UMI Linear Probe Spatial Transcriptomics
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Solution Overview
Problem
Current methods fail to simultaneously detect all expressed genes on a subcellular level and obtain their sequence and spatial information with high resolution.
Innovation Solution
A method involving a linear probe with a Unique Molecular Identifier (UMI) is used to reverse transcribe and amplify specific mRNA regions, generating DNA nanoballs through Rolling Circle Amplification (RCA) for sequencing, allowing for the spatial localization and sequencing of target mRNA sequences on a tissue sample.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional spatial transcriptomics methods are used, then spatial location information can be obtained, but sequence information and mutation detection capability are lost
Solution Approach 1:
The patent combines spatial location information (via padlock probes with SUMI) and sequence information (via reverse transcribed cDNA with UMI) into a single integrated workflow. Both types of information are captured simultaneously during the same experimental process, allowing mutual reinforcement rather than trade-off
Solution Approach 2:
The linear probe acts as an intermediary that bridges spatial detection and sequencing. It contains both the binding region for spatial localization and the UMI for sequence information, serving dual functions that connect the two information types
2Measurement precision
If high resolution spatial detection is achieved, then subcellular level localization is possible, but simultaneous detection of all expressed genes becomes challenging
Solution Approach 1:
The linear probe design is universal and can bind to any mRNA target through its binding region while maintaining the same UMI and padlock probe compatibility. This allows the same workflow to detect all expressed genes simultaneously at high spatial resolution without requiring target-specific optimization
Solution Approach 2:
The probe system is segmented into modular components (binding region, UMI, padlock regions) that can independently function. The binding region provides target specificity while the UMI and padlock regions provide universal spatial and sequencing capabilities, enabling parallel detection of multiple genes
3Ease of manufacture
If pre-defined UMI sequences are used, then sequencing is simplified, but the ability to capture unique molecular information is reduced
Solution Approach 1:
The patent changes the UMI from a fixed pre-defined sequence to a random sequence generated during probe synthesis. This parameter change increases the information content (molecular uniqueness) while maintaining sequencability through high-throughput sequencing technologies that can handle random sequences
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 method provides higher resolution in obtaining both spatial and sequence information of target sequences, enabling precise detection and localization of mRNA on a tissue sample.
Implementation Method 1
hybridizing the linear probe with its binding region to the m-RNA strand
Implementation Method 2
complementing the linear probe using the m-RNA strand as template thereby obtaining a reversed transcribed c-DNA strand
Implementation Method 3
hybridizing a locator molecule with its 3' and 5' ends to the first and second locator regions thereby creating a gap corresponding to the length of the UMI
Implementation Method 4
Filling the gap in the locator molecule with nucleotides complementary to the UMI using a non-strand displacement enzyme thereby creating a circular template
Implementation Method 5
multiplying the circular template molecule by RCA on the tissue sample, starting from a primer region thereby creating a rolony
Implementation Method 6
amplification of the primed single stranded oligomer by PCR
Data Source
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AI summary
The invention is directed to a method to obtain the spatial location and sequence information of a target sequence of at least one m-RNA strand on a tissue sample comprising the steps a. providing a linear probe, containing a) a binding region capable of binding to the at least one m-RNA strand and b) an anchor sequence comprising a UMI region located between a first and a second locator regions and c) a primer region; b. hybridizing the linear probe with its binding region to the m-RNA strand; c. complementing the linear probe using the m-RNA strand as template thereby obtaining a reversed transcribed c-DNA strand d. hybridizing a locator molecule with its 3' and 5' ends to the first and second locator regions thereby creating a gap corresponding to the length of the UMI of the linear probe e. Filling the gap in the locator molecule with nucleotides complementary to the UMI using a non-strand displacement enzyme thereby creating a circular template comprising a copy of the UMI region from the linear probe. f. multiplying the circular template molecule by RCA on the tissue sample, starting from a primer region thereby creating a rolony g. Sequencing at least the UMI portion of the rolonies thereby obtaining the spatial location of the m-RNA on the tissue h. removing the reversed transcribed c-DNA strand from the tissue and dehybridizing the m-RNA strand thereby obtaining a single stranded c-DNA oligomer i. providing the single stranded cDNA oligomer with a first and a second adaptor primer at the 3' and 5' ends obtaining a primed single stranded oligomer; amplification of the primed single stranded oligomer by PCR j. Sequencing the amplified primed single stranded oligomer and linking the spatial information of the rolonies with the sequence information of the amplified primed single stranded oligomer via the UMI sequence