Methods for selecting and combining sequencing results from a biological sample for neoantigen scoring

Abstract

Disclosed herein are methods of scoring the predicted immunogenicity of neoantigens from a biological sample from a subject. The methods can include the steps of preparing a biological sample for nucleic acid sequencing; nucleic acid sequencing; evaluating the initial sequencing results by analyzing (e.g., comparing) sequencing parameters of the results; combining the initial sequencing results to produce a joint sequencing result or selecting a representative biological sample based on the analysis (e.g., comparison) of sequencing parameters; and scoring the predicted immunogenicity of neoantigens in the biological sample based on the joint sequencing result or the sequencing results of the representative sample. The methods can also include the step of comparing sequencing parameters of the joint sequencing result and the initial sequencing results. The methods can also include the steps of producing a neoantigen vaccine comprising or encoding neoantigens scored for predicted immunogenicity and administering the neoantigen vaccine to the subject.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Application No. 18 / 542,383, filed December 15, 2023, the entire contents of which are incorporated herein by reference.

[0003] References to sequence lists

[0004] This application contains a sequence list that has been electronically submitted in XML format. The sequence list XML is incorporated herein by reference. The XML file was created on December 29, 2023, named 146401_093445_SL.xml, and has a size of 16,544 bytes. Background Technology

[0005] Since the Human Genome Project sequenced and assembled the first human genome in 2003, clinicians and scientists have envisioned treating a range of diseases through personalized genetic diagnostics and therapies. Cancer, a disease projected to cause an estimated 609,823 deaths in the United States by 2023 (see RL Siegel et al., Cancer statistics, 2023. CA Cancer J. Clin. (2023) 73, 17-48), is particularly well-suited for gene sequencing-based personalized genetic diagnostics and therapies because cancer originates from somatic mutations over time in the genome of a patient’s non-cancerous or precancerous cells (see ED Pleasance et al., A comprehensive catalogue of somatic mutations from a human cancer genome. Nature (2010), 463, 191-196; and MR Stratton et al., The cancer genome. Nature (2009), 458 (7239), 719-724).

[0006] Cancerous malignancies (e.g., solid tumors) consist of a heterogeneous mixture of subclones derived from common clonal cells. This is a result of selection, drift, and spatial segregation of the intratumoral clonal population (see M. Tarabichi et al., *Apractical guide to cancer subclonal reconstruction from DNA sequencing*, *Nat. Methods* (2021), 18(2), 144-155). Mutations present in different subclones are important for cancer diagnosis and treatment because they can confer resistance to chemotherapy agents or render personalized immunotherapies (e.g., CAR T-cells, neoantigen vaccines) ineffective (see DJ Craig et al., *Subclonal landscape of cancer drives resistance to immune therapy*, *Cancer Treat. Res. Commun.* (2022), 30, 100507, 1-6).

[0007] Unfortunately, tumor heterogeneity presents challenges to obtaining biological samples (e.g., tissue biopsies) that represent the genomic variants present throughout the tumor. Assessing the heterogeneity of genomic variants of nucleic acids from biological samples via sequencing is typically costly and time-consuming, requiring exhaustive sequencing of samples that may inevitably fail to represent the tumor and provide a representative basis for informing patients about treatment and / or developing personalized therapies. Collecting more, more representative samples through additional procedures is inconvenient for patients and carries additional risks of infection or other surgical complications. Therefore, there is an unmet need for methods to efficiently obtain sequencing results that represent tumor heterogeneity for ranking predictive immunogenicity and selecting neoantigens for patient treatment (e.g., generating neoantigen vaccines to inform clinicians of the most effective treatment for a patient's heterogeneous cancerous malignancy).

[0008] The heterogeneity of tumor somatic mutations and the spatial separation of related but distinct subclonal populations within tumors make it difficult to ensure that biological samples (e.g., tissue biopsy samples) represent the majority of subclonal populations. Samples representing genomic variations in tumor subpopulations are important for whole-cancer genomic analysis, diagnosis of driver mutations in cancer, selection of appropriate chemotherapy and immunotherapy agents for cancer treatment, and the development of personalized therapies (e.g., cell therapy, neoantigen vaccines). One solution to this problem is to collect multiple biological samples (e.g., tissue biopsy samples) from the patient (e.g., collected during surgery) to identify samples representing tumor subpopulations. Unfortunately, identifying and selecting representative biological samples (e.g., tissue biopsy samples) using conventional methods requires expensive, time-consuming, and exhaustive sequencing of each candidate biological sample (e.g., tissue sample).

[0009] This article describes methods for scoring (e.g., ranking, evaluating) the predictive immunogenicity of neoantigens in one or more biological samples from a subject in need. These methods may include the following steps: preparing biological samples (e.g., two or more biological samples) for nucleic acid sequencing; performing nucleic acid sequencing on the biological samples to produce initial sequencing results for each of the biological samples; evaluating the initial sequencing results by analyzing (e.g., comparing) sequencing parameters (e.g., one or more sequencing parameters) of the initial sequencing results; combining the initial sequencing results of the biological samples to produce combined sequencing results based on the analysis (e.g., comparison) of the sequencing parameters of the initial sequencing results of the biological samples; and scoring the predictive immunogenicity of neoantigens (e.g., one or more neoantigens) in the biological samples of a subject in need based on the combined sequencing results of the biological samples. The method may include the following steps: preparing biological samples (e.g., two or more biological samples) for nucleic acid sequencing; performing nucleic acid sequencing on the biological samples to produce initial sequencing results for each biological sample; evaluating the initial sequencing results by analyzing (e.g., comparing) sequencing parameters of the initial sequencing results; selecting representative biological samples based on the analysis (e.g., comparison) of sequencing parameters of the initial sequencing results of the biological samples; and scoring the predicted immunogenicity of neoantigens in representative samples of subjects in need based on the initial sequencing results of the representative biological samples.

[0010] The biological samples used in the methods described herein can be any biological sample from the subject. Each biological sample can be independently an excisional biopsy, liquid biopsy, incisional biopsy, needle aspiration biopsy, perforation biopsy, or curettage biopsy. The biological sample can originate from any region of the subject's body (e.g., any region of the body, any region of a tumor, any region of an organ). At least two of the biological samples can originate from different regions of the subject's body. At least two of the biological samples can originate from different regions of the subject's tumor. The biological sample can originate from any tissue or organ of the subject. The biological sample can originate from any type of tumor. The biological sample (e.g., at least one of the biological samples) can originate from the subject's primary tumor. The biological sample (e.g., at least one of the biological samples) can originate from the subject's secondary (e.g., metastatic) tumor.

[0011] Biological samples can be collected at different times, and the duration of time between the collection of each sample is arbitrary. Biological samples can be collected from the subject at different times that are at least about one day apart (e.g., at least two biological samples).

[0012] The sequencing parameters used in the methods described herein (e.g., combined sequencing results, subsequent sequencing results, initial sequencing results) can be any type of sequencing parameter. Sequencing parameters can be the number of reads, the number of sequencing variants, tumor purity, sequencing depth, the number of protein-modified sequencing variants, RNA confirmation rate, RNA quality, the number of identified long neoantigen peptides, the number of identified short neoantigen peptides, the predicted immunogenicity of the neoantigen peptide, or any combination thereof.

[0013] Any type of sequencing result (e.g., initial sequencing results, combined sequencing results, sequencing results of representative biological samples) can be obtained and used in the methods described herein. Sequencing results (e.g., initial sequencing results) can be sequencing reads, sequencing variants, encoded peptides, or combinations thereof.

[0014] The method described herein may also include a step of comparing the co-sequencing parameters of the co-sequencing results (e.g., results obtained by combining initial sequencing results of biological samples) with the sequencing parameters of the initial sequencing results.

[0015] The nucleic acid sequencing described herein can be the sequencing of any type of nucleic acid. The type of nucleic acid can be RNA, DNA, or a combination thereof. The sequencing in this method (e.g., nucleic acid sequencing, subsequent sequencing) can use any sequencing technology. Sequencing can be whole exome sequencing, whole genome sequencing, RNA sequencing, single-cell sequencing, targeted panel sequencing, or a combination thereof. Any number of different sequencing technologies (e.g., nucleic acid sequencing) can be used in the sequencing in this method. At least two different sequencing technologies can be used in the sequencing steps of this method. Any number of biological samples (e.g., at least two biological samples) can be sequenced using different sequencing technologies, such as in the nucleic acid sequencing step. Subsequent sequencing can be performed on any sample (e.g., biological sample, representative biological sample, biological sample combined to produce joint sequencing results).

[0016] The neoantigen can be a peptide of any length. The neoantigen peptide can be a long peptide, a short peptide, or a combination thereof. The method may also include a step of generating a neoantigen vaccine. The neoantigen vaccine may contain or encode a neoantigen for predicting immunogenicity scoring using the methods described herein. The method may also include a step of administering the neoantigen to subjects such as cancer patients. Attached Figure Description

[0017] Figure 1 This is a flowchart illustrating the steps of an exemplary method for scoring the predictive immunogenicity of neoantigens as described herein. Tissue biopsies are collected from the tumor 1 and 2 ( Figure 1 The left side of the sample was sequenced, and after preparation for sequencing, sequencing technologies such as whole exome sequencing (WES), whole genome sequencing (WGS), and RNA sequencing were used. Genomic variants (ai, ...) were identified in the sequencing results from each tissue biopsy sample. Figure 1 (Central region). Sequencing results can be combined to produce joint sequencing results (e.g., "chip-to-chip"). Figure 1 The lower right portion of the sample can be used, or a representative tissue biopsy sample can be selected (e.g., "core selection") to score the predicted immunogenicity of neoantigens in the sample. Combined or selected samples can be sequenced using sequencing technologies (e.g., deeper WGS, ...). Figure 1 The right-middle portion of the sample is then sequenced. Sequencing parameters based on the initial sequencing results, combined sequencing results, or sequencing results from selected representative samples can lead to improvements in the sequencing coverage and / or depth of variants for downstream applications (such as neoantigen vaccine production).

[0018] Figure 2This is an exemplary result of scoring the predicted immunogenicity of a neoantigen. Experimental details that produced this result are presented in Example 1. Initial sequencing results from whole-exome sequencing of two core needle biopsies (i.e., core #1 and core #2) from patient (i.e., patient A) each produced a list of encoded neoantigen peptides. Figure 2 The left and middle columns). Sequencing results are combined to produce joint sequencing results, which serve as a list of encoded neoantigen peptides (the left and middle columns). Figure 2 (The right column). The predicted immunogenicity score is displayed to the left of each neoantigen peptide sequence. Each list is sorted according to the predicted immunogenicity score. In each list, exemplary neoantigen peptides that overlap or contain each other are indicated by the symbol "*" or "#". Figure 2 From top to bottom, the sequences of “chip #1” are disclosed as SEQ ID NO 4-11, 2 and 12-13, the sequences of “chip #2” are disclosed as SEQ ID NO 4-6, 14, 8-11, 3, 15-17 and 12-13, and the sequences of “combined” are disclosed as SEQ ID NO 4-6, 14, 8, 1, 9-11, 15, 18, 16-17 and 12-13.

[0019] Figure 3A and Figure 3B These are Venn diagrams of long neoantigen peptides identified from core needle biopsies of two patients. Figure 3A It comes from and Figure 2 The diagram shows a Venn diagram of identified long neoantigen peptides from core needle biopsies of the same patient (i.e., Patient A). The diagram shows that only one neoantigen peptide was specific to each initial sequencing result (i.e., "Core 1", "Core 2") and combination of sequencing results (i.e., "Core Combination") for each core biopsy sample. The combination of sequencing results represents the majority of the identified long neoantigen peptides in both Core 1 and Core 2 tissue biopsy samples. Figure 3B It comes from a different Figure 2 The results shown are a Venn diagram of the identified long neoantigen peptides from the core needle biopsy of patient B (i.e., patient B). The diagram shows that six neoantigen peptides were specific to only one of the core needle biopsy samples (i.e., “Core 2”). Core 2 contained 55 of the 58 identified long neoantigen proteins. In this example, Core 2 could be further sequenced, and Cores 1 and 2 could not be combined using the methods described herein.

[0020] Figure 4A and Figure 4B This is a Venn diagram showing the selection of neoantigen peptides for neoantigen vaccine production from core needle biopsies of two patients. Figure 4A It is based on the top 48 identified peptides ranked according to predicted immunogenicity, from which... Figure 2 and Figure 3A The diagram shows a Venn diagram of the subset of neoantigen peptides selected for neoantigen vaccine production from core needle biopsies of patients (i.e., Patient A) with the same results. The diagram shows that several neoantigen peptides are dedicated to combinations of biopsy samples (i.e., "Core 1", "Core 2") and sequencing results (i.e., "Core Combination"). Figure 4B It is based on the top 48 identified peptides ranked by predicted immunogenicity, derived from […]. Figure 3B The diagram shows a Venn diagram of the subset of long neoantigen peptides identified in core needle biopsies from patients (i.e., Patient B) with the same results. The diagram illustrates that several neoantigen peptides are specific to only one of the core needle biopsy samples (i.e., "Core 2") and combinations of sequencing results (i.e., "Core Combination"). Detailed Implementation

[0021] The heterogeneity of tumor somatic mutations and the spatial separation of related but distinct subclonal populations within tumors make it difficult to ensure that biological samples (e.g., tissue biopsy samples) represent the majority of subclonal populations. Samples representing genomic variations in tumor subpopulations are important for whole-cancer genomic analysis, diagnosis of driver mutations in cancer, selection of appropriate chemotherapy and immunotherapy agents for cancer treatment, and the development of personalized therapies (e.g., cell therapy, neoantigen vaccines). One solution to this problem is to collect multiple biological samples (e.g., tissue biopsy samples) from the patient (e.g., collected during surgery) to identify samples representing tumor subpopulations. Unfortunately, identifying and selecting representative biological samples (e.g., tissue biopsy samples) using conventional methods requires expensive, time-consuming, and exhaustive sequencing of each candidate biological sample (e.g., tissue sample).

[0022] This article describes a method for scoring (e.g., sorting, assessment) the predictive immunogenicity of neoantigens in one or more biological samples from subjects in need. This method may include n initial biological sample sequencing steps for assessing sequencing parameters (e.g., sample quality, tumor purity, RNA quality, number of identified neoantigen peptides) for each biological sample (see [link to documentation]). Figure 1In one aspect, sequencing parameters of biological samples can be analyzed (e.g., compared), and based on this analysis (e.g., comparison), biological samples can be selected as representative biological samples. In this respect, based on the sequencing results of representative biological samples, the method described herein can score the predicted immunogenicity of neoantigens present in the representative biological samples. In another aspect, based on the analysis (e.g., comparison) of sequencing parameters of biological samples, sequencing results of two or more biological samples can be combined to produce joint sequencing results. In this respect, based on the joint sequencing results of two or more biological samples, the method described herein can score the predicted immunogenicity of neoantigens present in the biological samples. It is believed that, without being bound by any particular theory, through the initial sequencing of biological samples and the analysis (e.g., comparison) of sequencing parameters, biological samples representing multiple (e.g., most) subclones within a tumor can be selected, or sequencing results of two or more biological samples can be combined to form joint sequencing results representing multiple (e.g., most) subclones within a cancer. Advantageously, the method described herein limits the cost and time associated with selecting biological samples because initial sequencing techniques can be performed on all collected samples, and these initial sequencing techniques can be limited to initial shallow, targeted, and / or rapid sequencing techniques. The method described herein allows for the selection of representative samples or the combination of two or more sequencing results to generate representative sequencing results for tumor subclonal populations, while reducing the required time, data processing requirements, and costs compared to exhaustive sequencing of each collected biological sample.

[0023] The methods described herein may include steps for preparing biological samples (e.g., two or more biological samples) for nucleic acid sequencing. As described herein, any number of biological samples (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more) may be prepared, and any type of biological sample may be used, including but not limited to biopsy samples (e.g., liquid biopsy samples), surgically removed tissue samples, or combinations thereof. Preparation steps may include, but are not limited to, weighing the biological sample, isolating nucleic acids (e.g., DNA, RNA) from the biological sample (e.g., isolating nucleic acids by any method, including but not limited to organic extraction, solid-phase extraction, Chelex extraction, DNA precipitation, or combinations thereof), measuring the amount of nucleic acids in the sample, adding an RNase inhibitor, purifying the nucleic acids in the sample (e.g., by any nucleic acid purification method, such as agarose gel extraction, column-based nucleic acid purification, etc.), determining the purity of the isolated nucleic acids, aliquoting the isolated nucleic acids, or combinations thereof.

[0024] The methods described herein may include the step of performing nucleic acid sequencing on biological samples (e.g., two or more biological samples) to produce initial sequencing results for each biological sample. As described herein, any nucleic acid sequencing technology may be used in the nucleic acid sequencing or subsequent sequencing in this method. Suitable sequencing technologies include, but are not limited to, whole-genome sequencing, RNA sequencing, single-cell sequencing (e.g., single-cell RNA sequencing, single-cell DNA sequencing, single-cell whole-exome sequencing, single-cell whole-genome sequencing), real-time PCR, deep sequencing, high-throughput sequencing (e.g., next-generation sequencing), targeted combinatorial sequencing, or combinations thereof. The method may use any number (e.g., at least two) of different sequencing technologies at any step of the method. The method may use the same sequencing technology on the biological sample at any step of the method. Any type of nucleic acid may be sequenced in any step of the method (e.g., in the nucleic acid sequencing step, in the subsequent sequencing step), including but not limited to DNA, RNA, or combinations thereof.

[0025] The methods described herein may include steps for evaluating initial sequencing results (e.g., initial sequencing results for each biological sample) by analyzing (e.g., comparing) one or more sequencing parameters of the initial sequencing results. As described herein, sequencing parameters of sequencing results (e.g., initial sequencing results, subsequent sequencing results, combined sequencing results) can be any sequencing parameters. Suitable sequencing parameters include, but are not limited to, the number of sequence reads (e.g., total number of sequence reads), the number of sequencing variants (e.g., the number of DNA sequencing variants, the number of RNA sequencing variants), tumor purity, sequencing depth, the number of protein modification sequencing variants (e.g., the number of protein modification sequencing variants confirmed by RNA sequencing), RNA confirmation rate, RNA quality, the number of identified long neoantigen peptides, the number of identified short neoantigen peptides, the predicted immunogenicity of neoantigen peptides, or combinations thereof. Any number of sequencing parameters may be analyzed or compared between sequencing results of biological samples or between combined sequencing results and initial sequencing results of biological samples. Without being bound by any particular theory, it is believed that the analysis of sequencing parameters (e.g., comparison of sequencing parameters between biological samples, e.g., between two or more biological samples) can be used to determine whether to combine the initial sequencing results of two or more biological samples to produce a joint sequencing result or to select a representative biological sample. It is further believed that this method provides the most representative results of the genomic variations (e.g., somatic mutations) present in the source organism of the biological sample (e.g., combination of samples, selection of a representative sample), and that these representative results can be used to generate treatments for subjects (e.g., the production of neoantigen vaccines).

[0026] The methods described herein may include the step of combining the initial sequencing results of two or more biological samples to produce a co-sequencing result based on the analysis (e.g., comparison) of sequencing parameters of the initial sequencing results of biological samples. The methods described herein may also include the step of selecting a representative biological sample based on the analysis (e.g., comparison) of sequencing parameters of the initial sequencing results of biological samples. As described herein, the sequencing parameters of sequencing results (e.g., co-sequencing results, initial sequencing results, subsequent sequencing results) can be analyzed (e.g., compared) through any mathematical calculation. A suitable mathematical calculation is the ratio between the sequencing parameters of the sequencing results. The ratio may indicate that the sequencing results of two or more biological samples will be combined to form a co-sequencing result. The ratio may indicate that a biological sample (e.g., a single tissue sample) will be selected as the representative biological sample.

[0027] The methods described herein may include a step of scoring the predicted immunogenicity of a neoantigen (e.g., one or more neoantigens in a biological sample) based on combined sequencing results. The methods described herein may also include a step of scoring the predicted immunogenicity of a neoantigen (e.g., one or more neoantigens in a biological sample) based on sequencing results of representative biological samples. Any method for predicting the immunogenicity of a neoantigen may be used in the methods described herein.

[0028] In some implementations, the method for scoring the predictive immunogenicity of neoantigens in a subject's biological sample includes the following steps:

[0029] a) Prepare two or more biological samples for nucleic acid sequencing;

[0030] b) Perform nucleic acid sequencing on the two or more biological samples to produce initial sequencing results for each of the biological samples;

[0031] c) Evaluate the initial sequencing results by analyzing one or more sequencing parameters of the initial sequencing results;

[0032] d) Based on the analysis of the sequencing parameters of the initial sequencing results of the biological samples, combine the initial sequencing results of two or more biological samples to produce joint sequencing results; and

[0033] e) Based on the combined sequencing results of the biological samples, score the predicted immunogenicity of one or more neoantigens in the biological samples of the subjects in need.

[0034] In some implementations, the method for scoring the predictive immunogenicity of neoantigens in a subject's biological sample includes the following steps:

[0035] a) Prepare two or more biological samples for nucleic acid sequencing;

[0036] b) Perform nucleic acid sequencing on the two or more biological samples to produce initial sequencing results for each of the biological samples;

[0037] c) Evaluate the initial sequencing results by analyzing one or more sequencing parameters of the initial sequencing results;

[0038] d) Based on the analysis of the sequencing parameters of the initial sequencing results of the biological samples, select representative biological samples;

[0039] e) Based on the sequencing results of the representative biological samples, score the predicted immunogenicity of one or more neoantigens in the representative biological samples of the subjects in need.

[0040] The method described herein may also include a step of comparing the joint sequencing parameters of the joint sequencing results with the sequencing parameters of the initial sequencing results.

[0041] The method described herein may also include a step of subsequent sequencing of biological samples or representative biological samples.

[0042] The method described herein may also include the step of generating a neoantigen vaccine, wherein the neoantigen vaccine comprises or encodes a neoantigen for which the method scores a predicted immunogenicity. The neoantigen may be a peptide (e.g., an identified long peptide, an identified short peptide, a synthetic peptide, an isolated peptide), an RNA sequence encoding the peptide (e.g., mRNA, mRNA containing non-natural nucleotides (e.g., pseudouridine, N1-methylpseuuridine, 7-methylguanosine, N6-methyladenosine, 2'-O-methylnucleotide), mRNA containing inverse nucleotides, or combinations thereof), a DNA sequence encoding the peptide (e.g., plasmid DNA), or combinations thereof.

[0043] The method described herein may also include the step of administering a neoantigen vaccine to subjects in need. The neoantigen vaccine may be administered to subjects who have been diagnosed with cancer, already have cancer, have recurrent cancer (i.e., relapse), or are at risk of developing cancer.

[0044] The methods described herein can be performed on patients (e.g., human patients) as subjects. Patients may have any condition or disease, such as cancer, infection, or genetic disorder. The methods described herein can be repeated any number of times during patient treatment. The number of times the method can be repeated can be approximately 1, approximately 2, approximately 3, approximately 4, approximately 5, approximately 6, approximately 7, approximately 8, approximately 9, approximately 10, approximately 11, approximately 12, approximately 13, approximately 14, or approximately 15 times. As described herein, sample collection and method repetition can be separated at any time interval.

[0045] definition

[0046] All publications and patents referenced in this disclosure are incorporated herein by reference in their entirety. Where the referenced material conflicts with or is inconsistent with this specification, the specification shall supersede any such material. Reference to any reference herein does not constitute an admission that such reference is prior art to this disclosure. Various terms used throughout the specification and claims relate to various aspects of this specification. Unless otherwise stated, such terms shall have their ordinary meaning in the art. Other specifically defined terms will be interpreted in a manner consistent with the definitions provided herein.

[0047] When describing a range of values, it includes embodiments using any specific value within that range. Furthermore, references to numerical values ​​stated within a range include every and all values ​​within that range. All ranges include their endpoints and are composable. When a value is expressed as an approximation using the antecedent “about,” it should be understood that the specific value forms another embodiment. Unless the context clearly indicates otherwise, references to a specific numerical value include at least that specific value. Unless the specific context in which it is used indicates otherwise, the use of “or” will mean “and / or.”

[0048] Unless otherwise stated, the terms "at least," "less than," "about," and "at most," or similar terms preceding a series of elements or ranges, should be understood to refer to each element in the series or range. Those skilled in the art will recognize, or can determine, many equivalents of the specific embodiments of the invention described herein using only conventional experimentation. Such equivalents are intended to be covered by the following claims.

[0049] Unless the context clearly indicates otherwise, as used herein, the singular forms “a” and “the” include multiple indicators. Unless otherwise clearly indicated, the terms “comprising,” “such as,” etc., are intended to convey inclusion without limitation.

[0050] As used herein, the term "cancer" refers to a physiological condition in a subject characterized by uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rates, and / or certain morphological features. Typically, cancer may present as a tumor or mass, but can exist alone in a subject or circulate in the bloodstream as independent cells such as lymphoma cells. The term cancer encompasses all types of cancer and metastases, including hematologic malignancies, solid tumors, sarcomas, carcinomas, and other solid and non-solid tumors. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of this type of cancer include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell lung cancer, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer (e.g., triple-negative breast cancer, hormone receptor-positive breast cancer), osteosarcoma, melanoma, colon cancer, colorectal cancer, endometrial (e.g., serous) carcinoma or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatocellular carcinoma, and various types of head and neck cancer. Triple-negative breast cancer is breast cancer that is negative for estrogen receptor (ER), progesterone receptor (PR), and Her2 / neu gene expression. Hormone receptor-positive breast cancer is breast cancer that is positive for at least one of the following: ER or PR and negative for Her2 / neu (HER2).

[0051] As used herein, the term "subject" refers to any animal, such as any mammal, including but not limited to humans, non-human primates, dogs, cats, rodents (e.g., rats, mice, guinea pigs, hamsters), etc. In some embodiments, the mammal is a mouse. In some embodiments, the mammal is a human.

[0052] Any term not directly defined herein should be understood to have the meaning generally associated with it as understood within the field of this invention. Certain terms are discussed herein to provide additional guidance to practitioners in describing compositions, apparatuses, methods, etc., and how they are made or used in relation to aspects of the invention. It should be understood that the same thing can be expressed in more than one way. Therefore, alternative language and synonyms may be used for any one or more terms discussed herein. Whether a term is elaborated or discussed herein is not important. Several synonyms or alternative methods, materials, etc., are provided. Unless expressly stated, the use of one or more synonyms or equivalents does not preclude the use of other synonyms or equivalents. The use of examples, including instances of terms, is for illustrative purposes only and does not limit the scope and meaning of aspects of the invention.

[0053] biological samples

[0054] The biological samples used in the methods disclosed herein can be obtained by any method and contain tissue or fluid from any body part, organ, or tissue type. The biological sample may be a biopsy sample (e.g., a tissue biopsy sample). The biopsy sample may be any type of biopsy, including but not limited to excisional biopsy, liquid biopsy, excisional biopsy, needle aspiration biopsy, perforation biopsy, or scraping biopsy. Needle aspiration biopsy may be any type of needle aspiration biopsy, including but not limited to core needle biopsy, fine needle aspiration biopsy, or combinations thereof. Needle aspiration biopsy may have any needle size, including but not limited to 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. Needle biopsy specifications can be in the range of 9 to 18, 10 to 17, 11 to 16, 12 to 15, 13 to 14, 20 to 30, 21 to 30, 22 to 30, 23 to 30, 9 to 30, 9 to 29, 9 to 28, 9 to 27, 9 to 26, 9 to 25, 9 to 24, 9 to 23, 9 to 22, 9 to 21, 9 to 20, 9 to 19, 9 to 17, 9 to 16, 9 to 15, 9 to 14, 9 to 13, 9 to 12, 9 to 11, 9 to 10, 10 to 30, 11 to 30, 12 to 30, 13 to 30, 14 to 30, 15 to 30, 16 to 30, 17 to 30, 18 to 30, or 19 to 30.

[0055] Biological samples from subjects can be liquid biopsies. Liquid biopsies can be any biological fluid, including but not limited to amniotic fluid, blood, oral samples, cerebrospinal fluid, fecal samples, peritoneal fluid, pleural effusion samples (e.g., transudative pleural effusion, exudative pleural effusion), saliva, semen, synovial fluid, urine, or combinations thereof. Unlike tissue biopsies, which primarily consist of solid biological tissue and are collected surgically, liquid biopsies can be collected from a subject's vein or artery. Liquid biopsies may contain blood, circulating tumor cells, circulating tumor material (e.g., circulating tumor DNA (ctDNA), circulating tumor extracellular vesicles, circulating tumor proteins, circulating tumor RNA), plasma, serum, healthy blood cells, or combinations thereof. Liquid biopsies can be taken from subjects suffering from any type of illness or symptom, including but not limited to infections and cancers (e.g., hematologic malignancies, solid tumors).

[0056] Biological samples may include two or more different biological sample types (e.g., two or more different biopsy types). For example, the biological sample used in the methods described herein may include three biopsy samples: two liquid biopsy samples and one tissue needle biopsy sample. Biological samples may include two or more identical biopsy types. For example, the biological sample may include three tissue biopsy samples, which are core needle biopsies. The number of different biological sample types may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. The number of biological sample types may be at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10. Sequencing parameters of sequencing results from two or more different biological sample types may be analyzed (e.g., compared) using the methods described herein. Sequencing results from two or more different biological sample types may be combined using the methods described herein to produce joint sequencing results.

[0057] Biological samples (e.g., tissue samples) can be any tissue surgically removed from the subject, including but not limited to samples from adenoidectomy, adenoidectomy, adrenalectomy, apexification, appendectomy, auricle resection, bullae resection, bunionectomy, cecal resection, cervicectomy, cholecystectomy, colectomy, craniectomy, cystectomy, vertebral resection, intervertebral discectomy, diverticulum resection, duodenectomy, and esophageal resection. Samples of resection procedures, including: extrapleural lung resection, frenulum resection, gastric fundus resection, ganglion resection, gastrectomy, gingivectomy, tongue resection, gonadectomy, hemicolectomy, hemilaminectomy, hemipelvic resection, cerebral hemispherectomy, hemorrhoidectomy, hepatectomy, pituitary resection, hysterectomy, iridectomy, jejunectomy, corneal resection, laminectomy, laryngectomy, breast tumor resection, and lymph node dissection. Samples of various surgical procedures, including: nephrectomy, mastectomy, mastoidectomy, myocardectomy, myoma resection, necrotic tissue resection, nephrectomy, neurectomy, oophorectomy, orchiectomy, bone resection, pancreatectomy, pancreaticoduodenectomy, fat resection, parathyroidectomy, pericardectomy, pinealectomy, pulmonary resection, rectocolic resection, prostatectomy, endodontic resection, quartotomy, and rhinectomy. Surgical samples, including salpingectomy samples, salpingo-oophorectomy samples, nasal septum resection samples, splenectomy samples, stapes resection samples, sympathectomy samples, thymectomy samples, thyroidectomy samples, tonsillectomy samples, trabeculectomy samples, tumor samples (e.g., tumor resection, surgically removed tumor), turbinate resection samples, salpingectomy samples, hysterectomy samples, uvulectomy samples, vitrectomy samples, or combinations thereof. In some embodiments, the biological sample is a resected tumor sample.

[0058] Biological samples (e.g., tissue biopsies, surgically removed tissue) can come from any anatomical location. Anatomical locations include, but are not limited to, the abdomen, ankle, arm, back, upper arm, breast, buttocks, calf, chest, ear, elbow, eye, face, finger, foot, forearm, genitals, hand, head, hip, knee, leg, mouth, neck, nose, scalp, tibia, shoulder, thigh, toes, waist, and wrist. Biological samples can be derived from any organ, including but not limited to the adrenal glands, appendix, anus, arteries, bladder, bone marrow, brain, bronchi, bronchioles, capillaries, cervix, colon, ear, epididymis, esophagus, eye, fallopian tubes, gallbladder, enteroassociated lymphoid tissue, heart, interstitium, joints, kidneys, large intestine, larynx, ligaments, liver, lungs, lymph nodes, mammary glands, mesentery, mouth, muscle, nasal cavity, nerves, olfactory epithelium, ovary, pancreas, pharynx, pineal gland, pituitary gland, placenta, prostate, rectum, salivary glands, bones, skin, small intestine, spinal cord, spleen, stomach, subcutaneous tissue, tendons, testes, thymus, thyroid gland, tongue, trachea, ureter, urethra, uterus, vas deferens, ventricular system, and veins. Biological samples (e.g., tissue biopsy samples, surgically removed tissue) can contain any body tissue, including but not limited to connective tissue, epithelial tissue, muscle tissue, nerve tissue, or combinations thereof.

[0059] The method described herein can be applied to two or more biological samples from a subject. The number of biological samples can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. The number of biological samples can be at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, or at least about 15. Biological samples from the subject can originate from the same region of the subject's body. For example, three tissue samples can originate from the subject's left lung. Biological samples can originate from different regions of the subject's body. For example, the first tissue sample can originate from the pancreas, and the second tissue sample can originate from the subject's bile duct. The number of biological samples from different regions of the subject's body can be at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten. Biological samples may originate from the same region of the subject's tumor. Biological samples may originate from different regions of the subject's tumor. For example, two tissue samples may include a first tissue sample collected from the posterior portion of an adenocarcinoma tumor and a second tissue sample collected from the anterior portion of the same adenocarcinoma tumor. The number of biological samples from different regions of the subject's tumor can be at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten. Biological samples from the subject may originate from the subject's primary tumor. Biological samples from the subject may originate from the subject's secondary tumor (e.g., metastatic tumor). Two or more biological samples (e.g., tissue samples) may originate from different types of tumors, including primary and secondary tumors. For example, two tissue samples (e.g., a first tissue sample and a second tissue sample) may be collected, including a first tissue sample from a primary tumor of the liver, and a second tissue sample from a metastatic tumor in the subject's brain. In some embodiments, at least two biological samples in the biological sample are derived from different regions of the subject's body. In some embodiments, at least two biological samples in the biological sample are derived from different regions of the subject's tumor. In some embodiments, at least two biological samples in the biological sample are derived from different regions of the subject's tumor.

[0060] Biological samples (e.g., tissue biopsy samples, surgically removed tissue, liquid biopsy samples) may be collected and / or maintained at any temperature. For example, biological samples may be collected and / or maintained at room temperature. For example, biological samples may be collected and / or kept frozen. Biological samples may be collected and / or maintained at approximately -78°C, approximately -76°C, approximately -74°C, approximately -72°C, approximately -70°C, approximately -68°C, approximately -66°C, approximately -64°C, approximately -62°C, approximately -60°C, approximately -58°C, approximately -56°C, approximately -54°C, approximately -52°C, approximately -50°C, approximately -48°C, approximately -46°C, approximately -44°C, approximately -42°C, approximately -40°C, approximately -38°C, approximately -36°C, approximately -34°C, approximately -32°C, approximately -30°C. Approximately -28°C, approximately -26°C, approximately -24°C, approximately -22°C, approximately -20°C, approximately -18°C, approximately -16°C, approximately -14°C, approximately -12°C, approximately -10°C, approximately -8°C, approximately -6°C, approximately -4°C, approximately -2°C, approximately 0°C, approximately 2°C, approximately 4°C, approximately 6°C, approximately 8°C, approximately 10°C, approximately 12°C, approximately 14°C, approximately 16°C, approximately 18°C, approximately 20°C, approximately 22°C, approximately 24°C, approximately 26°C, approximately 28°C, approximately 30°C, approximately 32°C, or approximately 34°C. Biological samples may be collected and / or maintained at up to approximately -78°C, up to approximately -76°C, up to approximately -74°C, up to approximately -72°C, up to approximately -70°C, up to approximately -68°C, up to approximately -66°C, up to approximately -64°C, up to approximately -62°C, up to approximately -60°C, up to approximately -58°C, up to approximately -56°C, up to approximately -54°C, up to approximately -52°C, up to approximately -50°C, up to approximately -48°C, up to approximately -46°C, up to approximately -44°C, up to approximately -42°C, up to approximately -40°C, up to approximately -38°C, up to approximately -36°C, up to approximately -34°C, up to approximately -32°C, up to approximately -30°C, up to approximately -28°C. Temperatures up to approximately -26°C, up to approximately -24°C, up to approximately -22°C, up to approximately -20°C, up to approximately -18°C, up to approximately -16°C, up to approximately -14°C, up to approximately -12°C, up to approximately -10°C, up to approximately -8°C, up to approximately -6°C, up to approximately -4°C, up to approximately -2°C, up to approximately 0°C, up to approximately 2°C, up to approximately 4°C, up to approximately 6°C, up to approximately 8°C, up to approximately 10°C, up to approximately 12°C, up to approximately 14°C, up to approximately 16°C, up to approximately 18°C, up to approximately 20°C, up to approximately 22°C, up to approximately 24°C, up to approximately 26°C, up to approximately 28°C, up to approximately 30°C, up to approximately 32°C, or up to approximately 34°C. Biological samples can be collected and / or maintained in a temperature range of about -78°C to about -20°C, about -78°C to about 0°C, about -78°C to about 4°C, about -78°C to about 24°C, about -20°C to about 0°C, about -20°C to about 4°C, about -20°C to about 24°C, about 0°C to about 4°C, about 0°C to about 24°C, or about 4°C to about 24°C.

[0061] The biological samples used in the methods described herein (e.g., tissue samples, tissue biopsy samples, surgically removed tissue samples) may be derived from the subject's tumor. The tumor can be any type of cancerous tumor, including hematologic malignancies, solid tumors, sarcomas, carcinomas, and other solid and non-solid tumors. Typical examples of suitable cancers include, for example, adrenocortical carcinoma, anal cancer, appendiceal cancer, astrocytoma, basal cell carcinoma, brain tumors, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumors, cancers of unknown primary origin, cardiac tumors, cervical cancer, chordoma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma, embryonal tumors, endometrial cancer, ependymoma, esophageal cancer, olfactory neuroblastoma, fibrous histiocytoma, Ewing sarcoma, eye cancer, germ cell tumors, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, glioma, head and neck cancer, hepatocellular carcinoma, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors, Kaposi sarcoma, kidney cancer, and Langerhans cell histiocytosis. Histiocytosis, laryngeal cancer, leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, hairy cell leukemia, myelodysplastic syndrome, prolymphocytic leukemia, large granular lymphocytic leukemia, adult T-cell leukemia, clonal eosinophilia), lip and oral cancer, liver cancer, lobular carcinoma in situ, lung cancer, macroglobulinemia, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma. Carcinoma, mesothelioma, occult primary metastatic squamous neck carcinoma, midline carcinoma involving the NUT gene, oral cancer, multiple endocrine adenoma syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasms, nasal cavity and sinus carcinoma, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oropharyngeal carcinoma, osteosarcoma, ovarian cancer, pancreatic cancer, papilloma, paraganglioma, parathyroid carcinoma, penile cancer, pharyngeal carcinoma, pheochromocytoma, pituitary adenoma, pleural pulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureter cancer, retinoblastoma, rhabdomyosarcoma, salivary gland carcinoma, Sézary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumors, stomach cancer. Cancer), T-cell lymphoma, teratoma, testicular cancer, laryngeal cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer and nephroblastoma.

[0062] The biological samples (e.g., tissue biopsy samples) used in the methods described herein may be derived from the subject's primary and / or secondary (e.g., metastatic) tumors. Any number of biological samples (e.g., two or more biological samples) may be derived from primary tumors, such as at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10. Any number of biological samples (e.g., two or more biological samples) may be derived from secondary (e.g., metastatic) tumors, such as at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10. The tumor may be at any stage of cancer, including but not limited to stage 0, stage I, stage II, stage III, or stage IV. The tumor may be in remission (e.g., partial remission). The tumor may be a recurrent tumor (e.g., a tumor of recurrent cancer). The tumor may have any grade, including but not limited to grade X, 1, 2, 3, or 4. The tumor may be a refractory tumor. The tumor may be resistant to treatment (e.g., resistant to chemotherapy, resistant to immunotherapy). The tumor may be sensitive to treatment (e.g., sensitive to chemotherapy, sensitive to immunotherapy, sensitive to radiation therapy). The tumor may be benign. The tumor may be a precancerous lesion. In some embodiments, at least one biological sample in the biological sample is derived from the subject's primary tumor. In some embodiments, at least one biological sample in the biological sample is derived from the subject's secondary tumor.

[0063] Biological samples described herein can be collected at any point in time that is relevant to each other. Biological samples (e.g., two or more biological samples) can be collected simultaneously (e.g., during the same surgical procedure on the same day). Biological samples can be collected from the subject at different times that are spaced apart in time. The time interval between the collection of two biological samples can be approximately 1 minute, approximately 2 minutes, approximately 3 minutes, approximately 4 minutes, approximately 5 minutes, approximately 6 minutes, approximately 7 minutes, approximately 8 minutes, approximately 9 minutes, approximately 10 minutes, approximately 15 minutes, approximately 30 minutes, approximately 45 minutes, approximately 1 hour, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 7 hours, approximately 8 hours, approximately 9 hours, approximately 10 hours, approximately 11 hours, approximately 12 hours, approximately 14 hours, approximately 16 hours, approximately 18 hours, approximately 20 hours, approximately 22 hours, approximately 1 day, approximately 2 days, approximately 3 days, approximately 4 days, approximately 5 days, etc. Approximately 6 days, approximately 7 days, approximately 8 days, approximately 9 days, approximately 10 days, approximately 12 days, approximately 14 days, approximately 16 days, approximately 18 days, approximately 20 days, approximately 22 days, approximately 24 days, approximately 26 days, approximately 28 days, approximately 30 days, approximately 1 month, approximately 2 months, approximately 3 months, approximately 4 months, approximately 5 months, approximately 6 months, approximately 7 months, approximately 8 months, approximately 9 months, approximately 10 months, approximately 11 months, approximately 1 year, approximately 13 months, approximately 14 months, approximately 15 months, approximately 16 months, approximately 17 months, approximately 18 months, approximately 19 months, approximately 20 months, approximately 21 months, approximately 22 months, approximately 23 months, or approximately 2 years. The time interval between the collection of two biological samples may be at least about 1 minute, at least about 5 minutes, at least about 10 minutes, at least about 15 minutes, at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 6 hours, at least about 8 hours, at least about 10 hours, at least about 12 hours, at least about 14 hours, at least about 16 hours, at least about 18 hours, at least about 20 hours, at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 12 days, at least about 14 days, at least about 16 days, at least about 18 days, at least about 20 days, at least about 24 days, at least about 28 days, at least about 1 month, at least about 2 months, at least about 4 months, at least about 6 months, at least about 8 months, at least about 10 months, at least about 1 year, at least about 1.5 years, or at least about 2 years. In some implementations, at least two biological samples are collected from the subject at different times, at least about one day apart.

[0064] The methods described herein can be repeated during or after a patient's treatment (e.g., after chemotherapy). The number of times the method can be repeated during treatment may be approximately 1, approximately 2, approximately 3, approximately 4, approximately 5, approximately 6, approximately 7, approximately 8, approximately 9, approximately 10, approximately 11, approximately 12, approximately 13, approximately 14, or approximately 15.

[0065] The time interval between repetitions of this method can be approximately 1 day, approximately 2 days, approximately 3 days, approximately 4 days, approximately 5 days, approximately 6 days, approximately 7 days, approximately 8 days, approximately 9 days, approximately 10 days, approximately 12 days, approximately 14 days, approximately 16 days, approximately 18 days, approximately 20 days, approximately 22 days, approximately 24 days, approximately 26 days, approximately 28 days, approximately 30 days, approximately 1 month, approximately 2 months, approximately 3 months, approximately 4 months, approximately 5 months, approximately 6 months, approximately 7 months, approximately 8 months, approximately 9 months, approximately 10 months, approximately 11 months, approximately 1 year, approximately 13 months, approximately 14 months, approximately 15 months, approximately 16 months, approximately 17 months, approximately 18 months, approximately 19 months, approximately 20 months, approximately 21 months, approximately 22 months, approximately 23 months, or approximately 2 years. The time interval between repetitions of this method can be at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 12 days, at least about 14 days, at least about 16 days, at least about 18 days, at least about 20 days, at least about 24 days, at least about 28 days, at least about 1 month, at least about 2 months, at least about 4 months, at least about 6 months, at least about 8 months, at least about 10 months, at least about 1 year, at least about 1.5 years, or at least about 2 years.

[0066] sequencing

[0067] The methods described herein may include steps of nucleic acid sequencing of biological samples and subsequent sequencing of biological samples (e.g., representative biological samples). Any nucleic acid sequencing technology (e.g., any sequencing technology used for nucleic acid sequencing, any sequencing technology used for subsequent sequencing) may be used in the methods described herein. Suitable sequencing technologies include, but are not limited to, whole exome sequencing, whole genome sequencing, RNA sequencing, single-cell sequencing (e.g., single-cell RNA sequencing, single-cell DNA sequencing, single-cell whole exome sequencing, single-cell whole genome sequencing), targeted combinatorial sequencing (e.g., gene combinatorial sequencing), real-time PCR, deep sequencing, high-throughput sequencing (e.g., next-generation sequencing), or combinations thereof. Suitable sequencing technologies may also include, but are not limited to, pyrosequencing, sequencing-by-synthesis, single-molecule sequencing, nanopore sequencing, semiconductor sequencing, sequencing-by-synthesis, ligation-by-synthesis, hybridization sequencing, RNA-Sew (Illumina), digital gene expression (Helicos), next-generation sequencing, single-molecule sequencing-by-synthesis (SMSS) (Helicos), massively parallel sequencing, cloned single-molecule arrays (Solexa), shotgun sequencing, Maxam-Hilbery or Sanger sequencing, primer walking, sequencing using PacBio, SOLid, Ion Torrent platforms, nanopore platforms, or combinations thereof. In some implementations, nucleic acid sequencing is performed using sequencing technologies that are whole-exome sequencing, whole-genome sequencing, RNA sequencing, single-cell sequencing, targeted combinatorial sequencing, or combinations thereof. In some implementations, subsequent sequencing is performed using sequencing technologies that are whole-exome sequencing, whole-genome sequencing, RNA sequencing, single-cell sequencing, targeted combinatorial sequencing, or combinations thereof.

[0068] The method described herein may employ at least two different sequencing technologies at any step of the method (such as nucleic acid sequencing for biological samples or subsequent sequencing steps). The number of different sequencing technologies used in each step of the method may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. For example, the nucleic acid sequencing step may be performed by whole-genome sequencing and RNA sequencing of the biological sample. As another example, the subsequent sequencing step of the biological sample may be performed by whole-genome sequencing. As yet another example, the nucleic acid sequencing step may be performed by whole-genome sequencing, RNA sequencing, and whole-exome sequencing. The number of different sequencing technologies used in the steps of this method can be at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, or at least about 20. The number of different sequencing technologies used in the steps of this method can be approximately 1 to 20, approximately 1 to 19, approximately 1 to 18, approximately 1 to 17, approximately 1 to 16, approximately 1 to 15, approximately 1 to 14, approximately 1 to 13, approximately 1 to 12, approximately 1 to 11, approximately 1 to 10, approximately 1 to 9, approximately 1 to 8, approximately 1 to 7, approximately 1 to 6, approximately 1 to 5, approximately 1 to 4, approximately 1 to 3, approximately 1 to 2, approximately 2 to 20, approximately 3 to 20, approximately 4 to... Approximately 20, approximately 5 to approximately 20, approximately 6 to approximately 20, approximately 7 to approximately 20, approximately 8 to approximately 20, approximately 9 to approximately 20, approximately 10 to approximately 20, approximately 11 to approximately 20, approximately 12 to approximately 20, approximately 13 to approximately 20, approximately 14 to approximately 20, approximately 15 to approximately 20, approximately 16 to approximately 20, approximately 17 to approximately 20, approximately 18 to approximately 20, approximately 19 to approximately 20, approximately 2 to approximately 3, approximately 2 to approximately 4, approximately 2 to approximately 5, approximately 3 to approximately 4, approximately 3 to approximately 5, approximately 4 to approximately 5, or approximately 4 to approximately 6. In some embodiments, at least two different sequencing technologies are used in the nucleic acid sequencing step of the biological sample (e.g., two or more biological samples). In some embodiments, at least two different sequencing technologies are used in the subsequent sequencing step of the biological sample (e.g., one or more biological samples).

[0069] The methods described herein may use the same sequencing technology for each biological sample (e.g., each of two or more biological samples, a representative biological sample) at any step (e.g., a step of nucleic acid sequencing of the biological sample, a step of subsequent sequencing of the biological sample). The methods described herein may also use different sequencing technologies for biological samples (e.g., different sequencing technologies between two or more biological samples). For any number of biological samples, such as at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, or at least 15 biological samples, the sequencing technology may be different. For 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 biological samples, the sequencing technology may be different. For biological samples of approximately 1 to approximately 15, approximately 1 to approximately 14, approximately 1 to approximately 13, approximately 1 to approximately 12, approximately 1 to approximately 11, approximately 1 to approximately 10, approximately 1 to approximately 9, approximately 1 to approximately 8, approximately 1 to approximately 7, approximately 1 to approximately 6, approximately 1 to approximately 5, approximately 1 to approximately 4, approximately 1 to approximately 3, or approximately 1 to approximately 2, the sequencing technologies may be different. In some embodiments, at least two biological samples (e.g., from two or more biological samples) are sequenced using different sequencing technologies. In some embodiments, at least two biological samples (e.g., from two or more biological samples, or from representative biological samples) are sequenced using different sequencing technologies in a step of nucleic acid sequencing of biological samples. In some embodiments, at least two biological samples (e.g., from two or more biological samples, or from representative biological samples) are sequenced using different sequencing technologies in a step of subsequent sequencing of biological samples.

[0070] In the methods described herein, the step of subsequent sequencing can be performed on any biological sample. For example, a representative sample can be subsequently sequenced. As another example, two or more biological samples combined to form a joint sequencing result can be subsequently sequenced. The decision to perform subsequent sequencing on a sample can be based on sequencing parameters of the sequencing results (e.g., initial sequencing results, joint sequencing results, representative sequencing results of a representative biological sample, or a combination thereof). The sequencing technology used for subsequent sequencing can be different from the sequencing technology used for initial nucleic acid sequencing. For example, one method may include the steps of initial nucleic acid sequencing performed by whole exome sequencing and subsequent sequencing performed by whole genome sequencing. Subsequent sequencing of biological samples produces subsequent sequencing results. The methods described herein may include the step of combining the subsequent sequencing results of two or more biological samples to produce a joint sequencing result based on the analysis (e.g., comparison) of sequencing parameters (e.g., sequencing parameters of the subsequent sequencing results). The step of selecting a representative biological sample can be based on the sequencing parameters of the subsequent sequencing results. The scoring of the predicted immunogenicity of neoantigens in biological samples (e.g., representative biological samples, biological samples combined to form a joint sequencing result) can be based on the subsequent sequencing of the biological samples. In some implementations, the method includes a step of subsequent sequencing of the biological sample or a representative biological sample.

[0071] Nucleic acid

[0072] The sequencing described herein (e.g., initial sequencing, subsequent sequencing) can be any type of nucleic acid. Suitable nucleic acids include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), or combinations thereof. Nucleic acids (e.g., RNA, DNA) may include ribonucleic acid nucleotides (e.g., adenosine, uridine, cytidine, and guanosine) and / or deoxyribonucleic acid nucleotides (e.g., 2'-deoxyadenosine, thymidine, 2'-deoxycytidine, and 2'-deoxyguanosine). DNA and RNA may include any modified (e.g., post-translational modified, chemically modified) nucleotides, including but not limited to 1-methyladenosine, 6-methyladenosine, 2'-O-methyladenosine, 5-methylcytosine, 6-methylguanosine, 4-acetylcytidine, pseudouridine, N1-methylpseudouridine, 5-hydroxymethylcytidine, inosine, or combinations thereof.

[0073] The methods described herein can be used to sequence any type of DNA. Suitable types of DNA for sequencing include, but are not limited to, genomic DNA, complementary DNA (cDNA), plasmid DNA, mitochondrial DNA, or combinations thereof. The methods described herein can also be used to sequence any type of RNA. Suitable types of RNA for sequencing include, but are not limited to, transfer RNA (tRNA), small nuclear RNA (snRNA), Piwi-interacting RNA, ribosomal RNA (rRNA), messenger RNA (mRNA), non-coding RNA (ncRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNA), pre-mRNA, or combinations thereof.

[0074] Nucleic acid sequencing and / or subsequent sequencing steps for biological samples may include sequencing nucleic acids as RNA, DNA, or combinations thereof. In some embodiments, the nucleic acid sequencing step is sequencing nucleic acids as RNA, DNA, or combinations thereof. In some embodiments, the subsequent sequencing step (e.g., subsequent nucleic acid sequencing) is sequencing nucleic acids as RNA, DNA, or combinations thereof.

[0075] Analysis of sequencing parameters

[0076] The sequencing results obtained by the methods described herein (e.g., initial nucleic acid sequencing results, subsequent sequencing results) can be any type of nucleic acid sequencing result, including but not limited to raw nucleic acid sequencing reads (e.g., raw DNA sequence reads, raw RNA sequence reads, raw exome sequence reads, raw whole genome sequence reads), sequencing variants (e.g., DNA sequencing variants compared to healthy tissue of a subject, RNA sequencing variants compared to healthy tissue of a subject, protein-modified sequencing variants), predicted RNA sequences transcribed from DNA sequence reads, predicted neoantigen peptides (e.g., neoantigen peptides based on RNA sequence reads, neoantigen peptides based on DNA sequence reads), or combinations thereof. Sequencing variants can be somatic cell variants. Sequencing variants can be germline variants. Sequencing variants can be a combination of somatic cell and germline variants. Sequencing results can be sequencing reads, sequencing variants, encoded peptides, or combinations thereof. In some embodiments, initial sequencing results (e.g., results of nucleic acid sequencing of a biological sample, results of subsequent sequencing of a biological sample) can be sequencing reads, sequencing variants, encoded peptides, or combinations thereof. In some implementations, subsequent sequencing results (e.g., the results of subsequent sequencing steps of a biological sample, the results of nucleic acid sequencing steps of a biological sample) may be sequencing reads, sequencing variants, encoded peptides, or combinations thereof.

[0077] The sequencing results (e.g., combined sequencing results, initial sequencing results, subsequent sequencing results) described herein may have one or more sequencing parameters based on the sequencing technology used and the type of sequencing results obtained. Sequencing parameters can be any parameter of the sequencing results (e.g., initial nucleic acid sequencing results, subsequent sequencing results), including but not limited to the number of sequence reads (e.g., total number of sequence reads), the number of sequencing variants (e.g., the number of DNA sequencing variants, the number of RNA sequencing variants), tumor purity, sequencing depth, the number of protein modification sequencing variants (e.g., the number of protein modification sequencing variants confirmed by RNA), RNA confirmation rate, RNA quality, the number of identified long neoantigen peptides, the number of identified short neoantigen peptides, the predicted immunogenicity of neoantigen peptides, or combinations thereof. The number of sequence reads parameter can be the number of DNA sequence reads, the number of RNA sequence reads, or combinations thereof. The number of sequencing variants (e.g., DNA sequencing variants, RNA sequencing variants, encoded peptide variants, or combinations thereof) can be variants identified when comparing two samples from a subject (e.g., tumor samples and non-tumor samples) or when comparing a subject's sample with a reference sample (e.g., comparing a subject's tumor sample with a reference human genome). Tumor purity is the percentage or ratio of tumor sequence reads to healthy cell sequence reads (e.g., healthy tissue sequence reads). Higher tumor purity indicates the presence of less non-tumor genetic material and / or sequence reads in a biological sample. Sequencing depth indicates the number of nucleotides that contribute to a portion of the assembly. For example, a sequencing depth of 10X indicates that each nucleobase was sequenced an average of 10 times across 10 different read sequences. The number of protein modification sequencing variants can be the number of RNA variants, DNA variants, or combinations thereof that are predicted to encode protein variants. Protein variants can be determined by any method. For example, the number of protein variants can be determined by comparing proteins from a subject's healthy tissue with proteins from a subject's tumor tissue. As another example, the number of protein variants can be determined by comparing proteins or proteins encoded by a reference sequence (e.g., reference exome, reference genome, reference proteome) with proteins encoded by sequencing results from a subject's biological sample (e.g., proteins encoded by DNA sequencing results, RNA sequencing results, or combinations thereof from the biological sample). RNA confirmation rate is the ratio of protein modification variants identified by nucleic acid sequencing and confirmed by RNA sequencing (e.g., ratio,Percentage (e.g., the number of protein modification variants confirmed by RNA sequencing (e.g., batch RNA sequencing) divided by the total number of sequenced variants). For example, if 80 protein modification variants are observed by DNA sequencing and 25 protein modification variants are confirmed by RNA sequencing, the RNA confirmation rate is 0.3125 (i.e., 31.25%). RNA quality can be assessed by any method, including but not limited to the RNA integrity index (RIN, e.g., see Schroeder, A. et al. The RIN: an RNA integrity number for assigning integrity values ​​to RNA measurements. (2006) BMC Bioinformatics 7, 3, 1-14), the transcript integrity index (TIN, e.g., see Wang, L. et al. Measure transcript integrity using RNA-seq data. (2016) BMC Bioinformatics 17, 58, 1-16), or combinations thereof. The number of identified neoantigen peptides (e.g., identified long neoantigen peptides, identified short neoantigen peptides, and combinations of identified long and short neoantigen peptides), such as the number of identified long neoantigen peptides and / or the number of identified short neoantigen peptides, can be sequencing parameters used to analyze (e.g., compare) sequencing results of biological samples. The predicted immunogenicity of any number of neoantigen peptides can be a sequencing parameter. The number of neoantigen peptides with predictive immunogenicity can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. The number of neoantigenic peptides with predictive immunogenicity can be approximately 1 to approximately 3500, approximately 1 to approximately 3200, approximately 1 to approximately 2800, approximately 1 to approximately 2600, approximately 1 to approximately 2400, approximately 1 to approximately 2200, approximately 1 to approximately 2000, approximately 1 to approximately 1900, approximately 1 to approximately 1800, approximately 1 to approximately 1700, approximately 1 to approximately 1600, approximately 1 to approximately 1500, approximately 1 to approximately 1400, approximately 1 to approximately 1300, approximately 1 to approximately 1200, approximately 1 to approximately 1000, approximately 1 to approximately 1000, approximately 1 to approximately 950, approximately 1 to approximately 900.About 1 to about 850, about 1 to about 800, about 1 to about 750, about 1 to about 700, about 1 to about 650, about 1 to about 600, about 1 to about 550, about 1 to about 500, about 1 to about 480, about 1 to about 460, about 1 to about 440, about 1 to about 420, about 1 to about 400, about 1 to about 380, about 1 to about 360, about 1 to about 340, about 1 to about 320, about 1 to about 300, about 1 to about 280, about 1 to about 260, about 1 to about 240, about 1 to about 220, about 1 to about 200, about 1 to about 190, about 1 to about 18 0, about 1 to about 170, about 1 to about 160, about 1 to about 150, about 1 to about 140, about 1 to about 130, about 1 to about 120, about 1 to about 110, about 1 to about 100, about 1 to about 90, about 1 to about 85, about 1 to about 80, about 1 to about 75, about 1 to about 70, about 1 to about 65, about 1 to about 60, about 1 to about 55, about 1 to about 50, about 1 to about 45, about 1 to about 40, about 1 to about 35, about 1 to about 30, about 1 to about 29, about 1 to about 28, about 1 to about 27, about 1 to about 26, about 1 to about 25, about 1 About 24, about 1 to about 23, about 1 to about 22, about 1 to about 21, about 1 to about 20, about 1 to about 19, about 1 to about 18, about 1 to about 17, about 1 to about 16, about 1 to about 15, about 1 to about 14, about 1 to about 13, about 1 to about 12, about 1 to about 11, about 1 to about 10, about 1 to about 9, about 1 to about 8, about 1 to about 7, about 1 to about 6, about 1 to about 5, about 1 to about 4, about 1 to about 3, about 1 to about 2, about 2 to about 100, about 3 to about 100, about 4 to about 100, about 5 to about 100, about 6 to about 100, about 7 Approximately 100, approximately 8, approximately 9, approximately 10, approximately 11, approximately 12, approximately 13, approximately 14, approximately 15, approximately 16, approximately 17, approximately 18, approximately 19, approximately 20, approximately 22, approximately 24, approximately 26, approximately 28, approximately 30, approximately 33, approximately 36, approximately 40, approximately 44, approximately 44, approximately 100.Approximately 48 to approximately 100, approximately 50 to approximately 100, approximately 55 to approximately 100, approximately 60 to approximately 100, approximately 65 to approximately 100, approximately 70 to approximately 100, approximately 75 to approximately 100, approximately 80 to approximately 100, approximately 85 to approximately 100, approximately 90 to approximately 100, approximately 95 to approximately 100, or approximately 2 to approximately 100. The number of neoantigenic peptides with predictive immunogenicity can be at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 12, at least about 14, at least about 16, at least about 18, at least about 20, at least about 24, at least about 28, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, up to The number of sequences is approximately 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 160, 180, 200, 240, 280, 300, 350, 400, 450, or 500. In some embodiments, one or more sequencing parameters are the number of sequence reads, the number of sequencing variants, tumor purity, sequencing depth, the number of protein-modified sequencing variants, RNA confirmation rate, RNA quality, the number of identified long neoantigenic peptides, the number of identified short neoantigenic peptides, the predicted immunogenicity of the neoantigenic peptides, or combinations thereof.

[0078] As used herein, the terms “identified short neoantigen peptide” and “short peptide” can refer to peptides with an amino acid length of about 3 to about 15, about 3 to about 14, about 3 to about 13, about 3 to about 12, about 3 to about 11, about 3 to about 10, about 3 to about 9, about 3 to about 8, about 3 to about 7, about 3 to about 6, about 3 to about 5, about 3 to about 4, about 4 to about 15, about 5 to about 15, about 6 to about 15, about 7 to about 15, about 8 to about 15, about 9 to about 15, about 10 to about 15, about 11 to about 15, about 12 to about 15, about 13 to about 15, about 14 to about 15, about 8 to about 11, about 8 to about 10, about 8 to about 9, about 9 to about 11, about 10 to about 11, about 7 to about 11, about 6 to about 11, about 5 to about 11, about 4 to about 11, about 3 to about 11, about 8 to about 12, about 8 to about 13, or about 8 to about 14. "Identified short neoantigen peptide" and "short peptide" may refer to peptides with an amino acid length of about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15. "Identified short neoantigen peptide" and "short peptide" may refer to peptides with an amino acid length of up to about 15, up to about 14, up to about 13, up to about 12, up to about 11, up to about 10, up to about 9, up to about 8, up to about 7, up to about 6, up to about 5, up to about 4, up to about 3, or up to about 2. As used herein, the terms "identified long neoantigen peptide" and "long peptide" may refer to peptides with an amino acid length of about 13 to about 30, about 13 to about 29, about 13 to about 28, or about 15. 13 to 27, about 13 to 26, about 13 to 25, about 13 to 24, about 13 to 23, about 13 to 22, about 13 to 21, about 13 to 20, about 13 to 19, about 13 to 18, about 13 to 17, about 13 to 16, about 13 to 15, about 13 to 14, about 14 to 30, about 15 to 30, about 16 to 30, about 17 to 30, about 18 to 30, about 19 to 3 0, about 20 to about 21, about 22 to about 30, about 23 to about 30, about 24 to about 30, about 25 to about 30, about 26 to about 30, about 27 to about 30, about 28 to about 30, about 29 to about 30, or about 13 to about 25 peptides. "Identified long neoantigen peptides" and "long peptides" can refer to peptides with amino acid lengths of about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 25, about 26, about 27, about 30, about 28, about 30, about 29, about 30, or about 13 to about 25. 5. Peptides of about 26, about 27, about 28, about 29, or about 30. “Identified long neoantigen peptide” and “long peptide” may refer to peptides with an amino acid length of at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, or at least about 30.

[0079] Any number of sequencing parameters (e.g., comparisons between biological samples, e.g., between two or more biological samples) can be analyzed from sequencing results (e.g., initial sequencing results). Without wishing to be bound by any particular theory, it is believed that the analysis of sequencing parameters (e.g., comparative sequencing parameters between biological samples) can be used to determine the combination of initial sequencing results from two or more biological samples to produce joint sequencing results or to select representative biological samples. It is further believed that the method provides the most representative results of genomic variations in the source tissue of the biological samples (e.g., a combination of samples of similar quality, or the selection of samples of optimal quality), and that such representative results can be used to generate treatments for subjects (e.g., the production of neoantigen vaccines). Any sequencing parameters can be analyzed (e.g., compared between biological samples), including but not limited to the number of sequence reads (e.g., total number of sequence reads), the number of sequencing variants (e.g., the number of DNA sequencing variants, the number of RNA sequencing variants), tumor purity, sequencing depth, the number of protein modification sequencing variants (e.g., the number of protein modification sequencing variants confirmed by RNA), RNA confirmation rate, RNA quality, the number of identified long neoantigen peptides, the number of identified short neoantigen peptides, the predicted immunogenicity of neoantigen peptides, or combinations thereof. The number of different sequencing parameters being analyzed (e.g., for comparisons between biological samples) can be approximately 1, approximately 2, approximately 3, approximately 4, approximately 5, approximately 6, approximately 7, approximately 8, approximately 9, approximately 10, approximately 11, approximately 12, approximately 13, approximately 14, or approximately 15. (The repetition of the first instance is likely an error in the original text.) The number of different sequencing parameters analyzed (e.g., for comparison between biological samples) can be at least about 1, at least about 2, at least about 3, at least about 2, at least about 4, at least about 2, at least about 5, at least about 2, at least about 6, at least about 2, at least about 7, at least about 2, at least about 8, at least about 2, at least about 9, at least about 2, at least about 10, at least about 3, at least about 4, at least about 3, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, or at least about 4, at least about 10.For example, sequencing results of a first biological sample and a second biological sample can be compared based on the number of identified long neoantigenic peptides in the first and second biological samples. Continuing this example, if the number of identified long neoantigenic peptides between the first and second biological samples is similar (e.g., the difference is less than 20%, and the ratio between sequencing parameters is less than 1.20), the initial sequencing results of the first and second samples are combined to produce joint sequencing results. As another example, sequencing results of a first biological sample and a second biological sample can be analyzed (e.g., compared) based on tumor purity, number of protein modification variants, and RNA quality. Continuing this example, the sample with the highest tumor purity, number of protein modification variants, and RNA quality can be selected as the representative biological sample.

[0080] Sequencing parameters that are similar between biological samples can indicate whether it is appropriate to combine the sequencing results of biological samples to obtain a joint sequencing result. Sequencing parameters can be analyzed (e.g., compared) through any mathematical calculation. For example, the ratio between sequencing parameter values ​​that can produce sequencing results. In such a ratio, the larger sequencing parameter value among two sequencing parameter values ​​can be a molecular (e.g., A:B or...) The ratio of the sequencing parameter values ​​of the first biological sample to the sequencing parameter values ​​of the second biological sample (e.g., the ratio indicating that sequencing results will be combined to form a combined sequencing result) can be up to about 1.05, up to about 1.10, up to about 1.15, up to about 1.20, up to about 1.25, up to about 1.30, up to about 1.35, up to about 1.40, up to about 1.45, up to about 1.50, up to about 1.55, up to about 1.60, up to about 1.65, up to about 1.70, up to about 1.75, up to about 1.80, up to about 1.85, up to about 1.90, up to about 1.95, up to about 2.00, up to about 2.05, up to about 1. 2.10, up to approximately 2.15, up to approximately 2.20, up to approximately 2.25, up to approximately 2.30, up to approximately 2.35, up to approximately 2.40, up to approximately 2.45, up to approximately 2.50, up to approximately 2.55, up to approximately 2.60, up to approximately 2.65, up to approximately 2.70, up to approximately 2.75, up to approximately 2.80, up to approximately 2.85, up to approximately 2.90, up to approximately 2.95, up to approximately 3.00, up to approximately 3.10, up to approximately 3.20, up to approximately 3.30, up to approximately 3.40, up to approximately 3.50, up to approximately 3.60, up to approximately 3.70, up to approximately 3.80, up to approximately 3.90 or up to approximately 4.00. The ratio of sequencing parameter values ​​for the first biological sample to those for the second biological sample (e.g., the ratio indicating that sequencing results will be combined to form a combined sequencing result) can be approximately 1.00, approximately 1.05, approximately 1.10, approximately 1.15, approximately 1.20, approximately 1.25, approximately 1.30, approximately 1.35, approximately 1.40, approximately 1.45, approximately 1.50, approximately 1.55, approximately 1.60, approximately 1.65, approximately 1.70, approximately 1.75, approximately 1.80, approximately 1.85, approximately 1.90, approximately 1.95, or approximately 2.0. 0, approximately 2.05, approximately 2.10, approximately 2.15, approximately 2.20, approximately 2.25, approximately 2.30, approximately 2.35, approximately 2.40, approximately 2.45, approximately 2.50, approximately 2.55, approximately 2.60, approximately 2.65, approximately 2.70, approximately 2.75, approximately 2.80, approximately 2.85, approximately 2.90, approximately 2.95, approximately 3.00, approximately 3.10, approximately 3.20, approximately 3.30, approximately 3.40, approximately 3.50, approximately 3.60, approximately 3.70, approximately 3.80, approximately 3.90, or approximately 4.00.The ratio of sequencing parameter values ​​of the first biological sample to those of the second biological sample (e.g., the ratio indicating that sequencing results will be combined to form a combined sequencing result) can be from about 1.00 to about 4.00, from about 1.00 to about 3.80, from about 1.00 to about 3.60, from about 1.00 to about 3.40, from about 1.00 to about 3.20, from about 1.00 to about 3.00, from about 1.00 to about 2.80, from about 1.00 to about 2.60, from about 1.00 to about 4.00, from about 1.00 to about 3.80, from about 1.00 to about 2.60, from about 1.00 to about 3.60, from about 1.00 to about 3.0 ... 1.00 to about 2.40, about 1.00 to about 2.20, about 1.00 to about 2.00, about 1.00 to about 1.90, about 1.00 to about 1.80, about 1.00 to about 1.70, about 1.00 to about 1.60, about 1.00 to about 1.50, about 1.00 to about 1.40, about 1.00 to about 1.30, about 1.00 to about 1.20, about 1.00 to about 1.10, or about 1.00 to about 1.05. For example, the number of identified long neoantigen peptides may be 150 for the first biological sample and 100 for the second biological sample (i.e., a ratio of 1.50, a ratio of up to about 1.55, a ratio of about 1.00 to about 1.60), indicating that the sampling results should be combined to produce joint sequencing results.

[0081] Sequencing parameters that are similar between biological samples may indicate that combining the sequencing results of biological samples to obtain a joint sequencing result is inappropriate and / or that the biological sample should be selected as the representative biological sample in the methods described herein. Sequencing parameters can be analyzed (e.g., compared) through any mathematical calculation (such as a ratio). In such a ratio, the larger of two sequencing parameter values ​​can be a molecular (e.g., A:B or...) The ratio of the sequencing parameter values ​​of the first biological sample to the sequencing parameter values ​​of the second biological sample (e.g., the ratio indicating that sequencing results will not be combined to form a combined sequencing result, the ratio indicating that a biological sample will be selected as a representative biological sample) can be at least about 1.50, at least about 1.55, at least about 1.60, at least about 1.65, at least about 1.70, at least about 1.75, at least about 1.80, at least about 1.85, at least about 1.90, at least about 1.95, at least about 2.00, at least about 2.05, at least about 2.10, at least about 2.15, at least about 2.20, at least about 2.25, at least about 2.30, at least about 2.35, at least about 2.40, at least about 2.45, at least about 2.50, to Less than 2.55, at least 2.60, at least 2.65, at least 2.70, at least 2.75, at least 2.80, at least 2.85, at least 2.90, at least 2.95, at least 3.00, at least 3.10, at least 3.20, at least 3.30, at least 3.40, at least 3.50, at least 3.60, at least 3.70, at least 3.80, at least 3.90, at least 4.00, at least 4.50, at least 5.00, at least 5.50, at least 6.00, at least 6.50, at least 7.50, at least 8.00, at least 8.50, at least 9.00, at least 9.50 or at least 10.00. The ratio of sequencing parameter values ​​of the first biological sample to those of the second biological sample (e.g., the ratio of the indicator biological sample to be selected as the representative biological sample) can be approximately 1.50, approximately 1.55, approximately 1.60, approximately 1.65, approximately 1.70, approximately 1.75, approximately 1.80, approximately 1.85, approximately 1.90, approximately 1.95, approximately 2.00, approximately 2.05, approximately 2.10, approximately 2.15, approximately 2.20, approximately 2.25, approximately 2.30, approximately 2.35, approximately 2.40, approximately 2.45, approximately 2.50, approximately 2.55. Approximately 2.60, 2.65, 2.70, 2.75, 2.80, 2.85, 2.90, 2.95, 3.00, 3.10, 3.20, 3.30, 3.40, 3.50, 3.60, 3.70, 3.80, 3.90, 4.00, 4.50, 5.00, 5.50, 6.00, 6.50, 7.00, 7.50, 8.00, 8.50, 9.00, 9.50, or 10.00.The ratio of sequencing parameter values ​​of the first biological sample to those of the second biological sample (e.g., the ratio indicating that the biological sample will be selected as the representative biological sample) can be from about 1.50 to about 10.00, from about 1.75 to about 10.00, from about 2.00 to about 10.00, from about 2.25 to about 10.00, from about 2.50 to about 10.00, from about 3.00 to about 10.00, from about 3.50 to about 10.00, from about 4.00 to about 1 0.00, about 4.50 to about 10.00, about 5.00 to about 10.00, about 5.50 to about 10.00, about 6.00 to about 10.00, about 6.50 to about 10.00, about 7.00 to about 10.00, about 7.50 to about 10.00, about 8.00 to about 10.00, about 8.50 to about 10.00, about 9.00 to about 10.00, about 9.50 to about 10.00, about 1.50 From to approximately 9.50, from approximately 1.50 to approximately 9.0, from approximately 1.50 to approximately 8.50, from approximately 1.50 to approximately 8.00, from approximately 1.50 to approximately 7.50, from approximately 1.50 to approximately 7.00, from approximately 1.50 to approximately 6.50, from approximately 1.50 to approximately 6.00, from approximately 1.50 to approximately 5.50, from approximately 1.50 to approximately 5.00, from approximately 1.50 to approximately 4.50, from approximately 1.50 to approximately 4.00, from approximately 1.50 to approximately 3.80, from approximately 1. 50 to about 3.60, about 1.50 to about 3.40, about 1.50 to about 3.20, about 1.50 to about 3.00, about 1.50 to about 2.80, about 1.50 to about 2.60, about 1.50 to about 2.40, about 1.50 to about 2.20, about 1.50 to about 2.00, about 1.50 to about 1.90, about 1.50 to about 1.80, about 1.50 to about 1.70, or about 1.50 to about 1.60. For example, the number of long neoantigen peptides identified could be 1000 for the first biological sample and 200 for the second biological sample (i.e., a ratio of 5.00), indicating that the first biological sample would be selected as the representative biological sample.

[0082] Sequencing parameters can be used to assess the quality of sequencing results (e.g., sequencing results from biological samples from patients). A zero value for any sequencing parameter of the sequencing results described herein indicates that the corresponding biological sample should not be selected as a representative biological sample or should not be combined with another biological sample to produce a combined sequencing result. The value of a sequencing parameter can indicate the suitability of a sample for selection by the methods described herein, indicate that the biological sample is unsuitable for selection or combination with sequencing results from different biological samples, or indicate that another biological sample must be collected from the patient. A zero number of sequencing reads (e.g., total number of sequencing reads) in the sequencing results indicates that the sample is unsuitable for use in the methods or method steps described herein (e.g., unsuitable for selection or combination with sequencing results from different samples, indicating that another biological sample must be collected from the patient). A zero number of sequencing variants (e.g., number of DNA sequencing variants, number of RNA sequencing variants) in the sequencing results indicates that the sample is unsuitable for use in the methods or method steps described herein (e.g., unsuitable for selection of a representative biological sample or combination with sequencing results from different samples, indicating that another biological sample must be collected from the patient). A tumor purity of zero in the sequencing results indicates that the sample is unsuitable for use in the methods or steps described herein (e.g., unsuitable for selecting representative biological samples or combining with sequencing results from different samples, indicating that another biological sample must be collected from the patient). A sequencing depth of zero in the sequencing results indicates that the sample is unsuitable for use in the methods or steps described herein (e.g., unsuitable for selecting representative biological samples or combining with sequencing results from different samples, indicating that another biological sample must be collected from the patient). A protein modification sequencing variant number (e.g., the number of protein modification sequencing variants confirmed by RNA) of zero in the sequencing results indicates that the sample is unsuitable for use in the methods or steps described herein (e.g., unsuitable for selecting representative biological samples or combining with sequencing results from different samples, indicating that another biological sample must be collected from the patient). An RNA confirmation rate of zero in the sequencing results indicates that the sample is unsuitable for use in the methods or steps described herein (e.g., unsuitable for selecting representative biological samples or combining with sequencing results from different samples, indicating that another biological sample must be collected from the patient). A sequencing result with zero RNA quality indicates that the sample is unsuitable for use in the methods or steps described herein (e.g., unsuitable for selecting representative biological samples or combining sequencing results with different samples, indicating that another biological sample must be collected from the patient). A sequencing result with zero identified long neoantigenic peptides indicates that the sample is unsuitable for use in the methods or steps described herein (e.g., unsuitable for selecting representative biological samples or combining sequencing results with different samples, indicating that another biological sample must be collected from the patient). A sequencing result with zero identified short neoantigenic peptides indicates that the sample is unsuitable for use in the methods or steps described herein (e.g., unsuitable for selecting representative biological samples or combining sequencing results with different samples, indicating that another biological sample must be collected from the patient).A zero predicted immunogenicity of a neoantigen peptide in the sequencing results may indicate that the sample is not suitable for use in the methods or steps described herein (e.g., not suitable for selecting representative biological samples or for combining sequencing results with different samples, indicating that another biological sample must be collected from the patient).

[0083] Combinatorial sequencing results – combined sequencing results

[0084] Based on the analysis (e.g., comparison) of sequencing parameters (e.g., one or more sequencing parameters) of the initial sequencing results, sequencing results from two or more biological samples can be combined to produce joint sequencing results. Any method can be used to combine sequencing results, including but not limited to cascading of sequencing results (e.g., sequence reads), averaging of sequence read frequencies (e.g., weighted average of sequence read frequencies), joint determination of sequence reads, joint determination of variant sets, intersection of variant determination sets, or combinations thereof.

[0085] Combined sequencing results can be obtained by combining the sequencing results of any number of biological samples. The number of biological samples combined to produce combined sequence results (e.g., the number of biological sample sequencing results) can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. The number of biological samples combined to produce combined sequence results (e.g., the number of biological sample sequencing results) can be at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, or at least about 20. The number of biological samples combined to produce combined sequence results (e.g., the number of biological sample sequencing results) can be up to about 2, up to about 3, up to about 4, up to about 5, up to about 6, up to about 7, up to about 8, up to about 9, up to about 10, up to about 11, up to about 12, up to about 13, up to about 14, up to about 15, up to about 16, up to about 17, up to about 18, up to about 19, or up to about 20. The number of biological samples combined to produce the combined sequence results (e.g., the number of biological sample sequencing results) can be about 2 to about 20, about 2 to about 18, about 2 to about 16, about 2 to about 14, about 2 to about 12, about 2 to about 10, about 2 to about 9, about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2 to about 5, about 2 to about 4, about 2 to about 3, about 3 to about 20, about 4 to about 20, about 5 to about 20, about 6 to about 20, about 7 to about 20, about 8 to about 20, about 9 to about 20, about 10 to about 20, about 3 to About 10, about 3 to about 9, about 3 to about 8, about 3 to about 7, about 3 to about 6, about 3 to about 5, about 3 to about 4, about 4 to about 10, about 4 to about 9, about 4 to about 8, about 4 to about 7, about 4 to about 6, about 4 to about 5, about 5 to about 10, about 5 to about 9, about 5 to about 8, about 5 to about 7, about 5 to about 6, about 6 to about 10, about 6 to about 9, about 6 to about 8, about 6 to about 7, about 7 to about 10, about 7 to about 9, about 7 to about 8, about 8 to about 10, about 8 to about 9 or about 9 to about 10.

[0086] Co-sequencing results can be any type of nucleic acid sequencing result, including but not limited to raw nucleic acid sequencing reads (e.g., raw DNA sequence reads, raw RNA sequence reads, raw exome reads, raw whole genome reads), sequencing variants (e.g., DNA sequencing variants compared to the subject's healthy cells or tissues, RNA sequencing variants compared to the subject's healthy cells or tissues, protein modification sequencing variants, somatic mutant variants, germline mutant variants), predicted RNA sequences transcribed from DNA sequence reads, predicted neoantigen peptides (e.g., neoantigen peptides based on RNA sequence reads, neoantigen peptides based on DNA sequence reads), or combinations thereof. Co-sequencing results can be sequencing reads, sequencing variants, encoded peptides, or combinations thereof.

[0087] Any number of co-sequencing parameters from co-sequencing results (e.g., sequencing parameters from co-sequencing results obtained by combining initial sequencing results of two or more biological samples) may be compared with sequencing parameters from initial sequencing results as described herein (e.g., any sequencing parameters described herein). Without wishing to be bound by any particular theory, it is believed that comparison of one or more co-sequencing parameters with one or more sequencing parameters from initial sequencing results can be used to determine whether scoring the predicted immunogenicity of a neoantigen using the methods described herein is based on co-sequencing results of two or more biological samples or representative biological samples. Any co-sequencing parameters may be compared, including but not limited to the number of sequence reads (e.g., total number of sequence reads), the number of sequencing variants (e.g., the number of DNA sequencing variants, the number of RNA sequencing variants), tumor purity, sequencing depth, the number of protein modification sequencing variants (e.g., the number of protein modification sequencing variants confirmed by RNA), RNA confirmation rate, RNA quality, the number of identified long neoantigen peptides, the number of identified short neoantigen peptides, the predicted immunogenicity of the neoantigen peptide, or combinations thereof. The number of different combined sequencing parameters being compared can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The number of different combined sequencing parameters being compared can be approximately 1 to approximately 15, approximately 1 to approximately 14, approximately 1 to approximately 13, approximately 1 to approximately 12, approximately 1 to approximately 11, approximately 1 to approximately 10, approximately 1 to approximately 9, approximately 1 to approximately 8, approximately 1 to approximately 7, approximately 1 to approximately 6, approximately 1 to approximately 5, approximately 1 to approximately 4, approximately 1 to approximately 3, approximately 1 to approximately 2, approximately 2 to approximately 15, approximately 3 to approximately 15, approximately 4 to approximately 15, approximately 5 to approximately 15, approximately 6 to approximately 15, approximately 7 to approximately 15. Approximately 15, approximately 8 to approximately 15, approximately 2 to approximately 3, approximately 2 to approximately 4, approximately 2 to approximately 5, approximately 2 to approximately 6, approximately 2 to approximately 7, approximately 2 to approximately 8, approximately 2 to approximately 9, approximately 2 to approximately 10, approximately 3 to approximately 4, approximately 3 to approximately 5, approximately 3 to approximately 6, approximately 3 to approximately 7, approximately 3 to approximately 8, approximately 3 to approximately 9, approximately 3 to approximately 10, approximately 4 to approximately 5, approximately 4 to approximately 6, approximately 4 to approximately 7, approximately 4 to approximately 8, approximately 4 to approximately 9, or approximately 4 to approximately 10. The number of different combined sequencing parameters being compared can be at least approximately 1, at least approximately 2, at least approximately 3, at least approximately 4, at least approximately 5, at least approximately 6, at least approximately 7, at least approximately 8, at least approximately 9, or at least approximately 10. For example, the number of long neoantigen peptides identified in the combined sequencing results and the initial sequencing results of the first biological sample can be compared.Continuing this example, if the ratio of the number of identified long neoantigen peptides between the co-sequencing results and the initial sequencing results of the first biological sample is greater than 1.2 (e.g., the co-sequencing results contain 20% more identified long neoantigen peptides than the initial sequencing results), the co-sequencing results are used to score the predicted immunogenicity of the neoantigen using the methods described herein. As another example, the co-sequencing results and the initial sequencing results of the first biological sample can be compared based on tumor purity, number of protein modification variants, and RNA quality. Continuing this example, the sequencing results with the highest tumor purity, number of protein modification variants, and / or RNA quality can be selected as the basis for scoring the predicted immunogenicity of the neoantigen. In some embodiments, the method may also include a step of comparing one or more co-sequencing parameters of the co-sequencing results with one or more sequencing parameters of the initial sequencing results (e.g., sequencing parameters obtained by nucleic acid sequencing of the biological sample). In some implementations, one or more combined sequencing parameters are the number of sequence reads, the number of sequencing variants, tumor purity, sequencing depth, the number of protein-modified sequencing variants, RNA confirmation rate, RNA quality, the number of identified long neoantigen peptides, the number of identified short neoantigen peptides, the predicted immunogenicity of the neoantigen peptides, or a combination thereof.

[0088] Any ratio (e.g., combined-initial sequencing parameter ratio, combined:initial sequencing parameter ratio, etc.) The combined sequencing parameters of the biological sample and the sequencing parameters of the initial nucleic acid sequencing of the biological sample can indicate that the score for predicted immunogenicity will not be based on the combined sequencing results. The combined-initial sequencing parameter ratio (e.g., the ratio of the combined sequencing parameter value to the sequencing parameter value of the initial nucleic acid sequencing of the biological sample, the ratio indicating that the score for predicted immunogenicity will not be based on the combined sequencing results, the ratio indicating that the score for predicted immunogenicity will be based on the sequencing results of a representative biological sample) can be up to about 1.00, up to about 0.95, up to about 0.90, up to about 0.85, up to about 0.80, up to about 0.75, up to about 0.70, up to about 0.65, up to about 0.60, up to about 0.55, up to about 0.50, up to about 0.45, up to about 0.40, up to about 0.35, up to about 0.30, up to about 0.25, up to about 0.20, up to about 0.15, up to about 0.10, up to about 0.05, or up to about 0.01. The combined-initial sequencing parameter ratio (e.g., the ratio of combined sequencing parameter values ​​to the sequencing parameter values ​​of the initial nucleic acid sequencing of the biological sample, the ratio indicating that the score for predicted immunogenicity will not be based on the combined sequencing results, the ratio indicating that the score for predicted immunogenicity will be based on the sequencing results of a representative biological sample) can be about 0.95, about 0.90, about 0.85, about 0.80, about 0.75, about 0.70, about 0.65, about 0.60, about 0.55, about 0.50, about 0.45, about 0.40, about 0.35, about 0.30, about 0.25, about 0.20, about 0.15, about 0.10, about 0.05, or about 0.01.The combined-initial sequencing parameter ratio (e.g., the ratio of combined sequencing parameter values ​​to the sequencing parameter values ​​of the initial nucleic acid sequencing of the biological sample, the ratio indicating that the score for predicted immunogenicity will not be based on the combined sequencing results, the ratio indicating that the score for predicted immunogenicity will be based on the sequencing results of a representative biological sample) can be about 0.05 to about 0.95, about 0.10 to about 0.95, about 0.15 to about 0.95, about 0.20 to about 0.95, about 0.25 to about 0.95, about 0.30 to about 0.95, about 0.35 to about 0.95, about 0.40 to about 0.95, about 0.45 to about 0.95, about 0.50 to about 0.95, about 0.55 to about 0.95, about 0.60 to about 0.95, about 0.65 to about 0.95, about 0.70 to about 0. 0.95, about 0.75 to about 0.95, about 0.80 to about 0.95, about 0.85 to about 0.95, about 0.90 to about 0.95, about 0.05 to about 0.90, about 0.05 to about 0.85, about 0.05 to about 0.80, about 0.05 to about 0.75, about 0.05 to about 0.70, about 0.05 to about 0.65, about 0.05 to about 0.6 0, about 0.05 to about 0.55, about 0.05 to about 0.50, about 0.05 to about 0.45, about 0.05 to about 0.40, about 0.05 to about 0.35, about 0.05 to about 0.30, about 0.05 to about 0.25, about 0.05 to about 0.20, about 0.05 to about 0.15, about 0.05 to about 0.12, or about 0.05 to about 0.10. For example, the number of identified long neoantigen peptides could be 100 for co-sequencing results and 150 for initial sequencing results of a biological sample (i.e., a ratio of about 0.66), indicating that the score for predictive immunogenicity of one or more neoantigens would be based on sequencing results of a representative biological sample, rather than co-sequencing results.

[0089] Any ratio (e.g., the ratio of joint sequencing parameters to initial nucleic acid sequencing parameters of a biological sample) can indicate that the score for predicting immunogenicity will be based on the joint sequencing results. The combined-initial sequencing parameter ratio (e.g., the ratio of combined sequencing parameter values ​​to the sequencing parameter values ​​of the initial nucleic acid sequencing of the biological sample, or the ratio indicating that the score for predicting immunogenicity will be based on the combined sequencing results) can be at least about 1.05, at least about 1.10, at least about 1.15, at least about 1.20, at least about 1.25, at least about 1.30, at least about 1.35, at least about 1.40, at least about 1.45, at least about 1.50, at least about 1.55, at least about 1.60, at least about 1.65, at least about 1.70, at least about 1.75, at least about 1.80, at least about 1.85, at least about 1.90, at least about 1.95, at least about 2.00, at least about 2.05, at least about 2.10, at least about 2.15, at least about 2.20, at least about 2.25, at least about 2.30, at least about 2 .35, at least about 2.40, at least about 2.45, at least about 2.50, at least about 2.55, at least about 2.60, at least about 2.65, at least about 2.70, at least about 2.75, at least about 2.80, at least about 2.85, at least about 2.90, at least about 2.95, at least about 3.00, at least about 3.10, at least about 3.20, at least about 3.30, at least about 3.40, at least Approximately 3.50, at least approximately 3.60, at least approximately 3.70, at least approximately 3.80, at least approximately 3.90, at least approximately 4.00, at least approximately 4.50, at least approximately 5.00, at least approximately 5.50, at least approximately 6.00, at least approximately 6.50, at least approximately 7.00, at least approximately 7.50, at least approximately 8.00, at least approximately 8.50, at least approximately 9.00, at least approximately 9.50, or at least approximately 10.00.The combined-initial sequencing parameter ratio (e.g., the ratio of combined sequencing parameter values ​​to the sequencing parameter values ​​of the initial nucleic acid sequencing of the biological sample, indicating the ratio by which the score for predicting immunogenicity will be based on the combined sequencing results) can be approximately 1.05, approximately 1.10, approximately 1.15, approximately 1.20, approximately 1.25, approximately 1.35, approximately 1.40, approximately 1.45, approximately 1.50, approximately 1.55, approximately 1.60, approximately 1.65, approximately 1.70, approximately 1.75, approximately 1.80, approximately 1.85, approximately 1.90, approximately 1.95, approximately 2.00, approximately 2.05, approximately 2.10, approximately 2.15, approximately 2.20, approximately 2.25, approximately 2. 30, approximately 2.35, approximately 2.40, approximately 2.45, approximately 2.50, approximately 2.55, approximately 2.60, approximately 2.65, approximately 2.70, approximately 2.75, approximately 2.80, approximately 2.85, approximately 2.90, approximately 2.95, approximately 3.00, approximately 3.10, approximately 3.20, approximately 3.30, approximately 3.40, approximately 3.50, approximately 3.60, approximately 3.70, approximately 3.80, approximately 3.90, approximately 4.00, approximately 4.50, approximately 5.00, approximately 5.50, approximately 6.00, approximately 6.50, approximately 7.00, approximately 7.50, approximately 8.00, approximately 8.50, approximately 9.00, approximately 9.50, or approximately 10.00.The combined-initial sequencing parameter ratio (e.g., the ratio of combined sequencing parameter values ​​to the sequencing parameter values ​​of the initial nucleic acid sequencing of the biological sample, an indicator that the score for predicting immunogenicity will be based on the combined sequencing results) can be from about 1.05 to about 10.00, from about 1.10 to about 10.00, from about 1.15 to about 10.00, from about 1.20 to about 10.00, from about 1.25 to about 10.00, from about 1.30 to about 10.00, from about 1.35 to about 10.00, from about 1.40 to about 10.00, from about 1.45 to about 10.00, from about 1.50 to about 10.00, Approximately 1.75 to approximately 10.00, approximately 2.00 to approximately 10.00, approximately 2.25 to approximately 10.00, approximately 2.50 to approximately 10.00, approximately 3.00 to approximately 10.00, approximately 3.50 to approximately 10.00, approximately 4.00 to approximately 10.00, approximately 4.50 to approximately 10.00, approximately 5.00 to approximately 10.00, approximately 5.50 to approximately 10.00, approximately 6.00 to approximately 10.00, approximately 6.50 to approximately 10.00, approximately 7.00 to approximately 10.00, approximately 7.50 to approximately 10.00, approximately 8.00 to approximately 10.00, approximately 8.50 to approximately 10.00, about 9.00 to about 10.00, about 9.50 to about 10.00, about 1.05 to about 9.50, about 1.05 to about 9.00, about 1.05 to about 8.50, about 1.05 to about 8.00, about 1.05 to about 7.50, about 1.05 to about 7.00, about 1.05 to about 6.50, about 1.05 to about 6.00, about 1.05 to about 5.50, about 1.05 to about 5.00, about 1.05 to about 4.50, about 1.05 to about 4.00, about 1.05 to about 3.80, about 1.05 to about 3.60, about 1 The ratios are approximately 0.05 to 3.40, approximately 1.05 to 3.20, approximately 1.05 to 3.00, approximately 1.05 to 2.80, approximately 1.05 to 2.60, approximately 1.05 to 2.40, approximately 1.05 to 2.20, approximately 1.05 to 2.00, approximately 1.05 to 1.90, approximately 1.05 to 1.80, approximately 1.05 to 1.70, approximately 1.05 to 1.60, approximately 1.05 to 1.50, approximately 1.05 to 1.40, approximately 1.05 to 1.30, approximately 1.05 to 1.20, or approximately 1.05 to 1.10. For example, the number of identified long neoantigen peptides could be 150 for the combined sequencing results and 100 for the initial sequencing results of the biological sample (i.e., a ratio of approximately 1.50), indicating that the score for the predicted immunogenicity of one or more neoantigens would be based on the combined sequencing results.

[0090] neoantigen

[0091] Neoantigens are autoantigens produced by tumor cells due to genomic mutations or dysregulated RNA splicing. Neoantigens identified by sequencing as described herein can be in any form of sequencing result, including but not limited to sequence reads (DNA sequence reads, RNA sequence reads), sequence variants (e.g., peptide-modified sequence variants), encoded peptides, or combinations thereof. Neoantigens can be any type of peptide, including but not limited to long peptides, short peptides, or combinations thereof. In some embodiments, one or more neoantigens are peptides, which are long peptides, short peptides, or combinations thereof. Neoantigens described herein can be tumor-specific (e.g., the neoantigen is present only in cancer or tumors and not in the subject's healthy cells or germline cells).

[0092] Scoring the predictive immunogenicity of neoantigens

[0093] Sequencing results obtained by the methods described herein can be used to identify peptide sequences encoded by nucleic acids (e.g., encoded by DNA, encoded by RNA). The resulting peptide sequences can be analyzed to determine whether the encoded peptide is a neoantigen based on the presence of sequence variants (e.g., sequence variants in the peptide amino acid sequence compared to a subject's healthy cells or tissues, sequence variants in the peptide amino acid sequence compared to a reference genome). Neoantigens (e.g., neoantigen peptides) identified by the methods described herein can be analyzed to determine predicted immunogenicity based on any factors, including but not limited to whether the neoantigen is immunogenic (e.g., whether the neoantigen can elicit an immune response in a subject, prediction of major histocompatibility complex (MHC) binding affinity, whether the predicted peptide is presented on the cell surface via MHC molecules), whether the tumor expresses a sufficient amount of the neoantigen to elicit an immune response, whether the neoantigen is expressed on a sufficient proportion of tumor cells, or a combination thereof. Any method for predicting the immunogenicity of a neoantigen can be used in the methods described herein. For example, the number of sequencing variants (e.g., DNA sequence variants, RNA sequence variants, encoded peptide variants) can be used to predict the expression of neoantigens in biological samples and infer their expression in the subject's tissue of origin (e.g., in the subject's tumor). For several examples of predicting the immunogenicity of neoantigens, see WO2022 / 159176 A1, US 20230197192A1, US 20230173045A1, and US20220093209A1, all of which are incorporated herein by reference in full. Predicted immunogenicity can be used to score neoantigens. The scores of predicted immunogenicity can be sorted (e.g., neoantigens can be sorted in order of predicted immunogenicity). The scored neoantigens can be further selected for inclusion in a neoantigen vaccine (e.g., containing a neoantigen peptide, containing a nucleic acid encoding a neoantigen peptide). The scored neoantigens can be further selected for exclusion from a neoantigen vaccine (e.g., excluding a neoantigen peptide, excluding a nucleic acid encoding a neoantigen peptide).

[0094] Neoantigen vaccines and administration methods

[0095] The methods described herein may include steps for producing a neoantigen vaccine. The neoantigen vaccine may be any immunogenic composition, such as those described in WO2022 / 170067 A1, US2023 / 0173045 A1, WO2022 / 159176 A1, WO2022 / 251034 A1, or US2023 / 0173046 A1, the entire contents of all of which are incorporated herein by reference. The neoantigen vaccine may contain or encode a neoantigen (e.g., one or more neoantigens) for scoring predicted immunogenicity using the methods described herein. Neoantigen vaccines may contain any form of neoantigen, including but not limited to peptides (e.g., identified long peptides, identified short peptides, synthetic peptides, isolated peptides), RNA sequences encoding peptides (e.g., mRNA, mRNA containing non-natural nucleotides (e.g., pseudouridine, N1-methylpseudorabolic acid, 7-methylguanosine, N6-methyladenosine, 2'-O-methylnucleotide), mRNA containing inverse nucleotides, or combinations thereof), DNA sequences encoding peptides (e.g., plasmid DNA, viral DNA), or combinations thereof. Neoantigen vaccines containing nucleic acids encoding peptides (e.g., RNA, DNA) may be in the form of viruses (e.g., adenovirus, lentivirus, fowlpox, cowpox, self-replicating alphavirus, Maraba virus).

[0096] The nucleic acid (e.g., RNA, DNA) of a neoantigen vaccine can encode any number of peptide antigens. The number of peptide antigens encoded by the nucleic acid can be approximately 1, approximately 2, approximately 3, approximately 4, approximately 5, approximately 6, approximately 7, approximately 8, approximately 9, approximately 10, approximately 11, approximately 12, approximately 13, approximately 14, approximately 15, approximately 16, approximately 17, approximately 18, approximately 19, or approximately 20. The number of peptide antigens encoded by the nucleic acid can be up to approximately 1, up to approximately 2, up to approximately 3, up to approximately 4, up to approximately 5, up to approximately 6, up to approximately 7, up to approximately 8, up to approximately 9, up to approximately 10, up to approximately 11, up to approximately 12, up to approximately 13, up to approximately 14, up to approximately 15, up to approximately 16, up to approximately 17, up to approximately 18, up to approximately 19, or up to approximately 20. In some embodiments, the method includes the step of generating a neoantigen vaccine, wherein the neoantigen vaccine comprises or encodes one or more neoantigens for scoring predictive immunogenicity by the method.

[0097] Neoantigen vaccines may contain or encode any number of neoantigens (e.g., neoantigen proteins, DNA sequences encoding neoantigen proteins, RNA sequences encoding neoantigen proteins). Neoantigen vaccines may contain or encode approximately 1, approximately 2, approximately 3, approximately 4, approximately 5, approximately 6, approximately 7, approximately 8, approximately 9, approximately 10, approximately 11, approximately 12, approximately 13, approximately 14, approximately 15, approximately 16, approximately 17, approximately 18, approximately 19, approximately 20, approximately 21, approximately 22, approximately 23, approximately 24, approximately 25, approximately 26, approximately 27, approximately 28, approximately 29, or approximately 30 neoantigens. The number of neoantigens contained in or encoded by a neoantigen vaccine can be one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, nineteen or more, twenty or more, eleven or more, twentieth or thirteenth, thirteenth or thirteenth, thirteenth or thirteenth, thirteenth or thirteenth, thirteenth or thirteenth, twentieth or thirteenth, thirteenth or thirteenth, twentieth or thirteenth, thirteenth or thirteenth, twentieth or thirteenth, thirteenth or thirteenth, thirteenth or thirteenth, thirteenth or thirteenth, thirteenth or thirteenth, thirteenth or thirteenth, or thirteenth or thirteenth. The number of neoantigens contained in or encoded by a neoantigen vaccine may be at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, or at least about 30.The number of neoantigens contained or encoded in a neoantigen vaccine can be approximately 1 to approximately 50, approximately 2 to approximately 50, approximately 3 to approximately 50, approximately 4 to approximately 50, approximately 5 to approximately 50, approximately 6 to approximately 50, approximately 7 to approximately 50, approximately 8 to approximately 50, approximately 9 to approximately 50, approximately 10 to approximately 50, approximately 11 to approximately 50, approximately 12 to approximately 50, approximately 13 to approximately 50, approximately 14 to approximately 50, approximately 15 to approximately 50, approximately 16 to approximately 50, approximately 17 to approximately 50, approximately 18 to approximately 50, approximately 19 to approximately 50, approximately 20 to approximately 50, approximately 22 to approximately 50, and so on. 50, about 24 to about 50, about 26 to about 50, about 28 to about 50, about 30 to about 50, about 33 to about 50, about 36 to about 50, about 40 to about 50, about 45 to about 50, about 1 to about 45, about 1 to about 40, about 1 to about 36, about 1 to about 33, about 1 to about 30, about 1 to about 28, about 1 to about 26, about 1 to about 24, about 1 to about 22, about 1 to about 20, about 1 to about 18, about 1 to about 16, about 1 to about 14, about 1 to about 12, about 1 to about 10 Approximately 1 to approximately 9, approximately 1 to approximately 8, approximately 1 to approximately 7, approximately 1 to approximately 6, approximately 1 to approximately 5, approximately 1 to approximately 4, approximately 1 to approximately 3, approximately 1 to approximately 2, approximately 2 to approximately 14, approximately 2 to approximately 12, approximately 2 to approximately 10, approximately 2 to approximately 8, approximately 2 to approximately 6, approximately 2 to approximately 4, approximately 3 to approximately 14, approximately 3 to approximately 12, approximately 3 to approximately 10, approximately 3 to approximately 8, approximately 3 to approximately 6, approximately 3 to approximately 4, approximately 4 to approximately 14, approximately 4 to approximately 12, approximately 4 to approximately 10, approximately 4 to approximately 8, approximately 4 to approximately 6, approximately 5 to approximately 14 About 5 to about 12, about 5 to about 10, about 5 to about 8, about 5 to about 6, about 6 to about 14, about 6 to about 12, about 6 to about 10, about 6 to about 8, about 7 to about 14, about 7 to about 12, about 7 to about 10, about 7 to about 8, about 8 to about 14, about 8 to about 12, about 8 to about 10, about 9 to about 14, about 9 to about 12, about 9 to about 10, about 10 to about 14, about 10 to about 12, about 11 to about 14, about 11 to about 12, about 12 to about 14 or about 13 to about 14.

[0098] The neoantigen vaccine described herein may contain up to about 50 neoantigen long peptides and / or short peptides. A neoantigen vaccine may contain about 10 to about 20 neoantigen long peptides and / or short peptides. In some embodiments, the neoantigen vaccine contains about 19 neoantigen long peptides and / or short peptides.

[0099] Neoantigen vaccines may contain at least about 2 or more neoantigen long peptides. Neoantigen vaccines may contain about 2 to about 18 neoantigen long peptides. Neoantigen vaccines may contain at least about 10 to about 15 neoantigen long peptides. Neoantigen vaccines may contain at least about 2 or more neoantigen short peptides. Neoantigen vaccines may contain at least about 2 to about 10 neoantigen short peptides.

[0100] As an example, three peptide libraries of a neoantigen vaccine may contain approximately five neoantigen long peptides and / or short peptides, and one peptide library may contain four neoantigen long peptides and / or short peptides, as well as an accessory peptide. Each peptide library may contain different neoantigen long peptides and / or short peptides. The length of the neoantigen long peptides may be approximately 15 to approximately 30 amino acids. The length of the neoantigen short peptides may be approximately 5 to approximately 15 amino acids.

[0101] Neoantigen vaccines may contain at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50 or more neoantigen peptides (e.g., neoantigen long peptides and / or short peptides). Neoantigen vaccines may contain up to about 100 neoantigen peptides. Neoantigen vaccines may contain approximately 10-20 neoantigens, approximately 10-30 neoantigens, approximately 10-40 neoantigens, approximately 10-50 neoantigens, approximately 10-60 neoantigens, approximately 10-70 neoantigens, approximately 10-80 neoantigens, approximately 10-90 neoantigens, or approximately 10-100 neoantigens. In some embodiments, a neoantigen vaccine contains at least approximately 10 neoantigens. In some embodiments, the neoantigen vaccines disclosed herein may contain 10 to approximately 20 neoantigens. For example, a neoantigen vaccine may contain approximately 10, approximately 11, approximately 12, approximately 13, approximately 14, approximately 15, approximately 16, approximately 17, approximately 18, approximately 19, or approximately 20 neoantigens. In some embodiments, a neoantigen vaccine may contain approximately 19 neoantigens. In some embodiments, a neoantigen vaccine may contain approximately 20 neoantigens. Each neoantigen in a neoantigen vaccine can be different.

[0102] The neoantigen vaccines described herein may also contain adjuvants. An adjuvant is any substance incorporated into a neoantigen vaccine to increase or otherwise enhance and / or strengthen the immune response against tumor-specific neoantigens, but which, when administered alone, does not produce an immune response against tumor-specific neoantigens. Adjuvants can produce an immune response against neoantigens without causing allergic reactions or other adverse reactions. This document considers that adjuvants may be administered before, together with, simultaneously with, or after the administration of the neoantigen vaccine.

[0103] Adjuvants can enhance immune responses through several mechanisms, including, for example, lymphocyte recruitment, stimulation of B and / or T cells, and stimulation of macrophages. When the neoantigen vaccine described herein contains or is administered with one or more adjuvants, the adjuvants that may be used include, but are not limited to, mineral salt adjuvants or mineral salt gel adjuvants, particulate adjuvants, microparticle adjuvants, mucosal adjuvants, immunostimulatory adjuvants, or combinations thereof. Examples of adjuvants include, but are not limited to, aluminum salts (alum) (e.g., aluminum hydroxide, aluminum phosphate, and aluminum sulfate), 3-de-O-acylated monophosphoryl lipid A (MPL) (see GB 2220211), MF59 (Novartis), AS03 (Glaxo SmithKline), AS04 (Glaxo SmithKline), polysorbate 80 (Tween 80; ICL Americas, Inc.), imidazopyridine compounds (see International Application No. PCT / US2007 / 064857, published under International Publication No. WO2007 / 109812), imidazoquinoxaline compounds (see International Application No. PCT / US2007 / 064858, published under International Publication No. WO2007 / 109813), and saponins, such as QS21 (see Kensil et al., Vaccine Design: The Subunit and Adjuvant Approach (edited by Powell and Newman, Plenum Press, NY). (1995); U.S. Patent No. 5,057,540). In some embodiments, the adjuvant is Freund's adjuvant (complete or incomplete). Other adjuvants are oil-in-water emulsions (such as squalene or peanut oil), which are optionally combined with an immunostimulant such as monophospholipid A (see Stoute et al., N. Engl. J. Med. 336, 86-91 (1997)).

[0104] CpG immunostimulatory oligonucleotides have been reported to enhance the effect of adjuvants in the vaccine setting. Other TLR-binding molecules, such as RNA-binding TLR 7, TLR 8, and / or TLR 9, may also be used.

[0105] Other examples of useful adjuvants include, but are not limited to, chemically modified CpGs (e.g., CpR, Idera), poly(I:C) (e.g., polyi:CI2U), polyICLCs, non-CpG bacterial DNA or RNA, and immunologically active small molecules and antibodies such as cyclophosphamide, sunitinib, bevacizumab, Celebrex (celecoxib), NCX-4016, sildenafil, tadalafil, vardenafil, sorafenib, XL-999, CP-547632, pazopanib, ZD2171, AZD2171, ipilimumab, tremelimumab, and SC58175, which may act therapeutically and / or as adjuvants.

[0106] The neoantigen vaccine described herein may be administered to subjects who have been diagnosed with cancer, already have cancer, have recurrent cancer (i.e., relapse), or are at risk of developing cancer. The neoantigen vaccine described herein may be administered to subjects resistant to other forms of cancer treatment (e.g., chemotherapy, immunotherapy, or radiation). The neoantigen vaccine may be administered to subjects prior to other standard-of-care cancer therapies (e.g., chemotherapy, immunotherapy, or radiation). The neoantigen vaccine may be administered to subjects concurrently with, after, or in combination with other standard-of-care cancer therapies (e.g., chemotherapy, immunotherapy, or radiation). In some embodiments, the method includes the step of administering the neoantigen vaccine to a subject in need.

[0107] The neoantigen vaccine described herein is administered to subjects in an amount sufficient to elicit an immune response against a tumor-specific neoantigen and to disrupt or at least partially prevent symptoms and / or complications. In embodiments, the neoantigen vaccine can provide a durable immune response. A durable immune response can be established by administering a booster dose of the neoantigen vaccine to the subject. An immune response to the neoantigen vaccine can be prolonged by administering a booster dose to the subject. In embodiments, at least one, at least two, at least three, or more booster doses can be administered to reduce cancer. A first booster dose can increase the immune response by at least 50%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, or at least 1000%. A second booster dose can increase the immune response by at least 50%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, or at least 1000%. A third booster dose can increase the immune response by at least 50%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, or at least 1000%.

[0108] The amount sufficient to elicit an immune response is defined as the "therapeutic effective dose." The effective dose for this purpose will depend on factors such as the composition, method of administration, stage and severity of the disease being treated, the patient's weight and overall health, and the prescribing physician's judgment. It should be remembered that neoantigen vaccines are generally used for severe disease states, i.e., life-threatening or potentially life-threatening conditions, especially when cancer has metastasized. In such cases, given the minimization of foreign substances and the relatively non-toxic nature of neoantigens, the treating physician may feel the need to administer a significantly excessive dose of the neoantigen vaccine.

[0109] The neoantigen vaccine described herein may be administered to subjects alone or in combination with other therapeutic agents. These therapeutic agents may be, for example, chemotherapy agents, radiation, or immunotherapy. Any suitable therapeutic treatment for a specific cancer may be administered. Exemplary chemotherapy agents include, but are not limited to, adeleukin, hexamethicone, amifostine, asparaginase, bleomycin, capecitabine, carboplatin, carmustine, cladribine, cisapride, cisplatin, cyclophosphamide, cytarabine, dacarbazine (DTIC), dextrin, docetaxel, doxorubicin, dronabinol, epoetin alfa, etoposide, filgrastim, fludarabine, fluorouracil, gemcitabine, granisetron, and hydroxyurea. Idarubicin, ifosfamide, interferon-alpha, irinotecan, lansoprazole, levamisole, leucovorin, megestrol acetate, mesna, methotrexate, metoclopramide, mitomycin, mitotane, mitoxantrone, omeprazole, ondansetron, paclitaxel (Taxol®), pilocarpine, prochlorperazine, rituximab, tamoxifen, topotecan hydrochloride, trastuzumab, vinblastine, vincristine, and vinorelbine tartrate. Small molecule or targeted therapies (e.g., kinase inhibitors) may be administered to subjects. Anti-CTLA antibodies, anti-PD-1 antibodies, or anti-PD-L1 antibodies may be further administered to subjects. Blocking CTLA-4 or PD-L1 with antibodies can enhance the patient's immune response to cancer cells.

[0110] Example

[0111] Example 1. Analysis of initial sequencing results and combined sequencing results from patient tissue biopsies.

[0112] Core needle biopsies (i.e., cores #1 and #2) were collected from the tumor of patient A and processed for nucleic acid sequencing via whole-exome sequencing (WES). The raw sequencing reads from each core needle biopsy were analyzed for the encoded neoantigenic peptides. The predicted immunogenicity of the observed neoantigenic peptides was scored, and they were ranked according to their predicted immunogenicity scores (see [link to relevant documentation]). Figure 2(Left and middle columns). Predicted immunogenicity scoring was performed in a manner similar to that described in US 20230197192A1. Raw sequencing reads (e.g., WES DNA and bulk RNA sequencing reads) from two core needle biopsy samples were tandemly analyzed, and the encoded neoantigenic peptides were analyzed (e.g., co-sequencing results). Predicted immunogenicity of observed neoantigenic peptides from the co-sequencing results was scored, and similarly, single core needle biopsy cores were sorted according to predicted immunogenicity (see [reference]). Figure 2 (Right column). In Figure 2 In the diagram, overlapping or contained exemplary peptides are indicated by "*" and "#". Notably, the combined sequencing results identified a novel antigenic peptide ( LKGQAFLPLVLEPRR LPVGPL (SEQ ID NO: 1, score 0.859), whose predicted immunogenicity score was higher than any core (represented by "#", core #1: P LKGQAFLPLVLEPRR (SEQ ID NO: 2), Core #2: P LKGQAFLPLVLEPRRL (SEQ ID NO: 3), where underline The overlapping amino acids were indicated, with scores of 0.73 and 0.784, respectively. Similar sequencing and analysis were performed on individual core needle biopsy samples from different patients (i.e., patient B, results not shown).

[0113] Visualize the number of identified long neoantigen peptides from patients A and B using Venn diagrams of each core needle biopsy result and each co-sequencing result (see [link to Venn diagram]). Figure 3A and Figure 3B Results from patient A indicate that each of core 1, core 2, and the combined sequencing results contains only one uniquely identified long neoantigen peptide not found in the other sequencing results (see [link]). Figure 3A In contrast, sequencing results from patient B indicated that core 2 contained six uniquely identified long neoantigen peptides (see [link to relevant documentation]). Figure 3B Samples potentially included in neoantigen vaccines were selected using a cutoff value of the top 48 peptides sorted by predicted immunogenicity. A Venn diagram using this cutoff value is shown in [the diagram / illustration]. Figure 4A and Figure 4B In, respectively corresponding to Figure 3A and Figure 3B A subset of the long neoantigen peptides identified in the overall identification.

[0114] Example 2. Sequencing parameter analysis of initial nucleic acid sequencing results from patient tissue samples.

[0115] Two core needle biopsy samples were collected from each of three patients (i.e., patients C, D, and E) and prepared for whole-exome sequencing of their DNA. Sequencing parameters for each initial sequencing result are shown in Table 1. In some cases (e.g., the total number of variants in patient D, the number of identified long neoantigenic peptides in patient E, or the number of protein modification variants confirmed by RNA), the sequencing parameter values ​​between the two core needle biopsy samples from a particular patient were nearly equal (e.g., the ratio of sequencing parameter values ​​was approximately 1.0). In other cases (e.g., the number of protein modification variants or RNA confirmation rate in patient C), the sequencing parameter values ​​between the two core needle biopsy samples from a particular patient were not equal (e.g., the ratio of sequencing parameter values ​​was not approximately 1.0, but at least 1.5). Table 1 shows the samples selected as representative biological samples from a set of biological samples.

[0116] Neoantigen vaccines containing the identified neoantigen peptides were generated for each patient. The neoantigen vaccine for patient D was based on the predicted immunogenicity score of a combination of cores 1 and 2 (i.e., combined sequencing results). Neoantigen vaccines for patients C and E were based on the predicted immunogenicity scores of representative cores 1 and 2, respectively. The decision of which core to use as the representative core for each patient, or whether to combine the sequencing results of cores 1 and 2, was based on the sequencing parameters in Table 1, and a Venn diagram (similar to...) of the identified neoantigen peptides was generated. Figure 3A , Figure 3B , Figure 4A and Figure 4B Those shown; that is, the number of identified neoantigenic peptides in each sequencing result, whether shared between sequencing results or unique to each sequencing result.

[0117] Table 1. Sequencing parameters of initial sequencing results for patients C, D, and E

[0118]

Claims

1. A method for scoring the predictive immunogenicity of one or more neoantigens in one or more biological samples from a subject, comprising the following steps: a) Prepare two or more biological samples for nucleic acid sequencing; b) Perform nucleic acid sequencing on the two or more biological samples to produce initial sequencing results for each of the biological samples; c) Evaluate the initial sequencing results by analyzing one or more sequencing parameters of the initial sequencing results; d) Based on the analysis of the sequencing parameters of the initial sequencing results of the biological samples, combine the initial sequencing results of the two or more biological samples to produce joint sequencing results; as well as e) Based on the combined sequencing results of the biological samples, score the predicted immunogenicity of one or more neoantigens in the biological samples of the subjects in need.

2. A method for scoring the predictive immunogenicity of one or more neoantigens in one or more biological samples from a subject, comprising the following steps: a) Prepare two or more biological samples for nucleic acid sequencing; b) Perform nucleic acid sequencing on the two or more biological samples to produce initial sequencing results for each of the biological samples; c) Evaluate the initial sequencing results by analyzing one or more sequencing parameters of the initial sequencing results; d) Based on the analysis of the sequencing parameters of the initial sequencing results of the biological samples, select representative biological samples; e) Based on the sequencing results of the representative biological samples, score the predicted immunogenicity of one or more neoantigens in the representative biological samples of the subjects in need.

3. The method of claim 1, wherein each of the biological samples is independently a biopsy sample selected from the group consisting of excisional biopsy, liquid biopsy, excisional biopsy, needle aspiration biopsy, perforation biopsy, and scraping biopsy.

4. The method of claim 1, wherein at least two of the biological samples are derived from different regions of the subject's body.

5. The method of claim 1, wherein at least two of the biological samples are derived from different regions of the subject's tumor.

6. The method of claim 1, wherein at least one of the biological samples is derived from the primary tumor of the subject.

7. The method of claim 1, wherein at least one of the biological samples is derived from a secondary tumor of the subject.

8. The method of claim 1, wherein at least two of the biological samples are collected from the subject at different times at least about one day apart.

9. The method of claim 1, wherein the sequencing parameters are selected from the group consisting of: number of sequence reads, number of sequencing variants, tumor purity, sequencing depth, number of protein-modified sequencing variants, RNA confirmation rate, RNA quality, number of identified long neoantigen peptides, number of identified short neoantigen peptides, predicted immunogenicity of neoantigen peptides, and combinations thereof.

10. The method of claim 1, wherein the initial sequencing results are selected from the group consisting of sequencing reads, sequencing variants, encoded peptides, and combinations thereof.

11. The method of claim 1, further comprising the step of: One or more sequencing parameters of the combined sequencing results are compared with one or more sequencing parameters of the initial sequencing results of step c).

12. The method of claim 11, wherein the combined sequencing parameters are selected from the group consisting of: number of sequence reads, number of sequencing variants, tumor purity, sequencing depth, number of protein-modified sequencing variants, RNA confirmation rate, RNA quality, number of identified long neoantigen peptides, number of identified short neoantigen peptides, predicted immunogenicity of neoantigen peptides, and combinations thereof.

13. The method of claim 1, wherein the nucleic acid sequencing is the sequencing of nucleic acids as RNA, DNA, or a combination thereof.

14. The method of claim 1, wherein the nucleic acid sequencing is selected from the group consisting of whole exome sequencing, whole genome sequencing, RNA sequencing, single-cell sequencing, targeted combinatorial sequencing, and combinations thereof.

15. The method of claim 1, wherein at least two different sequencing technologies are used in the nucleic acid sequencing step of the biological sample.

16. The method of claim 1, wherein in the nucleic acid sequencing step, at least two biological samples of the biological samples are sequenced using different sequencing technologies.

17. The method of claim 1, further comprising the step of: The biological sample was then sequenced.

18. The method of claim 17, wherein the subsequent sequencing is selected from the group consisting of whole exome sequencing, whole genome sequencing, RNA sequencing, single-cell sequencing, targeted combinatorial sequencing, and combinations thereof.

19. The method of claim 17, wherein in the subsequent sequencing step, at least two biological samples of the biological samples are sequenced using different sequencing technologies.

20. The method of claim 1, wherein the one or more neoantigens are peptides selected from the group consisting of long peptides, short peptides, and combinations thereof.

21. The method of claim 1, further comprising the step of: A neoantigen vaccine is produced, wherein the neoantigen vaccine comprises or encodes one or more neoantigens that are scored against the predicted immunogenicity.

22. The method of claim 21, further comprising the step of: The neoantigen vaccine was administered to the subjects who needed it.

23. The method of claim 2, wherein the sequencing parameters are selected from the group consisting of: number of reads, number of sequencing variants, tumor purity, sequencing depth, number of protein-modified sequencing variants, RNA confirmation rate, RNA quality, number of identified long neoantigen peptides, number of identified short neoantigen peptides, predicted immunogenicity of neoantigen peptides, and combinations thereof.

24. The method of claim 2, wherein the nucleic acid sequencing is selected from the group consisting of whole exome sequencing, whole genome sequencing, RNA sequencing, single-cell sequencing, targeted combinatorial sequencing, and combinations thereof.

25. The method of claim 2, further comprising the following step: The representative biological samples were then sequenced.

26. The method of claim 25, wherein the subsequent sequencing is selected from the group consisting of whole exome sequencing, whole genome sequencing, RNA sequencing, single-cell sequencing, targeted combinatorial sequencing, and combinations thereof.

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