Multimodal Profiling via Unified Tn5 and Reverse Transcriptase Workflow
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Solution Overview
Problem
Current sequencing techniques, such as mcSCRB-Seq and SHARE-Seq, provide limited depth of coverage and require large amounts of tissue, necessitating the need for methods and systems that can offer increased depth of coverage and efficiency for multimodal profiling.
Innovation Solution
The method involves stabilizing a target using agents like Tn5 transposome complexes or Tn5 transposase reagents, combined with reverse transcriptase enzymes that can transcribe DNA and RNA templates, and perform template switching to generate libraries, allowing for simultaneous profiling of transcriptomes and chromatin accessibility through RNA and ATAC sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional sequencing techniques (mcSCRB-Seq, SHARE-Seq) are used for multimodal profiling, then cellular identity and regulatory mechanisms can be identified, but the depth of coverage is limited and large amounts of tissue are required
Solution Approach 1:
The patent combines RNA sequencing and ATAC sequencing into a single unified library preparation workflow. By merging these two modalities into one process using a single Tn5 transposase reaction and unified library construction, the method achieves deep coverage of both transcriptome and chromatin accessibility from the same input material, eliminating the need for separate parallel experiments that would consume more tissue
Solution Approach 2:
The Tn5 transposase-based library preparation method serves multiple functions simultaneously: it performs chromatin accessibility profiling (ATAC-seq), RNA capture and conversion, and library construction in a single universal protocol. This multi-functional approach allows the same reagent system to handle both DNA and RNA modalities, maximizing the information extracted from minimal input tissue
2Adaptability or versatility
If multiple separate sequencing experiments are performed for transcriptome and chromatin accessibility profiling, then comprehensive cellular profiling is achieved, but time consumption and cost increase
Solution Approach 1:
The patent merges RNA-seq and ATAC-seq workflows into a single integrated experiment. The unified library preparation protocol processes both RNA and chromatin accessibility data from the same cell population in parallel, reducing the number of separate experimental steps, reagent additions, and library construction procedures from two independent experiments to one coordinated process
Solution Approach 2:
The method performs preliminary actions by simultaneously preparing both RNA and chromatin libraries during the same Tn5 transposase incubation and library construction steps. Cell-specific barcodes are introduced early in the process to label both modalities, enabling downstream multiplexed sequencing and analysis without requiring separate experimental timelines
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 higher coverage of the transcriptome and chromatin accessibility, scalable to large numbers of cells, with improved data quality and reduced sequencing costs, outperforming existing technologies by achieving deeper RNA and ATAC profiles with higher cell recovery and stringent QC filtering.
Implementation Method 1
performing a tagmentation on the target using an agent, wherein the agent can include a Tn5 transposome complex, a Tn5 transposase reagent
Implementation Method 2
generating a library using a reverse transcriptase enzyme, wherein the reverse transcriptase enzyme can be configured to transcribe DNA from both a DNA template and an RNA template and perform a terminal transferase activity by a template switching
Implementation Method 3
placing the target on a solution, wherein the solution comprises an effective amount of phosphate-buffered saline (PBS), RNase inhibitor, sodium dodecyl sulphate (SDS)
Implementation Method 4
performing polymerase chain reaction (PCT) amplification on the cDNA and the chromatin of the target at a predetermined cycle
Data Source
AI summary
The present disclosure relates to the methods and systems for multimodal profiling. The method can include stabilizing a target, performing a tagmentation on the target using an agent, and generating a library using a reverse transcriptase enzyme. The agent can include a Tn5 transposome complex, a Tn5 transposase reagent, or a combination thereof, and the Tn5 transposome complex or the Tn5 transposase reagent can be loaded with an adapter. The reverse transcriptase enzyme can be configured to transcribe DNA from both a DNA template and an RNA template and perform a terminal transferase activity by template switching and an introduction of an adapter sequence into a cDNA and a transposed chromatin of the target.