Patent Description:
In the complex cellular environment, cells of a tissue often display multiple phenotypes, which is reflected in RNA expression profiles of the cells. In the prior art, RNA expression profiling has been applied to single cells in suspensions, typically utilizing microwells or emulsion droplets where the single cells are encapsulated together with microbeads coated with unique cell barcode sequence and poly-T which capture the poly-A mRNA of the lysing cell, labelling the <NUM>' end of the mRNA with the cell barcode sequence in reverse transcription reaction and cDNA synthesis (see e.g. Macosko et al <NUM> and <CIT>). After sequencing the mRNA transcripts derived from same original cell can be grouped together based on the cell barcode, and separate gene expression profiles of all cells generated. The current cell barcoding beads, however, are targeted only to cells in suspension.

To understand the tissue structure, cell functions and cell-to-cell interactions within the tissues, there is a need to link RNA expression data with the cell image and spatial information gained from examination of cells or tissue sections with single cell resolution and high throughput manner. <CIT> discloses a system based on optically readable barcodes for characterization of molecular interactions of single cells. The system is relying on the use of oligonucleotide probes with multiple fluorescent labels. Improved methods are still in need.

The present invention as defined in the appended claims is based on a novel design of microbeads with visible labels coupled with label specific barcodes that make it possible to link the obtained mRNA or DNA sequencing data to spatial coordinates of the cells in a biological sample profiled for RNA expression.

One object of the invention is to provide construct for detecting nucleic acid targets, said construct comprising a bead, preferably a magnetic bead, wherein said bead comprises one or several visually detectable features and/or said bead is coupled to one or several visually detectable entities, and wherein said bead comprises an oligonucleotide, and said oligonucleotide comprises a) an amplification handle sequence, b) a barcode sequence specific to one or several of said visually detectable features and/or entities and c) a <NUM>' terminal capture or anchor sequence, and wherein said oligonucleotide is arranged to be detachable from said bead. Preferably, said terminal capture or anchor sequence comprises a poly-A sequence for hybridizing to a complementary poly-T sequence on commercially available cell barcoding beads.

Alternatively, also the cell barcoding sequence can be placed on the same construct with visible labels.

Another object of the invention is to provide a composition comprising several constructs as defined above preferably with a mixture of distinct visually detectable features or entities.

Another object of the invention is to provide a well plate or chip comprising the composition according to the present invention loaded to the wells of said plate or chip.

Another object of the invention is to provide a kit comprising the well plate or chip as defined above and a computer readable entity comprising a file of a photograph or scan of said plate or chip with the composition of the present invention loaded to the wells of said plate or chip.

Another object of the invention is to provide a use of said construct or composition for identifying desired nucleic acid targets or mRNA sequencing products originating from a single cell or position in biological sample.

Another object of invention is to provide a method for profiling biological samples on a single cell level, the method comprising the steps of:.

The constructs, compositions and methods described in detail below provides means to generate location data for a single cell of a biological sample, such as a tissue section, among single-cell transcriptome data, on the scale of tens of thousands of cells per experiment. The efficiency of any number of diagnostic techniques and applications for assaying various disease states can be enhanced by use of the compositions described herein. The methods and compositions described herein greatly expand the power of single-cell phenotyping by combining location information and transcripts from the same single cells at an unprecedented scale.

As used herein, the term "construct" refers to a chemically synthesized and optionally genetically engineered assembly that comprises a bead attached (preferably covalently) to at least one oligonucleotide sequence by a linker. Each oligonucleotide sequence preferably comprises several functional elements: an amplification handle sequence; a barcode sequence, an optional unique molecular barcode (UMB) sequence that is positioned adjacent to the barcode on its <NUM>' or <NUM>' end, and an anchor sequence for hybridizing to a capture sequence that comprises a sequence complementary to the anchor.

The term "bead" refers herein to a solid support which may encompass any type of solid, porous, or hollow sphere, ball, bearing, cylinder, or other similar configuration composed of plastic, ceramic, metal, or polymeric material (e.g., hydrogel) onto which a nucleic acid can be immobilized (e.g., covalently or non-covalently). The bead may comprise a discrete particle that may be spherical (e.g., microspheres) or have a non-spherical or irregular shape, such as cubic, cuboid, pyramidal, cylindrical, conical, oblong, or disc-shaped, and the like.

The term "amplification handle" generally refers to a functional component of the oligonucleotide sequence in the construct of the present disclosure that provides an annealing site for amplification of the oligonucleotide sequence. In some embodiments, when present in first or additional oligonucleotide sequences, the amplification handle can be the same or different, depending upon the techniques intended to be used for amplification.

The term "barcode" (BC) generally describes a defined oligonucleotide sequence, which when it is a functional element of the construct of the present disclosure, can be used for identifying a particular cell or well in a substrate.

The term "unique molecular barcode" (UMB) generally refers to a random nucleotide, which when it is a functional element of the construct of the present disclosure, is specific for that construct. The UMB permits identification of amplification duplicates of the construct oligonucleotide sequence with which it is associated.

The "linker" refers to any moiety used to attach or associate the bead to the oligonucleotide sequence portion of the constructs. Thus in one embodiment, the linker is a covalent bond. In another embodiment, the linker is a non-covalent bond. The linkers used in the constructs of the compositions and methods are in one embodiment cleavable. The linkers used in the constructs of the compositions and methods are in one embodiment non-cleavable. Without limitation, in one embodiment, the linker is a cleavable linker, e.g., disulfide bond or photocleavable bond. In an example, the parts of the construct are linked to each other via a streptavidin-biotin linkage. In another example, the linker comprises a complex of biotin bound to the construct oligonucleotide sequence by a disulfide bond, with streptavidin fused and coated to the bead. In another embodiment, the biotin is coated to the bead and the streptavidin is fused to the construct oligonucleotide sequence. In another embodiment the bead surface is carboxylated and the oligonucleotide sequence is aminated and linked to the bead with EDC reaction. Photocleavable amino group can be used to enable photocleavage with UV light.

By the term "substrate" is preferably meant a slide, a multi-well plate or a chip. The substrates are conventional and can be glass, polymer or of any conventional materials suitable for the particular assay or diagnostic protocols. The substrates can comprise a matrix of microwells for positioning cells from a biological sample. The substrate can be also a membrane with well structure and with microscopic pores in the well bottoms, to enable easier washing and bead loading.

The terms "visible" and "visually detectable" are used herein to refer to a feature of the present constructs or an entity coupled to said construct that are observable by visual inspection, with or without prior illumination, or chemical or enzymatic activation. In an embodiment of the invention, such visually detectable features may refer to color, size or shape of the construct. Alternatively, such visually detectable features can absorb and emit light in a region of the spectrum ranging from about <NUM> to about <NUM>. For purposes of the invention, the detection of the present construct by its visible properties means that the construct is preferably observed, with or without illumination or chemical or enzymatic activation, with the aid of an optically based detection device, including, without limitation, absorption spectrophotometers, transmission light microscopes, fluorescence microscopes, digital cameras and scanners.

As used herein, a "biological sample" as used in the methods described herein refers to a naturally-occurring sample or deliberately prepared sample or library containing one or more cells. In one embodiment, a sample contains a population of cells or cell fragments, including without limitation cell membrane components, exosomes, and sub-cellular components. The cells may be a homogenous population of cells, such as isolated cells of a particular type, or a mixture of different cell types, such as from a biological fluid or tissue of a human or mammalian or other species subject. Still other samples for use in the methods and with the compositions include, without limitation, blood samples, including serum, plasma, whole blood, and peripheral blood, saliva, and urine. In one embodiment, the sample is a "tissue section" referring to a piece of tissue that has been obtained from a subject, fixed, sectioned, and mounted on a planar surface, e.g., a microscope slide. Tissue sections can also be classified as "planar cellular samples" referring to a substantially planar, i.e., two dimensional, material that contains cells. A planar cellular sample can be made by, e.g., cutting a three dimensional tissue biopsy that contains cells into sections and mounting the sections onto a planar surface. The cells may be fixed using any number of reagents including formalin, ethanol, methanol, DSP, paraformaldehyde, methanol:acetic acid and other similar fixing or tissue/RNA stabilizing reagents.

The constructs and methods described herein can be used to analyze cells from a subject to determine, for example, what kind of cell types exists in the tissue, in which locations and proportions they exist, whether the cell is normal or not, or to determine whether the cells are responding to a treatment. In one embodiment, the method may be employed to determine the degree of dysplasia in cancer cells. In these embodiments, the cells may be a sample from a multicellular organism. A biological sample may be isolated from an individual, e.g., from a soft tissue. In particular cases, the method may be used to distinguish different types of cancer cells in fresh frozen, cryopreserved and methanol fixed, or formalin fixed paraffin embedded (FFPE) samples. In alternative embodiments, the method described above can be practiced on planar cellular samples that have been fixed in other ways.

In an embodiment, the present invention is directed to a construct for detecting nucleic acid targets, said construct comprising a bead, preferably a magnetic bead, wherein said bead comprises one or several visually detectable features and/or said bead is coupled to one or several visually detectable entities, and wherein said bead comprises an oligonucleotide, and said oligonucleotide comprises a) an amplification handle sequence, b) a barcode sequence specific to one or several of said visually detectable features and/or entities and c) a <NUM>' terminal capture or anchor sequence, and wherein said oligonucleotide is arranged to be detachable from said bead.

In a preferred embodiment, said visually detectable feature is the size of the bead, color of the bead, shape of the bead or a combination thereof.

In another preferred embodiment, said oligonucleotide is linked to said bead via a cleavable linkage.

In another preferred embodiment, said cleavable linkage is a photocleavable, streptavidin-biotin, or carboxylate-amine linkage.

In another preferred embodiment, said bead is coupled to said one or several visually detectable entities via a streptavidin-biotin or carboxylate-amine linkage or via any other linkage.

In another preferred embodiment, said one or several visually detectable entities comprise a fluorescent label, preferably, without limitation, selected from the group consisting of: cyanine dyes, Texas Red dyes and fluorescein amidite dyes.

In another preferred embodiment, said bead is further coupled to an antigen or an antibody.

In another preferred embodiment, said amplification handle sequence is complementary to a PCR primer comprising a next generation sequencing compatible adapter sequence.

In another preferred embodiment, said construct comprises a second oligonucleotide coupled to said bead, said second oligonucleotide comprising a) a second amplification handle sequence preferably complementary to a PCR primer comprising a next generation sequencing compatible adapter sequence, b) a barcode sequence specific to one or several of visually detectable features and/or entities of the bead, c) a barcode sequence specific to the bead, d) an optional unique molecular barcode sequence, and e) a terminal poly-T sequence.

In another embodiment, the present invention is directed to a composition comprising the construct as defined above.

In a preferred embodiment, said composition comprises a mixture of several constructs providing a mixture of distinct visually detectable features or entities.

In a preferred embodiment, said mixture of several constructs comprises at least <NUM>, <NUM>, <NUM>, <NUM> or <NUM>, preferably at least <NUM>, separate visually detectable features and/or entities.

In a preferred embodiment, said composition comprises multiplicity of said constructs having at least <NUM> variable sets of visually detectable features such as fluorescent labels coupled to said constructs, wherein.

In another preferred embodiment, said first fluorescent dye is Cy5, the second fluorescent dye is Cy3, the third fluorescent dye is Atto425 and the fourth fluorescent dye is FAM.

In another preferred embodiment, different sizes, shapes or visible (non-fluorescent) colours are used in beads, with or without fluorescent dyes, to create multiple distinct bead constructs.

In another preferred embodiment, said composition further comprises a second construct (i.e. a CellBC bead) comprising a bead coupled to a second oligonucleotide, said second oligonucleotide comprising a) a second amplification handle sequence, b) a barcode sequence specific to the bead, c) an optional unique molecular barcode sequence, and d) a second anchor sequence, preferably a poly-T sequence, for hybridizing to a complementary sequence.

In another preferred embodiment, said second oligonucleotide comprising a) a second amplification handle sequence, b) a barcode sequence specific to the bead, c) an optional unique molecular barcode sequence, and d) a second anchor sequence, preferably a poly-T sequence, can be attached or immobilized directly to a bottom and/or wall of a well in a substrate (see, e.g., <FIG>).

In another embodiment, the present invention is directed to a use of the construct or the composition as defined above for identifying mRNA sequencing products originating from a single cell.

In another embodiment, the present invention is directed to a method for profiling biological samples on a single cell level, the method comprising the step of:.

The determination of the location or coordinate of the wells/samples is achieved by examining the photograph or the scanned picture obtained in the method as the visual properties of the constructs of the present invention are visible in the photograph or the scanned picture and thus the location of each type of constructs or mixtures thereof can be determined. In a preferred embodiment, the wells/samples in a substrate are photographed or scanned only once in the present method, i. e the present method does not require taking consecutive photos or scannings of the substrate with changing sets of labels, such as labelled oligonucleotide probes or labelled beads.

In a preferred embodiment, the method comprises the steps of:.

The invention thus can be used for creating a single-cell sequencing library by merging uniquely barcoded mRNA capturing microbeads with a single cell in a micro-well; lysing the cell thereby capturing the mRNA on the mRNA capturing oligonucleotides on the microbead; performing a reverse transcription reaction in said micro-well to convert the cells' mRNA to first strand cDNA containing said unique barcodes that record the location of each mRNA at the micro-well plate and subsequently sequencing the cDNAs produced.

In a more preferred embodiment, the method further comprises the steps of:.

In another preferred embodiment, the method comprises the steps of:.

In a preferred embodiment of the invention, the detection of the construct of the present invention is not dependent on addition of oligonucleotide probe(s) to said wells/samples, said probe(s) comprising an optically readable label and having complementary sequence(s) to said first and/or second oligonucleotide.

Thus, appearances of the phrases "in a preferred embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment.

The following examples disclose four versions of the present method to create visual bead barcoded coordinates for the spatial RNAseq analysis of single cells or tissues. These examples should not be construed to encompass any and all variations that become evident as a result of the teaching provided herein.

<NUM> different Colour Barcode Microbead constructs were designed, where <NUM> diameter sized magnetic and streptavidin coated Dynabeads (M-<NUM>, ThermoFisher Scientific, catalog number 11205D), were used as bead backbones. For single or multicolour staining of the beads, four short <NUM>' biotinylated oligos with stretch of <NUM> C's as a spacer and each with different fluorescent blue (Atto425), green (FAM), yellow (Cy3) and red (Cy5) labels at <NUM>' end were ordered from Integrated DNA Technologies (SEQ ID NO: <NUM>-<NUM>). Sequence of the staining oligos is irrelevant and can be replaced by other sequences or spacer molecules. Another set of <NUM> different Colour barcode tagging oligos with photocleavable biotin (PCbio) (released by UV exposure at <NUM>-<NUM> wavelengths) in the <NUM>' end were designed (SEQ ID NO: <NUM>-<NUM>) with the following characteristics: a <NUM>' end generic amplification handle (PCR handle2) for next-generation sequencing library preparation, a unique barcode sequence specific for each bead colour construct, and a polyA stretch at the <NUM>' end (<FIG> and table of <FIG>). <NUM> batches of beads were prepared, each with one of the <NUM> Colour barcode oligos. <NUM> of the batches were stained also with a different combination of <NUM>, <NUM>, <NUM> or <NUM> of the fluorescently labelled biotinylated oligos. The 16th batch of beads was used as non-fluorescent, labeled only with the Colour barcode oligo.

Streptavidin Dynabeads have a capacity of binding ~200pmols of biotinylated DNA oligos per mg of beads and the labelled and unlabelled oligos were added on beads in <NUM>:<NUM> to <NUM>:<NUM> ratios depending on each fluorochrome's signal intensities. After incubation of <NUM> at RT in rolling tubes the beads were washed thoroughly <NUM> times with PBS and pelleted by magnet in each washing round. 1X Dynabead binding and washing (B&W) Buffer was used in these steps (<NUM> Tris-HCl (pH <NUM>), <NUM> EDTA, <NUM> NaCl). Fluorescence intensities and bead integrities were checked with Zeiss AxioImager microscope. The selection of labels, staining intensities and exposure times need to be adjusted at each user lab to be compatible with their fluorescent microscope specifications. Proper binding and release of the Colour barcode oligo was tested from each <NUM> bead batches by incubating them with commercial Cell barcoding beads (MACOSKO <NUM>-<NUM> B, ChemGenes, USA, hereafter called CellBC or Cell barcoding beads) with <NUM> UV exposure (Bio-Rad ChemiDoc Imaging system) to release the oligo, followed by <NUM> hybridization of polyA to polyT, subsequent separation of non-magnetic CellBC beads from the magnetic Colour barcode beads with magnet, <NUM> rounds of thorough SSC buffer (Sigma Aldrich, catalog number S6639) washing and centrifugation steps of CellBC beads, PCR amplification at bulk reaction (SEQ ID NO: <NUM> and <NUM> ) and gel electrophoresis detection of the correct sized (~180bp) product. After the quality check, the prepared <NUM> bead batches were mixed in equal proportions and this stock bead mix was stored at +<NUM> in Tris-PBS buffer and protected from light.

Aliquot of ColourBC bead stock mix containing ~<NUM><NUM> beads was pelleted with magnet and washed once, and suspended to 200µl PBS with thorough but gentle pipet mixing at least <NUM> times, and then loaded randomly to microwell array area <NUM> x <NUM> consisting of <NUM><NUM> microwells of the size of <NUM> diameter and <NUM> depth (customized geometry designed and fabricated of polydimethyl sulfoxide (PDMS) but otherwise functionalized according to the Seq-Well protocol by Gierahn et al <NUM>). Removable silicone frames around the array area and a glass cover slip slide on top of the buffer was used to help in equal loading efficiency and prevention of buffer evaporation. Flat magnet was placed under the array slide to fasten the bead loading. During the <NUM> loading the magnet was carefully removed twice and the chip shaked gently horizontally to enhance the remaining beads between the microwells to settle down to the wells. Loading quality was also checked under light microscope. Extra buffer was removed, keeping the slide array still on magnet, and slides were let to dry for at least <NUM> protected from light. Loaded and dried arrays were stored in covered petri dishes at +<NUM> protected from light, until the next step.

Fluorescent microscope scanner Zeiss AxioImager was used to scan the whole array area for the <NUM> fluorescent colours and their combinations used on beads, and the associated Zen <NUM> Lite program to form a single stitched large image of the area. Image was saved as <NUM> fluorescent and <NUM> light microscope channels in a stacked multilayer image format. Image analysis and machine learning algorithm was created, trained and validated with a specific machine learning software designed for microscope image analysis. Algorithm was trained to recognize the <NUM> different classes of beads (constructs) based on their colour combinations, to recognize the <NUM> diameter sized microwells, to count the number of each type of these bead classes within each microwell, and to give a X-Y positioned coordinate on array for each such feature composition. Nearly all wells loaded with this loading protocol contained from <NUM> to over <NUM> beads per well, with most microwells having <NUM>-<NUM> beads as predicted by Poisson distribution. Out of the <NUM> possible features, total of theoretical <NUM> combinations are possible (<FIG>), and the AI algorithm mapped and recorded the wells with unique composition of bead features. List of non-unique combinations were also marked to the map.

Unless proceeding immediately to the next step, the pre-loaded and image coordinated slides can be prepared in stock beforehand and stored at +<NUM> dry with magnet underneath if handled, and protected from light and dust.

Pre-loaded and imaged chips were wetted for <NUM> hours with PBS, with magnet under the chip and light protected. <NUM><NUM> commercially available Cell barcoding (CellBC) beads, custom synthetized on <NUM> diameter polystyrene beads (ChemGenes, MACOSKO-<NUM>-<NUM> B with SEQ ID NO: <NUM>, synthesis described in Macosko et al <NUM>) were prewashed and loaded in 200µl volume of bead loading buffer (recipe on Gierahn et al <NUM>) on the desired microwell area with gentle horizontal shaking for <NUM>, and using silicon frames and light protection. After this <NUM> peripheral blood mononuclear cells in 200µl RPMI medium were added and cells let to settle in similar manner for <NUM>. The wells were sealed with semipermeable membrane and cells lysed with lysis buffer, adapted from Seq-Well protocol (Gierahn et al <NUM>). <NUM> UV light exposure was used to release the photocleavable oligos from the Colour barcode beads. After the <NUM> incubation the membrane was removed, CellBC beads were collected and washed, 1st strand cDNA synthesis was performed with reverse transcription with template switching oligo (SEQ ID NO: <NUM>) followed by ExoI treatment, PCR amplification of full length cDNA (SEQ ID NO: <NUM>) and tagmentation based Illumina library preparation (SEQ ID NO: <NUM> and standard Illumina Nextera i7 indexing primer) with appropriate washing, purification and quantitation steps, following the DropSeq (Macosko et al <NUM>) and Seq-Well (Gierahn et al <NUM>) protocols. Size selection of <200bp fragments and separate library preparation (with SEQ ID NO:<NUM> and SEQ ID NO:<NUM>) of the Colour barcodes linked to Cell barcodes, followed the CITE-seq protocol for ADT (antibody determining tag) libraries (Stoeckius et al <NUM>).

The libraries were sequenced with Illumina NextSeq500 platform and High Output <NUM> cycle sequencing kit. Custom read <NUM> sequencing primer (SEQ ID NO: <NUM>) was used to sequence 20bp for read <NUM> covering the 12bp cell barcode and 8bp molecular barcode (UMB) derived from the CellBC beads. Standard Illumina read <NUM> sequencing primers included in the sequencing cartridge produced 55bp reads of the gene sequences derived from captured mRNAs by the CellBC beads, as well as the colour bead barcodes derived from the UV-detached feature oligos that were also captured by the CellBC beads. Processing of raw fastq files followed the publicly available Drop-Seq data analysis pipeline (Macosko et al <NUM>) and, for the colour barcode, the CITE-seq pipeline (Stoeckius et al <NUM>). A combined gene expression matrix was created from the counts of transcripts aligned to the reference genome and the colour barcode counts linking to the same cell barcode. This matrix was further matched with the visual map of the spatial X-Y coordinates of the cells and beads in the wells, created earlier with the machine learning algorithm from the microscope scanner image.

Another variation of the protocol described in Example <NUM> was tested with different sizes (<NUM>, <NUM>, <NUM> or <NUM>) and visual colours of the beads, combined to just a single fluorescence label FAM. Such set of beads is easier to use with large variety of end-user microscopes as they need less optimization compared to multi-fluorescence excitation, emission and filter sets, and possible autofluorescence limitations of some beads. Beads in various sizes, colours and surface coatings or functionalizations are available from many vendors (e.g. Spherotech, Bangs Laboratories, PolySciences, Abvigen). When carboxylated beads were used instead of streptavidin coating, the FAM labelled staining oligo was ordered with <NUM>' amine group instead of biotin, and respectively the Colour barcode oligos were ordered with photocleavable <NUM>' end amine group (GeneLink). EDC activation was used to form amide bond between the carboxyl groups on beads and the primary amine of the oligo, following the instructions of the reagent provider (ThermoFisher Scientific, catalog number <NUM>). In case of non-magnetic beads, they were magnetized with aminated magnetic nanoparticles (Merck Life Science, catalog number <NUM>) in the same EDC reaction. The linked beads were washed thoroughly at least <NUM> times to remove the unbound oligos and labels, before mixing the bead batches. Otherwise the protocol was similar to Example <NUM>, the loaded beads were scanned with AxioImager fluorescence microscope equipped with a colour camera and the stitched colour image stacked with the green fluorescence image (for FAM label) were analyzed with a machine learning algorithm that was taught to recognize the following <NUM> classes of beads and recognize and count them from each microwell. Random sets of beads from the pool of <NUM> bead types produces over a million unique combinations of bead types per well (https://stattrek. com/online-calculator/combinations-permutations. aspx) as the sum of all possible <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM> bead combinations out of <NUM> bead types totals to <NUM>+<NUM>+<NUM>+<NUM>+<NUM>+<NUM>+<NUM>+<NUM>+<NUM>+<NUM>+<NUM>+<NUM>+ <NUM>+<NUM>+<NUM>+<NUM>+<NUM>+<NUM>+<NUM>+<NUM>=<NUM> possible combinations per well. For example, in an array with <NUM> wells and loading with <NUM> beads randomly in Poisson distribution, the proportion of non-informative/non-unique wells with <NUM>, <NUM> or <NUM> beads only can be kept in less than <NUM>%.

<NUM> bead types created in example <NUM>:.

Another variation of the protocol is the method without the commercial Cell barcode beads, but instead the Cell barcode oligo is carried by the coloured or other visibly featured beads, along with the ColourBC oligos (see <FIG>). Optionally, the new CellBC oligo carried on the featured beads carries also an additional ColourBC sequence matching with the colour or feature combination of the beads (SEQ ID NO: <NUM>). It is essential, though, that the featured beads also carry a separate cleavable ColourBC oligo that is released and captured randomly by all beads in a given well. The unique CellBC oligo sequence was created to each bead batch with biotinylated backbone oligo consisting PCRhandle1 and optional ColourBC. To create them, the split-and-pool synthesis of 12bp bead specific CellBC, 8bp totally random molecular barcode and 30bp poly-T sequence was adapted from the protocol described in Macosko et al <NUM>. After this, the ColourBC oligos with poly-A end, and photocleavable biotin on <NUM>' and identical to example <NUM>, were attached to the bead batches, washed <NUM> times, and then all bead featured batches were mixed and used in the experiments on microwells. Beads were loaded and imaged as in Example <NUM>, but no commercial CellBC beads were added in following steps. Cell or tissue loading and further analysis followed otherwise the examples <NUM> and <NUM>. Multiple beads per well created multiple cell barcodes per well, but as the colour or feature combination was unique, it allowed matching of multiple Cell barcodes to a single unique ColourBC combination in each well, and this was allowed in the bioinformatics analysis pipeline with this method version.

Another variation of the method is to use the bead barcoded microwells for direct spatial capture of the RNA from cryopreserved or formalin fixed paraffin embedded (FFPE) tissue sections, instead of dissociated cells in suspension. For this, snap frozen and TissueTek OCT Compound (Sakura) embedded mouse mammary tumor tissues were sliced with cryomicrotome to <NUM> thick sections on standard microscope glass slide, and kept in -<NUM> until used. Sections were fixed with methanol and stained with standard hematoxylin-eosin (HE) staining protocol, followed by light microscope image scan to visualize the tissue structure. A previously prepared microwell slide with ColourBC beads pre-loaded and fluorescently imaged (as described in examples <NUM> and <NUM>) was kept on magnet and wetted with cell permeabilization buffer (<NUM>% pepsin in <NUM> N HCl). The wetted array was placed on a humidified table-top chamber to prevent evaporation of the small liquid volumes from the microwells in the following steps. Keeping the slides within the chamber, the excess lysis buffer was removed from the top of the slide followed by immediate placing of the tissue section slide on top of it so that the lysis buffer filled wells and the tissue got in contact. Thin piece of metal taped on the backside of the tissue slide helped to hold it tightly and still in place with the power of the magnet under the microwell slide. Alternatively, a mechanical clip could be used. Cells were let to lyse and mRNA to hybridize on the beads for <NUM> at room temperature. <NUM> UV exposure was introduced in the beginning as in the Example <NUM> to release the Colour barcode oligos from the beads and hybridize them with the Cell barcoding oligos along with the mRNA from the lysing cells. After this, the tissue slide was quickly moved and the microwell array, while still on magnet, was immediately washed <NUM> times with SSC buffer to remove the excess of non-hybridized RNA and the UV-released oligos. Beads were collected from the microwell slide by placing the slide upside down to a slide chamber with SSC buffer and pulling the beads with magnet under the chamber. In case of Example <NUM> protocol with commercial, non-magnetic CellBC beads a slide centrifugation step was also needed. Beads in buffer were collected to a single Eppendorf tube and pelleted. The following reverse transcription and library preparation steps were done in bulk reaction on the beads, as referred in example <NUM>, following closely the protocols described in Macosko et al <NUM>, Gierahn et al <NUM> and Stoeckius et al <NUM>. Data analysis was otherwise similar to the one described in example <NUM>, but in addition, the microscope image of the original HE-stained tissue section was overlaid on top of the spatial sequencing information of the gene expression derived from the bead coordinated well positions. Matching the tissue and array images for the overlay was guided by the shape of the sequencing data producing area from the microwell slide and with inbuilt assisting marking spots on both slides recorded with microscope and aligned from the images.

Claim 1:
A construct for detecting nucleic acid targets, said construct comprising a bead, preferably a magnetic bead, wherein said bead comprises one or several visually detectable features and/or said bead is coupled to one or several visually detectable entities, and wherein said bead comprises an oligonucleotide, and said oligonucleotide comprises a) an amplification handle sequence, b) a barcode sequence specific to one or several of said visually detectable features and/or entities and c) a <NUM>' terminal capture or anchor sequence, and wherein said oligonucleotide is arranged to be detachable from said bead.