The disclosure provides for methods, compositions, and kits for multiplex nucleic acid analysis of single cells. The methods, compositions and systems may be used for massively parallel single cell sequencing. The methods, compositions and systems may be used to analyze thousands of cells concurrently. The thousands of cells may comprise a mixed population of cells (e.g., cells of different types or subtypes, different sizes).

Provided are immune cell RNA sequencing methods. In some embodiments, the methods comprise producing a circularized DNA comprising a complementary DNA (cDNA) and a known heterologous sequence, wherein the cDNA is produced from an immune cell RNA. Such methods further comprise performing rolling circle amplification using the circularized DNA as template to produce a concatemer comprising repeating segments comprising the cDNA and the known heterologous sequence. Such methods further comprise sequencing the concatemer or fragments thereof. Also provided are methods comprising producing immune cell RNA sequencing reads using a R2C2 sequencing method, extracting HLA reads from the sequencing reads, and producing allele-specific HLA sequences from the extracted HLA reads. Also provided are computer-readable media, systems, compositions and kits that find use, e.g., in practicing the methods of the present disclosure.

Provided are immune cell RNA sequencing methods. In some embodiments, the methods comprise producing a circularized DNA comprising a complementary DNA (cDNA) and a known heterologous sequence, wherein the cDNA is produced from an immune cell RNA. Such methods further comprise performing rolling circle amplification using the circularized DNA as template to produce a concatemer comprising repeating segments comprising the cDNA and the known heterologous sequence. Such methods further comprise sequencing the concatemer or fragments thereof. Also provided are methods comprising producing immune cell RNA sequencing reads using a R2C2 sequencing method, extracting HLA reads from the sequencing reads, and producing allele-specific HLA sequences from the extracted HLA reads. Also provided are computer-readable media, systems, compositions and kits that find use, e.g., in practicing the methods of the present disclosure.

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

The majority of glioblastomas can be classified into molecular subgroups based on mutations in the TERT promoter (TERTp) and isocitrate dehydrogenase 1 or 2 (IDH). These molecular subgroups utilize distinct genetic mechanisms of telomere maintenance, either TERTp mutation leading to telomerase activation or ATRX-mutation leading to an alternative lengthening of telomeres phenotype (ALT). However, about 20% of glioblastomas lack alterations in TERTp and IDH. These tumors, designated TERTp.sup.WT-IDH.sup.WT glioblastomas, did not have well-established genetic biomarkers or defined mechanisms of telomere maintenance. The genetic landscape of TERTp.sup.WT-IDH.sup.WT glioblastoma includes tumors that have chromosomal rearrangements upstream of TERT. These rearrangements define a novel molecular subgroup of glioblastoma, that is a telomerase-positive subgroup driven by TERT-structural rearrangements (IDH.sup.WT-TERT.sup.SV).

The majority of glioblastomas can be classified into molecular subgroups based on mutations in the TERT promoter (TERTp) and isocitrate dehydrogenase 1 or 2 (IDH). These molecular subgroups utilize distinct genetic mechanisms of telomere maintenance, either TERTp mutation leading to telomerase activation or ATRX-mutation leading to an alternative lengthening of telomeres phenotype (ALT). However, about 20% of glioblastomas lack alterations in TERTp and IDH. These tumors, designated TERTp.sup.WT-IDH.sup.WT glioblastomas, did not have well-established genetic biomarkers or defined mechanisms of telomere maintenance. The genetic landscape of TERTp.sup.WT-IDH.sup.WT glioblastoma includes tumors that have chromosomal rearrangements upstream of TERT. These rearrangements define a novel molecular subgroup of glioblastoma, that is a telomerase-positive subgroup driven by TERT-structural rearrangements (IDH.sup.WT-TERT.sup.SV).

The present disclosure provides methods of determining one or more tissues and/or cell-types contributing to cell-free DNA (“cfDNA”) in a biological sample of a subject. In some embodiments, the present disclosure provides a method of identifying a disease or disorder in a subject as a function of one or more determined more tissues and/or cell-types contributing to cfDNA in a biological sample from the subject.

The present disclosure provides methods of determining one or more tissues and/or cell-types contributing to cell-free DNA (“cfDNA”) in a biological sample of a subject. In some embodiments, the present disclosure provides a method of identifying a disease or disorder in a subject as a function of one or more determined more tissues and/or cell-types contributing to cfDNA in a biological sample from the subject.

A method for visualizing multi-antigen binding capabilities of a set of clonotype groups. Clonotype data that identifies a clonotype group derived from an immune cell sequence dataset (e.g., single cell or spatial dataset) is obtained. A set of interactions for the clonotype group is identified. An interaction in the set of interactions is between a set of cells in the clonotype group and a plurality of antigens in which each cell of the set of cells binds to the plurality of antigens. A binding diagram is generated for the clonotype group based on the set of interactions that has been identified. The binding diagram includes a set of interaction representations that visually represents the set of interactions for the clonotype group. An interaction representation in the set of interaction representations visually relates the plurality of antigens and visually indicates a number of cells in the set of cells that bind to the plurality of antigens.