Paired macromolecule abundance and t-cell receptor sequencing with high spatial resolution

A method for obtaining from a tissue sample spatially-resolvable TCR sequence that spans TCR transcript variable regions and spatially-resolvable bulk poly-A-tailed RNA expression data, the method comprising:

• (i) obtaining a tissue sample from a subject;
• (ii) preparing a section of the tissue sample;
• (iii) obtaining a solid support;
• (iv) contacting the solid support with a capture material, thereby forming a capture material-coated solid support;
• (v) contacting the capture material-coated solid support with a population of 1-100 μm diameter beads, wherein each bead has at least 1000 attached oligonucleotides and wherein at least 1000 attached oligonucleotides of each bead each comprises:
• (a) a bead identification sequence that is common to all at least 1000 oligonucleotides on each bead and (b) a poly-dT tail of sufficient length to allow for capture of poly-A-tailed RNAs via hybridization wherein the bead identification sequence that is common to all at least 1000 oligonucleotides on each bead is either a bead identification sequence that is unique to each bead within the population of 1-100 μm diameter beads or is a bead identification sequence that is a member of a population of bead identification sequences that is sufficiently degenerate to the population of 1-100 μm diameter beads that a majority of beads within the population of 1-100 μm diameter beads each possesses a unique bead identification sequence, thereby capturing a subpopulation of the population of 1-100 μm diameter beads upon the solid support;
• (vi) identifying the bead identification sequence and associated two-dimensional position on the solid support of individual beads of the subpopulation of beads attached to the solid support;
• (vii) contacting the subpopulation of 1-100 μm diameter beads captured upon the solid support with the section of the tissue sample;
• (viii) performing a reverse transcription reaction upon poly-A-tailed RNAs captured by the bead subpopulation, thereby generating a cDNA population;
• (ix) performing PCR amplification upon the cDNA subpopulation in a manner that does not specifically enrich for TCR-alpha and TCR-beta sequences, thereby generating a PCR-amplified nucleic acid population not specifically enriched for TCR-alpha and TCR-beta sequences;
• (x) contacting the PCR-amplified nucleic acid population not specifically enriched for TCR-alpha and TCR-beta sequences, or a subpopulation thereof, with (a) RNase H-dependent PCR primers designed for specific amplification of TCR-alpha and TCR-beta cDNAs and (b) RNase H, and performing PCR amplification, thereby generating a PCR-amplified nucleic acid population enriched for TCR-alpha and TCR-beta sequences;
• (xi) combining the PCR-amplified nucleic acid population enriched for TCR-alpha and TCR-beta sequences and the PCR-amplified nucleic acid population not specifically enriched for TCR-alpha and TCR-beta sequences into a single PCR-amplified nucleic acid population;
• and
• (xii) obtaining sequence from the PCR-amplified nucleic acid population using a sequencing process for TCR sequence-containing PCR-amplified nucleic acids having an average read length on at least one end in excess of 200 nucleotides, thereby obtaining (a) TCR sequences that span TCR transcript variable regions for substantially all TCR sequences obtained;
• (b) sequences of bead identification sequences associated with TCR sequences;
• and (c) sequences of a population of poly-A-tailed RNAs bound to the bead oligonucleotides and associated bead identification sequences for sequenced poly-A-tailed RNAs,
• thereby obtaining from the tissue sample spatially-resolvable T cell receptor (TCR) sequence that spans TCR transcript variable regions and spatially-resolvable bulk poly-A-tailed RNA expression data.

A method selected from the group consisting of:

• A method for obtaining from a tissue sample spatially-resolvable TCR sequence that spans TCR transcript variable regions, the method comprising:
• (i) generating a well array, wherein each well of the array can hold exactly one bead;
• (ii) depositing beads into the wells of the well array, optionally by evaporation in a centrifuge;
• (iii) brushing the well array to remove all of the beads not present in wells;
• (iv) obtaining a tissue sample from a subject;
• (v) preparing a section of the tissue sample;
• (vi) depositing the section onto the well array and centrifuging, thereby forcing the section into the wells of the well array;
• (vii) adding digestion buffer, thereby lysing the section and causing the RNA of cells of the section to transfer onto the beads in the wells;
• (viii) performing a reverse transcription reaction upon the beads in the wells, thereby generating a cDNA population;
• (ix) contacting a selection of or all of the cDNA population with (a) RNase H-dependent PCR primers designed for specific amplification of TCR-alpha and TCR-beta cDNAs and (b) RNase H, and performing PCR amplification upon the cDNA population, thereby generating a PCR-amplified nucleic acid population comprising TCR-alpha and TCR-beta sequences;
• and
• (x) obtaining sequence from the PCR-amplified nucleic acid population using a sequencing process for TCR sequence-containing PCR-amplified nucleic acids having an average read length on at least one end in excess of 200 nucleotides, thereby obtaining TCR sequences that span TCR transcript variable regions for substantially all TCR sequences obtained and obtaining sequence from the PCR-amplified nucleic acid population of bead identification sequences associated with TCR sequences,
• optionally further comprising removing beads from the wells by sonication or by photocleavage after step (vii), optionally before performing step (viii),
• thereby obtaining from the tissue sample spatially-resolvable T cell receptor (TCR) sequence that spans TCR transcript variable regions;
• A method for obtaining from a tissue sample spatially-resolvable TCR sequence that spans TCR transcript variable regions, the method comprising:
• (i) obtaining a tissue sample from a subject;
• (ii) preparing a section of the tissue sample;
• (iii) obtaining a solid support;
• (iv) adhering clusters of oligonucleotides in an array attached to the solid support, optionally wherein the array comprises barcoded clusters of oligonucleotides on a surface;
• (v) identifying oligonucleotide cluster identification sequences and associated two-dimensional positions on the solid support of individual oligonucleotide clusters attached to the solid support, wherein the individual oligonucleotides are designed to capture RNA or DNA from the section of the tissue sample, optionally wherein at least one of the individual oligonucleotides of each cluster is designed for specific capture of TCR mRNA from the section of the tissue sample;
• (vii) contacting the array with the section of the tissue sample;
• (viii) performing RNase H-dependent PCR upon captured mRNAs of the section of the tissue sample, thereby generating a PCR-amplified DNA population comprising TCR-alpha and TCR-beta sequences;
• and
• (ix) obtaining sequence from the PCR-amplified DNA population and an associated oligonucleotide cluster identification sequence for each DNA sequenced using a sequencing process for TCR sequence-containing PCR-amplified nucleic acids having an average read length on at least one end in excess of 200 nucleotides, thereby obtaining TCR sequences that span TCR transcript variable regions for substantially all TCR sequences obtained and obtaining sequence from the PCR-amplified DNA population of oligonucleotide cluster identification sequences associated with TCR sequences,
• thereby obtaining from the tissue sample spatially-resolvable TCR sequence that spans TCR transcript variable regions;
• A method for obtaining from a tissue sample spatially-resolvable TCR sequence that spans TCR transcript variable regions and macromolecule abundance data comprising:
• (i) obtaining a tissue sample from a subject;
• (ii) preparing a section of the tissue sample and adhering said section to a solid support;
• (iii) forming an array of barcoded oligonucleotide clusters and/or an array of beads attached to barcoded oligonucleotides and contacting the section adhered to the solid support with the array;
• (iv) identifying oligonucleotide cluster and/or bead array identification sequences and associated two-dimensional positions on the array of the barcoded oligonucleotide clusters and/or the array of beads attached to barcoded oligonucleotides;
• and
• (v) obtaining the sequences of a population of macromolecules bound to the array(s) for each macromolecule sequenced, wherein the population of macromolecules comprises TCR RNA sequences, wherein TCR sequences are obtained by a process comprising RNase H-dependent PCR amplification of captured TCR RNA, thereby generating a PCR-amplified cDNA population comprising TCR-alpha and TCR-beta sequences, and obtaining sequence of the PCR-amplified cDNA population and an associated oligonucleotide cluster identification sequence for each cDNA sequenced using a sequencing process for TCR sequence-containing PCR-amplified nucleic acids having an average read length on at least one end in excess of 200 nucleotides, thereby obtaining TCR sequences that span TCR transcript variable regions for substantially all TCR sequences obtained and obtaining sequence from the PCR-amplified cDNA population of oligonucleotide cluster and/or bead array identification sequences associated with TCR sequences, thereby obtaining from the tissue sample spatially-resolvable TCR sequence that spans TCR transcript variable regions and macromolecule abundance data;
• and
• A method for obtaining from a tissue sample spatially-resolvable T cell receptor (TCR) sequence that spans TCR transcript variable regions, the method comprising:
• (i) obtaining a tissue sample from a subject;
• (ii) preparing a section of the tissue sample;
• (iii) providing a solid support;
• (iv) contacting the solid support with a capture material, thereby forming a capture material-coated solid support;
• (v) contacting the capture material-coated solid support with a population of 1-100 μm diameter beads, wherein each bead has at least 1000 attached oligonucleotides and wherein at least one attached oligonucleotide of each bead each comprises:
• (a) a bead identification sequence that is common to all at least 1000 oligonucleotides on each bead and (b) a poly-dT tail of sufficient length to allow for capture of poly-A-tailed RNAs via hybridization, wherein the bead identification sequence that is common to all at least 1000 oligonucleotides on each bead is either a bead identification sequence that is unique to each bead within the population of 1-100 μm diameter beads or is a bead identification sequence that is a member of a population of bead identification sequences that is sufficiently degenerate to the population of 1-100 μm diameter beads that a majority of beads within the population of 1-100 μm diameter beads each possesses a unique bead identification sequence, thereby capturing a subpopulation of the population of 1-100 μm diameter beads upon the solid support;
• (vi) identifying the bead identification sequence and associated two-dimensional position on the solid support of individual beads of the subpopulation of beads attached to the solid support;
• (vii) contacting the subpopulation of 1-100 μm diameter beads captured upon the solid support with of the tissue sample;
• (viii) performing a reverse transcription reaction upon poly-A-tailed RNAs captured by the bead subpopulation, thereby generating a cDNA population;
• (ix) contacting a selection of or all of the cDNA population with biotinylated probes capable of specifically annealing to TCR-alpha or TCR-beta sequences, and enriching for biotinylated probe-TCR complexes, thereby generating a nucleic acid population enriched for TCR-alpha and TCR-beta sequences;
• and
• (x) obtaining sequence from the nucleic acid population enriched for TCR-alpha and TCR-beta sequences using a sequencing process for TCR sequence-containing nucleic acids having an average read length on at least one end in excess of 200 nucleotides, thereby obtaining TCR sequences that span TCR transcript variable regions for substantially all TCR sequences obtained and obtaining sequence from the nucleic acid population enriched for TCR-alpha and TCR-beta sequences of bead identification sequences associated with TCR sequences,
• thereby obtaining from the tissue sample spatially-resolvable T cell receptor (TCR) sequence that spans TCR transcript variable regions.