# US 11,111,487 B2 · Method and apparatus for encoding cellular spatial position information

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- Number: US 11,111,487 B2
- Document ID: US11111487B2
- Status: Granted
- Kind: B2
- Application number: 15/771,607
- Filed: 2016-10-27
- Published: 2018-10-25
- Granted: 2021-09-07
- Earliest claimed: 2015-10-28
- Assignees: Silicon Valley Scientific, Inc.
- Applicants: Silicon Valley Scientific, Inc.
- Inventors: Stevan B. Jovanovich; Peter Wagner
- Web page: https://patentgrep.com/patent/US11111487B2

## 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 1

A spatial preparation system comprising:
a) a spatial sampler subsystem comprising:
(1) a specimen holder comprising a plate having a flat surface, and, on the flat surface, a biological specimen comprising a tissue, and
(2) a multifunctional head comprising a transfer head comprising a plurality of extraction channels, wherein the extraction channels comprise openings covered with one or more transfer membranes, and wherein the extraction channels are in communication with a first liquid source under positive and/or negative pressure, and wherein the multifunctional head is mounted on a three axis stage to position the multifunctional head over the specimen holder at different spatial positions; and
b) a spatial encoder subsystem comprising:
(1) a plurality of spatial encoder microchannels, each microchannel having an inlet, and each communicating with a common output microchannel at different first junctions;
(2) a reagent rail comprising a plurality of containers, each container comprising different marker molecules comprising different spatial information, wherein the reagent rail communicates with the common output microchannel;
(3) a microdroplet generator comprising a source of immiscible liquid in communication with the common output microchannel at a second junction downstream of the first junctions; and
(4) one or more pumps communicating with the spatial encoder microchannels, the common output microchannel, the reagent rail and the source of immiscible liquid; and
wherein:
(1) the spatial sampler subsystem is configured:
(i) to extract, by contact adhesion or by vacuum, a plurality of microsamples from each of a plurality of different original spatial positions on the biological specimen onto the one or more transfer membranes;
(ii) to move the multifunctional head using the stage to the inlets of the spatial encoder microchannels; and
(iii) to deliver through application of positive or negative pressure, each extracted microsample to an inlet of one of the spatial encoder microchannels in a pattern based on their original spatial position in the biological specimen; and
(2) the spatial encoder subsystem is configured:
(i) to move each microsample from a spatial encoder microchannel to the common output microchannel through application of positive or negative pressure;
(ii) to move different marker molecules from containers in the reagent rail into the common output microchannel through application of positive or negative pressure; and
(iii) to generate microdrops in the common output channel, wherein the microdrops are spatially separated by the immiscible fluid, wherein a plurality of the microdrops comprise a microsample and a marker molecule.

### Claim 2 (depends on 1)

The system of claim 1, wherein the multifunctional head further comprises a dispense head configured to dispense liquid onto the biological specimen.

### Claim 3 (depends on 1)

The system of claim 1, wherein the extraction channels are arrayed in a linear array or a planar array.

### Claim 4 (depends on 1)

The system of claim 1, wherein the multifunctional head comprises a gas source.

### Claim 5 (depends on 1)

The system of claim 1, wherein the spatial encoder subsystem comprises:
a microsample encoder assembly comprising a plurality of reservoirs, each comprising a different spatial marker and each communicating with the spatial encoder microchannel, and, reservoirs comprising a reverse transcriptase, a polymerase or a ligase.

### Claim 6 (depends on 1)

The system of claim 1, wherein the transfer membranes have attached thereto a plurality of capture elements, each capture element comprising a particle having attached thereto one or more antibodies that bind into cells in the biological specimen, and nucleic acid markers comprising positional barcodes comprising spatial information where the spatial information calling to the position of the particle on the multifunctional head.

### Claim 7 (depends on 6)

The system of claim 6, wherein, the particle is paramagnetic.

### Claim 8 (depends on 6)

The system of claim 6, wherein, the nucleic acid markers further comprise cell markers identifying the cell to which particle binds, and/or molecular barcodes that differently label different nucleic acid molecules and a single cell.

### Claim 9 (depends on 1)

The system of claim 1, wherein the spatial sampler subsystem comprises a vacuum source that applies vacuum to the multifunctional head for extracting cells from tissue deposited on the specimen holder and a pressure source to push extracted cells from the multifunctional head.

### Claim 10 (depends on 1)

The system of claim 1, wherein the spatial markers comprise oligonucleotide barcodes attached to solid supports.

### Claim 11 (depends on 2)

The system of claim 2, wherein the multifunctional head comprises cell dissociation solution comprising at least one protease that digests extracellular matrix.

### Claim 12 (depends on 1)

The system of claim 1, further comprising:
c) a spatial librarian subsystem configured to perform a series of biochemical reactions on an emulsion comprising microdrops produced by the spatial preparation subsystem, wherein the spatial librarian subsystem comprises:
a reaction device comprising an inlet communicating with an outlet of the common output microchannel, at least one reaction chamber, and an outlet;
a reagent rail communicating with the reaction device through a microchannel and comprising a reverse transcriptase or a polymerase and;
one or more pumps configured to move the reagents from the reagent rail through the microchannel to the reaction chamber of the reaction device.

### Claim 13 (depends on 12)

The system of claim 12, wherein the spatial librarian subsystem further comprises:
a temperature controller configured to control temperature in the reaction chamber.

### Claim 14 (depends on 12)

The system of claim 12, wherein the spatial librarian subsystem further comprises:
a magnet configured to reversibly immobilize paramagnetic particles contained in the reaction chamber.

### Claim 15 (depends on 12)

The system of claim 12, wherein the biochemical reactions comprise at least:
(i) reverse transcription of messenger RNA into cDNA; and
(ii) amplification of cDNA.

### Claim 16 (depends on 12)

The system of claim 12, wherein the biochemical reactions comprise at least:
(i) primer extension of a primer hybridized to a DNA template to create an extension product; and
(ii) amplification of the extension product.

### Claim 17 (depends on 2)

The system of claim 2, wherein the fluid comprises an imaging reagent or dissociation solution.

### Claim 18 (depends on 1)

The system of claim 1, wherein the extraction channels each comprise ends covered with an air permeable, cell impermeable transfer membrane.

### Claim 19 (depends on 18)

The system of claim 18, wherein a single membrane covers the ends of the plurality of extraction channels.

### Claim 20 (depends on 1)

The system of claim 1, wherein the extraction channels are arrayed in a line or a plane.

### Claim 21 (depends on 1)

The system of claim 1, wherein the spatial sampler subsystem is configured to deliver each microsample to an inlet of one of the spatial encoder microchannels in one step.