EP-4739795-A1 - COMPOSITIONS FOR SPATIAL OMICS

Abstract

The present invention relates to a composition comprising a population of capture barcode units and a population of positioning barcode units, and its use for determining the spatial location of any analyte from a biological sample.

Inventors

• Fernandez, Nicolas
• KOMATSU, JUN
• ANDRE, Barbara

Assignees

• Scipio Bioscience

Dates

Publication Date

20260513

Application Date

20240705

Claims (1)

1. CLAIMS 1. A composition comprising: - a population of capture barcode units, wherein each capture barcode unit comprises a population of capture oligonucleotides, wherein each capture oligonucleotide comprises a barcode sequence, and a capture sequence capable of binding an analyte from a biological sample; - a population of positioning barcode units, wherein each positioning barcode unit comprises a population of positioning oligonucleotides, wherein each positioning oligonucleotide comprises a barcode sequence, and a first connection sequence that can hybridize on a capture oligonucleotide; wherein each barcode unit comprises a distinct barcode sequence, and all oligonucleotides on a same barcode unit comprise the same barcode sequence. 2. The composition according to claim 1, wherein said capture sequence cannot hybridize with said capture sequence, and wherein said first connection sequence cannot hybridize with said first connection sequence. 3. The composition according to claim 1 or 2, wherein said first connection sequence of the positioning oligonucleotides can hybridize on said capture sequence of the capture oligonucleotides. 4. The composition according to claims 1 to 3, wherein said capture oligonucleotides comprise a second connection sequence, wherein said second connection sequence is distinct from said barcode sequence and said capture sequence, and wherein said first connection sequence of the positioning oligonucleotides can hybridize on said second connection sequence of the capture oligonucleotides. 5. The composition according to any one of claims 1 to 4, wherein said capture sequence is identical for all capture barcode units, and said first connection sequence is identical for all positioning barcode units. 6. The composition according to any one of claims 1 to 5, wherein said capture oligonucleotides and/or said positioning oligonucleotides further comprise at least one PCR priming sequence. 7. The composition according any one of claims 1 to 6, wherein each oligonucleotide from said population of capture oligonucleotides and/or said population of positioning oligonucleotides further comprise at least one unique molecular identifier (UMI). 8. The composition according to any one of claims 1 to 7, wherein said capture oligonucleotides comprise, in the 5’ to 3’ order: optionally at least one linker, optionally at least one PCR priming sequence, said barcode sequence, optionally at least one UMI, and said capture sequence; and wherein said positioning oligonucleotides comprise, in the 5’ to 3’ order: optionally at least one linker, optionally at least one PCR priming sequence, said barcode sequence, and said first connection sequence. 9. The composition according any one of claims 1 to 8, wherein said barcode units are synthetic barcode units. 10. The composition according any one of claims 1 to 9, wherein said barcode units are beads or DNA nanoballs. 11. The composition according any one of claims 1 to 10, wherein said population of capture barcode units and said population of positioning barcode units are assembled on a substrate. 12. The composition according any one of claim 1 to 11, wherein said capture barcode units are in contact with at least one, preferably at least 2, more preferably at least 3 said positioning barcode units. 13. A method for determining the spatial location of a population of capture barcode units, comprising the steps of: (i) contacting the population of capture barcode units with the population of positioning barcode units from the composition according to any one of claims 1 to 12; (ii) forming a plurality of contact points between the populations of capture and positioning barcode units, wherein each contact point involves one capture oligonucleotide and one positioning oligonucleotide; (iii) at the plurality of contact points formed at step (ii), hybridizing at least one connection sequence with at least one complementary sequence on said capture oligonucleotide, thereby obtaining a plurality of hybridized oligonucleotides; (iv) extending the plurality of hybridized oligonucleotides obtained at step (iii), thereby obtaining a plurality of nucleic acid sequences comprising the barcode sequence of a capture barcode unit, and the barcode sequence of a positioning barcode unit; (v) optionally, amplifying the plurality of nucleic acid sequences obtained at step (iv), thereby obtaining a plurality of amplicons; (vi) sequencing the plurality of nucleic acid sequences obtained at step (iv) or the plurality of amplicons obtained at step (v); (vii) deducing the neighborhood relationships between the capture barcode units and the positioning barcode units; (viii) deducing the relative position of each capture barcode unit and/or positioning barcode unit; and (ix) optionally, deducing the absolute position of each capture barcode unit and/or positioning barcode unit. 14. A method for determining the spatial location of a plurality of analytes in a biological sample comprising thereof, comprising the steps of: (i) contacting the population of capture barcode units with the population of positioning barcode units from the composition according to any one of claims 1 to 12; (ii) forming a plurality of contact points between the populations of capture and positioning barcode units, wherein each contact point involves one capture oligonucleotide and one positioning oligonucleotide; (iii) at the plurality of contact points formed at step (ii), hybridizing at least one connection sequence with at least one complementary sequence on said capture oligonucleotide, thereby obtaining a plurality of hybridized oligonucleotides; (iv) extending the plurality of hybridized oligonucleotides obtained at step (iii), thereby obtaining a plurality of nucleic acid sequences comprising the barcode sequence of a capture barcode unit, and the barcode sequence of a positioning barcode unit; (v) optionally, amplifying the plurality of nucleic acid sequences obtained at step (iv), thereby obtaining a plurality of amplicons; (vi) sequencing the plurality of nucleic acid sequences obtained at step (iv) or the plurality of amplicons obtained at step (v); (vii) deducing the neighborhood relationships between the capture barcode units and the positioning barcode units; (viii) deducing the relative position of each capture barcode unit and/or positioning barcode unit; and (ix) optionally, deducing the absolute position of each capture barcode unit and/or positioning barcode unit; and further comprising the steps of: a) at any step before step (v), contacting the populations of capture and positioning barcode units with said biological sample; b) at any step after step (a), and before step (v), capturing said plurality of analytes from said biological sample by binding said plurality of analytes to the capture sequence of a plurality of capture barcode units; c) optionally, after step (b), extending said capture sequence on the sequence of said plurality of analytes, thereby obtaining a plurality of nucleic acids comprising the barcode sequence of a capture barcode unit and the sequence of the analyte; d) optionally, after step (c), amplifying the plurality of nucleic acids, thereby obtaining a plurality of amplicons; e) sequencing the plurality of nucleic acid sequences obtained at step (c) or the plurality of amplicons obtained at step (d); f) at any step after step (viii), deducing the relative position of each analyte; and optionally, deducing the absolute position of each analyte in said biological sample. 15. The method according to claim 14, wherein said biological sample is selected from the group comprising or consisting of a tissue, a tissue section, a monolayer of cells, multiple layers of cells, an organoid, a syncytium or a single cell.

Description

COMPOSITIONS FOR SPATIAL OMICS FIELD OF INVENTION [0001] The present invention relates to the spatial localization of barcode units, and spatial omics. BACKGROUND OF INVENTION [0002] Omics approaches are powerful tools that enable in-depth characterization of a cell, or population of cells, by identifying and quantifying its biological components (e.g., RNA, proteins) in a dynamic manner. [0003] There is growing interest in developing these approaches further when applied to tissues or other complex biological samples, in order to map the variations of the transcriptome, proteome, metabolome and the like, depending on the position of the cell in the sample. In other words, spatial omics aims to add spatial information to omics analysis. [0004] Several approaches are readily available in the art for implementing spatial omics. [0005] For example, Visium Spatial Gene Expression technology developed by 10x Genomics (see, e.g., 10x Genomics, 2019, “Visium Spatial Gene Expression Solution”; or WO2021/102039) relies on oligonucleotides microarrays. Typically, a microarray is formed by “spotting” droplets comprising oligonucleotides on a slide; the position of each oligonucleotide is known, thus enabling recapitalization of the spatial information for the RNA captured by the oligonucleotide. [0006] However, this approach is limited by the resolution of the microarray itself, due to local diffusion of the droplets during the spotting. [0007] Other approaches aim to overcome this limitation. For exemple, Rodriques, et al. (Science vol. 363,6434 (2019): 1463-1467) describe a slide-seq approach wherein barcode beads are randomly deposited on a slide, and then indexed, i.e., the barcodes are sequenced in order to associate an oligonucleotide to a given position on the slide. The slide is subsequently contacted with a sample (tissue slice); the captured analytes are then sequenced. Another example is Liao et al.2023 (bioRxiv preprint doi: https://doi.org/10.1101/2023.04.28.538364) that describes a stereo-seq approach based on a similar method except for barcoded beads that are replaced with DNA nanoballs. [0008] While these approaches improve resolution, they compulsorily involve, for each slide, indexing the position of each oligonucleotide on the slide with a first sequencing step, followed by a second sequencing once the analytes are captured, thereby complexifying the process. [0009] The patent US,11,624,088 provides an alternative, with beads comprising 2 types of barcoded oligonucleotides on the same bead: one type is responsible for capturing a analyte, and the other type is responsible for connecting beads together, thereby associating barcode information on 2 or more distinct beads and reconstituting relative spatial location of the barcoded bead array. This approach thus circumvents the need to perform 2 sequencing steps, as the spatial information is deduced from the connections between beads rather than by a preliminary indexing. [0010] However, in this approach, nothing prevents the self-hybridization of the connection oligonucleotides. This is of importance because a significant part of the library produced for the sequencing of these barcode beads will not provide spatial information. Moreover, having 2 species of oligonucleotides on each bead increases manufacturing costs and complexity. [0011] It is also worth mentioning another approach disclosed by Greenstreet et al. (bioRxiv preprint doi: https://doi.org/10.1101/2022.03.22.485380) called GPS- seq, based on a single layer of barcoded beads that is contacted with a slice of hydrogel containing spots (or satellites) of spatially indexed oligonucleotide, then contacted with a biological sample. The spatial indexes diffuse on to the beads from their initial spatial location in the hydrogel. Thus, neighborhood relationships are reconstituted based on shared spatial indexes. [0012] This approach seems, however, technically challenging, and difficult to implement. In particular, the quality of the resolution is strongly dependent on the homogeneity of diffusion of the spatially indexed oligonucleotide, which is a parameter that is well known to be hard to control. [0013] There is therefore an unmet need for an improved solution for spatial omics, in particular spatial transcriptomics, that is easy to manufacture and implement, while maintaining a high resolution. The present invention provides such a solution. SUMMARY [0014] This invention thus relates to a composition comprising: - a population of capture barcode units, wherein each capture barcode unit comprises a population of capture oligonucleotides, wherein each capture oligonucleotide comprises a barcode sequence, and a capture sequence capable of binding an analyte from a biological sample; - a population of positioning barcode units, wherein each positioning barcode unit comprises a population of positioning oligonucleotides, wherein each positioning oligonucleotide comprises a barcode sequence