# US 2022/0033802 A1 · Method and apparatus for encoding cellular spatial position information

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- Number: US 2022/0033802 A1
- Document ID: US20220033802A1
- Status: Published application
- Kind: A1
- Application number: 17/381,796
- Filed: 2021-07-21
- Published: 2022-02-03
- Earliest claimed: 2015-10-28
- Assignees: Silicon Valley Scientific, Inc.
- Applicants: Silicon Valley Scientific, Inc.
- Inventors: Stevan Bogdan Jovanovich; Peter Wagner
- Web page: https://patentgrep.com/patent/US20220033802A1

## CPC

- C12N 15/1003: {Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor}
- C12N 15/1017: {by filtration, e.g. using filters, frits, membranes}
- C12N 15/1093: {General methods of preparing gene libraries, not provided for in other subgroups}
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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

A system, methods, and apparatus are described to collect and prepare single cells and groups of cells from microsamples of specimens and encode spatial information of the physical position of the cells in the specimen. In some embodiment, beads or surfaces with oligonucleotides containing spatial barcodes are used to analyze DNA or RNA. The spatial barcodes allow the position of the cell to be defined and the nucleic acid sequencing information, such as target sequencing, whole genome, gene expression, used to analyze the cells in a microsample for cell type, expression pattern, DNA sequence, and other information, in the context of the cell's physical position in the specimen. In other embodiment, markers such as isotopes are added to a microsample to encode spatial position with mass spectoscopy or other analysis. The spatial encoded information is then readout by analysis such as DNA sequencing, mass spectrometry, fluorescence, or other methods.

## Claims

### Claim at position 1

-13. (canceled)

### Claim 14

A method comprising:
adding, to each of a plurality of different microsamples from a biological specimen, a marker comprising spatial information that encodes the original spatial position of the microsample within the biological specimen.

### Claim 15 (depends on 14)

The method of claim 14, further comprising dissociating the microsamples from the biological specimen.

### Claim 16 (depends on 15)

The method of claim 15, comprising adding the markers to the microsamples before dissociating the microsamples from the biological specimen.

### Claim 17 (depends on 15)

The method of claim 15, comprising adding the markers to the microsamples after dissociating the microsamples from the biological specimen.

### Claim 18 (depends on 15)

The method of claim 15, wherein each microsample comprises a single cell.

### Claim 19 (depends on 15)

The method of claim 15, wherein each microsample comprises a plurality of cells.

### Claim 20 (depends on 15)

The method of claim 15, wherein dissociating the microsamples comprises extracting the microsamples in a raster pattern across the biological specimen.

### Claim 21 (depends on 15)

The method of claim 15, wherein the microsamples are dissociated in a 3-D pattern.

### Claim 22 (depends on 15)

The method of claim 15, wherein dissociating comprises contacting the biological specimen with a membrane, applying vacuum to the membrane to hold a layer comprising the microsamples; and removing the microsamples held by the membrane from the biological specimen.

### Claim 23 (depends on 22)

The method of claim 22, comprising removing a second layer of the microsamples from the biological specimen after a first layer is removed.

### Claim 24 (depends on 15)

The method of claim 15, further comprising moving the dissociated microsamples into a fluidic stream.

### Claim 25 (depends on 24)

The method of claim 24, wherein the microsamples are moved into the fluidic stream in an order correlated with their original spatial position in the biological specimen.

### Claim 26 (depends on 24)

The method of claim 24, wherein microsamples are incorporated into microdrops (e.g., nanodroplets or boluses) in the fluidic stream.

### Claim 27 (depends on 26)

The method of claim 26, wherein the microdrops contain one or more beads.

### Claim 28 (depends on 27)

The method of claim 27, wherein the beads are paramagnetic.

### Claim at position 17

-33. (canceled)

### Claim 34

The method of claim Error! Reference source not found., wherein the oligonucleotide further comprises a poly T tail, and the method comprises capturing mRNA molecules from the microsamples having a poly T tail; and reverse transcribing the mRNA molecules to produce cDNA molecules comprising the barcode where the barcode provides the spatial information.

### Claim 35

The method of claim Error! Reference source not found.7, wherein the oligonucleotide further comprises a capture sequence complementary to a target sequence, and the method comprises capturing DNA molecules from the microsample having the target sequence; and extending the oligonucleotide to produce a nucleic acid molecule having a copy of the target sequence and comprising the barcode, wherein the barcode provides the spatial information.

### Claim at position 20

-38. (canceled)

### Claim 39 (depends on 27)

The method of claim 27, further comprising decoding the spatial information in the microsamples to determine the original spatial position of each microsamples.

### Claim at position 22

-42. (canceled)

### Claim 43

A spatial preparation system configured to entrain in a fluidic stream a plurality of microsamples from a biological specimen, wherein the microsamples are contained in spatially separated microdrops in a fluidic stream and positioned in an order based on their original spatial position within the biological specimen, wherein the system comprises:
a) a spatial sampler subsystem configured to extract a plurality of microsamples from different original spatial positions in a biological specimen; and
b) a spatial encoder subsystem comprising one or more spatial encoder microchannels, each having an inlet and an outlet;
wherein the spatial sampler subsystem delivers the microsamples to the spatial encoder microchannel inlets in a predetermined order based on their original spatial position in the biological specimen, and the spatial encoder subsystem incorporates the microsamples into spatially separated microdrops in a fluidic stream.

### Claim at position 24

-54. (canceled)

### Claim 55

A method comprising entraining in a fluidic stream a plurality of microsamples from a biological specimen, wherein the microsamples are contained in spatially separated microdrops in the fluidic stream and positioned in an order based on their original spatial position within the biological specimen.

### Claim 56 (depends on 55)

The method of claim 55, comprising:
a) providing a biological specimen;
b) collecting microsamples from each of a plurality of different spatial positions in the biological specimen;
c) introducing the microsamples in a predetermined order into a fluidic stream in a fluidic channel;
d) dividing the fluidic stream into microdrops by introducing boluses of immiscible liquid into the fluidic channel, whereby the microsamples are incorporated into microdrops that are spatially separated from each other in the fluidic stream.

### Claim 57 (depends on 55)

The method of claim 55, further comprising:
(i) introducing into the fluidic stream a plurality of different spatial markers encoding spatial information, wherein the different spatial markers are incorporated into different microdrops in the fluidic stream, thereby encoding each microdrop with spatial information.

### Claim 58 (depends on 57)

The method of claim 57, wherein the analytes comprise nucleic acids and the spatial markers comprise nucleic acids comprising nucleic acid barcodes, wherein the method further comprises:
(e) combining microdrops in a container in the form of an emulsion;
(f) generating spatially tagged nucleic acids by tagging nucleic acid analytes with the nucleic acid barcodes;
(g) breaking the emulsion;
(h) amplifying the tagged nucleic acids.

### Claim 59 (depends on 58)

The method of claim 58, wherein the analytes comprise polyadenylated mRNA and the nucleic acid markers further comprise polyT tail, and generating spatially tagged nucleic acids comprises:
hybridizing the polyT tail to polyadenylated mRNA nucleic acid markers to the mRNA molecules barcodes and reverse transcribing the polyadenylated messenger RNA to produce that spatially tagged cDNA molecules;
performing second strand synthesis on the spatially tagged cDNA molecules to produce tagged double stranded cDNA molecules.

### Claim 60 (depends on 58)

The method of claim 58, wherein the analytes comprise DNA molecules and the nucleic acid markers further comprise a nucleotide sequence complementary to a target sequence, and generating spatially tagged nucleic acids comprises:
hybridizing the complementary nucleotide sequence to a target sequence in the nucleic acid molecules and extending the nucleic acid markers to produce a double-stranded DNA molecule.

### Claim 61 (depends on 57)

The method of claim 57, wherein further comprising applying imaging reagent to the biological sample; imaging the biological sample to which the imaging reagent has been applied; based on the imaging selecting features of interest at predetermined spatial positions in the biological sample; and extracting the microsamples including the selected features of interest.

### Claim 62 (depends on 57)

The method of claim 57, further comprising, based on spatial information encoded in the microsamples determining the initial spatial position of the selected features in the biological specimen.