Methods and materials for assessing and treating cancers

Abstract

This document provides methods and materials for assessing and / or treating subjects (e.g., humans) suspected of having cancer. For example, this document provides methods and materials for a nucleic acid sequence analysis which can determine a sequence of B cell receptor. In some cases, determining a sequence of B cell receptor (and, optionally, identifying the presence of one or more mutations and / or identifying the presence of aneuploidy) in DNA (e.g., cell-free DNA (cfDNA)) in a fluid sample (e.g., a cerebrospinal fluid sample) obtained from a subject (e.g., a human subject such as a human suspected of having cancer) can be used to identify the subject as having cancer.

Description

[0001] Attorney Docket No. 44807-0503WO1 / C18573

[0002] METHODS AND MATERIALS FOR ASSESSING AND TREATING CANCERS

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of U. S. Patent Application Serial No. 63 / 722,908, filed on November 20, 2024. The disclosure of the prior application is considered part of, and is incorporated by reference in, the disclosure of this application.

[0005] STATEMENT REGARDING FEDERAL FUNDING

[0006] This invention was made with government support under GM136577 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0007] TECHNICAL FIELD

[0008] This document relates to methods and materials for assessing and / or treating subjects (e.g., humans) suspected of having cancer. For example, this document provides methods and materials for a nucleic acid sequence analysis which can determine a sequence of B cell receptor. In some cases, determining a sequence of B cell receptor (and, optionally, identifying the presence of one or more mutations and / or identifying the presence of aneuploidy) in DNA (e.g., cellular DNA and cell-free DNA (cfDNA)) in a fluid sample (e.g., a cerebrospinal fluid sample) obtained from a subject (e.g., a human subject such as a human suspected of having cancer) can be used to identify the subject as having cancer.

[0009] BACKGROUND

[0010] Brain cancers represent a heterogenous but highly aggressive class of neoplasms. They can be broadly categorized as primary or metastatic. Glioblastoma and medulloblastoma represent the most common types of primary brain cancers in adults and children respectively (Ostrom et al. Neuro-Oncol. 22:ivl-iv96 (2020); Weller et al. Nat. Rev. Dis. Primer 10:33 (2024); and Cohen, N. Engl. J. Med. 386:1922-1931 (2022)). Brain metastases occur in over 200,000 individuals in the United States every year and can described as parenchymal, which occur in the substance of the brain and are more common, or leptomeningeal, which occur in the lining of the brain in up to 10% of all cancer patients (Lamba, Neuro-Oncol. 23:1447-1456 (2021); and Wilcox et al. Neuro-Oncol. 26:1781-1804 Attorney Docket No. 44807-0503WO1 / C18573

[0011] (2024)). Lung, breast, colon, melanoma, and renal cancers represent the most common cancer types to metastasize to the brain. Despite aggressive multi-modality treatments, both primary and metastatic brain cancers are associated with abysmal long-term survival, with many forms of brain cancer being incurable (Schaff, JAMA 329:574-587 (2023); van den Bente / a / . Lancet Loud. Engl. 402:1564-1579 (2023); Girardi etal. Neuro-Oncol. 25:593-606 (2023); and Girardi et al. Neuro-Oncol. 25:580-592 (2023)).

[0012] The primary and almost exclusive methodology to diagnose brain cancers remains neurosurgical biopsy, which has significant inherent risks and expense. A brain biopsy is unlike tissue sampling in any other organ. It typically requires general anesthesia with inpatient hospitalization, and it carries a 5-10% risk for neurological decline, a 1% risk for catastrophic hemorrhage, and a 5-10% risk for a non-diagnostic result (Malone et al. World Neurosurg. 84:1084-1089 (2015); Pasternak et al. Neurosurg. Rev. 44:2597-2609 (2021); Katzendobler et al. Front. Neurol. 13:822362 (2022); Ragel et al. J. Neurooncol. 125:481— 501 (2015); Riche et al. Neurosurg. Rev. 44:301-307 (2021); Bex, Neurosurg. Rev. 46:5 (2022); and Riche et al. J. Neurosurg. 136:867-876 (2022)). In select cases, such as CNS lymphoma or leptomeningeal disease, CSF cytology can aid in diagnosis but the sensitivity is often relatively low (Rahimi, Handb. Clin. Neurol. 145:563-571 (2017); Morell et al. Neuro-Oncol. Pract. 6:415-423 (2019); Hoang-Xuan et al. Lancet Oncol. 16:e322-332 (2015); Tabatabai et al. Arch. Pathol. Lab. Med. 142:833-837 (2018); andNakasu et al. Neuro-Oncol. Adv. 5:vdad002 (2023)). In addition, outside of tissue sampling, there is no methodology to understand the molecular and cellular composition of CNS cancers.

[0013] SUMMARY

[0014] This document provides methods and materials for assessing and / or treating subjects (e.g., humans) having cancer. In some cases, this document provides methods and materials for sequencing a B cell receptor (BCR). For example, provided herein are methods for determining a sequence of a double-stranded DNA molecule of a BCR gene or a fragment thereof using primers designed to amplify the J segments of the IGH gene. In some cases, this document provides methods and materials for identifying a subject as having cancer. For example, a sequence of a BCR (and, optionally, identifying the presence of one or more Attorney Docket No. 44807-0503WO1 / C18573

[0015] mutations and / or identifying the presence of aneuploidy) in DNA (e.g., cellular DNA and cfDNA) in a fluid sample (e.g., a cerebrospinal fluid (CSF) sample) obtained from a subject suspected of having cancer can be used to identify the subject as having cancer. In some cases, this document provides methods and materials for treating a subject identified as having cancer. For example, a subject identified as having cancer as described herein (e.g., based, at least in part, on a sequence of a BCR (and, optionally, identifying the presence of one or more mutations and / or identifying the presence of aneuploidy) in DNA (e.g., cellular DNA and cfDNA) in a fluid sample (e.g., a CSF sample) obtained from the subject can be administered one or more cancer treatments.

[0016] As demonstrated herein, a “CSF-BAM” assay can simultaneously identify B-cell receptor rearrangements, Aneuploidy, and Mutations using polymerase chain reaction (PCR)-mediated amplification of both strands of the DNA from CSF samples.

[0017] Also as demonstrated herein, DNA (e.g., cellular DNA and cfDNA) in CSF samples from humans having cancer include (a) clonal compositions of BCRs, (b) aneuploidy, and / or (c) one or more mutations that are not present in CSF samples from humans that do not have cancer (e.g., healthy subjects).

[0018] Having the ability to sequence BCRs in a fluid sample (e.g., a CSF sample) as described herein provides a unique and unrealized opportunity to identify a subject (e.g., a human subject) as having a cancer. Further, the methods and materials provided herein (e.g., for determining a sequence of BCR (and, optionally, identifying the presence of one or more mutations and / or identifying the presence of aneuploidy)) can enable identification of BCR clonal expansions, chromosomal copy number changes, and somatic mutations thus allowing accurate diagnoses of central nervous system neoplasms quickly and without the need for surgical biopsy.

[0019] In general, one aspect of this document features methods for determining a sequence of a nucleic acid encoding a portion of a BCR in a sample. The methods can include, or consist essentially of, (a) attaching a 3' adapter fragment to each 3' end of a double-stranded DNA molecule and a 5' adapter fragment to each 5' end of the double-stranded DNA molecule to generate an adapted double-stranded DNA molecule, where the adapted doublestranded DNA molecule includes an adapted Watson strand and an adapted Crick strand, Attorney Docket No. 44807-0503WO1 / C18573

[0020] where the 3' adapter fragment includes a molecular barcode, a primer sequence, and an adapter sequence, and where the molecular barcode of the adapted Watson strand is the reverse complement of the molecular barcode of the adapted Crick strand; (b) copying both strands of the adapted double-stranded DNA molecule, where the copying includes performing around of linear extension of the adapted double- stranded DNA molecule, generating an adapted double-stranded Watson template and an adapted double-stranded Crick template, thereby generating a duplex sequencing library; (c) generating a first population of analyte DNA fragments from the adapted double-stranded Watson template in the duplex sequencing library by amplifying the adapted double-stranded Watson template with a first set of a Watson-target selective primer pair including (i) a first Watson target-selective primer including a sequence complementary to the 3' adapter sequence, and (ii) a second Watson target-selective primer including a sequence complementary to the J segment of the BCR gene sequence, and generating a first set of sequencing reads for at least one member of the first population of analyte DNA fragments; (d) generating a second population of analyte DNA fragments from the adapted double-stranded Crick template in the duplex sequencing library by amplifying the adapted double-stranded Crick template with a first set of a Crick-target selective primer pair including (i) a first Crick target-selective primer having a sequence complementary to the 3' adapter sequence, and (ii) a second Crick target-selective primer having a sequence complementary to the J segment of a BCR gene sequence and generating a second set of sequencing reads for at least one member of the second population of analyte DNA fragments; (e) grouping the first sequencing reads according to the molecular barcode present on the at least one member of the first population of analyte DNA fragments to generate a first analyte DNA family; (f) grouping the second sequencing reads according to the molecular barcode present on the at least one member of the second population of analyte DNA fragments to generate a second analyte DNA family; (g) analyzing the first sequencing reads of the first analyte DNA family; and (h) analyzing the second sequencing reads of the second analyte DNA family, thus, determining the sequence of the nucleic acid encoding the BCR. The 3' adaptor fragment can include a partially double-stranded molecular barcode. The partially double-stranded molecular barcode can include an endogenous barcode, an exogenous barcode, or both. Step (b) can include Attorney Docket No. 44807-0503WO1 / C18573

[0021] performing the round of linear extension of the adapted double-stranded DNA molecule with (i) a first primer complementary to the 3' adapter sequence, and (ii) a second primer complementary to the complement of the 5' adapter sequence. Steps (c) and (d) can be performed under PCR conditions. The second Watson target-selective primer can comprise, consist essentially of, or consist of a sequence set forth in Table 1 A. The second Crick target-selective primer can comprise, consist essentially of, or consist of a sequence set forth in Table 1 A. The double-stranded DNA molecule can include a V(D)J sequence of the BCR. The sample can be a blood sample, a CSF sample, a urine sample, or a tissue sample.

[0022] In another aspect, this document features methods for assessing a sample. The methods can include, or consist essentially of: 1) determining a sequence of a nucleic acid encoding a portion of a BCR, the determining including: (a) attaching a 3' adapter fragment to each 3' end of a double-stranded DNA molecule and a 5' adapter fragment to each 5' end of the double-stranded DNA molecule to generate an adapted double-stranded DNA molecule, where the adapted double-stranded DNA molecule includes an adapted Watson strand and an adapted Crick strand, where the 3’ adapter fragment includes a molecular barcode, a primer sequence, and an adapter sequence, and where the molecular barcode of the adapted Watson strand is the reverse complement of the molecular barcode of the adapted Crick strand; (b) copying both strands of the adapted double-stranded DNA molecule, where the copying includes performing a round of linear extension of the adapted double-stranded DNA molecule, generating an adapted double-stranded Watson template and an adapted double-stranded Crick template, thereby generating a duplex sequencing library; (c) generating a first population of analyte DNA fragments from the adapted double-stranded Watson template in the duplex sequencing library by amplifying the adapted double-stranded Watson template with a first set of a Watson-target selective primer pair including (i) a first Watson target-selective primer having a sequence complementary to the 3' adapter sequence, and (ii) a second Watson target-selective primer having a sequence complementary to the J segment of the BCR gene sequence, and generating a first set of sequencing reads for at least one member of the first population of analyte DNA fragments; (d) generating a second population of analyte DNA fragments from the adapted double-stranded Crick template in the duplex sequencing library by amplifying the adapted double-stranded Crick template with a Attorney Docket No. 44807-0503WO1 / C18573

[0023] first set of a Crick-target selective primer pair including (i) a first Crick target-selective primer having a sequence complementary to the 3' adapter sequence, and (ii) a second Crick target- selective primer having a sequence complementary to the J segment of a BCR gene sequence and generating a second set of sequencing reads for at least one member of the second population of analyte DNA fragments; (e) grouping the first sequencing reads according to the molecular barcode present on the at least one member of the first population of analyte DNA fragments to generate a first analyte DNA family; (f) grouping the second sequencing reads according to the molecular barcode present on the at least one member of the second population of analyte DNA fragments to generate a second analyte DNA family; (g) analyzing the first sequencing reads of the first analyte DNA family; and (h) analyzing the second sequencing reads of the second analyte DNA family, thus, determining the sequence of the nucleic acid encoding the BCR; 2) identifying the presence of aneuploidy within the sample; and 3) identifying the presence of a mutation within the sample.

[0024] In another aspect, this document features methods for assessing a subject suspected of having a cancer where the methods can include, or consist essentially of: (1) assessing a sample obtained from the subject where the assessing includes: (a) determining a sequence of a nucleic acid encoding a portion of a BCR, (b) identifying the presence of aneuploidy within the sample; and (c) identifying the presence of a mutation within the sample; (2) identifying the presence of a cancer within the subject when: (a) the analyte DNA fragments including the nucleic acid encoding the portion of the BCR have a clonal fraction that is at least 0.3, (b) a presence of aneuploidy is identified, and / or (c) a present of at least one mutation shown in Table 7 is identified. The sample can be a blood sample, a CSF sample, a urine sample, or a tissue sample. The subject can be a human. The cancer can be a B-cell cancer. The B-cell cancer can be a diffuse large B-cell lymphoma (DLBCL), a follicular lymphoma, a chronic lymphocytic leukemia (CLL), a small lymphocytic lymphoma (SLL), a mantle cell lymphoma (MCL), a Burkitt lymphoma, or a primary central nervous system lymphoma. The cancer can be a brain cancer. The brain cancer can be a high-grade glioma, a medulloblastoma, a spinal ganglioglioma, a diffuse midline glioma, a CNS lymphoma, an ependymoma, or a metastatic lesion to the brain. Attorney Docket No. 44807-0503WO1 / C18573

[0025] In another aspect, this document features methods for assessing a subject suspected of having a cancer where the methods can include, or consist essentially of: (1) assessing a sample obtained from the subject where the assessing includes: (a) determining a sequence of a nucleic acid encoding a portion of a BCR, (b) identifying the presence of aneuploidy within the sample; and (c) identifying the presence of a mutation within the sample; (2) identifying the lack of a cancer within the subject when: (a) the analyte DNA fragments including the nucleic acid encoding the portion of the BCR is identified as having a clonal fraction that is less than 0.3, (b) an absence of aneuploidy is identified, and (c) an absence of the mutations shown in Table 7 is identified. The sample can be a blood sample, a CSF sample, a urine sample, or a tissue sample. The subject can be a human. The cancer can be a B-cell cancer. The B-cell cancer can be a diffuse large B-cell lymphoma (DLBCL), a follicular lymphoma, a chronic lymphocytic leukemia (CLL), a small lymphocytic lymphoma (SLL), a mantle cell lymphoma (MCL), a Burkitt lymphoma, or a primary central nervous system lymphoma. The cancer can be a brain cancer. The brain cancer can be a high-grade glioma, a medulloblastoma, a spinal ganglioglioma, a diffuse midline glioma, a CNS lymphoma, an ependymoma, or a metastatic lesion to the brain.

[0026] In another aspect, this document features methods for treating a subject having a cancer. The methods can include, or consist essentially of: (1) assessing a sample obtained from the subject where the assessing includes: (a) determining a sequence of a nucleic acid encoding a portion of a BCR, (b) identifying the presence of aneuploidy within the sample; and (c) identifying the presence of a mutation within the sample; (2) administering a cancer treatment to the subject when: (a) the analyte DNA fragments including the nucleic acid encoding the portion of the BCR have a clonal fraction that is at least 0.3, (b) a presence of aneuploidy is identified, and / or (c) a presence of at least one mutation shown in Table 7 is identified. The sample can be a blood sample, a CSF sample, a urine sample, or a tissue sample. The subject can be a human. The cancer can be a B-cell cancer. The B-cell cancer can be a diffuse large B-cell lymphoma (DLBCL), a follicular lymphoma, a chronic lymphocytic leukemia (CLL), a small lymphocytic lymphoma (SLL), a mantle cell lymphoma (MCL), a Burkitt lymphoma, or a primary central nervous system lymphoma. The cancer can be a brain cancer. The brain cancer can be a high-grade glioma, a Attorney Docket No. 44807-0503WO1 / C18573

[0027] medulloblastoma, a spinal ganglioglioma, a diffuse midline glioma, a CNS lymphoma, an ependymoma, or a metastatic lesion to the brain. The cancer treatment can include administering chemotherapy, subjecting the subject to radiation therapy, and / or subjecting the subject to tumor resection surgery.

[0028] In another aspect, this document features methods for treating a cancer. The methods can include, or consist essentially of, administering a cancer treatment to a subject having a sample assessed for: (a) a sequence of a nucleic acid encoding a portion of a BCR, (b) the presence of aneuploidy within the sample; and (c) the presence of a mutation within the sample, where: (a) the analyte DNA fragments including the nucleic acid encoding the portion of the BCR are identified as having a clonal fraction that is at least 0.3, (b) a presence of aneuploidy is identified, and / or (c) a presence of at least one of the mutations shown in Table 7 is identified. The sample can be a blood sample, a CSF sample, a urine sample, or a tissue sample. The subject can be a human. The cancer can be a B-cell cancer. The B-cell cancer can be a diffuse large B-cell lymphoma (DLBCL), a follicular lymphoma, a chronic lymphocytic leukemia (CLL), a small lymphocytic lymphoma (SLL), a mantle cell lymphoma (MCL), a Burkitt lymphoma, or a primary central nervous system lymphoma. The cancer can be a brain cancer. The brain cancer can be a high-grade glioma, a medulloblastoma, a spinal ganglioglioma, a diffuse midline glioma, a CNS lymphoma, an ependymoma, or a metastatic lesion to the brain. The cancer treatment can include administering chemotherapy, subjecting the subject to radiation therapy, and / or subjecting the subject to tumor resection surgery.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Attorney Docket No. 44807-0503WO1 / C18573

[0030] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0031] BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1: CSF-BAM overview. CSF was obtained and DNA was extracted from the entire sample. CSF-BAM examined three analytes simultaneously: B-cell receptor rearrangements, aneuploidy, and mutations using PCR-meditated amplification of both strands of the DNA.

[0033] Figure 2: Schematic of CSF-BAM. Panel A) Independent libraries were generated from both strands of the original DNA template molecules. Panel B) BCRs were evaluated with SafeBSeqS. Illustrated here is amplification of BCRs with a primer targeting the IGHJ1 segment, among the multiplex set of 4 total IGHJ primers. Panel C) Aneuploidy was evaluated with WGS through amplification of total libraries using adapter-specific primers. Panel D) Mutations were evaluated with SaferSeqS. Illustrated here is amplification of IDH1 using and / / 9 / 7 / -specific primer, among the multiplex set of 120 total gene-specific primers.

[0034] Figure 3: Performance of CSF-BAM and each analyte. The sensitivity of each analyte within CSF-BAM is demonstrated across the major class of tumors tested. The composite sensitivity of CSF-BAM is demonstrated in orange.

[0035] Figures 4A-4C: Figure 2A) BCR clonality, Figure 2B) estimated tumor fraction, and Figure 4C) mutant allele frequency for each sample evaluated with CSF-BAM.

[0036] Figure 5: SafeBSeqS amplification. On-target reads and UIDs recovered for each IGHJ gene segment.

[0037] Figures 6A-6B: Aneuploidy reproducibility from two independent aliquots and libraries. Figure 6A) A representative non-cancer individual that was predicted diploid in both replicates. Figure 6B) A representative individual with cancer that was predicted aneuploid in both replicates.

[0038] Figure 7: Metrics for targeted panel. The number of duplex, Watson UIDs, and Crick UIDs, as well as Watson and Crick on-target rates are plotted. Attorney Docket No. 44807-0503WO1 / C18573

[0039] Figure 8: Correlation of predicted aneuploidy neoplastic content to mutation neoplastic content.

[0040] Figure 9: BCR UIDs recovered for each sample.

[0041] Figure 10: B cell clonality for evaluable samples with total UIDs >=20.

[0042] Figure 11 A: ROC for classification of lymphoma samples versus samples of all other cancer types for evaluable samples with total UIDs >=20. AUC = 0.97, 95% confidence interval 0.93-1.00.

[0043] Figure 11B: IGHV4-34 gene segment usage. Number of samples with the top clone using the IGHV4-34 gene segment versus all other IGHV segments for lymphoma versus all other sample types. P = 0.0029 by Fisher's exact test.

[0044] Figure 12A: CSF-BAM in setting of pseudoprogression. Gadolinium enhanced axial MRI demonstrating increasing enhancement in right frontal lobe of GLIA793. Patient had previously been treated with chemotherapy and radiation therapy. Repeat resection demonstrated pseudoprogression and no active tumor. CSF-BAM was negative in a CSF sample obtained prior to repeat resection.

[0045] Figure 12B: CSF-BAM identified targetable mutation. Gadolinium enhanced sagittal MRI demonstrating increasing size of a spinal ganglioglioma. CSF-BAM detected aBRAF V600E mutation that can be targeted therapeutically.

[0046] Figure 13. Performance of CSF-BAM and each analyte. The sensitivity of each analyte within CSF-BAM is demonstrated across the major classes of tumors tested. The composite sensitivity of CSF-BAM is demonstrated in black.

[0047] Figure 14. A, BCR clonality (clonality for non-evaluable samples with total UIDs <20 defined as 0), B, estimated tumor fraction by aneuploidy, and C, mutant allele frequency for each sample evaluated with CSF-BAM.

[0048] Figure 15. SafeBSeqS amplification. A, on-target reads and B, UIDs recovered for each IGHJ gene segment.

[0049] Figure 16. Aneuploidy reproducibility from two independent aliquots and libraries. A, A representative sample from a non-cancer individual that is predicted diploid in both replicates. B, A representative sample from an individual with cancerthat is predicted aneuploid in both replicates. Attorney Docket No. 44807-0503WO1 / C18573

[0050] Figure 17. Metrics for targeted mutation panel. The number of A, duplex, B, Watson, and C, Crick UIDs, as well as D, Watson and E, Crick on-target rates are plotted.

[0051] Figure 18. Correlation of predicted aneuploidy neoplastic content to mutation neoplastic content.

[0052] Figure 19. BCR UIDS recovered for each CSF sample.

[0053] Figure 20. BCR clonality for evaluable samples with total UIDs >20. B cell clonality variation P < 0.001 by Kruskal-Wallis test.

[0054] Figure 21. BCR clonality ROC and BCR IGHV4-34 gene segment usage. A, ROC for classification of CNS lymphoma samples versus samples of all other cancer types for evaluable samples with total UIDs >20. AUC = 0.95, 95% confidence interval 0.89-1.00. B, IGHV4-34 gene segment usage. Number of samples with the top clone using the IGHV4-34 gene segment versus all other IGHV segments for CNS lymphoma versus all other sample types. P < 0.0001 by Fisher’s exact test.

[0055] Figure 22. Case reports demonstrating CSF-BAM clinical applicability. A, CSF-BAM in the setting of pseudoprogression. Gadolinium enhanced axial MRI demonstrating increasing enhancement in right frontal lobe of patient GLIA793. The patient had previously been treated with chemotherapy and radiation therapy. Repeat resection demonstrated pseudoprogression and no active tumor. CSF-BAM was negative in a CSF sample obtained prior to the repeat resection where only pseudoprogression was detected. B, CSF-BAM can identify targetable mutations. Gadolinium enhanced sagittal MRI is shown demonstrating increasing size of a spinal ganglioglioma. CSF-BAM prior to resection detected a BRAF p. V600E mutation that can be targeted therapeutically.

[0056] DETAILED DESCRIPTION

[0057] High throughput sequencing can be used for the characterization of BCR repertoires. Existing methods for library preparation that begin with RNA as a template generally use adapter ligation or 5' RACE strategies. These methods can incorporate unique identifiers (UIDs) to increase accuracy. However, because cells can contain multiple BCR transcripts, quantification of clone abundance is confounded. In addition, RNA templates may not be obtainable from samples with decreased nucleotide quality, including fixed specimens. Attorney Docket No. 44807-0503WO1 / C18573

[0058] Methods that begin with DNA as a template for library preparation use multiplex PCR schemes or gene capture schemes. These methods are subject to bias from sources that include primer competition and differential amplification efficiencies. Complex methods are required to account for bias such as computational corrections, the use of spike-in standards, and primer balancing. Accordingly, existing methods for BCR sequencing are expensive, complex, require sophisticated or elaborate library preparation methods, or exhibit elements of all of these limitations. Moreover, even advanced methods still display systematic biases along with limitations in sensitivity, reproducibility, and quantification accuracy.

[0059] This document provides methods and materials for sequencing a BCR. For example, provided herein are methods for determining a sequence of a double-stranded DNA molecule of a BCR using primers designed to amplify the J segment of a BCR gene (e.g., the J segment of a IGH gene, also referred to herein as a IGHJ gene) or a fragment thereof (e g., to generate an amplicon to be assessed, also referred to as an analyte DNA fragment). The methods and materials provided herein can be useful for accurately identifying BCR sequences present in a nucleic acid sample. The methods for sequencing a BCR provided herein (also referred to as SafeBSeqS) include identifying the BCR sequences by using both Watson and Crick strands of a double-stranded DNA molecule. Such methods are particularly useful for characterizing and quantifying BCR sequences and allowing for the identification of BCR repertoires with high confidence.

[0060] In some cases, the methods and materials provided herein (e.g., for determining a sequence of BCR (and, optionally, identifying the presence of one or more mutations and / or identifying the presence of aneuploidy)) can be used to independently assess each strand of a double-stranded DNA molecule.

[0061] In some cases, the methods and materials provided herein (e.g., for determining a sequence of BCR (and, optionally, identifying the presence of one or more mutations and / or identifying the presence of aneuploidy)) can be performed without the need for amplifying all possible V and J gene segment pairs of a BCR.

[0062] In some cases, the methods and materials provided herein (e.g., for determining a sequence of BCR (and, optionally, identifying the presence of one or more mutations and / or identifying the presence of aneuploidy)) can determine BCR sequences with a low error rate. Attorney Docket No. 44807-0503WO1 / C18573

[0063] For example, the methods and materials described herein can be used to determine BCR sequences in a double-stranded DNA molecule (e.g., in an analyte DNA fragment amplified from a double-stranded DNA molecule) with an error rate of less than about 1% (e.g., less than about 0.1%, less than about 0.05%, or less than about 0.01%). In some cases, the methods and materials described herein can be used to determine BCR sequences in a double-stranded DNA molecule with an error rate of from about 0.001% to about 0.01%. In some cases, the error rate associated with the identification of BCR sequences in analyte DNA fragments according to a method described herein is no more than 1×10-2, no more than 1×10-3, no more than 1×10-4, no more than 1×10-5, no more than 1×10-6, no more than 5×10-6, or no more than 1×10-7. In some cases, the error rate associated with the identification of BCR sequences in analyte DNA fragments according to a method described herein is reduced by at least 2-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold (e.g., as compared to an error rate of a method for sequencing a BCR that does not require the use of both Watson and Crick strands of an analyte DNA fragment).

[0064] In some cases, the methods and materials provided herein (e.g., for determining a sequence of BCR (and, optionally, identifying the presence of one or more mutations and / or identifying the presence of aneuploidy)) can be used to achieve efficient duplex recovery. For example, methods provided herein can be used to recover PCR amplification products derived from both the Watson strand and the Crick strand of a double-stranded DNA molecule. In some cases, the methods described herein can be used to achieve at least 50% (e.g., about 50%, about 60%, about 70%, about 75%, about 80%, about 82%, about 85%, about 88%, about 90%, about 93%, about 95%, about 97%, about 99%, or 100%) duplex recovery.

[0065] In some cases, the methods and materials provided herein (e.g., for determining a sequence of BCR (and, optionally, identifying the presence of one or more mutations and / or identifying the presence of aneuploidy)) can be used to determine BCR sequences having low allele frequency. For example, methods provided herein can be used to determine BCR sequences having low allele frequency of less than about 1% (e.g., less than about 0.1%, less Attorney Docket No. 44807-0503WO1 / C18573

[0066] than about 0.05%, or less than about 0.01%). In some cases, the methods provided herein can be used to determine BCR sequences having low allele frequency of about 0.001%.

[0067] In some cases, the methods provided herein can be used to determine BCR sequences that are present in double-stranded DNA molecule (e.g., a double-stranded DNA molecule present in a sample such as a fluid sample obtained from a subject such as a human subject) at a frequency of 0.1% or less. In some cases, the methods described herein can be used to determine BCR sequences that are present in double-stranded DNA molecule at a frequency of 0.1% to 0.00001%. In some cases, the methods described herein can be used to determine BCR sequences that are present in a double-stranded DNA molecule at a frequency of 0.1% to 0.01%.

[0068] The methods and materials provided herein (e.g., for determining a sequence of BCR (and, optionally, identifying the presence of one or more mutations and / or identifying the presence of aneuploidy)) can be used to determine a BCR sequence from any appropriate double-stranded DNA molecule. Examples of double-stranded DNA molecules that can be used as a template for the methods provided herein include, without limitation, cellular DNA (e.g., genomic DNA) and cfDNA (e.g., circulating cell-free DNA (ccfDNA), circulating tumor DNA (ctDNA), and cell-free fetal DNA (cfDNA)). In some cases, double-stranded DNA molecule can be obtained from DNA (e.g., cfDNA) present in a biological sample. For example, double-stranded DNA molecules can be obtained from DNA present in a fluid sample obtained from a subject (e.g., a human subject).

[0069] A double-stranded DNA molecule that can serve as a template for the materials and methods provided herein can be any appropriate length (e.g., can include any appropriate number of nucleotides). In some cases, a double-stranded DNA molecule can include from about 4 to about 1000 nucleotides (e.g., about 10 to about 1000, about 20 to about 1000, about 30 to about 1000, about 40 to about 1000, about 50 to about 1000, about 60 to about 1000, about 70 to about 1000, about 80 to about 1000, about 90 to about 1000, about 100 to about 1000, about 250 to about 1000, about 500 to about 1000, about 750 to about 1000, about 4 to about 750, about 10 to about 750, about 20 to about 750, about 30 to about 750, about 40 to about 750, about 50 to about 750, about 60 to about 750, about 70 to about 750, about 80 to about 750, about 90 to about 750, about 100 to about 750, about 250 to about Attorney Docket No. 44807-0503WO1 / C18573

[0070] 750, about 500 to about 750, about 4 to about 500, about 10 to about 500, about 20 to about 500, about 30 to about 500, about 40 to about 500, about 50 to about 500, about 60 to about 500, about 70 to about 500, about 80 to about 500, about 90 to about 500, about 100 to about 500, about 250 to about 500, about 4 to about 250, about 10 to about 250, about 20 to about 250, about 30 to about 250, about 40 to about 250, about 50 to about 250, about 60 to about 250, about 70 to about 250, about 80 to about 250, about 90 to about 250, about 100 to about 250, about 4 to about 100, about 10 to about 100, about 20 to about 100, about 30 to about 100, about 40 to about 100, about 50 to about 100, about 60 to about 100, about 70 to about 100, about 80 to about 100, about 90 to about 100, about 4 to about 90, about 10 to about 90, about 20 to about 90, about 30 to about 90, about 40 to about 90, about 50 to about 90, about 60 to about 90, about 70 to about 90, about 80 to about 90, about 4 to about 80, about 10 to about 80, about 20 to about 80, about 30 to about 80, about 40 to about 80, about 50 to about 80, about 60 to about 80, about 70 to about 80, about 4 to about 70, about 10 to about 70, about 20 to about 70, about 30 to about 70, about 40 to about 70, about 50 to about 70, about 60 to about 70, about 4 to about 60, about 10 to about 60, about 20 to about 60, about 30 to about 60, about 40 to about 60, about 50 to about 60, about 4 to about 50, about 10 to about 50, about 20 to about 50, about 30 to about 50, about 40 to about 50, about 4 to about 40, about 10 to about 40, about 20 to about 40, about 30 to about 40, about 4 to about 30, about 10 to about 30, about 20 to about 30, about 4 to about 20, about 10 to about 20, or about 4 to about 10). In some cases, the length of the nucleic acid fragment to be analyzed may be less than 1000 (e.g., less than 750, less than 500, less than 250, less than 100, less than 50, or less than 20) nucleotides.

[0071] In some cases, methods for determining BCR sequences of a double-stranded DNA molecule (e.g., a nucleic acid encoding a portion of a BCR) can include generating a duplex sequencing library having a duplex molecular barcode on each end (e.g., the 5' end and the 3' end) of each nucleic acid in the library, generating a library of single stranded Watson strand-derived sequences and a library of single stranded Crick-strand derived sequences from the duplex sequencing library, and determining BCR sequences of the double-stranded DNA molecule in each single stranded library. The presence of a first molecular barcode in a 3' duplex adapter and a second molecular barcode present in a 5' adapter can be used to Attorney Docket No. 44807-0503WO1 / C18573

[0072] distinguish amplification products derived from the Watson strand from amplification products derived from the Crick strand.

[0073] Any appropriate method can be used to generate a duplex sequencing library. In some cases, a duplex sequencing library can be generated as described in Example 1, and also can be referred to as SaferSeqS libraries. As used herein, a duplex sequencing library is a plurality of nucleic acid fragments including a duplex molecular barcode on at one end (e.g., the 5' end and / or the 3' end) of each nucleic acid fragment in the library and can allow both strands of a double-stranded DNA molecule to be sequenced. In some cases, a nucleic acid sample can be fragmented to generate nucleic acid fragments, and the generated nucleic acid fragments can be used to generate a duplex sequencing library. Nucleic acid fragments used to generate a duplex sequencing library can also be referred to herein as input nucleic acid. For example, when nucleic acid fragments used to generate a duplex sequencing library are DNA fragments, the DNA fragments can also be referred to herein as input DNA. A duplex sequencing library can include any appropriate number of nucleic acid fragments. In some cases, generating a duplex sequencing library can include fragmenting a double-stranded DNA molecule and ligating adapters to each end of each nucleic acid fragment in the library. In some cases, a duplex sequencing library can be generated as described elsewhere (see, e.g., International Patent Application Publication No. WO 2021 / 163546 and International Patent Application Publication No. WO 2023 / 150277).

[0074] When generating a duplex sequencing library, the methods and materials provided herein (e.g., for determining a sequence of BCR (and, optionally, identifying the presence of one or more mutations and / or identifying the presence of aneuploidy)) can use any appropriate molecular barcode. As used herein, the term “molecular barcode” or “barcode” refers to a label, or identifier, that conveys or is capable of conveying information (e.g., information about an analyte DNA fragment). A molecular barcode can be part of an analyte, or independent of an analyte. In some cases, a molecular barcode can be attached to an analyte. In some cases, a particular molecular barcode can be unique relative to other molecular barcodes. In some cases, molecular barcodes can have a variety of different formats. For example, molecular barcodes can include non-random, semi-random, and / or random nucleic acid and / or amino acid sequences, and synthetic nucleic acid and / or amino Attorney Docket No. 44807-0503WO1 / C18573

[0075] acid sequences. In some cases, a molecular barcode can be attached to an analyte DNA fragment or to another moiety or structure in a reversible or irreversible manner. In some cases, a molecular barcode can be added to, for example, a fragment of analyte DNA fragment before or during sequencing of the sample. In some cases, molecular barcodes can allow for identification and / or quantification of individual sequencing-reads. In some cases, a molecular barcode can refer to a unique identifier (UID) and the terms “molecular barcode” and “UID” can be used interchangeably. In some cases, a molecular barcode or UID sequence does not exist in the analyte DNA fragment. In some cases, a molecular barcode or UID sequence does not exist in a target region (e.g., a region of interest) to be amplified and / or assessed using the methods and materials provided herein. A “duplex molecule barcode” is a molecular barcode that tags both strands of DNA with two distinct molecular barcodes (i.e., two distinct UIDs).

[0076] In some cases, ends of analyte DNA fragments can be used as endogenous UIDs. A skilled artisan may determine the length of the endogenous UID needed to uniquely identify an analyte DNA fragment, using factors such as overall template length, complexity of an analyte DNA fragment in a partition or starting nucleic acid sample, and the like. In some cases, only one end of an analyte DNA fragment is used as an endogenous UID.

[0077] In some cases, a UID can be exogenous to analyte DNA fragments. For example, an exogenous UID can be unique to each double-stranded DNA fragment in the nucleic acid sample. For example, an exogenous UID is not unique to each double-stranded DNA fragment.

[0078] A UID (e.g., an endogenous UID or an exogenous UID) can be any appropriate length (e.g. can have any appropriate number of nucleotides). In some cases, a UID can have about 2-4000 nucleotides (e.g., about 6-100 nucleotides, about 8-50 nucleotides, about 10-20 nucleotides, or about 12-14 nucleotides). In some cases, the length of a UID can be sufficient to uniquely barcode the molecules and the length / sequence of the UID does not interfere with any downstream amplification steps.

[0079] A UID (e.g., an endogenous UID or an exogenous UID) can have any appropriate nucleic acid sequence. In some cases, the nucleic acid sequence of a UID is random (e.g., is a N-mer). For example, when a UID having a length of six nucleotides is random, the UID can Attorney Docket No. 44807-0503WO1 / C18573

[0080] be referred to as a random hexamer. For example, when a UID having a length of 12 nucleotides is random, the UID can be referred to as a random 12-mer.

[0081] In some cases, the nucleic acid sequence of a UID is not random but is selected from a predetermined set of UID sequences. In some cases, a UID can be as described elsewhere (see, e.g., International Patent Application Publication No. WO 2021 / 163546 and International Patent Application Publication No. WO 2023 / 150277).

[0082] When generating a duplex sequencing library, the methods and materials provided herein (e.g., for determining a sequence of BCR (and, optionally, identifying the presence of one or more mutations and / or identifying the presence of aneuploidy)) can use any appropriate adapter. As used herein, an “adapter,” an “adaptor,” and a “tag” are terms that are used interchangeably and refer to species that can be coupled to a polynucleotide sequence (e g., in a process referred to as “tagging”) using any one of many different techniques including, but not limited to, ligation, hybridization, and tagmentation. In some cases, adaptors can also be nucleic acid sequences that add a function (e.g., spacer sequences, primer sequences / sites, molecular barcode sequences, or unique molecular identifier sequences). In some cases, methods for generating a duplex sequencing library can include attaching adapters to a population of double-stranded DNA molecules to produce a population of adapter-attached, double-stranded DNA molecules, where the adapted doublestranded DNA molecule includes an adapted Watson strand and an adapted Crick strand, where the adapter fragment includes a molecular barcode, a primer sequence, and an adapter sequence, and where the molecular barcode of the adapted Watson strand is the reverse complement of the molecular barcode of the adapted Crick strand. In some cases, the primer sequence can be the reverse complement of the adapter sequence. In some cases, the adapter sequence can include specific sequences to allow sequencing when generating a sequence library. In some cases, the adapter sequence includes a sequencing primer sequence (e.g., Rl, R2).

[0083] When generating a duplex sequencing library, the methods and materials provided herein (e.g., for determining a sequence of BCR (and, optionally, identifying the presence of one or more mutations and / or identifying the presence of aneuploidy)) can use any appropriate primer pair that can target (e.g., target and bind to) an adapter sequence (e.g., an Attorney Docket No. 44807-0503WO1 / C18573

[0084] adapter sequence containing a molecular barcode) present in an amplification product generated as described herein (e.g., by ligating a 3' duplex adapter including a first molecular barcode and a 5' adapter including a second molecular barcode to a nucleic acid fragment in a duplex sequencing library prior to the amplification). Examples of primer pairs that can be used to generate a library of single stranded Watson strand-derived sequences and a library of single stranded Crick-strand derived sequences as described herein include, without limitation, a P5 primer and a P7 primer.

[0085] In some cases, methods for identifying BCR sequences can include (a) attaching a 3' adapter fragment to each 3' end of the double-stranded DNA molecule and a 5' adapter fragment to each 5' end of the double-stranded DNA molecule to generate an adapted doublestranded DNA molecule, where the adapted double-stranded DNA molecule including an adapted Watson strand and an adapted Crick strand, where the 3’ adapter fragment includes a molecular barcode, a primer sequence, and an adapter sequence, and where the molecular barcode of the adapted Watson strand is the reverse complement of the molecular barcode of the adapted Crick strand; (b) copying both strands of the adapted double-stranded DNA molecule, where the copying includes performing a round of linear extension of the adapted double-stranded DNA molecule, generating an adapted double-stranded Watson template and an adapted double-stranded Crick template; (c) generating a first population of analyte DNA fragments from the adapted double-stranded Watson template by amplifying the adapted double-stranded Watson template with a first set of a Watson-target selective primer pair including (i) a first Watson target-selective primer including a sequence complementary to the 3' adapter sequence, and (ii) a second Watson target- selective primer including a sequence complementary to a J segment of a BCR gene sequence, and generating a first sequencing read for at least one member of the first population of analyte DNA fragments; (d) generating a second population of analyte DNA fragments from the adapted doublestranded Crick template by amplifying the adapted double-stranded Crick template with a first set of a Crick-target selective primer pair including (i) a first Crick target-selective primer including a sequence complementary to the 3' adapter sequence, and (ii) a second Crick target-selective primer including sequence complementary to the V(D)J sequence of the BCR sequence and generating a second sequencing read for at least one member of the Attorney Docket No. 44807-0503WO1 / C18573

[0086] second population of analyte DNA fragments; (e) grouping the first sequencing reads according to the molecular barcode present on the at least one member of the first population of analyte DNA fragments to generate a first analyte DNA family; (f) grouping the second sequencing reads according to the molecular barcode present on the at least one member of the second population of analyte DNA fragments to generate a second analyte DNA family; (g) analyzing the first sequencing read of the first analyte DNA family; and (h) analyzing the second sequencing read of the second analyte DNA family, thus, determining the sequence of the double-stranded DNA molecule.

[0087] Methods for determining BCR sequences of a double-stranded DNA molecule can include any appropriate BCR-selective primers. As used herein, a “primer” generally refers to a polynucleotide molecule including a nucleotide sequence (e.g., an oligonucleotide), generally with a free 3'-OH group, that hybridizes with a template sequence (such as a target polynucleotide, or a primer extension product) and is capable of promoting polymerization of a polynucleotide complementary to the template. In some cases, a primer can be a biotinylated primer. In some cases, BCR-selective primers can amplify (e.g., can be designed to amplify) the J segment of a BCR gene (e.g., the J segment of a IGH gene, also referred to herein as a IGH.) gene) or a fragment thereof. In some cases, a BCR-specific primer that can be used in the methods provided herein can target (e.g., can be designed to target) IGHJ1, IGHJ2, IGH3, IGHJ4, IGHJ5, or IGHJ6. For example, a BCR-selective primer pair can include a first primer that can amplify IGHJ1, IGHJ4, and IGHJ5, and can include a second primer that can amplify IGHJ2, IGH3, and IGHJ6. BCR-selective primers can have any appropriate nucleotide sequence. In some cases, a BCR-selective primer can have a nucleotide sequence that is complementary to a J segment of a BCR gene sequence (e.g., a IGHJ gene sequence). In some cases, a BCR-selective primer can have a nucleotide sequence set forth in any one of SEQ ID NOs: 1-12 (see, e.g., Table 1A). In some cases, a BCR-selective primer be a variant of (e.g., can have one, two, or three nucleotides preceding the articulated sequence, can have, one, two, or three nucleotides following the articulated sequence, or can have one, two, or three nucleotides substitutions within the articulated sequence) a nucleotide sequence set forth in any one of SEQ ID NOs: 1-12 (see, e.g., Table Attorney Docket No. 44807-0503WO1 / C18573

[0088] 1 A) provided that the variant maintains the ability to amplify the J segment of a BCR gene (e.g., a IGHJ gene) or a fragment thereof.

[0089] In some cases, methods provided herein also can include determining the presence or absence of BCR clonality in a double-stranded DNA molecule. For example, a duplex sequencing library described herein can be assessed to determine the sequence of a BCR in a double-stranded DNA molecule. In some cases, the sequence of a BCR can be used to determine the presence or absence of B-cell clonality. Because any B cell cancer is derived from a single B cell, a B cell cancer can be characterized by a single VDJ rearrangement.

[0090] Any appropriate method can be used to determine the presence or absence of B-cell clonality based on the sequence of a BCR in a double-stranded DNA molecule. In some cases, the presence or absence of clonality can be determined as described in Example 1. In some cases, when the total UIDs present in the analyte DNA fragments used to sequence a BCR are at least 20 and when the ratio of top clone UIDs / total UIDs (e.g., the clonal fraction) is at least 0.3, the sample can be identified as having a BCR sequence that is indicative of a clonal VDJ rearrangement, thus indicating the presence of B-cell clonality.

[0091] In some cases, methods provided herein also can include determining the presence or absence of aneuploidy (e.g., a gain or loss on at least one chromosome arm) in a doublestranded DNA molecule. For example, a duplex sequencing library described herein can be assessed for the presence or absence of aneuploidy in a double-stranded DNA molecule.

[0092] Any appropriate method can be used to determine the presence or absence of aneuploidy in a double-stranded DNA molecule. In some cases, the presence or absence of aneuploidy can be determined as described in Example 1. In some cases, a duplex sequencing library described herein can be converted to a form suitable for whole genome sequencing (WGS), and WGS data can be mapped to a comparable genome. For example, when a duplex sequencing library is generated from a sample obtained from a human, the WGS data would be mapped to a human genome (e.g., the hg!9 genome).

[0093] Any appropriate primers can be used to convert a duplex sequencing library described herein to a form that is suitable for WGS. In some cases, primers that can be used to convert a duplex sequencing library described herein to a form that is suitable for WGS can have a nucleotide sequence set forth in any one of SEQ ID NOs: 13-14 (see, e.g., Table IB). In some Attorney Docket No. 44807-0503WO1 / C18573

[0094] cases, a primer that can be used to convert a duplex sequencing library described herein to a form that is suitable for WGS can be a variant of (e.g., can have one, two, or three nucleotides preceding the articulated sequence, can have, one, two, or three nucleotides following the articulated sequence, or can have one, two, or three nucleotides substitutions within the articulated sequence) a nucleotide sequence set forth in any one of SEQ ID NOs: 13-14 (see, e.g., Table IB) provided that the variant maintains the ability to convert a duplex sequencing library described herein to a form that is suitable for WGS.

[0095] In some cases, aneuploidy can be assessed as described elsewhere (see, e.g., Douville et al., Proc. Natl. Acad. Sci. 117:4858-4863 (2020); International Patent Application Publication No. WO / 2013 / 148496; and International Patent Application Publication No. WO 2020 / 236625).

[0096] In some cases, methods provided herein also can include determining the presence or absence of one or more mutations (e.g., one or more somatic mutations) in a double-stranded DNA molecule. Examples of mutations include, without limitation, insertions, deletions, substitutions, deletion-insertions, duplications, inversions, frameshifts, repeat expansions, translocations, and combinations thereof. For example, a duplex sequencing library described herein can be assessed for the presence or absence of one or more mutations in a doublestranded DNA molecule. A mutation in a double-stranded DNA molecule can be in a Watson strand, a Crick strand, or both the Watson strand and the Crick strand.

[0097] Any appropriate method can be used to determine the presence or absence of one or more mutations in a double-stranded DNA molecule. In some cases, the presence or absence of aneuploidy can be determined as described in Example 1. In some cases, a target region present in a duplex sequencing library described herein can be amplified and sequenced to determine whether or not a particular mutation is present in that target region. Examples of target regions that can be amplified and sequence to determine whether or not there is a particular mutation within that target region include, without limitation, target regions within NRAS, TP53, APC, VHL, FBXW7, PIK3CA, RUNX1, ERBB2, EGFR, NFE2L2, FGFR3, GNAS, PTEN, IDH1, BRAF, PPP2R1A, CDH1, ERBB3, CDKN2A, ERCC2, IDH2, KMT2C, FGFR2, SMAD4, PAX3, FUS, SPOP, AKT1, CTNNB1, GATA3, RAFI, KRAS, TGFBR2, STK11, HRAS, TERT, H3F3A, NRG1, CD79B, or MYD88. In some cases, a Attorney Docket No. 44807-0503WO1 / C18573

[0098] particular mutation that can be present in the target regions set forth above can be as shown in Table 7.

[0099] Any appropriate primers can be used to amplify (e.g., amplify and sequence) a target region (e.g., a region of interest) present in a duplex sequencing library to determine whether or not a particular mutation is present in that target region. In some cases, primers of a primer pair that can be used to amplify a target region present in a duplex sequencing library to determine whether or not a particular mutation is present in that target region can have a nucleotide sequence set forth in any one of SEQ ID NOs: 15-256 (see, e.g., Table 1 C). In some cases, a primer of a primer pair that can be used to amplify a target region present in a duplex sequencing library to determine whether or not a particular mutation is present in that target region can be a variant of (e.g., can have one, two, or three nucleotides preceding the articulated sequence, can have, one, two, or three nucleotides following the articulated sequence, or can have one, two, or three nucleotides substitutions within the articulated sequence) a nucleotide sequence set forth in any one of SEQ ID NOs: 15-256 (see, e.g., Table 1C) provided that the variant maintains the ability to amplify the target region.

[0100] In some cases, the presence or absence of one or more mutations (e.g., one or more somatic mutations) in a double-stranded DNA molecule can be assessed as described elsewhere (see, e.g., International Patent Application Publication No. WO 2012 / 142213 and International Patent Application Publication No. WO 2021 / 163546).

[0101] In some cases, one or both primers of a primer pair used in a PCR amplification step used in the methods and materials provided herein (e.g., for determining a sequence of BCR (and, optionally, identifying the presence of one or more mutations and / or identifying the presence of aneuploidy)) can include one or more molecular barcodes.

[0102] In some cases, one or both primers of a primer pair used in a PCR amplification step used in the methods and materials provided herein (e.g., for determining a sequence of BCR (and, optionally, identifying the presence of one or more mutations and / or identifying the presence of aneuploidy)) can one or more graft sequences (e.g. graft sequences for next generation sequencing).

[0103] PCR amplification steps used in the methods and materials provided herein (e.g., for determining a sequence of BCR (and, optionally, identifying the presence of one or more Attorney Docket No. 44807-0503WO1 / C18573

[0104] mutations and / or identifying the presence of aneuploidy)) can include any appropriate PCR conditions. PCR amplification can include a denaturing phase, an annealing phase, and an extension phase. Each phase of an amplification cycle can include any appropriate conditions. In some cases, a denaturing phase can include a temperature of about 90°C to about 105°C (e.g., about 94°C to about 98°C), and a time of about 1 second to about 5 minutes (e.g., about 10 seconds to about 1 minute). For example, a denaturing phase can include a temperature of about 98°C for about 10 seconds. In some cases, an annealing phase can include a temperature of about 50°C to about 72°C, and a time of about 30 seconds to about 90 seconds. In some cases, an extension phase can include a temperature of about 55°C to about 80°C, and a time of about 15 seconds per kb of the amplicon to be generated to about 30 seconds per kb of the amplicon to be generated. In some cases, annealing and extension phases can be performed in a single cycle. For example, an annealing and phase extension phase can include a temperature of about 65°C for about 75 seconds.

[0105] PCR amplification can include any appropriate number of PCR amplification cycles. In some cases, PCR amplification can include from about 1 to about 50 cycles (e.g., from about 1 to about 35, from about 1 to about 30, from about 1 to about 25, from about 1 to about 20, from about 1 to about 18, from about 1 to about 15, from about 8 to about 50, from about 18 to about 50, from about 22 to about 50, from about 30 to about 50, from about 5 to about 30, from about 6 to about 25, from about 7 to about 20, from about 8 to about 15, from about 9 to about 12, from about 5 to about 15, from about 8 to about 12, from about 10 to about 25, or from about 15 to about 32 cycles).

[0106] In some cases, PCR amplification also can include an initialization step. For example, when PCR conditions include a heat-activated polymerase, the PCR amplification can include an initialization step. In some cases, PCR amplification can include an initialization step prior to performing the PCR amplification cycles. In some cases, an initialization step can include a temperature of about 94°C to about 98°C, and a time of about 15 seconds to about 1 minute. For example, an initialization step can include a temperature of about 98°C for about 30 seconds.

[0107] In some cases, PCR amplification also can include a hold step. For example, PCR amplification can include a hold step after performing the PCR amplification cycles, an Attorney Docket No. 44807-0503WO1 / C18573

[0108] optionally after performing any final extension step. In some case, a hold step can include a temperature of about 4°C to about 15°C, for an indefinite amount of time.

[0109] Sequencing steps used in the methods and materials provided herein (e.g., for determining a sequence of BCR (and, optionally, identifying the presence of one or more mutations and / or identifying the presence of aneuploidy)) can include any appropriate sequencing methods. In some cases, sequencing steps used in the methods and materials provided herein can be used to determine the sequence of a BCR in a double-stranded DNA molecule obtained from DNA (e.g., cellular DNA and cfDNA) present in a biological sample such as a fluid sample obtained from a subject (e.g., a human subject). In some cases, sequencing steps used in the methods and materials provided herein can be used to determine the presence or absence of aneuploidy in a double-stranded DNA molecule obtained from DNA (e.g., cellular DNA and cfDNA) present in a biological sample such as a fluid sample obtained from a subject. In some cases, sequencing steps used in the methods and materials provided herein can be used to determine the presence or absence of one or more mutations in a double-stranded DNA molecule obtained from DNA (e.g., cellular DNA and cfDNA) present in a biological sample such as a fluid sample obtained from a subject. Examples of sequencing methods that can be used in the methods and materials provided herein include, without limitation, single read sequencing, paired-end sequencing, WGS, NGS, and deep sequencing. In some cases, single read sequencing can include sequencing across the entire length of the templates to generate the sequence reads. In some cases, sequencing can be performed with a massively parallel sequencer. For example, a massively parallel sequencer can be configured to determine sequence reads from both ends of template polynucleotides.

[0110] In some cases, sequence reads can be mapped to a reference genome.

[0111] In some cases, the sequence reads can be assigned into UID families. A UID family can include sequence reads from analyte DNA fragments originating from an original template (e.g., original double-stranded DNA fragment from a nucleic acid sample). In some cases, each member of a UID family can include the same exogenous UID sequence. For example, each member of a UID family can include the same exogenous UID sequence and the same endogenous UID sequence. In some cases, the combination of the exogenous UID sequence and endogenous UID sequence can be unique to the UID family. In some cases, the Attorney Docket No. 44807-0503WO1 / C18573

[0112] combination of the exogenous UID sequence and endogenous UID sequence does not exist in another UID family represented in the nucleic acid sample.

[0113] In some cases, sequence reads of an individual UID family can be assigned to a Watson subfamily and a Crick subfamily. For example, sequence reads of an individual UID family can be assigned to the Watson and Crick subfamilies based on the orientation of the insert (e.g., the analyte DNA fragment containing aDNA segment of interest (e.g., a nucleic acid encoding a portion of a BCR gene, a nucleic acid containing one or more mutations, or nucleic acid containing a region of the genome to be assessed for aneuploidy)) relative to the adapter sequences. In some cases, the orientation of the insert relative to the adapter sequences can be resolved by how the sequence reads were aligned as “read pairs” or “mate pairs”.

[0114] In some cases, the assignment of sequence reads into the Watson and Crick subfamilies can be based on spatial relationship of the exogenous UID sequence to the R1 and R2 read sequence. For example, members of the Watson subfamily can be characterized by the exogenous UID sequence being downstream of the R2 sequence and upstream of the R1 sequence, by the exogenous UID sequence being in greater proximity to the R2 sequence and lesser proximity to the R1 sequence, or by the exogenous UID sequence being immediately downstream or within 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, or 1-5 nucleotides of the R2 sequence. For example, members of the Crick subfamily can be characterized by the exogenous UID sequence being downstream of the R1 sequence and upstream of the R2 sequence, by the exogenous UID sequence being in greater proximity to the R1 sequence and in lesser proximity to the R2 sequence, or by the exogenous UID sequence being immediately downstream or within 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, or 1-5 nucleotides of the R1 sequence.

[0115] In some cases, a UID subfamily (e.g., Watson subfamily and / or Crick subfamily) can include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 members. For example, a UID subfamily can include Attorney Docket No. 44807-0503WO1 / C18573

[0116] about 2-500 members, about 2-100 members, about 2-50 members, about 2-20 members, or about 2-10 members.

[0117] This document also provides methods and materials for assessing and / or treating a subject (e.g., a human subject) suspected of having cancer. In some cases, the methods and materials provided herein (e.g., for determining a sequence of BCR (and, optionally, identifying the presence of one or more mutations and / or identifying the presence of aneuploidy)) can include identifying a subject as having cancer. For example, a sequence of a BCR (and, optionally, identifying the presence of one or more mutations and / or identifying the presence of aneuploidy) in DNA (e.g., cellular DNA and cfDNA) in a fluid sample (e.g., a CSF sample) obtained from a subject suspected of having cancer can be used to identify the subject as having cancer. In some cases, a subject identified as having cancer as described herein (e.g., based, at least in part, on a sequence of a BCR (and, optionally, identifying the presence of one or more mutations and / or identifying the presence of aneuploidy) in DNA (e.g., cellular DNA and cfDNA) in a fluid sample (e.g., a CSF sample) obtained from the subject) can be administered one or more cancer treatments.

[0118] Any appropriate subject can be assessed and / or treated as described herein. In some cases, a subject can be any subject that contains B cells. For example, a subject can be a mammal. Examples of subjects that can be assessed and / or treated as described herein include, without limitation, humans, non-human primates (e.g., monkeys), dogs, cats, horses, cows, pigs, sheep, mice, rats, birds, fish, and reptiles. In some cases, a human (e.g., a human suspected of having cancer) can be assessed and / or treated as described herein.

[0119] When assessing a subject (e.g., a human subject such as a human suspected of having cancer) as described herein and / or treating a subject (e.g., a human subject such as a human identified as having cancer) as described herein, the cancer can be any type of cancer. In some cases, a cancer assessed and / or treated as described herein can include one or more solid tumors. In some cases, a cancer assessed and / or treated as described herein can be a blood cancer. In some cases, a cancer assessed and / or treated as described herein can be a primary cancer. A cancer assessed and / or treated as described herein can be a metastatic cancer. Examples of cancers that can be assessed and / or treated as described herein include, without limitation, diffuse large B-cell lymphomas (DLBCLs), follicular lymphomas, chronic Attorney Docket No. 44807-0503WO1 / C18573

[0120] lymphocytic leukemias (CLLs), small lymphocytic lymphomas (SLLs), mantle cell lymphomas (MCLs), Burkitt lymphomas, primary central nervous system lymphomas, highgrade gliomas, medulloblastomas, spinal gangliogliomas, diffuse midline gliomas, CNS lymphomas, ependymomas, and metastatic lesions to the brain. In some cases, a cancer that can be assessed and / or treated as described herein can be as described elsewhere (see, e.g., Alaggio et al., Leukemia, 36(7): 1720-1748 (2022); and Louis et al., Neuro. Oncol., 23(8): 1231-1251 (2021)).

[0121] Any appropriate fluid sample from a subject (e.g., a human subject such as a human suspected of having cancer) can be assessed as described herein (e.g., for a sequence of BCR (and, optionally, the presence of one or more mutations and / or the presence of aneuploidy)) in DNA (e.g., cellular DNA and cfDNA) in the fluid sample). In some cases, a fluid sample can be a biological sample. In some cases, a fluid sample can contain one or more biological molecules (e.g., nucleic acids such as DNA and RNA, polypeptides, carbohydrates, lipids, hormones, and / or metabolites). Examples of fluid samples that can be assessed as described herein include, without limitation, CSF samples, blood samples (e.g., whole blood samples plasma samples, and serum samples), and urine samples. In some cases, one or more biological molecules can be isolated from a fluid sample. For example, nucleic acid (e.g., DNA such as cfDNA) can be isolated from a fluid sample and can be assessed as described herein. In some cases, a tissue sample (e.g., a tissue sample obtained by a biopsy) can be used instead of a fluid sample.

[0122] Any appropriate method can be used to obtain a fluid sample from a subject (e.g., a human subject such as a human suspected of having cancer). In some cases, a fluid sample obtained from a subject can be referred to as a liquid biopsy.

[0123] In some cases, a fluid sample obtained from a subject and assessed as described herein (e.g., for determining a sequence of BCR (and, optionally, identifying the presence of one or more mutations and / or identifying the presence of aneuploidy)) can include low amounts of nucleic acid (e.g., DNA such as cfDNA). For example, a fluid sample obtained from a subject and assessed as described herein can include less than about 1 nanograms (ng) of nucleic acid (e.g., less than 0.9, less than 0.8, less than 0.7, less than 0.6, less than 0.5, less than 0.4, less than 0.3, or less than 0.2 ng of nucleic acid). For example, a fluid sample Attorney Docket No. 44807-0503WO1 / C18573

[0124] obtained from a subject and assessed as described herein can include from about 0.1 ng to about 1 ng of nucleic acid (e.g., from about 0.1 ng to about 0.8 ng, from about 0.1 ng to about 0.6 ng, from about 0.1 ng to about 0.5 ng, from about 0.1 ng to about 0.3 ng, from about 0.2 ng to about 1 ng, from about 0.3 ng to about 1 ng, from about 0.5 ng to about 1 ng, from about 0.7 ng to about 1 ng, from about 0.2 ng to about 0.9 ng, from about 0.3 ng to about 0.8 ng, from about 0.4 ng to about 0.7 ng, from about 0.5 ng to about 0.6 ng, from about 0.2 ng to about 0.5 ng, from about 0.3 ng to about 0.6 ng, from about 0.4 ng to about 0.7 ng, from about 0.5 ng to about 0.8 ng, or from about 0.6 ng to about 0.9 ng of nucleic acid).

[0125] In some cases, the methods provided herein can be performed without the need for any neurosurgical biopsy.

[0126] As described herein, a sequence of a BCR (and, optionally, the presence of one or more mutations and / or identifying the presence of aneuploidy) in DNA (e.g., cellular DNA and cfDNA) in a fluid sample (e.g., a CSF) sample obtained from a subject (e.g., a human subject such as a human suspected of having cancer) can be used to identify that subject as having cancer. In some cases, when a fluid sample obtained from a subject is determined to have the presence of a BCR clonal fraction that is at least 0.3 in DNA in the fluid sample, the subject can be identified as having cancer. For example, when a fluid sample obtained from a subject is determined to have the presence of one or more of (a) a BCR clonal fraction that is at least 0.3, (b) aneuploidy, and (c) one or more mutations, the subject can be identified as having cancer. In some cases, the absence of each of (a) a clonal fraction that is at least 0.3, (b) aneuploidy, and (c) one or more mutations in DNA in a fluid sample obtained from a subject can be used to identify that subject as not having cancer.

[0127] In some cases, when the methods provided herein are used to identify a subject (e.g., a human subject) as having cancer, a neurosurgical biopsy can be performed and the determination can be confirmed by assessment of the neurosurgical biopsy.

[0128] In some cases, a sequence of a BCR (and, optionally, the presence of one or more mutations and / or identifying the presence of aneuploidy) in DNA (e.g., cellular DNA and cfDNA) in a fluid sample (e.g., a CSF) obtained from a subject (e g., a human subject such as a human suspected of having cancer) can be used to select one or more actions for that subject. For example, a subject identified as having cancer as described herein (e.g., based, at Attorney Docket No. 44807-0503WO1 / C18573

[0129] least in part, on the presence of one or more of (a) a BCR clonal fraction that is at least 0.3, (b) aneuploidy, and (c) one or more mutations) can be selected for one or more cancer treatments. In some cases, when a fluid sample obtained from a subject is determined to have the presence of one or more of (a) a BCR clonal fraction that is at least 0.3, (b) aneuploidy, and (c) one or more mutations, the subject can be selected for one or more cancer treatments.

[0130] In some cases, a subject (e.g., a human subject) identified as having cancer as described herein (e.g., based, at least in part, on a sequence of a BCR (and, optionally, the presence of one or more mutations and / or identifying the presence of aneuploidy) in DNA (e.g., cellular DNA and cfDNA) in a fluid sample (e.g., a CSF) obtained from the subject) can be administered to instructed to self-administer one or more cancer treatments. For example, a subject identified as having cancer based, at least in part, on the presence of a BCR clonal fraction that is at least 0.3 can be administered to instructed to self-administer one or more cancer treatments. For example, when a fluid sample obtained from a subject is determined to have the presence of one or more of (a) a BCR clonal fraction that is at least 0.3, (b) aneuploidy, and (c) one or more mutations, the subject can be administered to instructed to self-administer one or more cancer treatments.

[0131] When a subject (e.g., a human subject) identified as having cancer as described herein (e.g., based, at least in part, on a sequence of a BCR (and, optionally, the presence of one or more mutations and / or identifying the presence of aneuploidy) in DNA (e g., cellular DNA and cfDNA) in a fluid sample (e.g., a CSF) obtained from the subject) is selected for and / or administered one or more cancer treatments, the cancer treatment(s) can be any appropriate cancer treatment(s). In some cases, a cancer treatment can include administering to the subject one or more (e.g., one, two, three, or more) anti-cancer agents. For example, an anticancer agent can be a chemotherapeutic agent. For example, an anti-cancer agent can be a cytotoxic agent. For example, an anti-cancer agent can be an angiogenesis inhibitor. For example, an anti-cancer agent can be an immuno-therapeutic agent (e.g., T cell such as a T cell expressing a chimeric T cell receptor (a CAR-T cell)). For example, an anti-cancer agent can be a targeted anti-cancer agent. In some cases, an anti-cancer agent can be a polypeptide (e.g., an antibody such as a bi-specific antibody). Examples of anti-cancer agents that can be administered to a subject identified as having cancer as described herein include, without Attorney Docket No. 44807-0503WO1 / C18573

[0132] limitation, temozolomide, procarbazine, lomustine, vincristine, vorasidenib, dabrafenib, trametinib, vemurafenib, cobimetinib, larotrectinib, selumetinib, bevacizumab, etoposide, regorafenib, methotrexate, and any combinations thereof (e.g., a combination of procarbazine, lomustine, and vincristine, a combination of dabrafenib and trametinib, or a combination of vemurafenib and cobimetinib). In some cases, a cancer treatment can include subjecting the subject to one or more (e.g., one, two, three, or more) cancer therapies.

[0133] Examples of cancer therapies that can be used to treat a subject identified as having cancer as described herein include, without limitation, radiation therapies and / or surgeries (e.g., surgical resection of one or more tumors). In some cases, a particular cancer treatment can be selected based, at least in part, on the specific type of the cancer a subject is identified has having. For example, a particular cancer treatment can be selected using the NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®).

[0134] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

[0135] EXAMPLES

[0136] Example 1: Genomic and B Cell Characterization of Cerebrospinal Fluid from Individuals with Brain Cancers

[0137] With these considerations in mind, it was sought to establish an approach using CSF that could supplement neurosurgical biopsy by providing a genetic and immune profile of brain tumor. There are several key capabilities that would be required for such an assay: 1) identification of driver mutations across a wide array of primary and metastatic cancers, 2) genome wide identification of chromosomal copy number alterations, 3) characterization of B cell populations, 4) robust compatibility with a relatively small amount of CSF DNA, 5) a uniform workflow that could use the same starting material for all analyses to reduce cost, improve ease, and maximize yield, and 6) an informatics pipeline capable of analyzing and integrating these heterogenous datasets. The design and execution of such an approach is described in this manuscript (Fig. 1). Attorney Docket No. 44807-0503WO1 / C18573

[0138] Results

[0139] Overview

[0140] Once DNA was purified from CSF, a DNA library was generated through a modified version of a SaferSeqS protocol (Cohen el al., Nat. Biotechnol. 39:1220-1227 (2021)), see Methods for details. This library preparation method resulted in a relatively high conversion efficiency of the original DNA template molecules to library DNA molecules. This conversion efficiency was particularly important when the quantity of CSF fluid was limited or when the DNA concentration in that fluid was low. Equally importantly, the SaferSeqS library preserved DNA from both the Watson and Crick strands of the original DNA templates. The ability to independently assess both strands of DNA exponentially increases the accuracy of the resulting sequencing data when the fraction of aberrant DNA molecules is low (Cohen et al., Nat. Biotechnol. 39: 1220-1227 (2021); Kinde et al., Proc. Natl. Acad. Set.

[0141] 108:9530-9535 (2011); and Salk et al., Nat. Rev. Genet. 19:269-285 (2018)). SaferSeqS libraries contains -200 copies of each of the original template molecule strands, and therefore can be used for multiple downstream assessments of DNA. For CSF-BAM, the clonal composition of DNA derived from malignant or normal B cells was analyzed, as well as chromosome copy number alterations and somatic mutations derived from the cancer cells (Fig. 2). The paradigm for evaluation of each of these three components was identical:

[0142] (i) Optimize the experimental procedures and bioinformatic analysis using DNA from blood leukocytes or cell-free DNA.

[0143] (ii) Use the optimized procedure to evaluate DNA from CSF or blood from a different cohort of patients with and without cancer to establish thresholds for specificity and estimate sensitivity at the chosen thresholds.

[0144] (iii) Use the optimized procedure to evaluate CSF from an independent cohort of patients to determine sensitivity at the pre-defined thresholds for positivity.

[0145] Development of the experimental procedures and bioinformatic pipelines for CSF-BAM The “B” component of CSF-BAM queries the B cell (BCR) receptor genes that are integral to the adaptive immune system. In any single individual, millions of BCRs in normal B are generated through imprecise joining of variable (V), diversity (D), and joining (J) Attorney Docket No. 44807-0503WO1 / C18573

[0146] segments of the BCR genes. The nature of these sequences and the degree of clonality provides a wealth of information about the adaptive immune system in that particular patient. Moreover, because any B cell cancer is derived from a single B cell, this neoplastic clone is characterized by a single VDJ rearrangement. The presence of malignant cells in CSF can thereby be detected by the over-representation of a single sequence in the CSF, implying a predominant clone. Although such clones can be detected through previously published methods that sequence either RNA or DNA templates, accurate detection and quantification of clonotypes is challenging. Among the reason for this is that sequencing from DNA templates has generally required multiplex combinations of primers to amplify all possible V and J gene segment pairs (Barennes etal., Nat. Biotechnol. 39:236-245 (2021); Frank etal., Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res. 29: 994-1008 (2023); and Carlson et al. I, Nat. Commun. 4: 2680 (2013)).

[0147] SafeBSeqS overcomes this challenge by requiring primers for only the J segments of one of the BCR (IGH) genes. Though these data cannot be used to analyze the constant region, they can be used to re-construct the entire VDJ sequence of the BCR. They are therefore adequate to determine the clonal representation of any population of B cells as well as to identify certain characteristics of the rearrangements associated with cancers or autoimmune disease (Pauken et al., Trends Immunol. 43:180-194 (2022)).

[0148] After extensive experimentation, it was found that four primers were sufficient to assess the entire BCR repertoire with SafeBSeqS (Table 1 A). One primer amplified gene segments IGHJ1, IGHJ4, and IGHJ5, and one primer each amplified IGHJ2, IGH3, and IGHJ6. These four primers were mixed together and used for hemi-nested amplification of the SaferSeqS libraries. They yielded uniform amplification of all the queried gene segments as tested on a sample of DNA derived from fibroblasts with a uniform representation of gene segments (Fig. 5). SafeBSeqS was then applied to the evaluation of DNA from peripheral white blood cells of 95 healthy control individuals and CSF from 25 individuals with CNS lymphoma. The summaries of results are listed in Table 2 and Table 3. Based on these results, the positive criteria for clonality was defined as total UIDs >=20 and top clone UIDs / total UIDS >=0.3. In total 1 / 95 samples from healthy individuals and 13 / 25 samples from patients with lymphoma in the set met these criteria (Tables 2 and 3). When SafeBSeqS was Attorney Docket No. 44807-0503WO1 / C18573

[0149] applied to CSF from a different cohort of individuals without known cancers, it was found that 0 met the positive criteria for clonality (Table 3). Similarly, 1 of the 202 CSF samples from patients with cancers other than CNS lymphomas scored positively in this (Table 3). Of CSF samples from 4 patients with B-cell lymphomas of the CNS, 2 scored positively in this assay with clonal fractions of 32% and 86% (Table 3). Attorney Docket No. 44807-0503W01 / Cl 8573

[0150] Table 2: Summary of SafeBSeqS Analysis in WBC Samples.

[0151] * indicates a stop codon

[0152] > indicates an incomplete codon (1 or 2 nucleotides on the DNA level).

[0153] IGH IGH nSeqCDR3 top clone IGH aaSeqCDR3 top clone IGH vGene top IGH clonality (SEQ ID NO) (SEQ ID NO) clone jGene top clone 0.004202 TGTGCGAGAGCCGATGGTGGCTACGTTCCTCGGGAGT CARADGGYVPRECW IGHV3-21*00 IGHJ4*00

[0154] GCTGG (SEQ ID NO:258)

[0155] (SEQ ID NO:257)

[0156] 0.071429 TGTGTGAGAGTTTTAGGATATTGTACTGGTGGTGTATG CVRVLGYCTGGVCYSAEYFQHW IGHV 1-17*00 IGHJl*00

[0157] CTATAGCGCTGAATACTTCCAGCACTGG (SEQ ID NO: 260)

[0158] (SEQ ID NO:259)

[0159] 0.009375 TGTGCGAGAGAAAAGGGAGAACTAACCTCCCGCCACC CAREKGELTSRHPYYFDYW IGHVl-3*00 IGHJ4*00

[0160] CGTACTACTTTGACTACTGG (SEQ ID NO: 262)

[0161] (SEQ ID NO: 261)

[0162] 0.007246 TGTGCGAGAAGGTAGACCTCTTTTGACTACTGG CARR*TSFDYW IGHV4-34*00 IGHJ4*00

[0163] (SEQ ID NO: 263) (SEQ ID NO: 264)

[0164] 0.0131 TGTGCGAGACAGCCTACGGGATCCATGGACTACTGG CARQPTGSMDYW IGHV4-39*00 IGHJ4*00

[0165] (SEQ ID NO:265) (SEQ ID NO: 266)

[0166] 0.0625 TGTGCGAGAGAGAAGGGACCGGTGGGAGCTAAATTTA CAREKGPVGAKFKVAYFQHW IGHV1-18*OO IGHJl*00

[0167] AGGTTGCATACTTCCAGCACTGG (SEQ ID NO:268)

[0168] (SEQ ID NO: 267)

[0169] 0.012295 TGTGCTTCGGTTCGCTATGATAGTGGTGGTTATTACCA CASVRYDSGGYYQDYW IGHV4-59*00 IGHJ4*00

[0170] AGACTACTGG (SEQ ID NO: 270)

[0171] (SEQ ID NO:269)

[0172]

[0173] 1 TGTGCGAGACGCCGAAACCAAGTAGTACCAGCTGCTC CARRRNQVVP CSLPLARDYW IGHV5-51*OO IGHJ4*00

[0174]

[0175] Attorney Docket No. 44807-0503WO1 / C18573

[0176] ATTACCGTTAGCACGTGACTACTGG (SEQ ID NO: 272)

[0177] (SEQ ID NO: 271)

[0178] 0.011111 TGTGCGAGAGTGGGAGTGGCTACATTCCGCCCCTTTG CARVGVATFRPFDSW IGHV4-34*00 IGHJ4*00

[0179] ACTCCTGG (SEQ ID NO: 274)

[0180] (SEQ ID NO: 273)

[0181] 0.009615 TGTGCGAGAGATTGGAACCCTATTGTAGTAGTACCAG CARDWNPIVWPAATGNNWFDP IGHVl-3*00 IGHJ5*00

[0182] CTGCTACCGGGAACAACTGGTTCGACCCCTGG W

[0183] (SEQ ID NO:275) (SEQ ID NO: 276)

[0184] 0.016575 TGTGCAAGAGATAACGGGTGGTTCGGGGATTATGGAA CARDNGWFGDYGTTGGWFAPW IGHV3-74*00 IGHJ5*00

[0185] CGACGGGAGGCTGGTTCGCCCCCTGG (SEQ ID NO:278)

[0186] (SEQ ID NO:277)

[0187] 0.012712 TGTGCGAGAGTCGGTGACTACGGACCCTACTACTACT CARVGDYGPYYYYGMDVW IGHV3-21*00 IGHJ6*00

[0188] ACGGTATGGACGTCTGG (SEQ ID NO:280)

[0189] (SEQ ID NO: 279)

[0190] 0.054545 TGTACCACAGACGGGGGGTACTTTGACTACTGG CTTDGGYFDYW IGHV3-15*OO IGHJ4*00

[0191] (SEQ ID NO:281) (SEQ ID NO:282)

[0192] 0.022556 TGTGCGAGAGATAGCCAGGATGCTGAGAATTACTACG CARDSQDAENYYAFDYW IGHV3-21*00 IGHJ4*00

[0193] CATTTGACTACTGG (SEQ ID NO:284)

[0194] (SEQ ID NO:283)

[0195] 0.032258 TGTGCAACAATCTTCTCAAGGCACGACGGGCACAGGA CATIFSRHDGHRNDYW IGHV 1-24*00 IGHJ4*00

[0196] ATGACTACTGG (SEQ ID NO:286)

[0197] (SEQ ID NO:285)

[0198] 0.02069 TGTGCGAGAGGGGTGGTTTTTGATATCTGG CARGWFDIW IGHV4-59*00 IGHJ3*00

[0199] (SEQ ID NO:287) (SEQ ID NO:288)

[0200] 0.006742 TGTGTGAGACTTTCTCTAAGGGGACTACAGTAACTAC CVRLSLRG YSNYGDW IGHV3-35*OO IGHJ4*00

[0201] GGGGACTGG (SEQ ID NO: 290)

[0202]

[0203] (SEQ ID NO:289)

[0204]

[0205] Attorney Docket No. 44807-0503WO1 / C18573

[0206] 0.016043 TGTGTGAAATTCATACCAGTGTCTGCCGGCAGTATCTA CVKFIPVSAGSIYNGMDVW IGHV3- IGHJ6*00

[0207] CAACGGTATGGACGTCTGG (SEQ ID NO: 292) 64D*00

[0208] (SEQ ID NO:291)

[0209] 0.090909 TGTGCGAGAGATCGGACTTACGGATTTTGGGGTCTGT CARDRTYGFWGLWAYGMDVW IGHV3-48*00 IGHJ6*00

[0210] GGGCTTACGGAATGGACGTCTGG (SEQ ID NO: 294)

[0211] (SEQ ID NO: 293)

[0212] 0.066667 TGTGCGAGAGGAGGCAGTGGCCTTACGCGAGATTTTG CARGGSGLTRDFDIW IGHV3-53*OO IGHJ3*00

[0213] ATATCTGG (SEQ ID NO: 296)

[0214] (SEQ ID NO:295)

[0215] 0.013699 TGTGCAAGAGTATAAGTATAGCAGCTCGTCGGTCGGG CARV*V*QL_RSGRPGGW IGHV3-13*OO IGHJ4*00

[0216] TCGACCTGGTGGGTGG (SEQ ID NO:298)

[0217] (SEQ ID NO:297)

[0218] 0.005792 TGTGCGTGCCACTCCGGATATAGTACCTCGCGATTTGA CACHSGYSTSRFDHW IGHV 1-46*00 IGHJ4*00

[0219] CCACTGG (SEQ ID NO: 300)

[0220] (SEQ ID NO:299)

[0221] 0.015075 TGTGCGAGATCGGTAAATTCGGGGGACCTCTACTACT CARSVNSGDLYYYYGMDVW IGHVl-8*00 IGHJ6*00

[0222] ACTACGGTATGGACGTCTGG (SEQ ID NO: 302)

[0223] (SEQ ID NO:301)

[0224] 0.01107 TGTGCGAGGCCCGGGTATAGCAGTGGCTGGCACCTCC CARPGYSSGWHLPFDLW IGHV 1-69* 00 IGHJ2*00

[0225] CCTTCGATCTCTGG (SEQ ID NO: 304)

[0226] (SEQ ID NO:303)

[0227] 0.007874 TGTGCGAGGGAGTTTAACTATGAGACTAGTGGTTATT CAREFNYETSGYYYFYW IGHV1 -69*00 IGHJ4*00

[0228] ACTACTTCTACTGG (SEQ ID NO: 306)

[0229] (SEQ ID NO:305)

[0230] 0.004622 TGTGCGAAAACCGTCGACCGTATAGCAGAAATAGAAG CAKTVDRIAEIEVFDYW IGHV3-23*00 IGHJ4*00

[0231] TCTTTGACTACTGG (SEQ ID NO:308)

[0232]

[0233] (SEQ ID NO: 307)

[0234]

[0235] Attorney Docket No. 44807-0503WO1 / C18573

[0236] 0.013825 TGTGCGAGAGACCTCGATAACCATAGTGGGAGCTATC CARDLDNHSGSYPGAFDIW IGHV3-48*00 IGHJ3*00

[0237] CAGGGGCTTTTGATATCTGG (SEQ ID NO: 310)

[0238] (SEQ ID NO:309)

[0239] 0.011407 TGTGCGAGAGATGCCGAGCAGTGGCTGGTACGGAGGG CARDAEQWLVRRGILTPDYW IGHV1-18*OO IGHJ4*00

[0240] GGATTTTGACTCCAGACTACTGG (SEQ ID NO: 312)

[0241] (SEQ ID NO:311)

[0242] 0.020134 TGTGCGAAATTTCAGCAGTGGCTGGCAGGCCCCTACT CAKFQQWLAGPYYFDYW IGHV3-23*00 IGHJ4*00

[0243] ACTTTGACTACTGG (SEQ ID NO: 314)

[0244] (SEQ IDNO:313)

[0245] 0.019608 TGTGCGAGAGTGCGGTCCGCTACGACCTGGGACTACT CARVRSATTWDYYYYGMDVW IGHVl-46*00 IGHJ6*00

[0246] ACTACTACGGTATGGACGTCTGG (SEQ ID NO: 316)

[0247] (SEQ ID NO:315)

[0248] 0.013699 TGTGCGAGACATTATTGTAGTGGTGGTAGCTGCTACTT CARHYCSGG AATCGLDYW IGHV5-51*OO IGHJ4*00

[0249] GCGGTTTGGACTACTGG (SEQ ID NO: 318)

[0250] (SEQ ID NO:317)

[0251] 0.045455 TGTGCGAGATTCTTCGGGGAGGACAATTTCTACTACTT CARFFGEDNFYYFDYW IGHVl-2*00 IGHJ4*00

[0252] TGACTACTGG (SEQ ID NO: 320)

[0253] (SEQ ID NO: 319)

[0254] 0.027027 TGTGCGAAGGAGGGGAAGGGGCCCGACTGG CAKEGKGPDW IGHV3-3O- IGHJ4*00

[0255] (SEQ ID NO:321) (SEQ ID NO: 322) 3*00

[0256] 0.009091 TGTGCACACAGACGAGGCCGGGGGGACACTGGCTGGC CAHRRGRGDTGWRAFDFW IGHV2-5*00 IGHJ4*00

[0257] GAGCCTTTGACTTTTGG (SEQ ID NO: 324)

[0258] (SEQ ID NO:323)

[0259] 0.00838 TGTGCGAGAGATCTAGTGGGAGCTACTAGGGGGGACT CARDLVGATRGDYYGMDVW IGHVl-2*00 IGHJ6*00

[0260] ACTACGGTATGGACGTCTGG (SEQ ID NO: 326)

[0261] (SEQ ID NO:325)

[0262] 0.022222 TGTGCGAGACTCGTGGGAGCAGCAGCTGGAAACTTTG CARLVGAAAGNFDYW IGHV4-34*00 IGHJ4*00

[0263]

[0264]

[0265] Attorney Docket No. 44807-0503WO1 / C18573

[0266] ACTACTGG (SEQ ID NO:328)

[0267] (SEQ ID NO: 327)

[0268] 0.004983 TGTGCGAGAGAGAAGGCTTATTGTAGTAGTACCAGCT CAREKAYCSS QLLSVCFDYW IGHV3-21*00 IGHJ4*00

[0269] GCTAAGCGTTTGTTTTGACTACTGG (SEQ ID NO: 330)

[0270] (SEQ ID NO: 329)

[0271] 0.018868 TGTGCGAGACGTGGTTCGGGGAGCATTGATGCTTTTG CARRGSGSIDAFDIW IGHV5-51*OO IGHJ3*00

[0272] ATATCTGG (SEQ ID NO:332)

[0273] (SEQ ID NO:331)

[0274] 0.006173 TGTGCGAAAGATCAACCCTGGAACTATTGTAGTAGTA CAKDQPWNYCSSTSCYFDYW IGHV3-ll*00 IGHJ4*00

[0275] CCAGCTGCTACTTTGACTACTGG (SEQ ID NO:334)

[0276] (SEQ ID NO:333)

[0277] 0.008929 TGTGCGAGAGACTTGGCAGCAACTGGTTATTATTACTT CARDLAATGYYYFDSW IGHV3-33*OO IGHJ4*00

[0278] TGACTCCTGG (SEQ ID NO: 336)

[0279] (SEQ ID NO:335)

[0280] 0.006726 TGTGCGAGAGCGGGGGCGTATTACTATGATAGTAGTG CARAGA YYYDS SGYYNYW IGHV1-18*OO IGHJ4*00

[0281] GTTATTATAACTACTGG (SEQ ID NO: 338)

[0282] (SEQ ID NO: 337)

[0283] 0.006897 TGTGCGAGACTCATGTATAGCAGTGGCTGGTTTTGTAT CARLMYSSGWFCMDVW IGHV4-39*00 IGHJ6*00

[0284] GGACGTCTGG (SEQ ID NO: 340)

[0285] (SEQ ID NO:339)

[0286] 0.006452 TGTGCGAGAGATCGACCCGACTATGATAGTAGTGGTT CARDRPDYDSSGYYQRSAFDYW IGHV3-21*00 IGHJ4*00

[0287] ATTACCAACGATCGGCCTTTGACTACTGG (SEQ ID NO: 342)

[0288] (SEQ ID NO:341)

[0289] 0.090909 TGTACTGGCTCCTTTCTTGCCTACTGG CTGSFLAYW IGHV3-15*OO IGHJ4*00

[0290] (SEQ ID NO:343) (SEQ ID NO: 344)

[0291] 0.009804 TGTGCGAAAATAGGCGTCATTGCCCTCTGGTACTTCGA CAKIGVIALWYFDIW IGHV3-23*00 IGHJ2*00

[0292]

[0293] TATCTGG (SEQ ID NO: 346)

[0294]

[0295] Attorney Docket No. 44807-0503WO1 / C18573

[0296] (SEQ ID NO:345)

[0297] 0.008264 TGTGCGAGAGCTGGGAATAGTGGGAGCTATAACATTG CARAGNSGSYNIDFDYW IGHV4-38- IGHJ4*00

[0298] ACTTTGACTACTGG (SEQ ID NO:348) 2*00

[0299] (SEQ ID NO:347)

[0300] 0.007026 TGTGCGAGCGACGATTTTTGGAGTGGTTATTATATAGG C ASDDFWSG II* GWFDPW IGHVl-2*00 IGHJ5*00

[0301] GCTGGTTCGACCCCTGG (SEQ ID NO:350)

[0302] (SEQ ID NO:349)

[0303] 0.009901 TGTACCACAGATTACCGGTATTACTATGATAGTAGTGG CTTDYRYYYDSSGYGGAYDAFDI IGHV3-15*OO IGHJ3*00

[0304] TTATGGAGGGGCGTATGATGCTTTTGATATCTGG W

[0305] (SEQ ID NO:351) (SEQ ID NO:352)

[0306] 0.005556 TGTGCGAGAGGGGGGATCGGGGAGTGGCCCTCCCCCC CARGGIGEWPSPLVVW IGHV3-21*00 IGHJ6*00

[0307] TGGTCGTCTGG (SEQ ID NO: 354)

[0308] (SEQ ID NO:353)

[0309] 0.014925 TGTGCGAGGACTAGGGGCTTCTTCAGCACCAGGGAGG CARTRGFFSTREGVWFDAW IGHV5-10- IGHJ5*00

[0310] GGGTCTGGTTCGACGCCTGG (SEQ ID NO:356) 1*00

[0311] (SEQ ID NO:355)

[0312] 0.018519 TGTGCGAGCGATCAAAGGGCCCTAGCATATTGTGGTG CASDQRALAYCGGDCYSGDYW IGHV1-18*OO IGHJ4*00

[0313] GTGACTGCTATTCTGGTGACTACTGG (SEQ ID NO: 358)

[0314] (SEQ ID NO: 357)

[0315] 0.022222 TGTGCAAGAGCAGGAGATAGTAGTGGTTATGGAGATG CARAGDSSGYGDAVDFW IGHV3-13*OO IGHJ3*00

[0316] CTGTAGATTTCTGG (SEQ ID NO: 360)

[0317] (SEQ ID NO: 359)

[0318] 0.013514 TGTGCAAGACAGTGGCTGCCACAATGGTATGCTTTTG CARQWLPQWYAFDIW IGHV3-74*00 IGHJ3*00

[0319] ATATCTGG (SEQ ID NO: 362)

[0320] (SEQ ID NO:361)

[0321] 0.055556 TGTGCGAAATTGCCAGATTACTATGATAGTAGTGGTTA CAKLPDYYDSSGYFIAFDIW IGHV3-3O*OO IGHJ3*00

[0322]

[0323] TTTTATTGCTTTTGATATCTGG (SEQ ID NO:364)

[0324]

[0325] Attorney Docket No. 44807-0503WO1 / C18573

[0326] (SEQ ID NO: 363)

[0327] 0.038462 TGTGCGAGAGATATTCTTGGGGGCAGTAACTGGTACG CARDILGGSNWYDGVDYW IGHV1-18*OO IGHJ4*00

[0328] ACGGTGTTGACTACTGG (SEQ ID NO: 366)

[0329] (SEQ ID NO:365)

[0330] 0.013575 TGTGCACGGATACCCGATAGTGGGAGCTACCATTTTG CARIPDSGSYHFDYW IGHV2-70*00 IGHJ4*00

[0331] ACTACTGG (SEQ ID NO:368)

[0332] (SEQ ID NO:367)

[0333] 0.009346 TGTGCGAGACATGATTACTATGGTTCGGGGAGTTATTA CARHDYYGSGSYYIDYW IGHV4-39*00 IGHJ4*00

[0334] TATTGACTACTGG (SEQ ID NO: 370)

[0335] (SEQ ID NO: 369)

[0336] 0.006757 TGTGCAAAGGATTGGGCGGCACGATACTACTACGGTA CAKDWAARYYYGMDVW IGHV3-9*00 IGHJ6*00

[0337] TGGACGTCTGG (SEQ ID NO: 372)

[0338] (SEQ ID NO:371)

[0339] 0.008746 TGTGCGGTACGCCTCCCGGGGTGGTCTTTTGACTACTG CAVRLPGWSFDYW IGHV3-23*00 IGHJ4*00

[0340] G (SEQ ID NO:374)

[0341] (SEQ ID NO:373)

[0342] 0.011194 TGTGCGAAAGCATATAGTGGGAGCTACTTCGGTGCTTT CAKAYSGSYFGAFDIW IGHV3-23*00 IGHJ3*00

[0343] TGATATCTGG (SEQ ID NO: 376)

[0344] (SEQ ID NO:375)

[0345] 0.011364 TGTGCGAGAGGGGGATATTGTAGTGGTGGTAGCTGCT CARGGYCSGGSCFMAVAGHYYY IGHV4-4*00 IGHJ6*00

[0346] TCATGGCAGTGGCTGGTCACTACTACTACGGTATGGA GMDVW CGTCTGG (SEQ ID NO: 378)

[0347] (SEQ ID NO: 377)

[0348] 0.02439 TGTGCGAGAGGGGAGTGGGTTCACGACTACTACTACT CARGEWVHDYYYYGMDVW IGHV4-38- IGHJ6*00

[0349] ACGGTATGGACGTCTGG (SEQ ID NO:380) 2*00

[0350] (SEQ ID NO: 379)

[0351] 0.009202 TGTGCGAGAGGCCTGAGTATCGATCTCTGG CARGLS1DLW 1GHV3-33*OO IGHJ3*00

[0352]

[0353]

[0354] Attorney Docket No. 44807-0503WO1 / C18573

[0355] (SEQ ID NO:381) (SEQ ID NO:382)

[0356] 0.013158 TGTGCAAGAGATCCTGCTATGATAGTAGTGGTTGGAA CARDPAMTV GWKYYFDYW IGHV3-47*00 IGHJ4*00

[0357] GTACTACTTTGACTACTGG (SEQ ID NO: 384)

[0358] (SEQ ID NO:383)

[0359] 0.007143 TGTGCGAAAGACCACCTGCTACCCCCTTACTACTACGG CAKDHLLPPYYYGMDVW IGHV3-23*00 IGHJ6*00

[0360] TATGGACGTCTGG (SEQ ID NO:386)

[0361] (SEQ ID NO:385)

[0362] 0.009404 TGTACCACAGCAGGCTGGTTACCGCACTTTGACTACTG CTTAGWLPHFDYW IGHV3-15*OO IGHJ4*00

[0363] G (SEQ ID NO: 388)

[0364] (SEQ ID NO:387)

[0365] 0.027778 TGTGCGAGGGGTTCGTATAGTGGGAGCTTGGTTGACT CARGSYSGSLVDYW IGHV3-21*00 IGHJ4*00

[0366] ACTGG (SEQ ID NO: 390)

[0367] (SEQ ID NO:389)

[0368] 0.0625 TGTGCGAGAGATCCCGTATAGCAGTGGCTGGTAGAGG CARDPV*QW GRGYFDYW IGHV4-4*00 IGHI4*00

[0369] CTACTTTGACTACTGG (SEQ ID NO: 392)

[0370] (SEQ ID NO:391)

[0371] 0.028571 TGTGCGAGAGATATGACCCATGGCTGCTTTGACTACTG CARDMTHGCFDYW IGHV3-21*00 IGHJ4*00

[0372] G (SEQ ID NO: 394)

[0373] (SEQ ID NO: 393)

[0374] 0.02381 TGTGCGACAGGCTGGGGCAGCTGCTTATACAACTACT CATGWGSCLYNYW IGHV3-53*OO IGHJ4*00

[0375] GG (SEQ ID NO: 396)

[0376] (SEQ ID NO:395)

[0377] 0.033333 TGTGCGAGTAGTACTATGGTTCAGGGAGTCATTGACT CAS STMVQGVIDYW IGHVl-46*00 IGHJ4*00

[0378] ACTGG (SEQ ID NO:398)

[0379] (SEQ ID NO:397)

[0380] 0.014634 TGTGCGAGAGATAACCCTATAGCAGCAGCTGGCGCTG CARDNPIAAAGAEW IGHV3-21*00 IGHJ4*00

[0381]

[0382] AGTGG (SEQ ID NO:400)

[0383]

[0384] Attorney Docket No. 44807-0503WO1 / C18573

[0385] (SEQ ID NO: 399)

[0386] 0.010949 TGTGCAAGATAGCAGGTCCGGCGAGGTGGCCCCCTGG CAR* Q VRR V APWFDY W IGHV 1-45*00 IGHJ4*00

[0387] TTTGACTACTGG (SEQ ID NO: 402)

[0388] (SEQ ID NO:401)

[0389] 0.011278 TGTGCGAGAGGGGTCGGGTATGGATATTGTAGTAGTA CARGVGYGYCSSTSCPEPAFDIW IGHVl-8*00 IGHJ3*00

[0390] CCAGCTGCCCGGAGCCTGCTTTTGATATCTGG (SEQ ID NO: 404)

[0391] (SEQ ID NO:403)

[0392] 0.006148 TGTGCAAGAGATAGGGAGAGTTGTAATAGGCGCCGAA CARDRESCN APKWFDPW IGHV3-13*OO IGHJ5*00

[0393] GTGGTTCGACCCCTGG (SEQ ID NO: 406)

[0394] (SEQ ID NO:405)

[0395] 0.0125 TGTGCGAGAGGCCGACGCTTTACCAGCGTTCGGGGCC CARGRRFTSVRGLILGRYIW IGHV4-34*00 IGHJ4*00

[0396] TCATTCTGGGTCGCTACATCTGG (SEQ ID NO:408)

[0397] (SEQ ID NO:407)

[0398] 0.006329 TGTGCCCTGACGGCCGTACCAGCTGCTAGATACTACAT CALTAVPAARYYMDVW IGHVl-2*00 IGHJ6*00

[0399] GGACGTCTGG (SEQ ID NO:410)

[0400] (SEQ ID NO:409)

[0401] 0.011494 TGTGCGAAAGATTCGGCGCGCGGGATACAGCTATGGT CAKDSARGIQLWLNWFDPW IGHV3-3O*OO IGHJ5*00

[0402] TAAATTGGTTCGACCCCTGG (SEQ ID NO:412)

[0403] (SEQ ID NO:411)

[0404] 0.022727 TGTGCGAGACCACATTACTATGATAGTAGTGGTTATTA CARPHYYDSSGYYPDAFDIW IGHV5-51*OO IGHJ3*00

[0405] CCCTGATGCTTTTGATATCTGG (SEQ ID NOAM)

[0406] (SEQ ID NO:413)

[0407] 0.694785 TGTGCGAGAGTGGAGATGGCTACAACCTCCTCTCCCA CARVEMATTSS HYGYYYGMDV IGHV3-7*00 IGHJ6*00

[0408] TTATGGCTACTACTACGGTATGGACGTCTGG W

[0409] (SEQ ID NO:415) (SEQ ID NO:416)

[0410] 0.011583 TGTGCGAGAGAGGGGTATAGCAGTACAGATGCTTTTG CAREGYSSTDAFDIW IGHV3-21*00 IGHJ3*00

[0411]

[0412] ATATCTGG (SEQ 1D NO:418)

[0413]

[0414] Attorney Docket No. 44807-0503WO1 / C18573

[0415] (SEQ ID NO:417)

[0416] 0.007407 TGTGTGAAAGACTTCCACCGCAAGGGGTTCGGCGGCC CVKDFHRKGFGGPFDYW IGHV3-3O*OO IGHJ4*00

[0417] CCTTTGACTACTGG (SEQ ID NO: 420)

[0418] (SEQ ID NO:419)

[0419] 0.030303 TGCGCGAGAGATTATCTGGGGAACCGGGATGCTTTTG CARDYLGNRDAFDIW IGHV 1-46*00 IGHJ3*00

[0420] ATATCTGG (SEQ ID NO: 422)

[0421] (SEQ ID NO:421)

[0422] 0.010274 TGTGCAAGAGCGTTTCAGGGAGTTAAGGGCTTCTGG CARAFQGVKGFW IGHV6-l*00 IGHJ4*00

[0423] (SEQ ID NO:423) (SEQ ID NO: 424)

[0424] 0.011765 TGTATTACTGTACCAGAGTTAGCAGCAGCTGGTTCGA CITVPELA SWFEFDSW IGHV3-41*00 IGHI4*00

[0425] ATTTGACTCCTGG (SEQ ID NO: 426)

[0426] (SEQ ID NO:425)

[0427] 0.008147 TGTGCGAGACATGATCGAGGTCATAGGAGTAGTTGGT CARHDRGHRSSWYESSNW IGHV4-55*00 IGHJ4*00

[0428] ACGAGAGTTCGAACTGG (SEQ ID NO:428)

[0429] (SEQ ID NO: 427)

[0430] 0.022222 TGTGCGAGAGAGCGTGCTGGCAACTTTGACTCCTGG CARERAGNFDSW IGHV6-l*00 IGHJ4*00

[0431] (SEQ ID NO:429) (SEQ ID NO: 430)

[0432] 0.013216 TGTGCGAAAAGGTGTAGTAGTACCAGCTGCCCCCCCG C AKRC S STSCPPDYW IGHV3-23*00 IGHJ4*00

[0433] ACTACTGG (SEQ ID NO:432)

[0434] (SEQ ID NO: 431)

[0435] 0.006452 TGTGCGAGAGATCCAGGGGTATATGGTTCGGGGAGTT CARDPGVYGSGSSWFDPW IGHVl-18*00 IGHJ5*00

[0436] CCTGGTTCGACCCCTGG (SEQ ID NO:434)

[0437] (SEQ ID NO: 433)

[0438] 0.010417 TGTGCACATTTGCATTATTACGATATAAACCCATACTT CAHLHYYDINPYFDYW IGHV2-5*00 IGHJ4*00

[0439] TGACTACTGG (SEQ ID NO: 436)

[0440] (SEQ IDNO:435)

[0441] 0.007557 TGTGCGTGCGCACACTTCTGG CACAHFW IGHV3-ll*00 IGHJ4*00

[0442]

[0443]

[0444] Attorney Docket No. 44807-0503WO1 / C18573

[0445] (SEQ ID NO: 437) (SEQ ID NO: 438)

[0446] 0.006849 TGTGCGAGAGTAGGGTTAATTATTCGGGGAGTTACCC CARVGLIIRGVT LYYYYYGMDV IGHV4-34*00 IGHJ6*00

[0447] CTTTACTACTACTACTACGGTATGGACGTCTGG W

[0448] (SEQ ID NO:439) (SEQ ID NO: 440)

[0449] 0.0131 TGTGCGAGAGATCTCTCTTGGATAGTGGCTACGGCTAT C ARDLS WIVAT LS * YNYYMDV IGHV4-31*00 IGHJ6*00

[0450] CCTAATACAACTACTACATGGACGTCTGG W

[0451] (SEQ ID NO:441) (SEQ ID NO: 442)

[0452] 0.044776 TGTGCGAGACTCCATATGACTACGGTGACTAACCGAC CARLHMTTVTNRPFDYW IGHV4-39*00 IGHJ4*00

[0453] CCTTTGACTACTGG (SEQ ID NO: 444)

[0454] (SEQ ID NO: 443)

[0455] 0.010453 TGTGCGAGAGCTCGGCGGGGTATAGCAGCAGCTGGTA CARARRGIAAAGTNTYNWFDPW IGHVl-46*00 IGHJ5*00

[0456] CCAACACCTACAACTGGTTCGACCCCTGG (SEQ ID NO: 446)

[0457]

[0458] (SEQ ID NO:445)

[0459] Table 3: Summary of CSF-BAM SafeBSeqS Analysis in CSF

[0460] IGH Clonality IGH top clone nSeqCDR3 IGH top clone aaSeqCDR3 IGH top clone IGH top clonality threshold- vGene clone (Top adjusted jGene clone (UIDs>=2

[0461] UDS / 0)

[0462] Total

[0463] UIDs)

[0464] 1 1 TGTGCGATGGCGGGCCTCGCTGAAAGCGG CAMAGLAESGLDPW IGHV4-31*00 IGHJ5*00

[0465] ACTTGACCCCTGG (SEQ ID NO:448)

[0466] (SEQ ID NO: 447)

[0467] 1 1 IGHV4-39*00 IGHJ4*00

[0468]

[0469] TGTGTGACACACGAGTCGCTTTTTGCCTAC CVTHESLFAYFDYW

[0470]

[0471] Attorney Docket No. 44807-0503WO1 / C18573

[0472] TTTGACTACTGG (SEQ ID NO:450)

[0473] (SEQ ID NO: 449)

[0474] 1 1 TGTGCGAACGGAAATGCTGTTGTGACCCTC CANGNAWTLHW IGHV3-23*00 IGHJ4*00

[0475] CACTGG (SEQ ID NO:452)

[0476] (SEQ ID NO:451)

[0477] 1 1 TGTGCGAGAGGGAATCAACACTGG CARGNQHW IGHV4-38- IGHJl*00

[0478] (SEQ ID NO:453) (SEQ ID NO:454) 2*00 1 1 TGTGCGAGCTCCTCCATCCCTTATTACGAT C AS S SI P Y Y DFSGGY Y Y YGM D V W IGHV1 -69*00 IGHJ6*00

[0479] TTTTCCGGAGGGTACTACTACTACGGTATG (SEQ ID NO:456)

[0480] GACGTCTGG

[0481] (SEQ ID NO:455)

[0482] 0.990487 0.990487 TGTGCGAAAATGACTGCCCAATACTTTGAG CAKMTAQYFESW IGHV4-34*00 IGHJ4*00

[0483] TCCTGG (SEQ ID NO:458)

[0484] (SEQ ID NO:457)

[0485] 0.981308 0.981308 TGTGCGAGAGGCCCCATACATTATTCGACC CARGPIHYSTVDYYGVDVW IGHV4-34*00 IGHJ6*00

[0486] GTCGACTATTACGGCGTGGACGTCTGG (SEQ ID NO:460)

[0487] (SEQ ID NO:459)

[0488] 0.971559 0.971559 TGTGTGAGAGGGGGGACTCGACATAATTG CVRGGTRHNWNFW IGHV4-31*00 IGHJ4*00

[0489] GAACTTCTGG (SEQ ID NO:462)

[0490] (SEQ ID NO: 461)

[0491] 0.895522 0.895522 TGTGCGCGCGTGAGGATCATGGGAGCCTC CARVRIMGASGDFNNW IGHV4-34*00 IGHJl*00

[0492] TGGAGACTTTAATAACTGG (SEQ ID NO:464)

[0493] (SEQ ID NO: 463)

[0494] 0.873016 0.873016 TGCGTGAGGGAGACCCAGGGACAGAGGAA CVRETQGQRNFDLW IGHV3-74*00 IGHJ2*00

[0495] CTTCGATCTCTGG (SEQ ID NO:466)

[0496] (SEQ ID NO:465)

[0497]

[0498] 0.869565 0.869565 TGTGCGAGACAGACTTCAAGAGGCTCCGA CARQTSRGSEHYRHKSEFDFW IGHV4-34*00 IGHJ4*00

[0499]

[0500] Attorney Docket No. 44807-0503WO1 / C18573

[0501] ACATTATAGACATAAGTCTGAATTTGACTT (SEQ ID NO:468)

[0502] CTGG

[0503] (SEQ ID NO: 467)

[0504] 0.86383 0.86383 TGTGCGAGTATTAGGTCGGGGAGTCCACA CASIRSGSPHSYW IGHV3-48*00 IGHJ4*00

[0505] TTCCTATTGG (SEQ ID NO:470)

[0506] (SEQ ID NO: 469)

[0507] 0.532258 0.532258 TGTGCGAGACGGCAAAAGTACGGTGGTGA CARRQKYGGEPEVW IGHV3-33*OO IGHJ6*00

[0508] ACCGGAAGTCTGG (SEQ ID NO:472)

[0509] (SEQ ID NO: 471)

[0510] 0.516484 0.516484 TGTGCGAAAGATGGGGGGTACAGCGGGAC CAKDGGYSGTSRYWYFDFW IGHV3-23*00 IGHJ2*00

[0511] CAGTCGATATTGGTACTTCGATTTCTGG (SEQ ID NO:474)

[0512] (SEQ ID NO: 473)

[0513] 0.478261 0.478261 TGTACGAGAGGCAACGATTACGTTTGGGG CTRGNDYVWGTYGEESW IGHV4-34*00 IGHJ4*00

[0514] GACTTATGGGGAGGAAAGCTGG (SEQ ID NO:476)

[0515] (SEQ ID NO:475)

[0516] 0.320755 0.320755 TGTGCGGCCTAGAGGGTACAATTTTTGGAG CAA*RVQFLEWFYVFDYW IGHV4-31*00 IGHJ4*00

[0517] TGGTTTTATGTCTTTGACTACTGG (SEQ ID NO:478)

[0518] (SEQ ID NO: 477)

[0519] 0.2 0.2 TGTGCGAGAGAAGAGGGCGGTTACGATAT CAREEGGYDIVTGYYAALDYW IGHV3-21*00 IGHJ4*00

[0520] TGTGACTGGTTATTATGCAGCCCTTGACTA (SEQ ID NO:480)

[0521] CTGG

[0522] (SEQ ID NO: 479)

[0523] 0.190476 0.190476 TGTGCGAGTTTACCGGTGGTGGTACCTGCC CASLPWVPATSNWFDPW IGHV4-4*00 IGHJ5*00

[0524] ACCTCAAACTGGTTCGACCCCTGG (SEQ ID NO:482)

[0525] (SEQ ID NO:481)

[0526] 0.15 0.15 TGTATCACTACTCCTAGTAGCGGCTGGCCC CITTPS SGWP AGGDFW IGHV3-15*OO IGHJ4*00

[0527]

[0528] GCGGGTGGCGACTTCTGG (SEQ ID NO:484)

[0529]

[0530] Attorney Docket No. 44807-0503WO1 / C18573

[0531] (SEQ ID NO:483)

[0532] 0.142857 0.142857 TGTGCGAGAGATTCGCCTTTTGCGTCCTGG CARDSPFASW IGHV4-38- IGHJ5*00

[0533] (SEQ ID NO:485) (SEQ ID NO:486) 2*00 0.136364 0.136364 TGTGTGAAAGATCGGAGCTTCAGCTGGGC CVKDRSFSWAFDYW IGHV3-3O*OO IGHJ4*00

[0534] CTTTGACTACTGG (SEQ ID NO:488)

[0535] (SEQ ID NO:487)

[0536] 0.135135 0.135135 TGTGCGAGGGCCCAGGGGTTCCAACTACC CARAQGFQLPYVGYCW IGHV3-33*OO IGHJ4*00

[0537] ATACGTGGGCTACTGTTGG (SEQ ID NO:490)

[0538] (SEQ ID NO:489)

[0539] 0.12 0.12 TGTGCGAGAGGGGTTGGAGTGGTTATTAT CARGVGVVIIGYYFDYW IGHV3-33*OO IGHJ4*00

[0540] AGGGTACTACTTTGACTACTGG (SEQ ID NO:492)

[0541] (SEQ ID NO: 491)

[0542] 0.111111 0.111111 TGTGCGAGAAGACGATATTACTTTGGTTCC CARRRYYFGSHNYYYYGMDVW IGHV3-35*OO IGHJ6*00

[0543] CATAACTACTACTACTACGGTATGGACGTC (SEQ ID NO:494)

[0544] TGG

[0545] (SEQ ID NO: 493)

[0546] 0.111111 0.111111 TGTGCGAGAGGATATATCCTTCGGATTGAC CARGYILRIDYW IGHV1 -46*00 IGHJ4*00

[0547] TACTGG (SEQ ID NO:496)

[0548] (SEQ ID NO:495)

[0549] 0.107143 0.107143 TGTGCGAGAGATATAGGTGGTTACGGTTA CARDIGGYGYFDYW IGHV4-59*00 IGHJ4*00

[0550] CTTTGACTACTGG (SEQ ID NO:498)

[0551] (SEQ ID NO: 497)

[0552] 0.1 0.1 TGTGCGAGAGGATCTAGTGATAGTAGTGG CARGSSDSSGYYFDYW IGHV4-34*00 IGHJ4*00

[0553] TTATTATTTTGACTACTGG (SEQ ID NO: 500)

[0554] (SEQ ID NO: 499)

[0555] 0.1 0.1 TGTGCGAGAGATCGTGGGAGCTACGTCTA CARDRGSYVYDYW IGHV1-18*OO IGHJ4*00

[0556]

[0557] TGACTACTGG (SEQ ID NO: 502)

[0558]

[0559] Attorney Docket No. 44807-0503WO1 / C18573

[0560] (SEQ ID NO:501)

[0561] 0.1 0.1 TGTGCGAGAGGAGGACGTGGATACAGCTA CARGGRGYSYGSKLYYFDYW IGHVl-8*00 IGHJ4*00

[0562] TGGTTCCAAATTGTACTACTTTGACTACTG (SEQ ID NO: 504)

[0563] G

[0564] (SEQ ID NO: 503)

[0565] 0.1 0.1 TGTGCGAAAGATTGTTGTGCGAAGCAGCT CAKDCCAKQLAVYYYYGIDVW IGHV3-ll*00 IGHJ6*00

[0566] GGCCGTCTACTACTACTACGGTATTGACGT (SEQ ID NO: 506)

[0567] CTGG

[0568] (SEQ ID NO: 505)

[0569] 0.096774 0.096774 TGTGCGGTGACCCCTCTCGAACGGCCCGGC CAVTPLERPGW IGHV1 -69*00 IGHJ4*00

[0570] TGG (SEQ ID NO: 508)

[0571] (SEQ ID NO: 507)

[0572] 0.090909 0.090909 TGTGCGAGAGATCAGAACAGGACGGCGGG CARDQNRTAGGYYYYYGMDVW IGHV1-18*OO IGHJ6*00

[0573] GGGATACTACTACTACTACGGTATGGACGT (SEQ ID NO: 510)

[0574] CTGG

[0575] (SEQ ID NO: 509)

[0576] 0.090909 0.090909 TGTGCGAGAGATTCGGGGAATCGACCGCC CARDSGNRPPKIAVAGYFDYW IGHV3-3O- IGHJ4*00

[0577] AAAAATAGCAGTGGCTGGATACTTTGACT (SEQ ID NO: 512) 3*00 ACTGG

[0578] (SEQ ID NO:511)

[0579] 0.083333 0.083333 TGTGCGAGAGACTCCTCGCCGGAAGGGTG CARDSSPEGCGDCYSGW IGHV1 -69*00 IGHJ4*00

[0580] TGGTGACTGCTATTCTGGCTGG (SEQ ID NO: 514)

[0581] (SEQ ID NO: 513)

[0582] 0.083333 0.083333 TGTGCGAGAGGATGCGCCTTATTGGGAGG CARGCALLGGNRP VSYYYYYGMD IGHVl-3*00 IGHJ6*00

[0583] AAACCGACCCCGTATCTTACTACTACTACT VW ACGGTATGGACGTCTGG (SEQ ID NO: 516)

[0584]

[0585] (SEQ ID NO: 515)

[0586]

[0587] Attorney Docket No. 44807-0503WO1 / C18573

[0588] 0.083333 0.083333 TGTGCGAGGGGAGAACTACCTAGAGGCTA CARGELPRGYDRYFDYW IGHV3-33*OO IGHJ4*00

[0589] CGATCGCTATTTTGACTACTGG (SEQ ID NO: 518)

[0590] (SEQ ID NO: 517)

[0591] 0.083333 0.083333 TGTGTGAGGACGAAAACGGGTGGGAGAAT CVRTKTGGRIF ITYYYYYMDVW IGHV7-56*00 IGHJ6*00

[0592] CTTCCATTACTTACTACTACTACTACATGG (SEQ ID NO: 520)

[0593] ACGTCTGG

[0594] (SEQ ID NO: 519)

[0595] 0.081081 0.081081 TGTGCAAGAGCGGTGGGAGCTACTTACTTT CARAVGATYFDYW IGHV3-74*00 IGHJ4*00

[0596] GACTACTGG (SEQ ID NO: 522)

[0597] (SEQ ID NO:521)

[0598] 0.07767 0.07767 TGTGTGAAGGATCGGGCGGGGTTTGGAAG CVKDRAGFGSHWHKDFDYW IGHV3-23*00 IGHJ4*00

[0599] TCACTGGCATAAGGACTTTGACTACTGG (SEQ ID NO: 524)

[0600] (SEQ ID NO: 523)

[0601] 0.076923 0.076923 TGTGCGAGAGATCGGAGGGATAGCAGTGG CARDRRDSSGWYPLDYW IGHV1-18*OO IGHJ4*00

[0602] CTGGTACCCCCTCGACTACTGG (SEQ ID NO: 526)

[0603] (SEQ ID NO: 525)

[0604] 0.076923 0.076923 TGTGCGAGAGGGGGATATAGCAGTGGCTG C ARGGYS SGWS V AQYYFDYW IGHV3-3O- IGHJ4*00

[0605] GTCGGTGGCCCAATACTACTTTGACTACTG (SEQ ID NO: 528) 3*00 G

[0606] (SEQ ID NO: 527)

[0607] 0.076923 0.076923 TGTGCGAGACATGTCGTATCTATGGTTCGG CARHVVSMVRGVRNWFDPW IGHV4-39*00 IGHJ5*00

[0608] GGAGTCCGTAACTGGTTCGACCCCTGG (SEQ ID NO:530)

[0609] (SEQ ID NO: 529)

[0610] 0.076923 0.076923 TGTGCGAGACATGGTACCCCTATTGTAGTA CARHGTPIVVVPAAIDYW IGHV4-39*00 IGHJ4*00

[0611] GTACCAGCTGCTATTGACTACTGG (SEQ ID NO: 532)

[0612] (SEQ ID NO: 531)

[0613] 0.076923 0.076923 TGTGCAAGACAGTTTTGTCTTACTACCAGT CARQFCLT Q*VKVD*R IGHV3-9*00 IGHJ4*00

[0614]

[0615]

[0616] Attorney Docket No. 44807-0503WO1 / C18573

[0617] AAGTCAAGGTGGACTAAAGG (SEQ ID NO: 534)

[0618] (SEQ ID NO:533)

[0619] 0.076923 0.076923 TGTACTAGACCTCGTGGATACAGCTATGGG CTRPRGYSYGGAGYW IGHV3-73*00 IGHJ4*00

[0620] GGTGCCGGCTACTGG (SEQ ID NO:536)

[0621] (SEQ ID NO: 535)

[0622] 0.071429 0.071429 TGTGCGAGAGATTCCTATGATAGTAGTGGC CARDSYDSSGPMDVW IGHV4-31*00 IGHJ6*00

[0623] CCTATGGACGTCTGG (SEQ ID NO: 538)

[0624] (SEQ ID NO: 537)

[0625] 0.071429 0.071429 TGTGCGAGAGTAAGTGGCCAACGGAGGGA CARVSGQRRDYW IGHV3-21*00 IGHJ4*00

[0626] CTATTGG (SEQ ID NO: 540)

[0627] (SEQ ID NO: 539)

[0628] 0.066667 0.066667 TGTGCAACAGATCCTATTGACTACGGTAGT CATDPIDYGSRSGTWDFW IGHV1 -24*00 IGHJ4*00

[0629] CGCTCCGGGACTTGGGACTTCTGG (SEQ ID NO: 542)

[0630] (SEQ ID NO: 541)

[0631] 0.066667 0.066667 TGTGCGAGATCACACTACGGTGACTACCTC CARSHYGDYLAYYMDVW IGHVl-2*00 IGHJ6*00

[0632] GCTTACTACATGGACGTCTGG (SEQ ID NO: 544)

[0633] (SEQ ID NO: 543)

[0634] 0.066667 0.066667 TGTGCGAAGAATACTGGGAGATTCCCGTA CAKNTGRFPYNWFDPW IGHV3-48*00 IGHJ5*00

[0635] TAACTGGTTCGACCCCTGG (SEQ ID NO: 546)

[0636] (SEQ ID NO: 545)

[0637] 0.066667 0.066667 TGTGCGCGGGACCGGGTGTCTTCACAGGG CARDRVSSQGYFQDW IGHV3-3O*OO IGHJl*00

[0638] ATACTTCCAGGACTGG (SEQ ID NO: 548)

[0639] (SEQ ID NO: 547)

[0640] 0.06383 0.06383 TGTGCAAACCTCTATGGGCGGGGCCCGGG CANLYGRGPGDYW IGHV3-74*00 IGHJ4*00

[0641] GGACTATTGG (SEQ ID NO:550)

[0642] (SEQ ID NO: 549)

[0643] 0.0625 0.0625 TGTGCGAGGCTGGACGTCTGG CARLDVW IGHVl-3*00 IGHJ6*00

[0644]

[0645]

[0646] Attorney Docket No. 44807-0503WO1 / C18573

[0647] (SEQ ID NO:551) (SEQ ID NO: 552)

[0648] 0.0625 0.0625 TGTGCGAAAGATCGCAGCAGCCCGTACTA CAKDRSSPYYFDYW IGHV3-3O*OO IGHJ4*00

[0649] CTTTGACTACTGG (SEQ ID NO: 554)

[0650] (SEQ ID NO:553)

[0651] 0.058824 0.058824 TGTGCGAGAGATCGATCGTTGTGGTGGTG CARDRSLWW*LSFGGDWFDPW IGHV3-53*OO IGHJ5*00

[0652] ATTGTCATTCGGAGGGGACTGGTTCGACCC (SEQ ID NO:556)

[0653] CTGG

[0654] (SEQ ID NO:555)

[0655] 0.057971 0.057971 TGTGCGCGACAAGGCTGGCTCGAATATTA CARQGWLEYYYDSW IGHV4-59*00 IGHJ4*00

[0656] CTATGATTCTTGG (SEQ ID NO: 558)

[0657] (SEQ ID NO: 557)

[0658] 0.057692 0.057692 TGTACTAGAGATTCGTATTACTATGATAGT CTRDSYYYDSSGFVSAFDIW IGHV3-49*00 IGHJ3*00

[0659] AGTGGTTTCGTATCGGCTTTTGATATCTGG (SEQ ID NO: 560)

[0660] (SEQ ID NO: 559)

[0661] 0.054795 0.054795 TGTGCGAGATTTGGGGGGAGCGGTGGCTG CARFGGSGGWYGPDDYW IGHV5-51*00 IGHJ4*00

[0662] GTACGGTCCCGATGACTACTGG (SEQ ID NO: 562)

[0663] (SEQ ID NO:561)

[0664] 0.052632 0.052632 TGTGCACACAAAGGAGTAGTACCAGCTGC CAHKGVVPAAIGSYYGMDVW IGHV2-5*00 IGHJ6*00

[0665] TATTGGGAGCTACTACGGTATGGACGTCTG (SEQ ID NO: 564)

[0666] G

[0667] (SEQ ID NO: 563)

[0668] 0.052632 0.052632 TGTGCGAGAGATTCCTGGCGGTACGATATT CARDSWRYDI DWLLNYFDYW IGHV1-18*OO IGHJ4*00

[0669] TTGACTGGTTATTGAACTACTTTGACTACT (SEQ ID NO: 566)

[0670] GG

[0671] (SEQ ID NO: 565)

[0672] 0.05 0.05 TGTGCGAGAGTATCTTACTATGGTTCGGGG CARVSYYGSGTYYYFDYW IGHV3-64*00 IGHJ4*00

[0673]

[0674] ACTTATTATTACTTTGACTACTGG (SEQ ID NO: 568)

[0675]

[0676] Attorney Docket No. 44807-0503WO1 / C18573

[0677] (SEQ ID NO: 567)

[0678] 0.05 0.05 TGTGCGAAAGATCTTGGGAGTTACTATGAT CAKDLGSYYDSIDYW IGHV3-23*00 IGHJ4*00

[0679] AGTATTGACTACTGG (SEQ ID NO: 570)

[0680] (SEQ ID NO: 569)

[0681] 0.04918 0.04918 TGTGCGAGAGGCAACTATGATAGTAGTGG CARGNYDSSGYYDYW IGHV1 -46*00 IGHJ4*00

[0682] TTATTACGACTACTGG (SEQ ID NO: 572)

[0683] (SEQ ID NO:571)

[0684] 0.044776 0.044776 TGTGCGAGAGACCTCAACTACGGTGACTC CARDLNYGDSTAAFDYW IGHVl-3*00 IGHJ4*00

[0685] AACCGCGGCATTTGACTACTGG (SEQ ID NO: 574)

[0686] (SEQ ID NO: 573)

[0687] 0.044444 0.044444 TGTGCGAAAGTCGAGGTGAACTACTACTA CAKVEVNYYYYMDVW IGHV3-23*00 IGHJ6*00

[0688] CTACATGGACGTCTGG (SEQ ID NO: 576)

[0689] (SEQ ID NO: 575)

[0690] 0.040486 0.040486 TGTGCGAGAGATCAGACTGCTGCCTGGAA CARDQTAAWNRGHFFDYW IGHV4-31*00 IGHJ4*00

[0691] CCGCGGACATTTCTTTGACTACTGG (SEQ ID NO: 578)

[0692] (SEQ ID NO: 577)

[0693] 0.036145 0.036145 TGTGCGAGGGGCCCTTACGATTTTTGGAGT CARGPYDFWSGIWFDPW IGHV3-21*00 IGHJ5*00

[0694] GGCATCTGGTTCGACCCCTGG (SEQ ID NO: 580)

[0695] (SEQ ID NO: 579)

[0696] 0.035714 0.035714 TGTGCGAGAGCGTCGGACTACGGGGACTA CARASDYGDYW IGHV3-21*00 IGHJ4*00

[0697] CTGG (SEQ ID NO: 582)

[0698] (SEQ ID NO:581)

[0699] 0.033333 0.033333 TGTGCGAGAGCTGCGCTCGGTAGAGACAG CARAALGRDSGTYAYW IGHV3-21*00 IGHJ4*00

[0700] TGGGACCTACGCCTACTGG (SEQ ID NO: 584)

[0701] (SEQ ID NO:583)

[0702] 0.030303 0.030303 TGTGTCAAAGCGGGGGACATCAGCTGGTA CVKAGDISWYEYW IGHV3-53*OO IGHJ4*00

[0703]

[0704] TGAGTACTGG (SEQ ID NO:586)

[0705]

[0706] Attorney Docket No. 44807-0503WO1 / C18573

[0707] (SEQ ID NO:585)

[0708] 0.025 0.025 TGTGCGAAAGATCGCCAGGACTACGGTGA CAKDRQDYGDSYYFDYW IGHV3-3O*OO IGHJ4*00

[0709] CTCGTACTACTTTGACTACTGG (SEQ ID NO:588)

[0710] (SEQ ID NO:587)

[0711] 0.025 0.025 TGTGTGAGAGATATGAATATTGTGGTGGTG CVRDMNIVW LRYPRFGYW IGHVl-17*00 IGHJ4*00

[0712] ACTGCGATATCCCCGCTTTGGGTACTGG (SEQ ID NO: 590)

[0713] (SEQ ID NO:589)

[0714] 0.02439 0.02439 TGTGCGAGAGGGGCGAGTGATTATATAAC CARGASDYITGYYFAYW IGHV4-59*00 IGHJ4*00

[0715] GGGCTACTACTTTGCCTACTGG (SEQ ID NO: 592)

[0716] (SEQ ID NO:591)

[0717] 0.022901 0.022901 TGTCGCACCCGGAAGGGGATAGTAGTGGT CRTRKGIWVPHAFDIW IGHV4-30- IGHJ3*00

[0718] CCCCCATGCTTTTGATATCTGG (SEQ ID NO: 594) 2*00 (SEQ ID NO: 593)

[0719] 0.022556 0.022556 TGTGGGAGAGGCCGAGGTCGTAGCAGTGG CGRGRGRSSGWGNYYYYGMDVW IGHVl-8*00 IGHJ6*00

[0720] CTGGGGGAATTACTACTACTACGGTATGG (SEQ ID NO: 596)

[0721] ACGTCTGG

[0722] (SEQ ID NO: 595)

[0723] 0.021053 0.021053 TGTGCGAGAGACTGGGGAGAGTTTGACTA CARDWGEFDYW IGHV1 -46*00 IGHJ4*00

[0724] CTGG (SEQ ID NO: 598)

[0725] (SEQ ID NO: 597)

[0726] 0.018868 0.018868 TGTGCGAGACGAAGGGGGCAGCCCCCCAA CARRRGQPPNFDYW IGHV5-51*OO IGHJ4*00

[0727] CTTTGACTACTGG (SEQ ID NO: 600)

[0728] (SEQ ID NO: 599)

[0729] 0.018519 0.018519 TGTGCGAGAGCGGCAACTCACTCTGGGGG CARAATHSGGG AVAGLPHNYW IGHV4-39*00 IGHJ4*00

[0730] TGGGAGCAGTGGCTGGTCTCCCCCACAACT (SEQ ID NO: 602)

[0731] ACTGG

[0732]

[0733] (SEQ ID NO: 601)

[0734]

[0735] Attorney Docket No. 44807-0503WO1 / C18573

[0736] 0.016043 0.016043 TGTGCGAGAGGAGCTTCCCGAACGATTTTT CARGASRTIFGVVIIRGRGFGWFDP IGHV3-33*OO IGHJ5*00

[0737] GGAGTGGTTATTATAAGGGGGAGGGGATT W CGGCTGGTTCGACCCCTGG (SEQ ID NO: 604)

[0738] (SEQ ID NO: 603)

[0739] 0.014925 0.014925 TGTGCGAGAGCCGCCGATAGTAGTGGTTTT CARAADSSGFDYW IGHV4-4*00 IGHJ4*00

[0740] GACTACTGG (SEQ ID NO:606)

[0741] (SEQ ID NO:605)

[0742] 0.014354 0.014354 TGTGCGAGAGGCCAAAGTAGAAGGTACCA CARGQSRRYQLLPGRAFDIW IGHV4-34*00 IGHJ3*00

[0743] GCTGCTACCTGGTCGTGCTTTTGATATCTG (SEQ ID NO: 608)

[0744] G

[0745] (SEQ ID NO: 607)

[0746] 0.014085 0.014085 TGTGCGAGTAGCTGGGGTATAGCAGTGGC CASSWGIAVAGTYLDYW IGHV3-33*OO IGHJ4*00

[0747] TGGTACTTATCTAGACTACTGG (SEQ ID NO: 610)

[0748] (SEQ ID NO:609)

[0749] 0.008523 0.008523 TGTGCGAAAGGGAATTATTATGGGTCGGG CAKGNYYGSGDLW IGHV4-59*00 IGHJ4*00

[0750] GGATCTCTGG (SEQ ID NO:612)

[0751] (SEQ ID NO:611)

[0752] 0.157895 0 TGTGCGAGGGGTAATGGTGGCTACGACCC CARGNGGYDPLDMDVW IGHV3-21*00 IGHJ6*00

[0753] CTTAGACATGGACGTCTGG (SEQ ID NO: 614)

[0754] (SEQ ID NO:613)

[0755] 0.157895 0 TGTGCGTTTGGTGGGGCCTCCTACGAGCCA CAFGGASYEPFDIW IGHVl-2*00 IGHJ3*00

[0756] TTTGATATCTGG (SEQ ID NO: 616)

[0757] (SEQ ID NO:615)

[0758] 0.388889 0 TGTGCACACAGAGGGAGCTACTATTTCATG CAHRGSYYFMDFDYW IGHV2-5*00 IGHJ4*00

[0759] GACTTTGACTACTGG (SEQ ID NO: 618)

[0760] (SEQ ID NO: 617)

[0761] 0.176471 0 TGTACTAGAGATATCCCCGGTGCCACACCA CTRDIPGATPEGDYW IGHV3-49*00 IGHJ4*00

[0762]

[0763]

[0764] Attorney Docket No. 44807-0503WO1 / C18573

[0765] GAGGGGGACTACTGG (SEQ ID NO: 620)

[0766] (SEQ ID NO: 619)

[0767] 0.176471 0 TGTGCGGGGGTTGGGAGCCACTACGAG CAGVGSHYE IGHV3-23*00 IGHJ4*00

[0768] (SEQ ID NO: 621) (SEQ ID NO: 622)

[0769] 0.125 0 TGTAGTCGGAGGTCGGGGTACTGTGGTGG CSRRSGYCGGGRCLNFDHW IGHV3-49*00 IGHJ4*00

[0770] TGGAAGGTGCTTGAACTTTGACCATTGG (SEQ ID NO: 624)

[0771] (SEQ ID NO: 623)

[0772] 0.125 0 TGTGCGAACGGATAGCAGCTCGTCGTTGA CANG*QLVVDCW IGHVl-3*00 IGHJ4*00

[0773] CTGCTGG (SEQ ID NO: 626)

[0774] (SEQ ID NO:625)

[0775] 0.125 0 TGTGCGAGAGGGGGGCAGCAGCAGTTGGT CARGGQQQLVPKTTYYYYGLDVW IGHVl-8*00 IGHJ6*00

[0776] TCCCAAGACAACCTACTACTACTACGGTCT (SEQ ID NO: 628)

[0777] GGACGTCTGG

[0778] (SEQ ID NO: 627)

[0779] 0.125 0 TGTGCGAGCCTCTGGGCCGGATACAGCTAT CASLWAGYSYGAPDYW IGHVl-3*00 IGHJ4*00

[0780] GGTGCCCCTGACTACTGG (SEQ ID NO: 630)

[0781] (SEQ ID NO: 629)

[0782] 0.125 0 TGTGCGGCGTCACTAGGGAGTATAGCAGT CAASLGSIAVAGSPPSGWW IGHVl-58*00 IGHJ4*00

[0783] GGCTGGTAGTCCCCCTTCGGGGTGGTGG (SEQ ID NO:632)

[0784] (SEQ ID NO: 631)

[0785] 0.125 0 TGTGCGAGACGGGGGATAGTGGGAGCTAC CARRGIVGATSAYYYGMDVW IGHV5-51*OO IGHJ6*00

[0786] TTCAGCCTACTACTACGGTATGGACGTCTG (SEQ ID NO:634)

[0787] G

[0788] (SEQ ID NO: 633)

[0789] 0.142857 0 TGTGCGAGACATATTCGTGTACTAATGGTG CARHIRVLMVYAPLGYYYGMDVW IGHV4-39*00 IGHJ6*00

[0790] TATGCCCCCCTTGGGTACTACTACGGTATG (SEQ ID NO: 636)

[0791]

[0792] GACGTCTGG

[0793]

[0794] Attorney Docket No. 44807-0503WO1 / C18573

[0795] (SEQ ID NO:635)

[0796] 0.461538 0 TGTGCGAGTCTTACGGTGGGCCCGGTACCA CASLTVGPVPAAIRVPRASMDVW IGHV4-39*00 IGHJ6*00

[0797] GCTGCAATTCGGGTTCCTCGGGCGAGTATG (SEQ ID NO: 638)

[0798] GACGTCTGG

[0799] (SEQ ID NO: 637)

[0800] 0.166667 0 TGTGCGAGAGTGGGGACGGGTTACCGACC CARVGTGYRPLDIW IGHV3-3O- IGHJ3*00

[0801] TTTGGATATCTGG (SEQ ID NO: 640) 3*00 (SEQ ID NO: 639)

[0802] 0.166667 0 TGTGCGAGATCGAGATTACTATGATAGTA CARSRLL DSSLPW IGHVl-3*00 IGHJ5*00

[0803] GTCTCCCCTGG (SEQ ID NO: 642)

[0804] (SEQ ID NO: 641)

[0805] 0.166667 0 TGTGCGAGAGAAGATGGCTACAAATTTGA CAREDGYKFDYW IGHV1 -46*00 IGHJ4*00

[0806] CTACTGG (SEQ ID NO: 644)

[0807] (SEQ ID NO: 643)

[0808] 0.166667 0 TGTGCGAGACACTGTGATAGTAGTGGTTAT CARHCDSSGYLRP DRLNRFGYYFD IGHV5-51*OO IGHJ4*00

[0809] TTGCGCCCGTTGACCGGTTAAACCGTTTTG YW GCTACTACTTTGACTACTGG (SEQ ID NO: 646)

[0810] (SEQ ID NO:645)

[0811] 0.166667 0 TGTGCAAGAGACTACGGTGACTACGTCTCT CARDYGDYVSGWNW IGHV3-74*00 IGHJ4*00

[0812] GGGTGGAACTGG (SEQ ID NO: 648)

[0813] (SEQ ID NO: 647)

[0814] 0.166667 0 TGTGCGAGAGCTCTAGCAGTGGCTGATCCC CARALA VADPYGMDVW IGHV1-18*OO IGHJ6*00

[0815] TACGGTATGGACGTCTGG (SEQ ID NO:650)

[0816] (SEQ ID NO: 649)

[0817] 0.363636 0 TGTGCGAGACTCGCGGGGAGAGCAGCAGC CARLAGRAAAGMDYW IGHV5-51*OO IGHJ4*00

[0818] TGGTATGGACTACTGG (SEQ ID NO:652)

[0819]

[0820] (SEQ ID NO:651)

[0821]

[0822] Attorney Docket No. 44807-0503WO1 / C18573

[0823] 0.272727 0 TGTGCGAGAGATGCGGAGCGGGATCATGC CARDAERDHAWLVSAFGYW IGHV1-18*OO IGHJ4*00

[0824] TGTAGTACTAGTGTCCGCATTCGGCTACTG (SEQ ID NO:654)

[0825] G

[0826] (SEQ ID NO:653)

[0827] 0.2 0 TGTGCGAGAGGCCGCGGGCATGACAATGG CARGRGHDNGWGSYYYYMDVW IGHV4-34*00 IGHJ6*00

[0828] CTGGGGGTCCTACTACTACTACATGGACGT (SEQ ID NO:656)

[0829] CTGG

[0830] (SEQ ID NO:655)

[0831] 0.2 0 TGTGCGAGAGATAGGCAGTGACTACCCTC CARDRQ*LPSDIW IGHV3-21*00 IGHJ3*00

[0832] AGATATCTGG (SEQ ID NO:658)

[0833] (SEQ ID NO:657)

[0834] 0.2 0 TGTGCGAGAGCTCCTAGCACCTTAAACTGG CARAPSTLNWFDPW IGHVl-3*00 IGHJ5*00

[0835] TTCGACCCCTGG (SEQ ID NO: 660)

[0836] (SEQ ID NO:659)

[0837] 0.2 0 TGTGCGAAAGATGCAGGATGTAGTAGTAC CAKDAGCSSTSCYFVGHPVFDYW IGHV3-23*00 IGHJ4*00

[0838] CAGCTGCTATTTTGTGGGTCATCCGGTCTT (SEQ ID NO: 662)

[0839] TGACTACTGG

[0840] (SEQ ID NO:661)

[0841] 0.333333 0 TGTGCGGGGGTTATGATAAGCTTGAGGAT CAGVMISLRMDFQHW IGHV4-34*00 IGHJl*00

[0842] GGACTTCCAGCACTGG (SEQ ID NO: 664)

[0843] (SEQ ID NO: 663)

[0844] 0.25 0 TGTGCGAGAGCGGATCATCCGCATATGGTT CARADHPHMVRARYYYYYGMDV IGHV4-4*00 IGHJ6*00

[0845] CGAGCGAGGTACTACTACTACTACGGTAT W GGACGTCTGG (SEQ ID NO: 666)

[0846] (SEQ ID NO:665)

[0847] 0.25 0 TGTGCGAAAGATGCGTGTAGTACCAGCTG CAKDACSTSCHEVHW IGHV3-ll*00 IGHJ4*00

[0848]

[0849] CCATGAGGTACACTGG (SEQ ID NO: 668)

[0850]

[0851] Attorney Docket No. 44807-0503WO1 / C18573

[0852] (SEQ ID NO: 667)

[0853] 0.25 0 TGTGCGAGATCCAGAATATTGTTCGGGGA CARSRILFGESSVFDFW IGHV4-55*00 IGHJ3*00

[0854] GTCTAGTGTTTTTGATTTCTGG (SEQ ID NO: 670)

[0855] (SEQ ID NO: 669)

[0856] 0.25 0 TGTGCGAGATGGGATAAAGAGGTCGGATT CARWDKEVGFIPSLDNW IGHV1 -46*00 IGHJ4*00

[0857] TATTCCCTCCTTAGACAACTGG (SEQ ID NO: 672)

[0858] (SEQ ID NO: 671)

[0859] 0.25 0 TGTGCGAGAGTCAGGCGGGAGTACCAGCT CARVRREYQLLGYFDYW IGHVl-3*00 IGHJ4*00

[0860] GCTGGGGTACTTTGACTACTGG (SEQ ID NO: 674)

[0861] (SEQ ID NO: 673)

[0862] 0.714286 0 TGTGCGAGAATACGCTTAAACGATATTTTG CAR1RLNDILTGYYNGPLDYYYGM IGHVl-2*00 IGHJ6*00

[0863] ACTGGTTATTATAACGGGCCCCTTGACTAC DVW TACTACGGTATGGACGTCTGG (SEQ ID NO: 676)

[0864] (SEQ ID NO:675)

[0865] 0.333333 0 TGTGCGAGAGATGGTAGTTCCGCCGCAGC CARDGSSAAAAGPQWDVW IGHV3-21*00 IGHJ6*00

[0866] AGCTGGCCCCCAGTGGGACGTCTGG (SEQ ID NO: 678)

[0867] (SEQ ID NO: 677)

[0868] 0.333333 0 TGTGCAAGGGATAGAGGTAGCGGCCAGTG CARDRGSGQWLVPGTLVSW IGHV3-9*00 IGHJ5*00

[0869] GCTGGTACCAGGGACCTTAGTCTCCTGG (SEQ ID NO:680)

[0870] (SEQ ID NO: 679)

[0871] 0.333333 0 TGTGCGAGAGTGCCGGACGCTTCTTACGAT CARVPDASYDFWSGYSPYYFDYW IGHV3-21*00 IGHJ4*00

[0872] TTTTGGAGTGGTTATTCCCCGTACTACTTT (SEQ ID NO: 682)

[0873] GACTACTGG

[0874] (SEQ ID NO:681)

[0875] 0.333333 0 TGTGCGACAGATGTCCCCACATGACTACA CATDVPT*LQ*LRDW IGHV1-18*OO IGHJ4*00

[0876] GTAACTACGGGACTGG (SEQ ID NO: 684)

[0877]

[0878] (SEQ ID NO:683)

[0879]

[0880] Attorney Docket No. 44807-0503WO1 / C18573

[0881] 0.333333 0 TGTGCGAGAGATCGCCTTGTCGTTACTATG CARDRLVVTM SCGQIPHDYW IGHV1-18*OO IGHJ4*00

[0882] ATAGTTGTGGTCAAATACCTCATGACTACT (SEQ ID NO:686)

[0883] GG

[0884] (SEQ ID NO:685)

[0885] 0.333333 0 TGACCGCGAATGCCCCCGAGACCTGGATTT *PRMPPRPGFDSW IGHV4-59*00 IGHJ4*00

[0886] GATTCCTGG (SEQ ID NO:688)

[0887] (SEQ ID NO:687)

[0888] 0.333333 0 TGTGCGAGAGTAGCTGATAGTAGTGGTTAT CARVADSSGYYQFDYW IGHV1 -69*00 IGHJ4*00

[0889] TACCAGTTTGACTACTGG (SEQ ID NO: 690)

[0890] (SEQ ID NO:689)

[0891] 0.333333 0 TGTGCGCGAGATCGTCTAAGCTCTATTAAT C ARDRLS SINGLWYGDGGYYYYG IGHV3-64*00 IGHJ6*00

[0892] GGCCTCTGGTACGGCGACGGGGGATACTA MNVW CTACTACGGTATGAACGTCTGG (SEQ ID NO: 692)

[0893] (SEQ ID NO:691)

[0894] 0.333333 0 TGTGCGAGACTGGGGGTCTTAGGGAACTG CARLGVLGNWFDPW IGHV5-10- IGHJ5*00

[0895] GTTCGACCCCTGG (SEQ ID NO: 694) 1*00 (SEQ ID NO: 693)

[0896] 0.333333 0 TGTGCGAGAGAACGTATTGGGTATGATAG CARERIGYDSSALGYLDYW IGHVl-3*00 IGHJ4*00

[0897] TAGTGCCCTCGGCTACTTGGACTACTGG (SEQ ID NO: 696)

[0898] (SEQ ID NO:695)

[0899] 0.333333 0 TGTGCGAGACTTAATTGGGCAATGGGTGA CARLNWAMGDSSGYSLYYFDYW IGHV4-39*00 IGHJ4*00

[0900] TAGTAGTGGTTACTCGCTCTACTACTTTGA (SEQ ID NO:698)

[0901] CTACTGG

[0902] (SEQ ID NO: 697)

[0903] 0.333333 0 TGTGCGAGACCGGTCCGACTTCTAGAAAA CARPVRLLENW IGHV3-21*00 IGHJ4*00

[0904] CTGG (SEQ ID NO: 700)

[0905]

[0906] (SEQ ID NO: 699)

[0907]

[0908] Attorney Docket No. 44807-0503WO1 / C18573

[0909] 0.333333 0 TGTGCGGTAATTACTACTCAATGTTCTACT CAVITTQCS YYHGMDVW IGHV3-23*00 IGHJ6*00

[0910] ACTACCACGGTATGGACGTCTGG (SEQ ID NO: 702)

[0911] (SEQ ID NO: 701)

[0912] 1 0 TGTGCGCGAGATGTTGGTAGTGGTTACTAC CARDVGSGYYFDYW IGHV4-4*00 IGHJ4*00

[0913] TTTGACTACTGG (SEQ ID NO: 704)

[0914] (SEQ ID NO: 703)

[0915] 0.6 0 TGTGCGAGATTTTCCTCTACGGCGACACGT CARFSSTAT SWWYFDLW IGHV4-55*00 IGHJ2*00

[0916] CCTGGTGGTACTTCGATCTCTGG (SEQ ID NO: 706)

[0917] (SEQ ID NO:705)

[0918] 0.5 0 TGTGCGAGAGAGGATGGAGAAGTGCCTGG CAREDGEVPGLYYFDSW IGHV3-20*00 IGHJ4*00

[0919] TCTGTACTACTTTGACTCCTGG (SEQ ID NO: 708)

[0920] (SEQ ID NO: 707)

[0921] 0.5 0 TGTGCACGGATACTGCTACTCTAAATGTAT CARILLL*M_ITIMGHYW IGHV2-26*00 IGHJ4*00

[0922] AACTATAATGGGACACTACTGG (SEQ ID NO: 710)

[0923] (SEQ ID NO: 709)

[0924] 0.5 0 TGTGCGAGCGCTTCAGCAGCTGGTACTATA CASASAAGTIIDYW IGHV4-59*00 IGHJ4*00

[0925] ATTGACTACTGG (SEQ ID NO:712)

[0926] (SEQ ID NO:711)

[0927] 0.5 0 TGTGTAAGGGGGGCCTATGGTGGAGCGTT CVRGAYGGAFDHW IGHV6-l*00 IGHJ4*00

[0928] TGACCACTGG (SEQ ID NO:714)

[0929] (SEQ ID NO:713)

[0930] 0.5 0 TGTGCGAAAGATATCCTATGGTGGTCCTTT CAKDILWWSFDYW IGHV3-23*00 IGHJ4*00

[0931] GACTACTGG (SEQ ID NO:716)

[0932] (SEQ ID NO:715)

[0933] 0.5 0 TGTGCCAGAGATCGGGGATATGACAGTAG CARDRGYDSSWSDYW IGHV3-33*OO IGHJ4*00

[0934] TTGGTCGGACTATTGG (SEQ ID NO: 718)

[0935]

[0936] (SEQ ID NO: 717)

[0937]

[0938] Attorney Docket No. 44807-0503WO1 / C18573

[0939] 0.5 0 TGTGCACGGACAATACTGGGGACGGGCAG CARTILGTGSIVARNYFFDYW IGHV2-70*00 IGHJ4*00

[0940] TATAGTGGCAAGGAATTACTTCTTTGACTA (SEQ ID NO: 720)

[0941] TTGG

[0942] (SEQ ID NO: 719)

[0943] 0.5 0 TGTGCGAGACGGGATGTGACTACGGTGAC CARRDVTTVTTYHFDYW IGHV4-39*00 IGHJ4*00

[0944] TACTTATCACTTTGACTACTGG (SEQ ID NO: 722)

[0945] (SEQ ID NO: 721)

[0946] 0.5 0 TGTGCGGAAGACACGGCTGTGGTCGCCCG CAEDTAVVARWEE_GI*K*KNGMD IGHV3-66*00 IGHJ6*00

[0947] GTGGGAGGAAGGGGAATTTGAAAATAGAA VW GAACGGTATGGACGTCTGG (SEQ ID NO: 724)

[0948] (SEQ ID NO: 723)

[0949] 0.5 0 TGTGCGAGACATCGTGATTACAGTCCCCGA CARHRDYSPRDYYYFAMDVW IGHV4-39*00 IGHJ6*00

[0950] GACTACTACTACTTCGCTATGGACGTCTGG (SEQ ID NO: 726)

[0951] (SEQ ID NO:725)

[0952] 0.5 0 TGTGCGAGCCTAGACGGGGATGGTTCGGG CASLDGD VRGVIP IGHV4-39*00 IGHJ4*00

[0953] GAGTTATTCCG (SEQ ID NO: 728)

[0954] (SEQ ID NO: 727)

[0955] 0.5 0 TGTTTTTCCTCGACCCTTTTGGAGTGGTTAT CFS STLLEWLLGIFPGATRFD YW IGHV3-23*00 IGHJ4*00

[0956] TGGGCATATTTCCTGGGGCCACCCGCTTTG (SEQ ID NO: 730)

[0957] ACTACTGG

[0958] (SEQ ID NO: 729)

[0959] 0.5 0 TGTGCGAGAGCAGAGGAGTACGATTTTTG CARAEEYDFWSGYSIYYYGMDVW IGHVl-2*00 IGHJ6*00

[0960] GAGTGGTTATTCTATCTACTACTACGGTAT (SEQ ID NO:732)

[0961] GGACGTCTGG

[0962] (SEQ ID NO: 731)

[0963] 0.5 0 TGTGCGAGACTTCCCCCTTCCCAATACTAT CARLPPSQYYGTGNPPDYW IGHV5-51*OO IGHJ4*00

[0964]

[0965] GGTACGGGTAATCCTCCTGACTACTGG (SEQ ID NO:734)

[0966]

[0967] Attorney Docket No. 44807-0503WO1 / C18573

[0968] (SEQ ID NO: 733)

[0969] 0.5 0 TGTGCGAGAGGAACCAGTGGGCCCGACTA CARGTSGPDYW IGHV3-23*00 IGHJ4*00

[0970] CTGG (SEQ ID NO: 736)

[0971] (SEQ ID NO:735)

[0972] 0.5 0 TGTGCGAGTTCCGGGGGCGATGTGGTAGTT C AS SGGDV WPA AREMAYW IGHV4-34*00 IGHJ4*00

[0973] CCGGCTGCTAGAGAGATGGCCTATTGG (SEQ ID NO: 738)

[0974] (SEQ ID NO: 737)

[0975] 0.5 0 TGTGCGAGAAGTAAACTCAGGGTGGTGAC CARSKLRWTARGYPPYFDYW IGHV4-34*00 IGHJ4*00

[0976] TGCTCGAGGATACCCCCCCTACTTTGACTA (SEQ ID NO: 740)

[0977] CTGG

[0978] (SEQ ID NO: 739)

[0979] 0.5 0 TGTGCGAAAGATCTAAGGGCAGTGGCTGG CAKDLRA GWSSDYW IGHV3-23*00 IGHJ4*00

[0980] TCAAGTGACTACTGG (SEQ ID NO: 742)

[0981] (SEQ ID NO: 741)

[0982] 0.5 0 TGTACCGCCACCAGGGGATATAGTGGCTC CTATRGYSGSVVDYW IGHV3-49*00 IGHJ4*00

[0983] CGTAGTTGACTACTGG (SEQ ID NO: 744)

[0984] (SEQ ID NO: 743)

[0985] 0.5 0 TGTGCGAGAGATGGGGAGGGGTATATAGC CARDGEGYI QWLVFDYW IGHV1 -46*00 IGHJ4*00

[0986] AGTGGCTGGTATTTGACTACTGG (SEQ ID NO: 746)

[0987] (SEQ ID NO:745)

[0988] 1 0 TGTGTAAAGGACATGGTCCCTTGTCGTGTT CVKDMVPCRVGGCYPKRYGMDVW IGHV3-9*00 IGHJ6*00

[0989] GGCGGCTGCTACCCTAAGCGCTATGGTATG (SEQ ID NO: 748)

[0990] GACGTCTGG

[0991] (SEQ ID NO: 747)

[0992] 1 0 TGTGCGACCCCAGGGGATTACGATTTTTGG CATPGDYDFWSGLLGLVY(SEQ ID IGHV3-23*00 IGHJ4*00

[0993] AGTGGGCTCTTAGGTCTTGTCTACTGG NO:750)

[0994]

[0995] (SEQ ID NO: 749)

[0996]

[0997] Attorney Docket No. 44807-0503WO1 / C18573

[0998] 1 0 TGTGCGAGAGATAATGGTAACTATGGGGG CARDNGNYGGYYYYGMDVW IGHV4-59*00 IGHJ6*00

[0999] TTACTACTACTACGGTATGGACGTCTGG (SEQ ID NO:752)

[1000] (SEQ ID NO: 751)

[1001] 1 0 GCCAGAGGGAGACTTAGGTATGATAGTAG ARGRLRYDSSGYYYLDYFDYW IGHV4-30- IGHJ4*00

[1002] TGGTTATTACTACTTGGACTACTTTGACTA (SEQ ID NO: 754) 4*00 CTGG

[1003] (SEQ ID NO: 753)

[1004] 1 0 TGTGCACACAGACGGCCCGGGCCTCCCAC CAHRRPGPPTLI CLGELSLLFDYW IGHV2-5*00 IGHJ4*00

[1005] CTTAATCCTGTTTGGGGGAGTTATCGTTAC (SEQ ID NO:756)

[1006] TATTTGACTACTGG

[1007] (SEQ ID NO:755)

[1008] 1 0 TGTGCGAGAGATTTCGGCCCCTATGATAGT CARDFGPYDSRGYADYWYFDLW IGHV1-18*OO IGHJ2*00

[1009] AGGGGTTATGCCGACTACTGGTACTTCGAT (SEQ ID NO: 758)

[1010] CTCTGG

[1011] (SEQ ID NO: 757)

[1012] 1 0 TGTGCGAGGCTTTTCCTTTGTGGTGGTGAC CARLFLCGGDCYSYFDYW IGHV3-21*00 IGHJ4*00

[1013] TGCTATTCCTACTTTGACTACTGG (SEQ ID NO: 760)

[1014] (SEQ ID NO:759)

[1015] 1 0 TGTGCAAAAGATATAGGGGAGGATATTGT CAKDIGEDIVWSALDVW IGHV3-9*00 IGHJ6*00

[1016] AGTAGTGTCTGCCCTGGACGTCTGG (SEQ ID NO: 762)

[1017] (SEQ ID NO: 761)

[1018] 1 0 TGTGCGAAAGATTACCAGGGACTGGTTCCT CAKDYQGLVPDAFDVW IGHV3-23*00 IGHJ3*00

[1019] GATGCTTTTGATGTCTGG (SEQ ID NO: 764)

[1020] (SEQ ID NO: 763)

[1021] 1 0 TGTGCGAGAGATTTCTACCAGCTGCCGGA CARDFYQLPELYS IGHV3-21*00 IGHJ4*00

[1022] ACTCTACTCT (SEQ ID NO: 766)

[1023]

[1024] (SEQ ID NO:765)

[1025]

[1026] Attorney Docket No. 44807-0503WO1 / C18573

[1027] 1 0 TGTGCGAGAGGGCATACTACTGGCTGGCA CARGHTTGWHSLAYW IGHV4-34*00 IGHJ4*00

[1028] CTCCCTTGCCTACTGG (SEQ ID NO:768)

[1029] (SEQ ID NO: 767)

[1030] 1 0 TGTGCGAGAGATGGGGTGGCAGCTCGATC CARDGVAARSENYFHFYYMDVW IGHV1-18*OO IGHJ6*00

[1031] CGAAAATTACTTCCACTTCTACTACATGGA (SEQ ID NO: 770)

[1032] CGTCTGG

[1033] (SEQ ID NO: 769)

[1034] 1 0 TGTGCGAAAGAAGGAGGCAGTGGCTGGAC CAKEGGSGWTYYFDSW IGHV4-34*00 IGHJ4*00

[1035] GTACTACTTTGACTCCTGG (SEQ ID NO: 772)

[1036] (SEQ ID NO: 771)

[1037] 1 0 TGTGCGAAACCGTCCTTCTCCGGTGACGCC CAKPSFSGDANFDYW IGHV3-3O*OO IGHJ4*00

[1038] AACTTTGACTACTGG (SEQ ID NO: 774)

[1039] (SEQ ID NO: 773)

[1040] 1 0 TGTGCGACCTCTGACTGGTTATTATATCCT CATSDWLLYPRPYYYYTEIW IGHV3-74*00 IGHJ6*00

[1041] CGCCCATACTACTACTACACAGAAATCTGG (SEQ ID NO: 776)

[1042] (SEQ ID NO:775)

[1043] 1 0 TGTGCGAGAGATCGCGCTCTGGGGGAAGA CARDRALGEDNWFDPW IGHV1-18*OO IGHJ5*00

[1044] CAACTGGTTCGACCCCTGG (SEQ ID NO: 778)

[1045] (SEQ ID NO: 777)

[1046] 1 0 TGTGCGGGTTTCACCCCCACATATAGCAGT CAGFTPTYSSGWYYFDYW IGHV3-66*00 IGHJ4*00

[1047] GGCTGGTACTACTTTGACTACTGG (SEQ ID NO: 780)

[1048] (SEQ ID NO: 779)

[1049] 1 0 TGTGCGAAAGATATTGTTGTAGTACCGGCT CAKDIVVVPAAGNWFDSW IGHV3-23*00 IGHJ5*00

[1050] GCGGGGAACTGGTTCGATTCCTGG (SEQ ID NO: 782)

[1051] (SEQ ID NO: 781)

[1052] 1 0 TGTGCGAGCAGAAGGTTCGGTGACAAGAC CASRRFGDKTKW IGHV3-53*OO IGHJ4*00

[1053]

[1054] AAAATGG (SEQ ID NO: 784)

[1055]

[1056] Attorney Docket No. 44807-0503WO1 / C18573

[1057] (SEQ ID NO:783)

[1058] 1 0 TGTGCGAGATGTGTCTCCCCCGGCTCCTAC CARCVSPGSYYSFYYFDHW IGHV1-18*OO IGHJ4*00

[1059] TACAGTTTTTACTACTTTGACCACTGG (SEQ ID NO: 786)

[1060] (SEQ ID NO:785)

[1061] 1 0 TGTGTGAAAGGCGGGCAGTGGCTGACGAC CVKGGQWLTTDW IGHV3-3O*OO IGHJ4*00

[1062] AGACTGG (SEQ ID NO:788)

[1063] (SEQ ID NO:787)

[1064] 1 0 TGTGCGAGAGCAAATCCAACAGTGGCTAC CARANPTVATTLLVFDIW IGHV1-18*OO IGHJ3*00

[1065] TACCCTGCTGGTTTTTGATATCTGG (SEQ ID NO: 790)

[1066] (SEQ ID NO:789)

[1067] 1 0 TGTGCGAGACAAACGTATTACTATGATAGT CARQTYYYDSGWFDPW IGHV4-39*00 IGHJ5*00

[1068] GGCTGGTTCGACCCCTGG (SEQ ID NO: 792)

[1069] (SEQ ID NO: 791)

[1070] 1 0 TGTGCCAGAGGTGGCTACGAAACCCCGGG CARGGYETPGYYYYYGMDVW IGHV4-30- IGHJ6*00

[1071] TTACTACTACTACTACGGTATGGACGTCTG (SEQ ID NO: 794) 2*00 G

[1072] (SEQ ID NO: 793)

[1073] 1 0 TGTGCGAGAGACTCAGGTCGCGGTTGTACT CARDSGRGCTDTNYYGLDVW IGHV3-66*00 IGHJ6*00

[1074] GATACCAACTACTACGGTCTGGACGTCTGG (SEQ ID NO: 796)

[1075] (SEQ ID NO:795)

[1076] 1 0 TGTGCGAGAGGTGCCCCGGATAGTAGTGG CARGAPDSSGKEDWYIDLW IGHV3-33*OO IGHJ2*00

[1077] C AAGGAAGACTGGTAC ATCGATCTCTGG (SEQ ID NO: 798)

[1078] (SEQ ID NO: 797)

[1079] 1 0 TGTGTGAGAGATTATCATACGTGGATACA CVRDYHTW1QPYYYYYMDVW IGHV3-ll*00 IGHJ6*00

[1080] GCCATATTACTACTACTACATGGACGTCTG (SEQ ID NO: 800)

[1081] G

[1082]

[1083] (SEQ ID NO: 799)

[1084]

[1085] Attorney Docket No. 44807-0503WO1 / C18573

[1086] 0.214286 0 TGTGCGAGGTCGGACGGGCAGTTCGTCTA CARSDGQFVYGMDVW IGHV4-34*00 IGHJ6*00

[1087] CGGTATGGACGTCTGG (SEQ ID NO: 802)

[1088] (SEQ ID NO:801)

[1089] 1 0 TGTGTGAAAGGGGGCAGTATGGACGTCTG CVKGGSMDVW IGHV3-48*00 IGHJ6*00

[1090] G (SEQ ID NO:452)

[1091] (SEQ ID NO:451)

[1092] 0.333333 0 TGTGCCAGAGAGGGGACTTTTTACTCTGAG CAREGTFYSEGSGFDAFDIW IGHV4-30- IGHJ3*00

[1093] GGTAGTGGATTTGATGCTTTTGATATCTGG (SEQ ID NO: 804) 2*00 (SEQ ID NO: 803)

[1094] 0.5 0 TGTGTGAGAAAGGCCCGCGGGTGGTTAGA CVRKARGWLEFDCW IGHV4-31*00 IGHJl*00

[1095] ATTTGACTGTTGG (SEQ ID NO:458)

[1096] (SEQ ID NO:457)

[1097] 1 0 TGTACGAAACAGAGTCGCCTTAAGTCGTA CTKQSRLKSYYVLEAW IGHV4-34*00 IGHJ6*00

[1098] CTATGTATTAGAGGCCTGG (SEQ ID NO: 806)

[1099] (SEQ ID NO: 805)

[1100] 1 0 TGTGCGAAAGAAGCCGATATTTCGAGTGG CAKEADISSGYFRDW IGHV3-3O*OO IGHJ4*00

[1101] TTACTTTAGGGACTGG (SEQ ID NO: 808)

[1102]

[1103] (SEQ ID NO: 807)

[1104]

[1105] Attorney Docket No. 44807-0503WO1 / C18573

[1106] The “A” component of CSF-BAM queries aneuploidy of throughout the genome. SaferSeqS libraries were converted to a form suitable for whole genome sequencing (WGS) by the addition of primers whose sequences match those of the Illumina NovaSeq flow cells (Fig. 2C and Table IB). The WGS data were mapped to the human genome through standard methods. A modified version of the ichorCNA algorithm was used to assess gains or losses on 34 chromosome arms. Sex chromosomes, acrocentric chromosomes, and arms with high background are excluded in the analysis (see Methods for details). Though Watson and Crick strands can be identified from the sequencing data, there is no need to couple the reads to ensure single bp accuracy to assess copy number alterations of entire arms.

[1107] This WGS assay was then applied to SaferSeqS libraries from CSF of 31 individuals without cancer (training cohort — Table 4). Two metrics of aneuploidy were used to derive a threshold for scoring samples as aneuploid. First, the number of arms altered was evaluated. In the training cohort, two of the 31 samples had exactly one arm altered while the remaining 29 samples had no arms altered. As a result, any sample with more than one arm altered would be scored as positive for aneuploidy. Next, the estimated tumor fraction as predicted by ichorCNA was evaluated. In the training cohort of individuals without cancer, the largest predicted tumor fraction was 1.4%. Any sample predicted to have a tumor fraction >1.5 would be scored as positive.

[1108] Table 4: Summary of Aneuploidy Analysis in Control Samples

[1109] Sample Name counts Tumor Fraction

[1110] GLIA 763 CSF 1A 1 0.008956

[1111] GLIA 443 CSF 1A 1 0.009581

[1112] GLIB 1012 N 0 0

[1113] GLIB 1018 N 0 0

[1114] GLIB 1017 N 0 0

[1115] GLIB 1016 N 0 0

[1116] GLIB 1014 N 0 0.01189

[1117] GLIB 1015 N 0 0.01811

[1118] GLIB 1011 N 0 0

[1119]

[1120] GLIA 517 CSF 1A 0 0.01042 Attorney Docket No. 44807-0503WO1 / C18573

[1121] GLIA 505 CSF 1A 0 0

[1122] GLIA 858 CSF 1 0 0

[1123] GLIA 857 CSF 1 0 0.0119

[1124] GLIA 805 CSF 1 0 0.01105

[1125] GLIA 817 CSF 1 0 0

[1126] GLIA 813 CSF 1 0 0

[1127] GLIA 718 CSF 1A 0 0

[1128] GLIA 719 CSF 1A 0 0.01399

[1129] GLIA 723 CSF 1A 0 0.008088

[1130] GLIA 714 CSF 1A 0 0

[1131] GLIA 717 CSF 1A 0 0

[1132] GLIA 567 CSF 1A 0 0

[1133] GLIA 510 CSF 1A 0 0

[1134] GLIA 445 CSF 1A 0 0

[1135] GLIA 442 CSF 1A 0 0

[1136] GLIA 503 CSF 1A 0 0

[1137] GLIA 505 CSF 1A 0 0

[1138] GLIA 439 CSF 1A 0 0

[1139] GLIA 440 CSF 1A 0 0

[1140] GLIA 438 CSF 1A 0 0

[1141]

[1142] GLIA 446 CSF 1A 0 0

[1143] When the same assay was applied to WGS data derived from SaferSeqS libraries of CSF from a different cohort (validation set) of individuals without known cancers, it was found that 0 of 33 scored positively (100% specificity, credible interval 89% to 100%) (Table 5). 121 of 206 CSF samples from patients with CNS cancers of various types had detectable aneuploidy. Of these, 55 of 81 samples from patients with high-grade gliomas were positive (67%, credible interval 56% to 76%). Patients with medulloblastomas or cancers outside the CNS that had metastasized to the brain also were often positive for aneuploidy using this measure (81%, credible interval 60% to 92% and 85%, credible interval 66% to 94%, respectively, Table 5).

[1144] Table 5: Summary of CSF-BAM Aneuploidy Analysis in CSF Samples

[1145]

[1146] Sample counts Tumor Attorney Docket No. 44807-0503WO1 / C18573

[1147] Fraction

[1148] Medulloblastoma 12 0.9574

[1149] Medulloblastoma 11 0.826

[1150] Medulloblastoma 11 0.6987

[1151] Metastasis 10 0.6421

[1152] High grade glioma 34 0.6137

[1153] High grade glioma 18 0.554

[1154] Metastasis 34 0.4999

[1155] Metastasis 31 0.4982

[1156] Medulloblastoma 34 0.479

[1157] Diffuse Midline Glioma 28 0.4783

[1158] Medulloblastoma 15 0.4761

[1159] High grade glioma 34 0.4751

[1160] Metastasis 16 0.4687

[1161] High grade glioma 13 0.4448

[1162] Glioma, low grade 24 0.4155

[1163] Metastasis 31 0.4067

[1164] Ependymoma 34 0.4057

[1165] Metastasis 31 0.3975

[1166] Metastasis 34 0.3899

[1167] High grade glioma 9 0.3825

[1168] High grade glioma 14 0.3719

[1169] High grade glioma 17 0.3696

[1170] High grade glioma 33 0.3672

[1171] Metastasis 26 0.3509

[1172] High grade glioma 27 0.3472

[1173] Ependymoma 34 0.3437

[1174] High grade glioma 10 0.3254

[1175] Metastasis 32 0.3196

[1176] Metastasis 19 0.3122

[1177] Medulloblastoma 2 0.2896

[1178] Diffuse Midline Glioma 10 0.2685

[1179] High grade glioma 29 0.253

[1180] Diffuse Midline Glioma 15 0.2524

[1181] Metastasis 13 0.2484

[1182] Medulloblastoma 23 0.2457

[1183] High grade glioma 33 0.2188

[1184] High grade glioma 7 0.2083

[1185]

[1186] Other CNS tumor type 28 0.1946 Attorney Docket No. 44807-0503WO1 / C18573

[1187] High grade glioma 29 0.1928

[1188] High grade glioma 19 0.1887

[1189] Medulloblastoma 18 0.1791

[1190] High grade glioma 18 0.1746

[1191] High grade glioma 10 0.1738

[1192] Metastasis 19 0.1628

[1193] High grade glioma 15 0.1597

[1194] High grade glioma 27 0.1528

[1195] Medulloblastoma 18 0.1503

[1196] Metastasis 24 0.1402

[1197] High grade glioma 8 0.1351

[1198] Metastasis 30 0.1285

[1199] High grade glioma 11 0.1135

[1200] High grade glioma 21 0.1082

[1201] Metastasis 25 0.1061

[1202] High grade glioma 21 0.1058

[1203] Medulloblastoma 1 0.1035

[1204] Medulloblastoma 15 0.09641

[1205] High grade glioma 5 0.0962

[1206] Ependymoma 18 0.09104

[1207] High grade glioma 25 0.08748

[1208] High grade glioma 9 0.08527

[1209] High grade glioma 14 0.08186

[1210] Medulloblastoma 17 0.08078

[1211] Medulloblastoma 6 0.07767

[1212] Metastasis 16 0.0725

[1213] High grade glioma 16 0.07066

[1214] Lymphoma 9 0.07052

[1215] Lymphoma 28 0.0698

[1216] High grade glioma 2 0.06864

[1217] High grade glioma 4 0.06385

[1218] High grade glioma 6 0.05963

[1219] Pilocytic astrocytoma 2 0.05838

[1220] High grade glioma 6 0.05671

[1221] Metastasis 24 0.05656

[1222] Metastasis 21 0.05601

[1223] High grade glioma 17 0.05207

[1224] Pilocytic astrocytoma 17 0.04195

[1225]

[1226] Medulloblastoma 8 0.04121 Attorney Docket No. 44807-0503WO1 / C18573

[1227] High grade glioma 6 0.04048

[1228] High grade glioma 5 0.0401

[1229] High grade glioma 6 0.03763

[1230] High grade glioma 7 0.03758

[1231] High grade glioma 13 0.03698

[1232] Lymphoma 15 0.03403

[1233] Metastasis 5 0.03193

[1234] High grade glioma 11 0.03049

[1235] Pilocytic astrocytoma 1 0.02707

[1236] High grade glioma 1 0.02668

[1237] High grade glioma 3 0.0266

[1238] Lymphoma 3 0.02635

[1239] Diffuse Midline Glioma 1 0.02606

[1240] Diffuse Midline Glioma 1 0.02543

[1241] High grade glioma 0 0.02405

[1242] High grade glioma 5 0.02393

[1243] High grade glioma 3 0.023

[1244] High grade glioma 0 0.02203

[1245] Ependymoma 4 0.02175

[1246] High grade glioma 1 0.02154

[1247] High grade glioma 0 0.02088

[1248] High grade glioma 0 0.02063

[1249] Diffuse Midline Glioma 0 0.02052

[1250] Metastasis 0 0.01986

[1251] High grade glioma 2 0.01927

[1252] Pilocytic astrocytoma 4 0.01919

[1253] Ependymoma 3 0.01911

[1254] High grade glioma 0 0.01902

[1255] Glioma, low grade 0 0.01841

[1256] Pilocytic astrocytoma 2 0.01835

[1257] Ependymoma 0 0.01797

[1258] High grade glioma 2 0.01776

[1259] High grade glioma 1 0.0177

[1260] Glioma, low grade 0 0.01756

[1261] Ependymoma 6 0.01699

[1262] Medulloblastoma 3 0.01696

[1263] Medulloblastoma 0 0.01632

[1264] High grade glioma 6 0.01628

[1265]

[1266] High grade glioma 0 0.0158 Attorney Docket No. 44807-0503WO1 / C18573

[1267] Metastasis 0 0.01552

[1268] Other CNS tumor type 0 0.01552

[1269] Metastasis 0 0.01544

[1270] Medulloblastoma 0 0.01529

[1271] Glioma, low grade 0 0.01444

[1272] Pilocytic astrocytoma 0 0.0143

[1273] High grade glioma 0 0.01415

[1274] Not a cancer 1 0.01395

[1275] Other CNS tumor type 0 0.01376

[1276] Not a cancer 0 0.0135

[1277] High grade glioma 1 0.01332

[1278] Metastasis 1 0.01329

[1279] Pilocytic astrocytoma 0 0.01327

[1280] Not a cancer 0 0.01324

[1281] Not a cancer 0 0.01279

[1282] High grade glioma 0 0.01266

[1283] Diffuse Midline Glioma 0 0.01251

[1284] Not a cancer 0 0.01249

[1285] High grade glioma 5 0.01245

[1286] Glioma, low grade 0 0.01236

[1287] Pilocytic astrocytoma 0 0.01227

[1288] High grade glioma 0 0.01217

[1289] Ependymoma 0 0.01211

[1290] Ependymoma 0 0.0121

[1291] Medulloblastoma 0 0.01193

[1292] High grade glioma 0 0.01192

[1293] Glioma, Low grade 0 0.01186

[1294] High grade glioma 0 0.01175

[1295] High grade glioma 0 0.01173

[1296] Ependymoma 0 0.01172

[1297] High grade glioma 0 0.01171

[1298] Ependymoma 0 0.01141

[1299] High grade glioma 0 0.01109

[1300] High grade glioma 0 0.0106

[1301] Metastasis 0 0.01055

[1302] Diffuse Midline Glioma 0 0.01049

[1303] Not a cancer 0 0.01028

[1304] High grade glioma 0 0.009919

[1305]

[1306] Not a cancer 0 0.009917 Attorney Docket No. 44807-0503WO1 / C18573

[1307] Ependymoma 0 0.009722

[1308] Ependymoma 0 0.009256

[1309] Not a cancer 0 0.009085

[1310] Not a cancer 0 0.008981

[1311] Not a cancer 0 0.00883

[1312] High grade glioma 0 0.008511

[1313] Not a cancer 0 0.008389

[1314] Not a cancer 0 0.008106

[1315] Diffuse Midline Glioma 0 0.007882

[1316] Diffuse Midline Glioma 0 0.007164

[1317] Diffuse Midline Glioma 0 0

[1318] Diffuse Midline Glioma 1 0

[1319] Ependymoma 0 0

[1320] Ependymoma 0 0

[1321] Ependymoma 0 0

[1322] Ependymoma 1 0

[1323] Ependymoma 0 0

[1324] Ependymoma 0 0

[1325] Ependymoma 0 0

[1326] Ependymoma 0 0

[1327] Ependymoma 0 0

[1328] Ependymoma 0 0

[1329] Ependymoma 0 0

[1330] Ependymoma 0 0

[1331] Ependymoma 0 0

[1332] Ependymoma 0 0

[1333] Ependymoma 0 0

[1334] High grade glioma 0 0

[1335] High grade glioma 0 0

[1336] High grade glioma 0 0

[1337] High grade glioma 0 0

[1338] High grade glioma 0 0

[1339] High grade glioma 0 0

[1340] High grade glioma 0 0

[1341] High grade glioma 0 0

[1342] High grade glioma 0 0

[1343] High grade glioma 0 0

[1344] High grade glioma 0 0

[1345]

[1346] Glioma, low grade 0 0 Attorney Docket No. 44807-0503WO1 / C18573

[1347] Glioma, low grade 1 0

[1348] Glioma, low grade 0 0

[1349] Glioma, low grade 0 0

[1350] Glioma, low grade 0 0

[1351] Glioma, low grade 0 0

[1352] Glioma, low grade 0 0

[1353] Glioma, low grade 0 0

[1354] High grade glioma 0 0

[1355] High grade glioma 0 0

[1356] High grade glioma 0 0

[1357] High grade glioma 0 0

[1358] Medulloblastoma 0 0

[1359] Medulloblastoma 1 0

[1360] Medulloblastoma 1 0

[1361] Metastasis 0 0

[1362] Metastasis 0 0

[1363] Not a cancer 0 0

[1364] Not a cancer 0 0

[1365] Not a cancer 0 0

[1366] Not a cancer 0 0

[1367] Not a cancer 0 0

[1368] Not a cancer 0 0

[1369] Not a cancer 0 0

[1370] Not a cancer 0 0

[1371] Not a cancer 0 0

[1372] Not a cancer 0 0

[1373] Not a cancer 0 0

[1374] Not a cancer 0 0

[1375] Not a cancer 0 0

[1376] Not a cancer 0 0

[1377] Not a cancer 0 0

[1378] Not a cancer 0 0

[1379] Not a cancer 0 0

[1380] Not a cancer 0 0

[1381] Not a cancer 0 0

[1382] Not a cancer 0 0

[1383] Other CNS tumor type 0 0

[1384] Other CNS tumor type 0 0

[1385]

[1386] Other CNS tumor type 0 0 Attorney Docket No. 44807-0503WO1 / C18573

[1387] Pilocytic astrocytoma 0 0

[1388] Pilocytic astrocytoma 0 0

[1389] Pilocytic astrocytoma 0 0

[1390] Pilocytic astrocytoma 0 0

[1391] Pilocytic astrocytoma 0 0

[1392]

[1393] Pilocytic astrocytoma 0 0

[1394] The reproducibility of the approach was assessed through the evaluation of 104 pairs of technical replicates derived from independent aliquots of CSF DNA from the same patient. Each aliquot had an independent SaferSeqS library. For the non-cancer controls (n=25) with a technical replicate, all arms were concordant while 98.5% of the arms in the cancer technical replicates (n=79) were concordant (Fig. 6; Methods). A subset of samples (n=30) were as described and evaluated using the Repetitive Element Aneuploidy Sequencing System (RealSeqS; Douville et al., Cell Rep. Med. 4:101148 (2023)). RealSeqS uses a single PCR primer to concomitantly amplify -350,000 loci spread through genome in order to evaluate aneuploidy (Douville etal., Proc. Natl. Acad. Sci. 117:4858-4863 (2020)). The chromosome arm level calls between the two assays were compared and it was found that 94.1% of the arms were concordant (Methods). Of the discordant calls, a majority could be explained as falling just below the threshold for one of the two assays.

[1395] The “M” component of CSF-BAM identifies subtle somatic mutations such as single base substitutions (SBS) or small insertions or deletions (indels). For this component, extremely high specificity was required to minimize errors during the experimental or bioinformatic constituents of this assay. The workflow was identical in principle to that described above for SafeBSeqS (Fig. 2B and 2D); the difference was in the primers used. Instead of 4 primers for SafeBSeqS, 120 primers were used for mutation analysis, each amplifying a region of the genome that is commonly mutated in cancers such as cancers of the CNS or cancers that metastasize to the CSF (Fig. 2D). These primers (Table 1C) were chosen after extensive experiments to maximize the uniformity of representation of the amplicons as well as to minimize the number of off-target reads upon sequencing. The uniformity of amplification of the 120 amplicons queried by the 120-plex is shown in Fig. 7). Attorney Docket No. 44807-0503WO1 / C18573

[1396] Mutations in cell-free DNA (cfDNA) from peripheral blood largely arise from either tumors or CHIP (Clonal Hematopoiesis of Indeterminate Potential) (Bolton et al., J. Clin. Oncol. 37:7-11 (2019)). To help ensure mutations identified in the CSF were not a result of CHIP, matched WBC DNA in available cases were analyzed.

[1397] CSF from a different cohort of 300 individuals without cancer was also evaluated (Table 6). These samples did not have matched peripheral blood to eliminate CHIP mutations and were not used for the evaluation of performance metrics. This cohort was used to tune the somatic mutation calling approach and determine thresholds for positivity. 20 mutations in TP53, 3 in KRAS, 5 NRAS, 8 mutations in FBXW7 in codon 505, and 7 in other amplicons were identified. Given the abundance of non-canonical mutations in KRAS and NRAS in the non-cancers, mutation calls within these genes were restricted to only KRAS codon 12 and NRAS codon 61, which represent the most commonly mutated hotspots in cancer.

[1398] Table 6: CSF-BAM Mutation in Non-Cancer CSF Samples

[1399] Sample Name Mutation Mutation 1 (CSF) Mutation 2 (CSF) Mutation 3 (CSF) found

[1400] (SCM>1)

[1401] CGLI 107 CSF 0 NA NA NA

[1402] CGLI 137 CSF 0 NA NA NA

[1403] CGLI 149 CSF 0 NA NA NA

[1404] CGLI 77 CSF 1 TP53 p. V216M NA NA

[1405] CGLI 78 CSF 0 NA NA NA

[1406] CGLI 79 CSF 0 NA NA NA

[1407] GLIA 437 CSF 1 0 NA NA NA

[1408] GLIA 438 CSF 1A 0 NA NA NA

[1409] GLIA 439 CSF 1A 0 NA NA NA

[1410] GLIA 440 CSF 1A 0 NA NA NA

[1411] GLIA 441 CSF 1A 2 FBXW7 p. R505C PAX3 p. T424M NA

[1412] GLIA 442 CSF 1A 0 NA NA NA

[1413] GLIA 443 CSF 1A 0 NA NA NA

[1414] GLIA 444 CSF 1A 0 NA NA NA

[1415] GLIA 445 CSF 1A 1 FBXW7 p. R505C NA NA

[1416] GLIA 446 CSF 1A 1 KRAS p. Q22K NA NA

[1417] GLIA 447 CSF 1A 0 NA NA NA

[1418]

[1419] GLIA 448 CSF 1A 0 NA NA NA Attorney Docket No. 44807-0503WO1 / C18573

[1420] GLIA 449 CSF 1A 0 NA NA NA

[1421] GLIA 499 CSF 1 0 NA NA NA

[1422] GLIA 500 CSF 1 0 NA NA NA

[1423] GLIA 501 CSF 1 0 NA NA NA

[1424] GLIA 503 CSF 1A 2 APC p. P1373X GNAS p. R844H NA

[1425] GLIA 504 CSF 1 0 NA NA NA

[1426] GLIA 505 CSF 1A 0 NA NA NA

[1427] GLIA 506 CSF 1 1 NRAS p. Q61R NA NA

[1428] GLIA 507 CSF 1 0 NA NA NA

[1429] GLIA 508 CSF 1 0 NA NA NA

[1430] GLIA 509 CSF 1 0 NA NA NA

[1431] GLIA 510 CSF 1A 0 NA NA NA

[1432] GLIA 511 CSF 1 1 TP53 p. H193R NA NA

[1433] GLIA 512 CSF 1 0 NA NA NA

[1434] GLIA 517 CSF 1A 1 APC p. R1399H NA NA

[1435] GLIA 532 CSF 1 1 TP53 p. Y220H NA NA

[1436] GLIA 535 CSF 1 0 NA NA NA

[1437] GLIA 536 CSF 1 0 NA NA NA

[1438] GLIA 541 CSF 1 0 NA NA NA

[1439] GLIA 580 CSF 1 0 NA NA NA

[1440] GLIA 583 CSF 1 0 NA NA NA

[1441] GLIA 716 CSF 1 0 NA NA NA

[1442] GLIA 724 CSF 1 0 NA NA NA

[1443] GLIA 725 CSF 1 0 NA NA NA

[1444] GLIA 726 CSF 1 0 NA NA NA

[1445] GLIA 727 CSF 1 0 NA NA NA

[1446] GLIA 728 CSF 1 0 NA NA NA

[1447] GLIA 729 CSF 1 0 NA NA NA

[1448] GLIA 730 CSF 1 0 NA NA NA

[1449] GLIA 732 CSF 1 0 NA NA NA

[1450] GLIA 733 CSF 1 0 NA NA NA

[1451] GLIA 736 CSF 1 0 NA NA NA

[1452] GLIA 737 CSF 1 0 NA NA NA

[1453] GLIA 739 CSF 1 0 NA NA NA

[1454] GLIA 741 CSF 1 0 NA NA NA

[1455] GLIA 743 CSF 1 0 NA NA NA

[1456] GLIA 745 CSF 1 0 NA NA NA

[1457] GLIA 748 CSF 1 0 NA NA NA

[1458]

[1459] GLIA 749 CSF 1 0 NA NA NA Attorney Docket No. 44807-0503WO1 / C18573

[1460] GLIA 751 CSF 1 0 NA NA NA

[1461] GLIA 753 CSF 1 0 NA NA NA

[1462] GLIA 754 CSF 1 0 NA NA NA

[1463] GLIA 755 CSF 1 0 NA NA NA

[1464] GLIA 759 CSF 1 0 NA NA NA

[1465] GLIA 760 CSF 1 0 NA NA NA

[1466] GLIA 762 CSF 1 0 NA NA NA

[1467] GLIA 943 CSF 1 0 NA NA NA

[1468] GLIA 973 CSF 1 0 NA NA NA

[1469] GLIA 977 CSF 1 0 NA NA NA

[1470] GLIA 989 CSF 1 0 NA NA NA GLIAB 1018 CSF1 0 NA NA NA GLIAB 1040 CSF1 0 NA NA NA GLIAB 1041 CSF1 0 NA NA NA GLIAB 1050 CSF1 0 NA NA NA GLIAB 1063 CSF1 0 NA NA NA GLIAB 1065 CSF1 0 NA NA NA GLIAB 1071 CSF1 0 NA NA NA GLIAB 1075 CSF1 1 TERT g.1295228G> A NA NA (promoter)

[1471] GLIAB 1085 CSF1 0 NA NA NA GLIAB 1086 CSF1 0 NA NA NA GLIAB 1087 CSF 1 1 NRAS p. Q61R NA NA GLIAB 1088 CSF1 2 TP53 p. N239S TP53 p. R181H NA GLIAB 1089 CSF1 1 TP53 g.7579311C> T NA NA

[1472] (splice site)

[1473] GLIAB 1090 CSF1 0 NA NA NA GLIAB 1091 CSF1 0 NA NA NA GLIAB 1092 CSF1 0 NA NA NA GLIAB 1093 CSF1 1 TP53 p. R181H NA NA GLIAB 1094 CSF1 0 NA NA NA GLIAB 1095 CSF1 0 NA NA NA GLIAB 1097 CSF1 0 NA NA NA GLIAB 1099 CSF1 0 NA NA NA GLIAB 1100 CSF1 2 KRAS p. G12D TP53 p. R248W NA GLIAB 1101 CSF1 0 NA NA NA GLIAB 1102 CSF1 2 VHL p. N78S FBXW7 p. R5O5C NA GLIAB 1103 CSF1 0 NA NA NA

[1474]

[1475] GLIAB 1105 CSF1 1 NRAS p. Q61R NA NA Attorney Docket No. 44807-0503WO1 / C18573

[1476] GLIAB 1106 CSF1 0 NA NA NA GLIAB 1107 CSF 1 0 NA NA NA

[1477] GLIB 1046 CSF 1 1 TP53 p. V122X NA NA

[1478] GLIB 1047 CSF 1 0 NA NA NA

[1479] GLIB 1048 CSF 1 0 NA NA NA

[1480] GLIB 1049 CSF 1 1 FBXW7 p. R505C NA NA

[1481] GLIB 1050 CSF 1 1 TP53 p. Y220H NA NA

[1482] GLIB 1051 CSF 1 0 NA NA NA

[1483] GLIB 1052 CSF 1 0 NA NA NA

[1484] GLIB 1053 CSF 1 0 NA NA NA

[1485] GLIB 1054 CSF 1 0 NA NA NA

[1486] GLIB 1055 CSF 1 0 NA NA NA

[1487] GLIB 1056 CSF 1 0 NA NA NA

[1488] GLIB 1057 CSF 1 0 NA NA NA

[1489] GLIB 1058 CSF 1 0 NA NA NA

[1490] GLIB 1059 CSF 1 1 FBXW7 p. R505C NA NA

[1491] GLIB 1060 CSF 1 0 NA NA NA

[1492] GLIB 1061 CSF 1 0 NA NA NA

[1493] GLIB 1062 CSF 1 0 NA NA NA

[1494] GLIB 1063 CSF 1 0 NA NA NA

[1495] GLIB 1064 CSF 1 0 NA NA NA

[1496] GLIB 1065 CSF 1 0 NA NA NA

[1497] GLIB 1066 CSF 1 0 NA NA NA

[1498] GLIB 1067 CSF 1 1 TP53 p. H193R NA NA

[1499] GLIB 1068 CSF 1 0 NA NA NA

[1500] GLIB 1069 CSF 1 0 NA NA NA

[1501] GLIB 1070 CSF 1 0 NA NA NA

[1502] GLIB 1071 CSF 1 0 NA NA NA

[1503] GLIB 1072 CSF 1 0 NA NA NA

[1504] GLIB 1073 CSF 1 0 NA NA NA

[1505] GLIB 1074 CSF 1 0 NA NA NA

[1506] GLIB 1075 CSF 1 0 NA NA NA

[1507] GLIB 1076 CSF 1 0 NA NA NA

[1508] GLIB 1077 CSF 1 1 FBXW7 p. R505C NA NA

[1509] GLIB 1078 CSF 1 0 NA NA NA

[1510] GLIB 1079 CSF 1 0 NA NA NA

[1511] GLIB 1080 CSF 1 0 NA NA NA

[1512] GLIB 1081 CSF 1 0 NA NA NA

[1513]

[1514] GLIB 1082 CSF 1 0 NA NA NA Attorney Docket No. 44807-0503WO1 / C18573

[1515] GLIB 1083 CSF 1 0 NA NA NA

[1516] GLIB 1084 CSF 1 1 KRAS p. G12D NA NA

[1517] GLIB 1085 CSF 1 0 NA NA NA

[1518] GLIB 1086 CSF 1 1 FBXW7 p. R505C NA NA

[1519] GLIB 1087 CSF 1 0 NA NA NA

[1520] GLIB 1088 CSF 1 1 NRAS p. G12D NA NA

[1521] GLIB 1089 CSF 1 1 BRAF p. D594G NA NA

[1522] GLIB 1090 CSF 1 1 TP53 p. C176R NA NA

[1523] GLIB 1091 CSF 1 0 NA NA NA

[1524] GLIB 1092 CSF 1 0 NA NA NA

[1525] GLIB 1093 CSF 1 0 NA NA NA

[1526] GLIB 1094 CSF 1 0 NA NA NA

[1527] GLIB 1095 CSF 1 0 NA NA NA

[1528] GLIB 1096 CSF 1 0 NA NA NA

[1529] GLIB 1097 CSF 1 0 NA NA NA

[1530] GLIB 1098 CSF 1 0 NA NA NA

[1531] GLIB 1099 CSF 1 0 NA NA NA

[1532] GLIB 1100 CSF 1 0 NA NA NA

[1533] GLIB 1101 CSF 1 0 NA NA NA

[1534] GLIB 1102 CSF 1 0 NA NA NA

[1535] GLIB 1103 CSF 1 0 NA NA NA

[1536] GLIB 1104 CSF 1 0 NA NA NA

[1537] GLIB 1105 CSF 1 0 NA NA NA

[1538] GLIB 1106 CSF 1 0 NA NA NA

[1539] GLIB 1107 CSF 1 0 NA NA NA

[1540] GLIB 1108 CSF 1 0 NA NA NA

[1541] GLIB 1109 CSF 1 0 NA NA NA

[1542] GLIB 1110 CSF 1 3 TP53 p. RE335-336X TP53 p. L130V TP53 p. A138V GLIB 1111 CSF 1 1 TP53 p. Y236D NA NA

[1543] GLIB 1112 CSF 1 0 NA NA NA

[1544] GLIB 1113 CSF 1 0 NA NA NA

[1545] GLIB 1114 CSF 1 0 NA NA NA

[1546] GLIB 1115 CSF 1 0 NA NA NA

[1547] GLIB 1116 CSF 1 0 NA NA NA

[1548] GLIB 1117 CSF 1 0 NA NA NA

[1549] GLIB 1118 CSF 1 0 NA NA NA

[1550] GLIB 1119 CSF 1 0 NA NA NA

[1551] GLIB 1120 CSF 1 0 NA NA NA

[1552]

[1553] GLIB 1121 CSF 1 0 NA NA NA Attorney Docket No. 44807-0503WO1 / C18573

[1554] GLIB 1123 CSF 1 0 NA NA NA

[1555] GLIB 1124 CSF 1 1 FBXW7 p. R505C NA NA

[1556] GLIB 1125 CSF 1 0 NA NA NA

[1557] GLIB 1126 CSF 1 0 NA NA NA

[1558] GLIB 1127 CSF 1 0 NA NA NA

[1559] GLIB 1128 CSF 1 0 NA NA NA

[1560] GLIB 1129 CSF 1 0 NA NA NA

[1561] GLIB 1130 CSF 1 0 NA NA NA

[1562] GLIB 1131 CSF 1 0 NA NA NA

[1563] GLIB 1132 CSF 1 0 NA NA NA

[1564] GLIB 1133 CSF 1 0 NA NA NA

[1565] GLIB 1134 CSF 1 0 NA NA NA

[1566] GLIB 1135 CSF 1 0 NA NA NA

[1567] GLIB 1136 CSF 1 0 NA NA NA

[1568] GLIB 1137 CSF 1 0 NA NA NA

[1569] GLIB 1138 CSF 1 0 NA NA NA

[1570] GLIB 1139 CSF 1 0 NA NA NA

[1571] GLIB 1140 CSF 1 0 NA NA NA

[1572] GLIB 1141 CSF 1 0 NA NA NA

[1573] GLIB 1142 CSF 1 0 NA NA NA

[1574] GLIB 1143 CSF 1 0 NA NA NA

[1575] GLIB 1144 CSF 1 0 NA NA NA

[1576] GLIB 1145 CSF 1 0 NA NA NA

[1577] GLIB 1146 CSF 1 0 NA NA NA

[1578] GLIB 1147 CSF 1 0 NA NA NA

[1579] GLIB 1148 CSF 1 1 TP53 p. C238F NA NA

[1580] GLIB 1149 CSF 1 0 NA NA NA

[1581] GLIB 1150 CSF 1 0 NA NA NA

[1582] GLIB 1151 CSF 1 0 NA NA NA

[1583] GLIB 1152 CSF 1 0 NA NA NA

[1584] GLIB 1153 CSF 1 0 NA NA NA

[1585] GLIB 1154 CSF 1 0 NA NA NA

[1586] GLIB 1155 CSF 1 0 NA NA NA

[1587] GLIB 1156 CSF 1 0 NA NA NA

[1588] GLIB 1157 CSF 1 0 NA NA NA

[1589] GLIB 1158 CSF 1 0 NA NA NA

[1590] GLIB 1159 CSF 1 0 NA NA NA

[1591] GLIB 1160 CSF 1 0 NA NA NA

[1592]

[1593] GLIB 1161 CSF 1 0 NA NA NA Attorney Docket No. 44807-0503WO1 / C18573

[1594] GLIB 1162 CSF 1 0 NA NA NA

[1595] GLIB 1163 CSF 1 0 NA NA NA

[1596] GLIB 1164 CSF 1 0 NA NA NA

[1597] GLIB 1165 CSF 1 0 NA NA NA

[1598] GLIB 1166 CSF 1 0 NA NA NA

[1599] GLIB 1167 CSF 1 0 NA NA NA

[1600] GLIB 1168 CSF 1 0 NA NA NA

[1601] GLIB 1169 CSF 1 0 NA NA NA

[1602] GLIB 1170 CSF 1 0 NA NA NA

[1603] GLIB 1171 CSF 1 0 NA NA NA

[1604] GLIB 1172 CSF 1 0 NA NA NA

[1605] GLIB 1173 CSF 1 0 NA NA NA

[1606] GLIB 1174 CSF 1 0 NA NA NA

[1607] GLIB 1175 CSF 1 0 NA NA NA

[1608] GLIB 1176 CSF 1 0 NA NA NA

[1609] GLIB 1177 CSF 1 0 NA NA NA

[1610] GLIB 1178 CSF 1 0 NA NA NA

[1611] GLIB 1179 CSF 1 0 NA NA NA

[1612] GLIB 1180 CSF 1 0 NA NA NA

[1613] GLIB 1181 CSF 1 0 NA NA NA

[1614] GLIB 1182 CSF 1 0 NA NA NA

[1615] GLIB 1183 CSF 1 0 NA NA NA

[1616] GLIB 1184 CSF 1 0 NA NA NA

[1617] GLIB 1185 CSF 1 1 NRAS p. Q61K NA NA

[1618] GLIB 1186 CSF 1 0 NA NA NA

[1619] GLIB 1187 CSF 1 0 NA NA NA

[1620] GLIB 1188 CSF 1 0 NA NA NA

[1621] GLIB 1189 CSF 1 0 NA NA NA

[1622] GLIB 1190 CSF 1 0 NA NA NA

[1623] GLIB 1191 CSF 1 0 NA NA NA

[1624] GLIB 1192 CSF 1 0 NA NA NA

[1625] GLIB 1193 CSF 1 0 NA NA NA

[1626] GLIB 1194 CSF 1 0 NA NA NA

[1627] GLIB 1195 CSF 1 0 NA NA NA

[1628] GLIB 1196 CSF 1 0 NA NA NA

[1629] GLIB 1197 CSF 1 0 NA NA NA

[1630] GLIB 1198 CSF 1 0 NA NA NA

[1631] GLIB 1199 CSF 1 0 NA NA NA

[1632]

[1633] GLIB 1200 CSF 1 0 NA NA NA Attorney Docket No. 44807-0503WO1 / C18573

[1634] GLIB 1201 CSF 1 0 NA NA NA

[1635] GLIB 1202 CSF 1 0 NA NA NA

[1636] GLIB 1203 CSF 1 0 NA NA NA

[1637] GLIB 1204 CSF 1 0 NA NA NA

[1638] GLIB 1205 CSF 1 0 NA NA NA

[1639] GLIB 1206 CSF 1 0 NA NA NA

[1640] GLIB 1207 CSF 1 0 NA NA NA

[1641] GLIB 1208 CSF 1 0 NA NA NA

[1642] GLIB 1209 CSF 1 0 NA NA NA

[1643] GLIB 1210 CSF 1 0 NA NA NA

[1644] GLIB 1211 CSF 1 0 NA NA NA

[1645] GLIB 1212 CSF 1 0 NA NA NA

[1646] GLIB 1213 CSF 1 0 NA NA NA

[1647] GLIB 1214 CSF 1 0 NA NA NA

[1648] GLIB 1215 CSF 1 0 NA NA NA

[1649] GLIB 1216 CSF 1 0 NA NA NA

[1650] GLIB 1217 CSF 1 1 TP53 p. R273L NA NA

[1651] GLIB 1218 CSF 1 0 NA NA NA

[1652] GLIB 1219 CSF 1 0 NA NA NA

[1653] GLIB 1220 CSF 1 0 NA NA NA

[1654] GLIB 1221 CSF 1 0 NA NA NA

[1655] GLIB 1222 CSF 1 0 NA NA NA

[1656] GLIB 1223 CSF 1 0 NA NA NA

[1657] GLIB 1224 CSF 1 1 TP53 p. P151R NA NA

[1658] GLIB 1225 CSF 1 0 NA NA NA

[1659] GLIB 1226 CSF 1 0 NA NA NA

[1660] GLIB 1227 CSF 1 0 NA NA NA

[1661] GLIB 1228 CSF 1 0 NA NA NA

[1662] GLIB 1229 CSF 1 0 NA NA NA

[1663] GLIB 1230 CSF 1 0 NA NA NA

[1664] GLIB 1231 CSF 1 0 NA NA NA

[1665] GLIB 1232 CSF 1 0 NA NA NA

[1666] GLIB 1233 CSF 1 0 NA NA NA

[1667] GLIB 1234 CSF 1 0 NA NA NA

[1668] GLIB 1235 CSF 1 0 NA NA NA

[1669] GLIB 1236 CSF 1 0 NA NA NA

[1670] GLIB 1237 CSF 1 0 NA NA NA

[1671] GLIB 1238 CSF 1 0 NA NA NA

[1672]

[1673] GLIB 1239 CSF 1 0 NA NA NA Attorney Docket No. 44807-0503WO1 / C18573

[1674] GLIB 1240 CSF 1 0 NA NA NA

[1675] GLIB 1241 CSF 1 0 NA NA NA

[1676] GLIB 1242 CSF 1 0 NA NA NA

[1677] GLIB 1243 CSF 1 0 NA NA NA

[1678] GLIB 1244 CSF 1 0 NA NA NA

[1679] GLIB 1245 CSF 1 2 TP53 g.7578560G> T APC p. E1397* NA

[1680] (splice site)

[1681] GLIB 1246 CSF 1 0 NA NA NA

[1682] GLIB 1247 CSF 1 0 NA NA NA

[1683] GLIB 1248 N1 0 NA NA NA

[1684] GLIB 1249 N1 0 NA NA NA

[1685]

[1686] GLIB 1250 N1 0 NA NA NA

[1687] The FBXW7 codon 505 mutations in the non-cancer samples were completely unexpected. This specific codon is not typically mutated in CHIP and other codons throughout FBXW7 are typically mutated in cancer. Upon closer inspection, every molecule with a mutant FBXW7 codon 505 also had mutations at codons 289, 299, 300, and 314. Blat analysis was performed to all possible non-human genomes. The sequence from the observed mutated FBXW7 molecules perfectly matched the bovine genome. It was evaluated whether bovine-derived hemostatic agents frequently used in neurosurgery (when CSF was collected for the trigeminal neuralgia samples) may have contributed minor amounts of DNA that amplified and incorrectly aligned to FBXW7.

[1688] On the basis of these data, it was chosen to positively score mutations that were present in more than one original template molecule and found in cancer patients in the COSMIC database (see Methods). Given the importance of TERT promoter mutations in CNS cancers, this metric was relaxed to score samples with even one mutant template molecule as positive. Using these thresholds, somatic mutations were identified in 0 of 32 healthy individuals (credible interval 89% to 100%) (Table 7).

[1689] Table 7: CSF-BAM Mutation Analysis in CSF Samples

[1690] Sample Name Mutation 1 Mutation 2 Mutation 3

[1691] GLIA 856 CSF 1 APC p. GA1357-1358X TP53 p. V216M KRAS p. G12A

[1692]

[1693] GLIA 886 CSF 1 BRAF p. V600E NA NA Attorney Docket No. 44807-0503WO1 / C18573

[1694] GLIA 913 CSF 1 CDKN2A p. P81L TP53 p. Q317* TP53 p. P278L GLIA 549 CSF 1A CTNNB1 p. D32A NA NA

[1695] GLIA 925 CSF 1 EGFRp. L858R TP53 p. R175H TERT g,1295250G> A (promoter)

[1696] CGLIA 301 CSF2A ERBB2 p. S310F TP53 p. G199V PIK3CA p. Ml 0431 GLIA 424 CSF 1A FBXW7 p. R465C NA NA

[1697] CGLI 184 CSF H3F3A p. K28M TP53 p. C275F NA

[1698] GLIA 935 CSF 1 NA NA NA

[1699] CGLI 163 CSF TERT g.1295250G> A NA NA

[1700] (promoter)

[1701] GLIA 522 CSF 1A H3F3A p. K28M TP53 p. C275F NA

[1702] GLIA 897 CSF 1 IDH1 p. R132C TERT NA

[1703] g.1295250G> A

[1704] (promoter)

[1705] CGLI 192 CSF IDH1 p. R132G TP53 p. R273C NA

[1706] GLIA 921 CSF 1 IDH1 p. R132H NA NA

[1707] GLIA 554 CSF 1A IDH1 p. R132H TERT TP53 p. R175H

[1708] g.1295228G> A

[1709] (promoter)

[1710] GLIA 797 CSF 1 IDH1 p. R132H TP53 p. R273C TP53 p. V274D GLIA 837 CSF 1 IDH2p. R172K TERT TERT g,1295250G> A g.1295228G> A (promoter) (promoter)

[1711] GLIA 537 CSF 3 KRAS p. G12C TP53 p. E287* NA

[1712] GLIA 934 CSF 1 KRAS p. G12V APC p. E1379* NA

[1713] GLIA 539 CSF 1A MYD88 p. L273P TP53 p. R209X NA

[1714] GLIA 928 CSF 1 NRAS p. Q61K TP53 p. T125R NA

[1715] CGLI 48 CSF TERT g.1295228G> A NA NA

[1716] (promoter)

[1717] CGLI 167 CSF PIK3CA p. F83S NA NA

[1718] CGLI 170 CSF PIK3CAp. R88Q TP53 p. G245S NA

[1719] CGLI 55 CSF PTEN p. R130* NA NA

[1720] CGLI 83 CSF TERT g.1295228G> A NA NA

[1721] (promoter)

[1722] CGLI 35 CSF TERT g,1295228G> A NA NA

[1723] (promoter)

[1724] CGLI 47 CSF TERT g.1295228G> A NA NA

[1725] (promoter)

[1726]

[1727] CGLI 50 CSF2 TERT g.1295228G> A NA NA Attorney Docket No. 44807-0503WO1 / C18573

[1728] (promoter)

[1729] GLIA 563 CSF 1A TERT g.1295228G> A NA NA

[1730] (promoter)

[1731] GLIA 569 CSF 1A TERT g.1295228G> A NA NA

[1732] (promoter)

[1733] GLIA 571 CSF 1A TERT g.1295228G> A NA NA

[1734] (promoter)

[1735] GLIA 893 CSF 1 TERT g.1295228G> A PTENp. K6X NA

[1736] (promoter)

[1737] CGLI 31 CSF2 TERT g.1295228G> A TP53 p. R248Q NA

[1738] (promoter)

[1739] GLIA 431 CSF 1A TERT g,1295228G> A TP53 p. R248Q NA

[1740] (promoter)

[1741] GLIA 559 CSF 1 TERT g.1295250G> A NA NA

[1742] (promoter)

[1743] CGLI 165 CSF TERT g,1295228G> A NA NA

[1744] (promoter)

[1745] CGLI 174 CSF TERT g.1295250G> A NA NA

[1746] (promoter)

[1747] GLIA 833 CSF 1 TERT g.1295250G> A NA NA

[1748] (promoter)

[1749] GLIA 922 CSF 1 TERT g.1295250G> A NA NA

[1750] (promoter)

[1751] GLIA 819 CSF 1 TERT g.1295228G> A NA NA

[1752] (promoter)

[1753] GLIA 550 CSF 1A TERT g.1295250G> A PTENp. R130G NA

[1754] (promoter)

[1755] GLIA 919 CSF 1 TERT g.1295250G> A TP53 p. R158X NA

[1756] (promoter)

[1757] GLIA 848 CSF 1 TERT g.1295250G> A TP53 p. R248W NA

[1758] (promoter)

[1759] CGLI 168 CSF TERT g.1295250G> A TP53 p. Y163C TP53 p. VRA157-159A (promoter)

[1760] GLIA 918 CSF 1 TP53 g.7577018C> T NA NA

[1761] (splice site)

[1762] GLIA 830 CSF 1 TP53 g.7579599G> T NA NA

[1763] (splice site)

[1764] GLIA 855 CSF 1 TP53 g.7579599G> T TP53 p. G245C TP53 p. S33S (splice (splice site) region variant)

[1765]

[1766] GLIA 794 CSF 1 TP53 g.7579599G> T VHL p. S183* TP53 p. T125T (splice Attorney Docket No. 44807-0503WO1 / C18573

[1767] (splice site) region variant) GLIA 590 CSF 1A TP53 p. C135Y NA NA

[1768] GLIA 820 CSF 1 TP53 p. C238F KRAS p. G12C NA

[1769] GLIA 914 CSF 1 TP53 p. E271* NA NA

[1770] GLIA 883 CSF 1 TP53 p. F134L IDH1 p. R132H PIK3CA p. E81K GLIA 823 CSF 1 TP53 p. G154V PIK3CA p. Q546P NA

[1771] GLIA 419 CSF 1A TP53 p. G245R NA NA

[1772] CGLI 97 CSF TP53 p. H179N H3F3A p. K28M NA

[1773] CGLI 28 C TP53 p. H214R TP53 p. R273C IDH1 p. R132H CGLI 41 CSF TP53 p. K132N NA NA

[1774] GLIA 859 CSF 1 TP53 p. L130R NA NA

[1775] GLIA 565 CSF 1 TP53 p. P190T NA NA

[1776] GLIA 906 CSF 1 TP53 p. R158G H3F3A p. K28M NA

[1777] GLIA 924 CSF 1 TERT g.1295250G> A NA NA

[1778] (promoter)

[1779] GLIA 582 CSF 2 TP53 p. R175H EGFRp. L858R PIK3CA p. E542K GLIA 552 CSF 1A TP53 p. R175H H3F3A p. K28M NA

[1780] GLIA 936 CSF 1 TP53 p. R175H H3F3A p. K28M PIK3CA p. R88Q GLIB 1005 CSF 1 TP53 p. R175H KRAS p. G12D TP53 p. V157L CGLI 164 CSF TP53 p. R175H TERT NA

[1781] g.1295228G> A

[1782] (promoter)

[1783] CGLI 14 CSF2 TP53 p. R213Q NA NA

[1784] GLIA 917 CSF 3 TERT g.1295250G> A NA NA

[1785] (promoter)

[1786] GLIA 804 CSF 1 TP53 p. R248L NA NA

[1787] GLIA 423 CSF 1A TP53 p. R248Q NA NA

[1788] GLIA 927 CSF 1 TP53 p. R248Q NA NA

[1789] GLIA 930 CSF 1 TP53 p. R248Q NA NA

[1790] CGLI 36 CSF TP53 p. R248W NA NA

[1791] CGLI 29 C TP53 p. R248W NA NA

[1792] GLIB 1004 CSF 1 TP53 p. R273C PIK3CA p. H1047R NA

[1793] CGLI 180 CSF TP53 p. R273C PIK3CA p. H1047R NA

[1794] GLIA 547 CSF 1A TP53 p. R273H NA NA

[1795] GLIB 1021 CSF 1 NA NA NA

[1796] GLIB 1023 CSF 1 NA NA NA

[1797] GLIB 1024 CSF 1 NA NA NA

[1798] GLIA 825 CSF 1 TP53 p. R337C NA NA

[1799]

[1800] GLIA 932 CSF 1 TP53 p. R337L NA NA Attorney Docket No. 44807-0503WO1 / C18573

[1801] 79 of 206 CSF samples from patients with CNS cancers of various types had detectable mutations (38%, credible interval 32% to 45%). Of these, 44 of 81 samples from patients with high-grade gliomas were positive (52%, credible interval 42% to 63%). Of the 5 high-grade gliomas that were scored positive based on the presence of only one mutant TERT molecule, all had the canonical gain on chr7 but 4 of 5 fell just below the threshold for aneuploid positivity. Given the heterogenous nature of medulloblastoma driver mutations, only 4 of 21 (19%, credible interval 8% to 40%) patients scored positive for mutations. 17 of 24 metastatic cancers (69%, credible interval 50% to 83%) had positive mutations.

[1802] Application of CSF-BAM to CSF samples

[1803] 239 CSF samples from 222 patients were evaluated; in each sample, peripheral blood WBC DNA was available to exclude any mutations due to CHIP. Clinical information including demographics are listed in Table 8. The amount of CSF available for these studies averaged 3.5 mL and ranged from 0.5 to 14 mL. The amount of DNA recovered from CSF averaged 25 ng (IQR 2.6 to 29.6 ng) (Table 8). Table 8 also includes summaries of the sequencing data obtained from all patients, and whether they scored positively in the B, A, or M components of CSF-BAM. If a patient scored positively in at least one of these assays using the pre-defined thresholds described above, the patient was considered positive for CSF-BAM. Attorney Docket No. 44807-0503W01 / Cl 8573

[1804] Table 8: CSF-BAM Summary Results

[1805] Diagnostic Volume Total CSF- SafeBSeqS clonality Aneuploidy Estimated Mutation Mutation MAF, Avg (Min-Max) Group (mL) DNA BAM positive (total UIDs>=20, Positive Tumor Positive found

[1806] (ng) clonality >=0.3)-- (l=Positive Fraction by (l=Positive

[1807] (l=Positive 0=Negative) 0=Negative) Aneuploidy 0=Negative)

[1808] Metastasis 3 14.5 1 0 1 5.7% 1 3 4.89% (0.35%-4.91%) Metastasis 3 3.1 1 0 1 14.0% 1 1 2.04% (2.04%-2.04%) Metastasis 3 1.9 1 0 1 3.2% 1 1 2.56% (2.56%-2.56%) High grade 3 20.7 1 0 0 0.0% 1 1 0.32% (0.32%-0.32%) glioma

[1809] High grade 5 25.8 1 0 1 1.2% 1 1 0.11% (0.11%-0.11%) glioma

[1810] High grade 3 50.3 1 0 0 0.0% 1 1 0.18% (0.18%-0.18%) glioma

[1811] High grade 3 9.8 1 0 0 1.2% 1 1 0.49% (0.49%-0.49%) glioma

[1812] High grade 3 89.9 1 0 0 0.9% 1 1 0.25% (0.25%-0.25%) glioma

[1813] Medulloblasto 2 30.0 1 0 1 47.9% 0 0 NA

[1814] ma

[1815] Pilocytic 1 0.3 1 0 1 4.2% 0 0 NA

[1816] astrocytoma

[1817] Low grade 4 71.0 0 0 0 0.0% 0 0 NA

[1818] glioma

[1819] Medulloblasto 6.5 29.6 1 0 1 1.6% 0 0 NA

[1820] ma

[1821]

[1822] Pilocytic 2 43.3 0 0 0 0.0% 0 0 NA Attorney Docket No. 44807-0503WO1 / C18573

[1823] astrocytoma

[1824] Low grade 9 10.2 1 0 1 1.8% 0 0 NA

[1825] glioma

[1826] Medulloblasto 8 21.1 1 0 1 47.6% 0 0 NA

[1827] ma

[1828] Low grade 9 6.5 0 0 0 1.2% 0 0 NA

[1829] glioma

[1830] Pilocytic 3 133.2 0 0 0 0.0% 0 0 NA astrocytoma

[1831] High grade 3 54.8 0 0 0 0.0% 0 0 NA

[1832] glioma

[1833] Pilocytic 1 2.9 1 0 1 1.8% 0 0 NA astrocytoma

[1834] Low grade 3 84.0 0 0 0 0.0% 0 0 NA

[1835] glioma

[1836] Pilocytic 0.5 11.0 0 0 0 0.0% 0 0 NA astrocytoma

[1837] Low grade 1 19.5 1 0 1 1.8% 0 0 NA

[1838] glioma

[1839] Ependymoma 4 2.0 1 0 1 1.7% 0 0 NA Ependymoma 4 2.0 0 0 0 0.0% 0 0 NA

[1840] Low grade 6.5 16.0 1 0 1 41.6% 1 1 22.10% (22.10%- glioma 22.10%) High grade 1 0.1 1 0 1 2.4% 0 0 NA

[1841] glioma

[1842] Ependymoma 5 12.2 0 0 0 1.1% 0 0 NA

[1843] 5 0.4 0 0 0 1.4% 0 0 NA

[1844]

[1845] Low grade

[1846]

[1847] Attorney Docket No. 44807-0503WO1 / C18573

[1848] glioma

[1849] High grade 1 24.3 1 0 1 7.1% 1 3 32.47% (31.98%- glioma 32.74%)

[1850] High grade 3.5 11.3 1 0 0 0.0% 1 1 0.17% (0.17%-0.17%) glioma

[1851] High grade 1 0.1 1 0 1 13.5% 0 0 NA

[1852] glioma

[1853] High grade 3.5 35.0 1 0 1 32.5% 1 2 9.95% (0.14%-19.77%) glioma

[1854] High grade 0.5 0.2 0 0 0 1.0% 0 0 NA

[1855] glioma

[1856] Medulloblasto 1.5 13.5 1 0 1 8.1% 0 0 NA

[1857] ma

[1858] High grade 1.5 0.2 1 0 1 1.6% 0 0 NA

[1859] glioma

[1860] High grade 2.5 58.8 1 0 1 9.6% 1 1 1.73% (1.73%-1.73%) glioma

[1861] High grade 5.5 0.7 1 0 1 3.8% 1 1 40.00% (40.00%- glioma 40.00%)

[1862] High grade 4.5 0.1 1 0 1 2.3% 0 0 NA

[1863] glioma

[1864] High grade 8 8.1 1 0 1 2.2% 0 0 NA

[1865] glioma

[1866] High grade 6.8 1.5 1 0 1 38.3% 1 1 15.71% (15.71%- glioma 15.71%)

[1867] Low grade 8.5 9.9 0 0 0 0.0% 0 0 NA

[1868]

[1869] glioma

[1870]

[1871] Attorney Docket No. 44807-0503WO1 / C18573

[1872] High grade 4.25 3.7 1 0 1 2.7% 1 1 1.02% (1.02%- 1.02%) glioma

[1873] Ependymoma 0.75 0.2 1 0 1 1.9% 0 0 NA

[1874] Low grade 4.5 3.4 0 0 0 0.0% 0 0 NA

[1875] glioma

[1876] Pilocytic 1.5 1.2 1 0 1 1.9% 0 0 NA

[1877] astrocytoma

[1878] Ependymoma 3 18.7 0 0 0 0.0% 0 0 NA Ependymoma 4 8.0 0 0 0 0.0% 0 0 NA

[1879] High grade 2 18.3 1 0 1 5.2% 1 2 2.67% (2.48%-2.87%) glioma

[1880] Medulloblasto 3 2.0 1 1 0 0.0% 1 1 0.08% (0.08%-0.08%) ma

[1881] Medulloblasto 3 30.0 1 0 1 29.0% 0 0 NA

[1882] ma

[1883] High grade 3 82.9 1 0 1 17.4% 1 2 2.08% (1.18%-2.98%) glioma

[1884] Medulloblasto 3 30.0 1 0 1 9.6% 1 1 36.99% (36.99%- ma 36.99%) Medulloblasto 5 26.6 1 0 0 0.0% 1 1 0.10% (0.10%-0.10%) ma

[1885] Other CNS 3 29.6 0 0 0 1.4% 0 0 NA

[1886] tumor type

[1887] Ependymoma 3 29.6 1 0 0 0.0% 1 1 0.13% (0.13%-0.13%) High grade 1 7.5 1 0 1 4.0% 1 4 0.61% (0.20%-1.27%) glioma

[1888] 5 72.2 0 0 0 0.0% 0 0 NA

[1889]

[1890] Metastasis

[1891]

[1892] Attorney Docket No. 44807-0503WO1 / C18573

[1893] Ependymoma 8 43.3 0 0 0 0.0% 0 0 NA Ependymoma 2.5 0.3 1 0 1 40.6% 0 0 NA Ependymoma 1 0.6 1 0 1 1.8% 0 0 NA

[1894] High grade 9 6.2 1 0 1 11.4% 1 1 55.26% (55.26%- glioma 55.26%) Metastasis 3 14.1 1 0 1 5.6% 1 3 3.67% (0.50%-6.51%) Not a cancer 3 15.9 0 0 0 0.0% 0 0 NA

[1895] High grade 3 25.8 1 0 1 8.2% 1 2 4.39% (2.34%-6.44%) glioma

[1896] Not a cancer 2 5.8 0 0 0 1.4% 0 0 NA

[1897] Not a cancer 2.5 2.6 0 0 0 0.0% 0 0 NA

[1898] Not a cancer 3.25 0.9 0 0 0 0.9% 0 0 NA

[1899] Diffuse 3 139.0 1 0 1 47.8% 1 2 29.86% (0.13%- Midline 59.58%) Glioma

[1900] Diffuse 3 39.0 1 0 0 0.7% 1 3 0.54% (0.41%-0.66%) Midline

[1901] Glioma

[1902] Not a cancer 3 1.9 0 0 0 0.0% 0 0 NA

[1903] Other CNS 3 30.0 1 0 1 19.5% 0 0 NA

[1904] tumor type

[1905] Not a cancer 3 2.9 0 0 0 0.0% 0 0 NA

[1906] Not a cancer 2.75 10.0 0 0 0 0.0% 0 0 NA

[1907] Not a cancer 3 2.2 0 0 0 0.0% 0 0 NA

[1908] Not a cancer 3 2.1 0 0 0 0.0% 0 0 NA

[1909] Not a cancer 3 40.7 0 0 0 0.0% 0 0 NA

[1910]

[1911] High grade 3 11.9 0 0 0 0.0% 0 0 NA Attorney Docket No. 44807-0503WO1 / C18573

[1912] glioma

[1913] Not a cancer 3 6.8 0 0 0 0.0% 0 0 NA

[1914] Not a cancer 3 3.3 0 0 0 0.0% 0 0 NA

[1915] Not a cancer 3 3.0 0 0 0 0.0% 0 0 NA

[1916] Not a cancer 3 3.6 0 0 0 0.0% 0 0 NA

[1917] Not a cancer 3 7.6 0 0 0 0.0% 0 0 NA

[1918] Not a cancer 1.60 14.3 0 0 0 1.3% 0 0 NA

[1919] Not a cancer 3 1.8 0 0 0 0.8% 0 0 NA

[1920] Not a cancer 3 0.1 0 0 0 0.0% 0 0 NA

[1921] Not a cancer 1.75 150.0 0 0 0 0.0% 0 0 NA

[1922] Not a cancer 3 39.3 0 0 0 0.0% 0 0 NA

[1923] Diffuse 3 9.6 0 0 0 1.3% 0 0 NA Midline

[1924] Glioma

[1925] High grade 3 2.4 1 0 1 1.9% 0 0 NA

[1926] glioma

[1927] Metastasis 14 44.4 1 0 1 32.0% 1 2 46.32% (41.17%- 51.47%) High grade 3 2.8 1 0 1 16.0% 0 0 NA

[1928] glioma

[1929] High grade 3 4.8 0 0 0 0.0% 0 0 NA

[1930] glioma

[1931] Pilocytic 3 26.0 0 0 0 1.3% 0 0 NA astrocytoma

[1932] Not a cancer 5.5 18.8 0 0 0 1.3% 0 0 NA

[1933] High grade 3 3.7 1 0 1 1.6% 0 0 NA

[1934]

[1935] glioma

[1936]

[1937] Attorney Docket No. 44807-0503WO1 / C18573

[1938] Other CNS 3 3.2 0 0 0 0.0% 0 0 NA

[1939] tumor type

[1940] Ependymoma 10 115.8 0 0 0 0.0% 0 0 NA

[1941] Pilocytic 10 25.0 0 0 0 0.0% 0 0 NA

[1942] astrocytoma

[1943] Not a cancer 2 9.6 0 0 0 0.0% 0 0 NA

[1944] Not a cancer 3 7.7 0 0 0 1.0% 0 0 NA Medulloblasto 1 0.4 1 0 1 95.7% 0 0 NA

[1945] ma

[1946] Medulloblasto 4 36.7 1 0 1 1.5% 0 0 NA

[1947] ma

[1948] High grade 4 5.6 1 0 1 21.9% 1 1 8.14% (8.14%-8.14%) glioma

[1949] High grade 3 3.1 0 0 0 0.0% 0 0 NA

[1950] glioma

[1951] High grade 3 0.2 1 0 1 44.5% 1 2 14.60% (14.49%- glioma 14.71%)

[1952] High grade 3 39.2 0 0 0 1.3% 0 0 NA

[1953] glioma

[1954] Metastasis 2.9 1.8 1 0 1 1.5% 0 0 NA

[1955] Not a cancer 1.5 0.4 0 0 0 1.4% 0 0 NA

[1956] Low grade 3 3.3 0 0 0 0.0% 0 0 NA

[1957] glioma

[1958] High grade 3 34.0 0 0 0 0.0% 0 0 NA

[1959] glioma

[1960] High grade 3 0.5 0 0 0 0.0% 0 0 NA

[1961]

[1962] glioma

[1963]

[1964] Attorney Docket No. 44807-0503WO1 / C18573

[1965] Metastasis 2.9 0.9 1 0 1 2.0% 0 0 NA

[1966] Other CNS 3 35.0 1 0 0 0.0% 1 4 0.29% (0.18%-0.50%) tumor type

[1967] High grade 3 0.4 1 0 1 3.8% 0 0 NA

[1968] glioma

[1969] High grade 3 0.8 0 0 0 1.3% 0 0 NA

[1970] glioma

[1971] High grade 3 2.7 1 0 0 1.1% 1 2 1.43% (0.58%-2.29%) glioma

[1972] High grade 3 0.4 0 0 0 1.4% 0 0 NA

[1973] glioma

[1974] Diffuse 3 29.3 1 0 1 26.9% 1 2 22.47% (0.23%- Midline 44.72%) Glioma

[1975] High grade 3 16.1 0 0 0 1.2% 0 0 NA

[1976] glioma

[1977] Pilocytic 3 1.1 0 0 0 1.2% 0 0 NA

[1978] astrocytoma

[1979] High grade 3 0.3 1 0 1 10.6% 1 2 11.17% (9.84%- glioma 12.50%)

[1980] High grade 3.5 1.4 1 0 1 2.2% 0 0 NA

[1981] glioma

[1982] Not a cancer 3 29.4 0 0 0 1.2% 0 0 NA Medulloblasto 3 224.0 1 0 1 1.7% 0 0 NA

[1983] ma

[1984] High grade 3 7.0 1 0 1 2.1% 0 0 NA

[1985]

[1986] glioma

[1987]

[1988] Attorney Docket No. 44807-0503WO1 / C18573

[1989] Low grade 3 1.6 0 0 0 0.0% 0 0 NA

[1990] glioma

[1991] Low grade 3 0.9 0 0 0 0.0% 0 0 NA

[1992] glioma

[1993] Low grade 3 1.0 0 0 0 1.2% 0 0 NA

[1994] glioma

[1995] Diffuse 3 0.6 0 0 0 0.0% 0 0 NA

[1996] Midline

[1997] Glioma

[1998] High grade 3 33.6 1 0 1 19.3% 1 2 16.12% (5.90%- glioma 26.35%) Diffuse 3 18.8 0 0 0 0.0% 0 0 NA

[1999] Midline

[2000] Glioma

[2001] Diffuse 3 97.3 1 0 1 2.5% 1 1 0.05% (0.05%-0.05%) Midline

[2002] Glioma

[2003] High grade 3 18.0 0 0 0 0.0% 0 0 NA

[2004] glioma

[2005] High grade 10.5 44.4 0 0 0 0.0% 0 0 NA

[2006] glioma

[2007] Metastasis 3 18.0 1 0 1 1.6% 0 0 NA

[2008] Pilocytic 2.8 0.2 1 0 1 2.7% 0 0 NA

[2009] astrocytoma

[2010] Metastasis 3 30.0 1 0 1 46.9% 1 4 21.26% (0.22%- 31.33%)

[2011]

[2012] Not a cancer 3 3.4 0 0 0 0.9% 0 0 NA

[2013]

[2014] Attorney Docket No. 44807-0503WO1 / C18573

[2015] Metastasis 3 29.6 1 0 0 1.3% 1 3 0.55% (0.17%-0.98%) Metastasis 3 5.3 1 0 1 40.7% 1 1 21.21% (21.21%- 21.21%) Metastasis 3 15.2 1 0 1 64.2% 0 0 NA

[2016] Metastasis 2 29.6 1 0 1 49.8% 1 1 59.62% (59.62%- 59.62%)

[2017] Not a cancer 3 2.7 0 0 0 0.8% 0 0 NA

[2018] Metastasis 2 17.0 1 0 1 24.8% 1 1 24.74% (24.74%- 24.74%)

[2019] High grade 3 41.4 1 0 1 55.4% 1 2 11.62% (5.05%- glioma 18.20%)

[2020] High grade 3 29.6 1 0 1 17.5% 1 1 56.87% (56.87%- glioma 56.87%)

[2021] High grade 3 29.6 1 0 1 61.4% 1 1 55.29% (55.29%- glioma 55.29%)

[2022] High grade 3 25.4 0 0 0 1.1% 0 0 NA

[2023] glioma

[2024] Metastasis 3 29.6 1 0 1 39.8% 1 1 49.33% (49.33%- 49.33%) Metastasis 3 1.6 0 0 0 0.0% 0 0 NA

[2025] Metastasis 3 29.6 1 0 1 35.1% 1 2 29.43% (28.32%- 30.55%) Metastasis 3 13.9 1 0 1 50.0% 0 0 NA

[2026] Metastasis 3 9.4 1 0 1 10.6% 1 1 3.73% (3.73%-3.73%) High grade 4.5 0.1 1 0 1 36.7% 1 3 37.72% (0.08%- glioma 68.50%)

[2027]

[2028] Medulloblasto 4 88.4 1 0 1 15.0% 0 0 NA

[2029]

[2030] Attorney Docket No. 44807-0503WO1 / C18573

[2031] ma

[2032] High grade 0.5 29.9 1 0 1 15.3% 1 2 21.79% (11.25%- glioma 32.33%) Medulloblasto 1 22.4 1 0 1 4.1% 0 0 NA

[2033] ma

[2034] Diffuse 1 0.4 1 0 1 2.1% 0 0 NA

[2035] Midline

[2036] Glioma

[2037] Medulloblasto 4 150.0 1 0 1 69.9% 0 0 NA

[2038] ma

[2039] CNS 3 3.3 1 0 1 7.1% 1 2 3.37% (2.68%-4.05%) lymphoma

[2040] Metastasis 3 29.6 1 0 1 12.9% 1 6 32.89% (0.14%- 77.10%)

[2041] High grade 3 4.5 0 0 0 1.2% 0 0 NA

[2042] glioma

[2043] CNS 1 16.6 1 1 1 3.4% 1 1 0.23% (0.23%-0.23%) lymphoma

[2044] High grade 10 0.0 1 0 1 6.9% 0 0 NA

[2045] glioma

[2046] High grade 3 42.6 1 0 1 37.0% 1 2 7.93% (0.01%-15.84%) glioma

[2047] Not a cancer 5 8.5 0 0 0 0.0% 0 0 NA

[2048] High grade 3 5.1 1 0 1 2.1% 1 1 0.70% (0.70%-0.70%) glioma

[2049] High grade 3 0.9 1 0 1 1.8% 0 0 NA

[2050]

[2051] glioma

[2052]

[2053] Attorney Docket No. 44807-0503WO1 / C18573

[2054] High grade 5 1.8 1 0 1 1.8% 0 0 NA

[2055] glioma

[2056] Metastasis 5 54.0 1 0 0 1.1% 1 1 0.07% (0.07%-0.07%) CNS 3 36.9 1 1 1 7.0% 0 0 NA

[2057] lymphoma

[2058] High grade 1 4.3 1 0 1 3.7% 1 1 0.75% (0.75%-0.75%) glioma

[2059] High grade 11 9.6 0 0 0 1.2% 0 0 NA

[2060] glioma

[2061] CNS 1 3.7 1 0 1 2.6% 0 0 NA

[2062] lymphoma

[2063] High grade 6 40.0 1 0 1 37.2% 1 4 7.43% (0.01%-17.58%) glioma

[2064] Not a cancer 3 30.0 0 0 0 0.0% 0 0 NA

[2065] Pilocytic 3 29.6 1 0 1 5.8% 1 2 3.57% (2.83%-4.31%) astrocytoma

[2066] Ependymoma 3 29.6 0 0 0 0.0% 0 0 NA

[2067] Not a cancer 3 32.5 0 0 0 0.9% 0 0 NA

[2068] Metastasis 2 8.3 1 0 1 7.3% 1 2 3.86% (1.62%-6.10%) High grade 4 23.5 1 0 1 3.0% 0 0 NA

[2069] glioma

[2070] Not a cancer 3 19.6 0 0 0 0.0% 0 0 NA

[2071] Metastasis 3 16.3 1 0 1 39.0% 1 2 42.29% (39.10%- 45.47%)

[2072] High grade 3 13.2 1 0 1 6.4% 1 1 3.59% (3.59%-3.59%) glioma

[2073]

[2074] High grade 3 29.6 1 0 1 8.5% 1 1 4.94% (4.94%-4.94%)

[2075]

[2076] Attorney Docket No. 44807-0503WO1 / C18573

[2077] glioma

[2078] High grade 3 0.4 1 0 1 6.0% 1 3 4.59% (4.05%-5.56%) glioma

[2079] High grade 3 4.0 0 0 0 0.0% 0 0 NA

[2080] glioma

[2081] Ependymoma 3 30.0 0 0 0 0.0% 0 0 NA

[2082] Diffuse 3 29.6 0 0 0 1.0% 0 0 NA

[2083] Midline

[2084] Glioma

[2085] Ependymoma 8 37.0 0 0 0 0.9% 0 0 NA Ependymoma 3 19.5 0 0 0 0.0% 0 0 NA

[2086] High grade 3 1.7 0 0 0 1.2% 0 0 NA

[2087] glioma

[2088] Ependymoma 4 56.6 0 0 0 0.0% 0 0 NA

[2089] Metastasis 3 37.7 1 0 1 16.3% 1 1 0.63% (0.63%-0.63%) High grade 3 29.6 1 0 1 2.7% 1 2 1.78% (1.62%- 1.93%) glioma

[2090] High grade 5 4.3 1 0 1 18.9% 1 1 6.26% (6.26%-6.26%) glioma

[2091] Diffuse 3 1.5 1 0 1 2.6% 0 0 NA

[2092] Midline

[2093] Glioma

[2094] High grade 5 53.6 1 0 1 2.4% 1 1 1.42% (1.42%- 1.42%) glioma

[2095] Ependymoma 3 150.0 0 0 0 0.0% 0 0 NA Ependymoma 3 29.6 0 0 0 0.0% 0 0 NA

[2096]

[2097] Not a cancer 3 23.1 0 0 0 0.0% 0 0 NA

[2098]

[2099] Attorney Docket No. 44807-0503WO1 / C18573

[2100] High grade 3 29.6 1 0 1 5.7% 0 0 NA

[2101] glioma

[2102] High grade 1 0.2 1 0 1 8.7% 0 0 NA

[2103] glioma

[2104] Ependymoma 3 6.4 1 0 1 2.2% 0 0 NA Ependymoma 3 29.5 0 0 0 0.0% 0 0 NA

[2105] Pilocytic 3 2.5 0 0 0 1.4% 0 0 NA

[2106] astrocytoma

[2107] High grade 8.5 35.2 1 0 1 34.7% 1 3 33.40% (0.35%- glioma 69.90%)

[2108] High grade 3 29.5 1 0 0 0.0% 1 3 0.26% (0.23%-0.29%) glioma

[2109] Pilocytic 3 11.3 0 0 0 1.2% 0 0 NA

[2110] astrocytoma

[2111] High grade 3 1.2 1 0 1 47.5% 1 2 22.08% (5.50%- glioma 38.66%)

[2112] High grade 5 0.1 1 0 1 1.9% 0 0 NA

[2113] glioma

[2114] Other CNS 3 366.0 1 0 1 1.6% 0 0 NA

[2115] tumor type

[2116] Ependymoma 3 1.5 0 0 0 1.2% 0 0 NA Ependymoma 3.5 44.4 1 0 1 9.1% 0 0 NA

[2117] Not a cancer 3 5.1 0 0 0 1.0% 0 0 NA Ependymoma 3 4.4 0 0 0 1.0% 0 0 NA

[2118] High grade 3 29.6 1 0 1 4.0% 1 3 1.45% (0.34%-2.44%) glioma

[2119]

[2120] Low grade 5 42.8 0 0 0 0.0% 0 0 NA

[2121]

[2122] Attorney Docket No. 44807-0503WO1 / C18573

[2123] glioma

[2124] Medulloblasto 3 1.6 0 0 0 1.2% 0 0 NA

[2125] ma

[2126] Ependymoma 3 29.6 1 0 1 31.2% 0 0 NA Ependymoma 3 24.7 0 0 0 0.0% 0 0 NA Medulloblasto 3 30.0 1 0 1 24.6% 0 0 NA

[2127] ma

[2128] Low grade 3 0.9 1 0 0 0.0% 1 1 0.25% (0.25%-0.25%) glioma

[2129] Medulloblasto 3 250.0 1 0 1 82.6% 0 0 NA

[2130] ma

[2131] Diffuse 5 44.4 1 0 1 25.2% 1 2 19.43% (0.15%- Midline 38.71%) Glioma

[2132] Ependymoma 3 25.6 0 0 0 1.2% 0 0 NA Medulloblasto 5 42.9 1 0 1 7.8% 0 0 NA

[2133] ma

[2134] Diffuse 3 30.6 1 0 0 0.8% 1 1 0.45% (0.45%-0.45%) Midline

[2135] Glioma

[2136] High grade 2 9.0 0 0 0 0.0% 0 0 NA

[2137] glioma

[2138] Medulloblasto 3 23.0 0 0 0 0.0% 0 0 NA

[2139] ma

[2140] Pilocytic 3 29.6 0 0 0 0.0% 0 0 NA

[2141] astrocytoma

[2142] 6 7.6 1 0 1 10.8% 1 2 3.57% (0.69%-6.46%)

[2143]

[2144] High grade

[2145]

[2146] Attorney Docket No. 44807-0503WO1 / C18573

[2147] glioma

[2148] Pilocytic 3 29.6 0 0 0 0.0% 0 0 NA

[2149] astrocytoma

[2150] Medulloblasto 6 5.4 1 0 1 10.4% 1 1 2.01% (2.01%-2.01%) ma

[2151] High grade 3 5.3 1 0 1 25.3% 1 1 44.08% (44.08%- glioma 44.08%) Medulloblasto 6 1.2 1 0 1 17.9% 0 0 NA

[2152] ma

[2153] Ependymoma 3 29.6 1 0 1 34.4% 0 0 NA

[2154] Low grade 3 1.8 0 0 0 0.0% 0 0 NA

[2155] glioma

[2156] High grade 5 56.6 1 0 1 20.8% 1 1 21.15% (21.15%-

[2157]

[2158] glioma 21.15%)

[2159]

[2160] Attorney Docket No. 44807-0503WO1 / C18573

[2161] 33 samples without cancer: None of the CSF samples from these patients scored positively in any of the three components, yielding 100% specificity (credible interval 89 to 100%) (Fig. 3 and 4 and Table 8).

[2162] 81 samples with high-grade glioma: 62 of these samples scored positively for at least one of the three components, yielding a sensitivity of 76% (credible interval 65% to 84%) (Fig. 3 and 4 and Table 8). No sample scored positively with the B component, as expected. The aneuploidy component was the predominant basis for sensitivity, with 67% positive (credible interval 56% to 76%). The mutation component scored positively in 44 patients, including 7 that were not scored positively by aneuploidy.

[2163] 21 samples with medulloblastomas: 19 of these samples scored positively for at least one of the three components, yielding a sensitivity of 90% (credible interval 71% to 97%) (Fig. 3 and 4 and Table 8). One scored positively with the B component. The aneuploidy component was the predominant basis for sensitivity, with 81% positive (credible interval 60% to 92%). The mutation component scored positively in 4 patients, including 2 that were not scored positively by aneuploidy.

[2164] 25 samples with metastatic lesions to the brain: 23 of these samples scored positively for at least one of the three components, yielding a sensitivity of 92% (credible interval 76% to 98%) (Fig. 3 and 4 and Table 8). No patient scored positively with the B component, as expected.

[2165] The aneuploidy component was the predominant basis for sensitivity, with 85% positive (credible interval 66% to 94%). The mutation component scored positively in 17 patients, including 1 that were not scored positively by aneuploidy.

[2166] 79 samples with other tumor types: These samples included those from patients with CNS lymphomas, gliomas other than high-grade, ependymomas, and various other primary brain tumor types (Table 8). 33 of these samples scored positively for at least one of the three components, yielding a sensitivity of 41% (credible interval 31% to 52%) (Fig. 3 and 4 and Table 8). As with high-grade gliomas, medulloblastomas, and metastatic cancers, the aneuploidy component was the predominant basis for sensitivity, with 35% positive Attorney Docket No. 44807-0503WO1 / C18573

[2167] (credible interval 25% to 35%). The mutation component scored positively in 13 patients, including 5 that were not scored positively by aneuploidy.

[2168] Other genetic observations of interest: When detectable by the A component, the median neoplastic DNA fraction based on the analysis of aneuploidy was 8.2% (IQR 2.5% to 26.9%). When detectable by the M component, the median mutant allele fraction based on the analysis of mutations was 3.9% (IQR 0.7% to 21.9%). The correlation between the two genetically altered fractions was high (R=0.55, P<5e-6; Fig. 8).

[2169] The nature of the genetic alterations provided some insight into the type of tumor present in the CNS. Mutations in IDH1 at codon 132 or 172 were observed in 8 patients, and all (100%) of these were patients with gliomas (2 with oligodendroglioma WHO Grade 3, 1 with astrocytoma WHO grade 3, and 5 with astrocytoma WHO grade 4). In all of these cases, standard sequencing of the resected gliomas, recorded in the patient's records, had revealed IDH mutations. In 3 additional samples from individuals with IDH mutant gliomas, no IDH mutations were observed in the CSF. Histone H3F3A mutations at codon 28 were detect in the CSF of 6 patients (3 with diffuse midline gliomas, 3 with GBM). Two of the six subjects had their tumor sequenced and in both cases the identical mutation was found in the matching CSF. However, no H3F3A mutations in the CSF were described in 5 other individuals who were diagnosed with H3.3 mutated gliomas. KRAS mutations at codon 12 were noted in 3 patients which harbored metastatic lesions from outside the CNS. Cytology was not routinely performed on all CSF given its poor performance but was available in 53 cases, 50 of which were from individuals with cancer (Table 8). The three cases without cancer were negative by cytology as well as by the CSF-BAM assay. Cytology was positive in nine (19%) of the cancer cases, and eight of (89%) were also scored as positive by the CSF-BAM assay. Of five cases recorded as “suspicious” on cytology, four (80%) were scored as positive by the CSF-BAM assay. Of 36 cases with cancers diagnosed as negative by cytology, 19 (53%) were scored as positive by CSF-BAM. Attorney Docket No. 44807-0503WO1 / C18573

[2170] Immune receptor repertoire profiling

[2171] A total of 264 CSF samples were evaluated using SafeBSeqS for the B component of CSF-BAM (Tables 3 and 10). A mean of 36 (range 0-1787) total UIDs representing original BCR DNA template molecules (Fig. 9). Because each unique UID can only be derived from a single cell in a SaferSeqS library, these data were used to determine the number of mature B cells present in the CSF when corrected for input amounts of CSF (see Methods).

[2172] B cell clonality varied substantially by cancer type (p<0.001 by Kruskal -Wallis test) (Fig. 10). Using clonality as a metric to classify CNS lymphomas against all other cancer types for evaluable samples produced a ROC curve with an AUC of 0.98 (Fig. 11 A). The proportion of samples for which the most frequently observed clone contained the IGHV4-34 gene segment was significantly enriched in CNS lymphomas compared to all other sample types (p=0.0029, Fisher's exact test) (Fig. 1 IB). The degree of IGHV4-34 gene segment representation was similar to that observed in prior studies of both peripheral and CNS lymphomas (Young et al., Proc. Natl. Acad. Sci. U. S. A. 112:13447-13454 (2015); and Montesinos-Rongen et al., J. Neuropathol. Exp. Neurol. 73: 1116-1125 (2014)).

[2173] Clinical Applicability of CSF-BAM

[2174] GLIA793 is a sample from a patient with grade 3 oligodendroglioma who underwent previous resection followed by adjuvant radiation and chemotherapy. CSF-BAM was negative on CSF obtained three weeks prior to repeat resection when there was a concern for tumor progression (Fig. 12A). Pathological examination of the surgical specimen revealed treatment effect without evidence of recurrent disease, a common finding in post-treatment gliomas. Another example is GLIA 914, an individual with metastatic breast cancer to the brain with suspicion of leptomeningeal disease. The patient had over five lumbar punctures for evaluation of LMD via cytology over a 12-month period and each was negative. The final CSF sample was tested via cytology and also CSF-BAM. The cytology was indeterminant with only rare, atypical cells. However, this sample was robustly positive via CSF-BAM with a tumor fraction estimated to be approximately 40% with both aneuploidy and mutations detected. A third case is GLIA 886, derived from an individual with a spinal cord ganglioglioma. The subject had a slowly recurrent spinal cord tumor which was initially Attorney Docket No. 44807-0503WO1 / C18573

[2175] resected many years ago prior to the advent of routine tumor sequencing and treated with carboplatin. The tumor was not responsive to chemotherapy and the individual underwent a repeat resection which demonstrated a BRAF mutant ganglioglioma (Fig. 12B). CSF-BAM identified the same disease defining canonical BRAF V600E mutation in CSF.

[2176] Methods

[2177] Sample processing and DNA purification

[2178] CSF samples were collected into standard CSF collection tubes. Blood samples were collected into Streck Cell-Free DNA BCT (#230469). DNA from CSF, plasma, or leukocytes was purified using the BioChain Cell-free DNA Extraction Kit (#K5011625). Control primary dermal fibroblasts were obtained from ATCC (#PCS-201-012) and DNA was purified using Qiagen QIAamp DNA Mini Kit (#51304).

[2179] Library construction

[2180] A library preparation workflow was developed that can efficiently recover input DNA and simultaneously incorporate double-stranded molecular barcodes. In brief, libraries were prepared using an Accel-NGS 2S DNA Library Kit (Swift Biosciences, 21024) with the following critical modifications: 1) DNA was pretreated with 3 U of USER enzyme (New England BioLabs, M5505L) for 15 minutes at 37 °C to excise uracil bases; 2) the SPRI bead / PEGNaCl ratios used after each reaction were 2.0x, 1.8x, 1.2x and 1.05x for end repair 1, end repair 2, ligation 1 and ligation 2, respectively; 3) a custom 50 pM 3' adapter was substituted for reagent Y2 and 4) a custom 42 pM 5' adapter was substituted for reagent B2. Libraries were subsequently PCR amplified in 50-pL reactions using primers targeting the ligated adapters. The following reaction conditions were used: 1× NEBNext Ultra II Q5 Master Mix (New England BioLabs, M0544L), 2 pM universal forward primer and 2 pM universal reverse primer. Libraries were amplified with 8 or 11 cycles of PCR, depending on how many experiments were planned, according to the following protocol: 98 °C for 30 s, cycles of 98 °C for 10 s, 65 °C for 75 s and hold at 4 °C. If eight cycles were used, the libraries were amplified in single 100-pL reactions. If 11 cycles were used, the libraries were divided into eight aliquots and amplified in eight 50-pL reactions, each supplemented with Attorney Docket No. 44807-0503WO1 / C18573

[2181] an additional 0.5 U of Q5 Hot Start High-Fidelity DNA Polymerase (New England BioLabs, M0493L), 1 pL of 10 mM dNTPs (New England BioLabs, N0447L) and 0.4 pL of 25 mM MgCh solution (New England BioLabs, B9021S). The products were purified with 1.8x SPRI beads (Beckman Coulter, B23317) and eluted in EB buffer (Qiagen).

[2182] The “B” component of CSF-BAM

[2183] BCR sequences were amplified from libraries using gene-specific primers for IGHJ segments as described for sequencing of somatic mutations (Cohen et al. Nat. Biotechnol. 39:1220-1227 (2021); and Wang etal., Proc. Natl. Acad. Set. U. S. A. 120:e2220704120 (2023)). 4 unique primers were used to cover all IGHJ gene segments in each amplification step (Table 1A). For clonotype analysis, demultiplexed reads were used to generate clonotype tables using the MiXCR 4.6.0 package with the function “analyze genericamplicon -with -umi” and specifications “—species hsa — dna —rigid-left-alignment-boundary --floating-right-alignment-boundary J.” The specification “-tag-pattern

[2184] A(R1:*)\A(UMI: N{ 14})(R2:*)” was used for Watson strands and the specification “—tagpattern "A(UMI: N{ 14})(R1:*)\A(R2:*)” was used for Crick strands. Clones with duplex support were assembled using the function “exportClonesOverlap” with the specifications criteria CDR3|NT|V|J” for each Watson and Crick pair. UIDs are reported as total UIDs for clonotypes with duplex support. Statistics were calculated using GraphPad Prism 10.

[2185] Statistical tests are specified in figure legends. To determine the relative yield for IGHJ gene segments, DNA from primary fibroblasts was evaluated. Non-rearranged IGHJ gene segments were analyzed using the computational pipeline as described from the “M” component below, with the modification of mapping to hg38. IGHJ1, IGHJ4, and IGHJ5 were computationally distinguished using primer sequences GAGGAGACGGTGACCAGGGTGCCCTGGCCCCAGTG (SEQ ID NO:809), GAGGAGACGGTGACCAGGGTTCCCTGGCCCCAGTA (SEQ ID NO: 810), and GAGGAGACGGTGACCAGGGTTCCCTGGCCCCAGGG (SEQ ID NO:811) respectively.

[2186] The “A” component of CSF-BAM (Aneuploidy)

[2187] Library DNA was amplified in 50 pL reactions in Ultra Q5 with primers at 2 pM for seven cycles with the following conditions: 98 °C for 30 s, then seven cycles of 98 °C for 10 Attorney Docket No. 44807-0503WO1 / C18573

[2188] s to denature, and 65 °C for 75 s to anneal and extend. WGS libraries were sequenced on a NovaSeq 6000 with paired-end 2x100 bp reads. The depth of sequencing averaged 40.0 M reads of 100 bp (IQR 33.8M to 45. IM, Table 5). Cutadapt was used to trim 27 base pairs from both reads and BWA-MEM was used to align reads to the hgl9 genome. Duplicate molecules were marked and removed using samtools. Reads with a quality >10 were binned into 500kb intervals and counted. IchorCNA was then used to perform GC correction and call the estimated tumor fraction using the following parameters: “ — chrs

[2189] “c(l, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 20, 21, 22) —normal “c(0.9, 0.95, 0.98)” -estimateScPrevalence FALSE -scStates “c()” -maxCN 3 -ploidy “c(2)” -normalPanel 20240722. CSF PON median. rds”. The panel of normal was based on 8 CSF sample without the presence of cancer and were not used in the study. Note: chr16 and chr19 were excluded due to the high variance on each of chromosomes. Increased GC content on these chromosomes can produce higher numbers of false positives. To generate chromosome level arm level calls, the median across each the GC corrected ichorCNA 500kb intervals was used and chr6p, which produced numerous false calls associated with alignment artifacts surrounding the MHC regions, was discarded.

[2190] The “M” component of CSF -BAM (Mutations)

[2191] Following library creation, two separate PCRs were designed to selectively enrich the Watson or Crick strand. Both PCRs used the same gene-specific primer, but each used a different anchoring primer. PCR duplicates derived from each strand could be distinguished by the orientation of the insert relative to the exogenous UID. Sequencing reads underwent initial processing by extracting the first 14 nucleotides as the exogenous barcode sequence (UIDs) and masking adapter sequencing Picard's IlluminaBasecallsToSam (broadinstitute. github.io / picard). Reads were then mapped to the hg19 reference genome using BWA-MEM and sorted by barcode sequence using Samtools. Duplex mutations were defined as mutations present in >80% of both the Watson and Crick families with the same UID. Several metrics for the interpretation of mutations were had: only genomic positions with at least 2 or more reported annotations in the Catalogue of Somatic Mutations in Cancer (COSMIC) in genome-wide studies and were confirmed somatic mutations were considered; Attorney Docket No. 44807-0503WO1 / C18573

[2192] only positions with at least two observations of mutant molecules were considered; only positions with at least 5x coverage were considered; only positions at least 30 bp away from the end of the molecule were considered.

[2193] During the analysis, two major outliers were noticed: FBXW7 codon 505 and KRAS codon 61. The frequency of mutations at these two codons was much higher than expected. Upon closer inspection, every molecule with a mutant FBXW7 codon 505 also had mutations at codons 289, 299, 300, and 314. Blat analysis was performed to compare the observed matches to any non-human genome. The observed sequence perfectly matched the bovine genome. It was hypothesized that bovine-derived hemostatic agents frequently used in neurosurgery may have contributed minor amounts of DNA that amplified and incorrectly aligned to FBXW7. KRAS codon 61 mutations in two patients with trigeminal neuralgia were also found. To ensure high specificity of the assay and given the low abundance of this particular mutation in CNS cancers, all mutations at this position were excluded in this study.

[2194] Example 2: Detection of human brain cancers using genomic and immune cell characterization of cerebrospinal fluid through CSF-BAM

[2195] The results in this Example re-present and expand on at least some of the results provided in other Examples.

[2196] Results

[2197] Overview

[2198] Once DNA was purified from CSF, a DNA library was generated through a modified version of a protocol described previously, named SaferSeqS, which produces a relatively high conversion efficiency of the original DNA template molecules into library DNA molecules. This conversion efficiency was particularly important when the quantity of CSF fluid was limited or when the DNA concentration in that fluid was low. Equally importantly, the SaferSeqS library preserved DNA from both the Watson and Crick strands of the original DNA templates. The ability to independently assess both strands of DNA exponentially increases the accuracy of the resulting sequencing data when the fraction of aberrant DNA molecules is low. SaferSeqS libraries contain -200 copies of each of the original template Attorney Docket No. 44807-0503WO1 / C18573

[2199] molecule strands, and therefore can be used for multiple downstream assessments of DNA. For CSF-BAM, the clonal composition of DNA derived from malignant or normal B cells was analyzed, as well as chromosome copy number alterations, and somatic mutations derived from the cancer cells (Fig. 2). The paradigm for evaluation of each of these three components was identical and was performed in distinct stages to maximize reproducibility and minimize overfitting:

[2200] (i) Analytical stage: optimize the experimental procedures and bioinformatic analysis using DNA from peripheral blood leukocytes or plasma cell-free DNA from healthy individuals.

[2201] (ii) Training stage: use the optimized procedure to evaluate DNA from CSF or blood from a different cohort of patients with and without cancer to establish thresholds for specificity and estimate sensitivity at the chosen thresholds.

[2202] (iii) Validation stage: use the optimized procedure to evaluate CSF from an independent cohort of patients to determine sensitivity and specificity at the pre-defined thresholds for positivity.

[2203] Development of the experimental procedures and bioinformatic pipelines for CSF-BAM The workflow for all three components of CSF-BAM begins with creation of libraries from original DNA template molecules (Fig. 2A). Copies of the DNA templates in the library, which are mappable to the original templates, are then split for analysis with each of the “B,” “A,” and “M” components (Table 1A-1C). Because essentially unlimited copies of DNA templates can be generated in the amplified library, material is not limiting after the initial library generation step and there are no input constraints on splitting copies between the different analytical components.

[2204] The “B” component of CSF-BAM queries the B cell (BCR) receptor genes that are integral to the adaptive immune system. In any single individual, millions of BCRs in normal B cells are generated through imprecise joining of variable (V), diversity (D), and joining (J) segments of the BCR genes. The nature of these sequences and the degree of clonality provides a wealth of information about the adaptive immune system in that particular patient. In comparison to flow cytometry, BCR sequencing provides comprehensive repertoire Attorney Docket No. 44807-0503WO1 / C18573

[2205] descriptions. Moreover, because any B cell cancer is derived from a single B cell, a neoplastic clone is characterized by a single VDJ rearrangement. The presence of malignant cells in CSF can thereby be detected by the over-representation of a single sequence in the CSF, implying a predominant clone. Although such clones can be detected through previously published methods that sequence either RNA or DNA templates, accurate detection and quantification of clonotypes is challenging. Among the reasons for this is that sequencing from DNA templates has generally required multiplex combinations of primers to amplify all possible V and J gene segment pairs.

[2206] SafeBSeqS overcomes this challenge by requiring primers for only the J segments of one of the BCR (IGH) genes (Fig. 2B). Though these data cannot be used to analyze the constant region, they can be used to re-construct the entire VDJ sequence of the BCR. They are therefore adequate to determine the clonal representation of any population of B cells or as well as to identify certain characteristics of the rearrangements associated with cancers or autoimmune disease.

[2207] Experimentation in the analytical stage was conducted to empirically test DNA shearing conditions, library preparation methods, primer sites, primer sequences, primer combinations, primer ratios, and reaction conditions using DNA from different primary cell types, cell lines, and synthetic constructs with a goal to maximize yield and uniformity of representation while minimizing errors. It was found that amplification using four primer sets was sufficient to assess the entire BCR repertoire with SafeBSeqS (Table 1A). One primer amplified gene segments IGHJ1, IGHJ4, and IGHJ5, and one primer each amplified IGHJ2, IGHJ3, and IGHJ6. These four primers were mixed together and used for hemi-nested amplification of the SaferSeqS libraries. They yielded uniform amplification of all the queried gene segments as tested on a sample of DNA derived from fibroblasts with a uniform representation of gene segments (Fig. 15A-15B). For the training stage, applied SafeBSeqS was then to CSF samples from 25 individuals with primary and secondary CNS lymphomas and peripheral blood samples from 95 healthy control individuals without cancer. Peripheral blood samples were used as negative controls because they enabled setting conservative thresholds to ensure high specificity while reserving the more limited number of CSF samples for use in the validation stage. The summaries of results are listed in Table 9 and Attorney Docket No. 44807-0503WO1 / C18573

[2208] Table 10. Based on these results, the positive criteria for clonality was defined as total UIDs >20 and top clone UIDs / total UIDS >0.3 (30%). In total, 1 / 95 samples from healthy individuals and 13 / 25 CSF samples from patients with CNS lymphoma in the set met these criteria (Tables 9 and Table 10). For the validation stage, when SafeBSeqS was applied to CSF from a different cohort of individuals without known cancers, it was found that 0 met the positive criteria for clonality (Table 10). Similarly, 1 of the 205 CSF samples from patients with cancers other than CNS lymphomas scored positively (Table 10). Of 4 CSF samples from patients with B cell lymphomas of the CNS, 2 scored positively in this assay with clonal fractions of 32% and 86% (Table 10). Attorney Docket No. 44807-0503W01 / Cl 8573

[2209] Table 9. Summary of SafeBSeqS analysis in WBC control samples.

[2210] IGH IGH IGH IGH IGH top clone CDR3 nucleotide sequence IGH top clone CDR3 amino IGH top IGH total total top clonality acid sequence clone V gene top clones UIDs clone clone total J gene UIDS

[2211] 16 32 2 0.0625 TGTGCGAGAGATCCCGTATAGCAGTGGCTGGTAGAG CARDPV* QW GRGYFDYW IGHV4-4 IGHJ4

[2212] GCTACTTTGACTACTGG (SEQ ID NO: 391) (SEQ ID NO:392)

[2213] 30 60 2 0.03333 TGTGCGAGTAGTACTATGGTTCAGGGAGTCATTGAC CASSTMVQGVIDYW (SEQ IGHV1-46 IGHJ4

[2214] TACTGG (SEQ ID NO:397) ID NO:398)

[2215] 79 159 3 0.01887 TGTGCGAGACGTGGTTCGGGGAGCATTGATGCTTTT CARRGSGSIDAFDIW (SEQ IGHV5-51 IGHJ3

[2216] GATATCTGG (SEQ ID NO: 331) ID NO: 332)

[2217] 26 52 2 0.03846 TGTGCGAGAGATATTCTTGGGGGCAGTAACTGGTAC CARDILGGSNWYDGVDYW IGHV1-18 IGHJ4

[2218] GACGGTGTTGACTACTGG (SEQ ID NO:365) (SEQ ID NO:366)

[2219] 55 111 3 0.02703 TGTGCGAAGGAGGGGAAGGGGCCCGACTGG (SEQ ID CAKEGKGPDW (SEQ ID IGHV3-30-3 IGHJ4

[2220] NO:321) NO:322)

[2221] 44 88 2 0.02273 TGTGCGAGACCACATTACTATGATAGTAGTGGTTATT CARPHYYDSSGYYPDAFDI IGHV5-51 IGHJ3

[2222] ACCCTGATGCTTTTGATATCTGG (SEQ ID NO:413) W (SEQ ID NO:414)

[2223] 33 66 2 0.0303 TGCGCGAGAGATTATCTGGGGAACCGGGATGCTTTT CARDYLGNRDAFDIW (SEQ IGHV1-46 IGHJ3

[2224] GATATCTGG (SEQ ID NO: 421) ID NO:422)

[2225] 16 32 2 0.0625 TGTGCGAGAGAGAAGGGACCGGTGGGAGCTAAATTT CAREKGPVGAKFKVAYFQ IGHV1-18 IGHJ1

[2226] AAGGTTGCATACTTCCAGCACTGG (SEQ ID NO:267) HW (SEQ ID NO:268)

[2227] 90 180 2 0.01111 TGTGCGAGAGTGGGAGTGGCTACATTCCGCCCCTTT CARVGVATFRPFDSW (SEQ IGHV4-34 IGHJ4

[2228] GACTCCTGG (SEQ ID NO:273) ID NO:274)

[2229] 27 55 3 0.05455 TGTACCACAGACGGGGGGTACTTTGACTACTGG (SEQ CTTDGGYFDYW (SEQ ID IGHV3-15 IGHJ4

[2230] ID NO:281) NO:282)

[2231] 11 22 2 0.09091 TGTGCGAGAGATCGGACTTACGGATTTTGGGGTCTG CARDRTYGFWGLWAYGM IGHV3-48 IGHJ6

[2232]

[2233]

[2234] Attorney Docket No. 44807-0503WO1 / C18573

[2235] TGGGCTTACGGAATGGACGTCTGG (SEQ ID NO 293) DVW (SEQ ID NO:294)

[2236] 22 44 2 0.04545 TGTGCGAGATTCTTCGGGGAGGACAATTTCTACTACT CARFFGEDNFYYFDYW IGHV1-2 IGHJ4

[2237] TTGACTACTGG (SEQ ID NO:319) (SEQ ID NO:320)

[2238] 18 36 2 0.05556 TGTGCGAAATTGCCAGATTACTATGATAGTAGTGGTT CAKLPDYYDSSGYFIAFDI IGHV3-30 IGHJ3

[2239] ATTTTATTGCTTTTGATATCTGG (SEQ ID NO:363) W (SEQ ID NO:364)

[2240] 33 67 3 0.04478 TGTGCGAGACTCCATATGACTACGGTGACTAACCGA CARLHMTTVTNRPFDYW IGHV4-39 IGHJ4

[2241] CCCTTTGACTACTGG (SEQ ID NO:443) (SEQ ID NO:444)

[2242] 35 70 2 0.02857 TGTGCGAGAGATATGACCCATGGCTGCTTTGACTACT CARDMTHGCFDYW (SEQ IGHV3-21 IGHJ4

[2243] GG (SEQ ID NO:393) ID NO:394)

[2244] 83 168 4 0.02381 TGTGCGACAGGCTGGGGCAGCTGCTTATACAACTAC CATGWGSCLYNYW (SEQ IGHV3-53 IGHJ4

[2245] TGG (SEQ ID NO:395) ID NO:396)

[2246] 112 224 2 0.00893 TGTGCGAGAGACTTGGCAGCAACTGGTTATTATTACT CARDLAATGYYYFDSW IGHV3-33 IGHJ4

[2247] TTGACTCCTGG (SEQ ID NO:335) (SEQ ID NO:336)

[2248] 74 148 2 0.01351 TGTGCAAGACAGTGGCTGCCACAATGGTATGCTTTT CARQWLPQWYAFDIW IGHV3-74 IGHJ3

[2249] GATATCTGG (SEQ ID NO: 361) (SEQ ID NO:362)

[2250] 109 219 3 0.0137 TGTGCGAGACATTATTGTAGTGGTGGTAGCTGCTACT CARHYCSGG AATCGLDY IGHV5-51 IGHJ4

[2251] TGCGGTTTGGACTACTGG (SEQ ID NO:317) W (SEQ ID NO:318)

[2252] 15 30 2 0.06667 TGTGCGAGAGGAGGCAGTGGCCTTACGCGAGATTTT CARGGSGLTRDFDIW (SEQ IGHV3-53 IGHJ3

[2253] GATATCTGG (SEQ ID NO 295) ID NO:296)

[2254] 135 270 2 0.00741 TGTGTGAAAGACTTCCACCGCAAGGGGTTCGGCGGC CVKDFHRKGFGGPFDYW IGHV3-30 IGHJ4

[2255] CCCTTTGACTACTGG (SEQ ID NO:419) (SEQ ID NO:420)

[2256] 121 244 3 0.0123 TGTGCTTCGGTTCGCTATGATAGTGGTGGTTATTACC CASVRYDSGGYYQDYW IGHV4-59 IGHJ4

[2257] AAGACTACTGG (SEQ ID NO:269) (SEQ ID NO:270)

[2258] 1 2 2 1 TGTGCGAGACGCCGAAACCAAGTAGTACCAGCTGCT CARRRNQVVP CSLPLARD IGHV5-51 IGHJ4

[2259] CATTACCGTTAGCACGTGACTACTGG (SEQ ID YW (SEQ ID NO:272)

[2260] NO:271)

[2261] 72 145 3 0.02069 TGTGCGAGAGGGGTGGTTTTTGATATCTGG (SEQ ID CARGVVFDIW (SEQ ID IGHV4-59 IGHJ3

[2262]

[2263] NO:287) NO:288)

[2264]

[2265] Attorney Docket No. 44807-0503WO1 / C18573

[2266] 73 146 2 0.0137 TGTGCAAGAGTATAAGTATAGCAGCTCGTCGGTCGG CARV*V*QL RSGRPGGW IGHV3-13 IGHJ4

[2267] GTCGACCTGGTGGGTGG (SEQ ID NO:297) (SEQ ID NO:298)

[2268] 76 153 3 0.01961 TGTGCGAGAGTGCGGTCCGCTACGACCTGGGACTAC CARVRSATTWDYYYYGM IGHV1-46 IGHJ6

[2269] TACTACTACGGTATGGACGTCTGG (SEQ ID NO:315) DVW (SEQ ID NO:316)

[2270] 67 135 3 0.02222 TGTGCAAGAGCAGGAGATAGTAGTGGTTATGGAGAT CARAGDSSGYGDAVDFW IGHV3-13 IGHJ3

[2271] GCTGTAGATTTCTGG (SEQ ID NO 359) (SEQ ID NO:360)

[2272] 143 287 3 0.01045 TGTGCGAGAGCTCGGCGGGGTATAGCAGCAGCTGGT CARARRGIAAAGTNTYNW IGHV1-46 IGHJ5

[2273] ACCAACACCTACAACTGGTTCGACCCCTGG (SEQ ID FDPW (SEQ ID NO:446)

[2274] NO:445)

[2275] 113 227 3 0.01322 TGTGCGAAAAGGTGTAGTAGTACCAGCTGCCCCCCC CAKRCSSTSCPPDYW (SEQ IGHV3-23 IGHJ4

[2276] GACTACTGG (SEQ ID NO:431) ID NO:432)

[2277] 88 176 2 0.01136 TGTGCGAGAGGGGGATATTGTAGTGGTGGTAGCTGC CARGGYCSGGSCFMAVAG IGHV4-4 IGHJ6

[2278] TTCATGGCAGTGGCTGGTCACTACTACTACGGTATGG HYYYGMDVW (SEQ ID

[2279] ACGTCTGG (SEQ ID NO:377) NO:378)

[2280] 161 326 3 0.0092 TGTGCGAGAGGCCTGAGTATCGATCTCTGG (SEQ ID CARGLSIDLW (SEQ ID IGHV3-33 IGHJ3

[2281] NO:381) NO:382)

[2282] 207 420 3 0.00714 TGTGCGAAAGACCACCTGCTACCCCCTTACTACTACG CAKDHLLPPYYYGMDVW IGHV3-23 IGHJ6

[2283] GTATGGACGTCTGG (SEQ ID NO:385) (SEQ ID NO:386)

[2284] 100 205 3 0.01463 TGTGCGAGAGATAACCCTATAGCAGCAGCTGGCGCT CARDNPIAAAGAEW (SEQ IGHV3-21 IGHJ4

[2285] GAGTGG (SEQ ID NO: 399) ID NO:400)

[2286] 11 22 2 0.09091 TGTACTGGCTCCTTTCTTGCCTACTGG (SEQ ID CTGSFLAYW (SEQ ID IGHV3-15 IGHJ4

[2287] NO:343) NO: 344)

[2288] 100 201 3 0.01493 TGTGCGAGGACTAGGGGCTTCTTCAGCACCAGGGAG CARTRGFFSTREGVWFDA IGHV5-10-1 IGHJ5

[2289] GGGGTCTGGTTCGACGCCTGG (SEQ ID NO:355) W (SEQ ID NO:356)

[2290] 130 263 3 0.01141 TGTGCGAGAGATGCCGAGCAGTGGCTGGTACGGAGG CARDAEQWLVRRGILTPDY IGHV1-18 IGHJ4

[2291] GGGATTTTGACTCCAGACTACTGG (SEQ ID NO:311) W (SEQ ID NO:312)

[2292] 129 259 3 0.01158 TGTGCGAGAGAGGGGTATAGCAGTACAGATGCTTTT CAREGYSSTDAFDIW (SEQ IGHV3-21 IGHJ3

[2293]

[2294] GATATCTGG (SEQ ID NO 417) ID NO:418)

[2295]

[2296] Attorney Docket No. 44807-0503WO1 / C18573

[2297] 145 292 3 0.01027 TGTGCAAGAGCGTTTCAGGGAGTTAAGGGCTTCTGG CARAFQGVKGFW (SEQ ID IGHV6-1 IGHJ4

[2298] (SEQ ID NO:423) NO:424)

[2299] 112 229 3 0.0131 TGTGCGAGACAGCCTACGGGATCCATGGACTACTGG CARQPTGSMDYW (SEQ ID IGHV4-39 IGHJ4

[2300] (SEQ ID NO 265) NO:266)

[2301] 104 208 2 0.00962 TGTGCGAGAGATTGGAACCCTATTGTAGTAGTACCA CARDWNPIVVVPAATGNN IGHV1-3 IGHJ5

[2302] GCTGCTACCGGGAACAACTGGTTCGACCCCTGG (SEQ WFDPW (SEQ ID NO:276)

[2303] ID NO:275)

[2304] 66 133 3 0.02256 TGTGCGAGAGATAGCCAGGATGCTGAGAATTACTAC CARDSQDAENYYAFDYW 1GHV3-21 1GHJ4

[2305] GCATTTGACTACTGG (SEQ ID NO 283) (SEQ ID NO:284)

[2306] 92 187 3 0.01604 TGTGTGAAATTCATACCAGTGTCTGCCGGCAGTATCT CVKFIPVSAGSIYNGMDVW IGHV3-64D IGHJ6

[2307] ACAACGGTATGGACGTCTGG (SEQ ID NO:291) (SEQ ID NO:292)

[2308] 163 330 3 0.00909 TGTGCACACAGACGAGGCCGGGGGGACACTGGCTGG CAHRRGRGDTGWRAFDFW IGHV2-5 IGHJ4

[2309] CGAGCCTTTGACTTTTGG (SEQ ID NO:323) (SEQ ID NO:324)

[2310] 109 221 3 0.01357 TGTGCACGGATACCCGATAGTGGGAGCTACCATTTT CARIPDSGSYHFDYW (SEQ IGHV2-70 IGHJ4

[2311] GACTACTGG (SEQ ID NO: 367) ID NO:368)

[2312] 113 229 3 0.0131 TGTGCGAGAGATCTCTCTTGGATAGTGGCTACGGCT CARDLSWIVAT LS*YNYY IGHV4-31 IGHJ6

[2313] ATCCTAATACAACTACTACATGGACGTCTGG (SEQ ID MDVW (SEQ ID NO:442)

[2314] NO:441)

[2315] 67 135 3 0.02222 TGTGCGAGAGAGCGTGCTGGCAACTTTGACTCCTGG CARERAGNFDSW (SEQ ID IGHV6-1 IGHJ4

[2316] (SEQ ID NO:429) NO:430)

[2317] 41 82 2 0.02439 TGTGCGAGAGGGGAGTGGGTTCACGACTACTACTAC CARGEWVHDYYYYGMDV IGHV4-38-2 IGHJ6

[2318] TACGGTATGGACGTCTGG (SEQ ID NO:379) W (SEQ ID NO:380)

[2319] 159 319 3 0.0094 TGTACCACAGCAGGCTGGTTACCGCACTTTGACTACT CTTAGWLPHFDYW (SEQ IGHV3-15 IGHJ4

[2320] GG (SEQ ID NO:387) ID NO:388)

[2321] 132 266 3 0.01128 TGTGCGAGAGGGGTCGGGTATGGATATTGTAGTAGT CARGVGYGYCSSTSCPEPA IGHV1-8 IGHJ3

[2322] ACCAGCTGCCCGGAGCCTGCTTTTGATATCTGG (SEQ FDIW (SEQ ID NO:404)

[2323] ID NO:403)

[2324] 221 446 3 0.00673 TGTGCGAGAGCGGGGGCGTATTACTATGATAGTAGT CARAGAYYYDSSGYYNYW IGHV1-18 IGHJ4

[2325]

[2326]

[2327] Attorney Docket No. 44807-0503WO1 / C18573

[2328] GGTTATTATAACTACTGG (SEQ ID NO 337) (SEQ ID NO:338)

[2329] 54 108 2 0.01852 TGTGCGAGCGATCAAAGGGCCCTAGCATATTGTGGT CASDQRALAYCGGDCYSG IGHV1-18 IGHJ4

[2330] GGTGACTGCTATTCTGGTGACTACTGG (SEQ ID DYW (SEQ ID NO:358)

[2331] NO:357)

[2332] 72 149 3 0.02013 TGTGCGAAATTTCAGCAGTGGCTGGCAGGCCCCTAC CAKFQQWLAGPYYFDYW IGHV3-23 IGHJ4

[2333] TACTTTGACTACTGG (SEQ ID NO:313) (SEQ ID NO:314)

[2334] 98 652 453 0.69479 TGTGCGAGAGTGGAGATGGCTACAACCTCCTCTCCC CARVEMATTSS HYGYYY IGHV3-7 IGHJ6

[2335] ATTATGGCTACTACTACGGTATGGACGTCTGG (SEQ GMDVW (SEQ ID NO:416)

[2336] ID NO:415)

[2337] 126 255 3 0.01176 TGTATTACTGTACCAGAGTTAGCAGCAGCTGGTTCG CITVPELA SWFEFDSW IGHV3-41 IGHJ4

[2338] AATTTGACTCCTGG (SEQ ID NO:425) (SEQ ID NO:426)

[2339] 20 42 3 0.07143 TGTGTGAGAGTTTTAGGATATTGTACTGGTGGTGTAT CVRVLGYCTGGVCYSAEY IGHV1-17 IGHJ1

[2340] GCTATAGCGCTGAATACTTCCAGCACTGG (SEQ ID FQHW (SEQ ID NO:260)

[2341] NO:259)

[2342] 116 236 3 0.01271 TGTGCGAGAGTCGGTGACTACGGACCCTACTACTAC CARVGDYGPYYYYGMDV IGHV3-21 IGHJ6

[2343] TACGGTATGGACGTCTGG (SEQ ID NO:279) W (SEQ ID NO:280)

[2344] 90 181 3 0.01657 TGTGCAAGAGATAACGGGTGGTTCGGGGATTATGGA CARDNGWFGDYGTTGGWF IGHV3-74 IGHJ5

[2345] ACGACGGGAGGCTGGTTCGCCCCCTGG (SEQ ID APW (SEQ ID NO:278)

[2346] NO:277)

[2347] 107 217 3 0.01382 TGTGCGAGAGACCTCGATAACCATAGTGGGAGCTAT CARDLDNHSGSYPGAFDIW IGHV3-48 IGHJ3

[2348] CCAGGGGCTTTTGATATCTGG (SEQ ID NO: 309) (SEQ ID NO:310)

[2349] 177 358 3 0.00838 TGTGCGAGAGATCTAGTGGGAGCTACTAGGGGGGAC CARDLVGATRGDYYGMDV IGHV1-2 IGHJ6

[2350] TACTACGGTATGGACGTCTGG (SEQ ID NO:325) W (SEQ ID NO:326)

[2351] 107 214 2 0.00935 TGTGCGAGACATGATTACTATGGTTCGGGGAGTTATT CARHDYYGSGSYYIDYW IGHV4-39 IGHJ4

[2352] ATATTGACTACTGG (SEQ ID NO:369) (SEQ ID NO:370)

[2353] 150 303 3 0.0099 TGTACCACAGATTACCGGTATTACTATGATAGTAGTG CTTDYRYYYDSSGYGGAY IGHV3-15 IGHJ3

[2354] GTTATGGAGGGGCGTATGATGCTTTTGATATCTGG DAFDIW (SEQ ID NO:352)

[2355]

[2356] (SEQ ID NO:351)

[2357]

[2358] Attorney Docket No. 44807-0503WO1 / C18573

[2359] 155 310 2 0.00645 TGTGCGAGAGATCCAGGGGTATATGGTTCGGGGAGT CARDPGVYGSGSSWFDPW IGHV1-18 IGHJ5

[2360] TCCTGGTTCGACCCCTGG (SEQ ID NO:433) (SEQ ID NO:434)

[2361] 133 268 3 0.01119 TGTGCGAAAGCATATAGTGGGAGCTACTTCGGTGCT CAKAYSGSYFGAFDIW IGHV3-23 IGHJ3

[2362] TTTGATATCTGG (SEQ ID NO:375) (SEQ ID NO:376)

[2363] 136 274 3 0.01095 TGTGCAAGATAGCAGGTCCGGCGAGGTGGCCCCCTG CAR* Q VRR VAPWFDYW IGHV1-45 IGHJ4

[2364] GTTTGACTACTGG (SEQ ID NO:401) (SEQ ID NO:402)

[2365] 36 72 2 0.02778 TGTGCGAGGGGTTCGTATAGTGGGAGCTTGGTTGAC CARGSYSGSLVDYW (SEQ IGHV3-21 IGHJ4

[2366] TACTGG (SEQ ID NO:389) ID NO:390)

[2367] 151 306 3 0.0098 TGTGCGAAAATAGGCGTCATTGCCCTCTGGTACTTCG CAKIGVIALWYFDIW (SEQ IGHV3-23 IGHJ2

[2368] ATATCTGG (SEQ ID NO: 345) ID NO:346)

[2369] 171 343 3 0.00875 TGTGCGGTACGCCTCCCGGGGTGGTCTTTTGACTACT CAVRLPGWSFDYW (SEQ IGHV3-23 IGHJ4

[2370] GG (SEQ ID NO:373) ID NO:374)

[2371] 297 602 3 0.00498 TGTGCGAGAGAGAAGGCTTATTGTAGTAGTACCAGC CAREKAYCSS QLLSVCFD IGHV3-21 IGHJ4

[2372] TGCTAAGCGTTTGTTTTGACTACTGG (SEQ ID NO:329) YW (SEQ ID NO:330)

[2373] 130 261 3 0.01149 TGTGCGAAAGATTCGGCGCGCGGGATACAGCTATGG CAKDSARGIQLWLNWFDP IGHV3-30 IGHJ5

[2374] TTAAATTGGTTCGACCCCTGG (SEQ ID NO:411) W (SEQ ID NO:412)

[2375] 244 491 4 0.00815 TGTGCGAGACATGATCGAGGTCATAGGAGTAGTTGG CARHDRGHRSSWYESSNW IGHV4-55 IGHJ4

[2376] TACGAGAGTTCGAACTGG (SEQ ID NO:427) (SEQ ID NO:428)

[2377] 238 476 2 0.0042 TGTGCGAGAGCCGATGGTGGCTACGTTCCTCGGGAG CARADGGYVPRECW (SEQ IGHV3-21 IGHJ4

[2378] TGCTGG (SEQ ID NO:257) ID NO:258)

[2379] 31 62 2 0.03226 TGTGCAACAATCTTCTCAAGGCACGACGGGCACAGG CATIFSRHDGHRNDYW IGHV1-24 IGHJ4

[2380] AATGACTACTGG (SEQ ID NO:285) (SEQ ID NO:286)

[2381] 121 242 2 0.00826 TGTGCGAGAGCTGGGAATAGTGGGAGCTATAACATT CARAGNSGSYNIDFDYW IGHV4-38-2 IGHJ4

[2382] GACTTTGACTACTGG (SEQ ID NO:347) (SEQ ID NO:348)

[2383] 251 508 4 0.00787 TGTGCGAGGGAGTTTAACTATGAGACTAGTGGTTAT CAREFNYETSGYYYFYW IGHV1-69 IGHJ4

[2384] TACTACTTCTACTGG (SEQ ID NO:305) (SEQ ID NO:306)

[2385] 206 414 3 0.00725 TGTGCGAGAAGGTAGACCTCTTTTGACTACTGG (SEQ CARR*TSFDYW (SEQ ID IGHV4-34 IGHJ4

[2386]

[2387] ID NO:263) NO:264)

[2388]

[2389] Attorney Docket No. 44807-0503WO1 / C18573

[2390] 99 199 3 0.01508 TGTGCGAGATCGGTAAATTCGGGGGACCTCTACTAC CARSVNSGDLYYYYGMDV IGHV1-8 IGHJ6

[2391] TACTACGGTATGGACGTCTGG (SEQ ID NO:301) W (SEQ ID NO:302)

[2392] 146 292 2 0.00685 TGTGCGAGAGTAGGGTTAATTATTCGGGGAGTTACC CARVGLIIRGVT LYYYYY IGHV4-34 IGHJ6

[2393] CCTTTACTACTACTACTACGGTATGGACGTCTGG GMDVW (SEQ ID NO:440)

[2394] (SEQ ID NO:439)

[2395] 119 240 3 0.0125 TGTGCGAGAGGCCGACGCTTTACCAGCGTTCGGGGC CARGRRFTSVRGLILGRYI IGHV4-34 IGHJ4

[2396] CTCATTCTGGGTCGCTACATCTGG (SEQ ID NO: 407) W (SEQ ID NO:408)

[2397] 145 290 2 0.0069 TGTGCGAGACTCATGTATAGCAGTGGCTGGTTTTGTA CARLMYSSGWFCMDVW 1GHV4-39 1GHJ6

[2398] TGGACGTCTGG (SEQ ID NO:339) (SEQ ID NO:340)

[2399] 45 90 2 0.02222 TGTGCGAGACTCGTGGGAGCAGCAGCTGGAAACTTT CARLVGAAAGNFDYW IGHV4-34 IGHJ4

[2400] GACTACTGG (SEQ ID NO:327) (SEQ ID NO:328)

[2401] 324 649 3 0.00462 TGTGCGAAAACCGTCGACCGTATAGCAGAAATAGAA CAKTVDRIAEIEVFDYW IGHV3-23 IGHJ4

[2402] GTCTTTGACTACTGG (SEQ ID NO: 307) (SEQ ID NO:308)

[2403] 268 540 3 0.00556 TGTGCGAGAGGGGGGATCGGGGAGTGGCCCTCCCCC CARGGIGEWPSPLVVW IGHV3-21 IGHJ6

[2404] CTGGTCGTCTGG (SEQ ID NO:353) (SEQ ID NO:354)

[2405] 76 152 2 0.01316 TGTGCAAGAGATCCTGCTATGATAGTAGTGGTTGGA CARDPAMIV GWKYYFDY IGHV3-47 IGHJ4

[2406] AGTACTACTTTGACTACTGG (SEQ ID NO:383) W (SEQ ID NO:384)

[2407] 162 324 2 0.00617 TGTGCGAAAGATCAACCCTGGAACTATTGTAGTAGT CAKDQPWNYCSSTSCYFD IGHV3-11 IGHJ4

[2408] ACCAGCTGCTACTTTGACTACTGG (SEQ ID NO:333) YW (SEQ ID NO:334)

[2409] 243 488 3 0.00615 TGTGCAAGAGATAGGGAGAGTTGTAATAGGCGCCGA CARDRESCN APKWFDPW IGHV3-13 IGHJ5

[2410] AGTGGTTCGACCCCTGG (SEQ ID NO:405) (SEQ ID NO:406)

[2411] 142 288 3 0.01042 TGTGCACATTTGCATTATTACGATATAAACCCATACT CAHLHYYDINPYFDYW IGHV2-5 IGHJ4

[2412] TTGACTACTGG (SEQ ID NO:435) (SEQ ID NO:436)

[2413] 222 445 3 0.00674 TGTGTGAGACTTTCTCTAAGGGGACTACAGTAACTA CVRLSLRG YSNYGDW IGHV3-35 IGHJ4

[2414] CGGGGACTGG (SEQ ID NO:289) (SEQ ID NO:290)

[2415] 148 296 2 0.00676 TGTGCAAAGGATTGGGCGGCACGATACTACTACGGT CAKDWAARYYYGMDVW IGHV3-9 IGHJ6

[2416] ATGGACGTCTGG (SEQ ID NO:371) (SEQ ID NO:372)

[2417] 213 427 3 0.00703 TGTGCGAGCGACGATTTTTGGAGTGGTTATTATATAG CASDDFWSG IDGWFDPW IGHV1-2 IGHJ5

[2418]

[2419]

[2420] Attorney Docket No. 44807-0503WO1 / C18573

[2421] GGCTGGTTCGACCCCTGG (SEQ ID NO:349) (SEQ ID NO:350)

[2422] 135 271 3 0.01107 TGTGCGAGGCCCGGGTATAGCAGTGGCTGGCACCTC CARPGYSSGWHLPFDLW IGHV1-69 IGHJ2

[2423] CCCTTCGATCTCTGG (SEQ ID NO:303) (SEQ ID NO:304)

[2424] 159 320 3 0.00938 TGTGCGAGAGAAAAGGGAGAACTAACCTCCCGCCAC CAREKGELTSRHPYYFDY IGHV1-3 IGHJ4

[2425] CCGTACTACTTTGACTACTGG (SEQ ID NO: 261) W (SEQ ID NO:262)

[2426] 257 518 3 0.00579 TGTGCGTGCCACTCCGGATATAGTACCTCGCGATTTG CACHSGYSTSRFDHW (SEQ IGHV1-46 IGHJ4

[2427] ACCACTGG (SEQ ID NO: 299) ID NO:300)

[2428] 198 397 3 0.00756 TGTGCGTGCGCACACTTCTGG (SEQ ID NO:437) CACAHFW (SEQ ID NO 438) IGHV3-11 IGHJ4 158 316 2 0.00633 TGTGCCCTGACGGCCGTACCAGCTGCTAGATACTAC CALTAVPAARYYMDVW IGHV1-2 IGHJ6

[2429] ATGGACGTCTGG (SEQ ID NO:409) (SEQ ID NO:410)

[2430] 155 310 2 0.00645 TGTGCGAGAGATCGACCCGACTATGATAGTAGTGGT CARDRPDYDS SGYYQRS AF IGHV3-21 IGHJ4

[2431] TATTACCAACGATCGGCCTTTGACTACTGG (SEQ ID DYW (SEQ ID NO:342)

[2432]

[2433] NO:341)

[2434] Table 10. Summary of CSF-BAM SafeBSeqS analysis in CSF

[2435] IGH IGH IGH IGH IGH top clone CDR3 nucleotide sequence IGH top clone CDR3 amino IGH top IGH top total total top clonality acid sequence clone V gene clone J clones UIDs clone gene UIDs

[2436] 0 0 0 #NA NA NA NA NA 4 9 3 0.33333 TGTGCCAGAGAGGGGACTTTTTACTCTGAGGGTAGT CAREGTFYSEGSGFDAFDI IGHV4-30-2 IGHJ3

[2437] GGATTTGATGCTTTTGATATCTGG (SEQ ID NO: 803) W (SEQ ID NO:804)

[2438] 12 268 240 0.89552 TGTGCGCGCGTGAGGATCATGGGAGCCTCTGGAGA CARVRIMGASGDFNNW IGHV4-34 IGHJ1

[2439] CTTTAATAACTGG (SEQ ID NO:463) (SEQ ID NO:464)

[2440] 48 103 8 0.07767 TGTGTGAAGGATCGGGCGGGGTTTGGAAGTCACTG CVKDRAGFGSHWHKDFD IGHV3-23 IGHJ4

[2441]

[2442] GCATAAGGACTTTGACTACTGG (SEQ ID NO:523) YW (SEQ ID NO:524)

[2443]

[2444] Attorney Docket No. 44807-0503WO1 / C18573

[2445] 0 0 0 NA NA NA NA NA 3 1787 1770 0.99049 TGTGCGAAAATGACTGCCCAATACTTTGAGTCCTGG CAKMTAQYFESW (SEQ ID IGHV4-34 IGHJ4

[2446] (SEQ ID NO:457) NO:458)

[2447] 5 14 3 0.21429 TGTGCGAGGTCGGACGGGCAGTTCGTCTACGGTAT CARSDGQFVYGMDVW IGHV4-34 IGHJ6

[2448] GGACGTCTGG (SEQ ID NO: 801) (SEQ ID NO: 802)

[2449] 2 182 94 0.51648 TGTGCGAAAGATGGGGGGTACAGCGGGACCAGTCG CAKDGGYSGTSRYWYFDF IGHV3-23 IGHJ2

[2450] ATATTGGTACTTCGATTTCTGG (SEQ ID NO:473) W (SEQ ID NO:474)

[2451] 2 63 55 0.87302 TGCGTGAGGGAGACCCAGGGACAGAGGAACTTCGA CVRETQGQRNFDLW (SEQ IGHV3-74 IGHJ2

[2452] TCTCTGG (SEQ ID NO:465) ID NO:466)

[2453] 1 20 20 1 TGTGCGAGCTCCTCCATCCCTTATTACGATTTTTCCG CASSSIPYYDFSGGYYYYG IGHV1-69 IGHJ6

[2454] GAGGGTACTACTACTACGGTATGGACGTCTGG (SEQ MDVW (SEQ ID NO:456)

[2455] ID NO:455)

[2456] 1 99 99 1 TGTGCGAACGGAAATGCTGTTGTGACCCTCCACTGG CANGNAVVTLHW (SEQ ID IGHV3-23 IGHJ4

[2457] (SEQ ID NO:451) NO:452)

[2458] 7 124 66 0.53226 TGTGCGAGACGGCAAAAGTACGGTGGTGAACCGGA CARRQKYGGEPEVW (SEQ 1GHV3-33 1GHJ6

[2459] AGTCTGG (SEQ ID NO:471) ID NO: 472)

[2460] 1 212 212 1 TGTGTGACACACGAGTCGCTTTTTGCCTACTTTGAC CVTHESLFAYFDYW (SEQ IGHV4-39 IGHJ4

[2461] TACTGG (SEQ ID NO: 449) ID NO: 450)

[2462] 1 11 11 1 TGTGTGAAAGGGGGCAGTATGGACGTCTGG (SEQ ID CVKGGSMDVW (SEQ ID IGHV3-48 IGHJ6

[2463] NO:451) NO:452)

[2464] 1 71 71 1 TGTGCGAGAGGGAATCAACACTGG (SEQ ID NO 453) CARGNQHW (SEQ ID IGHV4-38-2 IGHJ1

[2465] NO:454)

[2466] 1 309 309 1 TGTGCGATGGCGGGCCTCGCTGAAAGCGGACTTGA CAMAGLAESGLDPW (SEQ IGHV4-31 IGHJ5

[2467] CCCCTGG (SEQ ID NO:447) ID NO:448)

[2468] 7 879 854 0.97156 TGTGTGAGAGGGGGGACTCGACATAATTGGAACTT CVRGGTRHNWNFW (SEQ IGHV4-31 IGHJ4

[2469] CTGG (SEQ ID NO:461) ID NO: 462)

[2470] 0 0 0 #NA NA NA NA NA 3 8 4 0.5 TGTGTGAGAAAGGCCCGCGGGTGGTTAGAATTTGA CVRKARGWLEFDCW (SEQ IGHV4-31 IGHJ1

[2471]

[2472]

[2473] Attorney Docket No. 44807-0503WO1 / C18573

[2474] CTGTTGG (SEQ ID NO:457) ID NO:458)

[2475] 1 5 5 1 TGTACGAAACAGAGTCGCCTTAAGTCGTACTATGTA CTKQSRLKSYYVLEAW IGHV4-34 IGHJ6

[2476] TTAGAGGCCTGG (SEQ ID NO:805) (SEQ ID NO: 806)

[2477] 0 0 0 #NA NA NA NA NA 3 214 210 0.98131 TGTGCGAGAGGCCCCATACATTATTCGACCGTCGAC CARGPIHYSTVDYYGVDV IGHV4-34 IGHJ6

[2478] TATTACGGCGTGGACGTCTGG (SEQ ID NO:459) W (SEQ ID NO:460)

[2479] 2 23 20 0.86957 TGTGCGAGACAGACTTCAAGAGGCTCCGAACATTA CARQTSRGSEHYRHKSEF IGHV4-34 IGHJ4

[2480] TAGACATAAGTCTGAATTTGACTTCTGG (SEQ ID DFW (SEQ ID NO:468)

[2481] NO:467)

[2482] 0 0 0 #NA NA NA NA NA 0 0 0 #NA NA NA NA NA 71 142 2 0.01408 TGTGCGAGTAGCTGGGGTATAGCAGTGGCTGGTACT CASSWGIAVAGTYLDYW IGHV3-33 IGHJ4

[2483] TATCTAGACTACTGG (SEQ ID NO: 609) (SEQ ID NO:610)

[2484] 6 12 2 0.16667 TGTGCGAGAGTGGGGACGGGTTACCGACCTTTGGA CARVGTGYRPLDIW (SEQ IGHV3-30-3 IGHJ3

[2485] TATCTGG (SEQ ID NO: 639) ID NO: 640)

[2486] 1 2 2 1 TGTGCGACCCCAGGGGATTACGATTTTTGGAGTGGG CATPGDYDFWSGLLGLVY IGHV3-23 IGHJ4

[2487] CTCTTAGGTCTTGTCTACTGG (SEQ ID NO: 749) W (SEQ ID NO:750)

[2488] 79 159 3 0.01887 TGTGCGAGACGAAGGGGGCAGCCCCCCAACTTTGA CARRRGQPPNFDYW (SEQ IGHV5-51 IGHJ4

[2489] CTACTGG (SEQ ID NO: 599) ID NO:600)

[2490] 9 19 3 0.15789 TGTGCGAGGGGTAATGGTGGCTACGACCCCTTAGA CARGNGGYDPLDMDVW IGHV3-21 IGHJ6

[2491] CATGGACGTCTGG (SEQ ID NO:613) (SEQ ID NO:614)

[2492] 7 14 2 0.14286 TGTGCGAGACATATTCGTGTACTAATGGTGTATGCC CARHIRVLMVYAPLGYYY IGHV4-39 IGHJ6

[2493] CCCCTTGGGTACTACTACGGTATGGACGTCTGG GMDVW (SEQ ID NO:636)

[2494] (SEQ ID NO:635)

[2495] 119 247 10 0.04049 TGTGCGAGAGATCAGACTGCTGCCTGGAACCGCGG CARDQTAAWNRGHFFDY IGHV4-31 IGHJ4

[2496] ACATTTCTTTGACTACTGG (SEQ ID NO:577) W (SEQ ID NO:578)

[2497] 8 16 2 0.125 TGTAGTCGGAGGTCGGGGTACTGTGGTGGTGGAAG CSRRSGYCGGGRCLNFDH IGHV3-49 IGHJ4

[2498]

[2499] GTGCTTGAACTTTGACCATTGG (SEQ ID NO:623) W (SEQ ID NO: 624)

[2500]

[2501] Attorney Docket No. 44807-0503WO1 / C18573

[2502] 1 2 2 1 TGTGCGAGAGATAATGGTAACTATGGGGGTTACTA CARDNGNYGGYYYYGMD IGHV4-59 IGHJ6

[2503] CTACTACGGTATGGACGTCTGG (SEQ ID NO: 751) VW (SEQ ID NO:752)

[2504] 1 2 2 1 GCCAGAGGGAGACTTAGGTATGATAGTAGTGGTTA ARGRLRYDSSGYYYLDYF IGHV4-30-4 IGHJ4

[2505] TTACTACTTGGACTACTTTGACTACTGG (SEQ ID DYW (SEQ ID NO:754)

[2506] NO:753)

[2507] 3 6 2 0.33333 TGTGCGAGAGATGGTAGTTCCGCCGCAGCAGCTGG CARDGSSAAAAGPQWDV IGHV3-21 IGHJ6

[2508] CCCCCAGTGGGACGTCTGG (SEQ ID NO:677) W (SEQ ID NO:678)

[2509] 93 190 4 0.02105 TGTGCGAGAGACTGGGGAGAGTTTGACTACTGG CARDWGEFDYW (SEQ ID 1GHV1-46 1GHJ4

[2510] (SEQ ID NO:597) NO:598)

[2511] 1 2 2 1 TGTGCGAAAGATATTGTTGTAGTACCGGCTGCGGGG CAKDIVVVPAAGNWFDS IGHV3-23 IGHJ5

[2512] AACTGGTTCGATTCCTGG (SEQ ID NO:781) W (SEQ ID NO:782)

[2513] 2 4 2 0.5 TGTGCGAGAGAGGATGGAGAAGTGCCTGGTCTGTA CAREDGEVPGLYYFDSW IGHV3-20 IGHJ4

[2514] CTACTTTGACTCCTGG (SEQ ID NO:707) (SEQ ID NO:708)

[2515] 0 0 0 NA NA NA NA NA 0 0 0 NA NA NA NA NA 13 27 3 0.11111 TGTGCGAGAAGACGATATTACTTTGGTTCCCATAAC CARRRYYFGSHNYYYYG IGHV3-35 IGHJ6

[2516] TACTACTACTACGGTATGGACGTCTGG (SEQ ID MDVW (SEQ ID NO:494)

[2517] NO:493)

[2518] 1 2 2 1 TGTGCACACAGACGGCCCGGGCCTCCCACCTTAATC CAHRRPGPPTLI CLGELSL IGHV2-5 IGHJ4

[2519] CTGTTTGGGGGAGTTATCGTTACTATTTGACTACTG LFDYW (SEQ ID NO:756)

[2520] G (SEQ ID NO: 755)

[2521] 104 209 3 0.01435 TGTGCGAGAGGCCAAAGTAGAAGGTACCAGCTGCT CARGQSRRYQLLPGRAFDI IGHV4-34 IGHJ3

[2522] ACCTGGTCGTGCTTTTGATATCTGG (SEQ ID NO:607) W (SEQ ID NO:608)

[2523] 17 34 2 0.05882 TGTGCGAGAGATCGATCGTTGTGGTGGTGATTGTCA C ARDRSLWW* LSFGGDWF IGHV3-53 IGHJ5

[2524] TTCGGAGGGGACTGGTTCGACCCCTGG (SEQ ID DPW (SEQ ID NO:556)

[2525] NO:555)

[2526] 0 0 0 NA NA NA NA NA 0 0 0 NA NA NA NA NA

[2527]

[2528]

[2529] Attorney Docket No. 44807-0503WO1 / C18573

[2530] 33 66 2 0.0303 TGTGTCAAAGCGGGGGACATCAGCTGGTATGAGTA CVKAGDISWYEYW (SEQ IGHV3-53 IGHJ4

[2531] CTGG (SEQ ID NO:585) ID NO:586)

[2532] 4 8 2 0.25 TGTGCGAAAGATGCGTGTAGTACCAGCTGCCATGA CAKDACSTSCHEVHW IGHV3-11 IGHJ4

[2533] GGTACACTGG (SEQ ID NO:667) (SEQ ID NO:668)

[2534] 3 6 2 0.33333 TGTGCGAGAGAACGTATTGGGTATGATAGTAGTGC CARERIGYDS SALGYLDY IGHV1-3 IGHJ4

[2535] CCTCGGCTACTTGGACTACTGG (SEQ ID NO: 695) W (SEQ ID NO:696)

[2536] 0 0 0 NA NA NA NA NA 33 69 4 0.05797 TGTGCGCGACAAGGCTGGCTCGAATATTACTATGAT CARQGWLEYYYDSW IGHV4-59 IGHJ4

[2537] TCTTGG (SEQ ID NO:557) (SEQ ID NO: 558)

[2538] 2 4 2 0.5 TGTGCGAGACATCGTGATTACAGTCCCCGAGACTAC CARHRDYSPRDYYYFAM IGHV4-39 IGHJ6

[2539] TACTACTTCGCTATGGACGTCTGG (SEQ ID NO:725) DVW (SEQ ID NO:726)

[2540] 2 4 2 0.5 TGTGCACGGATACTGCTACTCTAAATGTATAACTAT CARILLL* M ITIMGHYW IGHV2-26 IGHJ4

[2541] AATGGGACACTACTGG (SEQ ID NO: 709) (SEQ ID NO:710)

[2542] 9 19 3 0.15789 TGTGCGTTTGGTGGGGCCTCCTACGAGCCATTTGAT CAFGGASYEPFDIW (SEQ IGHV1-2 IGHJ3

[2543] ATCTGG (SEQ ID NO:615) 1D NO:616)

[2544] 8 16 2 0.125 TGTGCGAACGGATAGCAGCTCGTCGTTGACTGCTGG CANG*QLVVDCW (SEQ ID IGHV1-3 IGHJ4

[2545] (SEQ ID NO:625) NO:626)

[2546] 0 0 0 NA NA NA NA NA 0 0 0 NA NA NA NA NA 0 0 0 NA NA NA NA NA 0 0 0 NA NA NA NA NA 0 0 0 NA NA NA NA NA 3 6 2 0.33333 TGTGCGAGAGTGCCGGACGCTTCTTACGATTTTTGG CARVPDASYDFWSGYSPY IGHV3-21 IGHJ4

[2547] AGTGGTTATTCCCCGTACTACTTTGACTACTGG (SEQ YFDYW (SEQ ID NO:682)

[2548] ID NO:681)

[2549] 0 0 0 NA NA NA NA NA 0 0 0 NA NA NA NA NA

[2550]

[2551]

[2552] Attorney Docket No. 44807-0503WO1 / C18573

[2553] 0 0 0 NA NA NA NA NA 17 37 5 0.13514 TGTGCGAGGGCCCAGGGGTTCCAACTACCATACGT CARAQGFQLPYVGYCW IGHV3-33 IGHJ4

[2554] GGGCTACTGTTGG (SEQ ID NO:489) (SEQ ID NO:490)

[2555] 35 73 4 0.05479 TGTGCGAGATTTGGGGGGAGCGGTGGCTGGTACGG CARFGGSGGWYGPDDYW IGHV5-51 IGHJ4

[2556] TCCCGATGACTACTGG (SEQ ID NO:561) (SEQ ID NO: 562)

[2557] 0 0 0 NA NA NA NA NA 55 114 3 0.02632 TGTGCGAAAGATGGGTTTGCCCCTATAGCAGCAGCT CAKDGFAPIAAAGHFDYW IGHV3-30 IGHJ4

[2558] GGGCACTTTGACTACTGG (SEQ ID NO:812) (SEQ ID NO:813)

[2559] 1 2 2 1 TGTGCGAGCAGAAGGTTCGGTGACAAGACAAAATG CASRRFGDKTKW (SEQ ID IGHV3-53 IGHJ4

[2560] G (SEQ ID NO:783) NO:784)

[2561] 0 0 0 NA NA NA NA NA 0 0 0 NA NA NA NA NA 13 26 2 0.07692 TGTGCGAGACATGTCGTATCTATGGTTCGGGGAGTC CARHVVSMVRGVRNWFD IGHV4-39 IGHJ5

[2562] CGTAACTGGTTCGACCCCTGG (SEQ ID NO:529) PW (SEQ ID NO:530)

[2563] 1 2 2 1 TGTGCGAGGCTTTTCCTTTGTGGTGGTGACTGCTATT CARLFLCGGDCYSYFDYW IGHV3-21 IGHJ4

[2564] CCTACTTTGACTACTGG (SEQ ID NO: 759) (SEQ ID NO:760)

[2565] 8 16 2 0.125 TGTGCGAGAGGGGGGCAGCAGCAGTTGGTTCCCAA CARGGQQQLVPKTTYYYY IGHV1-8 IGHJ6

[2566] GACAACCTACTACTACTACGGTCTGGACGTCTGG GLDVW (SEQ ID NO:628)

[2567] (SEQ ID NO:627)

[2568] 19 39 3 0.07692 TGTGCGAGAGATCGGAGGGATAGCAGTGGCTGGTA CARDRRDSSGWYPLDYW IGHV1-18 IGHJ4

[2569] CCCCCTCGACTACTGG (SEQ ID NO 525) (SEQ ID NO:526)

[2570] 0 0 0 NA NA NA NA NA 13 26 2 0.07692 TGTGCGAGAGGGGGATATAGCAGTGGCTGGTCGGT CARGGYS SGWS VAQYYF IGHV3-30-3 IGHJ4

[2571] GGCCCAATACTACTTTGACTACTGG (SEQ ID NO:527) DYW (SEQ ID NO:528)

[2572] 0 0 0 NA NA NA NA NA 0 0 0 NA NA NA NA NA 6 12 2 0.16667 TGTGCGAGATCGAGATTACTATGATAGTAGTCTCCC CARSRLL DSSLPW (SEQ IGHV1-3 IGHJ5

[2573]

[2574] CTGG(SEQ ID NO:641) ID NO:642) Attorney Docket No. 44807-0503WO1 / C18573

[2575] 0 0 0 NA NA NA NA NA 0 0 0 NA NA NA NA NA 2 4 2 0.5 TGTGCGAGCGCTTCAGCAGCTGGTACTATAATTGAC CASASAAGTIIDYW (SEQ IGHV4-59 IGHJ4

[2576] TACTGG (SEQ ID NO: 711) ID NO:712)

[2577] 1 2 2 1 TGTGCGAAAGATTACCAGGGACTGGTTCCTGATGCT CAKDYQGLVPDAFDVW IGHV3-23 IGHJ3

[2578] TTTGATGTCTGG (SEQ ID NO:763) (SEQ ID NO:764)

[2579] 1 2 2 1 TGTGCGAGAGATTTCTACCAGCTGCCGGAACTCTAC CARDFYQLPELYS (SEQ ID IGHV3-21 IGHJ4

[2580] TCT (SEQ ID NO:765) NO:766)

[2581] 0 0 0 NA NA NA NA NA 0 0 0 NA NA NA NA NA 1 2 2 1 TGTGCGAGAGGGCATACTACTGGCTGGCACTCCCTT CARGHTTGWHSLAYW IGHV4-34 IGHJ4

[2582] GCCTACTGG (SEQ ID NO: 767) (SEQ ID NO:768)

[2583] 0 0 0 NA NA NA NA NA 33 67 3 0.04478 TGTGCGAGAGACCTCAACTACGGTGACTCAACCGC CARDLNYGDSTAAFDYW 1GHV1-3 1GHJ4

[2584] GGCATTTGACTACTGG (SEQ ID NO:573) (SEQ ID NO:574)

[2585] 0 0 0 NA NA NA NA NA 3 6 2 0.33333 TGTGCGACAGATGTCCCCACATGACTACAGTAACTA CATDVPT*LQ*LRDW (SEQ IGHV1-18 IGHJ4

[2586] CGGGACTGG (SEQ ID NO:683) ID NO:684)

[2587] 3 6 2 0.33333 TGTGCGAGAGATCGCCTTGTCGTTACTATGATAGTT CARDRLVVTM SCGQIPH IGHV1-18 IGHJ4

[2588] GTGGTCAAATACCTCATGACTACTGG (SEQ ID DYW (SEQ ID NO:686)

[2589] NO:685)

[2590] 10 22 3 0.13636 TGTGTGAAAGATCGGAGCTTCAGCTGGGCCTTTGAC CVKDRSFSWAFDYW (SEQ IGHV3-30 IGHJ4

[2591] TACTGG (SEQ ID NO:487) ID NO:488)

[2592] 1 2 2 1 TGTGCAAAAGATATAGGGGAGGATATTGTAGTAGT CAKDIGEDIVVVSALDVW IGHV3-9 IGHJ6

[2593] GTCTGCCCTGGACGTCTGG (SEQ ID NO: 761) (SEQ ID NO:762)

[2594] 175 352 3 0.00852 TGTGCGAAAGGGAATTATTATGGGTCGGGGGATCT CAKGNYYGSGDLW (SEQ IGHV4-59 IGHJ4

[2595] CTGG(SEQ ID NO:611) ID NO:612)

[2596] 13 26 2 0.07692 TGTGCGAGACATGGTACCCCTATTGTAGTAGTACCA CARHGTPIVVVPAAIDYW IGHV4-39 IGHJ4

[2597]

[2598] Attorney Docket No. 44807-0503WO1 / C18573

[2599] GCTGCTATTGACTACTGG (SEQ ID NO:531) (SEQ ID NO:532)

[2600] 1 2 2 1 TGTGCGAGAGATGGGGTGGCAGCTCGATCCGAAAA CARDGVAARSENYFHFYY IGHV1-18 IGHJ6

[2601] TTACTTCCACTTCTACTACATGGACGTCTGG (SEQ ID MDVW (SEQ ID NO:770)

[2602] NO:769)

[2603] 2 4 2 0.5 TGTGCGAAAGATATCCTATGGTGGTCCTTTGACTAC CAKDILWWSFDYW (SEQ IGHV3-23 IGHJ4

[2604] TGG (SEQ ID NO 715) ID NO:716)

[2605] 22 45 3 0.06667 TGTGCAACAGATCCTATTGACTACGGTAGTCGCTCC CATDPIDYGSRSGTWDFW IGHV1-24 IGHJ4

[2606] GGGACTTGGGACTTCTGG (SEQ ID NO:541) (SEQ ID NO:542)

[2607] 12 24 2 0.08333 TGTGCGAGAGGATGCGCCTTATTGGGAGGAAACCG CARGCALLGGNRP VSYY IGHV1-3 IGHJ6

[2608] ACCCCGTATCTTACTACTACTACTACGGTATGGACG YYYGMDVW (SEQ ID

[2609] TCTGG(SEQ ID NO:515) NO:516)

[2610] 12 25 3 0.12 TGTGCGAGAGGGGTTGGAGTGGTTATTATAGGGTA CARGVGVVIIGYYFDYW IGHV3-33 IGHJ4

[2611] CTACTTTGACTACTGG (SEQ ID NO:491) (SEQ ID NO:492)

[2612] 3 6 2 0.33333 TGACCGCGAATGCCCCCGAGACCTGGATTTGATTCC *PRMPPRPGFDSW (SEQ ID IGHV4-59 IGHJ4

[2613] TGG (SEQ ID NO:687) NO:688)

[2614] 0 0 0 NA NA NA NA NA 1 2 2 1 TGTGCGAAAGAAGGAGGCAGTGGCTGGACGTACTA CAKEGGSGWTYYFDSW IGHV4-34 IGHJ4

[2615] CTTTGACTCCTGG (SEQ ID NO:771) (SEQ ID NO:772)

[2616] 8 17 3 0.17647 TGTACTAGAGATATCCCCGGTGCCACACCAGAGGG CTRDIPGATPEGDYW (SEQ IGHV3-49 IGHJ4

[2617] GGACTACTGG (SEQ ID NO:619) ID NO: 620)

[2618] 40 80 2 0.025 TGTGCGAAAGATCGCCAGGACTACGGTGACTCGTA CAKDRQDYGDSYYFDYW IGHV3-30 IGHJ4

[2619] CTACTTTGACTACTGG (SEQ ID NO:587) (SEQ ID NO: 588)

[2620] 54 108 2 0.01852 TGTGCGAGAGCGGCAACTCACTCTGGGGGTGGGAG CARAATHSGGG AVAGLP IGHV4-39 IGHJ4

[2621] CAGTGGCTGGTCTCCCCCACAACTACTGG (SEQ ID HNYW (SEQ ID NO: 602)

[2622] NO:601)

[2623] 0 0 0 NA NA NA NA NA 4 8 2 0.25 TGTGCGAGAGCGGATCATCCGCATATGGTTCGAGC CARADHPHMVRARYYYY IGHV4-4 IGHJ6

[2624]

[2625] GAGGTACTACTACTACTACGGTATGGACGTCTGG YGMDVW (SEQ ID NO: 666)

[2626]

[2627] Attorney Docket No. 44807-0503WO1 / C18573

[2628] (SEQ ID NO:665)

[2629] 0 0 0 NA NA NA NA NA 0 0 0 NA NA NA NA NA 0 0 0 NA NA NA NA NA 7 23 11 0.47826 TGTACGAGAGGCAACGATTACGTTTGGGGGACTTAT CTRGNDYVWGTYGEESW IGHV4-34 IGHJ4

[2630] GGGGAGGAAAGCTGG (SEQ ID NO:475) (SEQ ID NO:476)

[2631] 0 0 0 NA NA NA NA NA 12 24 2 0.08333 TGTGCGAGGGGAGAACTACCTAGAGGCTACGATCG CARGELPRGYDRYFDYW IGHV3-33 IGHJ4

[2632] CTATTTTGACTACTGG (SEQ ID NO: 517) (SEQ ID NO:518)

[2633] 41 83 3 0.03614 TGTGCGAGGGGCCCTTACGATTTTTGGAGTGGCATC CARGPYDFWSGIWFDPW IGHV3-21 IGHJ5

[2634] TGGTTCGACCCCTGG (SEQ ID NO:579) (SEQ ID NO:580)

[2635] 28 56 2 0.03571 TGTGCGAGAGCGTCGGACTACGGGGACTACTGG CARASDYGDYW (SEQ ID IGHV3-21 IGHJ4

[2636] (SEQ ID NO:581) NO:582)

[2637] 3 6 2 0.33333 TGTGCGAGAGTAGCTGATAGTAGTGGTTATTACCAG CARVADS SGY YQFDYW 1GHV1-69 1GHJ4

[2638] TTTGACTACTGG (SEQ ID NO:689) (SEQ ID NO:690)

[2639] 65 133 3 0.02256 TGTGGGAGAGGCCGAGGTCGTAGCAGTGGCTGGGG CGRGRGRS SGWGNYYYY IGHV1-8 IGHJ6

[2640] GAATTACTACTACTACGGTATGGACGTCTGG (SEQ GMDVW (SEQ ID NO:596)

[2641] ID NO 595)

[2642] 16 32 2 0.0625 TGTGCGAGGCTGGACGTCTGG (SEQ ID NO: 551) CARLDVW (SEQ ID IGHV1-3 IGHJ6

[2643] NO:552)

[2644] 1 2 2 1 TGTGCGAGAGATCGCGCTCTGGGGGAAGACAACTG CARDRALGEDNWFDPW IGHV1-18 IGHJ5

[2645] GTTCGACCCCTGG (SEQ ID NO:777) (SEQ ID NO:778)

[2646] 8 17 3 0.17647 TGTGCGGGGGTTGGGAGCCACTACGAG (SEQ ID CAGVGSHYE (SEQ ID IGHV3-23 IGHJ4

[2647] NO:621) NO: 622)

[2648] 0 0 0 NA NA NA NA NA 0 0 0 NA NA NA NA NA 4 8 2 0.25 TGTGCGAGATGGGATAAAGAGGTCGGATTTATTCCC CARWDKEVGFIPSLDNW IGHV1-46 IGHJ4

[2649]

[2650] TCCTTAGACAACTGG (SEQ ID NO: 671) (SEQ ID NO:672) Attorney Docket No. 44807-0503WO1 / C18573

[2651] 15 30 2 0.06667 TGTGCGAGATCACACTACGGTGACTACCTCGCTTAC CARSHYGDYLAYYMDVW IGHV1-2 IGHJ6

[2652] TACATGGACGTCTGG (SEQ ID NO:543) (SEQ ID NO:544)

[2653] 0 0 0 NA NA NA NA NA 1 2 2 1 TGTGCGAGATGTGTCTCCCCCGGCTCCTACTACAGT CARCVSPGSYYSFYYFDH IGHV1-18 IGHJ4

[2654] TTTTACTACTTTGACCACTGG (SEQ ID NO:785) W (SEQ ID NO:786)

[2655] 0 0 0 NA NA NA NA NA 0 0 0 NA NA NA NA NA 0 0 0 NA NA NA NA NA 0 0 0 NA NA NA NA NA 0 0 0 NA NA NA NA NA 8 16 2 0.125 TGTGCGGCGTCACTAGGGAGTATAGCAGTGGCTGG CAASLGSIAVAGSPPSGW IGHV1-58 IGHJ4

[2656] TAGTCCCCCTTCGGGGTGGTGG (SEQ ID NO:631) W (SEQ ID NO:632)

[2657] 0 0 0 NA NA NA NA NA 0 0 0 NA NA NA NA NA 2 4 2 0.5 TGTGCGAGACGGGATGTGACTACGGTGACTACTTAT CARRDVTTVTTYHFDYW IGHV4-39 IGHJ4

[2658] CACTTTGACTACTGG (SEQ ID NO: 721) (SEQ ID NO:722)

[2659] 2 4 2 0.5 TGTGTAAGGGGGGCCTATGGTGGAGCGTTTGACCA CVRGAYGGAFDHW (SEQ IGHV6-1 IGHJ4

[2660] CTGG(SEQ ID NO:713) ID NO:714)

[2661] 5 10 2 0.2 TGTGCGAGAGGCCGCGGGCATGACAATGGCTGGGG CARGRGHDNGWGSYYYY IGHV4-34 IGHJ6

[2662] GTCCTACTACTACTACATGGACGTCTGG (SEQ ID MDVW (SEQ ID NO: 656)

[2663] NO:655)

[2664] 2 4 2 0.5 TGTGCGGAAGACACGGCTGTGGTCGCCCGGTGGGA CAEDTAV VARWEE Gl* K* 1GHV3-66 1GHJ6

[2665] GGAAGGGGAATTTGAAAATAGAAGAACGGTATGGA KNGMDVW (SEQ ID

[2666] CGTCTGG (SEQ ID NO:723) NO: 724)

[2667] 0 0 0 NA NA NA NA NA 67 134 2 0.01493 TGTGCGAGAGCCGCCGATAGTAGTGGTTTTGACTAC CARAADSSGFDYW (SEQ IGHV4-4 IGHJ4

[2668]

[2669] TGG (SEQ ID NO: 605) ID NO: 606)

[2670]

[2671] Attorney Docket No. 44807-0503WO1 / C18573

[2672] 2 4 2 0.5 TGTTTTTCCTCGACCCTTTTGGAGTGGTTATTGGGCA CFSSTLLEWLLGIFPGATRF IGHV3-23 IGHJ4

[2673] TATTTCCTGGGGCCACCCGCTTTGACTACTGG (SEQ DYW (SEQ ID NO:730)

[2674] ID NO: 729)

[2675] 14 28 2 0.07143 TGTGCGAGAGATTCCTATGATAGTAGTGGCCCTATG CARDSYDSSGPMDVW IGHV4-31 IGHJ6

[2676] GACGTCTGG (SEQ ID NO:537) (SEQ ID NO: 538)

[2677] 19 53 17 0.32075 TGTGCGGCCTAGAGGGTACAATTTTTGGAGTGGTTT CAA* RVQFLEWFYVFDYW IGHV4-31 IGHJ4

[2678] TATGTCTTTGACTACTGG (SEQ ID NO: 477) (SEQ ID NO:478)

[2679] 8 16 2 0.125 TGTGCGAGCCTCTGGGCCGGATACAGCTATGGTGCC CASLWAGYSYGAPDYW 1GHV1-3 1GHI4

[2680] CCTGACTACTGG (SEQ ID NO: 629) (SEQ ID NO:630)

[2681] 0 0 0 NA NA NA NA NA 10 20 2 0.1 TGTGCGAGAGGATCTAGTGATAGTAGTGGTTATTAT CARGSSDSSGYYFDYW IGHV4-34 IGHJ4

[2682] TTTGACTACTGG (SEQ ID NO: 499) (SEQ ID NO: 500)

[2683] 1 5 5 1 TGTGCGCGAGATGTTGGTAGTGGTTACTACTTTGAC CARDVGSGYYFDYW (SEQ IGHV4-4 IGHJ4

[2684] TACTGG (SEQ ID NO:703) ID NO: 704)

[2685] 14 28 2 0.07143 TGTGCGAGAGTAAGTGGCCAACGGAGGGACTATTG CARVSGQRRDYW (SEQ ID 1GHV3-21 1GHJ4

[2686] G (SEQ ID NO:539) NO:540)

[2687] 0 0 0 NA NA NA NA NA 42 90 4 0.04444 TGTGCGAAAGTCGAGGTGAACTACTACTACTACAT CAKVEVNYYYYMDVW IGHV3-23 IGHJ6

[2688] GGACGTCTGG (SEQ ID NO:575) (SEQ ID NO: 576)

[2689] 0 0 0 NA NA NA NA NA 0 0 0 NA NA NA NA NA 0 0 0 NA NA NA NA NA 0 0 0 NA NA NA NA NA 1 2 2 1 TGTGCGAGAGATTTCGGCCCCTATGATAGTAGGGGT CARDFGPYDSRGYADYW IGHV1-18 IGHJ2

[2690] TATGCCGACTACTGGTACTTCGATCTCTGG (SEQ ID YFDLW (SEQ ID NO:758)

[2691] NO:757)

[2692] 9 21 4 0.19048 TGTGCGAGTTTACCGGTGGTGGTACCTGCCACCTCA CASLPVVVPATSNWFDPW IGHV4-4 IGHJ5

[2693]

[2694] AACTGGTTCGACCCCTGG (SEQ ID NO:481) (SEQ ID NO:482) Attorney Docket No. 44807-0503WO1 / C18573

[2695] 6 12 2 0.16667 TGTGCGAGAGCTCTAGCAGTGGCTGATCCCTACGGT CARALA VADPYGMDVW IGHV1-18 IGHJ6

[2696] ATGGACGTCTGG (SEQ ID NO: 649) (SEQ ID NO:650)

[2697] 19 38 2 0.05263 TGTGCGAGAGATTCCTGGCGGTACGATATTTTGACT CARDSWRYDI DWLLNYF IGHV1-18 IGHJ4

[2698] GGTTATTGAACTACTTTGACTACTGG (SEQ ID DYW (SEQ ID NO:566)

[2699] NO:565)

[2700] 0 0 0 NA NA NA NA NA 0 0 0 NA NA NA NA NA 3 6 2 0.33333 TGTGCGAGACTGGGGGTCTTAGGGAACTGGTTCGA CARLGVLGNWFDPW (SEQ IGHV5-10-1 IGHJ5

[2701] CCCCTGG (SEQ ID NO:693) ID NO:694)

[2702] 1 2 2 1 TGTGCGAGACAAACGTATTACTATGATAGTGGCTGG CARQTYYYDSGWFDPW IGHV4-39 IGHJ5

[2703] TTCGACCCCTGG (SEQ ID NO:791) (SEQ ID NO:792)

[2704] 3 6 2 0.33333 TGTGCAAGGGATAGAGGTAGCGGCCAGTGGCTGGT CARDRGSGQWLVPGTLVS IGHV3-9 IGHJ5

[2705] ACCAGGGACCTTAGTCTCCTGG (SEQ ID NO:679) W (SEQ ID NO:680)

[2706] 10 21 3 0.14286 TGTGCGAGAGATTCGCCTTTTGCGTCCTGG (SEQ ID CARDSPFASW (SEQ ID IGHV4-38-2 IGHJ5

[2707] NO:485) NO:486)

[2708] 0 0 0 NA NA NA NA NA 13 28 3 0.10714 TGTGCGAGAGATATAGGTGGTTACGGTTACTTTGAC CARDIGGYGYFDYW (SEQ IGHV4-59 IGHJ4

[2709] TACTGG (SEQ ID NO: 497) ID NO:498)

[2710] 2 4 2 0.5 TGTGCGAGCCTAGACGGGGATGGTTCGGGGAGTTA CASLDGD VRGVIP (SEQ IGHV4-39 IGHJ4

[2711] TTCCG (SEQ ID NO: 727) ID NO:728)

[2712] 3 6 2 0.33333 TGTGCGCGAGATCGTCTAAGCTCTATTAATGGCCTC CARDRLSSINGLWYGDGG IGHV3-64 IGHJ6

[2713] TGGTACGGCGACGGGGGATACTACTACTACGGTAT YYYYGMNVW (SEQ ID

[2714] GAACGTCTGG (SEQ ID NO: 691) NO:692)

[2715] 4 9 3 0.33333 TGTGCGGGGGTTATGATAAGCTTGAGGATGGACTTC CAGVMISLRMDFQHW IGHV4-34 IGHJ1

[2716] CAGCACTGG (SEQ ID NO:663) (SEQ ID NO:664)

[2717] 17 36 3 0.08333 TGTGCGAGAGACTCCTCGCCGGAAGGGTGTGGTGA CARDSSPEGCGDCYSGW IGHV1-69 IGHJ4

[2718] CTGCTATTCTGGCTGG (SEQ ID NO:513) (SEQ ID NOAM)

[2719] 4 8 2 0.25 TGTGCGAGATCCAGAATATTGTTCGGGGAGTCTAGT CARSRILFGESSVFDFW IGHV4-55 IGHJ3

[2720]

[2721]

[2722] Attorney Docket No. 44807-0503WO1 / C18573

[2723] GTTTTTGATTTCTGG (SFQID NO 669)(SEQ ID NO:670)

[2724] 0 0 0 NA NA NA NA NA 16 32 2 0.0625 TGTGCGAAAGATCGCAGCAGCCCGTACTACTTTGAC CAKDRSSPYYFDYW (SEQ IGHV3-30 IGHJ4

[2725] TACTGG (SEQ ID NO:553) ID NO:554)

[2726] 0 0 0 NA NA NA NA NA 1 2 2 1 TGTGCGGGTTTCACCCCCACATATAGCAGTGGCTGG CAGFTPTYSSGWYYFDYW IGHV3-66 IGHJ4

[2727] TACTACTTTGACTACTGG (SEQ ID NO: 779) (SEQ ID NO: 780)

[2728] 10 20 2 0.1 TGTGCGAGAGATCGTGGGAGCTACGTCTATGACTA CARDRGSYVYDYW (SEQ IGHV1-18 IGHJ4

[2729] CTGG(SEQ ID NO:501) ID NO: 502)

[2730] 11 22 2 0.09091 TGTGCGAGAGATTCGGGGAATCGACCGCCAAAAAT CARDSGNRPPKIAVAGYF IGHV3-30-3 IGHJ4

[2731] AGCAGTGGCTGGATACTTTGACTACTGG (SEQ ID DYW (SEQ ID NO: 512)

[2732] NO:511)

[2733] 12 24 2 0.08333 TGTGTGAGGACGAAAACGGGTGGGAGAATCTTCCA CVRTKTGGRIF ITYYYYY IGHV7-56 IGHJ6

[2734] TTACTTACTACTACTACTACATGGACGTCTGG (SEQ MDVW (SEQ ID NO:520)

[2735] ID NO:519)

[2736] 6 12 2 0.16667 TGTGCGAGACACTGTGATAGTAGTGGTTATTTGCGC CARHCDSSGYLRP DRLN 1GHV5-51 1GHJ4

[2737] CCGTTGACCGGTTAAACCGTTTTGGCTACTACTTTG RFGYYFDYW (SEQ ID

[2738] ACTACTGG (SEQ ID NO:645) NO:646)

[2739] 5 10 2 0.2 TGTGCGAGAGCTCCTAGCACCTTAAACTGGTTCGAC CARAPSTLNWFDPW (SEQ IGHV1-3 IGHJ5

[2740] CCCTGG (SEQ ID NO:659) ID NO: 660)

[2741] 0 0 0 NA NA NA NA NA 10 20 2 0.1 TGTGCGAGAGGAGGACGTGGATACAGCTATGGTTC CARGGRGYSYGSKLYYFD IGHV1-8 IGHJ4

[2742] CAAATTGTACTACTTTGACTACTGG (SEQ ID NO:503) YW (SEQ ID NO:504)

[2743] 15 31 3 0.09677 TGTGCGGTGACCCCTCTCGAACGGCCCGGCTGG CAVTPLERPGW (SEQ ID IGHV1-69 IGHJ4

[2744] (SEQ ID NO:507) NO:508)

[2745] 6 12 2 0.16667 TGTGCGAGAGAAGATGGCTACAAATTTGACTACTG CAREDGYKFDYW (SEQ ID IGHV1-46 IGHJ4

[2746] G (SEQ ID NO:643) NO: 644)

[2747] 2 4 2 0.5 TGTGCGAGAGCAGAGGAGTACGATTTTTGGAGTGG CARAEEYDFWSGYSIYYY IGHV1-2 IGHJ6

[2748]

[2749] Attorney Docket No. 44807-0503WO1 / C18573

[2750] TTATTCTATCTACTACTACGGTATGGACGTCTGG GMDVW (SEQ ID NO: 732)

[2751] (SEQ ID NO:731)

[2752] 1 2 2 1 TGTGTGAAAGGCGGGCAGTGGCTGACGACAGACTG CVKGGQWLTTDW (SEQ IGHV3-30 IGHJ4

[2753] G (SEQ ID NO:787) ID NO:788)

[2754] 6 12 2 0.16667 TGTGCAAGAGACTACGGTGACTACGTCTCTGGGTG CARDYGDYVSGWNW IGHV3-74 IGHJ4

[2755] GAACTGG(SEQ ID NO: 647) (SEQ ID NO:648)

[2756] 10 20 2 0.1 TGTGCGAAAGATTGTTGTGCGAAGCAGCTGGCCGT CAKDCCAKQLAVYYYYGI IGHV3-11 IGHJ6

[2757] CTACTACTACTACGGTATTGACGTCTGG (SEQ ID DVW (SEQ ID NO:506)

[2758] NO:505)

[2759] 0 0 0 NA NA NA NA NA 20 40 2 0.05 TGTGCGAGAGTATCTTACTATGGTTCGGGGACTTAT CARVSYYGSGTYYYFDY IGHV3-64 IGHJ4

[2760] TATTACTTTGACTACTGG (SEQ ID NO: 567) W (SEQ ID NO:568)

[2761] 13 26 2 0.07692 TGTACTAGACCTCGTGGATACAGCTATGGGGGTGCC CTRPRGYSYGGAGYW IGHV3-73 IGHJ4

[2762] GGCTACTGG (SEQ ID NO:535) (SEQ ID NO:536)

[2763] 15 30 2 0.06667 TGTGCGAAGAATACTGGGAGATTCCCGTATAACTG CAKNTGRFPYNWFDPW 1GHV3-48 1GHI5

[2764] GTTCGACCCCTGG (SEQ ID NO:545) (SEQ ID NO:546)

[2765] 41 82 2 0.02439 TGTGCGAGAGGGGCGAGTGATTATATAACGGGCTA CARGASDYITGYYFAYW IGHV4-59 IGHJ4

[2766] CTACTTTGCCTACTGG (SEQ ID NO:591) (SEQ ID NO: 592)

[2767] 15 30 2 0.06667 TGTGCGCGGGACCGGGTGTCTTCACAGGGATACTTC CARDRVSSQGYFQDW IGHV3-30 IGHJ1

[2768] CAGGACTGG (SEQ ID NO:547) (SEQ ID NO:548)

[2769] 3 6 2 0.33333 TGTGCGAGACTTAATTGGGCAATGGGTGATAGTAGT CARLNWAMGDSSGYSLY IGHV4-39 IGHJ4

[2770] GGTTACTCGCTCTACTACTTTGACTACTGG (SEQ ID YFDYW (SEQ ID NO:698)

[2771] NO:697)

[2772] 5 11 3 0.27273 TGTGCGAGAGATGCGGAGCGGGATCATGCTGTAGT CARDAERDHAVVLVSAFG IGHV1-18 IGHJ4

[2773] ACTAGTGTCCGCATTCGGCTACTGG (SEQ ID NO:653) YW (SEQ ID NO:654)

[2774] 4 11 4 0.36364 TGTGCGAGACTCGCGGGGAGAGCAGCAGCTGGTAT CARLAGRAAAGMDYW IGHV5-51 IGHJ4

[2775] GGACTACTGG (SEQ ID NO:651) (SEQ ID NO:652)

[2776] 1 2 2 1 TGTGCGAGAGCAAATCCAACAGTGGCTACTACCCT CARANPTVATTLLVFDIW IGHV1-18 IGHJ3

[2777]

[2778]

[2779] Attorney Docket No. 44807-0503WO1 / C18573

[2780] GCTGGTTTTTGATATCTGG (SEQ ID NO:789) (SEQ ID NO:790)

[2781] 3 13 6 0.46154 TGTGCGAGTCTTACGGTGGGCCCGGTACCAGCTGCA CASLTVGPVPAAIRVPRAS IGHV4-39 IGHJ6

[2782] ATTCGGGTTCCTCGGGCGAGTATGGACGTCTGG MDVW (SEQ ID NO: 638)

[2783] (SEQ ID NO:637)

[2784] 2 7 5 0.71429 TGTGCGAGAATACGCTTAAACGATATTTTGACTGGT CARIRLNDILTGYYNGPLD IGHV1-2 IGHJ6

[2785] TATTATAACGGGCCCCTTGACTACTACTACGGTATG YYYGMDVW (SEQ ID

[2786] GACGTCTGG (SEQ ID NO:675) NO:676)

[2787] 0 0 0 NA NA NA NA NA 1 2 2 1 TGTGCCAGAGGTGGCTACGAAACCCCGGGTTACTA CARGGYETPGYYYYYGM 1GHV4-30-2 1GHJ6

[2788] CTACTACTACGGTATGGACGTCTGG (SEQ ID NO:793) DVW (SEQ ID NO: 794)

[2789] 2 4 2 0.5 TGTGCGAGACTTCCCCCTTCCCAATACTATGGTACG CARLPPSQYYGTGNPPDY IGHV5-51 IGHJ4

[2790] GGTAATCCTCCTGACTACTGG (SEQ ID NO:733) W (SEQ ID NO:734)

[2791] 8 16 2 0.125 TGTGCGAGACGGGGGATAGTGGGAGCTACTTCAGC CARRGIVGATSAYYYGMD IGHV5-51 IGHJ6

[2792] CTACTACTACGGTATGGACGTCTGG (SEQ ID NO:633) VW (SEQ ID NO:634)

[2793] 2 4 2 0.5 TGTGCGAGAGGAACCAGTGGGCCCGACTACTGG CARGTSGPDYW (SEQ ID IGHV3-23 IGHJ4

[2794] (SEQ ID NO:735) NO:736)

[2795] 13 26 2 0.07692 TGTGCAAGACAGTTTTGTCTTACTACCAGTAAGTCA CARQFCLT Q* VKVD* R IGHV3-9 IGHJ4

[2796] AGGTGGACTAAAGG (SEQ ID NO:533) (SEQ ID NO:534)

[2797] 0 0 0 NA NA NA NA NA 11 22 2 0.09091 TGTGCGAGAGATCAGAACAGGACGGCGGGGGGATA CARDQNRTAGGYYYYYG IGHV1-18 IGHJ6

[2798] CTACTACTACTACGGTATGGACGTCTGG (SEQ ID MDVW (SEQ ID NO: 510)

[2799] NO: 509)

[2800] 30 60 2 0.03333 TGTGCGAGAGCTGCGCTCGGTAGAGACAGTGGGAC CARAALGRDSGTYAYW IGHV3-21 IGHJ4

[2801] CTACGCCTACTGG (SEQ ID NO:583) (SEQ ID NO:584)

[2802] 5 18 7 0.38889 TGTGCACACAGAGGGAGCTACTATTTCATGGACTTT CAHRGSYYFMDFDYW IGHV2-5 IGHJ4

[2803] GACTACTGG (SEQ ID NO:617) (SEQ ID NO:618)

[2804] 0 0 0 NA NA NA NA NA 9 20 3 0.15 TGTATCACTACTCCTAGTAGCGGCTGGCCCGCGGGT CITTPS SGWPAGGDFW IGHV3-15 IGHJ4

[2805]

[2806] Attorney Docket No. 44807-0503WO1 / C18573

[2807] GGCGACTTCTGG (SEQ ID NO:483) (SEQ ID NO:484)

[2808] 0 0 0 NA NA NA NA NA 2 4 2 0.5 TGTGCCAGAGATCGGGGATATGACAGTAGTTGGTC CARDRGYDSSWSDYW IGHV3-33 IGHJ4

[2809] GGACTATTGG (SEQ ID NO: 717) (SEQ ID NO:718)

[2810] 0 0 0 NA NA NA NA NA 0 0 0 NA NA NA NA NA 2 4 2 0.5 TGTGCGAGAAGTAAACTCAGGGTGGTGACTGCTCG CARSKLRVVTARGYPPYF 1GHV4-34 1GHJ4

[2811] AGGATACCCCCCCTACTTTGACTACTGG (SEQ ID DYW (SEQ ID NO: 740)

[2812] NO:739)

[2813] 0 0 0 NA NA NA NA NA 1 2 2 1 TGTGCGAAACCGTCCTTCTCCGGTGACGCCAACTTT CAKPSFSGDANFDYW IGHV3-30 IGHJ4

[2814] GACTACTGG (SEQ ID NO:773) (SEQ ID NO:774)

[2815] 2 5 3 0.6 TGTGCGAGATTTTCCTCTACGGCGACACGTCCTGGT CARFSSTAT SWWYFDLW IGHV4-55 IGHJ2

[2816] GGTACTTCGATCTCTGG (SEQ ID NO:705) (SEQ ID NO:706)

[2817] 18 37 3 0.08108 TGTGCAAGAGCGGTGGGAGCTACTTACTTTGACTAC CARAVGATYFDYW (SEQ IGHV3-74 IGHJ4

[2818] TGG (SEQ ID NO:521) ID NO: 522)

[2819] 1 2 2 1 TGTGCGACCTCTGACTGGTTATTATATCCTCGCCCA CATSDWLLYPRPYYYYTEI IGHV3-74 IGHJ6

[2820] TACTACTACTACACAGAAATCTGG (SEQ ID NO 775) W (SEQ ID NO:776)

[2821] 0 0 0 NA NA NA NA NA 1 2 2 1 TGTGCGAGAGACTCAGGTCGCGGTTGTACTGATACC CARDSGRGCTDTNYYGLD IGHV3-66 IGHJ6

[2822] AACTACTACGGTCTGGACGTCTGG (SEQ ID NO: 795) VW (SEQ ID NO:796)

[2823] 2 4 2 0.5 TGTGCACGGACAATACTGGGGACGGGCAGTATAGT CARTILGTGSIVARNYFFD IGHV2-70 IGHJ4

[2824] GGCAAGGAATTACTTCTTTGACTATTGG (SEQ ID YW (SEQ ID NO:720)

[2825] NO:719)

[2826] 1 2 2 1 TGTGTAAAGGACATGGTCCCTTGTCGTGTTGGCGGC CVKDMVPCRVGGCYPKR IGHV3-9 IGHJ6

[2827] TGCTACCCTAAGCGCTATGGTATGGACGTCTGG YGMDVW (SEQ ID NO:748)

[2828] (SEQ ID NO:747)

[2829] 0 0 0 NA NA NA NA NA

[2830]

[2831] Attorney Docket No. 44807-0503WO1 / C18573

[2832] 0 0 0 NA NA NA NA NA 5 10 2 0.2 TGTGCGAGAGATAGGCAGTGACTACCCTCAGATAT CARDRQ*LPSDIW (SEQ ID IGHV3-21 IGHJ3

[2833] CTGG (SEQ ID NO:657) NO:658)

[2834] 0 0 0 NA NA NA NA NA 5 10 2 0.2 TGTGCGAAAGATGCAGGATGTAGTAGTACCAGCTG CAKDAGCSSTSCYFVGHP IGHV3-23 IGHJ4

[2835] CTATTTTGTGGGTCATCCGGTCTTTGACTACTGG VFDYW (SEQ ID NO:662)

[2836] (SEQ ID NO:661)

[2837] 2 4 2 0.5 TGTGCGAGTTCCGGGGGCGATGTGGTAGTTCCGGCT CASSGGDVVVPAAREMA IGHV4-34 IGHJ4

[2838] GCTAGAGAGATGGCCTATTGG (SEQ ID NO:737) YW (SEQ ID NO:738)

[2839] 20 40 2 0.05 TGTGCGAAAGATCTTGGGAGTTACTATGATAGTATT CAKDLGSYYDSIDYW IGHV3-23 IGHJ4

[2840] GACTACTGG (SEQ ID NO:569) (SEQ ID NO:570)

[2841] 4 8 2 0.25 TGTGCGAGAGTCAGGCGGGAGTACCAGCTGCTGGG CARVRREYQLLGYFDYW IGHV1-3 IGHJ4

[2842] GTACTTTGACTACTGG (SEQ ID NO:673) (SEQ ID NO:674)

[2843] 25 52 3 0.05769 TGTACTAGAGATTCGTATTACTATGATAGTAGTGGT CTRDSYYYDSSGFVSAFDI IGHV3-49 IGHJ3

[2844] TTCGTATCGGCTTTTGATATCTGG (SEQ ID NO: 559) W (SEQ ID NO:560)

[2845] 28 57 3 0.05263 TGTGCACACAAAGGAGTAGTACCAGCTGCTATTGG CAHKGVVPAAIGSYYGMD IGHV2-5 IGHJ6

[2846] GAGCTACTACGGTATGGACGTCTGG (SEQ ID VW (SEQ ID NO: 564)

[2847] NO:563)

[2848] 23 47 3 0.06383 TGTGCAAACCTCTATGGGCGGGGCCCGGGGGACTA CANLYGRGPGDYW (SEQ IGHV3-74 IGHJ4

[2849] TTGG (SEQ ID NO:549) ID NO:550)

[2850] 0 0 0 NA NA NA NA NA 92 187 3 0.01604 TGTGCGAGAGGAGCTTCCCGAACGATTTTTGGAGTG CARGASRTIFGVVIIRGRGF IGHV3-33 IGHJ5

[2851] GTTATTATAAGGGGGAGGGGATTCGGCTGGTTCGA GWFDPW (SEQ ID NO: 604)

[2852] CCCCTGG (SEQ ID NO:603)

[2853] 0 0 0 NA NA NA NA NA 0 0 0 NA NA NA NA NA 2 4 2 0.5 TGTGCGAAAGATCTAAGGGCAGTGGCTGGTCAAGT CAKDLRA GWSSDYW IGHV3-23 IGHJ4

[2854]

[2855] GACTACTGG (SEQ ID NO: 741) (SEQ ID NO:742) Attorney Docket No. 44807-0503WO1 / C18573

[2856] 1 2 2 1 TGTGTGAGAGATTATCATACGTGGATACAGCCATAT CVRDYHTWIQPYYYYYM IGHV3-11 IGHJ6

[2857] TACTACTACTACATGGACGTCTGG (SEQ ID NO: 799) DVW (SEQ ID NO: 800)

[2858] 0 0 0 NA NA NA NA NA 1 2 2 1 TGTGCGAGAGGTGCCCCGGATAGTAGTGGCAAGGA CARGAPDSSGKEDWYIDL IGHV3-33 IGHJ2

[2859] AGACTGGTACATCGATCTCTGG (SEQ ID NO: 797) W (SEQ ID NO:798)

[2860] 40 80 2 0.025 TGTGTGAGAGATATGAATATTGTGGTGGTGACTGCG CVRDMNIVVV LRYPRFG IGHV1-17 IGHJ4

[2861] ATATCCCCGCTTTGGGTACTGG (SEQ ID NO:589) YW (SEQ ID NO:590)

[2862] 17 235 203 0.86383 TGTGCGAGTATTAGGTCGGGGAGTCCACATTCCTAT CASIRSGSPHSYW (SEQ ID IGHV3-48 IGHJ4

[2863] TGG (SEQ ID NO: 469) NO:470)

[2864] 0 0 0 NA NA NA NA NA 0 0 0 NA NA NA NA NA 0 0 0 NA NA NA NA NA 2 4 2 0.5 TGTACCGCCACCAGGGGATATAGTGGCTCCGTAGTT CTATRGYSGSVVDYW IGHV3-49 IGHJ4

[2865] GACTACTGG (SEQ ID NO:743) (SEQ ID NO: 744)

[2866] 3 6 2 0.33333 TGTGCGAGACCGGTCCGACTTCTAGAAAACTGG CARPVRLLENW (SEQ ID IGHV3-21 IGHJ4

[2867] (SEQ ID NO:699) NO:700)

[2868] 2 4 2 0.5 TGTGCGAGAGATGGGGAGGGGTATATAGCAGTGGC CARDGEGYI QWLVFDYW IGHV1-46 IGHJ4

[2869] TGGTATTTGACTACTGG (SEQ ID NO:745) (SEQ ID NO:746)

[2870] 0 0 0 NA NA NA NA NA 30 61 3 0.04918 TGTGCGAGAGGCAACTATGATAGTAGTGGTTATTAC CARGNYDSSGYYDYW IGHV1-46 IGHJ4

[2871] GACTACTGG (SEQ ID NO:571) (SEQ ID NO: 572)

[2872] 0 0 0 NA NA NA NA NA 13 30 6 0.2 TGTGCGAGAGAAGAGGGCGGTTACGATATTGTGAC CAREEGGYDIVTGYYAAL IGHV3-21 IGHJ4

[2873] TGGTTATTATGCAGCCCTTGACTACTGG (SEQ ID DYW (SEQ ID NO:480)

[2874] NO:479)

[2875] 65 131 3 0.0229 TGTCGCACCCGGAAGGGGATAGTAGTGGTCCCCCA CRTRKGIVVVPHAFDIW IGHV4-30-2 IGHJ3

[2876] TGCTTTTGATATCTGG (SEQ ID NO:593) (SEQ ID NO: 594)

[2877] 13 27 3 0.11111 TGTGCGAGAGGATATATCCTTCGGATTGACTACTGG CARGYILRIDYW (SEQ ID IGHV1-46 IGHJ4

[2878]

[2879] Attorney Docket No. 44807-0503WO1 / C18573

[2880] (SEQ ID NO:495) NO:496)

[2881] 3 6 2 0.33333 TGTGCGGTAATTACTACTCAATGTTCTACTACTACC CAVITTQCS YYHGMDVW IGHV3-23 IGHJ6

[2882]

[2883] ACGGTATGGACGTCTGG (SEQ ID NO:701) (SEQ ID NO:702)

[2884]

[2885] Attorney Docket No. 44807-0503WO1 / C18573

[2886] The initial validation stage for CSF-BAM was limited in that it included only 4 samples derived from patients with B cell cancers, the type relevant for the SafeBSeqS components. The SafeBSeqS assay component was therefore applied to a second validation set of CSF samples. The samples in this set could not be used for validation with full CSF-BAM because they lacked matched peripheral white blood cell samples necessary for the mutation analysis component. This second validation set consisted of CSF samples from 74 patients with peripheral B cell cancers and 14 patients with peripheral T cell cancers (Table 11). The set was divided into three groups. The first group consisted of samples that were interpreted to be true positives for CNS disease because the patients had concurrent evidence for active CNS disease on cytopathology, flow cytometry, or MRI. For these, 18 / 29 samples from patients with B cell cancers were positive for clonality on CSF-BAM for sensitivity of 62%. The second group consisted of patients with no evidence of active CNS disease on cytopathology, flow cytometry, or MRI and who had no documented CNS relapse subsequent to CSF sampling. These samples were interpreted to be true negatives. Of these, 0 / 41 samples from patients with B cell cancers were positive on CSF-BAM for specificity of 100%. The third group consisted of patients with no evidence for active CNS disease at the time of CSF collection but who did have subsequent CNS relapse, meaning that there was possible but uncertain low level CNS involvement at the time of CSF sampling. For these, 0 / 4 B cell cancer samples were positive on CSF-BAM. Attorney Docket No. 44807-0503W01 / Cl 8573

[2887] Table 11. SafeBSeqS analysis for CSF validation set 2

[2888] Diagnostic IGH IGH IGH top IGH IGH IGH top clone CDR3 IGH top clone CDR3 IGH top IGH top group total total clone clonality CSF- nucleotide sequence amino acid sequence clone V clone J gene clones UIDs UIDs BAM gene

[2889] positive

[2890] B cell cancer 0 0 0 NA 0 NA NA NA NA B cell cancer 1 2 2 1 0 TGTGCCAAACATTATTACT CAKHYYYGGSYAMDY IGHV1-45 IGHJ6 ACGGTGGTAGCTATGCTA W (SEQ ID NO:815)

[2891] TGGACTACTGG (SEQ ID

[2892] NO:814)

[2893] B cell cancer 0 0 0 NA 0 NA NA NA NA B cell cancer 0 0 0 NA 0 NA NA NA NA B cell cancer 0 0 0 NA 0 NA NA NA NA B cell cancer 0 0 0 NA 0 NA NA NA NA T cell cancer 138 282 3 0.0106383 0 TGTGCGAGATCATCCGGA CARSSGNYYDSSGYWT IGHV3-21 IGHJ1 AATTACTATGATAGTAGT MAEYFQHW (SEQ ID GGTTATTGGACCATGGCT NO:817)

[2894] GAATACTTCCAGCACTGG

[2895] (SEQ ID NO:816)

[2896] B cell cancer 0 0 0 NA 0 NA NA NA NA B cell cancer 1 792 792 1 1 TGTGCGAGAAATAATTCG CARNNSGPQVGSLDYW IGHV1-18 IGHJ4 GGGCCCCAGGTGGGGTCA (SEQ ID NO:819)

[2897] CTTGACTACTGG (SEQ ID

[2898] NO:818)

[2899] B cell cancer 2 5 3 0.6 0 TGTGCCCGGATGAATATA CARMNIASRWFYFDYW IGHV2-26 IGHJ4 GCGTCTCGTTGGTTCTACT (SEQ ID NO:821)

[2900]

[2901] TTGACTACTGG (SEQ ID Attorney Docket No. 44807-0503WO1 / C18573

[2902] NO:820)

[2903] B cell cancer 0 0 0 NA 0 NA NA NA NA B cell cancer 0 0 0 NA 0 NA NA NA NA B cell cancer 0 0 0 NA 0 NA NA NA NA B cell cancer 0 0 0 NA 0 NA NA NA NA B cell cancer 0 0 0 NA 0 NA NA NA NA B cell cancer 0 0 0 NA 0 NA NA NA NA B cell cancer 28 5376 5316 0.9888393 1 TGTGCGAGAGGTGGGGGG CARGGGDRGN GSTYN IGHV4-34 IGHJ5

[2904] GATCGGGGAAACTGGGTC WFDPW (SEQ ID CACCTACAACTGGTTCGA NO:823)

[2905] CCCCTGG (SEQ ID NO: 822)

[2906] B cell cancer 27 7453 7395 0.9922179 1 TGTGCGAGAGGTGGGGGG CARGGGDRGN GSTYN IGHV4-34 IGHJ5

[2907] GATCGGGGAAACTGGGTC WFDPW (SEQ ID CACCTACAACTGGTTCGA NO:823)

[2908] CCCCTGG (SEQ ID NO: 822)

[2909] B cell cancer 1 3 3 1 0 TGTGCGAATTTGAGTACG CANLSTLTTGYW (SEQ IGHV3-23 IGHJ4

[2910] CTGACGACGGGCTATTGG ID NO: 825)

[2911] (SEQ ID NO: 824)

[2912] B cell cancer 2 309 307 0.9935275 1 TGTGCGAGAGGTGGGGGG CARGGGDRGN GSTYN IGHV4-34 IGHJ5

[2913] GATCGGGGAAACTGGGTC WFDPW (SEQ ID CACCTACAACTGGTTCGA NO:823)

[2914] CCCCTGG (SEQ ID NO: 822)

[2915] B cell cancer 0 0 0 NA 0 NA NA NA NA B cell cancer 1 13162 13162 1 1 TGTGCGAATTTGAGTACG CANLSTLTTGYW (SEQ IGHV3-23 IGHJ4

[2916] CTGACGACGGGCTATTGG ID NO:825)

[2917] (SEQ ID NO: 824)

[2918] B cell cancer 1 2 2 1 0 TGTGCGAGAGATTTCCGA CARDFRTYSGYDYW IGHV3-33 IGHJ4

[2919]

[2920] ACCTATAGTGGCTACGAT (SEQ ID NO: 827) Attorney Docket No. 44807-0503WO1 / C18573

[2921] TATTGG (SEQ ID NO:826)

[2922] B cell cancer 2 6546 6542 0.9993889 1 TGTGCGAATTTGAGTACG CANLSTLTTGYW (SEQ IGHV3-23 IGHJ4

[2923] CTGACGACGGGCTATTGG ID NO:825)

[2924] (SEQ ID NO: 824)

[2925] B cell cancer 0 0 0 NA 0 NA NA NA NA B cell cancer 0 0 0 NA 0 NA NA NA NA B cell cancer 1 2 2 1 0 TGTGCGAGAGAAGGTGTA CAREGVVVVAATPTPG IGHV4-4 IGHJ2

[2926] GTGGTGGTAGCTGCTACT YFDLW (SEQ ID NO: 829) CCAACCCCCGGGTACTTC GATCTCTGG (SEQ ID

[2927] NO:828)

[2928] B cell cancer 0 0 0 NA 0 NA NA NA NA B cell cancer 7 5249 5237 0.9977139 1 TGTGCGACCAAAAGTACC CATKSTNWFDPW (SEQ IGHV4-34 IGHJ5

[2929] AACTGGTTCGACCCCTGG ID NO:831)

[2930] (SEQ ID NO: 830)

[2931] B cell cancer 2 139 137 0.9856115 1 TGTGCCAGAGACCTCTGG CARDLWGSGYDYW IGHV4-30- IGHJ4

[2932] GGGAGTGGTTACGACTAC (SEQ ID NO:833) 2

[2933] TGG (SEQ ID NO:832)

[2934] B cell cancer 0 0 0 NA 0 NA NA NA NA B cell cancer 0 0 0 NA 0 NA NA NA NA B cell cancer 0 0 0 NA 0 NA NA NA NA B cell cancer 0 0 0 NA 0 NA NA NA NA B cell cancer 1 2 2 1 0 TGTGCGAGAGATCTAGTG CARDLVVFGLGELFVD IGHV3-48 IGHJ6

[2935] GTTTTTGGATTGGGGGAA YFYGMDVW (SEQ ID CTATTTGTCGACTACTTCT NO:835)

[2936] ACGGCATGGACGTCTGG

[2937] (SEQ ID NO: 834)

[2938] T cell cancer 0 0 0 NA 0 NA NA NA NA

[2939]

[2940] Attorney Docket No. 44807-0503WO1 / C18573

[2941] T cell cancer 0 0 0 NA 0 NA NA NA NA B cell cancer 0 0 0 NA 0 NA NA NA NA B cell cancer 1 1460 1460 1 1 TGTGCGACCAAAAGTACC CATKSTNWFDPW (SEQ IGHV4-34 IGHJ5

[2942] AACTGGTTCGACCCCTGG ID NO:831)

[2943] (SEQ ID NO: 830)

[2944] B cell cancer 0 0 0 NA 0 NA NA NA NA B cell cancer 0 0 0 NA 0 NA NA NA NA B cell cancer 22 781 430 0.5505762 1 TGTGCGAGGGACGCTTGT CARDACFSWQLVGWE IGHV4-34 IGHJ4

[2945] TTTTCTTGGCAGCTGGTAG TVTSRVVRCPNFDYW GGTGGGAGCTACGGTTAC (SEQ ID NO:836) CTCTAGAGTAGTTCGGTGT CCGAACTTTGACTACTGG

[2946] (SEQ ID NO:835)

[2947] T cell cancer 0 0 0 NA 0 NA NA NA NA B cell cancer 1 6209 6209 1 1 TGTGCGAGCCTCCTCCTGA CASLLLRGT HGNWFD IGHV5-51 IGHJ5

[2948] GGGGGACAACACGGGAAC PW (SEQ ID NO: 838) TGGTTCGACCCCTGG (SEQ

[2949] ID NO:837)

[2950] B cell cancer 0 0 0 NA 0 NA NA NA NA B cell cancer 0 0 0 NA 0 NA NA NA NA B cell cancer 7 14 2 0.1428571 0 TGTGCGAGAGATGAAGGC CARDEGP DAWFDPW IGHV4-34 IGHJ5

[2951] CCCACGACGCGTGGTTCG (SEQ ID NO: 840)

[2952] ACCCCTGG (SEQ ID

[2953] NO:839)

[2954] T cell cancer 0 0 0 NA 0 NA NA NA NA T cell cancer 0 0 0 NA 0 NA NA NA NA B cell cancer 0 0 0 NA 0 NA NA NA NA

[2955]

[2956]

[2957] Attorney Docket No. 44807-0503WO1 / C18573

[2958] T cell cancer 2 7 5 0.7142857 0 TGTACTAGAGTAGCCCCT CTRVAPSFWSGYYYFD IGHV3-49 IGHJ4

[2959] AGTTTTTGGAGTGGTTATT YW (SEQ ID NO:842) ACTACTTTGACTACTGG

[2960] (SEQ ID NO: 841)

[2961] T cell cancer 3 8 3 0.375 0 TGTGCGAATTTGAGTACG CANLSTLTTGYW (SEQ IGHV3-23 IGHJ4

[2962] CTGACGACGGGCTATTGG ID NO:825)

[2963] (SEQ ID NO: 824)

[2964] B cell cancer 56 116 3 0.0258621 0 TGTGCGGGAGCTTTGTGG CAGALWNSPYNW (SEQ IGHV4-38- IGHJ4

[2965] AACTCGCCTTATAACTGG ID NO: 844) 2

[2966] (SEQ ID NO: 843)

[2967] B cell cancer 1 2 2 1 0 TGTGCGAGAGTTAGGAAC CARVRNYYYYMDVW IGHV3-33 IGHJ6

[2968] TACTACTACTACATGGAC (SEQ ID NO: 846)

[2969] GTCTGG (SEQ ID NO: 845)

[2970] B cell cancer 0 0 0 NA 0 NA NA NA NA B cell cancer 1 2 2 1 0 TGTGCGAGAGATCAGAAC CARDQN LVFDYW IGHV3-11 IGHJ4

[2971] TACTGGTTTTTGACTACTG (SEQ ID NO: 848)

[2972] G (SEQ ID NO: 847)

[2973] B cell cancer 1 413 413 1 1 TGTGCGAGCCTCCTCCTGA CASLLLRGT HGNWFD IGHV5-51 IGHJ5

[2974] GGGGGACAACACGGGAAC PW (SEQ ID NO: 838) TGGTTCGACCCCTGG (SEQ

[2975] ID NO:837)

[2976] B cell cancer 1 2 2 1 0 TGTGCGAGAGACTCGAGA CARDSRGAGYSSGRAF IGHV3-11 IGHJ4

[2977] GGGGCGGGTTATAGCAGT DYW (SEQ ID NO:850) GGCCGGGCCTTTGACTAC TGG (SEQ ID NO: 849)

[2978] B cell cancer 0 0 0 NA 0 NA NA NA NA T cell cancer 4 12 5 0.4166667 0 TGTGCGAGAGATGCCCAT CARDAHSGGGSCYVY IGHV3-21 IGHJ4

[2979]

[2980] AGTGGTGGTGGTAGTTGC W (SEQ ID NO:852)

[2981]

[2982] Attorney Docket No. 44807-0503WO1 / C18573

[2983] TACGTCTACTGG (SEQ ID

[2984] NO:851)

[2985] B cell cancer 0 0 0 NA 0 NA NA NA NA B cell cancer 1 6922 6922 1 1 TGTGCGAGACAGGTGGGA CARQVGNIGIYNFFDPW IGHV4-39 IGHJ5

[2986] AATATTGGGATATATAAC (SEQ ID NO: 854) TTCTTCGACCCCTGG (SEQ

[2987] ID NO:853)

[2988] B cell cancer 1 2 2 1 0 TGTGCGAGAGACCACACG CARDHTGGIAIFGVIMG IGHV4-34 IGHJ5

[2989] GGAGGAATTGCAATTTTT TWFDPW (SEQ ID GGAGTGATTATGGGGACC NO:856)

[2990] TGGTTCGACCCCTGG (SEQ

[2991] ID NO:855)

[2992] T cell cancer 0 0 0 NA 0 NA NA NA NA T cell cancer 0 0 0 NA 0 NA NA NA NA B cell cancer 22 70 13 0.1857143 0 TGTGTGAGAGGGGGAGGC CVRGGGNSFVAMDYW IGHV4-39 IGHJ4

[2993] AATAGTTTTGTCGCAATG (SEQ ID NO:858)

[2994] GATTACTGG (SEQ ID

[2995] NO:857)

[2996] B cell cancer 7 18 4 0.2222222 0 TGTGCGAGGGGCGATGGC CARGDGWCDSW (SEQ IGHV4-38- IGHJ5

[2997] TGGTGTGATTCCTGG (SEQ ID NO: 860) 2

[2998] ID NO:859)

[2999] T cell cancer 1 3 3 1 0 TGTGCGAGAGGTTTAAGT CARGLSSGWSKFYFDS IGHV3-21 IGHJ4

[3000] AGTGGGTGGAGCAAATTC W (SEQ ID NO:862) TACTTTGACTCCTGG (SEQ

[3001] ID NO:861)

[3002] B cell cancer 33 66 2 0.030303 0 TGTGCAAAAGATATGACA CAKDMTVTGYSYHYG IGHV3-43 IGHJ6

[3003] GTGACTGGTTATTCCTACC VDVW (SEQ ID NO: 864)

[3004]

[3005] ATTACGGTGTGGACGTCT

[3006]

[3007] Attorney Docket No. 44807-0503WO1 / C18573

[3008] GG (SEQ ID NO:863)

[3009] T cell cancer 1 2 2 1 0 TGTGCACACAGTTGGAGC CAHSWSS RPTDYW IGHV2-5 IGHJ4

[3010] AGCTCGTCCAACGGACTA (SEQ ID NO: 866)

[3011] CTGG (SEQ ID NO: 865)

[3012] T cell cancer 1 3 3 1 0 TGTAAGAAAGATCGCGAG CKKDREY GFFFGYW IGHV3-30 IGHJ4

[3013] TATGAGGTTTCTTTTTTGG (SEQ ID NO: 868)

[3014] CTACTGG (SEQ ID NO: 867)

[3015] B cell cancer 5 25 16 0.64 1 TGTGTGAGAGGGGGAGAC CVRGGDNSFVAMDYW IGHV3-74 IGHJ4

[3016] AATAGTTTTGTCGCAATG (SEQ ID NO: 870)

[3017] GATTACTGG (SEQ ID

[3018] NO:869)

[3019] B cell cancer 5 14 4 0.2857143 0 TGTGCGAGACCGTTAAAC CARPLNYGDSLADIW IGHV4-59 IGHJ3

[3020] TACGGTGACTCCCTCGCC (SEQ ID NO: 872)

[3021] GATATCTGG (SEQ ID

[3022] NO:871)

[3023] B cell cancer 1 19 19 1 0 TGTGCGAGCCTCCTCCTGA CASLLLRGT HGNWFD IGHV5-51 IGHJ5

[3024] GGGGGACAACACGGGAAC PW (SEQ ID NO: 838) TGGTTCGACCCCTGG (SEQ

[3025] ID NO:837)

[3026] B cell cancer 2 91 89 0.978022 1 TATATCAGAGAAATAAGG YIREIRGYDYFSAMDV IGHV3-65 IGHJ6

[3027] GGATATGACTACTTCTCCG W (SEQ ID NO:874) CTATGGACGTCTGG (SEQ

[3028] ID NO:873)

[3029] B cell cancer 2 241 138 0.5726141 1 TGTGCAAGGTAGTAGTAC CAR**YQLLK (SEQ ID IGHV6-1 IGHJ5

[3030] CAGCTGCTAAAG (SEQ ID NO:876)

[3031] NO: 875)

[3032] B cell cancer 0 0 0 NA 0 NA NA NA NA B cell cancer 106 5907 5688 0.9629253 1 TGTGCGAGGACGAAATCT CARTKSP YYGMDVW IGHV4-31 IGHJ6

[3033]

[3034]

[3035] Attorney Docket No. 44807-0503WO1 / C18573

[3036] CCGGCTACTACGGTATGG (SEQ ID NO: 878)

[3037] ACGTCTGG (SEQ ID

[3038] NO:877)

[3039] B cell cancer 1 4 4 1 0 TGTGCGAGCCTCCTCCTGA CASLLLRGT HGNWFD IGHV5-51 IGHJ5

[3040] GGGGGACAACACGGGAAC PW (SEQ ID NO: 838) TGGTTCGACCCCTGG (SEQ

[3041] ID NO:837)

[3042] B cell cancer 0 0 0 NA 0 NA NA NA NA B cell cancer 0 0 0 NA 0 NA NA NA NA B cell cancer 0 0 0 NA 0 NA NA NA NA B cell cancer 0 0 0 NA 0 NA NA NA NA B cell cancer 1 10068 10068 1 1 TGTGCGAGCCTCCTCCTGA CASLLLRGT HGNWFD IGHV5-51 IGHJ5

[3043] GGGGGACAACACGGGAAC PW (SEQ ID NO: 838) TGGTTCGACCCCTGG (SEQ

[3044] ID NO:837)

[3045] B cell cancer 0 0 0 NA 0 NA NA NA NA B cell cancer 35 83 10 0.1204819 0 TGTGCGAGAGGGGGAGAC CARGGDNSFVAMDNW IGHV4-34 IGHJ4

[3046] AATAGTTTTGTCGCAATG (SEQ ID NO: 880)

[3047] GATAATTGG (SEQ ID

[3048] NO:879)

[3049] B cell cancer 0 0 0 NA 0 NA NA NA NA B cell cancer 0 0 0 NA 0 NA NA NA NA

[3050]

[3051]

[3052] Attorney Docket No. 44807-0503WO1 / C18573

[3053] The “A” component of CSF-BAM queries aneuploidy throughout the genome.

[3054] SaferSeqS libraries were converted to a form suitable for whole genome sequencing (WGS) by the addition of primers whose sequences match those of the Illumina NovaSeq flow cells (Fig. 2C and Table IB). The WGS data were mapped to the human genome through standard methods. A modified version of the ichorCNA algorithm was used to assess gains or losses on 34 chromosome arms. Sex chromosomes, acrocentric chromosomes, and arms with high background are excluded in the analysis. Though Watson and Crick strands can be identified from the sequencing data, there was no need to couple the reads to ensure single base pair accuracy to assess copy number alterations of entire arms.

[3055] For the training stage this WGS assay was applied to SaferSeqS libraries from CSF of 31 individuals without cancer (training cohort, Table 12). Two metrics of aneuploidy were used to derive a threshold for scoring samples as aneuploid. First, the number of arms altered was evaluated. In the training cohort, two of the 31 samples had exactly one arm altered while the remaining 29 samples had no arms altered. As a result, any sample with more than one arm altered would be scored as positive for aneuploidy. Next, the estimated tumor fraction as predicted by ichorCNA was evaluated. In the training cohort of individuals without cancer, the largest predicted tumor fraction was 1.4%. Any sample predicted to have a tumor fraction >1.5% would be scored as positive.

[3056] Table 12. Summary of aneuploidy analysis in control samples

[3057] Sample name Annotation Reads Percent Percent Counts Tumor mapped properly fraction paired

[3058] GLIA 438 CSF 1A Negative Control 3.4E+07 0.9546 0.9239 0 0

[3059] GLIA 439 CSF 1A Negative Control 3E+07 0.9448 0.9174 0 0

[3060] GLIA 440 CSF 1A Negative Control 3.3E+07 0.9505 0.9225 0 0

[3061] GLIA 442 CSF 1A Negative Control 3.6E+07 0.9255 0.8985 0 0

[3062] GLIA 443 CSF 1A Negative Control 3.3E+07 0.9403 0.9137 1 0.00958 GLIA 445 CSF 1A Negative Control 4.5E+07 0.8942 0.8656 0 0

[3063] GLIA 446 CSF 1A Negative Control 3.3E+07 0.9622 0.9246 0 0

[3064]

[3065] Attorney Docket No. 44807-0503WO1 / C18573

[3066] GLIA 503 CSF 1A Negative Control 4.1E+07 0.9293 0.9019 0 0

[3067] GLIA 505 CSF 1A Negative Control 3.4E+07 0.9767 0.96 0 0

[3068] GLIA 505 CSF 1A Negative Control 3.8E+07 0.9517 0.9118 0 0

[3069] GLIA 510 CSF 1A Negative Control 3.7E+07 0.9497 0.9176 0 0

[3070] GLIA 517 CSF 1A Negative Control 3.4E+07 0.9771 0.96 0 0.01042 GLIA 567 CSF 1A Negative Control 2.9E+07 0.9283 0.9011 0 0

[3071] GLIA 714 CSF 1A Negative Control 3.5E+07 0.789 0.7584 0 0

[3072] GLIA 717 CSF 1A Negative Control 3.9E+07 0.8334 0.8005 0 0

[3073] GLIA 718 CSF 1A Negative Control 4.8E+07 0.5386 0.5198 0 0

[3074] GLIA 719 CSF 1A Negative Control 3.9E+07 0.6223 0.6019 0 0.01399 GLIA 723 CSF 1A Negative Control 4.2E+07 0.7135 0.6876 0 0.00809 GLIA 763 CSF 1A Negative Control 4E+07 0.9405 0.8878 1 0.00896 GLIA 805 CSF 1 Negative Control 5E+07 0.8494 0.8301 0 0.01105 GLIA 813 CSF 1 Negative Control 4.4E+07 0.9683 0.9469 0 0

[3075] GLIA 817 CSF 1 Negative Control 3.3E+07 0.9276 0.9055 0 0

[3076] GLIA 857 CSF 1 Negative Control 5.3E+07 0.9668 0.9436 0 0.0119 GLIA 858 CSF 1 Negative Control 5.4E+07 0.9596 0.9372 0 0

[3077] GLIB 1011 N Negative Control 4.1E+07 0.9818 0.9628 0 0

[3078] GLIB 1012 N Negative Control 5.4E+07 0.9818 0.8267 0 0

[3079] GLIB 1014 N Negative Control 4.2E+07 0.9833 0.9592 0 0.01189 GLIB 1015 N Negative Control 3.6E+07 0.9849 0.9607 0 0.01811 GLIB 1016 N Negative Control 4.3E+07 0.9817 0.9566 0 0

[3080] GLIB 1017 N Negative Control 3.9E+07 0.9807 0.9418 0 0

[3081] GLIB 1018 N Negative Control 4.7E+07 0.9813 0.9345 0 0

[3082]

[3083] When the same assay was applied for the validation stage to WGS data derived from SaferSeqS libraries of CSF from a different cohort of individuals without active cancers, it was found that 0 of 30 scored positively (100% specificity, credible interval 89% to 100%) (Table 13). 121 of 209 CSF samples from patients with CNS cancers of various types had detectable aneuploidy. Of these, 55 of 83 samples from patients with high grade gliomas were positive (66%, credible interval 56% to 76%). Patients with medulloblastomas or cancers outside the CNS that had metastasized to the brain also were often positive for aneuploidy using this measure (77%, credible interval 56% to 90% and 88%, credible interval 69% to 95%, respectively, Table 13). In addition to overall positivity or negativity, Attorney Docket No. 44807-0503W01 / C18573

[3084] the aneuploidy component also described chromosome arm changes, details that can further inform diagnosis, prognostication, and management (Table 13).

[3085] Table 13. Summary of CSF-BAM aneuploidy analysis in CSF samples

[3086] Sample name Reads Percent Percent Counts Tumor Aneuploid mapped properly fraction

[3087] paired

[3088] CGLI 02 CSF2 3.4E+07 0.9747 0.9343 0 0

[3089] CGLI 04 CSF4 4.7E+07 0.9798 0.9646 2 0.06864 Aneuploid

[3090] CGLT 06 C 3.5E+07 0.949 0.9146 2 0.01835 Aneuploid

[3091] CGLI 07 CSF2 3.1E+07 0.9771 0.9436 0 0

[3092] CGLI 08 C 4.7E+07 0.9612 0.9467 0 0

[3093] CGLI 09 C 3.5E+07 0.9807 0.9605 0 0.01841 Aneuploid

[3094] CGLI 11 CSF2 3.6E+07 0.9769 0.9381 6 0.01699 Aneuploid

[3095] CGLI 12 CSF2 4.2E+07 0.9504 0.936 0 0

[3096] CGLI 13 C 4.3E+07 0.9 0.8859 0 0.01797 Aneuploid

[3097] CGLI 14 CSF2 5.6E+07 0.9787 0.9599 24 0.4155 Aneuploid

[3098] CGLI 15 CSF2 3.5E+07 0.9757 0.8674 0 0

[3099] CGLI 157 CSF 3.2E+07 0.9786 0.9597 0 0.0121

[3100] CGLI 158 CSF 4.2E+07 0.8874 0.8734 0 0

[3101] CGLI 159 CSF 1A 3.2E+07 0.8866 0.8596 11 0.03049 Aneuploid

[3102] CGLI 16 C 6.2E+07 0.7225 0.7117 0 0.02405 Aneuploid

[3103] CGLI 160 CSF 5.1E+07 0.7778 0.7656 0 0

[3104] CGLI 161 CSF 3.7E+07 0.7874 0.7747 0 0.01186

[3105] CGLI 162 CSF 3.3E+07 0.936 0.9229 0 0

[3106] CGLI 163 CSF 3.6E+07 0.978 0.957 0 0.00851

[3107] CGLI 164 CSF 3.8E+07 0.965 0.9328 29 0.1928 Aneuploid

[3108] CGLI 165 CSF 3.9E+07 0.9625 0.9486 0 0.01175

[3109] CGLI 166 CSF 2.8E+07 0.9762 0.9592 1 0

[3110] CGLI 167 CSF 3.6E+07 0.9798 0.9606 1 0.02543 Aneuploid

[3111] CGLI 168 CSF 3.4E+07 0.9666 0.9509 5 0.0401 Aneuploid

[3112] CGLI 169 CSF 3.8E+07 0.9361 0.9196 0 0

[3113] CGLI 170 CSF 4.9E+07 0.6051 0.578 13 0.4448 Aneuploid

[3114] CGLI 171 CSF 3.9E+07 0.9801 0.959 1 0.01332

[3115] CGLI 172 CSF 4.1E+07 0.657 0.6442 1 0.01395

[3116] CGLI 173 CSF 3.6E+07 0.8826 0.8682 1 0

[3117]

[3118] Attorney Docket No. 44807-0503WO1 / C18573

[3119] CGLI 174 CSF 2.4E+07 0.969 0.9509 0 0

[3120] CGLI 175 CSF 2.1E+07 0.9682 0.946 0 0

[3121] CGLI 176 CSF 3.5E+07 0.9289 0.9154 0 0

[3122] CGLI 177 CSF 5E+07 0.7162 0.7056 7 0.03758 Aneuploid

[3123] CGLI 179 CSF 5E+07 0.8306 0.8178 0 0.01266

[3124] CGLI 180 CSF 4.5E+07 0.927 0.9128 0 0.0106

[3125] CGLI 182 CSF 6.3E+07 0.7049 0.6913 0 0.01415

[3126] CGLI 184 CSF 2E+07 0.9825 0.949 10 0.2685 Aneuploid

[3127] CGLI 186 CSF 2.7E+07 0.9663 0.9402 0 0.01173

[3128] CGLI 188 CSF 5.1E+07 0.8617 0.8475 0 0.01211

[3129] CGLI 19 CSF2 5.8E+07 0.6467 0.6371 34 0.4057 Aneuploid

[3130] CGLI 190 CSF 3.6E+07 0.9771 0.9584 0 0

[3131] CGLI 191 CSF 4.1E+07 0.9785 0.9589 0 0.00992

[3132] CGLI 192 CSF 5.2E+07 0.6801 0.6694 21 0.1058 Aneuploid

[3133] CGLI 193 CSF 1A 2.8E+07 0.9744 0.9536 2 0.01927 Aneuploid

[3134] CGLI 194 CSF 4.2E+07 0.8586 0.8445 0 0

[3135] CGLI 20 CSF2 4.4E+07 0.9827 0.9359 34 0.479 Aneuploid

[3136] CGLI 22 C 3.4E+07 0.3343 0.3273 17 0.04195 Aneuploid

[3137] CGLI 25 C 3.8E+07 0.9769 0.9624 0 0.01141

[3138] CGLI 26 C 6.3E+07 0.7165 0.7056 0 0.01444

[3139] CGLI 28 C 1.6E+07 0.9675 0.8312 16 0.07066 Aneuploid

[3140] CGLI 29 C 5.6E+07 0.9643 0.9488 0 0

[3141] CGLI 30 C 2.9E+07 0.2705 0.2648 8 0.1351 Aneuploid

[3142] CGLI 31 CSF2 1.9E+07 0.9753 0.833 10 0.3254 Aneuploid

[3143] CGLI 32 CSF 4.6E+07 0.561 0.552 0 0.00992

[3144] CGLI 34 CSF 2.7E+07 0.0695 0.0683 6 0.01628 Aneuploid

[3145] CGLI 35 CSF 1.5E+07 0.9787 0.9467 5 0.0962 Aneuploid

[3146] CGLI 36 CSF 3.5E+07 0.1636 0.1607 6 0.03763 Aneuploid

[3147] CGLI 37 CSF 2.4E+07 0.2149 0.2114 3 0.023 Aneuploid

[3148] CGLI 38 CSF 2.8E+07 0.9724 0.9572 1 0.02154 Aneuploid

[3149] CGLI 39 CSF 3.3E+07 0.9753 0.9543 0 0

[3150] CGLI 40 CSF 4.4E+07 0.9735 0.9586 17 0.08078 Aneuploid

[3151] CGLI 41 CSF 3.3E+07 0.9355 0.9215 3 0.0266 Aneuploid

[3152] CGLI 42 CSF 2.5E+07 0.152 0.1494 3 0.01911 Aneuploid

[3153] CGLI 43 CSF 4.2E+07 0.9268 0.9121 0 0

[3154] CGLI 44 CSF 4.5E+07 0.7849 0.773 4 0.01919 Aneuploid

[3155] CGLI 45 CSF 3.5E+07 0.9789 0.9642 0 0

[3156] CGLI 47 CSF 3.9E+07 0.9247 0.9111 9 0.3825 Aneuploid

[3157]

[3158] Attorney Docket No. 44807-0503WO1 / C18573

[3159] CGLI 48 CSF 1.9E+07 0.9679 0.8794 5 0.01245 Aneuploid

[3160] CGLI 49 CSF 3.8E+07 0.9788 0.9613 1 0

[3161] CGLI 50 CSF2 2.3E+07 0.9695 0.8998 0 0.02088 Aneuploid

[3162] CGLI 55 CSF 3.7E+07 0.9494 0.9344 11 0.1135 Aneuploid

[3163] CGLI 56 CSF 2E+07 0.976 0.8999 0 0.01632 Aneuploid

[3164] CGLI 57 CSF 4.2E+07 0.9817 0.9653 0 0

[3165] CGLI 58 CSF 3.9E+07 0.9692 0.9533 0 0.01756 Aneuploid

[3166] CGLI 59 CSF 3.6E+07 0.9557 0.9411 0 0.01236

[3167] CGLI 60 CSF 2.7E+07 0.9661 0.8173 15 0.4761 Aneuploid

[3168] CGLI 61 CSF 3.5E+07 0.9759 0.9617 0 0

[3169] CGLI 62 CSF 2.4E+07 0.9796 0.9602 0 0

[3170] CGLI 63 CSF 2.7E+07 0.9736 0.9513 0 0.01529 Aneuploid

[3171] CGLI 83 CSF 3.3E+07 0.9577 0.92 33 0.2188 Aneuploid

[3172] CGLI 87 CSF 3.5E+07 0.947 0.9333 0 0.01544 Aneuploid

[3173] CGLI 88 CSF 4.7E+07 0.7398 0.7243 0 0.01986 Aneuploid

[3174] CGLI 89 CSF 3.1E+07 0.9809 0.9462 11 0.6987 Aneuploid

[3175] CGLI 90 CSF 3.3E+07 0.98 0.9549 18 0.1503 Aneuploid

[3176] CGLI 91 CSF 6.9E+07 0.2857 0.2775 1 0.02707 Aneuploid

[3177] CGLI 97 CSF 3.2E+07 0.9833 0.9152 27 0.1528 Aneuploid CGLIA 100 CSF 5E+07 0.5936 0.5834 0 0.02052 Aneuploid CGLIA 301 CSF2A 3.2E+07 0.9444 0.9156 16 0.4687 Aneuploid

[3178] GLI 101 CSF 3.9E+07 0.9103 0.897 0 0.02203 Aneuploid

[3179] GLIA 419 CSF 1A 4.1E+07 0.9414 0.8906 0 0

[3180] GLIA 420 CSF 2 4.1E+07 0.973 0.9464 3 0.01696 Aneuploid

[3181] GLIA 422 CSF 1A 3.1E+07 0.955 0.9198 2 0.2896 Aneuploid

[3182] GLIA 423 CSF 1A 3.7E+07 0.9534 0.9018 15 0.09641 Aneuploid

[3183] GLIA 424 CSF 1A 2.6E+07 0.9697 0.9472 1 0

[3184] GLIA 425 CSF 1A 3.2E+07 0.884 0.8576 1 0

[3185] GLIA 426 CSF 1A 3E+07 0.9785 0.9567 18 0.1791 Aneuploid

[3186] GLIA 427 CSF 1A 3.3E+07 0.9581 0.8821 23 0.2457 Aneuploid

[3187] GLIA 428 CSF 1A 3.3E+07 0.9853 0.9524 11 0.826 Aneuploid

[3188] GLIA 430 CSF 1A 3.8E+07 0.979 0.9557 6 0.07767 Aneuploid

[3189] GLIA 431 CSF 1A 3.6E+07 0.9797 0.9569 17 0.3696 Aneuploid

[3190] GLIA 433 CSF 1A 3E+07 0.8867 0.8616 0 0

[3191] GLIA 435 CSF 1A 2.9E+07 0.9704 0.9452 0 0.01279

[3192] GLIA 436 CSF 1A 3.6E+07 0.9458 0.9179 0 0

[3193] GLIA 452 CSF 1 2.7E+07 0.9727 0.8553 8 0.04121 Aneuploid

[3194] GLIA 515 CSF 2 4.4E+07 0.9197 0.901 0 0

[3195]

[3196] Attorney Docket No. 44807-0503WO1 / C18573

[3197] GLIA 522 CSF 1A 2.8E+07 0.985 0.9563 15 0.2524 Aneuploid

[3198] GLIA 525 CSF 1 8.4E+07 0.0578 0.0563 25 0.08748 Aneuploid

[3199] GLIA 528 CSF 1 2.6E+07 0.8591 0.8192 3 0.02635 Aneuploid

[3200] GLIA 533 CSF 1A 4E+07 0.8265 0.7991 0 0.02063 Aneuploid

[3201] GLIA 534 CSF 2 4.2E+07 0.9013 0.8824 0 0.01251

[3202] GLIA 537 CSF 3 4.1E+07 0.9886 0.9459 32 0.3196 Aneuploid

[3203] GLIA 539 CSF 1A 4.3E+07 0.7837 0.7677 9 0.07052 Aneuploid

[3204] GLIA 547 CSF 1A 2.6E+07 0.9546 0.9285 7 0.2083 Aneuploid

[3205] GLIA 549 CSF 1A 2.9E+07 0.7671 0.7435 1 0.1035 Aneuploid

[3206] GLIA 550 CSF 1A 3.8E+07 0.9717 0.9481 10 0.1738 Aneuploid

[3207] GLIA 552 CSF 1A 2.8E+07 0.9327 0.9038 21 0.1082 Aneuploid

[3208] GLIA 554 CSF 1A 2.9E+07 0.9817 0.9536 14 0.3719 Aneuploid

[3209] GLIA 555 CSF 1A 4.5E+07 0.8084 0.7881 2 0.01776 Aneuploid

[3210] GLIA 559 CSF 1 4.5E+07 0.9583 0.7053 13 0.03698 Aneuploid

[3211] GLIA 561 CSF 1A 3E+07 0.9561 0.9338 0 0.01171

[3212] GLIA 563 CSF 1A 3.6E+07 0.9771 0.9591 9 0.08527 Aneuploid

[3213] GLIA 565 CSF 1 3.7E+07 0.9733 0.8999 15 0.03403 Aneuploid

[3214] GLIA 569 CSF 1A 3.4E+07 0.97 0.9469 5 0.02393 Aneuploid

[3215] GLIA 571 CSF 1A 4.6E+07 0.9333 0.9106 19 0.1887 Aneuploid

[3216] GLIA 573 CSF 1A 2.7E+07 0.9688 0.9484 0 0

[3217] GLIA 582 CSF 2 3.3E+07 0.9513 0.9247 21 0.05601 Aneuploid

[3218] GLIA 590 CSF 1A 2.9E+07 0.9498 0.9254 0 0.01055

[3219] GLIA 793 CSF 2 4E+07 0.9639 0.9243 0 0.01249

[3220] GLIA 794 CSF 1 4.9E+07 0.9774 0.9589 0 0

[3221] GLIA 795 CSF 1 2.1E+07 0.5236 0.5118 0 0.01217

[3222] GLIA 796 CSF 2 4.6E+07 0.7845 0.7651 15 0.1597 Aneuploid

[3223] GLIA 797 CSF 1 5.7E+07 0.4682 0.4598 6 0.05963 Aneuploid

[3224] GLIA 798 CSF 1 4.9E+07 0.8231 0.8049 0 0

[3225] GLIA 800 CSF 1 4.2E+07 0.8369 0.8163 4 0.02175 Aneuploid

[3226] GLIA 802 CSF 1 3.9E+07 0.9486 0.9272 0 0.01327

[3227] GLIA 804 CSF 1 4.1E+07 0.9665 0.9412 19 0.1628 Aneuploid

[3228] GLIA 806 CSF 2 4.6E+07 0.9849 0.9632 0 0

[3229] GLIA 807 CSF 1 4.9E+07 0.5822 0.5693 0 0.01172

[3230] GLIA 808 CSF 1 4.7E+07 0.716 0.7 0 0

[3231] GLIA 810 CSF 1 3.6E+07 0.9415 0.9201 0 0

[3232] GLIA 819 CSF 1 3.8E+07 0.9471 0.9231 0 0

[3233] GLIA 820 CSF 1 4E+07 0.9441 0.9032 34 0.3899 Aneuploid

[3234] GLIA 821 CSF 1 4.4E+07 0.9482 0.9265 0 0

[3235]

[3236] Attorney Docket No. 44807-0503WO1 / C18573

[3237] GLIA 822 CSF 1 5.1E+07 0.7672 0.7481 0 0.0143

[3238] GLIA 823 CSF 1 4.4E+07 0.9585 0.9358 2 0.05838 Aneuploid

[3239] GLIA 824 CSF 1 3.9E+07 0.9359 0.9143 0 0

[3240] GLIA 825 CSF 1 3.6E+07 0.9572 0.911 29 0.253 Aneuploid

[3241] GLIA 826 CSF 1 4.4E+07 0.9548 0.9231 19 0.3122 Aneuploid

[3242] GLIA 827 CSF 2 3.3E+07 0.986 0.9558 0 0

[3243] GLIA 828 CSF 1 4.3E+07 0.8962 0.8726 0 0.01227

[3244] GLIA 829 CSF 1 7.3E+07 0.5513 0.5388 0 0

[3245] GLIA 830 CSF 1 4.5E+07 0.9555 0.9352 0 0

[3246] GLIA 831 CSF 1 4.5E+07 0.8033 0.7863 0 0.00972

[3247] GLIA 833 CSF 1 4.2E+07 0.8969 0.8783 4 0.06385 Aneuploid

[3248] GLIA 834 CSF 1 4.5E+07 0.9555 0.935 0 0

[3249] GLIA 835 CSF 1 4.3E+07 0.9324 0.9119 0 0

[3250] GLIA 836 CSF 1 4.4E+07 0.9512 0.9297 0 0

[3251] GLIA 837 CSF 1 4.5E+07 0.9642 0.9413 6 0.04048 Aneuploid

[3252] GLIA 838 CSF 1 4.4E+07 0.9542 0.9353 0 0

[3253] GLIA 839 CSF 1 5.5E+07 0.5373 0.5261 1 0.02606 Aneuploid

[3254] GLIA 845 CSF 1 3.7E+07 0.964 0.9429 0 0

[3255] GLIA 846 CSF 1 3.8E+07 0.9628 0.9427 0 0

[3256] GLIA 848 CSF 1 4.7E+07 0.9801 0.9617 1 0.02668 Aneuploid

[3257] GLIA 850 CSF 1 4.5E+07 0.9802 0.9574 34 0.3437 Aneuploid

[3258] GLIA 853 CSF 1 5.6E+07 0.8795 0.8514 0 0.01028

[3259] GLIA 854 CSF 1 6.1E+07 0.9487 0.9245 0 0

[3260] GLIA 855 CSF 1 5E+07 0.9658 0.943 0 0

[3261] GLIA 856 CSF 1 5.6E+07 0.9759 0.943 30 0.1285 Aneuploid

[3262] GLIA 859 CSF 1 4.3E+07 0.9659 0.9423 0 0.00788

[3263] GLIA 882 CSF 1 5E+07 0.9606 0.9363 0 0

[3264] GLIA 883 CSF 1 4.6E+07 0.9822 0.9473 27 0.3472 Aneuploid

[3265] GLIA 884 CSF 1 5.1E+07 0.9536 0.9296 0 0

[3266] GLIA 886 CSF 1 4.2E+07 0.9515 0.9316 0 0

[3267] GLIA 888 CSF 1 4.3E+07 0.9733 0.9331 0 0.01192

[3268] GLIA 891 CSF 1 4.3E+07 0.9581 0.9373 0 0

[3269] GLIA 893 CSF 1 3.8E+07 0.9706 0.9369 14 0.08186 Aneuploid

[3270] GLIA 895 CSF 1 3.5E+07 0.815 0.796 0 0.0158 Aneuploid

[3271] GLIA 897 CSF 1 3.1E+07 0.9299 0.9091 17 0.05207 Aneuploid

[3272] GLIA 898 CSF 1 3E+07 0.9519 0.9323 0 0

[3273] GLIA 899 CSF 1 5.2E+07 0.5806 0.5695 1 0.0177 Aneuploid

[3274] GLIA 901 CSF 1 3.6E+07 0.9649 0.9418 0 0

[3275]

[3276] Attorney Docket No. 44807-0503WO1 / C18573

[3277] GLIA 902 CSF 1 3.6E+07 0.9787 0.9532 0 0.01552 Aneuploid

[3278] GLIA 904 CSF 1 4.5E+07 0.9678 0.9393 0 0.01049

[3279] GLIA 905 CSF 1 4E+07 0.9721 0.9499 0 0.01376

[3280] GLIA 906 CSF 1 3.1E+07 0.9783 0.9291 34 0.4751 Aneuploid

[3281] GLIA 907 CSF 1 4.6E+07 0.7444 0.7291 0 0.01902 Aneuploid

[3282] GLIA 908 CSF 1 4E+07 0.973 0.9394 6 0.05671 Aneuploid

[3283] GLIA 909 CSF1 3.6E+07 0.9787 0.9532 0 0.01552 Aneuploid

[3284] GLIA 910 CSF 1 3.8E+07 0.9679 0.94 0 0

[3285] GLIA 912 CSF 1 4.7E+07 0.7924 0.7744 0 0.00883

[3286] GLIA 913 CSF 1 4.4E+07 0.9592 0.9307 1 0.01329

[3287] GLIA 914 CSF 1 4.6E+07 0.8442 0.8241 31 0.4067 Aneuploid

[3288] GLIA 915 CSF 2 4.5E+07 0.9504 0.914 10 0.6421 Aneuploid

[3289] GLIA 916 CSF 1 5.2E+07 0.6714 0.6573 0 0.00811

[3290] GLIA 917 CSF 3 5.6E+07 0.7388 0.7241 5 0.03193 Aneuploid

[3291] GLIA 918 CSF 1 3.7E+07 0.9631 0.9219 31 0.4982 Aneuploid

[3292] GLIA 919 CSF 1 3.9E+07 0.9778 0.9016 18 0.554 Aneuploid

[3293] GLIA 921 CSF 1 4.1E+07 0.9795 0.875 18 0.1746 Aneuploid

[3294] GLIA 922 CSF 1 3.9E+07 0.9779 0.8724 34 0.6137 Aneuploid

[3295] GLIA 923 CSF 2 3.4E+07 0.9791 0.9522 0 0.01109

[3296] GLIA 924 CSF 1 5E+07 0.8666 0.8444 24 0.1402 Aneuploid

[3297] GLIA 925 CSF 1 4E+07 0.9659 0.9438 24 0.05656 Aneuploid

[3298] GLIA 926 CSF 1 4.5E+07 0.829 0.8125 0 0

[3299] GLIA 927 CSF 1 3.9E+07 0.9788 0.9389 31 0.3975 Aneuploid

[3300] GLIA 928 CSF 1 4.3E+07 0.9746 0.9469 26 0.3509 Aneuploid

[3301] GLIA 929 CSF 1 4.5E+07 0.976 0.9402 34 0.4999 Aneuploid

[3302] GLIA 930 CSF 1 4.7E+07 0.95 0.9297 25 0.1061 Aneuploid

[3303] GLIA 932 CSF 1 4E+07 0.9743 0.9522 13 0.2484 Aneuploid

[3304] GLIA 934 CSF 1 5.3E+07 0.9556 0.9324 16 0.0725 Aneuploid

[3305] GLIA 935 CSF 1 6.2E+07 0.6905 0.6672 0 0.00926

[3306] GLIA 936 CSF 1 4E+07 0.9835 0.9614 33 0.3672 Aneuploid

[3307] GLIA 937 CSF 1 5.6E+07 0.9822 0.9446 18 0.09104 Aneuploid

[3308] GLIB 1000 CSF 1 4.1E+07 0.8854 0.8643 0 0

[3309] GLIB 1001 CSF 1 4.1E+07 0.9485 0.926 0 0.0135

[3310] GLIB 1002 CSF 1 4E+07 0.9656 0.9447 0 0

[3311] GLIB 1003 CSF 1 3.1E+07 0.9628 0.9414 0 0.00898

[3312] GLIB 1004 CSF 1 3.6E+07 0.9701 0.9382 28 0.4783 Aneuploid

[3313] GLIB 1005 CSF 1 4.2E+07 0.9612 0.9408 0 0.00716

[3314] GLIB 1006 CSF 1 4.5E+07 0.4977 0.4874 0 0

[3315]

[3316] Attorney Docket No. 44807-0503WO1 / C18573

[3317] GLIB 1007 CSF 1 4.8E+07 0.7175 0.6935 28 0.1946 Aneuploid

[3318] GLIB 1008 CSF 1 3.5E+07 0.5385 0.5276 0 0

[3319] GLIB 1009 CSF 1 3.5E+07 0.8572 0.8377 0 0

[3320] GLIB 1010 CSF 1 6.2E+07 0.4013 0.3937 0 0

[3321] GLIB 1011 CSF 1 5.8E+07 0.5546 0.5439 0 0

[3322] GLIB 1012 CSF 1 4.4E+07 0.9753 0.9494 0 0

[3323] GLIB 1013 CSF 1 3.9E+07 0.9628 0.9435 28 0.0698 Aneuploid

[3324] GLIB 1014 CSF 1 2.9E+07 0.7658 0.7485 0 0

[3325] GLIB 1015 CSF 1 4.2E+07 0.6636 0.6505 0 0

[3326] GLIB 1016 CSF 1 4.3E+07 0.5172 0.5072 0 0

[3327] GLIB 1017 CSF 1 4.8E+07 0.6187 0.6059 0 0

[3328] GLIB 1018 CSF 1 4.5E+07 0.818 0.801 0 0

[3329] GLIB 1019 CSF 1 3.8E+07 0.887 0.8672 0 0.01324

[3330] GLIB 1020 CSF 1 5.1E+07 0.4594 0.4512 0 0.01193

[3331] GLIB 1021 CSF 1 4E+07 0.951 0.9325 0 0.00909

[3332] GLIB 1022 CSF 1 3.9E+07 0.3855 0.3782 0 0.00839

[3333] GLIB 1023 CSF 1 2.8E+07 0.9765 0.9564 0 0

[3334] GLIB 1024 CSF 1 3.7E+07 0.9722 0.9509 0 0

[3335] GLIB 1045 CSF 1 3.6E+07 0.9779 0.9582 0 0

[3336] MB 254 CSF 5.7E+07 0.9744 0.8713 12 0.9574 Ancuploid

[3337]

[3338] The reproducibility of the approach was assessed through the evaluation of independent aliquots of CSF DNA as technical replicates from the same patient (n = 104). Each aliquot had an independent SaferSeqS library. For the non-cancer controls (n=25) with a technical replicate, all arms were concordant while 98.5% of the arms in the cancer technical replicates (n=80) were concordant (Fig. 16A-16B). A subset of samples (n=30) were previously described and evaluated using the Repetitive Element Aneuploidy Sequencing System (RealSeqS). RealSeqS uses a single PCR primer to concomitantly amplify -350,000 loci spread through genome in order to evaluate aneuploidy. The chromosome arm level calls between the two assays were compared and 94.1% of the arms were found to be concordant. Of the discordant calls, a majority could be explained as falling just below the threshold for one of the two assays. Attorney Docket No. 44807-0503WO1 / C18573

[3339] The “M” component of CSF-BAM identifies subtle somatic mutations such as single base substitutions (SBS) or small insertions or deletions (indels). For this component, extremely high specificity is required to minimize errors during the experimental or bioinformatic components of this assay. The workflow was identical in principle to that described above for SafeBSeqS (Fig. 2B and 2D) but different primers were used. Instead of 4 primers for SafeBSeqS, 120 primers were used for mutation analysis (Fig. 2D). These primers (Table 1C) were chosen after extensive experiments in the analytical stage to maximize the uniformity of representation of the amplicons as well as minimize the number of off-target reads upon sequencing. The genomic regions for evaluation were selected based on a previously described algorithm that optimizes the number of cancers detectable with the fewest amplicons. The cancers of interest here included primary CNS tumors and common human cancers including those that frequently metastasize to the CNS. In sum, the panel consisted of 120 amplicons, encompassing regions in 40 genes. These span coding regions in oncogenes and tumor suppressor genes, splice sites in tumor suppressor genes, and promoter hotspots for TERT. The precise coordinates for each amplicon are listed in Table 1C. The uniformity of amplification of the 120 amplicons queried by the 120-plex is shown in Fig. 17A-17E.

[3340] It is well known that mutations in cell-free DNA (cfDNA) from peripheral blood largely arise from either tumors or CHIP (clonal hematopoiesis of indeterminate potential). To help ensure mutations identified in the CSF were not a result of CHIP, matched WBC DNA was analyzed in available cases.

[3341] For the training stage CSF from a different cohort of 300 individuals without cancer was evaluated (Table 6). These samples do not have matched peripheral blood to eliminate CHIP mutations and were not used for the evaluation of performance metrics. This cohort was used to tune our somatic mutation calling approach and determine thresholds for positivity (training set). Mutations identified include 20 in TP53, 3 in KRAS, 5 in NRAS, 8 in FBXW7 in codon 505, and 7 in other amplicons. Given the abundance of non-canonical mutations in KRAS and NRAS in the non-cancers, future mutation calls were restricted within these genes to only KRAS codon 12 and NRAS codon 61, which represent the most commonly mutated hotspots in cancer. Attorney Docket No. 44807-0503WO1 / C18573

[3342] The FBXW7 codon 505 mutations in the non-cancer samples were unexpected. This specific codon is not typically mutated in CHIP and other codons throughout FBXW7 are typically mutated in cancer. Upon closer inspection, every molecule with a mutant FBXW7 codon 505 also had mutations at codons 289, 299, 300, and 314. Blat analysis was performed for all possible non-human genomes. The sequence from the observed mutated FBXW7 molecules perfectly matched the bovine genome. Bovine-derived hemostatic agents frequently used in neurosurgery, when CSF was collected for the trigeminal neuralgia samples, may have contributed minor amounts of DNA that amplified and incorrectly aligned to FBXW7.

[3343] On the basis of these data, it was chosen to positively score mutations that were present in more than one original template molecule and found in cancer patients in the COSMIC database. Given the importance of TERT promoter mutations in CNS neoplasms, this metric was relaxed to score samples with even one mutant template molecule as positive.

[3344] Using these thresholds for the validation stage, somatic mutations were identified in 0 of 30 individuals without cancer (credible interval 89% to 100%) (Table 14). 79 of 209 CSF samples from patients with CNS cancers of various types had detectable mutations (38%, credible interval 31% to 45%). Of these, 44 of 83 samples from patients with high grade gliomas were positive (53%, credible interval 42% to 63%). Of the 5 high grade gliomas that were scored as positive based on the presence of only one mutant TERT molecule, one had the canonical gain on chr7 above the threshold for aneuploid positivity. Given the heterogenous nature of medulloblastoma driver mutations, only 4 of 22 (18%, credible interval 8% to 39%) samples scored positive for mutations. 18 of 24 metastatic cancer samples (75%, credible interval 56% to 88%) had mutations detected. Attorney Docket No. 44807-0503W01 / Cl 8573

[3345] Table 14. CSF-BAM mutation analysis in CSF samples

[3346] Sample name Mutation MAF, Avg (Min-Max), SCM > 1 Mutation 1 (CSF) Mutation 2 (CSF) Mutation 3 (CSF) found

[3347] (SCM>1)

[3348] OR TERT SCM=1

[3349] CGLI 02 CSF2 0 NA NA NA NA

[3350] CGLI 04 CSF4 0 NA NA NA NA

[3351] CGLI 06 C 0 NA NA NA NA

[3352] CGLI 07 CSF2 0 NA NA NA NA

[3353] CGLI 08 C 0 NA NA NA NA

[3354] CGLI 09 C 0 NA NA NA NA

[3355] CGLI 11 CSF2 0 NA NA NA NA

[3356] CGLI 12 CSF2 0 NA NA NA NA

[3357] CGLI 13 C 0 NA NA NA NA

[3358] CGLI 14 CSF2 1 22.10% (22.10%-22.10%) TP53 p. R213Q NA NA

[3359] CGLI 15 CSF2 0 NA NA NA NA

[3360] CGLI 157 CSF 0 NA NA NA NA

[3361] CGLI 158 CSF 0 NA NA NA NA

[3362] CGLI 159 CSF 1A 0 NA NA NA NA

[3363] CGLI 16 C 0 NA NA NA NA

[3364] CGLI 160 CSF 0 NA NA NA NA

[3365] CGLI 161 CSF 0 NA NA NA NA

[3366] CGLI 162 CSF 0 NA NA NA NA

[3367]

[3368]

[3369] Attorney Docket No. 44807-0503WO1 / C18573

[3370] CGLI 163 CSF 1 0.25% TERT g.1295250G> A NA NA

[3371] (promoter)

[3372] CGLI 164 CSF 2 16.12% (5.90%-26.35%) TP53 p. R175H TERT g,1295228G> A NA

[3373] (promoter)

[3374] CGLI 165 CSF 1 0.49% TERT g,1295228G> A NA NA

[3375] (promoter)

[3376] CGLI 166 CSF 0 NA NA NA NA

[3377] CGLI 167 CSF 1 0.05% (0.05%-0.05%) PIK3CA p. F83S NA NA

[3378] CGLI 168 CSF 4 0.61% (0.20%-1.27%) TERT g,1295250G> A TP53 p. Y163C TP53 p. VRA157-159A (promoter)

[3379] CGLI 169 CSF 0 NA NA NA NA

[3380] CGLI 170 CSF 2 14.60% (14.49%-14.71%) PIK3CA p. R88Q TP53 p. G245S NA

[3381] CGLI 171 CSF 0 NA NA NA NA

[3382] CGLI 172 CSF 0 NA NA NA NA

[3383] CGLI 173 CSF 0 NA NA NA NA

[3384] CGLI 174 CSF 0 0.18% TERT g,1295250G> A NA NA

[3385] (promoter)

[3386] CGLI 175 CSF 0 NA NA NA NA

[3387] CGLI 176 CSF 0 NA NA NA NA

[3388] CGLI 177 CSF 0 NA NA NA NA

[3389] CGLI 179 CSF 0 NA NA NA NA

[3390] CGLI 180 CSF 2 1.43% (0.58%-2.29%) TP53 p. R273C PIK3CA p. H1047R NA

[3391] CGLI 182 CSF 0 NA NA NA NA

[3392] CGLI 184 CSF 2 22.47% (0.23%-44.72%) H3F3A p. K28M TP53 p. C275F NA

[3393] CGLI 186 CSF 0 NA NA NA NA

[3394] CGLI 188 CSF 0 NA NA NA NA

[3395] CGLI 19 CSF2 0 NA NA NA NA

[3396]

[3397]

[3398] Attorney Docket No. 44807-0503WO1 / C18573

[3399] CGLI 190 CSF 0 NA NA NA NA CGLI 191 CSF 0 NA NA NA NA CGLI 192 CSF 2 11.17% (9.84%-12.50%) IDH1 p. R132G TP53 p. R273C NA CGLI 193 CSF 1A 0 NA NA NA NA CGLI 194 CSF 0 NA NA NA NA CGLI 20 CSF2 0 NA NA NA NA CGLI 22 C 0 NA NA NA NA CGLI 25 C 0 NA NA NA NA CGLI 26 C 0 NA NA NA NA CGLI 28 C 3 32.47% (31.98%-32.74%) TP53 p. H214R TP53 p. R273C IDH1 p. R132H CGLI 29 C 1 0.17% (0.17%-0.17%) TP53 p. R248W NA NA CGLI 30 C 0 NA NA NA NA CGLI 31 CSF2 2 9.95% (0.14%-19.77%) TERT g,1295228G> A TP53 p. R248Q NA (promoter)

[3400] CGLI 32 CSF 0 NA NA NA NA CGLI 34 CSF 0 NA NA NA NA CGLI 35 CSF 1 1.73% (1.73%-1.73%) TERT g,1295228G> A NA NA (promoter)

[3401] CGLI 36 CSF 1 40.00% (40.00%-40.00%) TP53 p. R248W NA NA CGLI 37 CSF 0 NA NA NA NA CGLI 38 CSF 0 NA NA NA NA CGLI 39 CSF 0 NA NA NA NA CGLI 40 CSF 0 NA NA NA NA CGLI 41 CSF 1 1.02% (1.02%-1.02%) TP53 p. K132N NA NA CGLI 42 CSF 0 NA NA NA NA CGLI 43 CSF 0 NA NA NA NA

[3402]

[3403]

[3404] Attorney Docket No. 44807-0503WO1 / C18573

[3405] CGLI 44 CSF 0 NA NA NA NA CGLI 45 CSF 0 NA NA NA NA CGLI 47 CSF 1 15.71% (15.71%-15.71%) TERT g,1295228G> A NA NA (promoter)

[3406] CGLI 48 CSF 0 0.11% TERT g,1295228G> A NA NA (promoter)

[3407] CGLI 49 CSF 0 NA NA NA NA CGLI 50 CSF2 1 0.70% (0.70%-0.70%) TERT g,1295228G> A NA NA (promoter)

[3408] CGLI 55 CSF 1 55.26% (55.26%-55.26%) PTEN p. R130* NA NA CGLI 56 CSF 0 NA NA NA NA CGLI 57 CSF 0 NA NA NA NA CGLI 58 CSF 0 NA NA NA NA CGLI 59 CSF 0 NA NA NA NA CGLI 60 CSF 0 NA NA NA NA CGLI 61 CSF 0 NA NA NA NA CGLI 62 CSF 0 NA NA NA NA CGLI 63 CSF 0 NA NA NA NA CGLI 83 CSF 1 8.14% (8.14%-8.14%) TERT g,1295228G> A NA NA (promoter)

[3409] CGLI 87 CSF 0 NA NA NA NA CGLI 88 CSF 0 NA NA NA NA CGLI 89 CSF 0 NA NA NA NA CGLI 90 CSF 0 NA NA NA NA CGLI 91 CSF 0 NA NA NA NA CGLI 97 CSF 2 21.79% (11.25%-32.33%) TP53 p. H179N H3F3A p. K28M NA

[3410]

[3411]

[3412] Attorney Docket No. 44807-0503WO1 / C18573

[3413] CGLIA 100 CSF 0 NA NA NA NA CGLIA 301 4 21.26% (0.22%-31.33%) ERBB2 p. S310F TP53 p. G199V PIK3CA p. M1043I CSF2A

[3414] GLI 101 CSF 0 NA NA NA NA

[3415] GLIA 419 CSF 1 0.08% (0.08%-0.08%) TP53 p. G245R NA NA

[3416] 1A

[3417] GLIA 420 CSF 2 0 NA NA NA NA

[3418] GLIA 422 CSF 0 NA NA NA NA

[3419] 1A

[3420] GLIA 423 CSF 1 36.99% (36.99%-36.99%) TP53 p. R248Q NA NA

[3421] 1A

[3422] GLIA 424 CSF 1 0.10% (0.10%-0.10%) FBXW7 p. R465C NA NA

[3423] 1A

[3424] GLIA 425 CSF 0 NA NA NA NA

[3425] 1A

[3426] GLIA 426 CSF 0 NA NA NA NA

[3427] 1A

[3428] GLIA 427 CSF 0 NA NA NA NA

[3429] 1A

[3430] GLIA 428 CSF 0 NA NA NA NA

[3431] 1A

[3432] GLIA 430 CSF 0 NA NA NA NA

[3433] 1A

[3434] GLIA 431 CSF 2 7.93% (0.01%-15.84%) TERT g,1295228G> A TP53 p. R248Q NA

[3435] 1A (promoter)

[3436] GLIA 433 CSF 0 NA NA NA NA

[3437] 1A

[3438] GLIA 435 CSF 0 NA NA NA NA

[3439]

[3440]

[3441] Attorney Docket No. 44807-0503WO1 / C18573

[3442] 1A

[3443] GLIA 436 CSF 0 NA NA NA NA

[3444] 1A

[3445] GLIA 452 CSF 1 0 NA NA NA NA GLIA 515 CSF 2 0 NA NA NA NA GLIA 522 CSF 2 19.43% (0.15%-38.71%) H3F3A p. K28M TP53 p. C275F NA

[3446] 1A

[3447] GLIA 525 CSF 1 0 NA NA NA NA GLIA 528 CSF 1 0 NA NA NA NA GLIA 533 CSF 0 NA NA NA NA

[3448] 1A

[3449] GLIA 534 CSF 2 0 NA NA NA NA GLIA 537 CSF 3 2 46.32% (41.17%-51.47%) KRAS p. G12C TP53 p. E287* NA GLIA 539 CSF 2 3.37% (2.68%-4.05%) MYD88 p. L273P TP53 p. R209X NA

[3450] 1A

[3451] GLIA 547 CSF 1 21.15% (21.15%-21.15%) TP53 p. R273H NA NA

[3452] 1A

[3453] GLIA 549 CSF 1 2.01% (2.01%-2.01%) CTNNB1 p. D32A NA NA

[3454] 1A

[3455] GLIA 550 CSF 2 2.08% (1.18%-2.98%) TERT g,1295250G> A PTEN p. R130G NA

[3456] 1A (promoter)

[3457] GLIA 552 CSF 2 3.57% (0.69%-6.46%) TP53 p. R175H H3F3A p. K28M NA

[3458] 1A

[3459] GLIA 554 CSF 4 7.43% (0.01%-17.58%) IDH1 p. R132H TERT g,1295228G> A TP53 p. R175H 1A (promoter)

[3460] GLIA 555 CSF 0 NA NA NA NA

[3461] 1A

[3462] GLIA 559 CSF 1 1 0.75% (0.75%-0.75%) TERT g,1295250G> A NA NA

[3463]

[3464]

[3465] Attorney Docket No. 44807-0503WO1 / C18573

[3466] (promoter)

[3467] GLIA 561 CSF 0 NA NA NA NA

[3468] 1A

[3469] GLIA 563 CSF 1 4.94% (4.94%-4.94%) TERT g,1295228G> A NA NA

[3470] 1A (promoter)

[3471] GLIA 565 CSF 1 1 0.23% (0.23%-0.23%) TP53 p. P190T NA NA

[3472] GLIA 569 CSF 1 1.42% (1.42%- 1.42%) TERT g,1295228G> A NA NA

[3473] 1A (promoter)

[3474] GLIA 571 CSF 1 6.26% (6.26%-6.26%) TERT g,1295228G> A NA NA

[3475] 1A (promoter)

[3476] GLIA 573 CSF 0 NA NA NA NA

[3477] 1A

[3478] GLIA 582 CSF 2 3 3.67% (0.50%-6.51%) TP53 p. R175H EGFRp. L858R PIK3CA p. E542K GLIA 590 CSF 1 0.07% (0.07%-0.07%) TP53 p. C135Y NA NA

[3479] 1A

[3480] GLIA 793 CSF 2 0 NA NA NA NA

[3481] GLIA 794 CSF 1 3 0.26% (0.23%-0.29%) TP53 g.7579599G> T VHL p. S183* TP53 p. T125T (splice (splice site) region variant) GLIA 795 CSF 1 0 NA NA NA NA

[3482] GLIA 796 CSF 2 0 NA NA NA NA

[3483] GLIA 797 CSF 1 3 4.59% (4.05%-5.56%) IDH1 p. R132H TP53 p. R273C TP53 p. V274D GLIA 798 CSF 1 0 NA NA NA NA

[3484] GLIA 800 CSF 1 0 NA NA NA NA

[3485] GLIA 802 CSF 1 0 NA NA NA NA

[3486] GLIA 804 CSF 1 1 0.63% (0.63%-0.63%) TP53 p. R248L NA NA

[3487] GLIA 806 CSF 2 0 NA NA NA NA

[3488] GLIA 807 CSF 1 0 NA NA NA NA

[3489]

[3490]

[3491] Attorney Docket No. 44807-0503WO1 / C18573

[3492] GLIA 808 CSF 1 0 NA NA NA NA

[3493] GLIA 810 CSF 1 0 NA NA NA NA

[3494] GLIA 819 CSF 1 1 0.32% TERT g,1295228G> A NA NA

[3495] (promoter)

[3496] GLIA 820 CSF 1 2 42.29% (39.10%-45.47%) TP53 p. C238F KRAS p. G12C NA

[3497] GLIA 821 CSF 1 0 NA NA NA NA

[3498] GLIA 822 CSF 1 0 NA NA NA NA

[3499] GLIA 823 CSF 1 2 3.57% (2.83%-4.31%) TP53 p. G154V PIK3CA p. Q546P NA

[3500] GLIA 824 CSF 1 0 NA NA NA NA

[3501] GLIA 825 CSF 1 1 44.08% (44.08%-44.08%) TP53 p. R337C NA NA

[3502] GLIA 826 CSF 1 0 NA NA NA NA

[3503] GLIA 827 CSF 2 0 NA NA NA NA

[3504] GLIA 828 CSF 1 0 NA NA NA NA

[3505] GLIA 829 CSF 1 0 NA NA NA NA

[3506] GLIA 830 CSF 1 1 0.13% (0.13%-0.13%) TP53 g.7579599G> T NA NA

[3507] (splice site)

[3508] GLIA 831 CSF 1 0 NA NA NA NA

[3509] GLIA 833 CSF 1 1 3.59% (3.59%-3.59%) TERT g,1295250G> A NA NA

[3510] (promoter)

[3511] GLIA 834 CSF 1 0 NA NA NA NA

[3512] GLIA 835 CSF 1 0 NA NA NA NA

[3513] GLIA 836 CSF 1 0 NA NA NA NA

[3514] GLIA 837 CSF 1 3 1.45% (0.34%-2.44%) IDH2 p. R172K TERT g,1295228G> A TERT g,1295250G> A (promoter) (promoter) GLIA 838 CSF 1 0 NA NA NA NA

[3515] GLIA 839 CSF 1 0 NA NA NA NA

[3516]

[3517]

[3518] Attorney Docket No. 44807-0503WO1 / C18573

[3519] GLIA 845 CSF 1 0 NA NA NA NA

[3520] GLIA 846 CSF 1 0 NA NA NA NA

[3521] GLIA 848 CSF 1 2 1.78% (1.62%-1.93%) TERT g,1295250G> A TP53 p. R248W NA

[3522] (promoter)

[3523] GLIA 850 CSF 1 0 NA NA NA NA

[3524] GLIA 853 CSF 1 0 NA NA NA NA

[3525] GLIA 854 CSF 1 0 NA NA NA NA

[3526] GLIA 855 CSF 1 4 0.29% (0.18%-0.50%) TP53 g.7579599G> T TP53 p. G245C TP53 p. S33S (splice (splice site) region variant) GLIA 856 CSF 1 6 32.89% (0.14%-77.10%) APC p. GA1357-1358X TP53 p. V216M KRAS p. G12A GLIA 859 CSF 1 1 0.45% (0.45%-0.45%) TP53 p. L130R NA NA

[3527] GLIA 882 CSF 1 0 NA NA NA NA

[3528] GLIA 883 CSF 1 3 33.40% (0.35%-69.90%) TP53 p. F134L IDH1 p. R132H PIK3CA p. E81K GLIA 884 CSF 1 0 NA NA NA NA

[3529] GLIA 886 CSF 1 1 0.25% (0.25%-0.25%) BRAF p. V600E NA NA

[3530] GLIA 888 CSF 1 0 NA NA NA NA

[3531] GLIA 891 CSF 1 0 NA NA NA NA

[3532] GLIA 893 CSF 1 2 4.39% (2.34%-6.44%) TERT g,1295228G> A PTEN p. K6X NA

[3533] (promoter)

[3534] GLIA 895 CSF 1 0 NA NA NA NA

[3535] GLIA 897 CSF 1 2 2.67% (2.48%-2.87%) IDH1 p. R132C TERT g,1295250G> A NA

[3536] (promoter)

[3537] GLIA 898 CSF 1 0 NA NA NA NA

[3538] GLIA 899 CSF 1 0 NA NA NA NA

[3539] GLIA 901 CSF 1 0 NA NA NA NA

[3540] GLIA 902 CSF 1 0 NA NA NA NA

[3541]

[3542]

[3543] Attorney Docket No. 44807-0503WO1 / C18573

[3544] GLIA 904 CSF 1 0 NA NA NA NA

[3545] GLIA 905 CSF 1 0 NA NA NA NA

[3546] GLIA 906 CSF 1 2 22.08% (5.50%-38.66%) TP53 p. R158G H3F3A p. K28M NA

[3547] GLIA 907 CSF 1 0 NA NA NA NA

[3548] GLIA 908 CSF 1 0 NA NA NA NA

[3549] GLIA 909 CSF1 0 NA NA NA NA

[3550] GLIA 910 CSF 1 0 NA NA NA NA

[3551] GLIA 912 CSF 1 0 NA NA NA NA

[3552] GLIA 913 CSF 1 3 0.55% (0.17%-0.98%) CDKN2A p. P81L TP53 p. Q317* TP53 p. P278L GLIA 914 CSF 1 1 21.21% (21.21%-21.21%) TP53 p. E271* NA NA

[3553] GLIA 915 CSF 2 0 NA NA NA NA

[3554] GLIA 916 CSF 1 0 NA NA NA NA

[3555] GLIA 917 CSF 3 1 2.56% TERT g,1295250G> A NA NA

[3556] (promoter)

[3557] GLIA 918 CSF 1 1 59.62% (59.62%-59.62%) TP53 g.7577018C> T NA NA

[3558] (splice site)

[3559] GLIA 919 CSF 1 2 11.62% (5.05%- 18.20%) TERT g,1295250G> A TP53 p. R158X NA

[3560] (promoter)

[3561] GLIA 921 CSF 1 1 56.87% (56.87%-56.87%) IDH1 p. R132H NA NA

[3562] GLIA 922 CSF 1 1 55.29% (55.29%-55.29%) TERT g,1295250G> A NA NA

[3563] (promoter)

[3564] GLIA 923 CSF 2 0 NA NA NA NA

[3565] GLIA 924 CSF 1 1 2.04% TERT g,1295250G> A NA NA

[3566] (promoter)

[3567] GLIA 925 CSF 1 3 4.89% (0.35%-4.91%) EGFRp. L858R TP53 p. R175H TERT g,1295250G> A (promoter) GLIA 926 CSF 1 0 NA NA NA NA

[3568]

[3569]

[3570] Attorney Docket No. 44807-0503WO1 / C18573

[3571] GLIA 927 CSF 1 1 49.33% (49.33%-49.33%) TP53 p. R248Q NA NA

[3572] GLIA 928 CSF 1 2 29.43% (28.32%-30.55%) NRAS p. Q61K TP53 p. T125R NA

[3573] GLIA 929 CSF 1 0 NA NA NA NA

[3574] GLIA 930 CSF 1 1 3.73% (3.73%-3.73%) TP53 p. R248Q NA NA

[3575] GLIA 932 CSF 1 1 24.74% (24.74%-24.74%) TP53 p. R337L NA NA

[3576] GLIA 934 CSF 1 2 3.86% (1.62%-6.10%) KRAS p. G12V APC p. E1379* NA

[3577] GLIA 935 CSF 1 0 NA NA NA NA

[3578] GLIA 936 CSF 1 3 37.72% (0.08%-68.50%) TP53 p. R175H H3F3A p. K28M PIK3CA p. R88Q GLIA 937 CSF 1 0 NA NA NA NA

[3579] GLIB 1000 CSF 1 0 NA NA NA NA

[3580] GLIB 1001 CSF 1 0 NA NA NA NA

[3581] GLIB 1002 CSF 1 0 NA NA NA NA

[3582] GLIB 1003 CSF 1 0 NA NA NA NA

[3583] GLIB 1004 CSF 1 2 29.86% (0.13%-59.58%) TP53 p. R273C PIK3CA p. H1047R NA

[3584] GLIB 1005 CSF 1 3 0.54% (0.41%-0.66%) TP53 p. R175H KRAS p. G12D TP53 p. V157L GLIB 1006 CSF 1 0 NA NA NA NA

[3585] GLIB 1007 CSF 1 0 NA NA NA NA

[3586] GLIB 1008 CSF 1 0 NA NA NA NA

[3587] GLIB 1009 CSF 1 0 NA NA NA NA

[3588] GLIB 1010 CSF 1 0 NA NA NA NA

[3589] GLIB 1011 CSF 1 0 NA NA NA NA

[3590] GLIB 1012 CSF 1 0 NA NA NA NA

[3591] GLIB 1013 CSF 1 0 NA NA NA NA

[3592] GLIB 1014 CSF 1 0 NA NA NA NA

[3593] GLIB 1015 CSF 1 0 NA NA NA NA

[3594] GLIB 1016 CSF 1 0 NA NA NA NA

[3595]

[3596] Attorney Docket No. 44807-0503WO1 / C18573

[3597] GLIB 1017 CSF 1 0 NA NA NA NA GLIB 1018 CSF 1 0 NA NA NA NA GLIB 1019 CSF 1 0 NA NA NA NA GLIB 1020 CSF 1 0 NA NA NA NA GLIB 1021 CSF 1 0 NA NA NA NA GLIB 1022 CSF 1 0 NA NA NA NA GLIB 1023 CSF 1 0 NA NA NA NA GLIB 1024 CSF 1 0 NA NA NA NA GLIB 1045 CSF 1 0 NA NA NA NA MB 254 CSF 0 NA NA NA NA

[3598]

[3599]

[3600] Attorney Docket No. 44807-0503WO1 / C18573

[3601] Application of CSF-BAM to CSF samples (validation stage)

[3602] 239 CSF samples from 222 patients were evaluated. For each sample, peripheral blood WBC DNA was available to exclude any mutations due to CHIP. Clinical information including demographics are described in Table 15. The sample categories presented here represent specific groupings where the number of samples for that category was sufficient to make aggregated conclusions. More detailed diagnostic information for each sample is presented in Table 15. The amount of CSF available for these studies averaged 3.5 mL and ranged from 0.5 to 14 mL. The amount of DNA recovered from CSF averaged 25 ng (IQR 2.6 to 29.6 ng) (Table 15). Table 15 also includes summaries of the sequencing data obtained from all patients, and whether they scored positively in the B, A, or M components of CSF-BAM. If a patient scored positively in at least one of these assays using the pre-defined thresholds described above, the patient was considered positive for CSF-BAM. Of note, 76% (93 / 122) of samples derived from individuals with grade 3 or 4 primary brain tumors were positive. Attorney Docket No. 44807-0503W01 / Cl 8573

[3603] Table 15. CSF-BAM results summary

[3604] Sample name Diagnostic group CSF-BAM SafeBSeqS Aneuploidy Estimated Mutation Mutation MAF, Avg (Min-Max) positive — clonality positive — tumor positive — found

[3605] (l=Positive, positive — (l=Positive, fraction by (l=Positive,

[3606] 0=Negative) (l=Positive. 0=Negative) aneuploidy 0=Negative)

[3607] 0=Negative)

[3608] CGLI 02 CSF2 High grade glioma 0 0 0 0.0% 0 0 NA

[3609] CGLI 04 CSF4 High grade glioma 1 0 1 6.9% 0 0 NA

[3610] CGLI 06 C Pilocytic astrocytoma 1 0 1 1.8% 0 0 NA

[3611] CGLI 07 CSF2 Low grade glioma 0 0 0 0.0% 0 0 NA

[3612] CGLI 08 C Pilocytic astrocytoma 0 0 0 0.0% 0 0 NA

[3613] CGLI 09 C Low grade glioma 1 0 1 1.8% 0 0 NA

[3614] CGLI 11 CSF2 Ependymoma 1 0 1 1.7% 0 0 NA

[3615] CGLI 12 CSF2 Ependymoma 0 0 0 0.0% 0 0 NA

[3616] CGLI 13 C Ependymoma 1 0 1 1.8% 0 0 NA

[3617] CGLI 14 CSF2 Low grade glioma 1 0 1 41.6% 1 1 22.10% (22.10%-22.10%) CGLI 15 CSF2 Ependymoma 0 0 0 0.0% 0 0 NA

[3618] CGLI 157 CSF Ependymoma 0 0 0 1.2% 0 0 NA

[3619] CGLI 158 CSF Low grade glioma 0 0 0 0.0% 0 0 NA

[3620] CGLI 159 CSF 1A High grade glioma 1 0 1 3.0% 0 0 NA

[3621] CGLI 16 C High grade glioma 1 0 1 2.4% 0 0 NA

[3622] CGLI 160 CSF Low grade glioma 0 0 0 0.0% 0 0 NA

[3623] CGLI 161 CSF Low grade glioma 0 0 0 1.2% 0 0 NA

[3624] CGLI 162 CSF Diffuse Midline Glioma 0 0 0 0.0% 0 0 NA

[3625] CGLI 163 CSF High grade glioma 1 0 0 0.9% 1 1 0.25% (0.25%-0.25%)

[3626]

[3627]

[3628] Attorney Docket No. 44807-0503WO1 / C18573

[3629] CGLI 164 CSF High grade glioma 1 0 1 19.3% 1 2 16.12% (5.90%-26.35%) CGLI 165 CSF High grade glioma 1 0 0 1.2% 1 1 0.49% (0.49%-0.49%) CGLI 166 CSF Diffuse Midline Glioma 0 0 0 0.0% 0 0 NA

[3630] CGLI 167 CSF Diffuse Midline Glioma 1 0 1 2.5% 1 1 0.05% (0.05%-0.05%) CGLI 168 CSF High grade glioma 1 0 1 4.0% 1 4 0.61% (0.20%-1.27%) CGLI 169 CSF High grade glioma 0 0 0 0.0% 0 0 NA

[3631] CGLI 170 CSF High grade glioma 1 0 1 44.5% 1 2 14.60% (14.49%-14.71%) CGLI 171 CSF High grade glioma 0 0 0 1.3% 0 0 NA

[3632] CGLI 172 CSF Not a cancer 0 0 0 1.4% 0 0 NA

[3633] CGLI 173 CSF Low grade glioma 0 0 0 0.0% 0 0 NA

[3634] CGLI 174 CSF High grade glioma 1 0 0 0.0% 1 1 0.18% (0.18%-0.18%) CGLI 175 CSF High grade glioma 0 0 0 0.0% 0 0 NA

[3635] CGLI 176 CSF High grade glioma 0 0 0 0.0% 0 0 NA

[3636] CGLI 177 CSF High grade glioma 1 0 1 3.8% 0 0 NA

[3637] CGLI 179 CSF High grade glioma 0 0 0 1.3% 0 0 NA

[3638] CGLI 180 CSF High grade glioma 1 0 0 1.1% 1 2 1.43% (0.58%-2.29%) CGLI 182 CSF High grade glioma 0 0 0 1.4% 0 0 NA

[3639] CGLI 184 CSF Diffuse Midline Glioma 1 0 1 26.9% 1 2 22.47% (0.23%-44.72%) CGLI 186 CSF High grade glioma 0 0 0 1.2% 0 0 NA

[3640] CGLI 188 CSF Pilocytic astrocytoma 0 0 0 1.2% 0 0 NA

[3641] CGLI 19 CSF2 Ependymoma 1 0 1 40.6% 0 0 NA

[3642] CGLI 190 CSF Not a cancer 0 0 0 0.0% 0 0 NA

[3643] CGLI 191 CSF Not a cancer 0 0 0 1.0% 0 0 NA

[3644] CGLI 192 CSF High grade glioma 1 0 1 10.6% 1 2 11.17% (9.84%-12.50%) CGLI 193 CSF 1A High grade glioma 1 0 1 1.9% 0 0 NA

[3645] CGLI 194 CSF High grade glioma 0 0 0 0.0% 0 0 NA

[3646]

[3647] Attorney Docket No. 44807-0503WO1 / C18573

[3648] CGLI 20 CSF2 Medulloblastoma 1 0 1 47.9% 0 0 NA

[3649] CGLI 22 C Pilocytic astrocytoma 1 0 1 4.2% 0 0 NA

[3650] CGLI 25 C Ependymoma 0 0 0 1.1% 0 0 NA

[3651] CGLI 26 C Low grade glioma 0 0 0 1.4% 0 0 NA

[3652] CGLI 28 C High grade glioma 1 0 1 7.1% 1 3 32.47% (31.98%-32.74%) CGLI 29 C High grade glioma 1 0 0 0.0% 1 1 0.17% (0.17%-0.17%) CGLI 30 C High grade glioma 1 0 1 13.5% 0 0 NA

[3653] CGLI 31 CSF2 High grade glioma 1 0 1 32.5% 1 2 9.95% (0.14%-19.77%) CGLI 32 CSF High grade glioma 0 0 0 1.0% 0 0 NA

[3654] CGLI 34 CSF High grade glioma 1 0 1 1.6% 0 0 NA

[3655] CGLI 35 CSF High grade glioma 1 0 1 9.6% 1 1 1.73% (1.73%-1.73%) CGLI 36 CSF High grade glioma 1 0 1 3.8% 1 1 40.00% (40.00%-40.00%) CGLI 37 CSF High grade glioma 1 0 1 2.3% 0 0 NA

[3656] CGLI 38 CSF High grade glioma 1 0 1 2.2% 0 0 NA

[3657] CGLI 39 CSF Low grade glioma 0 0 0 0.0% 0 0 NA

[3658] CGLI 40 CSF Medulloblastoma 1 0 1 8.1% 0 0 NA

[3659] CGLI 41 CSF High grade glioma 1 0 1 2.7% 1 1 1.02% (1.02%-1.02%) CGLI 42 CSF Ependymoma 1 0 1 1.9% 0 0 NA

[3660] CGLI 43 CSF Low grade glioma 0 0 0 0.0% 0 0 NA

[3661] CGLI 44 CSF Pilocytic astrocytoma 1 0 1 1.9% 0 0 NA

[3662] CGLI 45 CSF Low grade glioma 0 0 0 0.0% 0 0 NA

[3663] CGLI 47 CSF High grade glioma 1 0 1 38.3% 1 1 15.71% (15.71%-15.71%) CGLI 48 CSF High grade glioma 1 0 1 1.2% 1 1 0.11% (0.11%-0.11%) CGLI 49 CSF Ependymoma 0 0 0 0.0% 0 0 NA

[3664] CGLI 50 CSF2 High grade glioma 1 0 1 2.1% 1 1 0.70% (0.70%-0.70%) CGLI 55 CSF High grade glioma 1 0 1 11.4% 1 1 55.26% (55.26%-55.26%)

[3665]

[3666] Attorney Docket No. 44807-0503WO1 / C18573

[3667] CGLI 56 CSF Medulloblastoma 1 0 1 1.6% 0 0 NA

[3668] CGLI 57 CSF Pilocytic astrocytoma 0 0 0 0.0% 0 0 NA

[3669] CGLI 58 CSF Low grade glioma 1 0 1 1.8% 0 0 NA

[3670] CGLI 59 CSF Low grade glioma 0 0 0 1.2% 0 0 NA

[3671] CGLI 60 CSF Medulloblastoma 1 0 1 47.6% 0 0 NA

[3672] CGLI 61 CSF Pilocytic astrocytoma 0 0 0 0.0% 0 0 NA

[3673] CGLI 62 CSF Low grade glioma 0 0 0 0.0% 0 0 NA

[3674] CGLI 63 CSF Medulloblastoma 1 0 1 1.5% 0 0 NA

[3675] CGLI 83 CSF High grade glioma 1 0 1 21.9% 1 1 8.14% (8.14%-8.14%) CGLI 87 CSF Metastasis 1 0 1 1.5% 0 0 NA

[3676] CGLI 88 CSF Metastasis 1 0 1 2.0% 0 0 NA

[3677] CGLI 89 CSF Medulloblastoma 1 0 1 69.9% 0 0 NA

[3678] CGLI 90 CSF Medulloblastoma 1 0 1 15.0% 0 0 NA

[3679] CGLI 91 CSF Pilocytic astrocytoma 1 0 1 2.7% 0 0 NA

[3680] CGLI 97 CSF High grade glioma 1 0 1 15.3% 1 2 21.79% (11.25%-32.33%) CGLIA 100 CSF Diffuse Midline Glioma 1 0 1 2.1% 0 0 NA

[3681] CGLIA 301 Metastasis 1 0 1 46.9% 1 4 21.26% (0.22%-31.33%) CSF2A

[3682] GLI 101 CSF High grade glioma 1 0 1 2.2% 0 0 NA

[3683] GLIA 419 CSF Medulloblastoma 1 1 0 0.0% 1 1 0.08% (0.08%-0.08%) 1A

[3684] GLIA 420 CSF 2 Medulloblastoma 1 0 1 1.7% 0 0 NA

[3685] GLIA 422 CSF Medulloblastoma 1 0 1 29.0% 0 0 NA

[3686] 1A

[3687] GLIA 423 CSF Medulloblastoma 1 0 1 9.6% 1 1 36.99% (36.99%-36.99%)

[3688]

[3689] 1A

[3690]

[3691] Attorney Docket No. 44807-0503WO1 / C18573

[3692] GLIA 424 CSF Medulloblastoma 1 0 0 0.0% 1 1 0.10% (0.10%-0.10%) 1A

[3693] GLIA 425 CSF Medulloblastoma 0 0 0 0.0% 0 0 NA

[3694] 1A

[3695] GLIA 426 CSF Medulloblastoma 1 0 1 17.9% 0 0 NA

[3696] 1A

[3697] GLIA 427 CSF Medulloblastoma 1 0 1 24.6% 0 0 NA

[3698] 1A

[3699] GLIA 428 CSF Medulloblastoma 1 0 1 82.6% 0 0 NA

[3700] 1A

[3701] GLIA 430 CSF Medulloblastoma 1 0 1 7.8% 0 0 NA

[3702] 1A

[3703] GLIA 431 CSF High grade glioma 1 0 1 37.0% 1 2 7.93% (0.01%-15.84%) 1A

[3704] GLIA 433 CSF High grade glioma 0 0 0 0.0% 0 0 NA

[3705] 1A

[3706] GLIA 435 CSF Medulloblastoma 0 0 0 1.3% 0 0 NA

[3707] 1A

[3708] GLIA 436 CSF Not a cancer 0 0 0 0.0% 0 0 NA

[3709] 1A

[3710] GLIA 452 CSF 1 Medulloblastoma 1 0 1 4.1% 0 0 NA

[3711] GLIA 515 CSF 2 High grade glioma 0 0 0 0.0% 0 0 NA

[3712] GLIA 522 CSF Diffuse Midline Glioma 1 0 1 25.2% 1 2 19.43% (0.15%-38.71%) 1A

[3713] GLIA 525 CSF 1 High grade glioma 1 0 1 8.7% 0 0 NA

[3714] GLIA 528 CSF 1 CN S lymphoma 1 0 1 2.6% 0 0 NA

[3715] GLIA 533 CSF High grade glioma 1 0 1 2.1% 0 0 NA

[3716]

[3717] 1A

[3718]

[3719] Attorney Docket No. 44807-0503WO1 / C18573

[3720] GLIA 534 CSF 2 Diffuse Midline Glioma 0 0 0 1.3% 0 0 NA

[3721] GLIA 537 CSF 3 Metastasis 1 0 1 32.0% 1 2 46.32% (41.17%-51.47%) GLIA 539 CSF CNS lymphoma 1 0 1 7.1% 1 2 3.37% (2.68%-4.05%) 1A

[3722] GLIA 547 CSF High grade glioma 1 0 1 20.8% 1 1 21.15% (21.15%-21.15%) 1A

[3723] GLIA 549 CSF Medulloblastoma 1 0 1 10.4% 1 1 2.01% (2.01%-2.01%) 1A

[3724] GLIA 550 CSF High grade glioma 1 0 1 17.4% 1 2 2.08% (1.18%-2.98%) 1A

[3725] GLIA 552 CSF High grade glioma 1 0 1 10.8% 1 2 3.57% (0.69%-6.46%) 1A

[3726] GLIA 554 CSF High grade glioma 1 0 1 37.2% 1 4 7.43% (0.01%-17.58%) 1A

[3727] GLIA 555 CSF High grade glioma 1 0 1 1.8% 0 0 NA

[3728] 1A

[3729] GLIA 559 CSF 1 High grade glioma 1 0 1 3.7% 1 1 0.75% (0.75%-0.75%) GLIA 561 CSF High grade glioma 0 0 0 1.2% 0 0 NA

[3730] 1A

[3731] GLIA 563 CSF High grade glioma 1 0 1 8.5% 1 1 4.94% (4.94%-4.94%) 1A

[3732] GLIA 565 CSF 1 CNS lymphoma 1 1 1 3.4% 1 1 0.23% (0.23%-0.23%) GLIA 569 CSF High grade glioma 1 0 1 2.4% 1 1 1.42% (1.42%- 1.42%) 1A

[3733] GLIA 571 CSF High grade glioma 1 0 1 18.9% 1 1 6.26% (6.26%-6.26%) 1A

[3734] GLIA 573 CSF Low grade glioma 0 0 0 0.0% 0 0 NA

[3735]

[3736] 1A

[3737]

[3738] Attorney Docket No. 44807-0503WO1 / C18573

[3739] GLIA 582 CSF 2 Metastasis 1 0 1 5.6% 1 3 3.67% (0.50%-6.51%) GLIA 590 CSF Metastasis 1 0 0 1.1% 1 1 0.07% (0.07%-0.07%) 1A

[3740] GLIA 793 CSF 2 High grade glioma 0 0 0 1.2% 0 0 NA

[3741] GLIA 794 CSF 1 High grade glioma 1 0 0 0.0% 1 3 0.26% (0.23%-0.29%) GLIA 795 CSF 1 High grade glioma 0 0 0 1.2% 0 0 NA

[3742] GLIA 796 CSF 2 High grade glioma 1 0 1 16.0% 0 0 NA

[3743] GLIA 797 CSF 1 High grade glioma 1 0 1 6.0% 1 3 4.59% (4.05%-5.56%) GLIA 798 CSF 1 High grade glioma 0 0 0 0.0% 0 0 NA

[3744] GLIA 800 CSF 1 Ependymoma 1 0 1 2.2% 0 0 NA

[3745] GLIA 802 CSF 1 Pilocytic astrocytoma 0 0 0 1.3% 0 0 NA

[3746] GLIA 804 CSF 1 Metastasis 1 0 1 16.3% 1 1 0.63% (0.63%-0.63%) GLIA 806 CSF 2 Ependymoma 0 0 0 0.0% 0 0 NA

[3747] GLIA 807 CSF 1 Ependymoma 0 0 0 1.2% 0 0 NA

[3748] GLIA 808 CSF 1 Other CNS tumor type 0 0 0 0.0% 0 0 NA

[3749] GLIA 810 CSF 1 Not a cancer 0 0 0 0.0% 0 0 NA

[3750] GLIA 819 CSF 1 High grade glioma 1 0 0 0.0% 1 1 0.32% (0.32%-0.32%) GLIA 820 CSF 1 Metastasis 1 0 1 39.0% 1 2 42.29% (39.10%-45.47%) GLIA 821 CSF 1 Not a cancer 0 0 0 0.0% 0 0 NA

[3751] GLIA 822 CSF 1 Pilocytic astrocytoma 0 0 0 1.4% 0 0 NA

[3752] GLIA 823 CSF 1 Pilocytic astrocytoma 1 0 1 5.8% 1 2 3.57% (2.83%-4.31%) GLIA 824 CSF 1 Ependymoma 0 0 0 0.0% 0 0 NA

[3753] GLIA 825 CSF 1 High grade glioma 1 0 1 25.3% 1 1 44.08% (44.08%-44.08%) GLIA 826 CSF 1 Ependymoma 1 0 1 31.2% 0 0 NA

[3754] GLIA 827 CSF 2 High grade glioma 0 0 0 0.0% 0 0 NA

[3755] GLIA 828 CSF 1 Pilocytic astrocytoma 0 0 0 1.2% 0 0 NA

[3756]

[3757]

[3758] Attorney Docket No. 44807-0503WO1 / C18573

[3759] GLIA 829 CSF 1 Low grade glioma 0 0 0 0.0% 0 0 NA

[3760] GLIA 830 CSF 1 Ependymoma 1 0 0 0.0% 1 1 0.13% (0.13%-0.13%) GLIA 831 CSF 1 Ependymoma 0 0 0 1.0% 0 0 NA

[3761] GLIA 833 CSF 1 High grade glioma 1 0 1 6.4% 1 1 3.59% (3.59%-3.59%) GLIA 834 CSF 1 Ependymoma 0 0 0 0.0% 0 0 NA

[3762] GLIA 835 CSF 1 Ependymoma 0 0 0 0.0% 0 0 NA

[3763] GLIA 836 CSF 1 Ependymoma 0 0 0 0.0% 0 0 NA

[3764] GLIA 837 CSF 1 High grade glioma 1 0 1 4.0% 1 3 1.45% (0.34%-2.44%) GLIA 838 CSF 1 Ependymoma 0 0 0 0.0% 0 0 NA

[3765] GLIA 839 CSF 1 Diffuse Midline Glioma 1 0 1 2.6% 0 0 NA

[3766] GLIA 845 CSF 1 Pilocytic astrocytoma 0 0 0 0.0% 0 0 NA

[3767] GLIA 846 CSF 1 Ependymoma 0 0 0 0.0% 0 0 NA

[3768] GLIA 848 CSF 1 High grade glioma 1 0 1 2.7% 1 2 1.78% (1.62%-1.93%) GLIA 850 CSF 1 Ependymoma 1 0 1 34.4% 0 0 NA

[3769] GLIA 853 CSF 1 Not a cancer 0 0 0 1.0% 0 0 NA

[3770] GLIA 854 CSF 1 Ependymoma 0 0 0 0.0% 0 0 NA

[3771] GLIA 855 CSF 1 Other CNS tumor type 1 0 0 0.0% 1 4 0.29% (0.18%-0.50%) GLIA 856 CSF 1 Metastasis 1 0 1 12.9% 1 6 32.89% (0.14...

Claims

Attorney Docket No. 44807-0503W01 / C18573WHAT IS CLAIMED IS:

1. A method for determining a sequence of a nucleic acid encoding a portion of a B cell receptor (BCR) in a sample, the method comprising:(a) attaching a 3' adapter fragment to each 3' end of a double- stranded DNA molecule and a 5' adapter fragment to each 5' end of the double-stranded DNA molecule to generate an adapted double-stranded DNA molecule, wherein the adapted double-stranded DNA molecule comprises an adapted Watson strand and an adapted Crick strand, wherein the 3' adapter fragment comprises a molecular barcode, a primer sequence, and an adapter sequence, and wherein the molecular barcode of the adapted Watson strand is the reverse complement of the molecular barcode of the adapted Crick strand;(b) copying both strands of the adapted double-stranded DNA molecule, wherein the copying comprises performing a round of linear extension of the adapted double-stranded DNA molecule, generating an adapted double-stranded Watson template and an adapted double-stranded Crick template, thereby generating a duplex sequencing library;(c) generating a first population of analyte DNA fragments from the adapted doublestranded Watson template in the duplex sequencing library by amplifying the adapted double-stranded Watson template with a first set of a Watson-target selective primer pair comprising (i) a first Watson target-selective primer comprising a sequence complementary to the 3' adapter sequence, and (ii) a second Watson target-selective primer comprising a sequence complementary to the J segment of the BCR gene sequence, and generating a first set of sequencing reads for at least one member of the first population of analyte DNA fragments;(d) generating a second population of analyte DNA fragments from the adapted double-stranded Crick template in the duplex sequencing library by amplifying the adapted double-stranded Crick template with a first set of a Crick-target selective primer pair comprising (i) a first Crick target-selective primer comprising a sequence complementary to the 3' adapter sequence, and (ii) a second Crick target-selective primer comprising a sequence complementary to the J segment of a BCR gene sequence and generating a secondAttorney Docket No. 44807-0503W01 / C18573set of sequencing reads for at least one member of the second population of analyte DNA fragments;(e) grouping the first sequencing reads according to the molecular barcode present on the at least one member of the first population of analyte DNA fragments to generate a first analyte DNA family;(f) grouping the second sequencing reads according to the molecular barcode present on the at least one member of the second population of analyte DNA fragments to generate a second analyte DNA family;(g) analyzing the first sequencing reads of the first analyte DNA family; and(h) analyzing the second sequencing reads of the second analyte DNA family, thus, determining the sequence of the nucleic acid encoding the BCR.

2. The method of claim 1, wherein the 3' adaptor fragment comprises a partially doublestranded molecular barcode.

3. The method of claim 2, wherein the partially double-stranded molecular barcode comprises an endogenous barcode, an exogenous barcode, or both.

4. The method of any one of claims 1-3, wherein the copying step (b) further comprises performing the round of linear extension of the adapted double-stranded DNA molecule with (i) a first primer complementary to the 3' adapter sequence, and (ii) a second primer complementary to the complement of the 5' adapter sequence.

5. The method of any one of claims 1-4, wherein the generating steps (c) and (d) are performed under PCR conditions.

6. The method of any one of claim 1-5, wherein the second Watson target-selective primer comprises a sequence set forth in Table 1A.Attorney Docket No. 44807-0503WO1 / C185737. The method of any one of claims 1-5, wherein the second Crick target- selective primer comprises a sequence set forth in Table 1A.

8. The method of any one of claims 1-7, wherein the double-stranded DNA molecule comprises a V(D)J sequence of the BCR.

9. The method of any one of claims 1-8 further comprising:identifying the presence of aneuploidy within said sample; andidentifying the presence of a mutation within said sample.

10. A method for assessing a subject suspected of having a cancer, said method comprising:(1) assessing a sample obtained from said subject as set forth in claim 9;(2) identifying the presence of a cancer within the subject when: (a) the analyte DNA fragments comprising the nucleic acid encoding the portion of the BCR have a clonal fraction that is at least 0.3, (b) a presence of aneuploidy is identified, and / or (c) a present of at least one mutation shown in Table 7 is identified.

11. A method for assessing a subject suspected of having a cancer, said method comprising:(1) assessing a sample obtained from said subject as set forth in claim 9;(2) identifying the lack of a cancer within the subject when: (a) the analyte DNA fragments comprising the nucleic acid encoding the portion of the BCR is identified as having a clonal fraction that is less than 0.3, (b) an absence of aneuploidy is identified, and (c) an absence of the mutations shown in Table 7 is identified.

12. A method for treating a subject having a cancer, said method comprising:(1) assessing a sample obtained from said subject as set forth in claim 9;(2) administering a cancer treatment to said subject when: (a) the analyte DNA fragments comprising the nucleic acid encoding the portion of the BCR have a clonal fractionAttorney Docket No. 44807-0503W01 / C18573that is at least 0.3, (b) a presence of aneuploidy is identified, and / or (c) a presence of at least one mutation shown in Table 7 is identified.

13. A method for treating a cancer, said method comprising administering a cancer treatment to a subject having a sample assessed as set forth in claim 9, wherein: (a) the analyte DNA fragments comprising the nucleic acid encoding the portion of the BCR are identified as having a clonal fraction that is at least 0.3, (b) a presence of aneuploidy is identified, and / or (c) a presence of at least one of the mutations shown in Table 7 is identified.

14. The method of any one of claims 9-13, wherein the sample is selected from the group consisting of a blood sample, a cerebrospinal fluid (CSF) sample, a urine sample, and a tissue sample.

15. The method of any one of claims 9-14, wherein the subject is a human.

16. The method of any one of claims 10-15, wherein the cancer is a B-cell cancer.

17. The method of claim 16, wherein the B-cell cancer is a diffuse large B-cell lymphoma (DLBCL), a follicular lymphoma, a chronic lymphocytic leukemia (CLL), a small lymphocytic lymphoma (SLL), a mantle cell lymphoma (MCL), a Burkitt lymphoma, or a primary central nervous system lymphoma.

18. The method of any one of claims 10-15, wherein the cancer is a brain cancer.

19. The method of claim 18, wherein the brain cancer is a high-grade glioma, a medulloblastoma, a spinal ganglioglioma, a diffuse midline glioma, a CNS lymphoma, an ependymoma, or a metastatic lesion to the brain.Attorney Docket No. 44807-0503W01 / C1857320. The method of any one of claims 12-13, wherein the cancer treatment comprises administering chemotherapy, subjecting the subject to radiation therapy, and / or subjecting the subject to tumor resection surgery.

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