# US 2024/0203531 A1 · Cell type annotation

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- Number: US 2024/0203531 A1
- Document ID: US20240203531A1
- Status: Published application
- Kind: A1
- Application number: 18/544,824
- Filed: 2023-12-19
- Published: 2024-06-20
- Earliest claimed: 2022-12-20
- Assignees: Fluent Biosciences Inc.
- Applicants: Fluent Biosciences Inc.
- Inventors: Yigal Agam; Corey Hayford
- Web page: https://patentgrep.com/patent/US20240203531A1

## CPC

- G16B 40/30: Unsupervised data analysis
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6869: Methods for sequencing
- C12Q 1/6881: for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
- G16B 25/10: Gene or protein expression profiling; Expression-ratio estimation or normalisation
- C12Q 2600/158: Expression markers
- G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

The invention provides methods for annotating cell types using non-parametric statistical scoring of gene expression levels from RNA sequencing (RNA-seq). Expression levels for cells are measured by RNA-seq and a non-parametric statistic such as a Mann-Whitney U score or Wilcoxon score is generated for the expression levels and correlated to such scores from reference data from known cell types. When test cells in the RNA-seq data have a score that correlate highly with such a score from cells of a known type in the reference, those test cells are annotated as being of the known type from the reference.

## Claims

### Claim 1

A method of identifying cell types, the method comprising:
obtaining gene expression data for a plurality of cells;
assigning the cells to clusters based on the gene expression data;
determining non-parametric statistical scores of differential gene expression for each cluster versus other clusters;
calculating a correlation between the calculated score and annotated clusters in a reference; and
annotating each cluster with a cell type based on correlations to the annotated clusters.

### Claim 2 (depends on 1)

The method of claim 1, wherein the determining step comprises performing a Mann-Whitney test to calculate a U score for genes of each cluster.

### Claim 3 (depends on 2)

The method of claim 2, wherein the annotated clusters in the reference comprise reference U scores

### Claim 4 (depends on 1)

The method of claim 1, wherein the plurality of cells include peripheral blood monocytes and the clusters are annotated with cell types that include one or more of B cells, CD4 memory T cells, CD4 naïve T cells.

### Claim 5 (depends on 1)

The method of claim 1, wherein the assigning step is performed by obtaining a principal component analysis (PCA) matrix of the gene expression data; creating a k-nearest neighbor graph from the PCA matrix; and performing Leiden clustering on the graph.

### Claim 6 (depends on 1)

The method of claim 1, wherein the assigning, determining, calculating, and annotating are performed by a computer system comprising at least one processor coupled to a memory subsystem containing instructions executable by the processor to cause the computer system to perform the recited operations.

### Claim 7 (depends on 6)

The method of claim 6, wherein the reference includes the annotated clusters of reference cells of known type, wherein the annotated clusters were created by identifying reference genes with the highest differential expression among the reference cells and clustering the reference cells into the annotated clusters based on those reference genes.

### Claim 8 (depends on 7)

The method of claim 7, wherein the reference further includes reference U scores calculated by a non-parametrical statistical test for each annotated cluster.

### Claim 9 (depends on 8)

The method of claim 8, wherein the reference U scores are calculated by the computer system as the Wilcoxon ranked sum.

### Claim 10 (depends on 1)

The method of claim 1, wherein each cluster is given an annotation in one of three categories that include (i) definitive, (ii) primary and secondary, and (iii) no assignment.

### Claim 11 (depends on 6)

The method of claim 6, further comprising performing the obtaining step by single-cell RNA-sequencing (scRNA-seq) to obtain scRNA-seq data and performing the determining step to produce the non-parametric statistical scores of differential gene expression for each cluster from the scRNA-seq data.

### Claim 12 (depends on 1)

The method of claim 1, wherein the obtaining step comprises performing scRNA-Seq in droplets.

### Claim 13 (depends on 12)

The method of claim 12, further comprising
combining template particles with cells in a first fluid wherein the template particles are each linked to copies of a capture probe;
shearing the first fluid in the presence of a second immiscible fluid to thereby generate a plurality of droplets substantially simultaneously, wherein at least one droplet contains a single template particle and a single target cell;
lysing the target cell to release mRNA molecules in the droplet; and
capturing the mRNA molecule in the droplet with the capture probes of the single template particle.

### Claim 14 (depends on 13)

The method of claim 13, further comprising performing reverse transcription to yield a cDNA using a reverse transcriptase (RT).

### Claim 15 (depends on 14)

The method of claim 14, wherein the RT adds additional bases to a 3′ end the cDNA.

### Claim 16 (depends on 14)

The method of claim 14, wherein the reverse transcription leaves the cDNA linked to bead, the cDNA comprising first and second universal primer binding sites, at least one cell barcode, and at least one unique molecular identifier (UMI).

### Claim 17 (depends on 13)

The method of claim 13, wherein the capture probe comprises one or more of a template switching segment, a UMI, a droplet barcode, and a universal primer nucleotide sequence.

### Claim 18 (depends on 13)

The method of claim 13, wherein the first fluid is an aqueous fluid and the second fluid comprises an oil.

### Claim 19 (depends on 18)

The method of claim 18, wherein the template particles further comprise one or more compartments, recesses, or pores that contain one or more of a cell lysis reagent, a nucleic acid synthesis reagent, and a capture tag or oligonucleotide.