EP-4573210-B1 - METHOD FOR ANALYSING GENE EXPRESSION IN CELLS IN A PELLET

Inventors

• BELHOCINE, ZAHRA KAMILA
• FREEMAN, MELANIE
• GONZALEZ MUÑOZ, Veronica Emelina
• KRISHNAN, Sreenath
• MARKS, PATRICK J.
• SASAKI, HIROSHI

Dates

Publication Date

20260506

Application Date

20231115

Claims (15)

1. A method, comprising: (a) providing a test biological sample, wherein the test biological sample is a section of a cell pellet comprising a defined mixture of a first cell population and a second cell population; (b) contacting the test sample with a probe mixture comprising: (i) a first panel of probes or probe sets, wherein each probe or probe set of the first panel comprises a recognition sequence complementary to a target sequence of an RNA transcript or product thereof of a first set of genes expressed in the first cell population, and (ii) a second panel of probes or probe sets, wherein each probe or probe set of the second panel comprises a recognition sequence complementary to a target sequence of an RNA transcript or product thereof of a second set of genes expressed in the second cell population, and allowing the probes or probe sets to hybridize to their target sequences in the test biological sample, wherein the first cell population does not detectably express the second set of genes, and the second cell population does not detectably express the first set of genes; (c) detecting the hybridized probes or probe sets or products of the hybridized probes or probe sets at locations in the biological sample, thereby detecting RNA transcripts of the first and second sets of genes in the test biological sample; and (d) identifying discrete cells in the test biological sample and comparing the detected RNA transcripts of the first and second sets of genes in the cells to expected expression levels of the first and second sets of genes in the first and second cell populations.
2. The method of claim 1, wherein the probe mixture comprises: (iii) a third panel of probes or probe sets, wherein each probe or probe set of the third panel comprises a recognition sequence complementary to a target sequence of an RNA transcript or product thereof of a third set of genes expressed in both the first cell population and the second cell population, and wherein the method comprises allowing the probes or probe sets of the third panel to hybridize to their target sequences in the test biological sample, detecting the hybridized probes or probe sets or products of the hybridized probes or probe sets of the third panel at locations in the biological sample, thereby detecting RNA transcripts of the third set of genes in the test biological sample; and comparing the detected RNA transcripts of the third set of genes in the cells to expected expression levels of the third set of genes in the first and second cell populations.
3. The method of claim 1 or 2, wherein the first, and/or second sets of genes comprise genes having at least two different expression levels.
4. The method of any of claims 1-3, wherein the first panel of probes or probe sets and the second panel of probes or probe sets each is a panel of circularizable probes or probe sets, and the method comprises ligating the circularizable probes or probe sets hybridized to their target sequences, thereby generating circularized probes at locations in the test biological sample.
5. The method of any of claims 1-4, wherein the first panel of probes or probe sets and the second panel of probes or probe sets each comprise barcode sequences corresponding to the RNA transcripts comprising their corresponding target sequences.
6. The method of claim 4 or claim 5, wherein the method comprises performing rolling circle amplification of the circularized probes to generate rolling circle amplification products at locations in the test biological sample.
7. The method of claim 5, wherein detecting the hybridized probes or probe sets or products of the hybridized probes or probe sets comprises detecting the barcode sequences in the hybridized probes or probe sets.
8. The method of claim 6, wherein detecting the hybridized probes or probe sets or products of the hybridized probes or probe sets comprises detecting the rolling circle amplification products, optionally wherein detecting the rolling circle amplification products comprises detecting the complements of the barcode sequences.
9. The method of claim 7 or 8, wherein detecting the barcode sequences or complements thereof comprises (i) sequencing all or a portion of the barcode sequences or complements thereof, or (ii) sequential binding of detectably labeled probes directly or indirectly to the barcode sequences or complements thereof.
10. The method of claim 9, wherein the method comprises determining a quality score for the sequenced or decoded barcode sequence or complement thereof.
11. The method of any of claims 1-10, wherein identifying the discrete cells comprises performing cell segmentation, optionally wherein the cell segmentation comprises (i) detecting a nuclear stain; and/or (ii) detecting a membrane stain.
12. The method of any of claims 1-11, wherein: (a) the first cell population is a first cultured cell line, and the second cell population is a second cultured cell line; and/or (b) the first cell population is a T cell line, and the second population is a B cell line.
13. The method of any one of claims 1-12, wherein the first cell population are Jurkat cells, and the second cell population are Raji cells.
14. The method of any of claims 1-13, wherein the method comprises embedding the cell pellet in an embedding medium and sectioning the embedded cell pellet to provide the test biological sample.
15. The method of any of claims 11-14, wherein the detected RNA transcripts are assigned to the identified discrete cells, optionally wherein the detected RNA transcripts are assigned to the identified discrete cells for clustering the identified discrete cells.

Description

FIELD The present disclosure relates in some aspects to assays for transcriptomic profiling in situ. BACKGROUND Genomic, transcriptomic, and proteomic profiling of cells and tissue samples using microscopic imaging can resolve multiple analytes of interest at the same time, thereby providing valuable information regarding analyte abundance and localization in situ. Thus, in situ assays are important tools, for example, for understanding the molecular basis of cell identity and developing treatment for diseases. There is a need for improving in situ assays. Provided herein are methods and compositions that address such and other needs. SUMMARY The invention is defined by the appended claims. In some aspects, provided herein is a performance test for assessing the performance of in situ analyte detection and/or platforms (e.g., methods, assays, workflows, systems and/or instruments for in situ analyte detection). In some aspects, results from different in situ analyte detection and/or sequencing experiments can be variable, in part because the results depend on the analyzed biological sample (which can vary between experiments) as well as the specific in situ analyte detection and/or platform used to analyze the biological sample. This variability can make it difficult to assess the performance of in situ analyte detection and/or platforms based on the results, and/or to compare performance of different in situ analyte detection and/or platforms. Thus, there is a need for methods to assess and/or compare performance of in situ analyte detection and/or platforms. In some aspects, the performance test provided herein facilitates assessment and/or comparison of the performance of in situ analysis platforms. In some aspects, the assessment is quantitative and/or qualitative. In some aspects, the performance test depends on 1) a standardized test biological sample with known properties, 2) specific target analytes, and 3) specific methods of analysis, to produce results. In some aspects, the performance test produces expected results if performance is satisfactory. In some aspects, the performance test produces unexpected results if performance is not optimal and/or unsatisfactory. Provided herein is a method, comprising: (a) providing a test biological sample, wherein the test biological sample is a section of a cell pellet comprising a defined mixture of a first cell population and a second cell population; (b) contacting the test sample with a probe mixture comprising: (i) a first panel of probes or probe sets, wherein each probe or probe set of the first panel comprises a recognition sequence complementary to a target sequence of an RNA transcript or product thereof of a first set of genes expressed in the first cell population, and (ii) a second panel of probes or probe sets, wherein each probe or probe set of the second panel comprises a recognition sequence complementary to a target sequence of an RNA transcript or product thereof of a second set of genes expressed in the second cell population, and allowing the probes or probe sets to hybridize to their target sequences in the test biological sample, wherein the first cell population does not detectably express the second set of genes, and the second cell population does not detectably express the first set of genes; (c) detecting the hybridized probes or probe sets or products of the hybridized probes or probe sets at locations in the biological sample, thereby detecting RNA transcripts of the first and second sets of genes in the test biological sample; (d) identifying discrete cells in the test biological sample and comparing the detected RNA transcripts of the first and second sets of genes in the cells to expected expression levels of the first and second sets of genes in the first and second cell populations. In any of the embodiments herein, the probe mixture can comprise: (iii) a third panel of probes or probe sets, wherein each probe or probe set of the third panel comprises a recognition sequence complementary to a target sequence of an RNA transcript or product thereof of a third set of genes expressed in both the first cell population and the second cell population, and wherein the method comprises allowing the probes or probe sets of the third panel to hybridize to their target sequences in the test biological sample, detecting the hybridized probes or probe sets or products of the hybridized probes or probe sets of the third panel at locations in the biological sample, thereby detecting RNA transcripts of the third set of genes in the test biological sample; and comparing the detected RNA transcripts of the third set of genes in the cells to expected expression levels of the third set of genes in the first and second cell populations. In any of the embodiments herein, the first, and/or second sets of genes can comprise genes having at least two different expression levels. In any of the embodiments herein, the third set of genes can comprise