Patent Description:
The human body contains several hundred terminally different cell types such as muscle cells, enterocytes, and Purkinje neurons for example which basically share the same genome but exhibit strikingly different phenotypes. Ultimately these phenotypes are related to molecular interactions inside these cells between different classes of molecules such as the genome (DNA), the transcriptome (RNA), the proteome (protein), etc..

Document <CIT> discloses nucleic acid probes for sequencing nucleic acids. Single cell RNA sequencing and other single multi-omics technologies have followed the advent of next generation sequencing platforms and allow to make massively parallel measurements of these data layers measuring hundreds, thousands or even "genome-wide" (approx. <NUM>,<NUM> protein coding genes). These methods are collectively referred to as multi-omics. While these measurements are very useful to map the transcriptional cell state space and increasingly provide access to other data layers at scale, they rely on dissociating tissue into suspensions of cells. Tissue dissociation, however, removes cells from their surrounding taking away the auto-, para-, and jux-tracrine signalling inputs as well as severing the attachment of the cell to the extracellular matrix and as such has profound impact on the cell. A cell in suspension immediately rounds
and does no longer display a normal cell morphology. These changes also impact the various molecular layers described above.

This drawback has fuelled interest in technologies that can similarly perform multi-omics characterization, but in the tissue context. The currently available technologies include protein multiplexing, MIBI/IMC, various single-molecule FISH-based methods (e.g. MERFISH, SeqFISH), and spatial profiling methods such as the Visium platform from 10x Genomics or Nanostring's GeoMx. Neither of the aforementioned methods can measure the whole transcriptome at the single level within a tissue section as either the plexing or the spatial resolution is not high enough. , (<NUM>) propose a DNA origami comprising staple strand and scaffold strand. Rothemund P. (<NUM>) discloses nanostructure backbone comprising staple strand and scaffold strand. <CIT> relates to nucleic acid nanostructure comprising staple strand and scaffold strand.

It is an object to provide a marker and a method that enable tracking biological samples between different types of analyses in an efficient and fast way.

The aforementioned object is achieved by the subject-matter of the independent claims. Advantageous embodiments are defined in the dependent claims and the following description.

The present invention overcomes the limitations and provides a means to connect the cellular phenotypes with multi-omics in particular single cell RNA seq and ATAC seq measurements. Importantly the present invention uses markers that are compatible with tissue samples or cell suspension samples or particle suspension samples to mark and analyse those samples. A further aspect is, that a large number of unique markers may be generated, which can be readout by a microscope and by DNA sequencing like NGS. Thus, the present invention allows phenomics-<NUM>-multi-omics characterisation at scale.

In one aspect, a marker for marking a biological sample or a discrete entity comprising the biological sample is provided. The marker comprises an oligonucleotide nanostructure backbone with a plurality of attachment sites at predetermined positions. Further, the marker comprises a plurality of labels for attachment to at least some of the attachment sites, and at least a first orientation indicator and a second orientation indicator. Each label comprises at least one dye, an encoding oligonucleotide portion configured to encode characteristics of the at least one dye, and an attachment oligonucleotide portion configured to reversibly attach the label to one of the attachment sites. Further the attachment oligonucleotide portion of each label comprises a unique oligonucleotide sequence configured to bind to a complementary sequence of one of the attachment sites.

An oligonucleotide is, for example, a single stranded DNA or RNA molecule, that may be sequenced to determine its sequence of nucleotides. Complementary parts of oligonucleotides may hybridise or bind to each other. Preferably, the biological sample comprises at least one cell.

The orientation indicators are configured to attach to the backbone, and may be used to visually determine the orientation of the marker in space. The encoding oligonucleotide portion is a unique sequence of nucleic acids that are unique to the excitation/emission wavelength and/or the fluorescent lifetime of the at least one dye of the label. The marker is thus visually unambiguously identifiable by its unique combination of dyes in each label as well as their specific attachment sites. The same marker is unambiguously identifiable by sequencing the unique encoding oligonucleotide portions and the attachment oligonucleotide portions.

Preferably, the discrete entity is comprised of a polymeric compound. The polymeric compound can be polymerised to form the discrete entity. In particular, the polymeric compound can form a hydrogel. The diameter of the discrete entities or hydrogel beads may be in the range of <NUM> to <NUM>. Particularly preferred ranges are <NUM> to <NUM>, <NUM> to <NUM> and <NUM> to <NUM>. This enables embedding and culturing of the biological sample in the discrete entity in scaffold-based suspension 3D cell culture, which combines the benefits of 3D suspension cell culture and scaffold-based cell culture.

Preferably, the nanostructure backbone comprises scaffold strands, and staple strands configured to bind to the scaffold strands at predetermined positions to fold the scaffold strand into a predetermined shape. The nanostructure backbone may be a DNA-origami. These DNA origami structures may range in size from a few nanometres into the micron range. For the fabrication of such DNA origami-based structures longer DNA molecules (scaffold strands) are folded at precisely identified positions by so called staple strands. The DNA origami may be designed to provide a self-assembly nanostructure backbone of a particular predetermined shape. This enables an easy and reproducible synthesis and assembly of the backbone. Staple strands may be position-selectively functionalised. The positional resolution in this case is limited by the size of a nucleotide, which is in the range of a nanometre or below. This has been exploited in the prior art to generate fluorescent standards, wherein fluorescent dyes are connected to precisely located bands on the DNA origami. These standards are known as "nanoruler" and are used for the calibration of imaging systems like confocal or super resolution microscopes (e.g. STED), for example, as disclosed by <CIT>.

The DNA origami provides a scaffold for the labels. Preferably, the DNA origami structure comprises at least one scaffold strand and multiple staple strands, wherein the staple strands are complementary to at least parts of the scaffold strand and configured to bring the scaffold strand into a predetermined conformation. In particular, the strands are oligonucleotides. This enables generating nanostructure backbones with predetermined two- or three-dimensional shapes that can self-assemble. Further, this enables the site-specific placement of attachment sites on the backbone.

The attachment sites being unique nucleic acid sequences, preferably of the staple strands. Preferably, the labels may be attached to staple strands of the nanostructure backbone at predetermined attachment sites. Since the staple strands are located at predetermined positions the positions of the attachment sites may equally be predetermined. Thus, the attachment site is a unique oligonucleotide sequence complementary to the attachment oligonucleotide portion of one label.

Preferably, the largest spatial extent of the nanostructure backbone is in a range from <NUM> to <NUM>, preferably in a range from <NUM> to <NUM>. This enables a particularly compact marker.

Preferably, the attachment sites are spaced apart from each other in a range from <NUM> to <NUM>, preferably in a range from <NUM> to <NUM>. This enables a particularly dense arrangement of labels on the nanostructure backbone. The spacing between the attachment sites may be chosen depending on the resolving power of a readout device used to read out the labels. Particularly preferable ranges may correspond to the lateral resolution achievable with different microscopic modalities such as for example single molecule localization microscopy (<NUM> to <NUM>), structured illumination and STED microscopy (<NUM> to <NUM>), high NA (numerical aperture) light microscopy (around <NUM>), and low NA light microscopy (around <NUM>). Importantly, the labels may be distanced from each other such that the readout device can resolve the labels individually.

Preferably, the labels comprise primer sequences configured to enable sequencing of the attachment portion and the encoding portion, preferably, all labels comprise the same primer sequences. This enables particular efficient sequencing of the portions. Preferably, each label comprises a cleavage site configured to separate the dye from the label. The cleavage site may allow enzymatic, temperature, or light induced cleavage. This enables efficient removal of the dye from portions of the label, particularly prior to sequencing the portions.

Preferably, the dye is a fluorophore. Preferably each label has fluorophore(s) with different characteristics, such as at least one of excitation wavelength, emission wavelength and fluorescence lifetime, to enable generating a larger number of different markers.

Preferably, the marker comprises an anchor portion configured to attach the marker to a specific biological feature of the biological sample. For example, the anchor portion may be an oligonucleotide (part of the label or part of the nanostructure) configured to attach to a genomic sequence of the biological sample. This enables localisation of the marker in the cell nucleus, for example.

Preferably, the nanostructure backbone extends linearly in one dimension and the first orientation indicator and the second orientation indicator are spaced apart from each other, or arranged on opposite ends of the nanostructure backbone. This enables determining the orientation of the marker. The orientation indicators may comprise fluorescent dyes, for example. In particular, the first orientation indicator and the second orientation indicator have different properties, such as excitation wavelength, fluorescence emission wavelength, and/or fluorescence lifetime.

Preferably, the nanostructure backbone extends in two dimensions or three dimensions and the nanostructure backbone comprises at least a third orientation indicator. This enables determining the orientation of the marker.

In a further aspect a method is provided for analysing a biological sample comprising the following steps: introducing a plurality of markers according to one of the preceding claims into the biological sample or into a discrete entity comprising the biological sample; Acquiring at least one optical readout of the biological sample and the markers; determining in the optical readout for at least a first part of the biological sample the individual markers associated with the first part of the sample based on the at least one dye of each of the labels of each marker, in particular on the fluorescent characteristics of each label and its particular attachment site; dissociating the biological sample or the discrete entity into dissociated sample parts and separating the dissociated sample parts; sequencing a genetic content of at least one of the dissociated sample parts together with the encoding oligonucleotide portion and the attachment oligonucleotide portion of the respective associated markers to generate sequencing data; determining in the sequencing data a presence of the sequences of the attachment oligonucleotide portion and the encoding oligonucleotide portion; and correlating the sequencing data to the first part of the biological sample based on the presence of the sequences and the individual markers present in the first part of the sample.

The markers introduced preferably differ by their labels and/or the positions of label on nanostructure, that means the particular attachment sites of the labels. The markers may be introduced into the sample fully assembled or they may be assembled in situ. The optical readout is preferably an image stack or a 3D image.

Dissociation may comprise removing the discrete entity. The dissociated sample parts may be individual cells or small cell clusters.

This enables linking phenotypic information from the optical readout to genotyping information from the sequencing data.

When the biological sample are embedded in a discrete entity such as a polymeric compound, in particular a hydrogel, the associated markers and the biological sample are kept in close proximity. This enables particularly easy handling of the biological sample with the markers. For further examples of a discrete entity comprising a biological sample and a generalised method for imaging a discrete entity comprising a biological sample, reference is made to the applications <CIT>.

The optical readout may be an image-based readout, which may be acquired on a microscope like a point-scanning confocal or a camera-based/widefield imaging system for example a spinning disk microscope, a light sheet fluorescence microscope, a light field microscope, a stereomicroscope. Further the optical readout may be non-image based readouts for example in a cytometer or a flow-through based readout device with at least one point detector or a line detector. A readout may consist of a discrete readout, for example a single acquisition of an emission spectrum or image stack, a readout may be a readout data stream, for example in a point-scanning confocal or cytometer, which is substantially continuous. Further a readout may be a sequence of images for example a spectral or hyperspectral image stack, wherein in each image fluorescence emission of different wavelength bands is recorded.

The optical readout may be generated by a readout device used to perform fluorescence multi-colour reading or imaging. The readout device typically includes at least one excitation light source, a detection system including at least one detection channel and may further contain filters and/or dispersive optical elements such as prisms and/or gratings to route excitation light to the sample and/or to route emission light from the sample onto to a detector or onto an appropriate area of the detector. The detection system may comprise several detection channels, may be a spectral detector detecting multiple bands of the spectrum in parallel, or a hyperspectral detector detecting a contiguous part of the spectrum. The detection system contains at least one detector, which may be a point-detector (e.g. a photomultiplier, an avalanche diode, a hybrid detector), an array-detector, a camera, hyperspectral camera. The detection system may record intensities per channel as is typically the case in cytometers or may be an imaging detection system that records images as in the case of plate readers or microscopes. A readout device with one detector channel, for example a camera or a photomultiplier, may generate readouts with multiple detection channels using, for example, different excitation and emission bands.

<FIG> shows schematically oligonucleotide nanostructure backbones <NUM>, <NUM>, <NUM>, <NUM>, <NUM> with different geometries. Generally, the nanostructure backbones <NUM>, <NUM>, <NUM>, <NUM>, <NUM> comprise nucleic acids. In particular, the nanostructure backbones <NUM>, <NUM>, <NUM>, <NUM>, <NUM> are DNA-origami based, which allows generating predetermined, stable two- and three-dimensional shapes. Further, this allows generating a plurality of attachment sites at predetermined positions along the nanostructure backbones <NUM>, <NUM>, <NUM>, <NUM>, <NUM>. The attachment sites are stretches of oligonucleotide, that are unique and allow the hybridisation of complementary oligonucleotides, for example to attach labels to the nanostructure backbones <NUM>, <NUM>, <NUM>, <NUM>, <NUM> at these predetermined positions.

Nanostructure backbone <NUM> is linear or rod-like. It comprises a first orientation indicator <NUM> and a second orientation indicator <NUM>. The orientation indicators <NUM>, <NUM> may be used to determine the orientation, directionality or polarity of the nanostructure backbone <NUM>. The orientation indicators <NUM>, <NUM> may comprise a dye, in particular a fluorescent dye, such as fluorescein or a fluorescent protein. In addition, the dye of the first orientation indicator <NUM> has different characteristics than the dye of the second orientation indicator <NUM>. The characteristics may include fluorescent emission characteristics, excitation characteristics or lifetime characteristics. This enables differentiating between the first and the second orientation indicators <NUM>, <NUM> in an optical readout of the nanostructure <NUM>, for example generated by a microscope, a cytometer, or an imaging cytometer. The orientation indicators <NUM>, <NUM> are arranged spaced apart from each other. Preferably each orientation indicator <NUM>, <NUM> is arranged on the backbone <NUM> at opposite ends. Thus, the first and second orientation indicators <NUM>, <NUM> enable differentiating between a first end and a second end of the backbone <NUM>. Ultimately, this enables determining the orientation, directionality or polarity of the backbone <NUM>, for example from the first orientation indicator <NUM> on the first end to the second orientation indicator <NUM> on the second end.

The nanostructure <NUM> is sheet-like, which may be a large linear DNA molecule or an assembly of multiple DNA molecules. Sheet-like nanostructures may increase the number of available attachment sites substantially. In order to be able to determine the orientation of the nanostructure <NUM>, a third orientation indicator <NUM> is provided.

Further geometries are possible, for example, the tetrahedral nanostructure <NUM>, the cubic nanostructure <NUM>, or the polyhedral nanostructure <NUM>. These may comprise a fourth orientation indicator <NUM> in order to determine their orientation.

<FIG> shows schematically a label <NUM> for attachment to the attachment sites of the nanostructure backbones <NUM>, <NUM>, <NUM>, <NUM>, <NUM>. The label <NUM> comprises several fluorescent dyes 202a, 202b, 202c, 202d, 202e. The fluorescent dyes 202a to 202e may differ in their fluorescent properties, for example, excitation wavelengths, emission wavelengths and fluorescent lifetime characteristics. Preferably, the dyes 202a to 202e of the label <NUM> may be individually identified in a read-out in particular of the label <NUM>. Depending on the number of dyes in the used when generating the label <NUM>, a certain number of unique labels can be generated. Typically, the number of different dyes used in total may be in the range of <NUM>-<NUM>, for example. For <NUM> dyes and when using <NUM> dyes for each label it is easily possible to generate a set of labels with <NUM>,<NUM> unique labels.

The dyes 202a to 202e are individually attached to a label support <NUM>. The label support <NUM> may be an oligonucleotide and each of the dyes 202a to 202e may be specifically attached to the label support <NUM> via a unique hybridisation part 206a, 206b, 206c, 206d, 206e. Moreover, the one end <NUM> of the label support <NUM> may be specifically attached to the nanostructure backbones <NUM> to <NUM>, as described in more detail below. A cleavage site <NUM> is provided to cleave the label support <NUM>. This enables removing the dyes 202a to 202e from the end <NUM> of the label support <NUM>, for example, when the label <NUM> is attached to one of the nanostructure backbones <NUM> to <NUM>. The orientation indicators <NUM>, <NUM>, <NUM>, <NUM>, preferably have the same structure as described for the label <NUM>.

<FIG> shows schematically a variety of rod-shaped markers <NUM>, <NUM>, <NUM>, <NUM>, <NUM>. The markers <NUM> to <NUM> each comprise a first orientation indicator <NUM> and a second orientation indicator <NUM>, at a first attachment site and a second attachment site of the nanostructure backbone <NUM>, respectively. In addition, there are ten further attachment sites, one of which is indicated by reference sign <NUM>. In case of the marker <NUM>, labels <NUM>, <NUM>, <NUM> are attached at three further attachment sites <NUM>. The attachment sites <NUM> of each nanostructure backbone <NUM> is unique such that the labels <NUM>, <NUM>, <NUM> are specifically attachable to a particular attachment site <NUM>. The markers <NUM>, <NUM>, <NUM>, <NUM>, <NUM> are preferably between <NUM> to <NUM> in length.

The orientation indicators <NUM>, <NUM> generate a relative coordinate system for the markers <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, on which each attachment site <NUM> may be placed. For example, each attachment site <NUM> may be assigned an index n with n=<NUM>, <NUM>, <NUM>,. , based on the unique location of the respective attachment site <NUM>. Thus, the different markers <NUM> to <NUM> can all be distinguished visually, due to their use of labels with differing properties and/or the labels being attached at different, distinguishable attachment sites <NUM> along the nanostructure backbone <NUM>.

<FIG> shows schematically details of the rod-shaped marker <NUM>, in particular, the label <NUM> and its attachment to the nanostructure backbone <NUM>. The label <NUM> is attached to the nanostructure backbone <NUM> at a particular attachment site <NUM> of the nanostructure backbone <NUM>. This attachment site <NUM> has a unique oligonucleotide sequence that allows hybridisation of a complementary attachment oligonucleotide portion <NUM> of the label <NUM>. Thus, each attachment site of the marker <NUM> has a unique oligonucleotide sequence enabling to specifically target labels for each one of the attachment sites.

In addition, the label <NUM> comprises an encoding oligonucleotide portion <NUM>. The encoding portion <NUM> is an oligonucleotide sequence that is unique to the fluorescent dye or dyes <NUM> the label <NUM>. This means that the dyes of a particular label may be identified by the sequence of the encoding portion.

The label <NUM> also comprises a cleavage site <NUM> for removing the dye <NUM> from the encoding portion <NUM> and the attachment portion <NUM>.

Thus, each label has a unique sequence for the particular dyes (e.g. the encoding portion <NUM>) and a further unique sequence that hybridises to a particular one of the attachment sites of a nanostructure backbone (e.g. the attachment portion <NUM>).

The first and second orientation indicators <NUM>, <NUM> are similarly constructed. For example, the orientation indicator <NUM> is attached to an attachment site <NUM> of the nanostructure backbone <NUM> by a unique complementary attachment oligonucleotide portion <NUM>. Further, the orientation indictor <NUM> comprises an encoding portion <NUM>, that is unique to the dye or dyes <NUM> of the orientation indicator. The dyes <NUM> may be removed from the encoding portion <NUM> and the attachment portion <NUM> by cleaving a cleavage site <NUM>.

<FIG> shows steps for preparing the label <NUM> for sequencing, for example, to read the information of the attachment portion <NUM> and the encoding portion <NUM>. Initially, the label <NUM> is removed from the nanostructure backbone by heating to melt the hybridised attachment portion <NUM> off the nanostructure backbone. Next the dyes <NUM> are removed from the label <NUM> by cleaving the cleavage site <NUM>, for example, by enzymatic cleavage in case the cleavage site <NUM> is a restriction site. Alternatively, the cleavage site <NUM> may be cleaved by light or temperature. The remaining attachment portion <NUM> and encoding portion <NUM> may be separated, for example, by chromatography and subsequently sequenced. To that end, universal primers may be ligated to the remaining attachment portion <NUM> and encoding portion <NUM>. Alternatively, the universal primers may be provided with the label support.

<FIG> shows schematically an example of a cube-shaped marker <NUM> with a three-dimensional array nanostructure backbone <NUM>. The marker <NUM> comprises three orientation indicators <NUM>. Further, the marker <NUM> comprises a set of different labels <NUM>, <NUM>, <NUM>. Each label <NUM>, <NUM>, <NUM> is attached at a particular attachment site of the marker <NUM>. The attachment sites may, for example, be at the corners or edges of the array of the backbone <NUM>. The labels <NUM>, <NUM>, <NUM> differ in their fluorescent properties, such as fluorescent lifetime, emission wavelength and excitation wavelength.

Thus, similarly to the markers <NUM> to <NUM> in <FIG>, the marker <NUM> may be distinguished from other markers in an optical read-out by the placement of particular labels at specific attachment sites of the backbone <NUM>. In comparison to the rod-shaped markers <NUM> to <NUM>, the cube-shaped marker <NUM> provides for around <NUM> attachment sites for labels, which increases the number of possible combinations of labels and their position on the backbone <NUM>. The physical size of the cube-shaped marker <NUM> is in the range of <NUM> to <NUM> per side of the cube.

<FIG> shows schematically a biological sample <NUM> embedded in a discrete entity <NUM>. The discrete entity <NUM> may be a hydrogel bead. The discrete entity <NUM> further comprises a cube-shaped markers <NUM>, <NUM>. The markers <NUM>, <NUM> may be similar in structure to the marker <NUM>. However, the markers <NUM>, <NUM> may differ from each other in the specific labels attached to the respective backbone and/or in the specific attachment sites the labels are attached to. The biological sample <NUM> may be a single cell, for example.

<FIG> shows schematically a biological sample <NUM>. The sample <NUM> may be a section from a biopsy, for example, comprising a plurality of tissues and/or cells. The sample <NUM> may be stained with a plurality of markers, including the markers <NUM>, <NUM>, <NUM>. As an example, a cell <NUM> of an intestinal part <NUM> of the biological sample <NUM> is described in more detail. The markers <NUM>, <NUM>, <NUM>, represented here by their directionality as arrows, are introduced into the cell <NUM>. By using a plurality of markers, each cell of interest in the sample <NUM> may be stained by a unique combination of markers.

With reference to <FIG> and <FIG>, the cell <NUM> and the cell <NUM> may be one of a larger group of cells. In the case of the sample <NUM> and the cell <NUM>, the entire sample <NUM> may be stained with markers and therefore each cell of the plurality of cells the sample <NUM> is made up of may be stained by a unique combination of markers. The cell <NUM> may be one of a larger group of cells, each cell individually embedded in a discrete entity with a unique combination of markers. Thus, the markers enable identifying cells <NUM>, <NUM> visually in optical readouts of the discrete entity <NUM> or the sample <NUM>.

<FIG> shows schematically a particularly preferred method of staining a biological sample <NUM> with markers. Instead of introducing assembled markers into a biological sample, the markers may be assembled in situ. Exemplarily, the assembly of part of a marker <NUM> is shown. To that end, individual parts <NUM> of the marker <NUM> are introduced into the sample <NUM>. The individual parts <NUM> may include staple strands and scaffold strands of the DNA-origami backbone of the marker <NUM>, as well as the orientation indicators and the labels. Since each of the individual parts <NUM> of the marker <NUM> is smaller than the marker <NUM>, the individual parts <NUM> are easier to introduce into the sample <NUM>. The individual parts <NUM> of the marker <NUM> then hybridise at complementary oligonucleotide portions as indicated by the alpha-numeric notation of the individual parts <NUM>.

Optionally, the marker <NUM>, in particular its nanostructure backbone, may comprise an anchor portion <NUM>. The anchor portion <NUM> is an oligonucleotide sequence that is complementary to a genetic sequence <NUM> of the biological sample <NUM>. For example, the biological sample <NUM> may be a cell and the genetic sequence <NUM> is part of the genome of the cell. This allows anchoring the marker <NUM> inside the sample <NUM>.

<FIG> shows a flow chart of a method for analysing a biological sample. The biological sample is preferably a tissue, in particular a tissue section (as shown in <FIG>). Alternatively, the biological sample is embedded in a discrete entity, particularly a hydrogel bead, in which case the biological sample may be a single cell (as shown in <FIG>).

In a first step S1000, the biological sample is stained. This may include the introduction of a plurality of markers into the sample or the introduction of the individual parts of a plurality of markers for in situ assembly. The plurality of markers is generated with a plurality of labels with different fluorescent properties that are attached at predetermined attachment sites. This allows generating a large variety of distinguishable markers.

In case the biological sample is embedded in a discrete entity, the discrete entity may be stained instead or in addition of the biological sample. Specifically, the markers may be introduced into the discrete entity when the discrete entity is formed during embedding the biological sample in the discrete entity.

In step S1002, an optical readout, preferably an image or an image stack, of the biological sample together with the markers is generated. In case the biological sample is embedded in the discrete entity, the discrete entity may be imaged together with the biological sample and the markers.

In step S1004, the optical readout is analysed to determine the markers associated with at least a first part of the biological sample. The markers may each be identified by the particular labels attached to the particular attachment sites. This includes the fluorescent properties of the dyes of the labels. In case the markers were introduced into the biological sample the markers associated with the biological sample are the markers within the biological sample. In case the biological sample is embedded in a discrete entity, the associated markers are the markers within the discrete entity.

In step S1006, the biological sample is dissociated. This is to generate dissociated sample parts, which are separated subsequently. In case the biological sample is a tissue section, the dissociated sample parts may be individual cells or small cell clusters. In case the biological sample is embedded in a discrete entity, the discrete entity may be dissolved.

In step S1008, the genetic content of least one of the dissociated biological sample parts is sequenced together with the encoding oligonucleotide portion and the attachment oligonucleotide portion of the respective markers associated with the particular biological sample part. This generates sequence data comprising sequences of the encoding oligonucleotide portion and the attachment oligonucleotide portion as well as the genetic content.

In step S1010, the presence of the sequence of the attachment oligonucleotide portions and the sequence of the encoding portions is determined in the sequencing data. This enables determining the presence of respective markers in the dissociated sample part.

In step S1012, the sequencing data is correlated to the first part of the biological sample based on the presence of the sequences and the individual markers present in the first part of the sample. For each marker it is known which labels are attached at which attachment sites, therefore, in the optical readout, the markers present may be identified unambiguously. In the sequencing the identity of the markers present in the sequenced sample can be likewise identified by determining the presence of the respective encoding and attachment oligonucleotide sequences. By comparing these, an optical readout of a sample with particular markers may be correlated or assigned to sequencing data of that sample containing these particular markers. This enables directly linking data about the phenotype in the optical readout with data about the genotype in the sequencing data. The method ends in step S1014.

Claim 1:
A marker (<NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>) for marking a biological sample (<NUM>, <NUM>, <NUM>) or a discrete entity (<NUM>) comprising the biological sample, the marker comprising:
an oligonucleotide nanostructure backbone (<NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>) with a plurality of attachment sites (<NUM>, <NUM>, <NUM>) at predetermined positions,
a plurality of labels (<NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>) for attachment to at least some of the attachment sites (<NUM>, <NUM>, <NUM>),
at least a first orientation indicator and a second orientation indicator (<NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>), each configured to attach to the backbone and for visually determining the orientation of the marker,
wherein each label (<NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>) comprises at least one dye (202a, 202b, 202c, 202d, 202e, <NUM>, <NUM>), an encoding oligonucleotide portion (<NUM>, <NUM>) configured to encode characteristics of the at least one dye (202a, 202b, 202c, 202d, 202e, <NUM>, <NUM>), and an attachment oligonucleotide portion (<NUM>, <NUM>) configured to reversibly attach to one of the attachment sites (<NUM>, <NUM>, <NUM>), and
wherein the attachment oligonucleotide portion (<NUM>, <NUM>) of each label (<NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>) comprises a unique oligonucleotide sequence configured to bind to a complementary sequence of one of the attachment sites (<NUM>, <NUM>, <NUM>).