNA samples, such as FFPE samples, by reducing sequencing errors and enhancing the ability to analyze multiple cells or microparticles in parallel.
Implementation Method 1
Molecular barcoding generally involves attaching (for example, by ligation or by primer-extension) a unique nucleic acid label (a 'barcode') to several single target molecules
Implementation Method 2
followed by freezing and thawing steps to enhance lysis and annealing efficiency
Data Source
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.