Provided herein are methods, compositions, and kits for the detection of immune cell clonotypes and immune cell analytes within a biological sample.

Provided herein are methods, compositions, and kits for the detection of immune cell clonotypes and immune cell analytes within a biological sample.

The invention relates to methods for pairwise sequencing of a polynucleotide template which result in the sequential determination of nucleotide sequence in two distinct and separate regions of the polynucleotide template.

The invention relates to methods for pairwise sequencing of a polynucleotide template which result in the sequential determination of nucleotide sequence in two distinct and separate regions of the polynucleotide template.

The invention is directed to a method to obtain the spatial location and sequence information of a target sequence in a sample comprising at least one m-RNA strand comprising the steps a. providing a surface with a plurality of spacer units capable of binding at least one m-RNA strand and with at least one fiducial marker b. providing a sample comprising at least one m-RNA strand to the surface wherein at least one m-RNA strand of the sample binds to at least one spacer unit creating at least one single stranded oligomer c. taking a first image of the surface to obtain the spatial information of the sample relative to the fiducial marker d. removing sample form surface e. hybridizing at least one oligonucleotide comprising 50-1000 nucleic acids with its 5′ and 3′ ends to complementary parts of the single stranded oligomer thereby forming a padlock-shaped structure that is ligated to create a single strand circular template f. multiplying the single strand circular template by a polymerase capable of rolling circle amplification into a plurality of DNA concatemers thereby forming rolonies g. obtaining the sequence information of the rolonies h. linking the spatial information of the sample with the sequence information of the rolonies.

The invention is directed to a method to obtain the spatial location and sequence information of a target sequence in a sample comprising at least one m-RNA strand comprising the steps a. providing a surface with a plurality of spacer units capable of binding at least one m-RNA strand and with at least one fiducial marker b. providing a sample comprising at least one m-RNA strand to the surface wherein at least one m-RNA strand of the sample binds to at least one spacer unit creating at least one single stranded oligomer c. taking a first image of the surface to obtain the spatial information of the sample relative to the fiducial marker d. removing sample form surface e. hybridizing at least one oligonucleotide comprising 50-1000 nucleic acids with its 5′ and 3′ ends to complementary parts of the single stranded oligomer thereby forming a padlock-shaped structure that is ligated to create a single strand circular template f. multiplying the single strand circular template by a polymerase capable of rolling circle amplification into a plurality of DNA concatemers thereby forming rolonies g. obtaining the sequence information of the rolonies h. linking the spatial information of the sample with the sequence information of the rolonies.

Kits and methods of using the kits for analyzing macromolecules, including peptides, polypeptides, and proteins, employing nucleic acid encoding are disclosed. The sample analysis kits employ nucleic acid encoding and/or nucleic acid recording of a molecular interaction and/or reaction, such as recognition events (e.g., between an antigen and an antibody, between a modified terminal amino acid residue, or between a small molecule or peptide therapeutic and a target, etc.). Additional barcoding reagents, such as those for cycle-specific barcoding (e.g., “clocking”), compartment barcoding, combinatorial barcoding, spatial barcoding, or any combination thereof, may be included in the kits. The sample may comprise macromolecules, including peptides, polypeptides, and proteins, and the recording may generate molecular interaction and/or reaction information, and/or polypeptide sequence information. The kits may be used in high-throughput, multiplexed, and/or automated analysis, and are suitable for analysis of a proteome or subset thereof.

Kits and methods of using the kits for analyzing macromolecules, including peptides, polypeptides, and proteins, employing nucleic acid encoding are disclosed. The sample analysis kits employ nucleic acid encoding and/or nucleic acid recording of a molecular interaction and/or reaction, such as recognition events (e.g., between an antigen and an antibody, between a modified terminal amino acid residue, or between a small molecule or peptide therapeutic and a target, etc.). Additional barcoding reagents, such as those for cycle-specific barcoding (e.g., “clocking”), compartment barcoding, combinatorial barcoding, spatial barcoding, or any combination thereof, may be included in the kits. The sample may comprise macromolecules, including peptides, polypeptides, and proteins, and the recording may generate molecular interaction and/or reaction information, and/or polypeptide sequence information. The kits may be used in high-throughput, multiplexed, and/or automated analysis, and are suitable for analysis of a proteome or subset thereof.

An example of a flow cell includes a substrate and a pattern of two different silanes on at least a portion of a surface of the substrate. A first polymer is attached to a first of the two different silanes and a second polymer is attached to a second of the two different silanes. The first and second polymers respectively include a first functional group and a second functional group of a functional group pair, the functional group pair being selected from the group consisting of an activated ester functional group and an azide functional group, a tetrazine functional group and an activated ester functional group, and a tetrazine functional group and an azide functional group. A first primer set is grafted to the first polymer and a second primer set is grafted to the second polymer. The first and second primer sets are different.

An example of a flow cell includes a substrate and a pattern of two different silanes on at least a portion of a surface of the substrate. A first polymer is attached to a first of the two different silanes and a second polymer is attached to a second of the two different silanes. The first and second polymers respectively include a first functional group and a second functional group of a functional group pair, the functional group pair being selected from the group consisting of an activated ester functional group and an azide functional group, a tetrazine functional group and an activated ester functional group, and a tetrazine functional group and an azide functional group. A first primer set is grafted to the first polymer and a second primer set is grafted to the second polymer. The first and second primer sets are different.