Patent Application
20220403462
This application claims priority to EP21180189.9, filed in European Patent Office on Jun. 18, 2021
Not applicable.
Not applicable.
The invention is directed to a method for obtaining sequencing and spatial information of genes on a subcellular level on tissue.
It has been challenging to detect all expressed genes on a sub cellular level on tissue by keeping the spatial information and afterwards sequences those genes for potential variants. It is especially challenging to reach sub cellular resolution. The described method in this application allows a theoretical resolution down to a few tens of nanometers and allows to link the spatial information on the tissue with the sequenced information of the expressed gene.
Such technology is developed by the company Spatial Transcriptomics and 10× genomics. Here the spatial information is maintained as the tissue RNA molecules are tagged with a spatial identifier pre-spotted on an array. The resulting libraries are later sequenced by standard in vitro NGS sequencing approaches on Illumina for example. With the spotting process the position of the spatial identifier on the array is known before the sequencing process takes place. After sequencing of the spatial identifier, the linked RNA sequence of interest can be assigned to the tissue location. One major limitation of this approach is the resolution of the technology as it is dependent on the feature size of the spots on the array which is currently on a multicellular level only. Spatial Transcriptomics has multiple granted patents whereas the molecules are barcode labeled and are sequenced off substrate. It is also necessary to do a library preparation of all collected barcoded molecules. The presented method here does not remove the molecules from the substrate instead directly determines the sequence on the substrate. References are: US20150344942A1 METHODS AND PRODUCT FOR OPTIMISING LOCALISED OR SPATAL DETECTION OF GENE EXPRESSION IN A TISSUE and W02016162309A1 SPATIALLY DISTINGUISHED, MULTIPLEX NUCLEIC ACID ANALYSIS OF BIOLOGICAL SPECIMENS.
Another approach to combine genetic and special information are disclosed in WO2012/140224, by Fredrik Salmén et al in Nature protocols 2018 “Barcoded solid-phase RNA capture for Spatial Transcriptomics profiling in mammalian tissue” and Sanja Vickovic et al. “High-density spatial transcriptomics arrays for in situ tissue profiling”, Nature methods, Sep. 9, 2019.
The described method is being used to detect mRNA on tissue with a high spatial resolution of 300-500 nm with a simplified workflow compared to other competitive solutions in the market right now and without the requirement for a spatial identifier.
Object of the invention is therefore a method to obtain the spatial location and sequence information of a target sequence in a sample comprising at least one m-RNA strand comprising the steps
In a first embodiment of the invention as shown in
In a second embodiment of the invention as shown in
Various exemplary details are described with reference to the following figures, wherein:
The method of the invention is conducted in several steps which are explained in detail in the following:
First, a solid, flat two-dimensional substrate is provided with a functionalized surface, for example with an activated carboxylate group or succinimidyl ester. To this activated surface, the spacer groups are attached. In the variant of poly-T groups, this can be achieved by amine-modified oligonucleotides; in another variant using antibodies provided with amino groups can be used. The solid substrate also contains at least one, preferable 2 independent fiducial marks.
Afterwards the m-RNA molecules are loaded on the surface with a buffer solution. The m-RNA molecules will interact with the functionalized surface and randomly spatially distribute on the entire area of the surface. The spatial or surface density of the m-RNA molecules can be controlled by the loaded concentration.
The spacer units and therefore in turn the m-RNA molecules are preferable randomly distributed on the substrate. The distribution density may be controlled via concentration, temperature, pH and surface functionalization. Preferable, the sample is permeabilized after providing to the surface.
A tissue sample is brought in contact with the solid substrate where all the single molecules are located. The 5-50 bases long poly T tail will then be hybridized to the expressed mRNA in the tissue.
The tissue can optionally be stained for example with DAPI or hematoxylin or eosin and imaged. Images are also taken of those fiducial marks and X-Y position are recorded. Each sequence of each individual single molecule is recorded and the spatial location relative to the fiducial marks is stored as X-Y distances.
After providing the sample to the surface, the sample may be stained to obtain the spatial location relative to the fiducial markers. Optionally, the sample is stained allowing to determine morphology of the sample.
The tissue is then removed from the substrate enzymatically or chemically, for example with proteinase K. Afterwards the single molecules with the mRNA on may then be reversely transcribed into cDNA.
As a next step the solid substrate with single molecules on it is denatured so that the double stranded DNA molecule is forming a single stranded oligonucleotide.
Optionally, the spatial location of the sample and the spatial location of the sequenced rolonies are superimposed relative to the location of the fiducial marker.
Padlock probes targeting various genes with a gap are added. The padlock probes will bind to the different mRNA if the gene is expressed. After that the gap is also reversely transcribed and the gap filled padlock are ligated into circle templates. Then rolling circle amplification is carried out on padlocks.
In a variant, the method of the invention is performed by hybridizing the at least one oligonucleotide to complementary parts of the at least one single stranded oligomer thereby creating a padlock unit with a gap between the 5′ and the 3′ end of the oligonucleotide and filling the gap of the padlock unit with complementary nucleic acids as target sequence and ligate them to generate the single strand circular template.
In another variant, the method of the invention is performed by hybridizing the at least one oligonucleotide to complementary parts of the at least one single stranded oligomer and ligate the 5′ and the 3′ end of the oligonucleotide to generate the single strand circular template, wherein the complementary parts of the at least one single stranded oligomer define the target sequence.
Preferable, the oligonucleotide comprises at least one primer sequence for the polymerase capable of rolling circle amplification. In alternative the oligonucleotide may be provided with at least one primer sequence for the polymerase capable of rolling circle amplification by ligation of a primer oligonucleotide.
In another variant, the oligonucleotide is provided with or comprises with at least two different primer sequences can be used which can be consecutively sequenced and help to avoid optical crowding if multiple rolonies light up next to each other.
The location of each rolony on the substrate can be correlated with the original position of the tissue by taking one or more second images.
Finally, a sequencing primer is added and then the padlock with the filled gap is sequenced and each base is determined. The location of each rolony on the substrate can later on correlated with the original position of the tissue. That means the location of the gene on the tissue can be determined and it can be checked if the gene had a mutation or not via sequencing.
Preferable, sequencing information is obtained by sequencing by synthesis process. Sequencing by synthesis is performed by subsequently hybridizing fluorescently labelled nucleotides to the rollonies wherein the hybridized fluorescently labelled nucleotide provides a detectable fluorescence signal.
Exemplary Workflow Based on Variant with polyT Tail as shown in
Exemplary workflow based on variant with antibody capturing cap-end of mRNA as shown in
While various details have been described in conjunction with the exemplary implementations outlined above, various alternatives, modifications, variations, improvements, and/or substantial equivalents, whether known or that are or may be presently unforeseen, may become apparent upon reviewing the foregoing disclosure. While the systems and methods above are directed to the separation of biological materials such as cells, they may also be applied to the separation of other sorts of inert particles suspended in a fluid, such as paints or slurries. And while the systems and methods are described above with respect to separating a single, individual particle or cell, it should be understood that a plurality of cells or particles may be separated. Accordingly, the exemplary implementations set forth above, are intended to be illustrative, not limiting.
| Number | Date | Country | Kind |
|---|---|---|---|
| 21180189.9 | Jun 2021 | EP | regional |
