Methods of identifying and using biomarkers

Abstract

Methods for generating RNA vaccines for subjects suffering from diseases and for generating a database of disease biomarkers are provided. Methods for discovering disease biomarkers and diagnosing diseases are also provided. This invention enables frequent monitoring of subjects and adjustment of treatment. The invention also provides methods for preparing processed nucleic acid samples with a more uniform sequence distribution, which are easier to analyze. These methods can be combined to further improve the ability to generate RNA vaccines and discover disease biomarkers.

Description

Technical Field

[0001] This invention relates to methods for analyzing nucleic acid samples, including methods for identifying and using biomarkers. The invention also relates to methods for generating RNA vaccines, generating databases of disease biomarkers, and methods for the diagnosis, prognosis, and monitoring of diseases. Background Technology

[0002] Disease screening can take many forms, and molecular screening methods have recently gained increasing attention. For example, detecting cancer-specific RNA in a simple blood test has the potential to provide an easily acceptable and relatively non-invasive screening method, as well as prognostic biomarkers (Larson, MH, et al. A comprehensive characterization of the cell-free transcriptome reveals tissue- and subtype-specific biomarkers for cancer detection. 1-11). However, the handling and processing of samples used for RNA sequencing is a challenge in a clinical setting. Furthermore, developing sufficiently sensitive and specific methods has proven to be a challenge.

[0003] RNA expression is characterized by a wide range of expression levels across unique genes. A group of highly expressed genes exists, known as housekeeping genes. These genes do not provide useful information, but they typically account for more than half of the total RNA in any given sample. This problem is even more pronounced in blood, where globin RNA and ribosomal RNA often account for over 95% of the total RNA (Harrington, CA, et al. RNA-Seq of human whole blood: Evaluation of globin RNA depletion on Ribo-Zero library method. Sci. Rep. 10, 1-12 (2020)). Therefore, a major challenge for current detection methods is achieving the breadth of detection across the entire blood transcriptome.

[0004] Another issue is how representative these data are of the actual complexity and biological variation. While several RNA assays exist, until recently these methods only allowed the detection of small fragments of each RNA molecule. This is problematic because each gene can be transcribed into many different RNA isoforms. This is likely due to the multiple combinations of transcription start sites, termination sites, and alternative splicing (Harrow, J. et al. GENCODE: producing a reference annotation for ENCODE. 7, 1-9 (2006)). These alternative transcripts often have different functions and are typically associated with specific cell and tissue types. Therefore, detecting only small fragments of RNA does not tell the whole story. Summary of the Invention

[0005] This invention is based on a method that utilizes the ability to generate full-length sequences from RNA extracted from blood without fragmenting the RNA or cDNA product prior to sequencing. This provides a transcriptome representing any RNA that enters the circulatory system. These include RNA from typical blood cells such as red blood cells, white blood cells, other immune cells, etc. They will also include any other cells not typically found in the circulatory system, such as cancer cells or cells that have somehow detached from the circulatory system. This also includes extracellular RNA that can originate from any cell in the body. By detecting full-length RNA from all these sources, this invention makes it possible to attribute each RNA to the cell / tissue of origin and the state of cell or tissue behavior. Transcription start sites, termination sites, and splicing are characteristic combinations typically used to represent different cell types. This information can also be used for the direct identification of protein isotypes translated from messenger RNA. The ability to detect protein isotypes and identify those associated with diseases allows for the development of treatments, such as RNA vaccines.

[0006] It should be remembered that multiple aspects have been designed for advantageous combination, and all such combinations are considered to be within the scope of the invention. It should also be understood that the options described in relation to one area of ​​improvement will be adapted, with necessary modifications, to other areas; for example, sample type, disease, etc., as the case may be.

[0007] This invention provides a method for analyzing nucleic acid samples, the method comprising:

[0008] (i) Processing nucleic acid samples; and

[0009] (ii) Sequencing the processed nucleic acids.

[0010] Nucleic acid samples can be obtained from any suitable form of biological sample, including any material, biofluid, tissue, or cell obtained from or otherwise derived from the subject. Nucleic acid samples can be obtained from (cancer) cells or genetic material (DNA or RNA) derived from (cancer) cells, including cell-free genetic material (e.g., genetic material present in peripheral blood). Nucleic acid samples can be obtained from biopsy samples, optionally solid and / or liquid biopsy samples. Nucleic acid samples can be obtained from biofluids or fluids or lysates produced from biological materials. Nucleic acid samples can be obtained from blood. Nucleic acid samples can be obtained by extracting RNA from a biological sample obtained from the subject (e.g., blood, optionally whole blood). The RNA can then be used as a template to synthesize cDNA (i.e., via reverse transcription).

[0011] Blood samples are readily and frequently available, allowing for repeated and non-invasive sampling of patients.

[0012] This invention provides a method for analyzing nucleic acid samples, the method comprising:

[0013] (i) Provide an RNA sample extracted from a blood sample obtained from the subject;

[0014] (ii) Processing RNA samples, optionally including using RNA as a template to synthesize cDNA; and

[0015] (iii) Sequencing the processed RNA or cDNA.

[0016] The blood sample can be whole blood. The subject may have cancer.

[0017] Sequencing the processed RNA or cDNA produces sequencing output. The sequencing output provides a transcriptome representing any RNA in the blood. Therefore, sequencing output from the methods defined herein can contain sequences of RNA from blood cells, other cells shed into the bloodstream, and / or cancer cells, as well as extracellular RNA. The sequencing output can be used to attribute each RNA sequence / molecule to the cell or tissue of origin and / or the state of cellular or tissue behavior.

[0018] This invention provides a method for analyzing nucleic acid samples, the method comprising:

[0019] (i) Provide a nucleic acid sample containing RNA molecules, optionally wherein the nucleic acid sample is extracted from a blood sample obtained from the subject;

[0020] (ii) Processing nucleic acid samples containing RNA molecules, optionally including using RNA molecules as templates to synthesize cDNA; and

[0021] (iii) Sequencing the processed RNA or cDNA, wherein the sequencing output is used to identify the cell type and / or tissue type from which the RNA molecule originated.

[0022] This invention provides a method for analyzing nucleic acid samples, the method comprising:

[0023] (i) Provide a nucleic acid sample containing RNA molecules, optionally wherein the nucleic acid sample is extracted from a blood sample obtained from the subject;

[0024] (ii) Processing nucleic acid samples containing RNA molecules, optionally including using RNA molecules as templates to synthesize cDNA; and

[0025] (iii) Sequencing the processed RNA or cDNA, wherein the sequencing output is used to identify the protein isotype encoded by the RNA molecule.

[0026] Sequencing outputs from the methods defined herein can be used to identify transcription start sites, termination sites, and / or splicing junctions. The methods defined herein may include identifying at least one transcription start site, termination site, and / or splicing junction after sequencing of treated RNA or cDNA (i.e., in the sequencing output), optionally identifying transcription start sites, termination sites, and all splicing junctions in one or more (2, 3, 4, 5, 10, 20, 100, or 1000 or more) transcripts. These features typically represent unique combinations used by different cell types, enabling cell type identification. This information can also be used to identify protein isotypes that will be translated from the RNA sequence / molecule / transcript. Therefore, sequencing outputs can be used to identify the presence of RNA transcript sequences. Sequencing outputs can be used to identify one or more protein isotypes. Proteins may have 2, 3, 4, 5, 10, 15, or 20 or more isotypes, and the methods can identify which protein isotype the RNA molecule encodes.

[0027] RNA (transcription) sequences / molecules or groups of RNA sequences may be associated with a disease or condition. A disease- or condition-associated RNA (transcription) sequence / molecule or group of RNA sequences is an RNA (transcription) sequence / molecule or group of RNA sequences associated with a disease or condition. A disease- or condition-associated RNA (transcription) sequence / molecule or group of RNA sequences may (uniquely) exist in subjects suffering from the disease or condition, and optionally not exist in subjects not suffering from the disease or condition. A disease- or condition-associated RNA (transcription) sequence / molecule or group of RNA sequences may (uniquely) not exist in subjects suffering from the disease or condition, and optionally exist in subjects not suffering from the disease or condition. The RNA (transcription) sequence may have a disease- (e.g., cancer)-specific coding sequence. The RNA (transcription) sequence may encode a protein isotype associated with the disease or condition. One or more peptide sequences may be identified in the protein isotype that are present only in cells from subjects suffering from the disease (optionally in cancer cells). One or more peptide sequences that are antigenic peptides may be identified in the protein isotype. These peptide sequences (antigenic peptides) are encoded by segments of the RNA (transcription) sequence.

[0028] This invention provides a method for identifying a set of RNA sequences associated with a disease or condition, the method comprising:

[0029] (a) Provide RNA samples obtained from the subject;

[0030] (b) Processing RNA samples, optionally including using RNA as a template to synthesize cDNA;

[0031] (c) Sequencing of processed RNA or cDNA, wherein the sequencing output is used to identify the presence of RNA transcript sequences associated with a disease or condition; and

[0032] (d) Identify RNA sequences in RNA transcripts.

[0033] RNA samples can be extracted from any material, biological fluid, tissue, or cells obtained from or otherwise derived from the subject. The sample can be a (solid) biopsy sample. The sample can be a liquid biopsy sample. The sample can be obtained from a biological fluid or a fluid or lysate produced from biological material. RNA samples can be extracted from a blood sample, optionally a whole blood sample, obtained from the subject.

[0034] This invention provides a method for identifying a set of RNA sequences associated with a disease or condition, the method comprising:

[0035] (a) Provide an RNA sample extracted from a blood sample obtained from the subject;

[0036] (b) Processing RNA samples, optionally including using RNA as a template to synthesize cDNA;

[0037] (c) Sequencing of processed RNA or cDNA, wherein the sequencing output is used to identify the presence of RNA transcript sequences associated with a disease or condition; and

[0038] (d) Identify RNA sequences in RNA transcripts.

[0039] The subject may have a disease or condition, or the subject may be pregnant and the fetus may have a disease or condition.

[0040] In this method, one or more RNA sequences in the group may encode an antigenic peptide, optionally each RNA sequence in the group encodes an antigenic peptide. Each RNA sequence in the group may (uniquely) be expressed in subjects with a disease or condition, and optionally not expressed in subjects without a disease or condition. "RNA sequence group" means two or more, optionally three, four, five, six, seven, eight, nine, ten, twenty, fifty, or one hundred or more RNA sequences. Each RNA sequence in the group may be greater than 10 bp, 20 bp, 50 bp, 100 bp, 500 bp, or 1000 bp in length, optionally from 10 to 10000 bp, 20 to 1000 bp, or 50 to 500 bp in length. Each RNA sequence in the group may be less than 10 bp, 20 bp, 50 bp, 100 bp, 500 bp, or 1000 bp in length. One or more RNA sequences from the group, optionally 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or 100 or more, may be used to generate a (first) RNA vaccine against a target. One or more RNA sequences from the group, optionally each RNA sequence in the group, may be about 10 to about 1000 nucleotides in length, about 10 to about 100 nucleotides in length, about 15 to about 80 nucleotides in length, or about 18 to about 75 nucleotides in length.

[0041] This invention provides a method for generating an RNA vaccine for a subject suffering from a disease, the method comprising:

[0042] (i) Provide RNA samples obtained from the subject;

[0043] (ii) Processing RNA samples, optionally including using RNA as a template to synthesize cDNA;

[0044] (iii) Sequencing the processed RNA or cDNA, wherein the sequencing output is used to identify the presence of disease-related RNA (transcription) sequences; and

[0045] (iv) Use the identified RNA (transcription) sequence to generate the first RNA vaccine for the target.

[0046] RNA samples can be extracted from any material, biological fluid, tissue, or cells obtained from or otherwise derived from the subject. The sample can be a (solid) biopsy sample. The sample can be a liquid biopsy sample. The sample can be obtained from a biological fluid or a fluid or lysate produced from biological material. RNA samples can be extracted from a blood sample, optionally a whole blood sample, obtained from the subject.

[0047] This invention provides a method for generating an RNA vaccine for a subject suffering from a disease, the method comprising:

[0048] (i) Provide an RNA sample extracted from a blood sample obtained from the subject;

[0049] (ii) Processing RNA samples, optionally including using RNA as a template to synthesize cDNA;

[0050] (iii) Sequencing the processed RNA or cDNA, wherein the sequencing output is used to identify the presence of disease-related RNA (transcription) sequences; and

[0051] (iv) Use the identified RNA (transcription) sequence to generate the first RNA vaccine for the target.

[0052] The use of certain sample types (e.g., blood samples) facilitates continuous monitoring of the subject. Therefore, the method may further include:

[0053] (v) Provide additional RNA samples (extracted from blood samples) obtained from the subject at a later time point;

[0054] (vi) Processing RNA samples, optionally including using RNA as a template to synthesize cDNA;

[0055] (vii) Sequencing of processed RNA or cDNA, wherein the sequencing output is used to identify the presence of disease-related RNA (transcriptional) sequences, and

[0056] (viii) Use the identified RNA (transcription) sequence to generate additional RNA vaccines for the target population.

[0057] The first RNA vaccine and other RNA vaccines can be the same. The first RNA vaccine and other RNA vaccines can also be different; optionally, they differ in the antigenic peptide they encode. Disease-related RNA (transcription) sequences can differ between the RNA sample and the other RNA sample. Disease-related RNA (transcription) sequences can be the same between the RNA sample and the other RNA sample.

[0058] RNA (transcription) sequences can be approximately 10 to 1000 nucleotides in length, approximately 10 to 100 nucleotides in length, approximately 15 to 80 nucleotides in length, or approximately 18 to 75 nucleotides in length.

[0059] RNA transcripts may encode protein isotypes present in diseased subjects. In the method, using an identified RNA transcript sequence to generate an RNA vaccine may include generating an RNA molecule containing at least a portion of the sequence, wherein the RNA molecule contains an open reading frame (ORF) optionally encoding at least one antigenic peptide. The RNA molecule may also contain a 5' UTR, a 3' UTR, a polyA tail, and / or a 5' cap. The 5' cap may have a Cap 0 structure or a Cap 1 structure. The Cap 0 structure may include a methyl-7-guanine nucleotide linked to the 5' position via a 5' triphosphate. The Cap 1 structure may be achieved by methylation of the first nucleotide of the mRNA at the 2'-O position of the ribose. The RNA molecule may contain one or two (optionally uridine-based) RNA strands, optionally having a non-coding sequence optimized for translational performance. Thus, the RNA molecule in the RNA vaccine may contain at least a portion of the wild-type sequence of the RNA transcript, or may contain a modified sequence. For example, the sequence may be adjusted according to its codon usage. Adjustments to codon usage can improve the translation efficiency and half-life of the RNA. In the method, at least 25%, preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or even 100% of the uridine present in the RNA sequence of each RNA molecule in the RNA vaccine may be replaced by pseudouridine or N1-methylpseudouridine or 15-methyluridine or 2-thiouridine.

[0060] RNA vaccines may contain conventional (non-replicating) mRNA, self-amplifying mRNA, and / or trans-amplifying mRNA (taRNA). Self-amplifying mRNA (saRNA) can be based on the addition of a viral replicase gene to enable the mRNA to replicate itself.

[0061] As used herein, the term "RNA vaccine" refers to a vaccine containing RNA molecules as defined herein. However, vaccines may contain other substances and molecules (e.g., pharmaceutical excipients) that are desired or advantageous when administered to an individual. RNA vaccines may contain RNA molecules in a buffer solution. RNA molecules may be formulated in lipid-based carriers, polymers, or peptides, optionally in lipid nanoparticles. RNA molecules may be formulated in lipid complex nanoparticles containing a synthetic cationic lipid (R)-N,N,N-trimethyl-2,3-dioleoyloxy-1-propanediamine chloride (DOTMA) and a phospholipid 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine (DOPE). RNA molecules may be formulated for intravenous delivery. RNA vaccines can be based on uridine mRNA-lipid complex nanoparticles (as described in Luis Rojas et al., Nature 2023; Jun 618(7963): 144-150 doi: 10.1038 / s41586-023-06063-y, which is incorporated herein by reference). RNA vaccines can be delivered by transfecting dendritic cells. RNA vaccines can encode more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 antigenic peptides.

[0062] RNA vaccines can be prepared as described in Sara Sousa Rosa et al. Vaccine. 2021 Apr 15;39(16):2190-2200 doi: 10.1016 / j.vaccine.2021.03.038, which is incorporated herein by reference.

[0063] In the method described, the disease can be cancer or an infectious disease (e.g., COVID-19). The RNA vaccine can be an RNA cancer vaccine. The RNA transcript sequence can have a disease (cancer)-specific coding region. The RNA transcript sequence can encode a protein isotype that is (uniquely) expressed in a subject with cancer. The RNA sequence set in the transcript can encode a neoantigen (cancer-specific peptide sequence). The neoantigen can be expressed on the cell surface via the MHC complex. The neoantigen can be generated by alternative splicing and / or RNA editing and can be predicted from RNA sequencing output (Jiyeon Park and Yeun-Jun Chung, Genomics and Informatics 2019;17(3):e23 DOI: https: / / doi.org / 10.5808 / GI.2019.17.3.e23, which is incorporated herein by reference). Neoantigens can be targets for therapeutic purposes (e.g., immunotherapy), optionally cancer vaccines, adoptive cell therapy, and / or antibody-based therapies (Na Xie et al., Signal Transduction and Targeted Therapy (2023) 8:9 https: / / doi.org / 10.1038 / s41392-022-01270-x, which is incorporated herein by reference). Neoantigens can be targets for one or more targeted therapies. Neoantigens can be targets for one or more antibody-drug conjugates. Neoantigens can be targets for one or more radiopharmaceuticals. Two or more, five or more, ten or more, or fifteen or more neoantigens can be targeted by RNA vaccines. Two or more, five or more, ten or more, or fifteen or more neoantigens can be targeted by one or more of the following: targeted therapy, immunotherapy, adoptive cell therapy, antibody-based therapies, antibody-drug conjugates, and / or radiopharmaceuticals.

[0064] This invention provides an RNA vaccine for treatment, wherein the RNA vaccine is produced using the methods defined herein. Subjects may receive an immune checkpoint inhibitor, optionally atezolizumab, prior to treatment with the RNA vaccine. The RNA vaccine may be administered to the subject 1, 2, 3, 4, 5, 6, 7, 8, or 9 times or more.

[0065] This invention provides a method for detecting biomarkers of disease, comprising:

[0066] (i) Provide RNA samples obtained from subjects suffering from the disease;

[0067] (ii) Processing RNA samples, optionally including using RNA as a template to synthesize cDNA;

[0068] (iii) Sequencing the processed RNA or cDNA; and

[0069] (iv) Use sequencing output to discover disease biomarkers.

[0070] RNA samples can be extracted from blood samples, optionally whole blood samples.

[0071] This invention provides a method for detecting biomarkers of disease, comprising:

[0072] (i) Provide a first RNA sample and a second RNA sample;

[0073] (ii) Processing the first RNA sample and the second RNA sample, optionally including using RNA as a template to synthesize cDNA;

[0074] (iii) Sequencing the processed RNA or cDNA; and

[0075] (iv) Compare the sequencing outputs of the first and second samples to discover disease biomarkers.

[0076] The first RNA sample and the second RNA sample can each be extracted from a blood sample. The first RNA sample can be obtained from a subject with the disease, and the second RNA sample can be obtained from a subject without the disease.

[0077] This invention provides a method for detecting biomarkers of disease, comprising:

[0078] (i) Provide a first RNA sample obtained from a subject with the disease and a second RNA sample obtained from a subject without the disease;

[0079] (ii) Processing the first RNA sample and the second RNA sample, optionally including using RNA as a template to synthesize cDNA;

[0080] (iii) Sequencing the processed RNA or cDNA; and

[0081] (iv) Compare the sequencing outputs of the first and second samples to discover disease biomarkers, wherein the comparison of the sequencing outputs of the first and second samples includes analyzing the sequencing outputs by:

[0082] (i) Identify one or more amino acid sequences corresponding to the RNA or cDNA sequences from the sequencing outputs of the first and second samples;

[0083] (ii) Dividing one or more amino acid sequences into multiple segments of defined length; and

[0084] (iii) Compare multiple segments from the first sample with multiple segments from the second sample to identify segments present in one sample but absent in the other, and / or identify segments whose levels differ between the samples.

[0085] The first and second RNA samples can be extracted from the blood sample, respectively.

[0086] Diseases can be autoimmune diseases. Therefore, methods for identifying biomarkers for autoimmune diseases are also provided, including:

[0087] (i) Provide a first RNA sample extracted from a blood sample obtained from a subject with the disease and a second RNA sample extracted from a blood sample obtained from a subject without the disease;

[0088] (ii) Processing the first RNA sample and the second RNA sample, optionally including using RNA as a template to synthesize cDNA;

[0089] (iii) Sequencing the processed RNA or cDNA; and

[0090] (iv) Compare the sequencing outputs of the first and second samples to discover disease biomarkers.

[0091] The method may include comparing the sequencing outputs of a first sample and a second sample by analyzing the sequencing outputs, optionally by:

[0092] (i) Identify one or more amino acid sequences corresponding to the RNA or cDNA sequences from the sequencing outputs of the first and second samples;

[0093] (ii) Dividing one or more amino acid sequences into multiple segments of defined length; and

[0094] (iii) Compare multiple segments from the first sample with multiple segments from the second sample, optionally to identify segments present in one sample but absent in the other, and / or to identify segments whose levels differ between the samples.

[0095] The method may include identifying a segment as a disease biomarker when it is present in one sample but not in another, and / or identifying a segment as a disease biomarker when the levels of the segment differ between samples.

[0096] The RNA or cDNA sequences from the sequencing outputs of the first and second samples can originate from the same gene / transcription. The segment length can be 1 to 50, 2 to 40, 3 to 35, 4 to 30, 5 to 28, or 6 to 25 amino acids. The segment length can be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids.

[0097] This invention provides a method for discovering disease biomarkers, comprising:

[0098] (i) Provide a first cDNA sample and a second cDNA sample;

[0099] (ii) Standardize the first cDNA sample and the second cDNA sample;

[0100] (iii) Sequencing of standardized first and second cDNA samples; and

[0101] (iv) Compare the sequencing outputs of the first and second cDNA samples to identify disease biomarkers.

[0102] The first RNA / cDNA sample and the second RNA / cDNA sample may come from the same subject. The first RNA / cDNA sample may come from the subject before disease treatment, and the second RNA / cDNA sample may come from the same subject after disease treatment. The first RNA / cDNA sample may come from the subject before disease treatment, and the second RNA / cDNA sample may come from the same subject after disease treatment has begun and / or after disease treatment has been completed.

[0103] The first RNA / cDNA sample and the second RNA / cDNA sample can be from different subjects. In the method, the first RNA / cDNA sample and the second RNA / cDNA sample can be from subjects suffering from the same disease at different grades or stages. The first RNA / cDNA sample and the second RNA / cDNA sample can be from subjects suffering from different diseases (e.g., different types of cancer). The first RNA / cDNA sample can be from a subject suffering from the disease, and the second RNA / cDNA sample can be from a subject not suffering from the disease.

[0104] This invention provides a method for discovering disease biomarkers, comprising:

[0105] (i) Provide a first cDNA sample from a subject with the disease and a second cDNA sample from a subject without the disease;

[0106] (ii) Standardize the first cDNA sample and the second cDNA sample;

[0107] (iii) Sequencing of standardized first and second cDNA samples; and

[0108] (iv) Compare the sequencing outputs of the first and second cDNA samples to identify disease biomarkers.

[0109] Discovering disease biomarkers means identifying new biomarkers (indicators of biological status) for a specific disease, such as discovering previously unknown biomarkers for development into tests for that disease. Disease biomarkers can be used to diagnose, characterize, predict response to treatment, detect trace residual disease, and / or predict disease prognosis. Characterization, in this context, refers to the classification and evaluation of the disease. Prognosis refers to predicting the likely outcome of a subject's disease. Characterization and / or prognosis of a disease may include determining the grade and / or stage of the disease. Characterization of a disease may include determining its subtype. Disease biomarkers can be used to indicate the likelihood that a subject with a specific disease will benefit from a particular treatment.

[0110] In the method, the disease can be cancer. Characterization and / or prognosis of the cancer may include determining the presence or absence of metastasis. Metastasis or metastatic disease is the spread of cancer from one organ or site to another non-adjacent organ or site. The resulting new occurrence of disease is called metastasis. Characterization and / or prognosis of the disease may also include predicting biochemical recurrence and / or determining whether the cancer is invasive and / or determining whether the cancer has spread to lymph nodes. Invasiveness refers to rapidly growing cancer that is more likely to spread, more likely to recur, and / or exhibits resistance to treatment.

[0111] This invention provides a method for monitoring an object, comprising:

[0112] (i) Provide a first RNA sample extracted from a blood sample obtained from the subject at a first time point and a second RNA sample extracted from a blood sample obtained from the subject at a second time point;

[0113] (ii) Processing the first RNA sample and the second RNA sample, optionally including using RNA as a template to synthesize cDNA;

[0114] (iii) Sequencing the processed RNA or cDNA; and

[0115] (iv) Compare the sequencing output of the first and second samples.

[0116] Methods for monitoring objects are also provided, including:

[0117] (i) Provide a first cDNA sample from the subject at a first time point and a second cDNA sample from the subject at a second time point;

[0118] (ii) Standardize the first cDNA sample and the second cDNA sample;

[0119] (iii) Sequencing of standardized first and second cDNA samples; and

[0120] (iv) Compare the sequencing output of the first cDNA sample and the second cDNA sample.

[0121] Monitoring targets may include monitoring the response to disease treatment, such as monitoring treatment success and / or monitoring adverse reactions / complications. The first time point may be before the start of treatment, and the second time point may be during or after treatment. Comparing the sequencing outputs of the first RNA / cDNA sample and the second RNA / cDNA sample can provide an indication of treatment success. For example, the presence or absence of disease biomarkers can indicate treatment success. Comparing the sequencing outputs of the first RNA / cDNA sample and the second RNA / cDNA sample may include comparisons between the two samples and / or comparisons with sequencing outputs from a reference sample.

[0122] In this method, immune system-related transcripts can be detected in the sequencing output. The first time point can be before the start of immunotherapy, and the second time point can be during or after immunotherapy.

[0123] In the method, the disease biomarker can be a cDNA sequence or an RNA sequence. The cDNA sequence will correspond to an RNA sequence. The cDNA / RNA sequence can correspond to a protein or peptide. The method may also include identifying RNA, transcripts, transcriptional models, genes, proteins, and / or peptides corresponding to the cDNA sequence. Therefore, a disease biomarker can be a cDNA molecule (of a specific sequence), a DNA molecule (of a specific sequence), an RNA molecule (of a specific sequence), a transcript, a transcriptional model, a protein, or a peptide.

[0124] The method may include discovering more than one disease biomarker, optionally more than 10, 100, 1000, 10000, 100000, 1 million, or 10 million disease biomarkers. The method may include discovering 1 to 10, 1 to 100, 1 to 1000, 1 to 10000, 1 to 100000, 1 to 1 million, or 1 to 10 million disease biomarkers.

[0125] Two or more disease biomarkers can be aggregated to form a database. This invention provides a method for generating a database of disease biomarkers, the method comprising:

[0126] (i) Provide two or more RNA samples obtained from one or more subjects suffering from the disease;

[0127] (ii) Processing RNA samples, optionally including using RNA as a template to synthesize cDNA;

[0128] (iii) Sequencing of processed RNA or cDNA, wherein the sequencing output is used to identify two or more disease biomarkers; and

[0129] (iv) Compile disease biomarkers to form a database.

[0130] RNA samples can be extracted individually from blood samples, optionally whole blood samples. This invention provides a method for generating a database of disease biomarkers, the method comprising:

[0131] (i) Provide two or more RNA samples extracted from blood samples obtained from one or more subjects suffering from the disease;

[0132] (ii) Processing RNA samples, optionally including using RNA as a template to synthesize cDNA;

[0133] (iii) Sequencing of processed RNA or cDNA, wherein the sequencing output is used to identify two or more disease biomarkers; and

[0134] (iv) Compile disease biomarkers to form a database.

[0135] Each disease biomarker can be an RNA transcript, optionally wherein the RNA transcript encodes a protein isotype. The protein isotype may or may not be present in subjects with the disease.

[0136] This invention provides a method for generating a database of RNA sequences associated with diseases or symptoms, the method comprising:

[0137] (i) Provide two or more RNA samples obtained from one or more subjects;

[0138] (ii) Processing RNA samples, optionally including using RNA as a template to synthesize cDNA;

[0139] (iii) Sequencing of processed RNA or cDNA, wherein the sequencing output is used to identify the presence of two or more RNA transcript sequences associated with a disease or condition;

[0140] (iv) Identify the RNA sequence set in each RNA transcript; and

[0141] (v) Compile RNA sequence sets to form a database.

[0142] RNA samples can be extracted individually from blood samples, optionally whole blood samples. This invention provides a method for generating a database of RNA sequences associated with diseases or conditions, the method comprising:

[0143] (i) Provide two or more RNA samples extracted from blood samples obtained from one or more subjects;

[0144] (ii) Processing RNA samples, optionally including using RNA as a template to synthesize cDNA;

[0145] (iii) Sequencing of processed RNA or cDNA, wherein the sequencing output is used to identify the presence of two or more RNA transcript sequences associated with a disease or condition;

[0146] (iv) Identify the RNA sequence set in each RNA transcript; and

[0147] (v) Compile RNA sequence sets to form a database.

[0148] In the method, one or more RNA sequences in the group may encode an antigenic peptide, optionally each RNA sequence in the group encodes an antigenic peptide.

[0149] Identifying RNA sequences in RNA transcripts may include:

[0150] (i) Identify one or more amino acid sequences corresponding to the RNA transcript;

[0151] (ii) Dividing one or more amino acid sequences into multiple segments of defined length;

[0152] (iii) Compare the segment with the amino acid sequence data to determine whether the segment is present or absent in the amino acid sequence data; and

[0153] (iv) Identify two or more corresponding RNA sequence sets in the segment.

[0154] Prior to the comparison in step (iii), the amino acid sequence data may be divided into multiple segments of defined length. The amino acid sequence data in step (iii) above may include amino acid sequence data obtained by sequencing RNA from a sample from a subject with a disease (e.g., cancer) and determining one or more amino acid sequences corresponding to the RNA sequence. The amino acid sequence data may also include amino acid sequence data obtained by sequencing RNA from a sample from a subject without a disease (e.g., cancer) and determining one or more amino acid sequences corresponding to the RNA sequence. A segment may be identified as a (disease) biomarker based on its presence or absence in the amino acid sequence data; for example, a segment may be identified as a disease biomarker when it is uniquely present in or at a high level in subjects with a specific disease. In step (iv) above, two or more segments can be segments that are present in amino acid sequence data obtained by sequencing RNA from a sample from a subject with a disease (e.g., cancer) and determining one or more amino acid sequences corresponding to the RNA sequence, and not present in amino acid sequence data obtained by sequencing RNA from a sample from a subject without a disease (e.g., cancer) and determining one or more amino acid sequences corresponding to the RNA sequence.

[0155] The length of a segment can be 1 to 50, 2 to 40, 3 to 35, 4 to 30, 5 to 28, or 6 to 25 amino acids. A segment can be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids long. Segments (k-mers) can overlap, for example, such that each amino acid of one or more amino acid sequences is the start of a segment (k-mer) (within the allowed length of one or more amino acid sequences and segment length).

[0156] Sequencing outputs from samples analyzed and / or processed according to the methods defined herein can be compared with databases generated using the methods defined herein to identify the presence of disease biomarkers or RNA sequences associated with diseases or symptoms.

[0157] Databases of disease biomarkers and databases of RNA sequences associated with diseases or symptoms can be used to guide treatment decisions and contribute to the development of new therapies. Disease biomarker and / or RNA sequence sets can be suitable targets for therapeutic agents (e.g., vaccines, RNA therapies, and / or gene editing). The discovery of disease biomarkers specific to cancer cells can be an initial step in identifying cancer-specific antigens targeted for cancer vaccines. Therefore, the method may also include identifying transcripts or proteins / peptides corresponding to disease biomarkers as targets for treatment, optionally cancer vaccine targets. The treatment may be an antibody-drug conjugate and / or a radiopharmaceutical. The method may also include developing a treatment targeting the target, such as a (cancer) vaccine, optionally an RNA vaccine. The method may also include developing an antibody-drug conjugate targeting the target. The method may also include developing a radiopharmaceutical targeting the target.

[0158] This invention provides a method for discovering cancer vaccine targets, comprising:

[0159] (i) Provide a first RNA / cDNA sample from a subject with cancer and a second RNA / cDNA sample from a subject without cancer;

[0160] (ii) Standardize the first RNA / cDNA sample and the second RNA / cDNA sample;

[0161] (iii) Sequencing of standardized first and second RNA / cDNA samples; and

[0162] (iv) Compare the sequencing outputs of the first RNA / cDNA sample and the second RNA / cDNA sample to identify cancer vaccine targets.

[0163] The method may include providing 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40 or more RNA / cDNA samples from different subjects suffering from the disease and / or providing 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40 or more RNA / cDNA samples from different subjects not suffering from the disease. Preferably, the method includes providing 30 or more RNA / cDNA samples from different subjects suffering from the disease (e.g., cancer) and providing 30 or more RNA / cDNA samples from different subjects not suffering from the disease (e.g., not having cancer).

[0164] In the method, the first RNA / cDNA sample and the second RNA / cDNA sample may be obtained from a biological fluid or a fluid or lysate produced from biological material. The first RNA / cDNA sample and the second RNA / cDNA sample may be obtained from blood, optionally whole blood. The first RNA / cDNA sample may be obtained by extracting RNA from a biological sample (e.g., blood) obtained from a subject with a disease, and the second RNA / cDNA sample may be obtained by extracting RNA from a biological sample (e.g., blood) obtained from a subject without the disease. Optionally, cDNA is then synthesized using RNA as a template (i.e., via reverse transcription). Therefore, the method may further include:

[0165] (a) Extraction of RNA from biological fluids (e.g., blood) or fluids or lysates produced from biological materials derived from both diseased and disease-free subjects; and

[0166] (b) Using RNA as a template to synthesize cDNA (i.e., converting RNA into cDNA).

[0167] In this way, a first cDNA sample and a second cDNA sample can be generated.

[0168] A subject with a disease is one that was diseased at the time the RNA / cDNA sample was obtained from the subject. A subject without a disease is one that was not diseased at the time the RNA / cDNA sample was obtained from the subject.

[0169] A person who does not suffer from a disease can be a healthy person.

[0170] Disease biomarkers can be cDNA molecules, RNA molecules, proteins, or peptides (of a specific sequence) that are detectable in samples from subjects with the disease but not in samples from subjects without the disease. Alternatively, disease biomarkers can be cDNA molecules, RNA molecules, proteins, or peptides (of a specific sequence) that are not detectable in samples from subjects with the disease but are detectable in samples from subjects without the disease.

[0171] Cancer vaccine targets can be cDNA molecules (specific sequences), RNA molecules (specific sequences), proteins, or peptides that are detectable in samples from subjects with cancer but not in samples from subjects without cancer.

[0172] Disease biomarkers can be RNA / cDNA sequences present in the first RNA / cDNA sample but not in the second RNA / cDNA sample, or RNA / cDNA sequences present in the second RNA / cDNA sample but not in the first RNA / cDNA sample. Disease biomarkers can also be transcripts that are (uniquely) present in subjects with a specific disease. Furthermore, disease biomarkers can be transcripts that are (uniquely) not present in subjects with a specific disease.

[0173] Cancer vaccine targets can be RNA / cDNA sequences present in a first RNA / cDNA sample but not in a second RNA / cDNA sample. Cancer vaccine targets can also be transcripts uniquely present in individuals with a specific cancer. These transcript / RNA / cDNA sequences may correspond to specific proteins or peptides. At least a portion of the protein or peptide may serve as an antigen included in the cancer vaccine.

[0174] Disease biomarkers can be transcripts that are uniquely present in subjects with a specific disease. Disease biomarkers can also be transcripts that are uniquely not present in subjects with a specific disease. Cancer vaccine targets can be transcripts that are uniquely present in subjects with a specific cancer. The transcript / RNA / cDNA sequence may correspond to a specific protein or peptide. At least a portion of the protein or peptide may serve as an antigen included in a cancer vaccine.

[0175] This invention enables the identification of transcripts present only in subjects with a disease (optionally cancer). Such a transcriptional model can be identified by comparison with subjects without the disease (e.g., benign patients) and, optionally, with public transcriptome annotation databases. Sequencing outputs and / or resulting transcriptome profiles from subjects with a specific disease (e.g., breast cancer) can be compared with sequencing outputs and / or resulting transcriptome profiles from subjects with different diseases (e.g., ovarian cancer and / or colorectal cancer) to determine whether the biomarker is unique for a specific disease (e.g., breast cancer).

[0176] This invention provides a method for diagnosing a disease in an object, comprising:

[0177] (i) Provide RNA samples obtained from the subject (extracted from blood samples);

[0178] (ii) Processing RNA samples, optionally including using RNA as a template to synthesize cDNA; and

[0179] (iii) Sequencing the processed RNA or cDNA, wherein the sequencing output is used to identify whether the subject has a disease.

[0180] The disease can be an autoimmune disease, cancer, diabetes, coronary heart disease, metabolic disease, Alzheimer's disease, dementia, and / or an infectious disease. The disease can be a viral infection, bacterial infection, and / or fungal infection. The disease can be COVID-19. The disease can be identified at a (very) early stage, optionally before the onset of symptoms. The disease can be cancer, and said cancer can be identified at a (very) early stage, optionally before significant tumor growth occurs.

[0181] The disease can be cancer (such as hematologic cancers like leukemia, lymphoma, or multiple myeloma), and diagnosing the disease may include detecting minimal residual disease (MRD). MRD can be defined as cancer cells remaining in the subject during or after treatment.

[0182] The disease can be an autoimmune disease.

[0183] This invention provides a method for diagnosing autoimmune diseases in subjects, comprising:

[0184] (i) Provide RNA samples obtained from the subject;

[0185] (ii) Processing RNA samples, optionally including using RNA as a template to synthesize cDNA; and

[0186] (iii) Sequencing the processed RNA or cDNA, wherein the sequencing output is used to identify whether the subject has a disease.

[0187] RNA samples can be extracted from blood samples, optionally whole blood samples.

[0188] This invention provides a method for diagnosing autoimmune diseases in subjects, comprising:

[0189] (i) Provide an RNA sample extracted from a blood sample obtained from the subject;

[0190] (ii) Processing RNA samples, optionally including using RNA as a template to synthesize cDNA; and

[0191] (iii) Sequencing the processed RNA or cDNA, wherein the sequencing output is used to identify whether the subject has a disease.

[0192] In this article, "autoimmune disease" refers to a disease or condition in which the immune system of an individual produces an immune response against the individual's own tissues. Autoimmune diseases can include arthritis, celiac disease, type 1 diabetes, Graves' disease, inflammatory bowel disease, multiple sclerosis, alopecia areata, Addison's disease, pernicious anemia, psoriasis, systemic lupus erythematosus, myasthenia gravis, Hashimoto's thyroiditis, vitiligo, Sjogren's syndrome, myositis, chronic inflammatory demyelinating polyneuropathy (CIDP), dermatomyositis, Guillain-Barré syndrome, ulcerative colitis, Crohn's disease, and / or vasculitis.

[0193] This invention provides a method for diagnosing a disease in an object, comprising:

[0194] (i) Provide RNA / cDNA samples from the subject;

[0195] (ii) Standardize RNA / cDNA samples; and

[0196] (iii) Sequencing of standardized RNA / cDNA samples, wherein the sequencing output is used to identify whether the subject has a disease.

[0197] The sequencing output can be used to identify whether an individual has a disease by comparing it with a database of biomarkers, optionally generated using the methods defined herein.

[0198] Diagnosis means determining that a subject has a disease during testing.

[0199] The methods defined herein may also include selecting a treatment appropriate to the disease, and optionally treating the disease with the selected treatment. Treatment of the subject may begin early in the course of the disease, optionally before the onset of symptoms or significant tumor growth. Treatment may be a vaccine, antibody-drug conjugate, radiopharmaceutical, immune checkpoint inhibitor, and / or immunotherapy.

[0200] This invention provides a method for characterizing and / or prognosing a disease in an object, comprising:

[0201] (i) Provide RNA / cDNA samples from the subject;

[0202] (ii) Processing (standardizing) RNA / cDNA samples; and

[0203] (iii) Sequencing of processed (standardized) RNA / cDNA samples, wherein the sequencing output is used to provide characterization and / or prognosis of the disease.

[0204] This invention provides a method for selecting a treatment for a disease in a subject, comprising:

[0205] (i) Provide RNA / cDNA samples from the subject;

[0206] (ii) Processing (standardizing) RNA / cDNA samples;

[0207] (iii) Sequencing of processed (standardized) RNA / cDNA samples, wherein the sequencing output is used to provide diagnosis, characterization, and / or prognosis of the disease; and

[0208] (iv) Select a treatment appropriate to the diagnosis, characterization and / or prognosis of the disease.

[0209] This invention provides a method for predicting the response of a subject suffering from a disease to a therapeutic agent, comprising:

[0210] (i) Provide RNA / cDNA samples from the subject;

[0211] (ii) Processing (standardizing) RNA / cDNA samples; and

[0212] (iii) Sequencing of processed (standardized) RNA / cDNA samples, wherein the sequencing output is used to predict the subject’s response to therapeutic agents.

[0213] The therapeutic agent may be an immune checkpoint inhibitor and / or immunotherapy, optionally CAR-T therapy. The therapeutic agent may be an antibody-drug conjugate and / or a radiopharmaceutical. RNA / cDNA samples may contain full-length RNA / cDNA, and / or treated RNA / cDNA samples may contain full-length RNA / cDNA.

[0214] The methods described herein may also include treating the subject. The subject may be treated with vaccines, immune checkpoint inhibitors, immunotherapy, CAR-T therapy, antibody-drug conjugates, and / or radiopharmaceuticals.

[0215] The method may include comparing the sequencing output of a processed (normalized) RNA / cDNA sample with the sequencing output of one or more reference sequences or with the sequencing output of one or more control samples, optionally one or more control samples from one or more subjects with and / or without the disease. Preferably, the method includes comparing the sequencing output of a processed (normalized) RNA / cDNA sample with the sequencing output of one or more control samples from one or more subjects with the disease.

[0216] Sequencing output refers to one or more sequences obtained by sequencing processed (normalized) RNA / cDNA. The sequences can be raw sequences or sequences that have undergone further processing. For example, low-quality reads can be filtered out and / or adaptor sequences can be filtered out and removed. The (processed) sequences can be aligned to a human reference genome (e.g., using Minimap2) to prepare a transcriptome profile. One or more transcriptional models can be identified in the transcriptome profile (sequences aligned to the genome). A transcriptional model represents a specific transcript, i.e., a specific RNA isotype or splice variant produced by a gene. The sequencing output used in the methods defined herein (e.g., compared to discover disease biomarkers or used to identify whether a subject has a disease) can be transcripts, transcriptome profiles, and / or transcriptional models.

[0217] Using sequencing output to identify whether a subject has a disease may include detecting disease biomarkers. Using sequencing output to identify whether a subject has a disease may include detecting more than one disease biomarker, optionally more than 10, 100, 1000, 10000, 100000, 1 million, or 10 million disease biomarkers. Using sequencing output to identify whether a subject has a disease may include detecting 1 to 10, 1 to 100, 1 to 1000, 1 to 10000, 1 to 100000, 1 to 1 million, or 1 to 10 million disease biomarkers. Detecting disease biomarkers may include determining the presence or absence of disease biomarkers. Using sequencing output to identify whether a subject has a disease may include determining the presence or absence of more than one disease biomarker, optionally determining the presence or absence of more than 10, 100, 1000, 10000, 100000, 1 million, or 10 million disease biomarkers. Using sequencing output to identify whether an individual has a disease may include determining the presence or absence of 1 to 10, 1 to 100, 1 to 1000, 1 to 10000, 1 to 100000, 1 to 1 million, or 1 to 10 million disease biomarkers.

[0218] The presence of specific RNA / cDNA sequences in the sequencing output can indicate that a subject has a disease, for example, when a specific transcript (corresponding to a cDNA molecule) is uniquely present in a subject with a specific disease. Similarly, the presence of specific RNA / cDNA sequences in the sequencing output can indicate the characterization and / or prognosis of a disease. The presence of specific RNA / cDNA sequences in the sequencing output can allow for the prediction of a subject's response to a therapeutic agent, for example, when a specific transcript is found to be associated with a subject's response to a specific therapeutic agent.

[0219] The absence of a specific RNA / cDNA sequence in the sequencing output can indicate that a subject has a disease, for example, when a specific transcript (corresponding to a cDNA molecule) is not present in a subject with a specific disease. Similarly, the absence of a specific RNA / cDNA sequence in the sequencing output can indicate the characterization and / or prognosis of a disease. The absence of a specific RNA / cDNA sequence in the sequencing output can allow for the prediction of a subject's response to a therapeutic agent, for example, when a specific transcript is found to be associated with a subject's response to a specific therapeutic agent.

[0220] Sequencing output can be analyzed to identify unique RNA sequences (transcriptions), optionally virtually all unique RNA sequences (transcriptions). Sequencing output can be analyzed to identify unique RNA sequences (transcriptions) as described in Kuo, RI, Cheng, Y., Zhang, R. et al., BMC Genomics 21, 751 (2020) https: / / doi.org / 10.1186 / s12864-020-07123-7 (which is incorporated herein by reference). One or more amino acid sequences that can be translated from the RNA sequence can be identified, optionally identifying three amino acid sequences corresponding to the three longest open reading frames (LOCs) of the RNA sequence (complete translation, first to last codon, no start or stop codon selection). One or more amino acid sequences can be divided into two or more (peptide) segments (k-mers). The length of the (peptide) segments (k-mers) can be 1 to 50, 2 to 40, 3 to 35, 4 to 30, 5 to 28, or 6 to 25 amino acids. The length of a peptide k-mer can be 6 to 25 amino acids, optionally 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. Peptide k-mers can overlap, for example, such that each amino acid of one or more amino acid sequences is the start of a peptide k-mer (within the allowed length of one or more amino acid sequences and the length of the k-mer). Peptide k-mers can be aggregated into a database. Peptide k-mers identified from sequencing output obtained from subjects with a disease (e.g., cancer) can be compared with peptide k-mers identified from sequencing output obtained from subjects without a disease (e.g., cancer). This comparison can be used to identify one or more peptide k-mers associated with a disease (e.g., cancer). This comparison can be used to identify one or more (peptide) segments (k-mers) present in subjects with a disease (e.g., cancer) and / or absent in subjects without a disease (e.g., cancer). Alternatively, this comparison can be used to identify one or more (peptide) segments (k-mers) at elevated levels in subjects with a disease (e.g., cancer) compared to subjects without a disease (e.g., cancer). One or more (peptide) segments (k-mers) identified by the comparison described above can be identified as biomarkers, neoantigen targets, targets for antibody-drug conjugates, targets for neoantigen therapy, targets for vaccines, and / or targets for radiopharmaceuticals.Two or more (peptide) segments (k-mers) identified by the comparison described above can be combined to form a longer amino acid sequence, which can be identified as a biomarker, a neoantigen target, a target for antibody-drug conjugates, a target for neoantigen therapy, a target for vaccines, and / or a target for radiopharmaceuticals. For example, two or more overlapping (peptide) segments (k-mers) identified by the comparison described above can be combined to form a longer amino acid sequence containing both overlapping and non-overlapping amino acids (i.e., the two or more overlapping (peptide) segments (k-mers) are not combined in tandem, but overlapped to regenerate a sequence from which they can be segmented).

[0221] The method may include analyzing sequencing output by:

[0222] (i) Identify one or more amino acid sequences corresponding to the RNA or cDNA sequence from the sequencing output;

[0223] (ii) Dividing the amino acid sequence into multiple segments of defined length; and

[0224] (iii) Compare one or more segments with the amino acid sequence data to determine whether the segment is present or absent in the amino acid sequence data.

[0225] Prior to the comparison in step (iii), the amino acid sequence data may be divided into multiple segments of defined length. The amino acid sequence data in step (iii) above may include amino acid sequence data obtained by sequencing RNA from a sample from a subject with a disease (e.g., cancer) and determining one or more amino acid sequences corresponding to the RNA sequence. The amino acid sequence data may also include amino acid sequence data obtained by sequencing RNA from a sample from a subject without a disease (e.g., cancer) and determining one or more amino acid sequences corresponding to the RNA sequence. A segment may be identified as a (disease) biomarker based on the presence or absence of one or more segments in the amino acid sequence data. For example, a segment may be identified as a disease biomarker when it is uniquely present in or at a high level in the amino acid sequence data of a subject with a specific disease. The presence of a specific segment in the amino acid sequence data may indicate that the subject has a disease, for example, when the specific segment is uniquely present in the amino acid sequence data of a subject with a specific disease. The absence of a specific segment in the amino acid sequence data may indicate that the subject has a disease, for example, when the specific segment is uniquely absent in the amino acid sequence data of a subject with a specific disease. Amino acid sequence data may be obtained from or derived from databases, such as public databases like GTex, TCGA, and / or CPTAC.

[0226] This invention provides a method for analyzing nucleic acid samples, the method comprising:

[0227] (i) Provide RNA samples obtained from the subject (extracted from blood samples);

[0228] (ii) Processing RNA samples, optionally including using RNA as a template to synthesize cDNA;

[0229] (iii) Sequencing the processed RNA or cDNA;

[0230] (iv) Identify one or more amino acid sequences corresponding to the RNA or cDNA sequence from the sequencing output;

[0231] (v) Divide the amino acid sequence into multiple segments of defined length.

[0232] The method may further include comparing one or more segments to amino acid sequence data to determine whether the segment is present or absent in the amino acid sequence data. The amino acid sequence data may comprise sequences from multiple segments of defined lengths. The amino acid sequence data may be obtained from subjects who have or do not have a disease or condition. The amino acid sequence data may be obtained from subjects who have or do not have cancer.

[0233] The subjects may have cancer. The method can be performed using samples from subjects with cancer and samples from subjects without cancer. The method may also include comparing one or more segments obtained by performing the method using samples from subjects with cancer with one or more segments obtained by performing the method using samples from subjects without cancer. Segments present in subjects with cancer but not in subjects without cancer, or present at higher levels in subjects with cancer than in subjects without cancer, may be identified as biomarkers or targets for treatment. Segments may be contained within a longer sequence identified as a biomarker or target for treatment. Treatment may be a vaccine, antibody-drug conjugate, and / or radiopharmaceutical. Two or more segments (k-mers) (identified by comparison as described above) may be combined to form a longer amino acid sequence, which may be identified as a biomarker or target for treatment. Targets for treatment may be neoantigen targets, targets for antibody-drug conjugates, targets for neoantigen therapy, targets for vaccines, and / or targets for radiopharmaceuticals. For example, two or more overlapping segments (k-mers) (identified by comparison as described above) can be combined to form a longer amino acid sequence containing overlapping and non-overlapping amino acids (i.e., the two or more overlapping segments (k-mers) are not combined in series, but overlapped to regenerate a sequence from which they can be segmented).

[0234] The length of a segment can be 1 to 50, 2 to 40, 3 to 35, 4 to 30, 5 to 28, or 6 to 25 amino acids. A segment can be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids long. Segments (k-mers) can overlap, for example, such that each amino acid of one or more amino acid sequences is the start of a segment (k-mer) (within the allowed range of one or more amino acid sequences and segment length). Multiple segments can be 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 20 or more, 50 or more, 100 or more, or 1000 or more segments. Multiple segments can be 2 or more segments.

[0235] RNA samples can be obtained from any suitable form of biological sample, including any material, biological fluid, tissue, or cell obtained from or otherwise derived from the subject. Samples may include cancer cells or genetic material (DNA or RNA) derived from cancer cells, including cell-free genetic material (e.g., present in peripheral blood). Samples may contain biopsy samples (e.g., formalin-fixed paraffin-embedded biopsy samples). Samples may contain fresh / frozen (FF) samples. Samples may contain tumor (cancer) tissue, optionally breast tumor (cancer) tissue. Samples may contain tumor (cancer) cells, optionally breast tumor (cancer) cells. Tissue samples can be obtained using any suitable technique. Some examples include biopsy procedures, optionally fine-needle aspiration biopsies. Body fluid samples may also be used. Suitable sample types include blood (including whole blood, white blood cells, peripheral blood mononuclear cells, erythrocyte sedimentation rate (ESR), plasma, and serum), sputum, tears, mucus, nasal wash, nasal aspirate, breath, urine, semen, saliva, meningeal fluid, amniotic fluid, glandular fluid, lymph, papillary aspirate, bronchial aspirate, synovial fluid, joint aspirate, ascites, cells, cell extracts, and cerebrospinal fluid. This also includes all of the aforementioned experimentally separated fractions. For example, blood samples may be graded into serum or fractions containing specific types of blood cells (e.g., red blood cells or white blood cells (leukocytes)). If desired, a sample may be a combination of samples from an object, such as a combination of tissue and fluid samples. The term "sample" also includes materials containing homogenized solid matter, such as samples from feces, tissue samples, or tissue biopsies. The term "sample" also includes materials derived from tissue cultures or cell cultures, including excised and biopsy samples. Exemplary methods for obtaining samples include, for example, venipuncture and swabs (e.g., buccal swabs). Samples may also be collected by, for example, microdissection (e.g., laser capture microdissection (LCM) or laser microdissection (LMD)), bladder irrigation, smears (e.g., PAP smears), or catheter irrigation. "Samples obtained from or derived from the subject" includes any such sample that has been processed in any suitable manner after being obtained from the subject. The methods of the invention as defined herein may begin with the obtained sample and therefore do not necessarily incorporate the step of obtaining a sample from a patient.

[0236] In the method, the RNA / cDNA sample may be obtained from a tumor (e.g., a solid biopsy) or from a biological fluid or a fluid or lysate produced from biological materials. The RNA / cDNA sample may be obtained from blood. The RNA / cDNA sample can be obtained by extracting RNA from a biological sample (e.g., blood) obtained from the subject. cDNA is then synthesized using the RNA as a template (i.e., via reverse transcription). Therefore, the method may further include:

[0237] (a) Extracting RNA from biological fluids (e.g., blood) derived from the object or from fluids or lysates produced from biological material derived from the object; and / or

[0238] (b) Using RNA as a template to synthesize cDNA (i.e., converting RNA into cDNA).

[0239] In this way, cDNA samples from the subject can be generated.

[0240] The method may also include reporting the outcome of the method to the subject. The outcome may be a diagnosis or prognosis of a disease. The outcome may be a specific grade or stage of a disease (e.g., cancer).

[0241] The term "sequence" can refer to all individual nucleic acid (e.g., cDNA or RNA) molecules that have 100% identical nucleotide sequences. Alternatively, the term "sequence" can refer to all individual nucleic acid (e.g., cDNA or RNA) molecules that have greater than 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% identity with each other. The "% identity" between a query nucleic acid sequence and a target nucleic acid sequence can be calculated using a suitable algorithm (e.g., BLASTN, FASTA, Needleman-Wunsch, Smith-Waterman, LALIGN, or GenePAST / KERR) or software (e.g., DNASTAR Lasergene, GenomeQuest, EMBOSS needle, or EMBOSS infoalign) over the entire length of the query sequence after a pairwise global sequence alignment using a suitable algorithm (e.g., BLASTN, FASTA, Needleman-Wunsch, Smith-Waterman, LALIGN, or GenePAST / KERR). The terms “unique sequence” or “unique cDNA sequence” or “unique RNA sequence” can refer to all individual nucleic acid (e.g., cDNA or RNA, as the case may be) molecules that meet or exceed a threshold percentage of identity (e.g., having 100%, 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% identity with each other). A “unique sequence” or “unique cDNA sequence” or “unique RNA sequence” can differ from other sequences present in a sample (e.g., differing from other sequences by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or 100 nucleotides, or differing from other sequences by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%).

[0242] In the method, the disease may not be an infectious disease. The disease may be cancer. The cancer may be epithelial cancer. The cancer may be breast cancer, ovarian cancer, and / or colorectal cancer. Preferably, the cancer is breast cancer. The first RNA / cDNA sample may be from a subject with breast cancer, and the second RNA / cDNA sample may be from a subject with benign breast disease.

[0243] The method can be used to diagnose more than one disease in a single process, for example by detecting multiple RNA or cDNA molecules (derived from transcripts) that are uniquely present in individuals with a specific disease. The method can also be used to diagnose more than one autoimmune disease.

[0244] This method can be used to diagnose more than one type of cancer. It can also be used to differentiate breast cancer from benign breast conditions.

[0245] In the method described, processing the RNA sample may include standardization (reducing the variability in the levels of different RNA or cDNA sequences in the sample). Therefore, the processed RNA or cDNA sample can be a standardized RNA or cDNA sample.

[0246] RNA sample processing may include homogenizing the sample. Therefore, the relative abundance of all unique RNA or cDNA sequences can be more uniform in the processed RNA or cDNA. For example, the variation in unique sequence levels in a processed RNA or cDNA sample may be less than 50%, less than 40%, less than 30%, less than 20%, or less than 10%.

[0247] In the method, treating the RNA or cDNA sample can reduce the variability of RNA or cDNA levels (e.g., by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%). Treating the RNA or cDNA can achieve a more uniform distribution of cDNA sequences. The abundance difference between the most abundant and least abundant RNA / cDNA in the sample can be reduced (e.g., by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%). In the method, treating the RNA or cDNA sample can reduce the number (copy number) of the most abundant RNA or cDNA molecules (1, 10, 100, 1000, or 10000) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%). The number (copy number) of the most abundant RNA or cDNA molecules in the (first and / or second) RNA or cDNA sample may be reduced by at least 50% in the treated RNA or cDNA. The relative abundance of the least abundant RNA or cDNA molecules (1, 10, 100, 1000, or 10000) may be increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. The number (copy number) of the least abundant RNA or cDNA molecules in the (first and / or second) cDNA sample may be increased by at least 50% in the treated RNA or cDNA.

[0248] Processed RNA or cDNA samples can be more easily analyzed. They can be sequenced more efficiently due to the increased relative representation or level of less abundant sequences.

[0249] Standardizing RNA or cDNA samples results in standardized RNA or cDNA samples. Standardization can include (selectively) increasing the relative abundance of less abundant sequences without targeting a specific sequence based on the nucleotide sequence of that sequence (i.e., identity or homology with known sequences).

[0250] "Standardization" means that the levels of RNA or cDNA sequences in a sample are more uniform. Therefore, a standardized RNA or cDNA sample can be one in which the amount of each unique RNA or cDNA sequence is more uniform than in the same sample before standardization; that is, a standardized RNA or cDNA sample is closer to achieving the same abundance (relative to other unique RNA or cDNA sequences in the standardized RNA or cDNA sample) for each unique RNA or cDNA sequence than the same sample before standardization. To achieve this, the relative presentation or level of less abundant sequences can be increased, and / or the relative presentation or level of more abundant sequences can be decreased. The increase / decrease of less abundant sequences is selective in the sense that if all sequences are increased / decreased to the same extent, the relative abundance will remain unchanged. However, when targeting a specific sequence (e.g., using a predefined probe) without basing it on the nucleotide composition of that sequence (i.e., on their identity or homology with known sequences), the relative presentation or level of less abundant sequences can be increased, and / or the relative presentation or level of more abundant sequences can be decreased. Less abundant sequences can be unique sequences whose quantity is below a threshold; for example, they are present in RNA or cDNA samples before standardization at a quantity lower than the average quantity of unique sequences in the sample. Less abundant sequences can be present in RNA or cDNA samples before standardization at a quantity 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% lower than the average quantity of unique sequences in the sample. More abundant sequences can be unique sequences whose quantity is above a threshold; for example, they are present in RNA or cDNA samples before standardization at a quantity higher than the average quantity of unique sequences in the sample. More abundant sequences can be present in RNA or cDNA samples before standardization at a quantity 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% higher than the average quantity of unique sequences in the sample. Relative abundance refers to the abundance relative to other unique sequences in the sample.

[0251] Standardized RNA or cDNA samples may contain RNA or cDNA sequences with substantially the same level. For example, the level difference of the standardized RNA or cDNA sequences is less than 50%, less than 40%, less than 30%, less than 20%, or less than 10%. Standardized RNA or cDNA may be a standardized RNA or cDNA sample in which at least a portion of the 10, 100, 1000, or 10000 most abundant (unique) sequences in the RNA or cDNA sample has been removed or reduced in copy number (removed or reduced by at least 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%). Standardized RNA or cDNA may be a standardized RNA or cDNA sample in which the level of at least a portion of the 10, 100, 1000, or 10000 least abundant (unique) sequences in the RNA or cDNA sample has been increased in copy number, for example, increased by at least 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. The methods described in this article for standardizing RNA or cDNA samples can be methods for making cDNA samples equal, that is, making the relative abundance of each unique sequence equal.

[0252] In the method, normalizing the (first and / or second) RNA or cDNA samples can increase the amount (copy number) of at least a portion of the low-abundance RNA or cDNA sequences in the (first and / or second) cDNA samples. The low-abundance RNA or cDNA sequences can be 50%, 40%, 30%, 20%, 10%, or 1% of the (unique) sequence with the lowest copy number. Therefore, normalization can include selectively increasing the amount of low-abundance RNA or cDNA in each RNA or cDNA sample.

[0253] In the method described, the standardized RNA or cDNA may be readily analyzable RNA or cDNA. It can be sequenced more efficiently due to the improved relative presentation of less abundant sequences. Standardization of the (first and / or second) RNA or cDNA sample may not include the removal of abundant (more abundant) RNA or cDNA molecules / sequences from the sample (e.g., those corresponding to albumin, IgG, apolipoprotein AI, transferrin, apolipoprotein A-II, α1-protease inhibitors, α1-acid glycoproteins, thyroxine transporters, haptoglobin, and / or hemoglobin-binding proteins) (e.g., using duplex-specific nucleases or sequence-targeting methods). Standardization may not include targeting specific (unique) sequences (e.g., those corresponding to albumin, IgG, apolipoprotein AI, transferrin, apolipoprotein A-II, α1-protease inhibitors, α1-acid glycoproteins, thyroxine transporters, haptoglobin, and / or hemoglobin-binding proteins). Standardization of RNA or cDNA samples can be non-targeted, meaning it can target a specific sequence without involving the nucleotide sequence of that sequence (e.g., it can target a specific sequence without involving the identity or homology of that specific sequence with a known sequence).

[0254] Processing RNA samples can improve the detection of low-abundance RNA or cDNA, optionally including increasing the amount of low-abundance RNA or cDNA in each sample.

[0255] Methods that can be used to process RNA or cDNA samples include exhaustion methods (e.g., CRISPR-based exhaustion methods), methods that include inhibiting reverse transcription of rich RNA sequences (e.g., inhibiting cDNA synthesis using oligonucleotide blockers), and normalization methods (e.g., cDNA normalization).

[0256] Depletion methods involve removing unwanted RNA or cDNA molecules / sequences. These can be contaminating RNA or cDNA molecules / sequences (e.g., bacterial transcripts, optionally bacterial ribosomal RNA) or abundant (more abundant) RNA or cDNA molecules / sequences from the sample (e.g., those corresponding to ribosomes, mitochondria, globins, and housekeeping genes, optionally corresponding to albumin, IgG, apolipoprotein AI, transferrin, apolipoprotein A-II, α1-protease inhibitors, α1-acid glycoproteins, thyroxine transporters, haptoglobins, and / or hemoglobin-binding proteins). CRISPR-Cas9 can be used to degrade the abundant sequences. Optionally, a CRISPR-Cas9 complex is formed together with a designed guide RNA library, and this complex is mixed with the cDNA sample. After the unwanted abundant sequences are cleaved, they cannot be used as substrates for PCR amplification and subsequent sequencing. Exemplary products include CRISPclean from Jumpcode Genomics with rRNA depletion. TM Stranded Total RNAPrep.

[0257] Methods including inhibiting reverse transcription can use high-affinity RNA-binding oligonucleotides to block reverse transcription and / or PCR amplification of specific RNA transcripts (see, for example, Everaert C et al., Biological Procedures Online 25, article number: 7 (2023) https: / / doi.org / 10.1186 / s12575-023-00193-3, which is incorporated herein by reference). LNA-modified oligonucleotides complementary to undesired RNA can be engineered to block reverse transcription and / or PCR amplification when they bind downstream of the priming site.

[0258] Complementary DNA (cDNA) normalization (Alex S. Shcheglov, Pavel A. Zhulidov, Ekaterina A. Bogdanova, DAS Normalization of cDNA Libraries, Nucleic Acids Hybrid. Chapter 5, (2014)) addresses the problem of reduced sampling efficiency for target genes due to high abundance of housekeeping genes. Since RNA sequencing typically relies on the conversion of RNA to double-stranded cDNA, cDNA normalization leverages the biochemical properties of cDNA to produce a uniform distribution of unique genes and isotypes within the cDNA library. Theoretically, maximum non-targeted sampling efficiency is achieved if all unique RNA sequences are represented with the same relative abundance. Therefore, the goal of normalization is to redistribute the cDNA library (sample) to meet this standard as closely as possible.

[0259] Complementary DNA (cDNA) normalization can be performed on full-length cDNA. CDNA normalization can be achieved using either a duplex-specific nuclease (DSN) method (see, for example, Zhulidov, PA et al. Simple cDNA normalization using kamchatka crab duplex-specific nuclease. Nucleic Acids Res. 32, e37 (2004), which is incorporated herein by reference) or a hydroxyapatite column method (see, for example, Andrews-Pfannkoch, C., Fadrosh, DW, Thorpe, J. & Williamson, SJ Hydroxyapatite-mediated separation of double-stranded DNA, single-stranded DNA, and RNA genomes from natural viral assemblages. Appl. Environ. Microbiol. 76, 5039-5045 (2010), which is incorporated herein by reference). Both methods rely on denaturation and rehybridization of the cDNA strand. As single-stranded cDNA moves throughout the solution, sequences with higher abundance are more likely to find complementary sequences to rehybridize with. Therefore, when rehybridization reaches its limit, the remaining single-stranded cDNA represents a standardized sequence library.

[0260] Therefore, processing RNA samples may include using RNA as a template to synthesize double-stranded cDNA, and then denaturing and rehybridizing the cDNA strands.

[0261] The difference between the DSN method and the hydroxyapatite column method lies in their methods of isolating single-stranded cDNA libraries from rehybridized double-stranded cDNA molecules.

[0262] In the DSN method, an enzyme that specifically cleaves double-stranded DNA is used to break down all double-stranded cDNA in solution. The solution is then purified, and size selection is performed on cDNA sequences exceeding a certain length. These sequences are then amplified using polymerase chain reaction (PCR).

[0263] In the column method, a denatured and rehybridized cDNA library is passed through a heated column packed with hydroxyapatite particles. Hydroxyapatite preferentially binds to larger DNA molecules. The size of the bound DNA is controlled by the concentration of the phosphate buffer used to dissolve the cDNA library. Therefore, the concentration of the phosphate buffer must be specifically adjusted for cDNA molecules within a certain sequence length range. The cDNA is eluted through the column using a higher concentration of phosphate buffer to extract the larger-sized DNA molecules. Since single-stranded cDNA is approximately half the size of the rehybridized cDNA, the elution of single-stranded portions can be controlled if the average cDNA sequence length is known. The resulting elution aims to enrich single-stranded cDNA, which is then amplified using PCR.

[0264] Complementary DNA (cDNA) normalization can be performed using selective amplification methods. Complementary DNA (cDNA) normalization can be performed via "Level-Up" (as described in WO2022 / 229128A1, which is incorporated herein by reference). Level-Up normalization makes it possible to obtain a cDNA library and equalize the relative abundance of each unique transcribed sequence. This is done without exhaustion or targeting. Essentially, this allows for the creation of an optimal cDNA library for detecting all RNA present in a sample.

[0265] Therefore, processing RNA samples may include using RNA as a template to synthesize cDNA and standardizing the cDNA sample using a selective amplification method of single-stranded cDNA, the method comprising:

[0266] (i) Provide a cDNA sample containing a double-stranded cDNA template, each template having a known 5' pre-attached adapter and a known 3' pre-attached adapter;

[0267] (ii) Denature the cDNA sample to generate a single-stranded cDNA template;

[0268] (iii) Reassociate the cDNA sample to produce a mixture of associated single-stranded cDNA template and associated double-stranded cDNA template;

[0269] (iv) Anneal the 5' adaptor complex with at least one 5' pre-attached adaptor of an associated single-stranded cDNA template and anneal the 3' adaptor complex with the 3' pre-attached adaptor of the same associated single-stranded cDNA template, wherein each adaptor complex contains at least one oligonucleotide.

[0270] (v) Ligating oligonucleotides from the 5' adaptor complex to the 5' pre-attached adaptor of the associated single-stranded cDNA template, and ligating oligonucleotides from the 3' adaptor complex to the 3' pre-attached adaptor of the same associated single-stranded cDNA template; and

[0271] (vi) Use primers that are specific to the oligonucleotides to selectively amplify cDNA samples.

[0272] In the method, processing the RNA sample may include using RNA as a template to synthesize cDNA to generate double-stranded cDNA templates, each cDNA template having a known 5' pre-attachment adaptor and a known 3' pre-attachment adaptor, and:

[0273] (i) Denature the cDNA to generate a single-stranded cDNA template;

[0274] (ii) Reassociate the cDNA to produce a mixture of associated single-stranded cDNA template and associated double-stranded cDNA template;

[0275] (iii) Annealing the 5' adaptor complex with at least one 5' pre-attached adaptor of an associated single-stranded cDNA template and annealing the 3' adaptor complex with the 3' pre-attached adaptor of the same associated single-stranded cDNA template, wherein each adaptor complex contains at least one oligonucleotide;

[0276] (iv) Ligating oligonucleotides from the 5' adaptor complex to the 5' pre-attached adaptor of the associated single-stranded cDNA template, and ligating oligonucleotides from the 3' adaptor complex to the 3' pre-attached adaptor of the same associated single-stranded cDNA template; and

[0277] (v) Use primers that are specific to the oligonucleotides to selectively amplify cDNA.

[0278] In the method:

[0279] (A) The 5' adaptor complex may be a pre-oligonucleotide dimer comprising:

[0280] (i) prelig-oligonucleotides for ligation to the 5' pre-attachment adaptor of the (post-association) single-stranded cDNA template; and

[0281] (ii) a frontlink-oligonucleotide for annealing with a 5' pre-attached adaptor and a front-lig-oligonucleotide, the frontlink-oligonucleotide comprising a region complementary to the 5' pre-attached adaptor and a region complementary to the front-lig-oligonucleotide.

[0282] Therefore, during annealing, the end of the pre-lig-oligonucleotide is adjacent to the end of the 5' pre-attached adapter, enabling the pre-lig-oligonucleotide to be attached to the 5' pre-attached adapter at the attachment site; and

[0283] (B) The 3' adaptor complex can be a post-oligonucleotide dimer, comprising:

[0284] (i) Post-lig-oligonucleotides for ligation to the 3' pre-attached adaptor of a (post-association) single-stranded cDNA template; and

[0285] (ii) a backlink-oligonucleotide for annealing with a 3' pre-attached adaptor and a post-lig-oligonucleotide, the backlink-oligonucleotide comprising a region complementary to the 3' pre-attached adaptor and a region complementary to the post-lig-oligonucleotide.

[0286] Therefore, during annealing, the end of the post-lig-oligonucleotide is adjacent to the end of the 3' pre-attached adapter, so that the post-lig-oligonucleotide can be attached to the 3' pre-attached adapter at the attachment site.

[0287] In the method:

[0288] (A) Pre-linking oligonucleotides may include:

[0289] (i) a template overhang region at the pre-connector oligonucleotide terminus near a region complementary to the 5' pre-attached adaptor, wherein the template overhang region is non-complementary to the corresponding region of the (post-association) single-stranded cDNA template; and / or

[0290] (ii) A lig-oligonucleotide overhang region at the proximal end of a pre-lig-oligonucleotide terminus that is complementary to the pre-lig-oligonucleotide region, wherein the lig-oligonucleotide overhang region is non-complementary to the corresponding region of the pre-lig-oligonucleotide; and / or

[0291] (B) Post-linked oligonucleotides may include:

[0292] (i) a template overhang region at the post-joint oligonucleotide terminus near a region complementary to the 3' pre-attached adaptor, wherein the template overhang region is non-complementary to the corresponding region of the (post-association) single-stranded cDNA template; and / or

[0293] (ii) A lig-oligonucleotide overhang region at the end of a post-lig-oligonucleotide near a region complementary to the post-lig-oligonucleotide, wherein the lig-oligonucleotide overhang region is non-complementary to the corresponding region of the post-lig-oligonucleotide.

[0294] In the method, the first cDNA sample and the second cDNA sample may contain a double-stranded cDNA template, each template having a known 5' pre-attached adapter and a known 3' pre-attached adapter;

[0295] Standardization of the first and second cDNA samples may include:

[0296] (i) Denature the cDNA sample to generate a single-stranded cDNA template;

[0297] (ii) Reassociate the cDNA sample to produce a mixture of associated single-stranded cDNA template and associated double-stranded cDNA template;

[0298] (iii) Annealing the 5' adaptor complex with at least one 5' pre-attached adaptor of an associated single-stranded cDNA template and annealing the 3' adaptor complex with the 3' pre-attached adaptor of the same associated single-stranded cDNA template, wherein each adaptor complex contains at least one oligonucleotide;

[0299] (iv) Ligating oligonucleotides from the 5' adaptor complex to the 5' pre-attached adaptor of the associated single-stranded cDNA template, and ligating oligonucleotides from the 3' adaptor complex to the 3' pre-attached adaptor of the same associated single-stranded cDNA template; and

[0300] (v) Use primers that are specific to the oligonucleotides to selectively amplify cDNA samples.

[0301] cDNA (sample) may contain a double-stranded cDNA template, each template having a known 5' pre-attached adapter and a known 3' pre-attached adapter;

[0302] Furthermore, the processing (standardization) of cDNA (samples) may include:

[0303] (i) Denature the cDNA (sample) to generate a single-stranded cDNA template;

[0304] (ii) Reassociate the cDNA (sample) to produce a mixture of associated single-stranded cDNA template and associated double-stranded cDNA template;

[0305] (iii) Annealing the 5' adaptor complex with at least one 5' pre-attached adaptor of an associated single-stranded cDNA template and annealing the 3' adaptor complex with the 3' pre-attached adaptor of the same associated single-stranded cDNA template, wherein each adaptor complex contains at least one oligonucleotide;

[0306] (iv) Ligating oligonucleotides from the 5' adaptor complex to the 5' pre-attached adaptor of the associated single-stranded cDNA template, and ligating oligonucleotides from the 3' adaptor complex to the 3' pre-attached adaptor of the same associated single-stranded cDNA template; and

[0307] (v) Use primers that are specific to the oligonucleotides to selectively amplify cDNA (sample).

[0308] The present invention also provides a method for discovering disease biomarkers (of autoimmune diseases), comprising:

[0309] (i) Provide a first RNA sample extracted from a blood sample from a subject with the disease and a second RNA sample extracted from a blood sample from a subject without the disease.

[0310] (ii) Using RNA as a template to synthesize cDNA to produce a first cDNA sample and a second cDNA sample containing a double-stranded cDNA template, each template having a known 5' pre-attached adapter and a known 3' pre-attached adapter;

[0311] (iii) Denature the first and second cDNA samples to generate a single-stranded cDNA template;

[0312] (iv) Reassociate the first cDNA sample and the second cDNA sample to produce a mixture of associated single-stranded cDNA template and associated double-stranded cDNA template;

[0313] (v) In each of the first cDNA sample and the second cDNA sample, the 5' adaptor complex is annealed with the 5' pre-attached adaptor of at least one associated single-stranded cDNA template, and the 3' adaptor complex is annealed with the 3' pre-attached adaptor of the same associated single-stranded cDNA template, wherein each adaptor complex contains at least one oligonucleotide.

[0314] (vi) The oligonucleotide from the 5' adaptor complex is ligated to the 5' pre-attached adaptor of the associated single-stranded cDNA template, and the oligonucleotide from the 3' adaptor complex is ligated to the 3' pre-attached adaptor of the same associated single-stranded cDNA template.

[0315] (vii) Use primers that are specific to the oligonucleotides being linked to selectively amplify the first cDNA sample and the second cDNA sample.

[0316] (viii) Sequencing of the selectively amplified first and second cDNA samples; and

[0317] (ix) Compare the sequencing outputs of the first and second cDNA samples to discover disease biomarkers (autoimmune diseases).

[0318] The first RNA / cDNA sample and the second RNA / cDNA sample can be kept separate and / or identified individually.

[0319] The present invention also provides a method for diagnosing (autoimmune) diseases in subjects, the method comprising:

[0320] (i) Provide an RNA sample extracted from a blood sample obtained from the subject;

[0321] (ii) Use RNA as a template to synthesize cDNA to produce cDNA samples containing double-stranded cDNA templates, each template having a known 5' pre-attached adapter and a known 3' pre-attached adapter;

[0322] (iii) Denature the cDNA sample to generate a single-stranded cDNA template;

[0323] (iv) Reassociate the cDNA sample to produce a mixture of associated single-stranded cDNA template and associated double-stranded cDNA template;

[0324] (v) Annealing a 5' adaptor complex with a 5' pre-attached adaptor of at least one associated single-stranded cDNA template and annealing a 3' adaptor complex with a 3' pre-attached adaptor of the same associated single-stranded cDNA template, wherein each adaptor complex contains at least one oligonucleotide;

[0325] (vi) The oligonucleotide from the 5' adaptor complex is ligated to the 5' pre-attached adaptor of the associated single-stranded cDNA template, and the oligonucleotide from the 3' adaptor complex is ligated to the 3' pre-attached adaptor of the same associated single-stranded cDNA template.

[0326] (vii) Using primers specific to the oligonucleotides being linked to selectively amplify cDNA samples; and

[0327] (viii) Sequencing of selectively amplified cDNA samples, wherein the sequencing output is used to identify whether the subject has an (autoimmune) disease.

[0328] This invention provides a method for generating a database of disease biomarkers, the method comprising:

[0329] (i) Provide two or more RNA samples extracted from blood samples obtained from one or more subjects suffering from the disease;

[0330] (ii) Processing RNA samples, optionally including using RNA as a template to synthesize cDNA;

[0331] (iii) Sequencing of processed RNA or cDNA, wherein the sequencing output is used to identify two or more disease biomarkers; and

[0332] (iv) Compile disease biomarkers to form a database;

[0333] The RNA sample processing involves using RNA as a template to synthesize cDNA to generate double-stranded cDNA templates, each cDNA template having a known 5' pre-attachment adaptor and a known 3' pre-attachment adaptor, and:

[0334] (i) Denature the cDNA to generate a single-stranded cDNA template;

[0335] (ii) Reassociate the cDNA to produce a mixture of associated single-stranded cDNA template and associated double-stranded cDNA template;

[0336] (iii) Annealing the 5' adaptor complex with at least one 5' pre-attached adaptor of an associated single-stranded cDNA template and annealing the 3' adaptor complex with the 3' pre-attached adaptor of the same associated single-stranded cDNA template, wherein each adaptor complex contains at least one oligonucleotide;

[0337] (iv) Ligating oligonucleotides from the 5' adaptor complex to the 5' pre-attached adaptor of the associated single-stranded cDNA template, and ligating oligonucleotides from the 3' adaptor complex to the 3' pre-attached adaptor of the same associated single-stranded cDNA template; and

[0338] (v) Use primers that are specific to the oligonucleotides to selectively amplify cDNA.

[0339] Two or more RNA samples can be kept separate and / or identified individually.

[0340] This invention provides a method for generating an RNA vaccine for a subject suffering from a disease, the method comprising:

[0341] (i) Provide an RNA sample extracted from a blood sample obtained from the subject;

[0342] (ii) Processing RNA samples, optionally including using RNA as a template to synthesize cDNA;

[0343] (iii) Sequencing the processed RNA or cDNA, wherein the sequencing output is used to identify the presence of disease-related RNA transcript sequences;

[0344] (iv) Use the identified RNA transcript sequences to generate the first RNA vaccine for the target population;

[0345] (v) Provide additional RNA samples extracted from blood samples obtained from the subject at a later time point;

[0346] (vi) Processing additional RNA samples, optionally including using RNA as a template to synthesize cDNA;

[0347] (vii) Sequencing of processed RNA or cDNA, wherein the sequencing output is used to identify the presence of disease-related RNA transcript sequences, and

[0348] (viii) Use the identified RNA transcript sequences to generate additional RNA vaccines for the target population;

[0349] The processing of RNA samples and other RNA samples includes using RNA as a template to synthesize cDNA to produce double-stranded cDNA templates, each cDNA template having a known 5' pre-attachment adaptor and a known 3' pre-attachment adaptor, and:

[0350] (i) Denature the cDNA to generate a single-stranded cDNA template;

[0351] (ii) Reassociate the cDNA to produce a mixture of associated single-stranded cDNA template and associated double-stranded cDNA template;

[0352] (iii) Annealing the 5' adaptor complex with at least one 5' pre-attached adaptor of an associated single-stranded cDNA template and annealing the 3' adaptor complex with the 3' pre-attached adaptor of the same associated single-stranded cDNA template, wherein each adaptor complex contains at least one oligonucleotide;

[0353] (iv) Ligating oligonucleotides from the 5' adaptor complex to the 5' pre-attached adaptor of the associated single-stranded cDNA template, and ligating oligonucleotides from the 3' adaptor complex to the 3' pre-attached adaptor of the same associated single-stranded cDNA template; and

[0354] (v) Use primers that are specific to the oligonucleotides to selectively amplify cDNA.

[0355] RNA samples and other RNA samples can be kept separate and / or identified individually.

[0356] In the method:

[0357] (A) The 5' adaptor complex may be a pre-oligonucleotide dimer comprising:

[0358] (i) prelig-oligonucleotides for ligation to the 5' pre-attachment adaptor of the (post-association) single-stranded cDNA template; and

[0359] (ii) A pre-linker oligonucleotide for annealing with a 5' pre-attached adaptor and a pre-lig-oligonucleotide, the pre-linker oligonucleotide comprising a region complementary to the 5' pre-attached adaptor and a region complementary to the pre-lig-oligonucleotide.

[0360] Therefore, during annealing, the end of the pre-lig-oligonucleotide is adjacent to the end of the 5' pre-attached adapter, enabling the pre-lig-oligonucleotide to be attached to the 5' pre-attached adapter at the attachment site; and

[0361] (B) The 3' adaptor complex can be a post-oligonucleotide dimer, comprising:

[0362] (i) Post-lig-oligonucleotides for ligation to the 3' pre-attached adaptor of a (post-association) single-stranded cDNA template; and

[0363] (ii) A post-linking oligonucleotide for annealing with a 3' pre-attached adaptor and a post-lig-oligonucleotide, the post-linking oligonucleotide comprising a region complementary to the 3' pre-attached adaptor and a region complementary to the post-lig-oligonucleotide.

[0364] Therefore, during annealing, the end of the post-lig-oligonucleotide is adjacent to the end of the 3' pre-attached adapter, so that the post-lig-oligonucleotide can be attached to the 3' pre-attached adapter at the attachment site.

[0365] In the method:

[0366] (A) Pre-linking oligonucleotides may include:

[0367] (i) a template overhang region at the pre-connector oligonucleotide terminus near a region complementary to the 5' pre-attached adaptor, wherein the template overhang region is non-complementary to the corresponding region of the (post-association) single-stranded cDNA template; and / or

[0368] (ii) A lig-oligonucleotide overhang region at the proximal end of a pre-lig-oligonucleotide terminus that is complementary to the pre-lig-oligonucleotide region, wherein the lig-oligonucleotide overhang region is non-complementary to the corresponding region of the pre-lig-oligonucleotide; and / or

[0369] (B) Post-linked oligonucleotides may include:

[0370] (i) a template overhang region at the post-joint oligonucleotide terminus near a region complementary to the 3' pre-attached adaptor, wherein the template overhang region is non-complementary to the corresponding region of the (post-association) single-stranded cDNA template; and / or

[0371] (ii) A lig-oligonucleotide overhang region at the end of a post-lig-oligonucleotide near a region complementary to the post-lig-oligonucleotide, wherein the lig-oligonucleotide overhang region is non-complementary to the corresponding region of the post-lig-oligonucleotide.

[0372] The template overhang and / or lig-oligonucleotide overhang can be from about 1 bp to about 20 bp in length. The template overhang and / or lig-oligonucleotide overhang can be 2 bp to 19 bp, 3 bp to 18 bp, 2 bp to 17 bp, 3 bp to 16 bp, 2 bp to 15 bp, 3 bp to 14 bp, 2 bp to 13 bp, 3 bp to 12 bp, 2 bp to 11 bp, 3 bp to 10 bp, 2 bp to 9 bp, 3 bp to 8 bp, 2 bp to 7 bp, 3 bp to 6 bp, 2 bp to 5 bp, 3 bp to 5 bp, or 2 bp to 4 bp. Preferably, the template overhang and / or lig-oligonucleotide overhang is 3 bp.

[0373] The template overhang and / or lig-oligonucleotide overhang may be at least 2 bp or at least 3 bp. Preferably, the template overhang and / or lig-oligonucleotide overhang is at least 3 bp.

[0374] The combined length of the pre-linker oligonucleotide and the pre-lig-oligonucleotide may be less than about 300 bp, and / or the combined length of the post-linker oligonucleotide and the post-lig-oligonucleotide may be less than about 300 bp. The combined length of the pre-linker oligonucleotide and the pre-lig-oligonucleotide may be at least about 200 bp, and / or the combined length of the post-linker oligonucleotide and the post-lig-oligonucleotide may be at least about 100 bp.

[0375] The length of the pre-linking oligonucleotide and / or post-linking oligonucleotide can be less than 200 bp. The length of the pre-linking oligonucleotide and / or post-linking oligonucleotide can be at least 50 bp.

[0376] The pre-oligonucleotide dimer and / or post-oligonucleotide dimer may have at least one non-flat end.

[0377] The pre-linking oligonucleotide and / or post-linking oligonucleotide can provide at least 5 bp of complementary binding on either side of the linker site.

[0378] The nucleotide sequence of the pre-oligonucleotide dimer may be different from and non-complementary to the nucleotide sequence of the post-oligonucleotide dimer.

[0379] At least one of the pre-oligonucleotide dimer and the post-oligonucleotide dimer can be annealed with the (associated) single-stranded cDNA template at a temperature exceeding 30°C.

[0380] The concentration of pre-oligonucleotide dimers and / or post-oligonucleotide dimers may exceed the predicted total single-stranded cDNA concentration or total cDNA concentration in the cDNA sample.

[0381] The duration of the reassociation of cDNA samples can be 0 to 24 hours, optionally 0 to 8 hours, 1 to 7 hours, 1 to 24 hours or 7 to 24 hours.

[0382] Sequencing can include the use of long-read sequencing. By using long-read sequencing, full-length RNA / cDNA can be detected, which provides better information for identifying the tissue origin and specific function of each RNA. Although long-read RNA sequencing is more expensive than other assays, Level-Up technology allows for a reduction in the amount of sequencing required, thereby reducing the overall cost.

[0383] In the method, sequencing may include long-read sequencing. Long-read sequencing may be single-molecule long-read sequencing (e.g., PacBio). ® HiFi or Oxford Nanopore Technologies nanopore sequencing. Long-read sequencing can be single-molecule nanopore sequencing. Long-read sequencing may include tagging (e.g., Illumina Complete Long Read sequencing technology). Long-read sequencing can produce reads greater than 1 kb, greater than 5 kb, greater than 10 kb, or greater than 20 kb.

[0384] RNA can be full-length RNA. Therefore, the processed RNA or cDNA sequenced can be full-length. "Full-length" means that the entire length of the RNA / cDNA molecule (or at least 99%, 98%, 95%, 90%, or 80% of its length) can be obtained, i.e., the RNA or cDNA molecule is not fragmented prior to sequencing. The entire splicing isotype can be directly observed. The method may not include the step of (actively) fragmenting RNA and / or cDNA prior to sequencing. RNA samples may contain full-length RNA.

[0385] Sequencing can include sequencing long reads and full-length RNA. This allows for direct observation of the entire splice isotype.

[0386] Level-up processing allows for the analysis of samples with RNA degradation by increasing the presence of low-abundance transcripts. However, it is preferable to minimize RNA degradation during the processing of biological samples. Blood samples (optionally whole blood samples) can be placed in blood tubes designed to maintain RNA integrity. The inventors have developed methods for processing blood samples on the day of blood collection when RNA extraction is not performed. Specific steps for freezing, storing, and thawing blood samples improve the condition of the samples, especially if they are to be sequenced for long reads.

[0387] This invention provides a method for processing blood samples, comprising:

[0388] (i) Store blood samples at or below -15°C;

[0389] (ii) Thaw the blood sample at 5°C to 30°C for at least 1 hour; and

[0390] (iii) Extract RNA from thawed blood samples.

[0391] Blood samples can be liquid (i.e., not dried) blood samples. Blood samples can be whole blood. Blood samples can be in sample tubes. Blood samples do not need to be absorbed into materials (such as sponges).

[0392] Blood samples can be stored at -15°C to -80°C, -15°C to -70°C, -15°C to -60°C, -15°C to -50°C, -15°C to -40°C, -15°C to -30°C, or -15°C to -20°C.

[0393] Blood samples can be stored at -15°C or below for 12 hours, 8 hours, 5 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, 5 minutes, or 1 minute after collection. Preferably, blood samples are stored at -15°C or below for 12 hours after collection (i.e., obtaining blood samples from the subject).

[0394] Blood samples can be stored at -20°C or below. Blood samples can be stored at -20°C or below for 12 hours, 8 hours, 5 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, 5 minutes, or 1 minute after collection. Preferably, blood samples are stored at -20°C or below for 12 hours after collection (i.e., obtaining blood samples from the subject).

[0395] Blood samples may be stored at -15°C or below (optionally -20°C or below) for at least 24 hours (and optionally no more than 72 hours, 1 week, 2 weeks, 4 weeks, 1 month, or 2 months), and then at -70°C or below (optionally -80°C or below) for no more than 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or 2, 3, 4, or 5 years. Preferably, they may be stored at -70°C or below (optionally -80°C or below) for no more than 5 years.

[0396] Preferably, the blood sample is thawed on the same day as the RNA extraction.

[0397] Blood samples can be thawed at 16°C to 29°C, 17°C to 28°C, 18°C ​​to 27°C, 18°C ​​to 26°C, or 18°C ​​to 25°C. Preferably, blood samples are thawed at 18°C ​​to 25°C. The duration of the thawing step can be 1 to 5 hours, 2 to 5 hours, 1 to 4 hours, 2 to 4 hours, 1 to 3 hours, 2 to 3 hours, or 1 to 2 hours. Blood samples can be thawed at 18°C ​​to 25°C for 1 to 3 hours. More preferably, blood samples are thawed at 18°C ​​to 25°C for 3 hours.

[0398] Once the blood sample is completely thawed, the sample tube can be inverted at least 5, 6, 7, 8, 9, or 10 times. Preferably, the sample tube should be inverted 10 times. The blood sample can then be incubated at 18°C ​​to 25°C for approximately 2 hours before RNA extraction.

[0399] RNA can be extracted from (thawed) blood samples using the Qiagen Paxgene Blood RNA Kit.

[0400] In this method, prior to step (i), blood can be collected in a container (optionally a Paxgene blood RNA tube) at room temperature (5°C to 30°C, preferably 18°C ​​to 25°C). The container can be inverted at least 5, 6, 7, 8, 9, or 10 times immediately after blood collection. Preferably, the container is inverted 10 times immediately after blood collection. The blood sample can be stored at -15°C or below (or -20°C or below) immediately after inversion.

[0401] Therefore, the present invention provides a method for processing blood samples, comprising:

[0402] (i) Receive blood samples in containers at 18°C ​​to 25°C;

[0403] (ii) Store blood samples at or below -20°C within 12 hours of collection;

[0404] (iii) Thaw the blood sample at 18°C ​​to 25°C for 1 to 3 hours; and

[0405] (iv) Extract RNA from thawed blood samples.

[0406] The blood sample may be no more than 5 ml, 3 ml, 2.5 ml or 2 ml. Preferably, the blood sample is no more than 2.5 ml. The blood sample may be 0.1 ml to 5 ml, 0.5 ml to 5 ml, 1 ml to 4 ml or 2 ml to 3 ml.

[0407] Methods for processing blood samples can be combined with methods for using RNA or cDNA samples as outlined herein. Therefore, RNA or cDNA samples can be obtained from blood by following the steps of the methods for processing blood samples as described herein. Additionally, the methods may include a step of synthesizing cDNA using the extracted RNA as a template (i.e., converting the extracted RNA into cDNA, reverse transcription). First RNA / cDNA samples and second RNA / cDNA samples can be obtained from blood by following the steps of the methods for processing blood samples as described herein. Additionally, the methods may include a step of synthesizing cDNA using the extracted RNA as a template (i.e., converting the extracted RNA into cDNA, reverse transcription).

[0408] This invention provides a method for discovering biomarkers for (autoimmune) diseases, comprising:

[0409] (i) At 18°C ​​to 25°C, a first blood sample from a subject suffering from the disease is received in a first container, and a second blood sample from a subject not suffering from the disease is received in a second container;

[0410] (ii) Store the first and second blood samples at or below -20°C within 12 hours of collection from the subject;

[0411] (iii) Thaw the first and second blood samples at 18°C ​​to 25°C for 1 to 3 hours;

[0412] (iv) RNA was extracted from the thawed first and second blood samples;

[0413] (v) Using the extracted RNA as a template to synthesize cDNA to form a first cDNA sample from a subject with the disease and a second cDNA sample from a subject without the disease;

[0414] (vi) Process (standardize) the first and second cDNA samples;

[0415] (vii) Sequencing of the processed (standardized) first and second cDNA samples; and

[0416] (viii) Compare the sequencing outputs of the first cDNA sample and the second cDNA sample to discover disease biomarkers.

[0417] This invention provides a method for diagnosing (autoimmune) diseases in subjects, comprising:

[0418] (i) Blood samples from the subject are received in a container at 18°C ​​to 25°C;

[0419] (ii) Store blood samples at or below -20°C within 12 hours of collection from the subject;

[0420] (iii) Thaw the blood sample at 18°C ​​to 25°C for 1 to 3 hours;

[0421] (iv) Extracting RNA from thawed blood samples;

[0422] (v) Using the extracted RNA as a template to synthesize cDNA to form a cDNA sample from the subject;

[0423] (vi) Processing (standardizing) cDNA samples; and

[0424] (vii) Sequencing of processed (standardized) cDNA samples, wherein the sequencing output is used to identify whether the subject has a disease.

[0425] This invention provides the use of oligonucleotide dimer compositions in methods for discovering biomarkers of autoimmune diseases and / or disease-associated RNA sequence sets (optionally wherein each of the RNA sequence sets encodes an antigenic peptide), said oligonucleotide dimer compositions for selectively amplifying single-stranded cDNA by linking oligonucleotides to the 5' and 3' ends of an associated single-stranded cDNA template having known 5' and 3' pre-attachment adaptors, said composition comprising:

[0426] (A) Pre-oligonucleotide dimer, comprising:

[0427] (i) prelig-oligonucleotides for ligation to the 5' pre-attachment adaptor of the associated single-stranded cDNA template; and

[0428] (ii) A pre-linker oligonucleotide for annealing with a 5' pre-attached adaptor and a pre-lig-oligonucleotide, the pre-linker oligonucleotide comprising a region complementary to the 5' pre-attached adaptor and a region complementary to the pre-lig-oligonucleotide.

[0429] Therefore, during annealing, the end of the pre-lig-oligonucleotide is adjacent to the end of the 5' pre-attached adapter, enabling the pre-lig-oligonucleotide to be attached to the 5' pre-attached adapter at the attachment site; and

[0430] (B) Post-oligonucleotide dimers, comprising:

[0431] (i) Post-lig-oligonucleotides for ligation to the 3' pre-attachment adaptor of the associated single-stranded cDNA template; and

[0432] (ii) A post-linking oligonucleotide for annealing with a 3' pre-attached adaptor and a post-lig-oligonucleotide, the post-linking oligonucleotide comprising a region complementary to the 3' pre-attached adaptor and a region complementary to the post-lig-oligonucleotide.

[0433] Therefore, during annealing, the end of the post-lig-oligonucleotide is adjacent to the end of the 3' pre-attached adapter, so that the post-lig-oligonucleotide can be attached to the 3' pre-attached adapter at the attachment site.

[0434] The present invention also provides the use of an oligonucleotide dimer composition in a method for diagnosing and / or prognosing autoimmune diseases in subjects, the oligonucleotide dimer composition being used to selectively amplify single-stranded cDNA by linking an oligonucleotide to the 5' and 3' ends of an associated single-stranded cDNA template having known 5' and 3' pre-attachment adaptors, wherein the composition comprises:

[0435] (A) Pre-oligonucleotide dimer, comprising:

[0436] (i) prelig-oligonucleotides for ligation to the 5' pre-attachment adaptor of the associated single-stranded cDNA template; and

[0437] (ii) A pre-linker oligonucleotide for annealing with a 5' pre-attached adaptor and a pre-lig-oligonucleotide, the pre-linker oligonucleotide comprising a region complementary to the 5' pre-attached adaptor and a region complementary to the pre-lig-oligonucleotide.

[0438] Therefore, during annealing, the end of the pre-lig-oligonucleotide is adjacent to the end of the 5' pre-attached adapter, enabling the pre-lig-oligonucleotide to be attached to the 5' pre-attached adapter at the attachment site; and

[0439] (B) Post-oligonucleotide dimers, comprising:

[0440] (i) Post-lig-oligonucleotides for ligation to the 3' pre-attachment adaptor of the associated single-stranded cDNA template; and

[0441] (ii) A post-linking oligonucleotide for annealing with a 3' pre-attached adaptor and a post-lig-oligonucleotide, the post-linking oligonucleotide comprising a region complementary to the 3' pre-attached adaptor and a region complementary to the post-lig-oligonucleotide.

[0442] Therefore, during annealing, the end of the post-lig-oligonucleotide is adjacent to the end of the 3' pre-attached adapter, so that the post-lig-oligonucleotide can be attached to the 3' pre-attached adapter at the attachment site.

[0443] The oligonucleotide dimer compositions defined herein can also be used in methods for characterizing diseases in subjects, methods for selecting treatments for diseases in subjects, and / or methods for predicting the responsiveness of subjects with diseases to therapeutic agents. Diseases can be autoimmune diseases.

[0444] In this article:

[0445] (A) Pre-linking oligonucleotides may include:

[0446] (i) a template overhang region at the pre-linker oligonucleotide terminus near a region complementary to the 5' pre-attached adaptor, wherein the template overhang region is non-complementary to the corresponding region of the associated single-stranded cDNA template; and / or

[0447] (ii) A lig-oligonucleotide overhang region at the proximal end of a pre-lig-oligonucleotide terminus that is complementary to the pre-lig-oligonucleotide region, wherein the lig-oligonucleotide overhang region is non-complementary to the corresponding region of the pre-lig-oligonucleotide; and / or

[0448] (B) Post-linked oligonucleotides may include:

[0449] (i) a template overhang region at the post-joint oligonucleotide terminus near a region complementary to the 3' pre-attached adaptor, wherein the template overhang region is non-complementary to the corresponding region of the associated single-stranded cDNA template; and / or

[0450] (ii) A lig-oligonucleotide overhang region at the end of a post-lig-oligonucleotide near a region complementary to the post-lig-oligonucleotide, wherein the lig-oligonucleotide overhang region is non-complementary to the corresponding region of the post-lig-oligonucleotide.

[0451] The template overhang and / or lig-oligonucleotide overhang can be from about 1 bp to about 20 bp in length. The template overhang and / or lig-oligonucleotide overhang can be 2 bp to 19 bp, 3 bp to 18 bp, 2 bp to 17 bp, 3 bp to 16 bp, 2 bp to 15 bp, 3 bp to 14 bp, 2 bp to 13 bp, 3 bp to 12 bp, 2 bp to 11 bp, 3 bp to 10 bp, 2 bp to 9 bp, 3 bp to 8 bp, 2 bp to 7 bp, 3 bp to 6 bp, 2 bp to 5 bp, 3 bp to 5 bp, or 2 bp to 4 bp. Preferably, the template overhang and / or lig-oligonucleotide overhang is 3 bp.

[0452] The template overhang and / or lig-oligonucleotide overhang may be at least 2 bp or at least 3 bp. Preferably, the template overhang and / or lig-oligonucleotide overhang is at least 3 bp.

[0453] The combined length of the pre-lig-oligonucleotide and the pre-lig-oligonucleotide can be less than about 300 bp, and / or the combined length of the post-lig-oligonucleotide and the post-lig-oligonucleotide can be less than about 300 bp.

[0454] The length of the pre-linked oligonucleotide and / or post-linked oligonucleotide can be less than 200 bp.

[0455] The pre-oligonucleotide dimer and / or post-oligonucleotide dimer may have at least one non-flat end.

[0456] In the use of the composition, the pre-linking oligonucleotide and / or post-linking oligonucleotide can provide complementary binding of at least 5 bp on either side of the linker site.

[0457] The nucleotide sequence of the pre-oligonucleotide dimer may be different from and non-complementary to the nucleotide sequence of the post-oligonucleotide dimer.

[0458] The pre-oligonucleotide dimer and / or post-oligonucleotide dimer can be annealed with the associated single-stranded cDNA template at temperatures exceeding 30°C.

[0459] A selective amplification kit is provided for selectively amplifying low-abundance RNA / cDNA from RNA / cDNA samples and / or for selectively amplifying cDNA containing known adaptor sequences, said cDNA samples containing cDNA templates having known 5' pre-attached adaptors and 3' pre-attached adaptors, said kit comprising tools for preparing oligonucleotide dimer compositions as described above and tools for performing the selective amplification method as described above.

[0460] This invention provides the use of the kit described herein in methods for diagnosing and / or prognosing diseases in subjects. It also provides the use of the kit described herein in methods for characterizing diseases in subjects, methods for selecting treatments for diseases in subjects, and / or methods for predicting the responsiveness of subjects with diseases to therapeutic agents. The disease may be an autoimmune disease.

[0461] Selective amplification kits are also provided for selectively amplifying low-abundance RNA / cDNA from a first RNA / cDNA sample from a diseased subject and a second RNA / cDNA sample from a disease-free subject, and / or for selectively amplifying cDNA containing known adaptor sequences, the first and second cDNA samples containing cDNA templates having known 5' and 3' pre-attached adaptors, the kits comprising tools for preparing oligonucleotide dimer compositions as described above and tools for performing the selective amplification methods as described above.

[0462] This invention provides the use of the kit as described herein in a method for discovering disease biomarkers and / or disease-related RNA sequence sets (optionally wherein each of the RNA sequence sets encodes an antigenic peptide).

[0463] Tools for preparing oligonucleotide dimer compositions may comprise pre-lig-oligonucleotides, pre-linked-oligonucleotides, post-lig-oligonucleotides, and / or post-linked-oligonucleotides as described herein.

[0464] Tools used to implement selective amplification methods may contain primers that are specific to pre-lig-oligonucleotides and / or post-lig-oligonucleotides.

[0465] The kit may also include hybridization buffer. Hybridization buffer may contain 1 M HEPES (pH = 7.5), 5 M NaCl, and H2O. The kit may also include ligase and / or ligase buffer. Any suitable ligase can be used. The ligase may be a nick repair ligase or a blunt-end ligase. Optionally, the ligase may be Taq DNA ligase. Suitable ligase buffers are also known and commercially available.

[0466] The kit may also contain primers for adding phosphate groups to cDNA before using it as a cDNA sample. These primers are based on known 5' pre-attachment adaptor sequences and known 3' pre-attachment adaptor sequences.

[0467] The kit may also contain suitable reagents for PCR, including one or more, or all of, the following: polymerase, dinucleotide triphosphate (dNTP), MgCl2, and buffer. Any suitable polymerase may be used. Typically, DNA polymerase is used to amplify the nucleic acid targets according to the invention. Some examples include thermostable polymerases such as Taq or Pfu polymerases and various derivatives of these enzymes. Suitable buffers are also known and commercially available and may be included in the PCR master mixture, which contains most of the components required for PCR amplification.

[0468] The kit may also contain suitable reagents for reverse transcription of RNA into cDNA, including reverse transcriptase. Any suitable reverse transcriptase can be used. Suitable buffers are also known and commercially available and can be included in the reverse transcription master mixture, which contains most of the components required for reverse transcription.

[0469] The kit may also include suitable reagents for processing blood samples, such as containers (optionally PAXgene blood RNA tubes), RNA stabilizing agents, and / or blood cell lysis buffer. The reagents may be RNase-free. RNA stabilizing agents are commercially available and include RNAlater® (Sigma-Aldrich) and RNAprotect (Qiagen). Suitable RNA stabilizing agents may contain EDTA, sodium citrate, and / or ammonium sulfate, such as 70% (w / v) ammonium sulfate, 25 mM sodium citrate, and / or 10 mM EDTA. The pH can be adjusted to 5.2 with sulfuric acid.

[0470] This invention provides the use of a reagent kit in a method as described herein, the reagent kit comprising:

[0471] Prelig-oligonucleotides, pre-linked-oligonucleotides, postlig-oligonucleotides, and / or post-linked-oligonucleotides; and

[0472] Primers that are specific to pre-lig-oligonucleotides and / or post-lig-oligonucleotides.

[0473] Prelig-oligonucleotides, pre-linked-oligonucleotides, postlig-oligonucleotides, and / or post-linked-oligonucleotides can be provided as oligonucleotide dimer compositions as described herein.

[0474] The reagent kit may also contain one or more, up to all of the following: hybridization buffer (optionally containing 1 M HEPES (pH = 7.5), 5 M NaCl and H2O), ligase, ligase buffer, primer pairs for adding phosphate groups to cDNA, DNA polymerase and / or dNTPs.

[0475] The reagent kit may also include suitable reagents for processing blood samples, such as containers (optionally PAXgene blood RNA tubes), RNA stabilizing agents, and / or blood cell lysis buffer. The reagents may be RNase-free. RNA stabilizing agents are commercially available and include RNAlater® (Sigma-Aldrich) and RNAprotect (Qiagen). Suitable RNA stabilizing agents may contain EDTA, sodium citrate, and / or ammonium sulfate, such as 70% (w / v) ammonium sulfate, 25 mM sodium citrate, and / or 10 mM EDTA. The pH can be adjusted to 5.2 with sulfuric acid. Incubation for cell lysis can be, for example, from 1 minute to 3 hours.

[0476] The reagent kit may include:

[0477] RNA stabilizing agent;

[0478] (Blood) cell lysis buffer;

[0479] Prelig-oligonucleotides, pre-linked-oligonucleotides, postlig-oligonucleotides, and / or post-linked-oligonucleotides;

[0480] Primers specific to pre-lig-oligonucleotides and / or post-lig-oligonucleotides;

[0481] Hybridization buffer;

[0482] Ligase;

[0483] Ligase buffer; and

[0484] Primer pairs used to add phosphate groups to cDNA.

[0485] The oligonucleotide dimer compositions for selectively amplifying single-stranded cDNA described herein have the ability to select cDNA with known adaptor sequences. This can be applied to single-cell sequencing, where the adaptor is necessary to assign reads to a single cell. In this application, cDNA sequences without cell-identifying barcodes / adaptors appear in the cDNA library. These are called template-switching oligo (TSO) artifacts and are undesirable in single-cell sequencing projects because they cannot be assigned to the source cells. The method of the present invention can be applied to select only cDNA sequences with the desired adaptor sequence, thereby effectively restricting sequencing of TSO artifacts. The method of the present invention can also be used with single-cell cDNA libraries, thereby both removing TSO artifacts and improving transcriptome coverage per cell.

[0486] TSO removal can also be performed without standardization, in which case the step of reassociating the cDNA sample to produce a mixture of associated single-stranded cDNA template and associated double-stranded cDNA template is not required.

[0487] This invention provides a method for diagnosing a disease in an object, the method comprising:

[0488] (i) Provide an RNA sample extracted from a blood sample obtained from the subject;

[0489] (ii) Use RNA as a template to synthesize cDNA to produce a cDNA sample containing a double-stranded cDNA template, a portion of which has a known 5' pre-attached adaptor and a known 3' pre-attached adaptor;

[0490] (iii) Denature the cDNA sample to generate a single-stranded cDNA template;

[0491] (iv) Anneal the 5' adaptor complex with the 5' pre-attached adaptor of at least one single-stranded cDNA template and anneal the 3' adaptor complex with the 3' pre-attached adaptor of the same single-stranded cDNA template, wherein each adaptor complex contains at least one oligonucleotide;

[0492] (v) The oligonucleotide from the 5' adaptor complex is ligated to the 5' pre-attached adaptor of the single-stranded cDNA template, and the oligonucleotide from the 3' adaptor complex is ligated to the 3' pre-attached adaptor of the same single-stranded cDNA template.

[0493] (vi) Using primers specific to the oligonucleotides being linked to selectively amplify cDNA samples; and

[0494] (vii) Sequencing of cDNA samples, wherein the sequencing output is used to identify whether the subject has a disease.

[0495] This invention provides a method for discovering disease biomarkers, the method comprising:

[0496] (i) Provide a first RNA sample extracted from a blood sample obtained from a subject with the disease and a second RNA sample extracted from a blood sample obtained from a subject without the disease;

[0497] (ii) Using RNA as a template to synthesize cDNA to produce a first cDNA sample and a second cDNA sample, the samples containing a double-stranded cDNA template, a portion of which has a known 5' pre-attached adapter and a known 3' pre-attached adapter;

[0498] (iii) Denature each cDNA sample to generate a single-stranded cDNA template;

[0499] (iv) Anneal the 5' adaptor complex with the 5' pre-attached adaptor of at least one single-stranded cDNA template and anneal the 3' adaptor complex with the 3' pre-attached adaptor of the same single-stranded cDNA template, wherein each adaptor complex contains at least one oligonucleotide;

[0500] (v) The oligonucleotide from the 5' adaptor complex is ligated to the 5' pre-attached adaptor of the single-stranded cDNA template, and the oligonucleotide from the 3' adaptor complex is ligated to the 3' pre-attached adaptor of the same single-stranded cDNA template.

[0501] (vi) Use primers specific to the oligonucleotides being linked to selectively amplify each cDNA sample; and

[0502] (vii) Sequencing of each cDNA sample and comparison of the sequencing output of the first and second cDNA samples to discover disease biomarkers.

[0503] The disease can be an autoimmune disease. RNA / cDNA samples can originate from a single cell.

[0504] The oligonucleotide dimer composition is suitable for use in methods for selectively amplifying cDNA containing a known adaptor sequence, as defined herein. Methods for selectively amplifying cDNA containing a known adaptor sequence, as defined herein, can be used to discover disease biomarkers and / or disease-associated RNA sequences, or in the diagnosis and / or prognosis of diseases. The kits and reagent sets defined herein are also suitable for use in methods for selectively amplifying cDNA containing a known adaptor sequence.

[0505] cDNA samples may contain no more than 800 ng, 700 ng, 500 ng, 100 ng, 20 ng, 10 ng, 5 ng, or 1 ng of starting cDNA. cDNA samples may contain 1 to 800 ng, 1 to 500 ng, 5 to 100 ng, or 10 to 50 ng of starting cDNA.

[0506] RNA from a sample can first be reverse transcribed into cDNA. Sample types include blood samples (particularly blood samples from plasma and serum), other bodily fluids such as saliva, urine, or lymph. Other sample types include solid tissues, including frozen tissues or formalin-fixed, paraffin-embedded (FFPE) materials. RNA can be messenger RNA (mRNA), microRNA (miRNA), etc. RNA can be reverse transcribed using reverse transcriptase to form complementary DNA (cDNA) molecules. Methods for reverse transcribing RNA into cDNA using reverse transcriptase are well known in the art. Any suitable reverse transcriptase can be used, and some examples of suitable reverse transcriptases are widely available in the art. The starting cDNA molecule can be single-stranded until a complementary strand is generated using DNA polymerase. Commercially available kits (such as the NEBNext® Single-Cell / Low-Input cDNA Synthesis & Amplification Module) can be used to convert RNA into double-stranded cDNA with 5' and 3' adaptors. Primers based on the 5' and 3' adaptors can be used to add phosphate groups to the cDNA. cDNA purification steps (e.g., using ProNex or Ampure beads) can be performed before using cDNA as a cDNA sample or before sequencing.

[0507] Because the method requires only a small amount of starting cDNA, this can be generated from a small amount of RNA and / or no additional PCR cycles are needed during cDNA generation. RNA samples may contain no more than 3.5 μg, 3 μg, 2 μg, 1 μg, 500 ng, 100 ng, 10 ng, or 1 ng of starting RNA. RNA samples may contain 1 ng to 3 μg, 10 ng to 2 μg, or 100 ng to 1 μg of starting RNA.

[0508] This invention provides a system or test kit for discovering disease biomarkers and / or disease-related RNA sequence sets, comprising:

[0509] (a) One or more testing devices for processing (normalizing) a first RNA / cDNA sample from a subject with a disease (cancer) and a second RNA / cDNA sample from a subject without a disease (cancer), and sequencing the processed (normalized) first RNA / cDNA sample and the second RNA / cDNA sample.

[0510] (b) processor; and

[0511] (c) A storage medium including a computer application, which, when executed by the processor, is configured to:

[0512] (i) Obtain the sequences of a first RNA / cDNA sample and a second RNA / cDNA sample determined on one or more testing devices;

[0513] (ii) Calculate whether there is an RNA / cDNA sequence present in the first RNA / cDNA sample but not in the second RNA / cDNA sample, or whether there is an RNA / cDNA sequence present in the second RNA / cDNA sample but not in the first RNA / cDNA sample; and

[0514] (iii) Output the result of step (ii) from the processor.

[0515] This invention provides a system or test kit for diagnosing and / or prognosing diseases in subjects, comprising:

[0516] (a) One or more testing devices for processing (normalizing) RNA / cDNA samples from the subject and sequencing the processed (normalized) cDNA samples.

[0517] (b) processor; and

[0518] (c) A storage medium containing a computer application, which, when executed by the processor, is configured to:

[0519] (i) Obtain the sequence of one or more RNA / cDNA samples identified on a testing device;

[0520] (ii) Calculate the presence or absence of RNA / cDNA sequences, wherein the presence or absence of RNA / cDNA sequences is associated with the disease; and

[0521] (iii) Output from the processor whether the object has a disease and / or the prognosis of the disease.

[0522] One or more assay devices may use / contain oligonucleotide dimer compositions as described herein. One or more assay devices may include long-read sequencers.

[0523] The system or test kit may also include a display for output from the processor.

[0524] It also provides computer applications or storage media containing computer applications as defined herein.

[0525] The present invention provides peptides or proteins comprising, consisting of, or substantially consisting of one or more amino acid sequences selected from SEQ ID NO: 9 to 23, their subsequences, portions, homologs, variants, and derivatives.

[0526] The present invention provides polynucleotides encoding peptides or proteins comprising, consisting of, or substantially consisting of one or more amino acid sequences selected from SEQ ID NO: 9 to 23, their subsequences, portions, homologs, variants, and derivatives.

[0527] One or more amino acid sequences may be selected from SEQ ID NO: 11, 14 and 23.

[0528] Polynucleotides can be RNA or DNA molecules.

[0529] Subsequences, portions, homologs, variants, or derivatives may have about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with the associated sequence, that is, with one or more amino acid sequences selected from SEQ ID NO: 9 to 23, that is, with 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity.

[0530] The "sequence identity percentage" is determined by comparing two best-aligned sequences in a comparison window. The portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (i.e., vacancies) compared to a reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. The percentage is calculated as follows: determine the number of positions in both sequences where the same nucleic acid base or amino acid residue appears to obtain the number of matching positions; divide the number of matching positions by the total number of positions in the comparison window; and multiply the result by 100 to obtain the sequence identity percentage.

[0531] The present invention provides a method for diagnosing cancer and / or prognosing cancer in a subject, comprising measuring the levels of PMS2, APC, or at least one peptide thereof in a sample from the subject, wherein the levels of the protein or peptide are used to provide a diagnosis and / or prognosis for cancer.

[0532] The present invention provides a method for diagnosing and / or prognosing cancer in a subject, comprising measuring the level of one or more amino acid sequences selected from SEQ ID NO: 9 to 23, or polynucleotides encoding such peptides or proteins in a sample from the subject, said peptides or proteins comprising one or more amino acid sequences selected from SEQ ID NO: 9 to 23, consisting of one or more amino acid sequences selected from SEQ ID NO: 9 to 23, or substantially consisting of one or more amino acid sequences selected from SEQ ID NO: 9 to 23, wherein the level of the amino acid sequence or polynucleotide is used to provide diagnosis and / or prognosis of cancer.

[0533] Polynucleotides can be RNA or DNA molecules.

[0534] This invention provides the use of the methods, apparatus, or kits described herein in methods for determining the composition of soil microbial communities.

[0535] Soil microbial flora composition can be determined by identifying the microorganisms and / or viruses present in soil samples. This can be achieved by identifying sequences in sequencing output.

[0536] This invention provides the use of the methods, devices, or kits described herein in ecological research.

[0537] Ecological research may include sequencing processed RNA or cDNA from one or more species. Ecological research may include obtaining information about the transcriptome of one or more species. Ecological research can improve our understanding of the overall biology of one or more species and / or how one or more species influence their local ecology.

[0538] This invention provides the use of the methods, apparatus, or kits described herein in methods for assessing water quality.

[0539] Water quality can be assessed by identifying / detecting microorganisms (e.g., bacteria and / or fungi) and / or viruses. Water quality can also be assessed by extracting RNA from water samples from a water source. This can be achieved by identifying sequences in the sequencing output.

[0540] This invention provides the use of the methods, apparatus, or kits described herein in methods for screening hazardous biological materials.

[0541] Hazardous biological materials may include fungi, bacteria, and / or viruses, optionally pathogenic fungi, bacteria, or viruses, and / or fungal, plant, or animal-derived toxins or drugs that may still contain trace amounts of nucleic acids (e.g., RNA). Screening can be conducted at travel gateways.

[0542] This invention provides the use of the methods, apparatus, or kits described herein in methods for determining the identity of an object.

[0543] Object identification can be achieved by identifying sequences in the sequencing output. Object identification may include DNA fingerprinting analysis or steps from DNA fingerprinting methods. Object identification can also be achieved by analyzing highly variable repetitive sequences (e.g., variable-number tandem repeats, optionally short tandem repeats). The method can be performed at a travel checkpoint.

[0544] The present invention is further defined in the following numbered clauses:

[0545] 1. A method for generating an RNA vaccine for a subject suffering from a disease, the method comprising:

[0546] (i) Provide an RNA sample extracted from a blood sample obtained from the subject;

[0547] (ii) Processing the RNA sample, optionally including using the RNA as a template to synthesize cDNA;

[0548] (iii) Sequencing the processed RNA or cDNA, wherein the sequencing output is used to identify the presence of RNA transcript sequences associated with the said disease; and

[0549] (iv) Use the identified RNA transcript sequence to generate a first RNA vaccine against the subject.

[0550] 2. The method described in Clause 1, further comprising:

[0551] (v) Provide additional RNA samples extracted from blood samples obtained from the subject at a later time point;

[0552] (vi) Processing the RNA sample, optionally including using the RNA as a template to synthesize cDNA;

[0553] (vii) Sequencing the processed RNA or cDNA, wherein the sequencing output is used to identify the presence of RNA transcript sequences associated with the said disease, and

[0554] (viii) Use the identified RNA transcript sequence to generate an additional RNA vaccine against the subject.

[0555] 3. The method of Clause 1 or Clause 2, wherein using the identified RNA transcript sequence to generate an RNA vaccine comprises generating an RNA molecule containing at least a portion of the sequence, wherein the RNA molecule contains an open reading frame (ORF) encoding at least one antigenic peptide.

[0556] 4. The method of Clause 3, wherein the RNA molecule further comprises a 5' UTR, a 3' UTR, a polyA tail, and / or a 5' cap.

[0557] 5. A method for generating a database of disease biomarkers, the method comprising:

[0558] (i) Provide two or more RNA samples extracted from blood samples obtained from one or more subjects suffering from the disease;

[0559] (ii) Processing the RNA sample, optionally including using the RNA as a template to synthesize cDNA;

[0560] (iii) Sequencing of processed RNA or cDNA, wherein the sequencing output is used to identify two or more disease biomarkers; and

[0561] (iv) Compile the aforementioned disease biomarkers to form a database.

[0562] 6. A method for detecting biomarkers of autoimmune diseases, comprising:

[0563] (i) Provide a first RNA sample extracted from a blood sample obtained from a subject suffering from said disease and a second RNA sample extracted from a blood sample obtained from a subject not suffering from said disease;

[0564] (ii) Processing the first RNA sample and the second RNA sample, optionally including using the RNA as a template to synthesize cDNA;

[0565] (iii) Sequencing the processed RNA or cDNA; and

[0566] (iv) Compare the sequencing outputs of the first sample and the second sample to discover disease biomarkers.

[0567] 7. A method for diagnosing autoimmune diseases in subjects, comprising:

[0568] (i) Provide an RNA sample extracted from a blood sample obtained from the subject;

[0569] (ii) Processing the RNA sample, optionally including using the RNA as a template to synthesize cDNA; and

[0570] (iii) Sequencing the processed RNA or cDNA, wherein the sequencing output is used to identify whether the subject has the disease.

[0571] 8. The method of Clause 7, wherein the sequencing output is used to identify whether the subject has the disease by comparison with a database of biomarkers, optionally wherein the database of biomarkers is generated using the method of Clause 5.

[0572] 9. The method described in any of the preceding clauses, wherein processing the RNA sample includes reducing the variability of different RNA or cDNA sequence levels in the sample.

[0573] 10. The method described in any of the preceding clauses, wherein the relative abundance of all unique RNA or cDNA sequences in the treated RNA or cDNA is more uniform.

[0574] 11. The method of any of the preceding clauses, wherein processing the RNA sample improves the detection of low-abundance RNA or cDNA, optionally wherein processing the RNA sample includes increasing the amount of low-abundance RNA or cDNA in each sample.

[0575] 12. The method described in any of the preceding clauses, wherein the RNA is a full-length RNA.

[0576] 13. The method described in any of the preceding clauses, wherein sequencing includes sequencing using long reads.

[0577] 14. The method of any one of Clauses 6 to 13, wherein the autoimmune disease is arthritis, celiac disease, type 1 diabetes, Graves' disease, inflammatory bowel disease, multiple sclerosis, alopecia areata, Addison's disease, pernicious anemia, psoriasis, systemic lupus erythematosus, myasthenia gravis, Hashimoto's thyroiditis, vitiligo, Sjögren's syndrome, myositis, chronic inflammatory demyelinating polyneuropathy (CIDP), dermatomyositis, Guillain-Barré syndrome, ulcerative colitis, and / or vasculitis.

[0578] 15. The method of any one of Clauses 1 to 4 or 9 to 13, wherein using the sequencing output to identify the presence of a sequence associated with the disease comprises identifying an RNA transcript encoding a protein isotype present in a subject suffering from the disease. Detailed Implementation

[0579] The invention will now be described in further detail by way of example only, with reference to the following examples and figures, wherein:

[0580] Figure 1 This is a schematic diagram illustrating the addition of a ligation sequence to the end of a single-stranded cDNA template;

[0581] Figure 2 This is a schematic diagram of one embodiment of the cDNA standardization process according to the present invention;

[0582] Figure 3 This is a schematic diagram illustrating the structures of the pre- and post-oligonucleotide dimers and the annealing of a single-stranded cDNA template, as shown. Figure 3A It is its details, and Figure 3B Illustrations are provided using some of the exemplary sequence representations described herein;

[0583] Figure 4 This is a graph showing the length distribution of gel electrophoresis from the input cDNA;

[0584] Figure 5 It shows that, as Figure 2 The diagram shows the length distribution of gel electrophoresis from standardized cDNA produced during the cDNA normalization process; and

[0585] Figure 6 This demonstrates the use of nanopore cDNA sequencing, as shown in the example. Figure 2 Saturation curves of standardized cDNA and input cDNA.

[0586] Figure 7 This is a 2D PCA plot from 13,573 transcripts, showing the clustering of cancer and control samples in the dataset.

[0587] Figure 8 An example of a cancer-specific transcript (a novel transcript) from PMS2 is shown, which has novel exons that produce cancer-specific peptide sequences.

[0588] Figure 9 An example of a cancer-specific transcript (a novel transcript) from APC is shown, which has novel exons that produce cancer-specific peptide sequences.

[0589] Figure 10 An exemplary workflow for identifying cancer-related k-mers is shown. RNA sequences from cancer patients and control patients are converted into possible peptide sequences that can be translated from RNA (i.e., the three possible open reading frames (ORFs)). The resulting peptide sequences are then segmented into k-mers. K-mers from cancer patients are compared with those present in control patients to identify k-mers that are present only in cancer patients or are highly enriched in cancer patients.

[0590] Figure 11 The overlapping k-mer sequences are shown. It is formed by multiple overlapping k-mers. The sequence can be divided into overlapping k-mers. Conversely, overlapping k-mers can be combined to form a sequence that constitutes a complete neoantigen region.

[0591] This invention is based on a method that utilizes the ability to generate full-length sequences from RNA extracted from blood without fragmenting the RNA or cDNA product prior to sequencing. This provides a transcriptome representing any RNA entering the circulatory system, including RNA from typical blood cells (such as red blood cells, white blood cells, other immune cells, etc.), RNA from any other cells not typically found in the circulatory system (such as cancer cells or cells that have somehow detached from the circulatory system), and extracellular RNA that can originate from any cell in the body. By detecting full-length RNA from all these sources, the inventors can attribute each RNA to the cell / tissue of origin and the state of cell or tissue behavior. Transcription start sites, termination sites, and splicing are characteristic combinations typically used by different cell types. This information can also be used to directly identify protein isotypes that will be translated from messenger RNA. The ability to detect low-level signals from full-length RNA (which can then be translated into protein isotypes) means that the inventors can detect unique protein isotypes expressed only by certain cells (such as cancer cells). These isotypes will have peptide sequence segments that are unique to a particular cell (such as cancer cells). Figure 8 and 9 These cancer-specific peptide sequences are also known as neoantigens because they are typically presented on the cell surface via the MHC complex.

[0592] Detecting isotype-based neoantigens offers unique advantages over mutation-based neoantigens. For example, this means that it is not necessary to design new vaccines for each patient based on the unique mutations present in each individual. The method of this invention allows for the identification of isotype-based neoantigen groups that are representative of a large proportion of the population. This means that RNA cancer vaccines can be based on such groups without having to design new vaccines for each patient. Under this paradigm, the development, safety testing, production, QC, and delivery of RNA cancer vaccines can be managed more economically and with a lower risk of adverse effects. This implies that RNA cancer vaccines can be more affordable and can be used more frequently because they will be safer and more cost-effective.

[0593] Using blood samples instead of tumor samples to find neoantigen targets is also advantageous. Tumor samples, being limited materials, may be difficult to obtain and only available at specific time points. This also means that targets are based on extracted primary cells, while the goal is to target remaining distant cells that may be genetically different. If a target covering all remaining cancer cells is not identified, selective pressure arises, causing cancer cells that do not present the target to proliferate and lead to recurrence. This can even promote cancers with higher mutation rates, which may be even more problematic.

[0594] Because the method of this invention allows for continuous monitoring of neoantigen targets in patients, and because it can help make RNA cancer vaccines cheaper and safer, RNA cancer vaccine therapy can be used earlier and more frequently. The neoantigen can be read out from the method of this invention and applied multiple times until no neoantigen is observed in the patient's blood.

[0595] Neoantigen targets also represent unique structures within cancer proteins, which can be used for new drug development. Since a large proportion of targeted cancer therapies interact with proteins, data obtained from the methods of this invention can be used to understand the usefulness of a wide range of cancer treatments, including immune checkpoint inhibitors.

[0596] This invention allows for the detection of disease at its earliest stages, with optimal treatment guided entirely by a single testing paradigm. This not only enables earlier treatment, thus increasing success rates, but also reduces the time from diagnosis to treatment. The process can also be applied to prevent the formation of tumors or destructive cancers by screening populations and administering RNA cancer vaccines before cancer cells have had the opportunity to grow to any meaningful size.

[0597] The data collected using the method of this invention can be used to aggregate a wide range of full-length RNA human databases, which can be used for data mining of new potential drug targets.

[0598] In summary, unlike mutation-based methods that must be designed for each patient, the method of this invention uses isotype-level targets that should be common in patients. This means that a set of targets that works for a large proportion of the population can be identified. Therefore, RNA cancer vaccines can be developed, tested, and produced targeting each target in this set, and applied alone or in combination when detected using the method described herein. This significantly reduces costs and improves safety, meaning that RNA cancer vaccines can be used earlier and more frequently. This means that it is not necessary to destroy all cancer cells at once. The method of this invention allows for monitoring and adjustment of treatment until all neoantigens in the blood have disappeared. The method of this invention can also guide complementary therapies, such as immune checkpoint inhibitors, thereby allowing for the application of combination therapy when needed. Therefore, the method of this invention can be applied to both preventing cancer formation and treating cancer that has already formed.

[0599] These concepts can be applied in very similar ways to a wide range of diseases, including autoimmune diseases, diabetes, coronary artery disease, metabolic diseases, and so on.

[0600] cDNA standardization

[0601] In RNA or cDNA samples, sequences from highly expressed genes typically dominate. Standardization to achieve a more uniform distribution of sequences can improve sequencing efficiency for transcript discovery and / or detection. First, genes and isotypes specific to the disease in question are more easily detected, and second, there is less redundancy in the generated data, reducing data storage requirements.

[0602] Therefore, the methods provided herein may include processing steps for cDNA normalization. To address problems in current cDNA normalization techniques, the inventors have developed an improved selective amplification method (referred to as "Level-Up"). This method uses the same denaturation and rehybridization processes as the DSN and column methods described herein. However, the method of the present invention differs from other methods by using a non-depletion or additive mechanism. In other words, Level-Up provides methods and tools for increasing the amount of low-abundance cDNA in a sample. These methods and tools can be used for cDNA normalization during sequencing processes or for other processes that will benefit from the amplification of low-abundance cDNA, such as the discovery, detection, or identification of biomarkers and the production of RNA vaccines.

[0603] In the context of this invention, the following terms and methods are provided for explanation to better describe this disclosure and to provide guidance in the practice of this disclosure.

[0604] The phrase "selective amplification" is used to describe a method developed by the inventors that preferentially amplifies a specific DNA template relative to other DNA templates (e.g., amplifying only single-stranded cDNA in a sample containing a mixture of single-stranded and double-stranded cDNA). This term is also used herein to describe the preferential amplification of specific types of DNA, such as low-abundance DNA.

[0605] The term "adaptor" is used to describe a short DNA sequence added to the end of a DNA template, such as those commonly used in RNA sequencing by ligating an adaptor to a cDNA template. A "3' pre-attached adaptor" is an adaptor with a known nucleotide sequence that has been added to the 3' end of a cDNA template. A "5' pre-attached adaptor" is an adaptor with a known nucleotide sequence that has been added to the 5' end of a cDNA template.

[0606] The terms “normalization” and “normalized score” refer to the process of leveling the abundance of different transcripts in a sample. This can be achieved by existing techniques of reducing the amount of high-abundance transcripts, or by selectively amplifying low-abundance transcripts using the methods described herein.

[0607] As used in the context of this invention, the phrase "post-association single-stranded cDNA template" refers to a single-stranded cDNA template produced by deassociating and reassociating (i.e., denaturing and rehybridizing) a double-stranded cDNA sample to form a mixture of single-stranded and double-stranded cDNA. Single-stranded cDNA that remains single-stranded after reassociation is referred to as post-association single-stranded cDNA. In embodiments defined herein, the phrases "post-association single-stranded cDNA template" and "single-stranded cDNA template" are interchangeable if the reassociation step is not performed.

[0608] The phrase “ligated adaptor-cDNA template” used in this article refers to the cDNA template formed by ligating an adaptor to a cDNA template.

[0609] This invention covers "adaptor complexes" suitable for end-annealing with associated single-stranded cDNA templates. The term "adaptor complex" refers to an adaptor comprising more than one component.

[0610] As used in the context of this invention, the terms "pre-oligonucleotide dimer," "pre-k-linker," and "pre-dimer" refer to an adaptor complex that can anneal to the 5' end of a post-association single-stranded cDNA template. As used in the context of this invention, the terms "post-oligonucleotide dimer," "post-k-linker," and "post-dimer" refer to an adaptor complex that can anneal to the 3' end of a post-association single-stranded cDNA template.

[0611] In the context of this invention, the terms "prelig-oligonucleotide" and "prelig" refer to the oligonucleotide component of the predimer. In the context of this invention, the terms "postlig-oligonucleotide" or "postlig" refer to the oligonucleotide component of the postdimer.

[0612] In the context of this invention, the terms "front link" and "front link" refer to the oligonucleotide component of the pre-dimer. In the context of this invention, the terms "back link" and "back link" refer to the oligonucleotide component of the back-dimer.

[0613] The term "protruding end" is used in the context of this invention to describe the protruding end region of the dimer sequence of this invention, wherein the protruding end region is not complementary to its paired region and, upon annealing with the associated single-stranded cDNA template, prevents the protruding end region from binding to its paired region.

[0614] Single-stranded cDNA is selectively amplified using primers specific to the linked oligonucleotides. Since the single-stranded DNA serves as the template, the primer region of one primer in the specific primer pair is complementary to the single-stranded DNA molecule. The other primer in the specific primer pair contains a primer region complementary to and thus hybridizes with the complementary single-stranded DNA molecule formed during the amplification cycle. Therefore, one primer is complementary to one linked oligonucleotide, and the other primer (at least partially) contains the sequence of the other linked oligonucleotide.

[0615] Previously developed cDNA normalization methods

[0616] Two forms of full-length cDNA normalization have been previously developed: the double-stranded specific nuclease (DSN) method (Zhulidov, PA et al. Simple cDNA normalization using kamchatka crab duplex-specific nuclease. Nucleic Acids Res. 32, e37 (2004)) and the hydroxyapatite column method (Andrews-Pfannkoch, C., Fadrosh, DW, Thorpe, J. & Williamson, SJHydroxyapatite-mediated separation of double-stranded DNA, single-stranded DNA, and RNA genomes from natural viral assemblages. Appl. Environ. Microbiol. 76, 5039-5045 (2010)). Both methods rely on the denaturation and rehybridization of the cDNA strand. As single-stranded cDNA moves throughout the solution, the more abundant sequence has a greater chance of finding a matching complementary sequence to rehybridize with. Therefore, when rehybridization reaches its limit, the remaining single-stranded cDNA represents a standardized sequence library.

[0617] The difference between these two methods lies in their approach to separating single-stranded cDNA libraries from rehybridized double-stranded cDNA molecules.

[0618] In the DSN method, an enzyme that specifically cleaves double-stranded DNA is used to break down all double-stranded cDNA in solution. The solution is then purified, and size selection is performed on cDNA sequences exceeding a certain length. These sequences are then amplified using polymerase chain reaction (PCR).

[0619] In the column method, a denatured and rehybridized cDNA library is passed through a heated column packed with hydroxyapatite particles. Hydroxyapatite preferentially binds to larger DNA molecules. The size of the bound DNA is controlled by the concentration of the phosphate buffer in which the cDNA library is dissolved. Therefore, the concentration of the phosphate buffer must be specifically adjusted for cDNA molecules within a certain sequence length range. The cDNA is eluted through the column using a higher concentration of phosphate buffer to extract the larger-sized DNA molecules. Since single-stranded cDNA is approximately half the size of the rehybridized cDNA, the elution of the single-stranded portion can be controlled if the average cDNA sequence length is known. The resulting elution aims to enrich the single-stranded cDNA, which is then amplified using PCR.

[0620] In both the DSN and column methods, the known adaptor must be ligated to the end of the cDNA before standardization to facilitate PCR amplification (so that suitable primers can be used).

[0621] Since both methods are essentially subtraction methods that deplete most of the cDNA, the amount of starting cDNA usually needs to be higher than 1 μg for the DSN method and higher than 4 μg for the column method.

[0622] Because the DSN method uses an enzyme that cleaves all double-stranded cDNA, it can theoretically deplete low-abundance sequences with matching high-abundance sequences. This effect can also increase the likelihood of forming PCR chimeras. A PCR chimera is formed when an incomplete single-stranded cDNA sequence acts as a primer for other sequences, thus combining those sequences in a way that does not exist in nature. PCR chimeras represent false positives of novel allotypes and are difficult to distinguish from true surrogate allotypes. Validating PCR chimeras typically requires in-depth biochemical assays.

[0623] Because column methods only allow the separation of high-abundance and low-abundance fractions within a narrow size range, they exhibit a significant bias towards longer cDNA sequences. This results in a loss of representativeness for longer RNA sequences.

[0624] Column Method

[0625] As described above, the hydroxyapatite column method relies on the denaturation and rehybridization of cDNA strands. When single-stranded cDNA moves throughout the solution, higher-abundance sequences are more likely to find complementary sequences for rehybridization. In the column method, the denatured and rehybridized cDNA library passes through a heated column filled with hydroxyapatite particles. Hydroxyapatite preferentially binds to larger DNA molecules. Since single-stranded cDNA is approximately half the size of the rehybridized cDNA, the elution of single-stranded portions can be controlled if the average cDNA sequence length is known. The resulting elution aims to enrich the single-stranded cDNA, which is then amplified using PCR.

[0626] The hydroxyapatite column method was performed using 4 μg of cDNA as a starting sample, as described in Andrews-Pfannkoch et al. (Andrews-Pfannkoch, C., Fadrosh, DW, Thorpe, J. & Williamson, SJ Hydroxyapatite-mediated separation of double-stranded DNA, single-stranded DNA, and RNA genomes from natural viral assemblages. Appl. Environ. Microbiol. 76, 5039-5045 (2010)). When using 2 μg or less of cDNA, the hydroxyapatite column method did not produce a usable yield.

[0627] Because the column method is based on separation by size, it results in a loss of representativeness for longer RNA sequences (longer than 4 kb), which is observable in the length distribution before and after normalization.

[0628] DSN method

[0629] As for column methods, the DSN method relies on the denaturation and rehybridization of cDNA strands. Because single-stranded cDNA moves freely throughout the solution, sequences with higher abundance are more likely to find complementary sequences for rehybridization. In the DSN method, enzymes that specifically cleave double-stranded DNA are used to break down all double-stranded cDNA in solution.

[0630] The commercially available Evrogen Trimmer-2 cDNA normalization kit uses the DSN method. The kit is used with a starting sample of 1 μg cDNA according to the manufacturer's instructions. However, to generate sufficient material for long-read RNA sequencing, it was found necessary to use 2 μg of cDNA.

[0631] The DSN method was found to completely eliminate high-abundance RNA, and this was expected to be the case for RNAs with sequence similarity to high-abundance RNA. Therefore, over-depletion was observed, where high-abundance RNA was not only reduced in quantity but also completely removed from the sample. Table 1 shows the over-depletion levels 1 through 20, and also shows selections of lower levels (55, 64, 77, 92, and 98) where RNA was significantly reduced but not completely depleted.

[0632] Table 1 - Over-exhaustion of high-abundance RNA using the DSN method

[0633]

[0634]

[0635] In addition, the DSN method creates conditions that can generate artificial chimeric sequences, which can manifest as false positives in gene prediction.

[0636] Selective amplification method (Level-up)

[0637] This method is in Figures 1 to 3 As shown in the diagram. In general, refer to... Figure 2 The process involves denaturing a cDNA sample containing cDNA with known 5' and 3' pre-attached adapters to provide a single-stranded cDNA sample; the sample is then rehybridized or reassociated to provide a mixture of single-stranded and double-stranded cDNA. This single-stranded cDNA represents low-abundance cDNA. The single-stranded cDNA in the reassociated sample is then modified by adding oligonucleotides to the 5' and 3' pre-attached adapters. The cDNA sample is amplified using primers targeting these oligonucleotides. This process selectively increases the amount of single-stranded cDNA alone in the reassociated sample, thereby increasing the amount of low-abundance cDNA in the sample and providing a standardized sample.

[0638] The input cDNA library used in the selective amplification method is double-stranded, and each double-stranded template contains a 5' pre-attached adapter with a known nucleotide sequence and a 3' pre-attached adapter with a known nucleotide sequence. Because the method of this invention is an addition method, it requires a lower amount of starting cDNA compared to standardized methods in the prior art. In the DSNase method, a minimum of 1 μg of input cDNA is required, while in the column method, a minimum of 4 μg of input cDNA is required. In testing, it was found that the method of this invention can use as little as 20 ng of starting cDNA.

[0639] The input cDNA is combined with hybridization buffer, and the solution is heated to a denaturation temperature—approximately 98 degrees Celsius—to produce a denatured single-stranded cDNA template. After 5 to 10 minutes, the solution is then lowered to a rehybridization temperature—approximately 68 degrees Celsius. Depending on the desired normalization amount, the solution is incubated at this temperature for 0 to 24 hours, for example, 3 to 10 hours. Seven hours is a typical duration for the reassociation step. This step produces a reassociated or rehybridized sample containing both a post-association double-stranded cDNA template and a post-association single-stranded cDNA template.

[0640] Following incubation, the oligonucleotide dimers of the present invention (referred to as K-linkers) are added. These oligonucleotide dimers are discussed in more detail below. At this point, the solution can be incubated at 68°C for 0 to 1 hour. Five minutes is a typical duration for this incubation step. The solution is then lowered to the annealing temperature of the K-linkers, which is typically between 40 and 60°C, for example, 44°C. The solution is incubated at this temperature for 10 minutes to 2 hours, for example, 25 minutes. This step anneals the K-linkers to the associated single-stranded cDNA template.

[0641] Following this incubation period, DNA ligase is added along with the ligation mixture. The solution is incubated at the same temperature for 0.5 to 2 hours, for example, 1 hour, and then cooled to room temperature. This step results in the formation of the ligation adaptor-cDNA template, in which oligonucleotides from the K-adaptor are ligated to each end of the associated single-stranded cDNA template. At this point, the cDNA can be purified (e.g., using Pronex or Ampure beads) or can be used directly for PCR amplification using primers based on the K-adaptor sequence.

[0642] After PCR amplification, the cDNA is then purified using any suitable tool, and the resulting cDNA represents a standardized cDNA library.

[0643] Before PCR amplification, the associated double-stranded cDNA template can be removed, but after testing, the inventors have shown that leaving the double-stranded cDNA in solution does not negatively affect the standardization process.

[0644] In practice, the post-associative double-stranded cDNA can also be analyzed and used, for example, to obtain estimates of gene expression. This involves an additional (PCR) selective amplification step using primers targeting known 5' and 3' pre-attached adaptors, where molecular barcoding is contained within the primers. PCR amplification is first performed using primers based on the K-adaptor sequence, followed by a single PCR cycle using primers targeting known 5' and 3' pre-attached adaptors. PCR can be paused to add additional primers for the final cycle. Alternatively, the cDNA can be purified using primers based on the K-adaptor sequence after PCR, and a new PCR cycle can be performed using primers targeting known 5' and 3' pre-attached adaptors. In both cases, the product will be a mixture of two distinguishable portions consisting of sequences derived from the post-associative double-stranded cDNA template and sequences derived from the post-associative single-stranded cDNA template. The addition of molecular barcoding allows for the identification of the source molecule during sequencing analysis. This aspect of the invention can be used for multiplex sequencing.

[0645] Design of oligonucleotide dimer complexes

[0646] The oligonucleotide dimer composition of the present invention comprises a pre-oligonucleotide dimer and a post-oligonucleotide dimer (pre-K-linker and post-K-linker), both of which are annealed to the same strand of the associated single-stranded cDNA.

[0647] Reference Figure 3 and 3A Each K-linker contains two oligonucleotide sequences; one is called a linker oligonucleotide (also known as a "linker"); in Figure 3 and 3A One is called the "LU adaptor linker" in Chinese, and the other is called lig-oligonucleotide (also known as "lig"). Figure 3 and 3A This is referred to as the "LU connector" in Chinese.

[0648] The linker sequence contains regions complementary to the known 3' / 5' pre-attached linker sequence (which was previously added to cDNA) and regions complementary to lig.

[0649] If still Figure 3 and 3A As described, the linker can be designed with a protruding region at one end that is non-complementary to a known pre-attached linker sequence; this region is called a "template protrusion." The opposite end of the linker can have a similar protrusion that is non-complementary to the lig sequence; this is called a "lig protrusion" or "lig-oligonucleotide protrusion."

[0650] The purpose of the linker is to anneal with both the pre-attached linker and the LIG (ligand) of the associated single-stranded cDNA template in such a way that, during use, one end of the cDNA template is adjacent to one end of the LIG. By positioning the cDNA template and LIG in this manner, DNA ligase can be used to ligate the cDNA template to the LIG, thereby adding the LIG sequence to the end of the cDNA template. The LIG sequence is added to both the 5' and 3' ends of the single-stranded cDNA template. Pre- and post-K-linkers are used to add these LIGs to the 5' and 3' ends of the cDNA template, respectively.

[0651] In the method described above, once lig sequences have been added to each end of the single-stranded cDNA template, primers based on the added lig sequences are used to selectively amplify the single-stranded cDNA portions that have been successfully linked to both the pre-lig and post-lig sequences. In this way, PCR can be used to amplify only the low-abundance associated single-stranded cDNA portions.

[0652] The specific structure of the K-connector provides an advantage in overall standardized performance, with the front and rear K-connectors offering different functions provided by their specific structural features.

[0653] A pre-K-linker (shown as the inverse complementary sequence of the original RNA sequence) that binds to the 5' end of a single-stranded cDNA template can be designed so that the K-linker does not act as a primer during PCR amplification.

[0654] To provide additional advantages, the pre-K-adaptor lacks a blunt end on the ligature side to allow the use of DNA ligases that can perform blunt-end ligation during selective amplification. Providing the ligature side of the K-adaptor complex with a non-blunt end also avoids ligation with other K-adaptor complexes or with double-stranded cDNA in solution.

[0655] Because the pre-ligand has a 5' to 3' orientation pointing away from the template, the adapter itself cannot act as a primer for the template. However, in some cases, the pre-lig or PCR primer can potentially anneal with the adapter during PCR amplification and undergo polymerase extension to present the template-side sequence of the linker. Providing a linker with a protruding end on the template side avoids the extended lig / primer acting as a primer without a cDNA sequence (i.e., a highly abundant sequence) to which the lig sequence is added to its end. Therefore, a template protruding end structure can be provided for the post-linker.

[0656] A post-K-connector (shown as the inverse complementary sequence of the original RNA sequence in the figure) that binds to the 3' end of a single-stranded cDNA template can be designed so that the K-connector does not act as a primer during PCR amplification. This can be achieved by using a template overhang, which, as described above, and Figure 3 and 3AAs shown in the image.

[0657] For the same reason that the proximal K-linker can be designed to have a protruding end on the lig side, the posterior K-linker can be provided without a blunt end on the lig side. If the posterior K-linker complex has a blunt end on the lig side, in some cases, the lig side may potentially ligate to other K-linker complexes or to double-stranded cDNA in the sample, which could lead to cDNA template ligation.

[0658] The overhang also serves to lower the annealing temperature of the K-connector complex, making it less likely to act as a primer for each other during PCR amplification using higher annealing temperatures. The overhang reduces unintended priming. As a supplement or alternative, the overhang can provide an indicator that allows measurement of the amount of unintended priming. For example, if a K-connector complex with its overhang is able to primate the template, the overhang sequence will be visible in the sequencing data, and it can be inferred that it is the product of unintended priming.

[0659] Ideally, all protrusions should be from about 1 bp to about 20 bp, preferably 3 bp. Longer protrusions can be used, but this will make the design more difficult because as the protrusions become longer, there are fewer sequence compositions to prevent unintended initiation.

[0660] The complementary regions between the cDNA template and the linker, and between the linker and the ligase, should be long enough to allow annealing at the activity temperature of the ligase to be used. Gap-repair ligases are particularly preferred for this process, typically requiring about five or more complementary bases on either side of the ligation site.

[0661] The combined length of the linker and the corresponding ligand should ideally be less than about 300 bp, preferably less than about 200 bp, to reduce carryover during the purification process.

[0662] Exemplary sequence representation:

[0663] All exemplary sequence structures are provided in the 5' to 3' orientation. Also... Figure 3B As shown in the image.

[0664] Pre-connector:

[0665]

[0666] front lig:

[0667]

[0668] Rear connector:

[0669]

[0670] After lig:

[0671]

[0672] X - A nucleotide complementary to the 5' / 3' pre-attached adaptor

[0673] O - Protruding end sequence

[0674] F - The sequence complementary to the preceding ligature to be joined.

[0675] F-prelig sequence

[0676] B - The sequence complementary to the postligature to be joined

[0677] B-postlig sequences

[0678] Examples of oligonucleotides and single-stranded cDNA templates used in this selective amplification method:

[0679] Primer sequences from the NEB / PacBio cDNA synthesis kit

[0680] Iso-Seq expression Fwd:

[0681]

[0682] Iso-Seq expression Rev:

[0683]

[0684] Pre-connector:

[0685]

[0686] Pre-Lig and primers:

[0687]

[0688] Rear connector:

[0689]

[0690] After Lig:

[0691]

[0692] Primers:

[0693]

[0694] Protrusions are indicated by underlining. Complementary regions between oligonucleotides are indicated by bold.

[0695] Single-stranded cDNA template:

[0696] (i.e., 5'-3': the sequence of the 5' pre-attached adaptor, the sequence of the cDNA represented by N, and the sequence of the 3' pre-attached adaptor; regions complementary to the pre-joint oligonucleotide and post-joint oligonucleotide are shown in bold).

[0697] Preparation of oligonucleotide dimers

[0698] Once the design is complete, the dimer can be prepared using standard techniques known in the field.

[0699] Amplification of oligonucleotide-cDNA template

[0700] After ligating the lig to the 3' and 5' ends of the cDNA template, the resulting cDNA solution can be purified using a suitable cDNA purification method. The purification step can be skipped, but skipping it may reduce the efficiency of PCR amplification.

[0701] After purification or ligation, the resulting material can be amplified by PCR using forward and reverse primers based on the LIG sequence. Primer sequences with higher annealing temperatures than the complementary regions of the template / linker / LIG can be selected to avoid unwanted priming.

[0702] If necessary, the optimal number of PCR cycles can be determined by first performing qPCR experiments to identify the inflection point of the amplification curve.

[0703] The cDNA obtained from PCR amplification can then be purified and used as input for any downstream process, such as sequencing.

[0704] Validation of selectively amplified samples

[0705] The effects of selective amplification or normalization can be measured indirectly by using gel electrophoresis to measure the length distribution of a cDNA library, or directly by using sequencing.

[0706] To assess the role of normalization using gel electrophoresis, the length distribution of the input cDNA can be compared to that of the normalized cDNA. The input cDNA will typically show peaks along its length distribution, corresponding to high-abundance transcript sequences (…). Figure 4 ).

[0707] Standardized cDNA will have a length distribution similar to a normal distribution without spikes. Figure 5 This represents a uniform distribution throughout the transcript sequence.

[0708] When using sequencing for direct measurement standardization, long-read sequencing is the preferred sequencing method. This allows for the identification of different isotypes. The results of direct measurement can be a graph of the number of reads per gene or a saturation graph showing the number of new genes identified as sequencing depth increases.

[0709] To verify the method of this invention, the inventors performed nanopore cDNA sequencing on both the input cDNA library and the cDNA library generated by the method of this invention. The saturation curves of each library were compared. Figure 6 The curve obtained by the method of this invention is many times higher than that obtained by inputting cDNA. This indicates that the cDNA library is more uniformly distributed.

[0710] Applications of selective amplification

[0711] The primary application of the selective amplification method of this invention is to improve the discovery and detection of low-abundance genes and isotypes. When combined with sequencing, the sampling efficiency for identifying all unique genes in a sample is improved.

[0712] The method of this invention can be applied to any double-stranded cDNA library with known adaptors at the ends and whose length can be amplified by PCR. This means that it can be used for DNA sequencing.

[0713] Another important aspect of the method of the present invention is the ability to select cDNAs with known LG sequences. This aspect can be applied to single-cell sequencing, where adaptors are necessary to allocate reads to a single cell. In this application, cDNA libraries contain cDNA sequences without cell identifier barcodes / adaptors. These are called template-changing oligonucleotide (TSO) artifacts and are undesirable in single-cell sequencing projects because they cannot be allocated to the cells of origin. The method of the present invention can be applied to a short rehybridization step to select only cDNA sequences with the desired LG sequences, thereby effectively limiting the sequencing of TSO artifacts. The method of the present invention can also be performed with single-cell cDNA libraries to both remove TSO artifacts and improve transcriptome coverage per cell.

[0714] discuss

[0715] The selective cDNA amplification method of this invention represents an innovative approach to achieving greater non-targeted discovery / detection of low-abundance nucleic acids. Because it is non-targeted, there is no need to know which sequences in a given sample are low- or high-abundance.

[0716] The difference between this invention and existing standardized methods lies in the use of an additive method, while current methods use an exhaustion method. The additive method allows the method of this invention to use significantly less starting cDNA. It facilitates the detection of low-abundance nucleic acids and provides greater confidence in determining the absence of specific nucleic acids. The additive method also prevents excessive exhaustion and artificial chimerism (characteristic of the DSNase method). The method of this invention also exhibits length deviation only to the extent that PCR amplification has length deviation. Therefore, it can be successfully run with longer cDNA molecules.

[0717] Exemplary workflow for identifying cancer treatment targets

[0718] Whole blood collected from cancer patients was placed in special blood tubes designed to preserve RNA integrity. Full-length RNA was then extracted from the whole blood sample. The RNA was converted into cDNA using a commercially available kit via cDNA synthesis. The resulting cDNA library was normalized (e.g., using Level-Up as described herein). The normalized cDNA library was then sequenced using a long-read sequencing platform. The resulting long-read RNA sequencing data was then analyzed to identify all unique RNA transcripts from the original sample. The RNA sequences were then converted into possible peptide sequences that could be translated from the RNA (i.e., the three possible open reading frames (ORFs)). Figure 10 These are complete translations, from the first to the last codon, with no start codon selection.

[0719] The resulting peptide sequence is then divided into k-mers (6 to 25 amino acids in length, k = peptide length). Overlapping k-mers are... Figure 11 As shown in the diagram, a database of these k-mers was created, where each k-mer represents a potential target. A count of the frequency of each k-mer in the data was also included. K-mers from cancer patients were compared with k-mers present in controls and / or other data sources to identify k-mers that are present only in cancer patients or highly enriched in cancer patients.

[0720] The resulting cancer-specific k-mers represent potential neoantigen targets, targets for antibody-drug conjugates, and targets for radiopharmaceuticals. Additional data from public sources were used to annotate the k-mers and their source genes to identify and rank them based on their suitability as cancer therapeutic targets. For example, for neoantigen targets, k-mers were compared with guidelines on MHC presentation characteristics (such as those described in Shao XM, Bhattacharya R, Huang J, SivakumarIKA, Tokheim C, Zheng L, Hirsch D, Kaminow B, Omdahl A, Bonsack M, Riemer AB, Velculescu VE, Anagnostou V, Pagel KA, Karchin R. Cancer Immunol Res. 2020Mar;8(3):396-408. doi: 10.1158 / 2326-6066.CIR-19-0464. Epub 2019 Dec 23. PMID:31871119; PMCID: PMC7056596, which are incorporated herein by reference) to rank k-mers based on their likelihood of being presented by the MHC and recognized by immune cells. Examine the k-mer to see if it is present in the translation of the corresponding RNA transcript produced using various protein translation tools (such as SQANTI).

[0721] Novel cancer-specific exons can encode peptide sequences that are already present in the control proteome of another gene. This phenomenon is known in gene paralogs, where similar transcript sequences exist at different loci in the genome, producing very similar proteins. However, by observing novel sequences in the context of peptide k-mers, it is possible to verify that the specific sequence is indeed absent in the control proteome.

[0722] The exemplary systems, methods, and actions described in the previously presented embodiments are illustrative, and in some alternative embodiments, without departing from the scope and spirit of the various embodiments, certain actions may be performed in a different order, in parallel with each other, omitted entirely, and / or combined among different example embodiments, and / or certain additional actions may be performed. Therefore, such alternative embodiments are included in the embodiments described herein.

[0723] Although specific implementation schemes have been described in detail above, this description is for illustrative purposes only. Therefore, it should be understood that, unless otherwise explicitly stated, many of the aspects described above are not intended to be essential or necessary elements.

[0724] In addition to those described above, those skilled in the art who benefit from this disclosure may modify the disclosed aspects of the exemplary embodiments and make equivalent components or actions corresponding to the disclosed aspects of the exemplary embodiments without departing from the spirit and scope of the embodiments defined in the appended claims, the scope of which is to be interpreted in the broadest sense to cover such modifications and equivalent structures.

[0725] Example

[0726] The invention will be further understood by referring to the following experimental examples.

[0727] Example 1

[0728] Using a novel transcriptome discovery platform to detect unique RNA signatures in epithelial cancer.

[0729] The inventors have validated the use of level-up RNA from cancer cell lines and blood samples. These preliminary studies have identified thousands of previously unannotated isotypes that have the potential to be used as cancer biomarkers. Figure 7 Clustering is shown based on 13,573 transcripts identified in these initial studies in cancer samples (blood samples from people diagnosed with breast cancer) and control samples (blood samples from people not diagnosed with breast cancer). Cancer samples can be easily distinguished from control samples.

[0730] An exemplary scheme is provided for discovering unique RNA signatures in epithelial cancer:

[0731] Phase 1

[0732] Blood processing validation was performed using 5 samples from 5 individuals (25 samples in total). Blood was collected and stored in PAXgene RNA blood tubes (2.5 ml per tube). After collection, the tubes were transported from the local collection center on the same day using dry ice.

[0733] For each biological replicate, five collected tubes were processed according to different simulated treatment scenarios. These included:

[0734] 1. Collect and process the tube on the same day.

[0735] 2. Process the tubes 72 hours after collection.

[0736] 3. Process the tubes two weeks after collection.

[0737] 4. Process the tubes 4 weeks after collection.

[0738] 5. Store the tubes at -80°C for extended periods and process them within 4 to 12 months.

[0739] RNA was extracted from PAXgene tubes using the Qiagen PAXgene Blood RNA Kit (catalog number / ID: 762164). The resulting RNA was converted into cDNA using the NEBNext® Single Cell / Low Input cDNA Synthesis & Amplification Module (E6421S). The resulting cDNA was normalized as described herein. The normalized cDNA library was then sequenced using an Oxford Nanopore Technologies Minion MK1C sequencer.

[0740] Phase 2

[0741] Sample processing was optimized and the quality of the generated data was ensured before the study proceeded to patient samples.

[0742] Inclusion and Exclusion Criteria

[0743] Inclusion criteria: All premenopausal women aged 18 years and older but under 50 years of age with invasive breast cancer or benign conditions (B2).

[0744] Exclusion criteria: Women with any history of cancer or other types of cancer, autoimmune diseases, or who are unable or unwilling to give informed consent.

[0745] Patient identification and recruitment for prospective cohort studies

[0746] Based on the results of the triple assessment, eligible patients are identified from multidisciplinary team (MDT) meetings. When patients review the results with their breast surgeon at the clinic, they are invited to participate in the study and given a patient information sheet (PIS).

[0747] The research team will follow up with patients who received PIS 72 hours later. If patients choose to participate in the study, they will meet with the research nurse and sign an informed consent form, or give informed consent remotely.

[0748] Anonymization

[0749] Once a patient is enrolled in the study, the research nurse assigns a unique ID number that is linked to background data through a secure database and used for sample collection and identification.

[0750] Background data collection

[0751] For each patient, the following data were collected: baseline demographics, menopausal status, medical history, medication history, drug and alcohol intake, BMI, and family medical history.

[0752] A triple assessment, including presentation, examination results, imaging, and biopsy results, was compiled. Postoperative histology was also collected. Any additional prognostic information, such as the use of biomolecular assays, was also collected.

[0753] Blood sample collection

[0754] On the morning of the surgery, blood samples are obtained and stored at -20°C before being transported to the processing center. For any patients not undergoing surgery, such as those with fibroadenomas, they are invited to the day unit where the same team performs blood tests. Sample volumes are 20 ml, with a maximum of 30 ml collected on the same day.

[0755] Blood sample processing

[0756] Blood samples from collection tubes were processed using the Qiagen RNA extraction kit in a laminar flow Class 2 biosafety cabinet. After RNA extraction, any remaining solid waste was autoclaved and disposed of by a human medical waste disposal service provider. Liquid waste was cleaned and disposed of in accordance with the University of Edinburgh Guideline on the use of human samples. Blood samples not processed on the day of arrival were stored in a secure -80°C freezer. Blood samples were transported between the freezer and biosafety cabinet using sealed containers.

[0757] Data Analysis

[0758] Transcriptomics data is stored and processed in secure Amazon Web Services (AWS) cloud repositories and servers. No personal information is stored in the same computing location.

[0759] An Oxford Nanopore Technologies (ONT) Minion sequencing machine was used to perform cDNA sequencing on the samples. This output the raw data as a FAST5 file. The latest high-precision basecaller (Bonito) from ONT was run to convert the data into FASTQ sequence files. The nanopore reads were quality filtered using seqkit, and adaptors were removed using pychopper. The pruned reads were then aligned to the HG38 (or later) human reference genome assembly using Minimap2.

[0760] Phase 3 to 4

[0761] The study was modified to initially increase the sample size and was subsequently expanded to include colorectal cancer and ovarian cancer.

[0762] Statistical considerations and sample size

[0763] For the first phase of the study, five samples were processed. This was sufficient to generate technical and biological replicates and to conduct an initial quality assessment.

[0764] Data processing and manipulation are carried out as in Phase 2.

[0765] For Phase 2 of the study, 30 samples were initially collected and processed from patients with benign conditions and 30 samples from patients with cancer. This data will inform the subsequent sample volumes for Phases 3 and 4 of the study.

[0766] Ethical Approval

[0767] Seek ethical approval from the National Research Ethics Service before starting the research.

[0768] Informed consent process

[0769] Written informed consent was obtained from all patients participating in this study to collect their demographic and clinical data for subsequent storage of blood samples, transcriptomic materials, and data. Patients may also give consent by telephone if they choose to do so. Patients may withdraw their consent at any time. Anyone deemed unable to give informed consent was excluded from this study.

[0770] Quality control

[0771] To assess whether samples were correctly labeled and whether there were any processing issues in each sample, a computer-based quality control test was performed. This test consisted of checking all samples for known genes that should be present and performing cluster analysis to assess outliers that might indicate sample contamination.

[0772] They also look for genes in the sample data that shouldn't be there. For example, genes from other species.

[0773] Example 2

[0774] Blood sample collection procedure for full-length RNA extraction

[0775] cDNA is DNA synthesized from an RNA template. Therefore, the quality of a cDNA sample is related to the RNA derived from that cDNA sample via reverse transcription. Existing methods for processing blood samples prior to RNA extraction involve overnight thawing of frozen blood samples. The inventors have found that this leads to significant RNA degradation, which negatively impacts long-read sequencing. The inventors use the following protocol to process blood samples prior to RNA extraction to minimize degradation and optimize RNA extraction for long-read sequencing.

[0776] Blood sample collection procedure for full-length RNA extraction

[0777] At room temperature (18°C to 25°C), draw 2.5 ml of blood into a PAXgene blood RNA tube. Immediately after blood collection, gently invert the blood tube 10 times.

[0778] If RNA is to be extracted on the same day the sample is collected, store the blood sample upright at room temperature (18°C to 25°C) for 2 to 3 hours, and then immediately proceed with RNA extraction using the PAXgene Blood RNA Kit.

[0779] If RNA extraction is not performed on the day of blood collection, follow these instructions regarding sample freezing, storage, and thawing:

[0780] Blood samples should be stored at -20°C or below immediately after collection. For long-term storage, freeze blood samples at -20°C for 24 hours, then transfer them to a freezer at -70°C or -80°C.

[0781] If the samples need to be transferred to different locations, they should be shipped with dry ice to ensure they remain frozen during transport.

[0782] On the day of RNA extraction, thaw the blood sample by placing the sample tube upright on a support and incubating at room temperature (18°C to 25°C) for 1 to 3 hours. Once the blood is completely thawed, gently invert the sample tube 10 times and incubate at room temperature for another 2 hours, then immediately use the PAXgene Blood RNA Kit for RNA extraction.

[0783] As shown in Table 2, a shorter thawing time (3 hours) improved RNA integrity compared to overnight thawing. The RNA integrity number was calculated according to Schroeder et al. (The RIN: an RNA integrity number for assigning integrity values ​​to RNA measurements. BMC Molecular Biology 7, 3 (2006). https: / / doi.org / 10.1186 / 1471-2199-7-3.). Samples were placed at -20°C for 12 hours for the “-20°C 2 weeks; overnight thawing” and “-20°C 1 month; 3-hour thawing” tests (second and third tests). Five individuals were used for each test. All thawing was performed at room temperature.

[0784] Table 2 - Blood Sample Storage Conditions and RNA Integrity

[0785]

[0786] The scope of this invention is not limited to the specific embodiments described herein. In fact, various modifications to the invention (in addition to those described herein) will become apparent to those skilled in the art from the foregoing description and drawings. Such modifications are intended to fall within the scope of the appended claims. Furthermore, all embodiments described herein are considered broadly applicable and can be suitably combined with any and all other consistent embodiments.

[0787] This article cites multiple publications, the published content of which is incorporated in its entirety through citation.

Claims

1. A method for generating an RNA vaccine for a subject suffering from a disease, the method comprising: (i) Provide an RNA sample extracted from a blood sample obtained from the subject; (ii) Processing the RNA sample, optionally including using the RNA as a template to synthesize cDNA; (iii) Sequencing the processed RNA or cDNA, wherein the sequencing output is used to identify the presence of RNA transcript sequences associated with the disease; as well as (iv) Use the identified RNA transcript sequence to generate a first RNA vaccine against the subject.

2. The method of claim 1, further comprising: (v) Provide additional RNA samples extracted from blood samples obtained from the subject at a later time point; (vi) Processing the RNA sample, optionally including using the RNA as a template to synthesize cDNA; (vii) Sequencing the processed RNA or cDNA, wherein the sequencing output is used to identify the presence of RNA transcript sequences associated with the said disease, and (viii) Use the identified RNA transcript sequence to generate an additional RNA vaccine against the subject.

3. The method of claim 1 or claim 2, wherein using the identified RNA transcript sequence to generate an RNA vaccine comprises generating an RNA molecule containing at least a portion of the sequence, wherein the RNA molecule contains an open reading frame (ORF) encoding at least one antigenic peptide.

4. The method of claim 3, wherein the RNA molecule further comprises a 5' UTR, a 3' UTR, a polyA tail, and / or a 5' cap.

5. A method for generating a database of disease biomarkers, the method comprising: (i) Provide two or more RNA samples extracted from blood samples obtained from one or more subjects suffering from the disease; (ii) Processing the RNA sample, optionally including using the RNA as a template to synthesize cDNA; (iii) Sequencing of processed RNA or cDNA, wherein the sequencing output is used to identify two or more disease biomarkers; and (iv) Compile the aforementioned disease biomarkers to form a database.

6. Methods for detecting biomarkers of disease, including: (i) Provide a first RNA sample obtained from a subject suffering from the disease and a second RNA sample obtained from a subject not suffering from the disease; (ii) Processing the first RNA sample and the second RNA sample, optionally including using the RNA as a template to synthesize cDNA; (iii) Sequencing the processed RNA or cDNA; as well as (iv) Comparing the sequencing outputs of the first sample and the second sample to discover disease biomarkers, wherein comparing the sequencing outputs of the first sample and the second sample includes analyzing the sequencing outputs by: (i) Determine one or more amino acid sequences corresponding to the RNA or cDNA sequences from the sequencing outputs of the first sample and the second sample; (ii) Dividing the one or more amino acid sequences into multiple segments of a defined length; as well as (iii) Compare multiple segments from the first sample with multiple segments from the second sample to identify segments present in one sample but absent in the other, and / or identify segments whose levels differ between the samples.

7. A method for diagnosing autoimmune diseases in subjects, comprising: (i) Provide an RNA sample extracted from a blood sample obtained from the subject; (ii) Processing the RNA sample, optionally including using the RNA as a template to synthesize cDNA; and (iii) Sequencing the processed RNA or cDNA, wherein the sequencing output is used to identify whether the subject has the disease.

8. The method of claim 7, wherein the sequencing output is used to identify whether the subject has the disease by comparison with a database of biomarkers, optionally wherein the database of biomarkers is generated using the method of claim 5.

9. A method for analyzing nucleic acid samples, the method comprising: (i) Provide an RNA sample extracted from a blood sample obtained from the subject; (ii) Processing the RNA sample, optionally including using the RNA as a template to synthesize cDNA; (iii) Sequencing the processed RNA or cDNA; (iv) Determine one or more amino acid sequences corresponding to the RNA or cDNA sequences derived from sequencing output; and (v) Divide the amino acid sequence into multiple segments of a defined length.

10. The method of any of the preceding claims, wherein processing the RNA sample includes reducing the variability of different RNA or cDNA sequence levels in the sample.

11. The method of any of the preceding claims, wherein the relative abundance of all unique RNA or cDNA sequences is more uniform in the treated RNA or cDNA.

12. The method of any of the preceding claims, wherein processing the RNA sample improves the detection of low-abundance RNA or cDNA, optionally wherein processing the RNA sample includes increasing the amount of low-abundance RNA or cDNA in each sample.

13. The method of any of the preceding claims, wherein the RNA is full-length RNA and / or wherein sequencing includes sequencing using long reads.

14. The method of any one of claims 7, 8, or 10 to 13, wherein the autoimmune disease is arthritis, celiac disease, type 1 diabetes, Graves' disease, inflammatory bowel disease, multiple sclerosis, alopecia areata, Addison's disease, pernicious anemia, psoriasis, systemic lupus erythematosus, myasthenia gravis, Hashimoto's thyroiditis, vitiligo, Sjögren's syndrome, myositis, chronic inflammatory demyelinating polyneuropathy (CIDP), dermatomyositis, Guillain-Barré syndrome, ulcerative colitis, and / or vasculitis.

15. The method of any one of claims 1 to 4 or 10 to 14, wherein using the sequencing output to identify the presence of a sequence associated with the disease comprises identifying an RNA transcript encoding a protein isotype present in a subject suffering from the disease.

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