Methods, compositions, and systems for mapping locations of single molecules in multi-dimensional space
Systems, methods, and compositions for generating a high-resolution spatial map of a distribution of targets of a sample are described. Processes for generating the spatial map can include: receiving the sample at a substrate having a distribution of functionalized particles, each having a stochastic barcode sequence paired with a position on the substrate; promoting interactions between the distribution of targets of the sample and the distribution of functionalized particles upon transmitting heat to a surface of the substrate opposite the distribution of functionalized particles; applying a set of reactions to the sample at the substrate, obtaining a set of sequences of a population of molecules generated from the set of reactions, the set of sequences associated with the distribution of targets labeled using the stochastic barcode sequences of the distribution of functionalized particles, and returning a set of positions of the distribution of targets upon processing the set of sequences.
1 . A method for whole transcriptome spatial analysis of a tissue sample, the method comprising:
(a) receiving the tissue sample at a substrate comprising a distribution of functionalized particles,
wherein each particle of the distribution of functionalized particles comprises an exonuclease-treated oligonucleotide comprising a stochastic barcode sequence paired with a position on the substrate, and
wherein the distribution of functionalized particles is coupled to the substrate with a hydrophobic and electrostatic adhesive polymer layer comprising ethylene;
(b) promoting interactions between target molecules of the tissue sample and the distribution of functionalized particles;
(c) applying a set of reactions to the tissue sample at the substrate, wherein the tissue sample is not subjected to a permeabilization reaction;
(d) obtaining a set of sequences of a population of molecules generated from the set of reactions, the set of sequences associated with the target molecules labeled using the stochastic barcode sequences of the distribution of functionalized particles;
(e) returning a set of positions of the target molecules upon processing the set of sequences; and
(f) generating a spatial map of a distribution of the target molecules based on the set of positions, wherein the spatial map is characterized by a signal-to-noise ratio (SNR) wherein the noise is minimized based on the non-permeabilized tissue sample.
2 . The method of claim 1 , wherein the tissue sample is comprises a frozen tissue sample.
3 . The method of claim 1 , wherein the set of reactions comprises hybridization between mRNA targets of the tissue sample and capture segments of the distribution of functionalized particles barcoded oligonucleotides.
4 . The method of claim 1 , wherein the spatial map comprises a resolution greater than 1 target per 50 μm 2 .
5 . The method of claim 1 , wherein the spatial map comprises single cell resolution.
6 . The method of claim 1 , wherein the set of reactions comprises probe-ligation and permeabilization of the tissue sample.
7 . The method of claim 1 , wherein each particle of the distribution of functionalized particles further barcoded oligonucleotides comprises a photocleavable linker.
8 . The method of claim 1 , wherein the tissue sample comprises a formalin-fixed paraffin-embedded tissue sample comprising analytes modified to have a polyadenylated tail.
9 . The method of claim 1 , wherein the spatial map comprises a characterization of single cell subtypes of the tissue sample based upon an analysis of spatial marker genes represented by the set of sequences.
10 . The method of claim 1 , wherein the spatial map comprises a mapping of greater than 20,000 targets.
11 . The method of claim 1 , wherein at least one of the set of reactions of the tissue sample comprises obtaining a nuclei in suspension from the tissue sample.
12 . The method of claim 1 , wherein receiving the tissue sample at the substrate comprises aligning the tissue sample with the distribution of functionalized particles.
13 . The method of claim 12 , further comprising staining the tissue sample.
14 . The method of claim 1 , wherein the SNR is determined by:
identifying a gene with a known expression profile across regions of the tissue sample, wherein the target molecules comprise the gene;
quantifying expression of the gene across the tissue sample based upon the set of positions of the distribution of the target molecules;
determining a signal value from expression of the gene in regions of the tissue sample that should express the gene, based upon the known expression profile;
determining a noise value from expression of the gene in regions of the tissue sample that should not express the gene, based upon the known expression profile; and
determining the SNR from the signal value and the noise value.
15 . The method of claim 1 , wherein capture segments of each of the exonuclease-treated oligonucleotides comprise polyT sequences.
16 . The method of claim 1 , wherein the distribution of functionalized particles barcoded oligonucleotides are attached to an intermediate particle layer, and wherein the intermediate particle layer is distributed onto the substrate.
17 . The method of claim 1 , wherein the hydrophobic and electrostatic adhesive polymer layer is to an intermediate particle layer distributed onto a glass substrate.
18 . The method of claim 1 , further comprising freezing the tissue sample at the substrate after receiving the tissue sample at the substrate.