Method and instrumentation for spatial multiomics using SUMI-technology

Patent metadata

Field	Value
Publication number	US-12286665-B2
Application number	US-202217588371-A
Country	US
Kind code	B2
Filing date	Jan 31, 2022
Priority date	Feb 4, 2021
Publication date	Apr 29, 2025
Grant date	Apr 29, 2025

Abstract

Microscopy imaging that allow for multiple mRNAs, proteins and metabolites to be spatially resolved at a subcellular level provides valuable molecular information which is a crucial factor for understanding tissue heterogeneity as for example within the tumor micro environment. The current invention describes a method (SUMI-Seq) which combines the use of Spatial Unique Molecular Identifier in situ sequencing and in vitro sequencing of rolonies derived from rolling circle amplification from padlock oligonucleotides targeting portion of RNA or cDNA transcript at a subcellular level with less limitation in the amount of transcripts and the length of the sequence that can be analyzed. Apart from padlocks oligonucleotides, the SUMI-Seq method can also be applied using circular oligonucleotides to spatially resolve proteins and metabolites to provide multiomics results.

First claim

What is claimed is:

1. A method for obtaining a spatial location and sequence information of a target sequence in a sample comprising at least one RNA or c-DNA strand comprising the steps;
a. providing an oligonucleotide having 50-1000 nucleotides that are complementary to the at least one RNA or c-DNA strand of the sample;
b. hybridizing the oligonucleotide at its 5′ and 3′ ends to complementary parts of the at least one RNA or c-DNA strand to create a padlock with a gap between the 5′ and the 3′ end of the padlock;
c. filling the gap of the padlock with the complementary nucleotides as target sequence and ligate them to generate a single strand circular template;
d. multiplying the single strand circular template by a polymerase capable of rolling circle amplification into a plurality of DNA concatemers thereby forming rolonies;
wherein providing the oligonucleotide with at least one spatial unique molecular identifier (SUMI) comprising at least 2 nucleotides;
e. determining the spatial location of the rolonies by in situ sequencing of the SUMI;
f. collecting the rolonies after in situ sequencing and transfer them for in vitro sequencing;
g. determining the sequence of the SUMIs and the target sequence by in vitro sequencing of the rolonies; and
h. linking the target sequence of the rolonies with the spatial location obtained from in situ sequencing via the sequence of the SUMIs.

2. The method according to claim 1 , wherein the oligonucleotide comprises at least one primer region with 5 to 50 nucleotides for the rolling circle amplification.

3. The method according to claim 1 , wherein at least one sequencing primer region is located between the SUMI region and the 5′ and/or the 3′ ends of the oligonucleotide.

4. The method according to claim 1 , wherein rolling circle amplification (RCA) to generate rolonies for in situ sequencing is activated by light and/or heat.

5. The method according to claim 1 , wherein after in situ sequencing cells are subjected to single cell sequencing.

6. The method according to claim 1 , wherein the oligonucleotide comprises at least one barcode-tag comprising at least 2 nucleotides.

7. The method according to claim 1 , wherein the rolony transfer includes nucleotide fragmentation and/or targeted enrichment and/or amplification.

Assignees

• Miltenyi Biotec Bv & Co Kg

Inventors

• Rothmann Thomas
• Pinard Robert
• Meyer Hansueli
• Bosio Andreas
• Spiecker Heinrich
• Hosono Seiyu
• Hindman Ryan
• Neil Emily
• Nehme Chris

Classifications

• G01N2458/10

  Oligonucleotides as tagging agents for labelling antibodies · CPC title

• G01N33/6803

  General methods of protein analysis not limited to specific proteins or families of proteins · CPC title

• C12Q1/6876

  Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes · CPC title

• C12Q1/682

  Signal amplification · CPC title

• C12Q1/6804 Primary

  Nucleic acid analysis using immunogens (immunoassay G01N33/53) · CPC title