High performance spatial mapping of individual targets using releasable handshake sequences
Systems, methods, and compositions for generating a spatial map of a distribution of targets of a sample are described. A system for mapping targets can include a substrate; and a distribution of functionalized features associated with the substrate, wherein a representative feature of the distribution of functionalized features comprises: one or more functionalized molecules coupled to the representative feature, the one or more molecules including at least: a handshake sequence comprising a reactive portion, a barcode segment serving as a spatial address, and a cleavable linker configured to allow the handshake sequence to be released from the representative feature in response to a stimulus. The system can be used to tag nuclei, cells, and/or other target components of a sample.
1 . A method comprising:
applying a layer of optimum cutting temperature (OCT) compound to a tissue sample;
tagging a distribution of nuclei from the tissue sample with a set of functionalized molecules, wherein a functionalized molecule of the set of functionalized molecules comprises:
a handshake sequence;
a barcode sequence that serves as a spatial address; and
a releasable linker,
wherein tagging comprises:
applying a stimulus to the releasable linkers of the set of functionalized molecules, wherein upon application of the stimulus, a set of handshake sequences and barcode sequences are cleaved from the set of functionalized molecules to allow diffusion of the set of handshake sequences and barcode sequences toward the distribution of nuclei to tag the distribution of nuclei;
isolating a set of nuclei from the tissue sample after the OCT compound is applied; and
generating and displaying a spatial map of the isolated set of nuclei from the tissue sample within an interface of an electronic display, wherein generating the spatial map comprises recovering and mapping positions of more than 30% of nuclei originally present in the tissue sample.
2 . A method comprising:
applying a layer of optimum cutting temperature (OCT) compound to a tissue sample;
tagging a distribution of nuclei from the tissue sample with a set of functionalized molecules, wherein a functionalized molecule of the set of functionalized molecules comprises:
a handshake sequence;
a barcode sequence that serves as a spatial address; and
a releasable linker,
wherein tagging comprises:
applying a stimulus to the releasable linkers of the set of functionalized molecules, wherein upon application of the stimulus, a set of handshake sequences and barcode sequences are cleaved from the set of functionalized molecules to allow diffusion of the set of handshake sequences and barcode sequences toward the distribution of nuclei to tag to the distribution of nuclei;
isolating a set of nuclei from the tissue sample after the OCT compound is applied; and
generating and displaying a spatial map of the isolated set of nuclei from the tissue sample within an interface of an electronic display, wherein generating the spatial map is performed within a duration of 2 hours.
3 . The method of claim 2 , further comprising decoding the set of barcode sequences by performing a set of iterations of sequencing with error-reduction by dynamic annealing and ligation (SEDAL) upon the set of barcode sequences coupled to a substrate, wherein decoding the set of barcode sequences has a passing rate greater than 90% as determined by a set of criteria, and wherein the set of criteria comprises a substrate coverage criterion and a criterion regarding amount of empty space on the substrate.
4 . A method comprising:
applying a layer of optimum cutting temperature (OCT) compound to a tissue sample;
tagging a distribution of nuclei from the tissue sample with a set of functionalized molecules, wherein a functionalized molecule of the set of functionalized molecules comprises:
a handshake sequence;
a barcode sequence that serves as a spatial address; and
a releasable linker,
wherein tagging comprises:
applying a stimulus to the releasable linkers of the set of functionalized molecules, wherein upon application of the stimulus, a set of handshake sequences and barcode sequences are cleaved from the set of functionalized molecules to allow diffusion of the set of handshake sequences and barcode sequences toward the distribution of nuclei to tag the distribution of nuclei;
isolating a set of nuclei from the tissue sample after the OCT compound is applied; and
generating and displaying a spatial map of the isolated set of nuclei from the tissue sample within an interface of an electronic display, upon releasing the set of handshake sequences and barcode sequences for diffusion toward the distribution of nuclei.
5 . A method comprising:
applying a layer of optimum cutting temperature (OCT) compound to a tissue sample;
generating and displaying a spatial map of a set of nuclei of the tissue sample upon:
processing the tissue sample comprising a distribution of nuclei with a substrate comprising a set of functionalized molecules, wherein a representative functionalized molecule of the set of functionalized molecules comprises:
a handshake sequence that binds to a nucleus of the distribution of nuclei,
a barcode sequence that serves as a spatial address, and
a releasable linker;
tagging the distribution of nuclei with handshake sequences of the set of functionalized molecules, wherein tagging comprises:
applying a stimulus to the releasable linkers of the set of functionalized molecules, wherein upon application of the stimulus, a set of handshake sequences and barcode sequences are cleaved from the set of functionalized molecules to allow diffusion of the set of handshake sequences and barcode sequences toward the distribution of nuclei to tag the distribution of nuclei;
isolating a set of nuclei of the tissue sample after the OCT compound is applied;
determining a set of sequences of resultant molecules generated from the set of nuclei and the set of functionalized molecules, thereby determining a set of spatial positions of the set of nuclei based upon the barcode sequences associated with each nucleus of the set of nuclei; and
generating and displaying the spatial map of the set of nuclei from the set of spatial positions within an interface of an electronic display.
6 . The method of claim 5 , wherein generating the spatial map is performed within a duration of 2 hours.
7 . The method of claim 5 , wherein isolating the set of nuclei comprises recovering more than 30% of nuclei originally present in the tissue sample.
8 . The method of claim 7 , wherein isolating the set of nuclei comprises trituration of the tissue sample with an isolation buffer.
9 . The method of claim 7 , wherein isolating the set of nuclei comprises isolating the set of nuclei with an irrigator.
10 . The method of claim 5 , wherein the releasable linker is responsive to a photocleaving mechanism.
11 . The method of claim 5 , wherein the set of functionalized molecules comprises a first subset of releasable linkers that cleave in response to a first stimulus, and a second subset of releasable linkers that cleave in response to a second stimulus.
12 . The method of claim 11 , wherein the first stimulus is a first wavelength of light, and wherein the second stimulus is a second wavelength of light.
13 . The method of claim 5 , wherein determining the set of spatial positions comprises determining a position of a nucleus from a centroid of a subset of spatial positions of a subset of barcode sequences of a subset of functionalized molecules that tagged the nucleus.
14 . The method of claim 5 , wherein the method is performed at or below 4 degrees C.
15 . The method of claim 5 , wherein the set of functionalized molecules is distributed across a distribution of particles coupled to the substrate by an adhesive.
16 . The method of claim 5 , further comprising covering the set of functionalized molecules of the substrate with a masking layer and removing the masking layer prior to applying the stimulus.