MASSIVELY PARALLEL CHARACTERIZATIONS OF MULTIPLE TARGET TYPES 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. Systems and methods can further include features configured to reduce observation of smearing artifacts in generated spatial maps. 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; applying a set of reactions to the sample at the substrate; and obtaining a set of sequences of a population of molecules generated from the set of reactions.
1 . A system comprising:
a substrate comprising a distribution of functionalized features,
wherein the distribution of functionalized features comprises a set of barcode sequences corresponding to a set of positions on the substrate, and
wherein the distribution of functionalized features comprises molecules functionalized to interact with a set of targets of a sample; and
a smear-prevention layer configured to be positioned over the sample when the sample is positioned on the distribution of functionalized features.
2 . The system of claim 1 , wherein the smear-prevention layer comprises optimum cutting temperature (OCT) compound.
3 . The system of claim 1 , wherein the smear-prevention layer comprises OCT compound combined with a sample processing reagent.
4 . The system of claim 1 , wherein the distribution of functionalized features comprises a set of functionalized particles coupled to the substrate in a random close packed configuration.
5 . The system of claim 1 , wherein the distribution of functionalized features comprises features with a center-to-center spacing less than 5 micrometers.
6 . The system of claim 1 , wherein the distribution of functionalized features comprises at least 20 million functionalized features.
7 . The system of claim 1 , wherein the sample comprises a tissue sample.
8 . The system of claim 1 , wherein the set of targets comprises mRNA targets of the sample.
9 . A method comprising:
generating a spatial map of a distribution of targets of a tissue sample, with a percent reduction in smearing artifacts in the spatial map greater than 60%, upon:
receiving the tissue sample at a substrate comprising a distribution of functionalized features, each of the distribution of functionalized features comprising a barcode sequence paired with a position on the substrate;
applying a smear-prevention layer over the tissue sample at the substrate;
promoting interactions between the distribution of targets of the tissue sample and the distribution of functionalized features;
applying a set of reactions, comprising a hybridization reaction, to the tissue sample at the substrate, with the tissue sample positioned between the smear-prevention layer and 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 barcode sequences of the distribution of functionalized features; and
returning a set of positions of the distribution of targets upon processing the set of sequences.
10 . The method of claim 9 , wherein the smear-prevention layer comprises optimum cutting temperature (OCT) compound.
11 . The method of claim 9 , wherein the smear-prevention layer comprises OCT compound combined with a processing reagent.
12 . The method of claim 9 , wherein the smear-prevention layer comprises a thickness greater than 5 micrometers thick.
13 . The method of claim 9 , wherein generating the spatial map of the distribution of targets of the tissue sample with the percent reduction in smearing artifacts further comprises:
omitting mapping of data from a first category of features of the distribution of functionalized features, wherein sequences acquired from the first category of features have unique molecular identifier (UMI) counts above a first threshold; and
omitting mapping of data from a second category of features of the distribution of functionalized features, wherein sequences acquired from the second category of features have an associated density greater than a second threshold.
14 . The method of claim 13 , wherein the first threshold is greater than 70 UMI counts.
15 . The method of claim 13 , wherein the second threshold is a density greater than 1 particle within an area of 20 micrometers.
16 . The method of claim 9 , wherein the tissue sample comprises one of a fresh frozen tissue sample and a formalin-fixed and paraffin-embedded (FFPE) tissue sample.
17 . The method of claim 9 , wherein the distribution of targets comprises mRNA targets.
18 . The method of claim 9 , wherein the distribution of functionalized features comprises at least 800,000 features with a center-to-center spacing less than 5 micrometers.
19 . A system comprising:
a substrate comprising a distribution of functionalized features;
a set of cells positioned at interstitial spaces of the distribution of functionalized features, wherein the distribution of functionalized features comprises a set of barcode sequences corresponding to a set of positions on the substrate, and wherein the distribution of functionalized features comprises molecules functionalized to interact with a set of targets of the set of cells; and
a layer configured to be positioned over the distribution of functionalized features.
20 . The system of claim 19 , wherein the layer comprises a layer of optimum cutting temperature (OCT) compound.