Transposase Barcoding for Single-Cell Structural Variation Detection
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Solution Overview
Problem
Current methods for processing biological samples, such as PCR and sequencing, lack efficient techniques for accurately analyzing and identifying genetic variations within individual biological particles.
Innovation Solution
The method involves generating template nucleic acid fragments using a transposase-nucleic acid complex within biological particles, followed by partitioning these particles into droplets or wells, where barcoded nucleic acid fragments are created. These barcoded fragments are then pooled and analyzed to determine allelic distributions and structural variations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional PCR and sequencing methods are used to process biological samples, then general genetic analysis can be performed, but accurate identification of genetic variations within individual biological particles cannot be achieved
Solution Approach 1:
The patent segments the biological sample into individual biological particles (cells), and further segments the genetic material into barcoded nucleic acid fragments. Each fragment receives a unique barcode identifier, enabling individual tracking and precise analysis of genetic variations at the single-cell level, thereby achieving high measurement precision without requiring overly complex processing systems.
Solution Approach 2:
The patent applies preliminary action by pre-attaching unique barcode sequences to nucleic acid fragments before pooling and sequencing. This pre-tagging enables direct identification and classification of genetic variations during data analysis, eliminating the need for complex post-processing methods and achieving accurate genetic variation identification through simplified workflows.
2Loss of information
If individual biological particles are analyzed separately to identify genetic variations, then precise allelic distribution data can be obtained, but processing time and complexity increase
Solution Approach 1:
The patent merges individual particle analysis with bulk processing by pooling barcoded nucleic acid fragments from multiple biological particles into a single sequencing reaction. The unique barcodes enable computational separation and reconstruction of allelic distributions after sequencing, achieving complete genetic information recovery while significantly reducing processing time compared to individual particle analysis.
Solution Approach 2:
The patent uses barcode sequences as information copies that are attached to each nucleic acid fragment. These barcode copies serve as identifiers that allow the system to track and reconstruct the origin of each sequenced fragment, enabling complete allelic distribution data recovery from pooled samples without requiring time-consuming individual particle processing.
3Productivity
If barcoded nucleic acid fragments are pooled and sequenced together, then processing efficiency increases, but accurate attribution of sequences to individual biological particles becomes challenging
Solution Approach 1:
The patent introduces barcode sequences as intermediary elements that mediate between the nucleic acid fragments and the sequencing analysis system. These barcodes act as unique identifiers attached to each fragment, enabling the system to accurately attribute sequenced reads to their original biological particles even when processed in pooled format, thereby maintaining high measurement precision while achieving high productivity.
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 precise identification of allelic distributions and structural variations within biological particles, facilitating accurate analysis and characterization of genetic material.
Implementation Method 1
generating, in each respective biological particle of a plurality of biological particles obtained from a biological sample, a corresponding plurality of template nucleic acid fragments using a transposase-nucleic acid complex comprising a transposase molecule and a transposon end nucleic acid molecule in the respective biological particle
Data Source
AI summary
Template nucleic acid fragments are generated in cells or nuclei using transposase-nucleic acid complexes. Partitions are formed, each comprising a single cell or nuclei, the corresponding plurality of template nucleic acid fragments and nucleic acid barcodes comprising a corresponding common barcode sequence unique to a respective cell or nuclei. Barcoded nucleic acid fragments are generated in each partition using the barcodes and the template fragments. The barcoded fragments in each partition collectively form a pool of barcoded nucleic acid fragments. A set of alleles for each locus in a plurality of loci are identified and, for each such locus, a subset of the pool of barcoded fragments mapping to the locus are aligned to determine an allelic identity of such fragments from among the set of alleles for the locus, thereby determining a corresponding allelic distribution at each respective locus. These distributions are used to identify a structural variation.