Single Cell ATAC-Seq Transposase Partitioning
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Solution Overview
Problem
Current methods for single-cell Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq) face limitations, including high variability, low read counts per cell, and throughput issues, which hinder clinical decision-making and fail to capture cell-to-cell variation effectively.
Innovation Solution
A method involving the generation of partitions containing biological particles, nucleic acid barcode molecules, transposon end nucleic acid molecules, and transposase molecules, which produce barcoded nucleic acid fragments through transposition and amplification, enabling sequencing and reducing mitochondrial contamination.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If single cell ATAC-seq methods utilize sample pooling, cell indexing, and cell sorting, then cell-to-cell variation can be captured, but high variability and low read counts per cell result
Solution Approach 1:
The patent divides the chromatin analysis into two distinct segments: first, transposase-based fragmentation of chromatin into accessible regions, and second, tagmentation-based amplification and barcoding. This segmentation allows separate optimization of each step, improving both cell-to-cell variation detection and read count reliability by preventing premature amplification bias.
Solution Approach 2:
The patent performs chromatin accessibility tagging with transposase before cell lysis and nucleic acid extraction. This preliminary action preserves the in vivo chromatin state and ensures that accessibility patterns are captured before any amplification or processing steps that could introduce variability or bias.
2Productivity
If programmable microfluidic devices are used to isolate single cells and perform scATAC-seq in nanoliter reaction chambers, then throughput is improved, but the assay cannot generate personal epigenomic profiles on a timescale compatible with clinical decision-making
Solution Approach 1:
The patent merges multiple steps into a single tagmentation reaction: chromatin accessibility tagging, DNA fragmentation, and library preparation occur simultaneously in one pot. This consolidation eliminates multiple processing steps and reduces total assay time, enabling clinical-scale throughput while maintaining single-cell resolution.
Solution Approach 2:
The patent employs a continuous tagmentation reaction that proceeds without interruption or intermediate purification steps. The transposase remains active throughout the reaction, continuously fragmenting and tagging accessible chromatin regions, which maintains reaction efficiency and reduces processing time compared to discontinuous methods.
3Quantity of substance
If traditional ATAC-seq methodology is used with large pools of cells processed in bulk, then data representative of entire cell population is obtained, but cell-to-cell variation information is lost
Solution Approach 1:
The patent applies local quality by assigning unique molecular identifiers (UMIs) and cell barcodes to individual chromatin fragments during tagmentation. This allows each fragment to carry information about its specific cell of origin and accessibility pattern, enabling reconstruction of cell-to-cell variation while maintaining the simplicity of bulk processing.
Solution Approach 2:
The patent creates molecular copies of chromatin accessibility patterns through transposase insertion of barcoded adapters. Each accessible region generates multiple tagged DNA copies that all carry the same cell-specific barcode, allowing amplification and sequencing while preserving the original single-cell accessibility information.
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 enhances the sensitivity and accuracy of ATAC-seq by reducing noise and increasing the median fragments per cell barcode, providing more comprehensive epigenomic profiles compatible with clinical timelines.
Implementation Method 1
a transposase-nucleic acid complex comprising a transposase molecule of the plurality of transposase molecules and a transposon end oligonucleotide molecule of the plurality of transposon end oligonucleotide molecules, wherein the transposase-nucleic acid complex generates a plurality of template nucleic acid fragments
Implementation Method 2
generating a barcoded nucleic acid fragment using a nucleic acid barcode molecule of the plurality of nucleic acid barcode molecules and a template nucleic acid fragment of the plurality of template nucleic acid fragments
Data Source
AI summary
Methods and systems for sample preparation techniques that allow amplification (e.g., whole genome amplification) and sequencing of chromatin accessible regions of single cells are provided. The methods and systems generally operate by forming or providing partitions (e.g., droplets) including a single biological particle and a single bead comprising a barcoded oligonucleotide. The preparation of barcoded next-generation sequencing libraries prepared from a single cell is facilitated by the transposon-mediated transposition and fragmentation of a target nucleic acid sequence. The methods and systems may be configured to allow the implementation of single-operation or multi-operation chemical and/or biochemical processing within the partitions.