Omics Core

K190661 · Nanthealth, Inc. · PZM · Nov 9, 2019 · Pathology

Device Facts

Record IDK190661
Device NameOmics Core
ApplicantNanthealth, Inc.
Product CodePZM · Pathology
Decision DateNov 9, 2019
DecisionSESE
Submission TypeTraditional
Regulation21 CFR 866.6080
Device ClassClass 2
AttributesReal-World Evidence

Real-World Evidence

SubmissionDeviceSponsorRWD SourcesRWE Use SummaryKey Tags
K190661 · Nov 9, 2019Omics CoreNanthealth, Inc.In-house historical clinical testing data (>2,000 samples); Clinical tumor sample cohort (n=401)Historical clinical data was used to determine pre-analytical invalid rates across 42 tumor types. A clinical cohort was used for method comparison to establish assay accuracy.Historical data; Clinical cohort; Method comparison; Invalid rates

Clinical Evidence

Study DesignPopulationComparatorKey Endpoints
Historical Invalid Rate Analysis; Retrospective analysis of historical clinical data; Follow-up/Duration: Not applicable; Study Period: HistoricalPatients with solid malignant neoplasms across 42 tumor types; Sample Size: >2,000; Number of Sites: 1 (NantHealth, Inc.)Not applicable for this studyInvalid rates (insufficient tumor, insufficient DNA, library prep failure, sequencing failure)
Method Comparison Study; Retrospective method comparison; Follow-up/Duration: Not applicableFFPE tumor samples from clinical cases; Sample Size: 401; Number of Sites: 1 (NantHealth, Inc.)Orthogonal methodAccuracy (PPA, PPV) for SNVs, insertions, and deletions

Indications for Use

The Omics Core assay is a qualitative in vitro diagnostic test that uses targeted next generation sequencing of formalin-fixed paraffin-embedded tumor tissue matched with normal specimens with solid malignant neoplasms to detect tumor gene alterations in a broad multi gene panel. The test is intended to provide informations (point mutations (point mutations and deletions) and tumor mutational burden (TMB) for use by qualified health care professionals in accordance with professional guidelines, and is not conclusive or prescriptive for labeled use of any specific therapeutic product. Omics Core is a single-site assay performed at NantHealth, Inc.

Device Story

Omics Core is a targeted whole exome sequencing assay performed at a single site (NantHealth, Inc.). Input: FFPE tumor tissue and patient-matched normal whole blood. Process: DNA extraction; library preparation (shearing, end repair, A-base addition, adapter ligation, PCR amplification); solution-phase exon capture using biotinylated probes; sequencing on Illumina NovaSeq 6000. Bioinformatics pipeline: FASTQ generation; alignment to reference human genome; variant calling via comparison of tumor to matched normal DNA; TMB calculation (mutations/Mb). Output: Report detailing somatic mutations and TMB. Used by oncologists/pathologists to inform clinical management; provides genomic information for cancer patients; not intended to guide specific drug therapy directly.

Clinical Evidence

Bench testing only. Precision assessed using 12 FFPE clinical samples and 1 commercial cell line (98.4% positive call rate; TMB %CV <10%). Analytical sensitivity (LoD) established at 5% mutant allele frequency for SNVs/indels. Accuracy compared to orthogonal method across 401 FFPE samples (100% accuracy; PPA 99.76% for SNVs). TMB accuracy showed high correlation (R^2=0.9899) with orthogonal method.

Technological Characteristics

Targeted whole exome sequencing; hybrid capture; Illumina NovaSeq 6000 sequencer. Analyzes 468 genes for SNVs/indels and 19,396 protein-coding genes for TMB. Requires 50-300ng DNA input. Software-based bioinformatics pipeline for alignment and variant calling. Single-site laboratory assay.

Indications for Use

Indicated for patients with solid malignant neoplasms. Qualitative in vitro diagnostic test using targeted NGS of FFPE tumor tissue matched with normal specimens to detect somatic mutations and TMB.

Regulatory Classification

Identification

A next generation sequencing (NGS) based tumor profiling test is a qualitative in vitro diagnostic test intended for NGS analysis of tissue specimens from malignant solid neoplasms to detect somatic mutations in a broad panel of targeted genes to aid in the management of previously diagnosed cancer patients by qualified health care professionals.

Special Controls

*Classification.* Class II (special controls). The special controls for this device are:(1) Premarket notification submissions must include the following information: (i) A detailed description of all somatic mutations that are intended to be detected by the test and that are adequately supported in accordance with paragraph (b)(1)(v) of this section and reported in the test results in accordance with paragraph (b)(2)(iv) of this section, including: (A) A listing of mutations that are cancer mutations with evidence of clinical significance. (B) As appropriate, a listing of mutations that are cancer mutations with potential clinical significance. (ii) The indications for use must specify the following: (A) The test is indicated for previously diagnosed cancer patients. (B) The intended specimen type(s) and matrix ( *e.g.,* formalin-fixed, paraffin-embedded tumor tissue).(C) The mutation types ( *e.g.,* single nucleotide variant, insertion, deletion, copy number variation or gene rearrangement) for which validation data has been provided.(D) The name of the testing facility or facilities, as applicable. (iii) A detailed device description including the following: (A) A description of the test in terms of genomic coverage, as follows: ( *1* ) Tabulated summary of all mutations reported, grouped according to gene and target region within each gene, along with the specific cDNA and amino acid positions for each mutation.( *2* ) A description of any within-gene targeted regions that cannot be reported and the data behind such conclusion.(B) Specifications for specimen requirements including any specimen collection devices and preservatives, specimen volume, minimum tumor content, specimen handling, DNA extraction, and criteria for DNA quality and quantity metrics that are prerequisite to performing the assay. (C) A detailed description of all test components, reagents, instrumentation, and software required. Detailed documentation of the device software including but not limited to, software applications and hardware-based devices that incorporate software. (D) A detailed description of the methodology and protocols for each step of the test, including description of the quality metrics, thresholds, and filters at each step of the test that are implemented for final result reporting and a description of the metrics for run-failures, specimen-failures, invalids, as applicable. (E) A list of links provided by the device to the user or accessed by the device for internal or external information ( *e.g.,* decision rules or databases) supporting clinical significance of test results for the panel or its elements in accordance with paragraphs (b)(1)(v) and (b)(2)(vi) of this section.(F) A description of internal and external controls that are recommended or provided and control procedures. The description must identify those control elements that are incorporated into the testing procedure. (iv) Information demonstrating analytical validity of the device according to analytical performance characteristics, evaluated either specifically for each gene/mutation or, when clinically and practically justified, using a representative approach based on other mutations of the same type, including: (A) Data that adequately supports the intended specimen type ( *e.g.,* formalin-fixed, paraffin-embedded tumor tissue), specimen handling protocol, and nucleic acid purification for specific tumor types or for a pan-tumor claim.(B) A summary of the empirical evidence obtained to demonstrate how the analytical quality metrics and thresholds were optimized. (C) Device precision data using clinical samples to adequately evaluate intra-run, inter-run, and total variability. The samples must cover all mutation types tested (both positive and negative samples) and include samples near the limit of detection of the device. Precision must be assessed by agreement within replicates on the assay final result for each representative mutation, as applicable, and also supported by sequencing quality metrics for targeted regions across the panel. (D) Description of the protocols and/or data adequately demonstrating the interchangeability of reagent lots and multiplexing barcodes. (E) A description of the nucleic acid assay input concentration range and the evidence to adequately support the range. (F) A description of the data adequately supporting the limit of detection of the device. (G) A description of the data to adequately support device accuracy using clinical specimens representing the intended specimen type and range of tumor types, as applicable. ( *1* ) Clinical specimens tested to support device accuracy must adequately represent the list of cancer mutations with evidence of clinical significance to be detected by the device.( *2* ) For mutations that are designated as cancer mutations with evidence of clinical significance and that are based on evidence established in the intended specimen type (*e.g.,* tumor tissues) but for a different analyte type (*e.g.,* protein, RNA) and/or a measurement (*e.g.,* incorporating a score or copy number) and/or with an alternative technology (*e.g.,* IHC, RT-qPCR, FISH), evidence of accuracy must include clinically adequate concordance between results for the mutation and the medically established biomarker test (*e.g.,* evidence generated from an appropriately sized method comparison study using clinical specimens from the target population).( *3* ) For qualitative DNA mutations not described in paragraph (b)(1)(iv)(G)(*2* ) of this section, accuracy studies must include both mutation-positive and wild-type results.(H) Adequate device stability information. (v) Information that adequately supports the clinical significance of the panel must include: (A) Criteria established on what types and levels of evidence will clinically validate a mutation as a cancer mutation with evidence of clinical significance versus a cancer mutation with potential clinical significance. (B) For representative mutations of those designated as cancer mutations with evidence of clinical significance, a description of the clinical evidence associated with such mutations, such as clinical evidence presented in professional guidelines, as appropriate, with method comparison performance data as described in paragraph (b)(1)(iv)(G) of this section. (C) For all other mutations designated as cancer mutations with potential clinical significance, a description of the rationale for reporting. (2) The 21 CFR 809.10 compliant labeling and any product information and test report generated, must include the following, as applicable: (i) The intended use statement must specify the following: (A) The test is indicated for previously diagnosed cancer patients. (B) The intended specimen type(s) and matrix ( *e.g.,* formalin-fixed, paraffin-embedded tumor tissue).(C) The mutation types ( *e.g.,* single nucleotide variant, insertion, deletion, copy number variation or gene rearrangement) for which validation data has been provided.(D) The name of the testing facility or facilities, as applicable. (ii) A description of the device and summary of the results of the performance studies performed in accordance with paragraphs (b)(1)(iii), (b)(1)(iv), and (b)(1)(v) of this section. (iii) A description of applicable test limitations, including, for device specific mutations validated with method comparison data to a medically established test in the same intended specimen type, appropriate description of the level of evidence and/or the differences between next generation sequencing results and results from the medically established test ( *e.g.,* as described in professional guidelines).(iv) A listing of all somatic mutations that are intended to be detected by the device and that are reported in the test results under the following two categories or equivalent designations, as appropriate: “cancer mutations panel with evidence of clinical significance” or “cancer mutations panel with potential clinical significance.” (v) For mutations reported under the category of “cancer mutations panel with potential clinical significance,” a limiting statement that states “For the mutations listed in [cancer mutations panel with potential clinical significance or equivalent designation], the clinical significance has not been demonstrated [with adequate clinical evidence ( *e.g.,* by professional guidelines) in accordance with paragraph (b)(1)(v) of this section] or with this test.”(vi) For mutations under the category of “cancer mutations panel with evidence of clinical significance,” or equivalent designation, link(s) for physicians to access internal or external information concerning decision rules or conclusions about the level of evidence for clinical significance that is associated with the marker in accordance with paragraph (b)(1)(v) of this section.

Predicate Devices

Submission Summary (Full Text)

{0} # 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY A. 510(k) Number: K190661 B. Purpose for Submission: New Device C. Measurand: Somatic single nucleotide variants, insertions, deletions, and tumor mutational burden (TMB) in human genomic DNA obtained from formalin-fixed, paraffin-embedded tumor tissue. Refer to Appendix 1a for a complete list of the genes in the assay. D. Type of Test: Next generation sequencing tumor profiling test E. Applicant: NantHealth, Inc. F. Proprietary and Established Names: Trade Name: Omics Core Common Name: NantHealth Next Generating Sequencing Tumor Profiling Test G. Regulatory Information: 1. Regulation section: 21 CFR 866.6080 2. Classification: Class II 3. Product code: PZM 4. Panel: 88-Pathology 1 {1} ### H. Intended Use: #### 1. Indications for use: The Omics Core assay is a qualitative in vitro diagnostic test that uses targeted next generation sequencing of formalin-fixed paraffin-embedded tumor tissue matched with normal specimens from patients with solid malignant neoplasms to detect tumor gene alterations in a broad multi gene panel. The test is intended to provide information on somatic mutations (point mutations and small insertions and deletions) and tumor mutational burden (TMB) for use by qualified health care professionals in accordance with professional guidelines, and is not conclusive or prescriptive for labeled use of any specific therapeutic product. Omics Core is a single-site assay performed at NantHealth, Inc. #### 2. Special conditions for use statement(s): For in vitro diagnostic use only For prescription use only #### 3. Special instrument requirements: Illumina NovaSeq6000 (qualified by NantHealth) ### I. Device Description: A description of required equipment, software, reagents, vendors, and storage conditions were provided, and are described in the product labeling (NantHealth Omics Core manual). NantHealth assumes responsibility for the device. The device is a NGS-based tumor profiling assay, which sequences both tumor and matched normal specimens to detect somatic mutations. #### 1. Specimen Preparation The tumor volume and minimum tumor content needed to obtain sufficient DNA for testing to achieve the necessary quality performance are shown in the Table 1 below: Table 1: Specimen Handling and Processing FFPE Tissue | Tissue Type | Volume | Macrodissection requirements and Minimum Tumor Proportion; | Limitations | Storage | | --- | --- | --- | --- | --- | | Formalin-fixed, paraffin-embedded (FFPE) Tumor tissue blocks or slides. | 5-20 unstained section, 10 micron thick | Yes; more than 10% of tumor cells; sections containing >20% viable tumor are preferred. For TMB testing ≥ 20% tumor cells. | Decalcified tissues are unsuitable for analysis | Room temperature | Genomic DNA is extracted from tissue specimens using Qiagen's QIAsymphony DSP DNA MiniK it per manufacturer's protocol. Matched normal control samples are 2 {2} processed from blood (2 x 2.5ml in PAxGene Blood DNA collection tubes). DNA is quantified and concentrated if necessary. The amount of DNA required to perform the test is 50-300ng. Genomic DNA is stored at -20°C until assay initiation. DNA shearing is conducted per protocol and a quality control check is reviewed. Average fragment size for sheared DNA should be approximately ~200bp for tissue and approximately ~300bp for blood. Sheared DNA proceeds directly to library preparation. ## 2. Library Preparation Sequence libraries are prepared using KAPA Biosystems Library Preparation Reagents by first producing blunt-ended, 5'-phosphorylated fragments. To the 3' ends of the dsDNA library fragments, dAMP is added (A-tailing). Next, dsDNA adapters with 3'dTMP is ligated to the A-tailed library fragments. Library fragments with appropriate adapter sequences are amplified via ligation-mediated pre-capture PCR. A quality control review on the amplified DNA libraries is performed: Samples should be a smear with average fragment peak size for tissue as approximately ~200bp and average blood peak is approximately ~300bp with a fluorescence above 20. The concentration should be 10-300ng/μL to ensure adequate hybridization for capture. ## 3. Hybrid Capture Library capture is conducted using IDT Capture reagents. Sequencing libraries are hybridized to the vendor oligo pool. Capture beads are used to pull down the complex of capture oligos and genomic DNA fragments. Unbound fragments are washed away. The enriched fragment pool is amplified by ligation mediated-PCR (LM-PCR). The success of the enrichment is measured as a quality control step: Samples should be a smear with an average peak fragment size for tissue at approximately ~200bp and an average peak fragment size for blood at approximately ~300bp with a fluorescence above 20. The concentration of the amplified DNA library should be 1-45ng/μL; the LM-PCR yield should be ≥ 25ng. Reactions can be stored at 4°C until ready for purification, up to 72 hours. ## 4. Sequencing and Data Analysis Sequencing is conducted using the Illumina NovaSeq 6000 sequencing instruments and reagents along with PhiX Control v3. The sequencing process uses multiple quality checks. a. Data Management System (DMS): Automated sample tracking and archival of run-associated metadata (barcode, samples accession number, requisition number, source (class), and specimen type) is conducted with the following key functions: Tracking sample status through various stages of data analysis; tracking iterations of analysis applied to a given sample; recording versions of databases and algorithms used in analysis; archival of selected pipeline output files (FASTQ, BAM, VCF) and sequencing run statistics (e.g. yield, error rate, unassigned read indices). b. Demultiplexing and FASTQ Generation: The analysis pipeline uses software provided by Illumina. Multiple pairs of FASTQ files are generated per sample corresponding to full length forward and reverse reads. Demultiplexing quality 3 {3} control includes quality metrics for per-base sequence quality, sequence content, GC content and sequence length distribution, and relative percentages of unmatched indices. c. Indexing QC Check: The potential for index or sample contamination is managed during mutation calling and determined by looking for the presence of common polymorphisms in tumor samples that are different from the polymorphisms present in the matched-normal equivalent sample. d. Read Alignment and BAM Generation: Spurious adapter sequences are trimmed prior to read alignment. Reads are aligned in paired-end mode to the Genome Reference Consortium Human Build 37 (GRCh37, aka hg19) version of the human genome. Aligned reads are written to a Sequence Alignment Map (SAM) file, which is then converted into Binary Alignment Map (BAM) format. PCR duplicates are flagged and excluded. Each base within a read is assigned a base quality score by the sequencing software, which reflects the probability an error was made with the base call. To account for systemic biases that may not accurately reflect the actual error probabilities observed empirically, the analysis pipeline uses another tool to adjust the reported quality scores based on the selected covariates. Control for low quality bases during identification as part of variant calling results in low quality reads being excluded. e. Sample QC Checks: The baits used for hybridization capture include probes targeting >200,000 regions throughout the genome containing common single nucleotide polymorphisms (SNPs). The unique combination of SNPs specific to a given sample serves as a 'fingerprint' for the identity of the corresponding patient, and serves to identify potential sample mix-ups and contamination between samples and barcodes. QC checks involve the use of fingerprint' SNPs. i. Sample mix-up check: The analysis pipeline computes the % Similarity between the normal and tumor samples associated with the same patient, where % Similarity is defined as the percentage "compatible" genotypes shared by the two samples among a random set of up to 1,000 dbSNP loci with sufficient coverage (coverage depth ≥60 in both tumor and normal samples). If the % Similarity falls below 97%, the tumor and normal samples are flagged for review as potentially mismatched samples. ii. Sample contamination checks: The percentage of somatic variants that overlap common dbSNP loci (where "common" is defined as a polymorphism with a global population allele frequency > 5%) is calculated (% Common). If % Common exceeds 5% of the total somatic variant burden, the sample is flagged for review as potentially contaminated with unrelated DNA. iii. Check for presence of tumor in normal: Tumor contamination in matched-normal samples is assessed by calculating the number and percentage of somatic calls that meet the following criteria: 1. Two (2) or more reads supporting mutant allele in matched-normal, and 2. Mutant allele fraction > 5% in matched-normal sample. 4 {4} f. Mutation calling – SNVs and Indels: The analysis pipeline identifies two classes of mutations: (1) single nucleotide variants (SNVs) and (2) indels. Paired sample mutation calling is performed on tumor samples and their respective matched normal controls. Filtering is performed to remove low quality sequence data, sources of sequencing artifacts, and germline results. Summary of mutation filtering scheme is provided in Figure 1. Figure 1: Summary of the mutation filtering scheme for Omics Core ![img-0.jpeg](img-0.jpeg) 5 {5} i. Analysis of positive and negative controls: Data from controls is used to confirm lack of contamination as well as analytical sensitivity. ii. Filters on sample coverage: Sequencing coverage of ≥ 500X is required for tumor samples and sequencing coverage of ≥ 150X is required for normal samples. iii. Filtering for high confidence mutations: Raw SNV and indel calls are subjected to a series of filtering steps to ensure only high-confidence calls are reported. These parameters include (1) evidence of it being a somatic mutations (i.e., log odds of tumor data at site explained by presence of somatic mutation vs. sequencing error > 6.3 (>4.3 for indels) in tumor, log odds of normal data at site explained by sequencing error vs. presence of somatic mutation > 2.2, (2) reference set of common artifacts found in 3 or more normal samples from a panel of >4,000 normal blood samples, (3) technical characteristics that use coverage depth (DP), number of mutant reads (AD), mutation allele fraction (AF), strand bias (SB), characteristics of reads supporting mutant allele (mismatch quality sum, nearby indels, mapping quality), and characteristics of region where mutant is located (adjacent simple tandem repeat, overlapping dbSNP markers). Omics Core does not report mutations below 2%. g. Mutation Annotation Predicted functional effect and clinical interpretation for each mutation is curated by automated software using information from several databases. ### 5. Controls a. Matched Normal Control: Genomic DNA is extracted from patient-matched normal whole blood. NantHealth requires a normal sample to perform the test. b. Positive Control: The positive control sample (commercial vendor qualified by NantHealth) contains different confirmed mutations, representing a range of mutation allele frequencies. Results are compared against a negative control as an unmatched normal. Data generated from the positive control sample is analyzed using the pipeline, and frequencies of the detected mutations are reviewed to determine if (1) the known mutations are among those called, and (2) the observed frequencies for the known mutations match their expected values within 10%. The percentage agreement for scenarios (1) and (2) is expected to be 95% or higher. c. Negative Control: The negative control sample is a purchased reference sample, verified to be free of tumor contamination and germline copy number mutations in a majority of genes. Single Nucleotide Polymorphisms (SNPs) present in the negative control have been identified in prior analyses. The observed genotypes are compared against the expected genotypes for 420 common SNPs, and the degree of concordance is measured by identifying the percentage of SNPs that are identical or compatible. The percentage agreement between expected and observed mutations is expected to be 97% or higher. d. PCR Reagent Control – No Template Control (NTC): The NTC control should have a quantification measurement of < 1.0ng/μL. If this is not the case, the measurement may be high due to the presence of unincorporated primers carried over 6 {6} from the LM-PCR reaction and not an indication of possible contamination amplified sample libraries. If the quantification measurement is \( \geq \) 1ng/ \( \mu \) L and determined not to be due to dimers, the sample will be flagged and reviewed to determine if a re-run or sequencing run is necessary. ### 6. Result Reporting: - Oncopanel results are reported out under one of the two categories: “Cancer Mutations with Evidence of Clinical Significance” or “Cancer Mutations with Potential Clinical Significance”. The two categories are based on the supporting level of clinical evidence. Refer to the Clinical Performance Section for more information. - Results are reported for point mutations and small insertions and deletions in protein-coding exons of the 468 gene panel. Refer to Appendix 1a for a list of genes - The Omics Core does not report mutations in 203 exons due to low sequence coverage, poor mapping quality, or high GC content. Refer to Appendix 1b for a list of excluded exons. - Tumor Mutational Burden (TMB) Reporting: TMB is measured by counting the total number of somatic, non-synonymous exonic variants per the total number of genes (19,396) surveyed by the Omics Core assay. TMB is measured as mutations per megabase (mut/Mb), reported as a rate and included in the category for “Cancer Mutations with Potential Clinical Significance” Reporting takes in account the following quality metrics in the Table 2 below. Table 2. Flowcell and Sample Level Quality Control Metrics | QC Metrics | Acceptance Criteria | | --- | --- | | Flowcell Metrics | | | Sequencing Output S2 | >850 Gb | | Error Rate % | < 2 | | Base Quality Q30 % | >75 | | Sample Metrics | | | Average Target Coverage | ≥500X | | Coverage Uniformity | ≥95% target exons above 100X | | Mutation Calling Threshold | AF ≥ 0.02, Conf >15 | | Normal DNA: Homozygosity of X | Male >75% Female < 75% | | Tumor DNA: % Interchromosomal | < 16 | | Provenance: % Similarity | >90 | | Contamination: % Common | >5% total and >10 common site somatic mutations | | Criteria for calling test failure | If a sample presents with mean coverage across all exons < 50X and no mutations are detected due to the low overall coverage, the test is deemed “failed” for the sample. | 7 {7} # **J. Substantial Equivalence Information:** # **1. Predicate device name(s):** MSK-IMPACT (Integrated Mutation Profiling of Actionable Cancer Targets) # **2. Predicate 510(k) number(s):** DEN170058 # **3. Comparison with predicate:** **Table 3: Comparison of Omics Core with Predicate Device** | Characteristic | Predicate Device: MSK-IMPACT (DEN170058) | Subject Device: Omics Core | | --- | --- | --- | | **Similarities** | | | | **Indications For Use** | The MSK-IMPACT assay is a qualitative in vitro diagnostic test that uses targeted next generation sequencing of formalin-fixed paraffin-embedded tumor tissue matched with normal specimens from patients with solid malignant neoplasms to detect tumor gene alterations in a broad multi gene panel. The test is intended to provide information on somatic mutations (point mutations and small insertions and deletions) and microsatellite instability for use by qualified health care professionals in accordance with professional guidelines, and is not conclusive or prescriptive for labeled use of any specific therapeutic product. MSK-IMPACT is a single-site assay performed at Memorial Sloan Kettering Cancer Center. | The Omics Core assay is a qualitative in vitro diagnostic test that uses targeted next generation sequencing of formalin-fixed paraffin-embedded tumor tissue matched with normal specimens from patients with solid malignant neoplasms to detect tumor gene alterations in a broad multi gene panel. The test is intended to provide information on somatic mutations (point mutations and small insertions and deletions) and tumor mutational burden (TMB) for use by qualified health care professionals in accordance with professional guidelines, and is not conclusive or prescriptive for labeled use of any specific therapeutic product. Omics Core is a single-site assay performed at NantHealth, Inc. | | **Technology** | Hybrid Capture | Same | | **Specimen Types** | Formalin-fixed, paraffin-embedded (FFPE) tumor tissue matched with normal specimens from patients with solid malignant neoplasms | Same | | **Target Population** | Patients with solid malignant neoplasms | Same | 8 {8} | Characteristic | Predicate Device: MSK-IMPACT (DEN170058) | Subject Device: Omics Core | | --- | --- | --- | | **Similarities** | | | | **Genes on Panel** | 468 | Same | | **Test Environment** | Single-site assay (performed at Memorial Sloan Kettering Cancer Center) | Same. Single-site assay (performed at NantHealth, Inc.) | | **Controls** | - Matched normal - Positive control - Negative control - No template control (NTC) | Omics Core uses the same types of controls. | | **Result Report Format** | Oncopanel results are reported under one of these two categories: - “Cancer Mutations with Evidence of Clinical Significance” or - “Cancer Mutations with Potential Clinical Significance.” | Same | | Characteristic | Predicate (MSK IMPACT) | New Device (Omics Core) | | --- | --- | --- | | **Differences** | | | | **Black List** | 73 exons | 203 exons | | **Variant types** | Intended to provide information on somatic mutations (point mutations and small insertions and deletions), and microsatellite instability | Same except Omics Core provides information on tumor mutational burden (TMB) and not microsatellite instability (MSI). | | **Instrument** | Illumina HiSeq® 2500 Sequencing System | Illumina NovaSeq™ 6000 Sequencing System | | **Determination of Pipeline Thresholds** | - Based on >200X target coverage, - 100X for ≥ 98% target exons, - hotspot mutation calling threshold (mutation coverage (DP) ≥ 20, mutant reads (AD) ≥ 8, mutation frequency (VF) ≥ 2%, and non-hotspot mutation threshold (DP ≥ 20, AD ≥ 10, VF ≥ 5%) | Based on ≥ 500X target coverage, ≥ 100X for 95% of target exons, and a mutation calling threshold of allele frequency (AF) ≥ 2% with Conf > 15 and heuristic filters. The minimum read depth for variants in the Omics Core assay are allele depth (AD) ≥ 2 and overall depth (DP) ≥ 4. | | **Assay cut-off** | MSK-IMPACT does not report mutations below 2% for known hotspot mutations and 5% for non-hotspot mutations. | Omics Core does not report mutations below 2% for all mutations | 9 {9} | Clinical Evidence Curation | Classification criteria were developed by MSK using the in-house OncoKB database. OncoKB undergoes periodic updates through the review of new information by a panel of experts | Classification criteria were developed by NantHealth, Inc. NantHealth periodically updates Omics Core through the review of new information available. | | --- | --- | --- | # **K. Standard/Guidance Document Referenced (if applicable):** Not applicable # **L. Test Principle:** The NantHealth Omics Core assay is a custom targeted sequencing platform, utilizing solution-phase exon capture and sequencing, to detect somatic alterations (point mutations, small insertions and deletions), and overall tumor mutation burden in tumor specimens. The NantHealth Omics Core assay involves hybridization capture and deep sequencing of 468 cancer-associated genes for reporting of variants. TMB is reported on the basis of SNVs in the protein-coding exons of 19,396 genes including all known oncogenes and tumor suppressors. Omics Core reports on 468 genes from within the larger panel, reporting cancer mutations with evidence of clinical significance and cancer mutations with potential for clinical significance. The assay is not cleared for whole exome reporting of individual variants outside of the 468 genes. DNA probes are synthesized by a secondary manufacturer and are biotinylated to enable sequence enrichment through capture by streptavidin-conjugated beads. In total, the probes target approximately 39Mb of the human genome. Genomic DNA is extracted from both a tumor and a patient-matched normal control sample. Sequence libraries are prepared through a series of enzymatic steps including shearing of double-stranded DNA, end repair, A-base addition, ligation of barcoded sequence adaptors, and low cycle PCR amplification. Single barcoded sequence libraries are captured using the biotinylated probes. Captured DNA fragments are then pooled and sequenced on an Illumina NovaSeq 6000 as paired-end reads. Sequence reads are then aligned to the reference human genome. By comparing the identity of bases from the tumor DNA to the matched normal DNA and the reference human genome, somatic alterations are identified in the tumor. # **M. Performance:** # **1. Determination of pipeline thresholds** a. Requirements on exon coverage establishment: A power analysis to compute the coverage or total number of reads needed to detect a mutation with true underlying mutation frequency 2% or greater, for varying levels of power (0.8 to 0.99), assuming a fixed alpha (Type I error rate) of 0.05 was conducted. Additionally, the 95% confidence interval ranges of observed mutation frequency as a function of coverage was also calculated. When the mutation is present at 10%, the 95% confidence interval with a coverage of 500X is expected to fall between 7.5% and 13%. When the overall coverage is 100X, the 95% CI for a mutation at 10% is estimated to fall 10 {10} between 5.0% and 17.6%. To confirm these estimates, empirical data was obtained to measure the range of observed VF to expected VF using DNA from 10 normal FFPE samples from unrelated individuals which was mixed in equimass parts so as to create a range of SNPs with expected frequencies as low as 5%. A total of 672 common SNPs were considered for this experiment. A boxplot for one of the mixtures, mixture 6, showing the observed mutation frequencies for the 672 common SNPs genotyped in the pooled normal sample binned by their true underlying mutation frequency is shown in Figure 2. The results demonstrated that an observed VAF range of 4.2% to 20.3% for a SNP with true underlying mutation frequency of 10% when the mean coverage of the sample was 968X. This range in values is wider than the expected range of 7.5% to 13% and likely due to slight variations in the true mixing percentages. This data provided support for using a 5% as the lower limit for reporting mutations detected with true underlying frequency of 10%. The increased VAF range observed at the expected 10% frequency was also shown to be due to the different protocols and methods used for specimens sent to NantHealth e.g., fixation time, storage conditions, etc. These processing variations manifest as variable DNA damage and impact the sample DNA to library conversion rate (conversion efficiency), and thus result in differing sample representation within both a mixture of samples and the final sequencing data. The boxplot in Figure 2 shows a correlation of 0.983, with a slope of .997 and intercept of -0.0096. Consistent correlation is established as >0.9 as for the whole pool analyzed. Figure 2. Observed vs Expected Variant Frequency ![img-1.jpeg](img-1.jpeg) 11 {11} **b. Requirements on sample coverage:** Ten normal (diploid) FFPE samples were profiled in duplicate using the Omics Core assay (total = 20 replicates) to generate summary statistics across all targeted exons. The mean coverage across all targeted exons for the normal FFPE samples was 810X (SD = 106X). Summary statistics were also computed on coverage values per exon normalized by per-sample coverage. There were exons that presented with consistently low coverage values, due to sequence similarity with other loci and high GC content; however, none of those exons were among the clinical validation genes and were removed from the Omics Core assay. Of the remaining exons across all genes, 99.9% were sequenced to a depth of 100X or greater while 91.2% were sequenced to a depth of 250X or greater. This analysis of normal FFPE samples indicates that with a mean sample coverage of 810X, 98% of exons are sequenced with coverage greater than 170X, or with normalized coverage greater than 0.21. (The 'mean-normalized coverage' is the coverage of the mutation divided by the mean coverage across all exons; it serves as a measure of how deeply the validation exon was sequenced relative to the overall coverage of the sample. A mean-normalized coverage below 1 indicates the exon coverage is below average; conversely if greater than 1, it indicates above average coverage.) The data are shown in Figure 3a and 3b. **Figure 3a. Distribution of mean coverage values for targeted exons.** (Dashed line indicates coverage at 100X). ![img-2.jpeg](img-2.jpeg) 12 {12} Figure 3a. Distribution of mean coverage values for targeted exons normalized by per-sample coverage. (Dashed line indicates 20% of mean sample coverage). ![img-3.jpeg](img-3.jpeg) Based on the calculations, 98% of exons can be expected to be sequenced to coverage greater than 100X, when mean sample coverage is 476X (0.21 * 476X = 100X). (A 100X minimum coverage threshold per exon is required based on the power calculations, which showed 100X coverage was necessary to call mutations with true underlying mutation frequency 10% or greater, with 95% power at an alpha level of 0.05). To be conservative, a threshold of 500X on mean sample coverage is used to determine if a sample is sequenced to sufficient depth for subsequent analysis. A sample is flagged as being at increased risk of false negatives it its mean coverage is below 500X. To provide empirical data for these requirements, NantHealth utilizes the pooled normal FFPE sample with known expected single nucleotide mutations (n = 974) and the underlying mutation allele fractions (MAF). In silico downsampling analysis was conducted with a pooled normal mixture down to 46% where the sample coverage decreased from 968X to 450X. At this coverage level, 135 of the 139 (97%; 93-99% CI) mutations with expected underlying MAF of 10% were called. c. Requirements on mutation calling threshold: Permissive standard filters were used to intentionally generate false positives to identify suitable thresholds for parameters such as variant frequency (VF) and heuristic filters to optimize specificity. The following criteria allows optimal rejection of false positive SNVs and indel calls, while maintaining the ability to detect true positive events with underlying frequency of 10% (5-17.6% observable). Potential strand-bias and heuristic filters (ctl, laf, mq0, np, str, end, tri, idl, grm, phs) are also evaluated in the standard somatic mutation 13 {13} calling pipeline. An example of the number of false positive events detected pre and post filtering for the mixture sample using the standard somatic mutation calling pipeline is shown in Table 4. **Table 4: Sample Error Correction Pre and Post filtering on Metres** | | Mutations | | | --- | --- | --- | | | SNVs | Indels | | Pre-filter | 147 | 136 | | Post-filter | 1 | 0 | | Rejection Rate | 0.993 | 1.0 | ## 2. Pre-analytical Performance Table 5 below displays the invalid rates across 42 different tumor types (in FFPE) gathered from in-house historical data from testing >2000 samples. The data shows the separate invalid rates for the different steps involved in the assay including the percentage of specimens with insufficient tumor, the percentage with failed library prep and the percentage that failed the sequencing run per cancer type. The range of invalid rates was 0.0% to 33.33% which were principally due to insufficient tumor and related to the specimen source. Invalid results due to sequencing failures were below 7.2% except for pancreas which was twice that but still acceptable. The data shows that the DNA extraction is valid across tumor types and interference effects from different specimens are not significant across different tumor types supporting the performance of the pan-cancer specimen handling. **Table 5. Omics Core Invalid Rates from Historical Data** | Tumor Type | Number of Tests | Pre-Run Invalids | Pre-Run Invalids | Pre-Run Invalids | Post-Run Invalids | Percent Invalids | | --- | --- | --- | --- | --- | --- | --- | | | | Insufficient Tumor (Tumor % < 20%) | Insufficient DNA (DNA yield <50ng) | Library Prep QC Failure | Sequencing QC Failure | | | Adrenal | 8 | 0 | 2 | 0 | 0 | 25.00% | | Ampulla of Vater | 3 | 0 | 0 | 0 | 0 | 0.00% | | Anal | 12 | 2 | 0 | 0 | 1 | 25.00% | | Bile Duct (extrahepatic) | 11 | 1 | 0 | 0 | 0 | 9.09% | | Biliary Tract (intrahepatic) | 11 | 2 | 0 | 0 | 1 | 27.27% | | Bladder | 35 | 1 | 0 | 0 | 1 | 5.71% | 14 {14} | Tumor Type | Number of Tests | Pre-Run Invalids | Pre-Run Invalids | Pre-Run Invalids | Post-Run Invalids | Percent Invalids | | --- | --- | --- | --- | --- | --- | --- | | | | Insufficient Tumor (Tumor % < 20%) | Insufficient DNA (DNA yield <50ng) | Library Prep QC Failure | Sequencing QC Failure | | | Bone and Soft Tissue Cancers (including Sarcoma) | 120 | 4 | 4 | 0 | 5 | 10.83% | | Brain | 87 | 1 | 1 | 0 | 3 | 5.75% | | Breast | 362 | 33 | 10 | 1 | 26 | 19.34% | | Cervical | 27 | 0 | 0 | 0 | 1 | 3.70% | | Colon | 191 | 15 | 1 | 2 | 13 | 16.23% | | Esophageal | 41 | 4 | 1 | 0 | 2 | 17.07% | | Gall Bladder | 18 | 0 | 1 | 0 | 1 | 11.11% | | Gastric (Stomach) | 41 | 2 | 0 | 0 | 4 | 14.63% | | Gastrointestinal Stromal Tumor (GIST) | 3 | 1 | 0 | 0 | 0 | 33.33% | | Head and Neck | 76 | 4 | 1 | 0 | 5 | 13.16% | | Kidney | 21 | 1 | 1 | 0 | 3 | 23.81% | | Liver | 36 | 0 | 0 | 0 | 1 | 2.78% | | Lung | 234 | 58 | 5 | 1 | 14 | 33.33% | | Melanoma | 39 | 2 | 1 | 0 | 2 | 12.82% | | Mesothelioma | 4 | 0 | 0 | 0 | 0 | 0.00% | | Oral and Throat Cancers (Including Thyroid) | 31 | 0 | 1 | 1 | 3 | 16.13% | | Ovarian | 121 | 5 | 0 | 1 | 5 | 9.09% | | Pancreatic | 151 | 20 | 1 | 0 | 22 | 28.48% | | Penile | 2 | 0 | 0 | 0 | 0 | 0.00% | | Prostate | 96 | 11 | 4 | 0 | 4 | 19.79% | | Rectal | 56 | 7 | 0 | 2 | 3 | 21.43% | | Renal Pelvis and Ureter Cancers | 16 | 0 | 1 | 0 | 1 | 12.50% | | Skin (Non-Melanoma) | 16 | 2 | 0 | 0 | 1 | 18.75% | | Small Intestine | 12 | 0 | 0 | 0 | 0 | 0.00% | 15 {15} | Tumor Type | Number of Tests | Pre-Run Invalids | Pre-Run Invalids | Pre-Run Invalids | Post-Run Invalids | Percent Invalids | | --- | --- | --- | --- | --- | --- | --- | | | | Insufficient Tumor (Tumor % < 20%) | Insufficient DNA (DNA yield <50ng) | Library Prep QC Failure | Sequencing QC Failure | | | Thymic | 6 | 0 | 0 | 0 | 0 | 0.00% | | Thyroid | 2 | 0 | 0 | 0 | 0 | 0.00% | | Unknown Primary | 35 | 5 | 0 | 0 | 0 | 14.29% | | Uterine (Endometrial) | 77 | 2 | 0 | 0 | 4 | 7.79% | | Vulvar | 2 | 0 | 0 | 0 | 0 | 0.00% | | Urethral | 2 | 0 | 0 | 0 | 0 | 0.00% | | Eye | 1 | 0 | 0 | 0 | 0 | 0.00% | | Metastasis | 2 | 0 | 0 | 0 | 0 | 0.00% | | Testicular | 1 | 0 | 0 | 0 | 0 | 0.00% | | Vaginal | 3 | 0 | 0 | 0 | 0 | 0.00% | | Soft Tissue | 3 | 0 | 0 | 0 | 0 | 0.00% | | Totals | 2,189 | 204 | 38 | 9 | 141 | 17.91% | | % Invalid by Category | N/A | 9.32% | 1.74% | 0.41% | 6.44% | 17.91% | ### 3. Analytical Performance: The hybridization-capture-based targeted sequencing assay is designed to detect point mutations [also referred to as single nucleotide variants (SNVs)] as well as small insertions/deletions (indels) < 21 bp in length in the coding exons of 468 genes (See Appendix 1 and 2). A paired-sample analysis pipeline (tumor vs. matched normal blood) is used to identify somatic mutations in the targeted exons. A representative approach to validation of the SNVs and indels targeted in this panel was submitted at the variant level, and assesside quality metrics (data not shown). #### a. Precision Studies Reproducibility and repeatability were assessed in precision studies for Omics Core. Extracted DNA was run once per day for 3 non-consecutive days using different barcodes for inter-day assessment (n = 3). For one run, a sample was run in triplicate for intra-day assessment, resulting in a total of 3+1+1=5 replicates. For each replicate tested, all observed mutations in the selected genes were reported and assessed for precision. Details of the studies are described below. 16 {16} i. *Precision Panel:* The precision of Omics Core was assessed using 12 FFPE clinical tumor samples and 1 commercial cell line (n = 13). The panel represented different tumor types, different mutation types, and a range of mutant allele frequencies. The specimen panel was selected based on known mutations corresponding to “Cancer Mutations with Evidence of Clinical Significance.” Table 6 shows the list of specimens used in this study. A total of 530 variants were identified in the clinical and cell line samples that included 483 SNVs, 32 deletions and 15 insertions. Precision of each variant is reported across all samples to provide an assessment of the error rate. **Table 6. Summary of the Specimens and Allele Frequencies in the Precision Studies** | Tumor Types | Mutation Type | Mutation | Gene / Exon | cDNA Change | Amino Acid Change | Mutation Allele Frequency | | --- | --- | --- | --- | --- | --- | --- | | Non-Small Cell Lung Cancer | DEL | In-Frame Deletion | EGFR Exon 19 | c.2235_2249delGGAA | p.E746_A750del | ~8 | | Colon Cancer | SNV | Missense | BRAF Exon 15 | c.1799T>A | p.V600E | ~30 | | Pancreatic Cancer | SNV | Missense | KRAS Exon 2 | c.35G>A | p.G12D | ~19 | | Mullerian Ovarian Cancer | SNV | Missense | PIK3CA Exon 10 | c.1624G>A | p.E542K | ~80^{1} | | Endometrial Cancer | SNV | Missense | CTCF Exon 3 | c.263C>A | p.A88D | ~36 | | Adenoid Cystic Carcinoma | INS | In-Frame Insertion | BCOR Exon 4 | c.2936_2937insATTCAAATG | p.K978_F977ins* | ~31 | | Malignant Gastric Gist | INS | In-Frame Insertion | KIT Exon 9 | c.1504_1509dupGCCTAT | p.Y503_A502ins | ~36 | | Colon Cancer | INS | Frame Shift Ins. | PTEN Exon 5 | c.440dupA | p.A148Gfs*32 | ~57 | | Colon Cancer | DEL | Frame Shift Del. | PTEN Exon 5 | c.262_264delinsAG | p.Y88Sfs*11 | ~62 | | Metastatic Breast Cancer | DEL | In-Frame Deletion | PIK3CA Exon 2 | c.312_317delAGTAGG | p.V105_G106del | ~62 | | Glioblastoma | SNV | Missense | PIK3C2G Exon 20 | c.2090T>C | p.L697P | ~35 | | Melanoma | SNV | Missense | BRAF Exon 15 | c.1798_1799delinsAA | p.V600K | ~29 | $^{1}$ Due to copy number gain of the gene, the MAF was the same as the tumor purity. 17 {17} | Tumor Types | Mutation Type | Mutation | Gene / Exon | cDNA Change | Amino Acid Change | Mutation Allele Frequency | | --- | --- | --- | --- | --- | --- | --- | | Commercial FFPE cell line | DEL, SNV | Hotspot mutations in BRAF, KIT, EGFR, KRAS, NRAS, PIK3CA | | | | ~3 - 25 | ii. Precision – Panel-Wide Reproducibility: The precision was reported for all detected mutants in all replicates for each sample. The results demonstrate that the overall positive call rate for all variants analyzed across the 12 FFPE clinical samples and one commercial cell line was 2607/2650, or 98.4% (97.8-98.8% CI). The coefficient of variation (%CV) for the mutation allele frequency was calculated for all 5 replicates. Three-hundred and ninety-eight (398) of 521 (76%) mutations in the clinical specimens had %CV ≤ 10%; 91 of 521 (17%) were between 10 and 20%; and 32 of 521 (6%) were > 20%. Discordance was observed for variants in the following genes as summarized in Table 7 below. Each specimen is separated by a dark gray line. Known mutation within each specimen are in bold. Discordant cases are denoted in light grey. All runs passed the quality metrics criteria. Table7 : Panel-wide precision summary for all 5 replicates Abbreviations: NC (normalized coverage); MAF (Mutant allele frequency) | Gene/ Exon | Coding Change | Protein Change | NC Range | MAF Range | MAF Mean | MAF Median | MAF (SD) | MAF (%CV) | Positive/ Total Calls | Positive Call Rate (two-sided 95% CI) | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | EGFR Exon 19 | c.2235_2249delGGAATTAAGAGAAGC | p.E746_A 750del | 0.67-0.82 | 0.03-0.06 | 0.05 | 0.05 | 0.01 | 23.25 % | 5 of 5 | 100.0% (47.8%, 100.0%) | | TP53 Exon 8 | c.818G>A | p.R273H | 0.98-1.07 | 0.07-0.11 | 0.09 | 0.09 | 0.01 | 15.23 % | 5 of 5 | 100.0% (47.8%, 100.0%) | | ARID1B Exon 1^{a} | c.1379C>G | p.A460G | 2.43-2.92 | 0.01-0.03 | 0.02 | 0.01 | 0.01 | 39.35 % | 4 of 5 | 80.0% (28.4%, 99.5%) | | PIK3CA Exon 10 | c.1633G>A | p.E545K | 0.15-0.23 | 0.05-0.14 | 0.08 | 0.08 | 0.03 | 36.52 % | 5 of 5 | 100.0% (47.8%, 100.0%) | | STK11 Exon 1 | c.179dupA | p.Y60* | 1.45-1.56 | 0.08-0.11 | 0.10 | 0.10 | 0.01 | 13.40 % | 5 of 5 | 100.0% (47.8%, 100.0%) | | | | | | | | | | | | | | PHOX2B Exon 3 | c.756_776del GGCGGCAG CGGCAGCG GCGGC | p.A254_A 260del | 1.00-1.30 | 0.03-0.08 | 0.06 | 0.07 | 0.02 | 31.70 % | 5 of 5 | 100.0% (47.8%, 100.0%) | 18 {18} | Gene/ Exon | Coding Change | Protein Change | NC Range | MAF Range | MAF Mean | MAF Median | MAF (SD) | MAF (%CV) | Positive/ Total Calls | Positive Call Rate (two-sided 95% CI) | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | ARID5B Exon 10 | c.3131A>T | p.E1044V | 1.60-2.44 | 0.03-0.04 | 0.04 | 0.04 | 0.00 | 12.45% | 5 of 5 | 100.0% (47.8%, 100.0%) | | PARP1 Exon 4 | c.433G>C | p.D145H | 0.96-1.15 | 0.10-0.13 | 0.12 | 0.12 | 0.01 | 8.82% | 5 of 5 | 100.0% (47.8%, 100.0%) | | PTEN Exon 5 | c.440dupA | p.A148Gf s*32 | 0.17-0.52 | 0.51-0.59 | 0.55 | 0.55 | 0.03 | 4.64% | 5 of 5 | 100.0% (47.8%, 100.0%) | | NRAS Exon 2 | c.35G>A | p.G12D | 0.23-0.31 | 0.49-0.57 | 0.54 | 0.55 | 0.03 | 5.64% | 5 of 5 | 100.0% (47.8%, 100.0%) | | BMPR1A Exon 6 | c.423dupT | p.V142Cf s*7 | 0.16-0.43 | 0.39-0.63 | 0.53 | 0.55 | 0.08 | 14.46% | 5 of 5 | 100.0% (47.8%, 100.0%) | | MAP2K1 Exon 3 | c.383G>A | p.G128D | 0.69-0.82 | 0.11-0.15 | 0.13 | 0.12 | 0.02 | 14.02% | 5 of 5 | 100.0% (47.8%, 100.0%) | | EPHA5 Exon 14 | c.2402T>G | p.L801R | 0.29-0.36 | 0.27-0.33 | 0.30 | 0.31 | 0.02 | 7.17% | 5 of 5 | 100.0% (47.8%, 100.0%) | | ANKRD1 1 Exon 10 | c.3640A>C | p.T1214P | 1.07-1.68 | 0.12-0.17 | 0.15 | 0.15 | 0.02 | 11.84% | 5 of 5 | 100.0% (47.8%, 100.0%) | | | | | | | | | | | | | | PIK3CA Exon 2 | c.312_317del AGTAGG | p.V105_G106del | 0.64-0.76 | 0.51-0.54 | 0.52 | 0.52 | 0.01 | 2.08% | 5 of 5 | 100.0% (47.8%, 100.0%) | | DCUN1D 1 Exon 7 | c.748C>T | p.Q250* | 0.75-0.90 | 0.17-0.20 | 0.18 | 0.18 | 0.01 | 6.19% | 5 of 5 | 100.0% (47.8%, 100.0%) | | GATA3 Exon 6 | c.1087C>T | p.Q363* | 0.96-1.12 | 0.39-0.43 | 0.40 | 0.40 | 0.01 | 3.26% | 5 of 5 | 100.0% (47.8%, 100.0%) | | PRKD1 Exon 16a | c.2429A>C | p.H810P | 0.58-0.58 | 0.08-0.08 | 0.08 | 0.08 | 0.00 | 0.00% | 1 of 5 | 20.0% (0.5%, 71.6%) | | NCOR1 Exon 37 | c.5765G>C | p.R1922T | 1.13-1.27 | 0.34-0.40 | 0.38 | 0.39 | 0.02 | 5.62% | 5 of 5 | 100.0% (47.8%, 100.0%) | | TP53 Exon 6 | c.584T>C | p.I195T | 0.53-0.59 | 0.33-0.42 | 0.38 | 0.39 | 0.03 | 8.22% | 5 of 5 | 100.0% (47.8%, 100.0%) | | KEAP1 Exon 2 | c.233A>G | p.D78G | 1.31-1.65 | 0.21-0.23 | 0.22 | 0.22 | 0.01 | 4.03% | 5 of 5 | 100.0% (47.8%, 100.0%) | 19 {19} | Gene/ Exon | Coding Change | Protein Change | NC Range | MAF Range | MAF Mean | MAF Median | MAF (SD) | MAF (%CV) | Positive/ Total Calls | Positive Call Rate (two-sided 95% CI) | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | RB1 Exon 19 | c.1901C>A | p.S634* | 0.08-0.75 | 0.29-0.41 | 0.35 | 0.34 | 0.04 | 10.99% | 5 of 5 | 100.0% (47.8%, 100.0%) | | NSD1 Exon 5 | c.3768G>C | p.L1256F | 0.92-1.05 | 0.25-0.29 | 0.27 | 0.28 | 0.01 | 5.12% | 5 of 5 | 100.0% (47.8%, 100.0%) | | BAP1 Exon 5 | c.295G>A | p.V99M | 0.47-0.98 | 0.37-0.43 | 0.40 | 0.39 | 0.02 | 4.97% | 5 of 5 | 100.0% (47.8%, 100.0%) | | PIK3CG Exon 4 | c.2182A>T | p.M728L | 0.69-0.76 | 0.17-0.21 | 0.19 | 0.19 | 0.01 | 7.55% | 5 of 5 | 100.0% (47.8%, 100.0%) | | TBX3 Exon 7 | c.1339C>G | p.R447G | 1.74-2.14 | 0.26-0.29 | 0.27 | 0.27 | 0.01 | 3.79% | 5 of 5 | 100.0% (47.8%, 100.0%) | | PPM1D Exon 1 | c.254C>G | p.S85W | 1.63-2.80 | 0.21-0.24 | 0.22 | 0.22 | 0.01 | 3.27% | 5 of 5 | 100.0% (47.8%, 100.0%) | | | | | | | | | | | | | | KDM5A Exon 7 | c.820G>A | p.A274T | 0.26-0.33 | 0.27-0.37 | 0.31 | 0.30 | 0.04 | 11.54% | 5 of 5 | 100.0% (47.8%, 100.0%) | | ARID1A Exon 20 | c.5548delG | p.D1850T fs*33 | 2.04-2.33 | 0.28-0.30 | 0.30 | 0.30 | 0.01 | 2.87% | 5 of 5 | 100.0% (47.8%, 100.0%) | | PALB2 Exon 12 | c.3260G>T | p.S1087I | 0.23-0.58 | 0.13-0.26 | 0.20 | 0.20 | 0.04 | 21.07% | 5 of 5 | 100.0% (47.8%, 100.0%) | | BCL6 Exon 3 | c.76C>T | p.R26W | 0.79-1.65 | 0.27-0.31 | 0.29 | 0.29 | 0.01 | 4.60% | 5 of 5 | 100.0% (47.8%, 100.0%) | | MGA Exon 17 | c.5907_5911d elinsAAGAT | p.E1971* | 0.63-0.73 | 0.32-0.99 | 0.73 | 0.99 | 0.32 | 43.07% | 5 of 5 | 100.0% (47.8%, 100.0%) | | ARID1A Exon 1 | c.780_782del CTC | p.S265del | 2.06-2.65 | 0.30-0.35 | 0.32 | 0.31 | 0.02 | 5.09% | 5 of 5 | 100.0% (47.8%, 100.0%) | | TP53 Exon 8 | c.817C>T | p.R273C | 1.38-1.53 | 0.26-0.29 | 0.27 | 0.27 | 0.01 | 4.05% | 5 of 5 | 100.0% (47.8%, 100.0%) | | RNF43 Exon 9 | c.1976delG | p.G659Vf s*41 | 1.52-1.81 | 0.62-0.63 | 0.63 | 0.62 | 0.01 | 0.98% | 5 of 5 | 100.0% (47.8%, 100.0%) | | KMT2B Exon 37 | c.7964G>A | p.C2655Y | 1.42-1.67 | 0.28-0.32 | 0.30 | 0.30 | 0.01 | 3.63% | 5 of 5 | 100.0% (47.8%, 100.0%) | 20 {20} | Gene/ Exon | Coding Change | Protein Change | NC Range | MAF Range | MAF Mean | MAF Median | MAF (SD) | MAF (%CV) | Positive/ Total Calls | Positive Call Rate (two-sided 95% CI) | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | ZFHX3 Exon 9 | c.4805A>T | p.N1602I | 1.75-1.97 | 0.34-0.38 | 0.37 | 0.38 | 0.02 | 4.51% | 5 of 5 | 100.0% (47.8%, 100.0%) | | MAP3K1 Exon 14 | c.3651_3653delCAT | p.I1219del | 0.58-0.66 | 0.30-0.37 | 0.34 | 0.35 | 0.02 | 6.90% | 5 of 5 | 100.0% (47.8%, 100.0%) | | MAP3K14 Exon 12 | c.2101G>T | p.G701C | 1.55-2.00 | 0.30-0.32 | 0.32 | 0.32 | 0.01 | 2.24% | 5 of 5 | 100.0% (47.8%, 100.0%) | | SOX17 Exon 1 | c.167C>A | p.A56D | 1.61-2.05 | 0.33-0.36 | 0.34 | 0.34 | 0.02 | 4.50% | 5 of 5 | 100.0% (47.8%, 100.0%) | | MED12 Exon 42a | c.6226C>T | p.Q2076* | 0.99-1.02 | 0.01-0.02 | 0.02 | 0.02 | 0.00 | 26.55% | 2 of 5 | 40.0% (5.3%, 85.3%) | | PAK1 Exon 7 | c.607C>T | p.R203W | 0.47-0.60 | 0.23-0.29 | 0.26 | 0.26 | 0.02 | 8.07% | 5 of 5 | 100.0% (47.8%, 100.0%) | | BRAF Exon 15 | c.1799T>A | p.V600E | 0.17-0.56 | 0.28-0.37 | 0.32 | 0.32 | 0.04 | 11.14% | 5 of 5 | 100.0% (47.8%, 100.0%) | | PTPRD Exon 44 | c.5452G>T | p.E1818* | 1.34-1.47 | 0.29-0.34 | 0.32 | 0.33 | 0.02 | 4.98% | 5 of 5 | 100.0% (47.8%, 100.0%) | | PTPRS Exon 15 | c.2186C>T | p.A729V | 0.55-1.19 | 0.05-0.10 | 0.07 | 0.07 | 0.02 | 28.08% | 5 of 5 | 100.0% (47.8%, 100.0%) | | NOTCH1 Exon 34 | c.7455delC | p.S2486Rfs*103 | 1.58-2.01 | 0.31-0.35 | 0.33 | 0.33 | 0.02 | 4.63% | 5 of 5 | 100.0% (47.8%, 100.0%) | | FBXW7 Exon 10 | c.1429G>A | p.G477S | 0.52-0.59 | 0.33-0.38 | 0.36 | 0.36 | 0.02 | 5.43% | 5 of 5 | 100.0% (47.8%, 100.0%) | | BRD4 Exon 10 | c.1988C>G | p.T663R | 1.41-1.75 | 0.04-0.06 | 0.05 | 0.05 | 0.01 | 10.52% | 5 of 5 | 100.0% (47.8%, 100.0%) | | NBN Exon 13a | c.1932G>T | p.Q644H | 0.42-0.57 | 0.02-0.04 | 0.03 | 0.03 | 0.01 | 21.22% | 4 of 5 | 80.0% (28.4%, 99.5%) | | NCOR1 Exon 15 | c.1584delA | p.E529Kfs*26 | 0.52-0.70 | 0.27-0.32 | 0.29 | 0.28 | 0.02 | 7.77% | 5 of 5 | 100.0% (47.8%, 100.0%) | | PAK7 Exon 10 | c.1945delC | p.L649Sfs*15 | 0.54-0.60 | 0.27-0.32 | 0.31 | 0.32 | 0.02 | 6.03% | 5 of 5 | 100.0% (47.8%, 100.0%) | 21 {21} | Gene/ Exon | Coding Change | Protein Change | NC Range | MAF Range | MAF Mean | MAF Median | MAF (SD) | MAF (%CV) | Positive/ Total Calls | Positive Call Rate (two-sided 95% CI) | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | KMT2B Exon 27 | c.5636delG | p.G1879V fs*16 | 1.15-1.48 | 0.31-0.35 | 0.33 | 0.34 | 0.01 | 3.94% | 5 of 5 | 100.0% (47.8%, 100.0%) | | RBM10 Exon 10 | c.994C>A | p.R332S | 0.29-0.79 | 0.62-0.68 | 0.65 | 0.66 | 0.02 | 2.65% | 5 of 5 | 100.0% (47.8%, 100.0%) | | BRCA1 Exon 21* | c.5347A>T | p.M1783L | 0.19-0.42 | 0.04-0.10 | 0.06 | 0.05 | 0.02 | 40.44% | 4 of 5 | 80.0% (28.4%, 99.5%) | | ARID5B Exon 10 | c.2263G>A | p.V755I | 1.23-1.88 | 0.30-0.37 | 0.35 | 0.37 | 0.03 | 7.63% | 5 of 5 | 100.0% (47.8%, 100.0%) | | NF1 Exon 16 | c.1738delT | p.Y580Tf s*6 | 0.47-0.60 | 0.30-0.41 | 0.36 | 0.38 | 0.04 | 11.03% | 5 of 5 | 100.0% (47.8%, 100.0%) | | NKX2-1 Exon 3 | c.1015G>A | p.G339S | 1.32-1.61 | 0.24-0.35 | 0.30 | 0.32 | 0.04 | 13.52% | 5 of 5 | 100.0% (47.8%, 100.0%) | | GRIN2A Exon 3 | c.244C>A | p.L82I | 0.98-1.33 | 0.35-0.38 | 0.36 | 0.37 | 0.01 | 2.47% | 5 of 5 | 100.0% (47.8%, 100.0%) | | MDM2 Exon 11 | c.1111delA | p.T371Lfs *2 | 0.60-0.70 | 0.32-0.34 | 0.33 | 0.33 | 0.01 | 2.78% | 5 of 5 | 100.0% (47.8%, 100.0%) | | RRAGC Exon 2 | c.328G>A | p.V110M | 0.41-0.43 | 0.27-0.31 | 0.28 | 0.28 | 0.01 | 4.55% | 5 of 5 | 100.0% (47.8%, 100.0%) | | ATM Exon 58 | c.8558C>T | p.T2853M | 0.26-0.71 | 0.28-0.34 | 0.30 | 0.30 | 0.02 | 6.70% | 5 of 5 | 100.0% (47.8%, 100.0%) | | PHOX2B Exon 3 | c.451G>A | p.A151T | 0.68-0.93 | 0.02-0.05 | 0.03 | 0.03 | 0.01 | 29.43% | 5 of 5 | 100.0% (47.8%, 100.0%) | | BCL10 Exon 3 | c.686C>G | p.T229S | 0.29-0.63 | 0.29-0.38 | 0.34 | 0.34 | 0.03 | 9.63% | 5 of 5 | 100.0% (47.8%, 100.0%) | | ARID2 Exon 2 | c.109delA | p.I37Sfs* 21 | 0.70-1.33 | 0.29-0.38 | 0.34 | 0.33 | 0.03 | 9.91% | 5 of 5 | 100.0% (47.8%, 100.0%) | | KIT Exon 3 | c.590C>T | p.S197L | 0.98-1.09 | 0.17-0.20 | 0.19 | 0.19 | 0.01 | 5.77% | 5 of 5 | 100.0% (47.8%, 100.0%) | | FLT1 Exon 9 | c.1107-1G>A | None | 0.37-0.49 | 0.24-0.36 | 0.30 | 0.32 | 0.04 | 13.80% | 5 of 5 | 100.0% (47.8%, 100.0%) | 22 {22} | Gene/ Exon | Coding Change | Protein Change | NC Range | MAF Range | MAF Mean | MAF Median | MAF (SD) | MAF (%CV) | Positive/ Total Calls | Positive Call Rate (two-sided 95% CI) | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | CCND3 Exon 2 | c.373_374del TG | p.C125Hf s*34 | 0.99-1.23 | 0.25-0.32 | 0.29 | 0.29 | 0.02 | 8.24% | 5 of 5 | 100.0% (47.8%, 100.0%) | | MSH6 Exon 5 | c.3425C>T | p.T1142M | 0.65-0.73 | 0.31-0.36 | 0.35 | 0.35 | 0.02 | 5.01% | 5 of 5 | 100.0% (47.8%, 100.0%) | | STAT3 Exon 7 | c.641G>T | p.R214L | 1.10-1.32 | 0.30-0.34 | 0.31 | 0.30 | 0.01 | 4.82% | 5 of 5 | 100.0% (47.8%, 100.0%) | | BMPR1A Exon 4 | c.176T>A | p.L59* | 0.31-0.67 | 0.22-0.28 | 0.25 | 0.27 | 0.03 | 10.26% | 5 of 5 | 100.0% (47.8%, 100.0%) | | RNF43 Exon 9 | c.1586G>T | p.R529L | 1.52-1.81 | 0.05-0.07 | 0.06 | 0.05 | 0.01 | 13.32% | 5 of 5 | 100.0% (47.8%, 100.0%) | | ROS1 Exon 39 | c.6175C>A | p.L2059M | 0.23-0.25 | 0.27-0.31 | 0.30 | 0.30 | 0.02 | 5.69% | 5 of 5 | 100.0% (47.8%, 100.0%) | | ERCC3 Exon 6 | c.777A>T | p.E259D | 0.61-0.65 | 0.06-0.10 | 0.08 | 0.08 | 0.01 | 15.20% | 5 of 5 | 100.0% (47.8%, 100.0%) | | STAT3 Exon 3 | c.272A>G | p.Q91R | 0.80-0.93 | 0.03-0.08 | 0.06 | 0.06 | 0.01 | 26.29% | 5 of 5 | 100.0% (47.8%, 100.0%) | | KDR Exon 15 | c.2194G>A | p.E732K | 0.32-0.43 | 0.31-0.38 | 0.33 | 0.33 | 0.03 | 7.66% | 5 of 5 | 100.0% (47.8%, 100.0%) | | TBX3 Exon 7 | c.1292C>T | p.A431V | 2.06-2.40 | 0.08-0.09 | 0.08 | 0.08 | 0.01 | 7.40% | 5 of 5 | 100.0% (47.8%, 100.0%) | | PIK3CA Exon 2 | c.11G>A | p.R4Q | 0.42-0.49 | 0.22-0.26 | 0.23 | 0.23 | 0.02 | 7.09% | 5 of 5 | 100.0% (47.8%, 100.0%) | | MSI1 Exon 9 | c.587C>A | p.A196D | 1.04-1.26 | 0.28-0.32 | 0.30 | 0.30 | 0.01 | 4.97% | 5 of 5 | 100.0% (47.8%, 100.0%) | | MAP3K14 Exon 7 | c.1294G>A | p.G432S | 1.28-1.61 | 0.04-0.06 | 0.05 | 0.05 | 0.01 | 13.55% | 5 of 5 | 100.0% (47.8%, 100.0%) | | WT1 Exon N/A | c.20C>T | p.T7M | 0 | 0.37-0.43 | 0.39 | 0.39 | 0.02 | 5.81% | 5 of 5 | 100.0% (47.8%, 100.0%) | | TP53BP1 Exon 12 | c.2672C>T | p.A891V | 0.46-0.55 | 0.02-0.03 | 0.02 | 0.02 | 0.00 | 20.89% | 5 of 5 | 100.0% (47.8%, 100.0%) | 23 {23} | Gene/ Exon | Coding Change | Protein Change | NC Range | MAF Range | MAF Mean | MAF Median | MAF (SD) | MAF (%CV ) | Positive/ Total Calls | Positive Call Rate (two-sided 95% CI) | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | KMT2C Exon 38a | c.7443-2_7443-1insAA | None | 0.89-0.90 | 0.06-0.06 | 0.06 | 0.06 | 0.00 | 3.99% | 2 of 5 | 40.0% (5.3%, 85.3%) | | IGF1R Exon 3 | c.668C>T | p.A223V | 1.48-1.76 | 0.25-0.30 | 0.28 | 0.28 | 0.02 | 6.83% | 5 of 5 | 100.0% (47.8%, 100.0%) | | EPCAM Exon 5 | c.545C>T | p.T182M | 0.26-0.34 | 0.30-0.34 | 0.32 | 0.32 | 0.01 | 4.46% | 5 of 5 | 100.0% (47.8%, 100.0%) | | MGA Exon 3 | c.1626delA | p.K542Nf s*5 | 0.54-0.59 | 0.30-0.41 | 0.37 | 0.39 | 0.04 | 10.41 % | 5 of 5 | 100.0% (47.8%, 100.0%) | | SMARCA 4 Exon 35 | c.4933C>T | p.R1645 W | 1.17-1.43 | 0.35-0.39 | 0.36 | 0.35 | 0.02 | 4.40% | 5 of 5 | 100.0% (47.8%, 100.0%) | | SMARCA 4 Exon 26 | c.3727C>T | p.R1243 W | 1.00-1.15 | 0.31-0.37 | 0.34 | 0.35 | 0.02 | 6.01% | 5 of 5 | 100.0% (47.8%, 100.0%) | | AXIN2 Exon 8 | c.1994_1995insG | p.N666Qf s*41 | 1.18-2.32 | 0.30-0.33 | 0.31 | 0.31 | 0.01 | 2.92% | 5 of 5 | 100.0% (47.8%, 100.0%) | | MAP3K13 Exon 5 | c.806A>G | p.Y269C | 0.39-0.46 | 0.33-0.41 | 0.37 | 0.35 | 0.03 | 8.01% | 5 of 5 | 100.0% (47.8%, 100.0%) | | SMAD4 Exon 12 | c.1607T>C | p.L536P | 0.41-0.45 | 0.51-0.59 | 0.54 | 0.54 | 0.04 | 6.56% | 5 of 5 | 100.0% (47.8%, 100.0%) | | IRS1 Exon 1 | c.2059_2060insGCA | p.S686_S 680ins | 1.39-1.65 | 0.24-0.27 | 0.25 | 0.25 | 0.01 | 4.89% | 5 of 5 | 100.0% (47.8%, 100.0%) | | ARID1B Exon 1 | c.81G>T | p.E27D | 0.91-2.12 | 0.30-0.34 | 0.32 | 0.33 | 0.02 | 4.85% | 5 of 5 | 100.0% (47.8%, 100.0%) | | JAK3 Exon 16 | c.2172G>A | p.M724I | 1.37-1.63 | 0.29-0.34 | 0.32 | 0.33 | 0.02 | 5.94% | 5 of 5 | 100.0% (47.8%, 100.0%) | | TP53 Exon 6 | c.637C>T | p.R213* | 0.88-0.97 | 0.36-0.42 | 0.39 | 0.38 | 0.03 | 6.49% | 5 of 5 | 100.0% (47.8%, 100.0%) | | | | | | | | | | | | | | KMT2C Exon 43 | c.11248_1124 9insTAAGGT AGAAGGAA ACGCTGTA G | p.A3750V fs*3 | 1.09-1.17 | 0.11-0.16 | 0.13 | 0.12 | 0.02 | 11.97 % | 5 of 5 | 100.0% (47.8%, 100.0%) | 24 {24} | Gene/ Exon | Coding Change | Protein Change | NC Range | MAF Range | MAF Mean | MAF Median | MAF (SD) | MAF (%CV) | Positive/ Total Calls | Positive Call Rate (two-sided 95% CI) | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | ARID1B Exon 1 | c.1379C>G | p.A460G | 1.19-2.39 | 0.01-0.02 | 0.02 | 0.02 | 0.00 | 19.28% | 5 of 5 | 100.0% (47.8%, 100.0%) | | MUTYH Exon 16 | c.1613C>G | p.S538C | 1.23-1.51 | 0.35-0.43 | 0.38 | 0.36 | 0.03 | 7.10% | 5 of 5 | 100.0% (47.8%, 100.0%) | | BCOR Exon 4 | c.2936_2937insATTCAA ATG | p.K978_F 977ins* | 1.47-1.68 | 0.28-0.34 | 0.31 | 0.32 | 0.02 | 6.16% | 5 of 5 | 100.0% (47.8%, 100.0%) | | RHEB Exon 2 | c.104A>T | p.Y35F | 0.50-0.60 | 0.04-0.05 | 0.04 | 0.04 | 0.00 | 9.99% | 5 of 5 | 100.0% (47.8%, 100.0%) | | ASXL2 Exon 1 | c.38G>A | p.W13* | 1.21-1.53 | 0.76-0.80 | 0.77 | 0.77 | 0.02 | 2.05% | 5 of 5 | 100.0% (47.8%, 100.0%) | | | | | | | | | | | | | | EZH2 Exon 8 | c.893G>A | p.R298H | 0.34-0.38 | 0.31-0.36 | 0.34 | 0.33 | 0.02 | 4.92% | 5 of 5 | 100.0% (47.8%, 100.0%) | | PIK3CB Exon 18 | c.2638G>A | p.A880T | 0.25-0.32 | 0.25-0.29 | 0.26 | 0.26 | 0.01 | 4.73% | 5 of 5 | 100.0% (47.8%, 100.0%) | | RAD51 Exon 9 | c.868A>G | p.N290D | 0.24-0.89 | 0.26-0.42 | 0.32 | 0.32 | 0.05 | 15.51% | 5 of 5 | 100.0% (47.8%, 100.0%) | | FLT4 Exon 14 | c.2131T>C | p.W711R | 1.12-1.31 | 0.27-0.35 | 0.32 | 0.33 | 0.03 | 8.97% | 5 of 5 | 100.0% (47.8%, 100.0%) | | NOTCH2 Exon 24 | c.3933G>A | p.M1311I | 0.76-0.97 | 0.26-0.30 | 0.28 | 0.28 | 0.02 | 5.44% | 5 of 5 | 100.0% (47.8%, 100.0%) | | NSD1 Exon 23 | c.7975G>A | p.A2659T | 1.59-1.77 | 0.31-0.34 | 0.33 | 0.32 | 0.01 | 3.81% | 5 of 5 | 100.0% (47.8%, 100.0%) | | NCOR1 Exon 19 | c.2063G>A | p.R688Q | 0.29-0.32 | 0.31-0.38 | 0.34 | 0.34 | 0.03 | 7.88% | 5 of 5 | 100.0% (47.8%, 100.0%) | | MGA Exon 11 | c.3736C>T | p.R1246* | 0.73-0.84 | 0.29-0.35 | 0.31 | 0.30 | 0.02 | 7.83% | 5 of 5 | 100.0% (47.8%, 100.0%) | | PBRM1 Exon 4 | c.340G>A | p.A114T | 0.29-0.39 | 0.27-0.35 | 0.30 | 0.29 | 0.03 | 9.07% | 5 of 5 | 100.0% (47.8%, 100.0%) | | FUBP1 Exon 11 | c.857C>A | p.A286D | 0.13-0.21 | 0.20-0.37 | 0.30 | 0.33 | 0.06 | 18.92% | 5 of 5 | 100.0% (47.8%, 100.0%) | 25 {25} | Gene/ Exon | Coding Change | Protein Change | NC Range | MAF Range | MAF Mean | MAF Median | MAF (SD) | MAF (%CV) | Positive/ Total Calls | Positive Call Rate (two-sided 95% CI) | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | ARID2 Exon 15 | c.4640C>T | p.A1547V | 0.81-1.61 | 0.30-0.36 | 0.33 | 0.34 | 0.02 | 6.99% | 5 of 5 | 100.0% (47.8%, 100.0%) | | CSDE1 Exon 13a | c.1333A>G | p.M445V | 0.54-0.54 | 0.02-0.02 | 0.02 | 0.02 | 0.00 | 0.00% | 1 of 5 | 20.0% (0.5%, 71.6%) | | RNF43 Exon 5 | c.520G>A | p.E174K | 1.18-1.40 | 0.31-0.34 | 0.33 | 0.34 | 0.01 | 3.22% | 5 of 5 | 100.0% (47.8%, 100.0%) | | POLD1 Exon 7 | c.795G>A | p.W265* | 0.97-1.16 | 0.02-0.03 | 0.02 | 0.02 | 0.00 | 19.27% | 5 of 5 | 100.0% (47.8%, 100.0%) | | FOXP1 Exon 21 | c.1945C>A | p.L649M | 0.80-0.90 | 0.31-0.36 | 0.33 | 0.33 | 0.02 | 4.98% | 5 of 5 | 100.0% (47.8%, 100.0%) | | SMO Exon 12 | c.2014A>G | p.K672E | 1.02-1.24 | 0.31-0.34 | 0.33 | 0.33 | 0.01 | 2.99% | 5 of 5 | 100.0% (47.8%, 100.0%) | | APC Exon 5 | c.239G>A | p.S80N | 0.56-0.66 | 0.32-0.37 | 0.34 | 0.34 | 0.02 | 5.19% | 5 of 5 | 100.0% (47.8%, 100.0%) | | PTPRS Exon 37 | c.5695C>T | p.R1899 W | 0.88-1.41 | 0.02-0.03 | 0.02 | 0.02 | 0.01 | 22.12% | 5 of 5 | 100.0% (47.8%, 100.0%) | | ALOX12 B Exon 12 | c.1534T>C | p.Y512H | 1.30-1.55 | 0.32-0.36 | 0.34 | 0.33 | 0.02 | 4.70% | 5 of 5 | 100.0% (47.8%, 100.0%) | | DICER1 Exon 24 | c.5105A>G | p.Q1702R | 1.33-1.48 | 0.31-0.35 | 0.33 | 0.33 | 0.01 | 4.39% | 5 of 5 | 100.0% (47.8%, 100.0%) | | SOS1 Exon 10 | c.1575A>G | p.I525M | 0.21-0.27 | 0.29-0.37 | 0.33 | 0.32 | 0.03 | 8.75% | 5 of 5 | 100.0% (47.8%, 100.0%) | | RECQL Exon 15 | c.1679G>A | p.S560N | 0.16-0.22 | 0.24-0.34 | 0.29 | 0.30 | 0.03 | 11.62% | 5 of 5 | 100.0% (47.8%, 100.0%) | | GLI1 Exon 4 | c.299G>A | p.R100H | 0.60-0.69 | 0.29-0.33 | 0.31 | 0.31 | 0.02 | 5.50% | 5 of 5 | 100.0% (47.8%, 100.0%) | | TET2 Exon 3 | c.2548C>G | p.H850D | 0.77-0.89 | 0.29-0.35 | 0.32 | 0.32 | 0.02 | 6.65% | 5 of 5 | 100.0% (47.8%, 100.0%) | | KMT2B Exon 11 | c.3589C>T | p.P1197S | 0.87-0.98 | 0.30-0.34 | 0.32 | 0.33 | 0.01 | 3.78% | 5 of 5 | 100.0% (47.8%, 100.0%) | 26 {26} | Gene/ Exon | Coding Change | Protein Change | NC Range | MAF Range | MAF Mean | MAF Median | MAF (SD) | MAF (%CV ) | Positive/ Total Calls | Positive Call Rate (two-sided 95% CI) | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | DDR2 Exon 17 | c.2141G>A | p.R714Q | 0.81- 0.95 | 0.29- 0.34 | 0.31 | 0.31 | 0.02 | 5.46% | 5 of 5 | 100.0% (47.8%, 100.0%) | | SOS1 Exon 23 | c.3716G>A | p.G1239D | 1.36- 1.48 | 0.29- 0.33 | 0.31 | 0.31 | 0.01 | 4.74% | 5 of 5 | 100.0% (47.8%, 100.0%) | | ATM Exon 39 | c.5784delT | p.F1928Lf s*9 | 0.21- 0.65 | 0.29- 0.34 | 0.31 | 0.31 | 0.02 | 6.06% | 5 of 5 | 100.0% (47.8%, 100.0%) | | GLI1 Exon 12 | c.2030A>G | p.D677G | 0.93- 1.09 | 0.31- 0.36 | 0.33 | 0.33 | 0.02 | 5.14% | 5 of 5 | 100.0% (47.8%, 100.0%) | | MAP3K1 Exon 13 | c.2253G>A | p.W751* | 0.33- 0.38 | 0.29- 0.37 | 0.33 | 0.32 | 0.03 | 8.23% | 5 of 5 | 100.0% (47.8%, 100.0%) | | CASP8 Exon 3 | c.52G>T | p.D18Y | 0.71- 0.83 | 0.27- 0.35 | 0.31 | 0.31 | 0.03 | 8.45% | 5 of 5 | 100.0% (47.8%, 100.0%) | | SPEN Exon 11 | c.9808C>T | p.L3270F | 1.44- 1.57 | 0.31- 0.34 | 0.33 | 0.34 | 0.01 | 3.89% | 5 of 5 | 100.0% (47.8%, 100.0%) | | REL Exon 3 | c.254G>A | p.R85K | 0.41- 0.50 | 0.31- 0.34 | 0.33 | 0.33 | 0.01 | 3.41% | 5 of 5 | 100.0% (47.8%, 100.0%) | | GATA3 Exon 2 | c.178G>A | p.V60I | 1.58- 1.99 | 0.31- 0.34 | 0.32 | 0.32 | 0.01 | 3.50% | 5 of 5 | 100.0% (47.8%, 100.0%) | | NOTCH2 Exon 5 | c.874+1G>A | None | 0.25- 0.35 | 0.28- 0.40 | 0.34 | 0.34 | 0.04 | 11.54 % | 5 of 5 | 100.0% (47.8%, 100.0%) | | CDK8 Exon 8 | c.818T>C | p.M273T | 0.16- 0.23 | 0.24- 0.37 | 0.30 | 0.29 | 0.05 | 15.36 % | 5 of 5 | 100.0% (47.8%, 100.0%) | | HRAS Exon 4 | c.295C>T | p.Q99* | 1.10- 1.44 | 0.28- 0.33 | 0.30 | 0.30 | 0.02 | 5.80% | 5 of 5 | 100.0% (47.8%, 100.0%) | | DICER1 Exon 22 | c.4103G>A | p.R1368H | 0.66- 0.77 | 0.28- 0.35 | 0.32 | 0.33 | 0.02 | 7.18% | 5 of 5 | 100.0% (47.8%, 100.0%) | | MTOR Exon 25 | c.3754delA | p.T1252Q fs*4 | 0.63- 0.72 | 0.30- 0.34 | 0.32 | 0.33 | 0.01 | 4.64% | 5 of 5 | 100.0% (47.8%, 100.0%) | | PREX2 Exon 23 | c.2626G>T | p.V876L | 0.36- 0.50 | 0.28- 0.36 | 0.32 | 0.31 | 0.03 | 9.82% | 5 of 5 | 100.0% (47.8%, 100.0%) | 27 {27} | Gene/ Exon | Coding Change | Protein Change | NC Range | MAF Range | MAF Mean | MAF Median | MAF (SD) | MAF (%CV ) | Positive/ Total Calls | Positive Call Rate (two-sided 95% CI) | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | JAK1 Exon 14 | c.1978G>A | p.D660N | 0.63- 0.75 | 0.30- 0.35 | 0.33 | 0.33 | 0.02 | 6.62% | 5 of 5 | 100.0% (47.8%, 100.0%) | | KMT2A Exon 3* | c.1102G>T | p.D368Y | 0.80- 0.90 | 0.30- 0.32 | 0.31 | 0.31 | 0.01 | 3.28% | 3 of 5 | 60.0% (14.7%, 94.7%) | | ATM Exon 25 | c.3637C>A | p.H1213N | 0.25- 0.65 | 0.23- 0.34 | 0.28 | 0.28 | 0.04 | 13.44 % | 5 of 5 | 100.0% (47.8%, 100.0%) | | RICTOR Exon 37 | c.4898G>A | p.R1633H | 0.32- 0.38 | 0.29- 0.42 | 0.32 | 0.30 | 0.05 | 15.83 % | 5 of 5 | 100.0% (47.8%, 100.0%) | | ARID1B Exon 1 | c.862G>A | p.E288K | 1.24- 2.63 | 0.28- 0.33 | 0.31 | 0.32 | 0.02 | 5.12% | 5 of 5 | 100.0% (47.8%, 100.0%) | | BRCA1 Exon 12 | c.4238A>G | p.E1413G | 0.21- 0.48 | 0.29- 0.36 | 0.31 | 0.31 | 0.02 | 7.49% | 5 of 5 | 100.0% (47.8%, 100.0%) | | AXIN1 Exon 6 | c.1321G>A | p.A441T | 1.17- 2.07 | 0.31- 0.35 | 0.34 | 0.34 | 0.01 | 3.85% | 5 of 5 | 100.0% (47.8%, 100.0%) | | ATM Exon 25 | c.3602T>C | p.F1201S | 0.25- 0.65 | 0.32- 0.38 | 0.35 | 0.35 | 0.02 | 5.57% | 5 of 5 | 100.0% (47.8%, 100.0%) | | NOTCH1 Exon 34 | c.7034G>T | p.G2345V | 1.92- 2.22 | 0.33- 0.36 | 0.34 | 0.35 | 0.01 | 2.77% | 5 of 5 | 100.0% (47.8%, 100.0%) | | STAG2 Exon 29 | c.2925-3C>T | None | 0.30- 0.36 | 0.27- 0.33 | 0.31 | 0.31 | 0.02 | 7.35% | 5 of 5 | 100.0% (47.8%, 100.0%) | | ANKRD1 1 Exon 10 | c.7468G>A | p.V2490 M | 1.23- 1.92 | 0.31- 0.36 | 0.33 | 0.33 | 0.02 | 5.38% | 5 of 5 | 100.0% (47.8%, 100.0%) | | IFNGR1 Exon 7 | c.1100C>T | p.P367L | 0.19- 0.23 | 0.26- 0.38 | 0.32 | 0.33 | 0.04 | 13.26 % | 5 of 5 | 100.0% (47.8%, 100.0%) | | ATRX Exon 9 | c.2999A>C | p.K1000T | 0.13- 0.32 | 0.31- 0.35 | 0.32 | 0.32 | 0.02 | 4.69% | 5 of 5 | 100.0% (47.8%, 100.0%) | | RUNX1 Exon 9 | c.1163C>T | p.S388L | 1.70- 2.02 | 0.34- 0.35 | 0.35 | 0.35 | 0.01 | 1.48% | 5 of 5 | 100.0% (47.8%, 100.0%) | | FLT4 Exon 30 | c.3982G>T | p.G1328* | 0.93- 1.17 | 0.31- 0.39 | 0.34 | 0.34 | 0.03 | 8.39% | 5 of 5 | 100.0% (47.8%, 100.0%) | 28 {28} | Gene/ Exon | Coding Change | Protein Change | NC Range | MAF Range | MAF Mean | MAF Median | MAF (SD) | MAF (%CV ) | Positive/ Total Calls | Positive Call Rate (two-sided 95% CI) | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | SF3B1 Exon 7 | c.809G>A | p.R270Q | 0.74- 0.86 | 0.29- 0.32 | 0.30 | 0.31 | 0.01 | 4.22% | 5 of 5 | 100.0% (47.8%, 100.0%) | | FLT1 Exon 11 | c.1523G>A | p.R508H | 0.21- 0.24 | 0.28- 0.38 | 0.33 | 0.31 | 0.04 | 11.31 % | 5 of 5 | 100.0% (47.8%, 100.0%) | | PPM1D Exon 6 | c.1573G>T | p.E525* | 0.21- 0.61 | 0.30- 0.36 | 0.32 | 0.31 | 0.02 | 7.49% | 5 of 5 | 100.0% (47.8%, 100.0%) | | INSR Exon 2 | c.211G>A | p.E71K | 0.79- 0.92 | 0.28- 0.36 | 0.32 | 0.32 | 0.03 | 8.69% | 5 of 5 | 100.0% (47.8%, 100.0%) | | ATM Exon 10 | c.1339C>T | p.R447* | 0.40- 0.84 | 0.28- 0.34 | 0.31 | 0.31 | 0.02 | 5.68% | 5 of 5 | 100.0% (47.8%, 100.0%) | | PTEN Exon 7 | c.146C>T | p.P49L | 0.23- 0.57 | 0.18- 0.32 | 0.27 | 0.28 | 0.05 | 17.50 % | 5 of 5 | 100.0% (47.8%, 100.0%) | | BCOR Exon 8 | c.3625C>T | p.R1209C | 1.22- 1.38 | 0.32- 0.33 | 0.33 | 0.33 | 0.01 | 1.81% | 5 of 5 | 100.0% (47.8%, 100.0%) | | TRAF2 Exon 2 | c.88G>A | p.A30T | 1.46- 1.66 | 0.30- 0.35 | 0.32 | 0.31 | 0.02 | 6.41% | 5 of 5 | 100.0% (47.8%, 100.0%) | | SLX4 Exon 3 | c.751G>A | p.A251T | 1.12- 1.27 | 0.02- 0.04 | 0.04 | 0.04 | 0.01 | 18.61 % | 5 of 5 | 100.0% (47.8%, 100.0%) | | IRS1 Exon 1 | c.2685A>C | p.E895D | 1.69- 1.96 | 0.35- 0.39 | 0.37 | 0.37 | 0.01 | 3.66% | 5 of 5 | 100.0% (47.8%, 100.0%) | | PTCH1 Exon 15 | c.2427A>C | p.K809N | 0.52- 1.34 | 0.29- 0.38 | 0.33 | 0.33 | 0.03 | 9.75% | 5 of 5 | 100.0% (47.8%, 100.0%) | | SETD2 Exon 12 | c.5721G>T | p.E1907D | 0.64- 0.71 | 0.30- 0.36 | 0.32 | 0.32 | 0.02 | 5.66% | 5 of 5 | 100.0% (47.8%, 100.0%) | | TSHR Exon 10 | c.2011T>A | p.S671T | 1.59- 1.76 | 0.33- 0.37 | 0.35 | 0.34 | 0.01 | 4.21% | 5 of 5 | 100.0% (47.8%, 100.0%) | | **CTCF Exon 3** | **c.263C>A** | **p.A88D** | **0.75- 0.85** | **0.29- 0.34** | **0.31** | **0.31** | **0.02** | **5.20%** | **5 of 5** | **100.0% (47.8%, 100.0%)** | | SYK Exon 2 | c.202C>T | p.R68W | 1.39- 1.69 | 0.33- 0.35 | 0.34 | 0.34 | 0.00 | 1.45% | 5 of 5 | 100.0% (47.8%, 100.0%) | 29 {29} | Gene/ Exon | Coding Change | Protein Change | NC Range | MAF Range | MAF Mean | MAF Median | MAF (SD) | MAF (%CV) | Positive/ Total Calls | Positive Call Rate (two-sided 95% CI) | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | TNFAIP3 Exon 7 | c.1477G>A | p.G493R | 1.94-2.02 | 0.31-0.34 | 0.32 | 0.32 | 0.01 | 3.54% | 5 of 5 | 100.0% (47.8%, 100.0%) | | GRIN2A Exon 3a | c.97_99delins ACA | p.A33T | 1.00-1.21 | 0.28-0.31 | 0.29 | 0.30 | 0.01 | 3.63% | 4 of 5 | 80.0% (28.4%, 99.5%) | | NF1 Exon 33 | c.4336C>A | p.L1446I | 0.20-0.23 | 0.22-0.36 | 0.29 | 0.31 | 0.05 | 16.96% | 5 of 5 | 100.0% (47.8%, 100.0%) | | FLT4 Exon 25a | c.3391G>A | p.G1131S | 0.64-0.79 | 0.02-0.02 | 0.02 | 0.02 | 0.00 | 11.82% | 4 of 5 | 80.0% (28.4%, 99.5%) | | ERBB4 Exon 28 | c.3581G>A | p.G1194D | 1.15-1.28 | 0.29-0.35 | 0.32 | 0.33 | 0.02 | 6.67% | 5 of 5 | 100.0% (47.8%, 100.0%) | | TET1 Exon 12 | c.6205G>A | p.E2069K | 1.70-1.85 | 0.33-0.36 | 0.34 | 0.34 | 0.01 | 3.23% | 5 of 5 | 100.0% (47.8%, 100.0%) | | DNMT1 Exon 41 | c.4879G>T | p.E1627* | 0.59-0.73 | 0.30-0.35 | 0.32 | 0.32 | 0.02 | 5.70% | 5 of 5 | 100.0% (47.8%, 100.0%) | | TCF3 Exon 2 | c.42G>T | p.K14N | 0.81-1.15 | 0.28-0.38 | 0.33 | 0.32 | 0.03 | 10.22% | 5 of 5 | 100.0% (47.8%, 100.0%) | | FLT1 Exon 19a | c.2669A>G | p.N890S | 0.70-0.85 | 0.02-0.03 | 0.02 | 0.02 | 0.00 | 21.87% | 4 of 5 | 80.0% (28.4%, 99.5%) | | INPPL1 Exon 21 | c.2339G>A | p.S780N | 0.59-0.69 | 0.27-0.34 | 0.30 | 0.30 | 0.03 | 9.21% | 5 of 5 | 100.0% (47.8%, 100.0%) | | MTOR Exon 13 | c.2153T>C | p.L718P | 1.21-1.37 | 0.31-0.36 | 0.33 | 0.33 | 0.02 | 5.02% | 5 of 5 | 100.0% (47.8%, 100.0%) | | ERG Exon 6 | c.413C>A | p.P138H | 0.95-1.12 | 0.29-0.36 | 0.32 | 0.33 | 0.02 | 7.05% | 5 of 5 | 100.0% (47.8%, 100.0%) | | MAP3K13 Exon 12 | c.2147G>A | p.G716D | 1.31-1.62 | 0.32-0.35 | 0.34 | 0.34 | 0.01 | 3.64% | 5 of 5 | 100.0% (47.8%, 100.0%) | | PGR Exon 1 | c.538G>A | p.A180T | 2.08-2.56 | 0.50-0.54 | 0.52 | 0.52 | 0.01 | 2.43% | 5 of 5 | 100.0% (47.8%, 100.0%) | | JAK3 Exon 9 | c.1208G>A | p.R403H | 0.44-0.60 | 0.27-0.33 | 0.31 | 0.31 | 0.02 | 7.41% | 5 of 5 | 100.0% (47.8%, 100.0%) | 30 {30} | Gene/ Exon | Coding Change | Protein Change | NC Range | MAF Range | MAF Mean | MAF Median | MAF (SD) | MAF (%CV) | Positive/ Total Calls | Positive Call Rate (two-sided 95% CI) | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | FOXO1 Exon 2 | c.1000G>A | p.E334K | 1.22-1.34 | 0.33-0.34 | 0.33 | 0.33 | 0.00 | 1.32% | 5 of 5 | 100.0% (47.8%, 100.0%) | | CREBBP Exon 2 | c.458C>T | p.P153L | 1.36-1.58 | 0.31-0.34 | 0.32 | 0.32 | 0.01 | 2.65% | 5 of 5 | 100.0% (47.8%, 100.0%) | | IGF2 Exon 3 | c.223G>A | p.A75T | 0.78-1.06 | 0.31-0.35 | 0.33 | 0.33 | 0.01 | 3.95% | 5 of 5 | 100.0% (47.8%, 100.0%) | | NOTCH2 Exon 33 | c.5978C>A | p.T1993N | 0.35-0.42 | 0.27-0.34 | 0.29 | 0.28 | 0.03 | 9.88% | 5 of 5 | 100.0% (47.8%, 100.0%) | | CDC73 Exon 14 | c.1159G>A | p.V387I | 0.50-0.72 | 0.29-0.37 | 0.32 | 0.32 | 0.03 | 9.88% | 5 of 5 | 100.0% (47.8%, 100.0%) | | AXIN2 Exon 4 | c.1016G>A | p.R339H | 0.42-0.91 | 0.21-0.26 | 0.24 | 0.26 | 0.02 | 8.82% | 5 of 5 | 100.0% (47.8%, 100.0%) | | GRIN2A Exon 14 | c.4357G>A | p.V1453 M | 0.94-1.08 | 0.27-0.33 | 0.31 | 0.32 | 0.02 | 6.25% | 5 of 5 | 100.0% (47.8%, 100.0%) | | EPCAM Exon 4 | c.458G>T | p.R153I | 0.30-0.37 | 0.33-0.38 | 0.36 | 0.36 | 0.02 | 6.49% | 5 of 5 | 100.0% (47.8%, 100.0%) | | APC Exon 6 | c.437C>T | p.A146V | 0.21-0.26 | 0.26-0.31 | 0.29 | 0.30 | 0.02 | 5.16% | 5 of 5 | 100.0% (47.8%, 100.0%) | | APC Exon 17 | c.7286C>A | p.S2429Y | 0.52-0.63 | 0.24-0.35 | 0.30 | 0.29 | 0.04 | 12.66% | 5 of 5 | 100.0% (47.8%, 100.0%) | | EPHB1 Exon 15 | c.2725G>T | p.D909Y | 1.17-1.44 | 0.31-0.34 | 0.33 | 0.33 | 0.01 | 3.22% | 5 of 5 | 100.0% (47.8%, 100.0%) | | KEAP1 Exon 2 | c.626T>C | p.M209T | 1.09-1.32 | 0.31-0.35 | 0.33 | 0.33 | 0.02 | 5.14% | 5 of 5 | 100.0% (47.8%, 100.0%) | | PTPRT Exon 4 | c.535G>A | p.V179M | 0.47-0.85 | 0.29-0.36 | 0.33 | 0.34 | 0.02 | 7.18% | 5 of 5 | 100.0% (47.8%, 100.0%) | | ARID1A Exon 11 | c.3177G>T | p.K1059N | 1.27-1.49 | 0.02-0.03 | 0.03 | 0.02 | 0.00 | 15.50% | 5 of 5 | 100.0% (47.8%, 100.0%) | | TSC2 Exon 17 | c.1832G>A | p.R611Q | 1.52-1.79 | 0.35-0.36 | 0.35 | 0.35 | 0.01 | 1.52% | 5 of 5 | 100.0% (47.8%, 100.0%) | 31 {31} | Gene/ Exon | Coding Change | Protein Change | NC Range | MAF Range | MAF Mean | MAF Median | MAF (SD) | MAF (%CV) | Positive/ Total Calls | Positive Call Rate (two-sided 95% CI) | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | BCL10 Exon 3 | c.595G>T | p.G199W | 0.29-0.70 | 0.28-0.34 | 0.31 | 0.31 | 0.02 | 6.36% | 5 of 5 | 100.0% (47.8%, 100.0%) | | DICER1 Exon 2 | c.58G>A | p.A20T | 0.34-0.38 | 0.25-0.35 | 0.29 | 0.30 | 0.04 | 12.07% | 5 of 5 | 100.0% (47.8%, 100.0%) | | EP300 Exon 31 | c.5458C>T | p.H1820Y | 2.03-2.27 | 0.32-0.35 | 0.33 | 0.33 | 0.01 | 2.73% | 5 of 5 | 100.0% (47.8%, 100.0%) | | HGF Exon 17 | c.1900C>A | p.L634I | 0.20-0.25 | 0.27-0.36 | 0.31 | 0.32 | 0.03 | 10.22% | 5 of 5 | 100.0% (47.8%, 100.0%) | | FAT1 Exon 27 | c.13382C>T | p.S4461F | 1.49-1.71 | 0.30-0.31 | 0.30 | 0.31 | 0.00 | 1.07% | 5 of 5 | 100.0% (47.8%, 100.0%) | | RAF1 Exon 2a | c.125C>T | p.A42V | 0.52-0.71 | 0.02-0.03 | 0.03 | 0.03 | 0.01 | 19.09% | 3 of 5 | 60.0% (14.7%, 94.7%) | | CIC Exon 3 | c.406G>T | p.G136* | 1.64-1.96 | 0.34-0.35 | 0.35 | 0.35 | 0.01 | 1.84% | 5 of 5 | 100.0% (47.8%, 100.0%) | | MTOR Exon 10a | c.1436_1438d elinsCA | p.D479Af s*35 | 1.20-1.20 | 0.92-0.92 | 0.92 | 0.92 | 0.00 | 0.00% | 1 of 5 | 20.0% (0.5%, 71.6%) | | CTNNB1 Exon 12 | c.1856G>C | p.C619S | 0.32-0.39 | 0.28-0.34 | 0.31 | 0.31 | 0.02 | 7.91% | 5 of 5 | 100.0% (47.8%, 100.0%) | | CHEK2 Exon 2 | c.153G>T | p.Q51H | 1.30-1.50 | 0.23-0.26 | 0.24 | 0.24 | 0.01 | 3.78% | 5 of 5 | 100.0% (47.8%, 100.0%) | | PTCH1 Exon 23 | c.4115C>T | p.T1372M | 1.36-2.56 | 0.32-0.35 | 0.33 | 0.33 | 0.01 | 3.08% | 5 of 5 | 100.0% (47.8%, 100.0%) | | SYK Exon 4 | c.650G>A | p.R217H | 1.63-1.81 | 0.32-0.35 | 0.33 | 0.33 | 0.01 | 3.24% | 5 of 5 | 100.0% (47.8%, 100.0%) | | SETD2 Exon 3 | c.2749_2750i nsA | p.S917Kfs *18 | 0.49-0.60 | 0.24-0.32 | 0.29 | 0.29 | 0.03 | 9.77% | 5 of 5 | 100.0% (47.8%, 100.0%) | | ERCC4 Exon 8 | c.1468C>T | p.R490W | 0.43-0.49 | 0.27-0.37 | 0.30 | 0.30 | 0.03 | 11.19% | 5 of 5 | 100.0% (47.8%, 100.0%) | | JUN Exon 1 | c.686C>T | p.P229L | 1.60-1.91 | 0.29-0.34 | 0.32 | 0.32 | 0.02 | 4.79% | 5 of 5 | 100.0% (47.8%, 100.0%) | 32 {32} | Gene/ Exon | Coding Change | Protein Change | NC Range | MAF Range | MAF Mean | MAF Median | MAF (SD) | MAF (%CV) | Positive/ Total Calls | Positive Call Rate (two-sided 95% CI) | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | EPAS1 Exon 16 | c.2512G>A | p.E838K | 1.49-1.88 | 0.32-0.34 | 0.33 | 0.33 | 0.01 | 2.00% | 5 of 5 | 100.0% (47.8%, 100.0%) | | KMT2B Exon 14 | c.3890G>A | p.C1297Y | 0.53-0.65 | 0.32-0.35 | 0.33 | 0.34 | 0.01 | 3.32% | 5 of 5 | 100.0% (47.8%, 100.0%) | | ERBB2 Exon 29 | c.2927G>A | p.R976H | 0.75-0.89 | 0.31-0.34 | 0.32 | 0.32 | 0.01 | 3.12% | 5 of 5 | 100.0% (47.8%, 100.0%) | | FGFR4 Exon 17 | c.2201C>A | p.P734H | 0.83-0.97 | 0.31-0.34 | 0.32 | 0.33 | 0.01 | 3.23% | 5 of 5 | 100.0% (47.8%, 100.0%) | | KMT2B Exon 14 | c.3896C>T | p.A1299V | 0.53-0.65 | 0.29-0.34 | 0.31 | 0.30 | 0.02 | 5.51% | 5 of 5 | 100.0% (47.8%, 100.0%) | | NTRK1 Exon 7 | c.844G>A | p.V282I | 1.02-1.21 | 0.31-0.35 | 0.34 | 0.35 | 0.02 | 4.84% | 5 of 5 | 100.0% (47.8%, 100.0%) | | KMT2D Exon 6 | c.804G>A | p.W268* | 1.31-1.51 | 0.30-0.33 | 0.32 | 0.32 | 0.01 | 3.79% | 5 of 5 | 100.0% (47.8%, 100.0%) | | ERBB4 Exon 25 | c.2999_3002d elinsGCAA | p.D1000_S1001delinsGN | 0.17-0.24 | 0.25-0.37 | 0.30 | 0.29 | 0.04 | 13.06% | 5 of 5 | 100.0% (47.8%, 100.0%) | | CARD11 Exon 8 | c.1094T>C | p.M365T | 0.83-0.99 | 0.30-0.36 | 0.32 | 0.31 | 0.02 | 7.69% | 5 of 5 | 100.0% (47.8%, 100.0%) | | ARID1B Exon 20 | c.5968C>T | p.R1990* | 1.16-2.18 | 0.32-0.34 | 0.33 | 0.33 | 0.01 | 3.02% | 5 of 5 | 100.0% (47.8%, 100.0%) | | CIC Exon 10 | c.1526delC | p.P509Hfs *14 | 1.62-1.93 | 0.31-0.33 | 0.32 | 0.33 | 0.01 | 2.26% | 5 of 5 | 100.0% (47.8%, 100.0%) | | DNAJB1 Exon 2 | c.293C>A | p.P98H | 1.74-1.91 | 0.30-0.33 | 0.31 | 0.32 | 0.01 | 4.65% | 5 of 5 | 100.0% (47.8%, 100.0%) | | HNF1A Exon 2 | c.341G>A | p.R114H | 0.89-1.11 | 0.29-0.33 | 0.31 | 0.31 | 0.02 | 5.28% | 5 of 5 | 100.0% (47.8%, 100.0%) | | PIK3CB Exon 12 | c.1771C>A | p.L591I | 0.43-0.51 | 0.27-0.29 | 0.28 | 0.28 | 0.01 | 2.28% | 5 of 5 | 100.0% (47.8%, 100.0%) | | CDK12 Exon 4 | c.2228A>C | p.K743T | 0.35-0.50 | 0.28-0.36 | 0.32 | 0.31 | 0.03 | 9.04% | 5 of 5 | 100.0% (47.8%, 100.0%) | 33 {33} | Gene/ Exon | Coding Change | Protein Change | NC Range | MAF Range | MAF Mean | MAF Median | MAF (SD) | MAF (%CV) | Positive/ Total Calls | Positive Call Rate (two-sided 95% CI) | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | MYD88 Exon 1 | c.17C>T | p.A6V | 1.96-2.42 | 0.32-0.34 | 0.33 | 0.33 | 0.01 | 2.37% | 5 of 5 | 100.0% (47.8%, 100.0%) | | KMT2D Exon 10 | c.1315C>T | p.P439S | 1.38-1.64 | 0.31-0.33 | 0.32 | 0.32 | 0.01 | 2.43% | 5 of 5 | 100.0% (47.8%, 100.0%) | | ANKRD1 1 Exon 10 | c.4610G>A | p.G1537D | 1.23-1.92 | 0.32-0.34 | 0.33 | 0.33 | 0.01 | 2.28% | 5 of 5 | 100.0% (47.8%, 100.0%) | | ETV1 Exon 8 | c.680C>T | p.A227V | 1.14-1.27 | 0.30-0.36 | 0.32 | 0.33 | 0.02 | 7.00% | 5 of 5 | 100.0% (47.8%, 100.0%) | | PIK3CG Exon 11 | c.3197delA | p.K1066S fs*42 | 0.24-0.27 | 0.25-0.35 | 0.30 | 0.29 | 0.03 | 11.69% | 5 of 5 | 100.0% (47.8%, 100.0%) | | CDC42 Exon 6 | c.402G>T | p.Q134H | 0.31-0.36 | 0.28-0.34 | 0.30 | 0.29 | 0.02 | 7.63% | 5 of 5 | 100.0% (47.8%, 100.0%) | | PPP2R1A Exon 4 | c.375G>T | p.E125D | 1.09-1.61 | 0.31-0.35 | 0.33 | 0.33 | 0.01 | 3.57% | 5 of 5 | 100.0% (47.8%, 100.0%) | | SH2B3 Exon 6 | c.1160G>A | p.G387E | 0.77-0.90 | 0.27-0.33 | 0.30 | 0.31 | 0.02 | 6.84% | 5 of 5 | 100.0% (47.8%, 100.0%) | | MPL Exon 5 | c.785G>T | p.G262V | 1.03-1.23 | 0.31-0.37 | 0.33 | 0.33 | 0.02 | 5.85% | 5 of 5 | 100.0% (47.8%, 100.0%) | | PAK1 Exon 14 | c.1468C>A | p.L490M | 0.20-0.22 | 0.27-0.36 | 0.31 | 0.31 | 0.03 | 9.87% | 5 of 5 | 100.0% (47.8%, 100.0%) | | BCL6 Exon 5 | c.1129A>G | p.K377E | 1.20-2.52 | 0.30-0.35 | 0.33 | 0.33 | 0.02 | 5.56% | 5 of 5 | 100.0% (47.8%, 100.0%) | | RIT1 Exon 6 | c.503G>A | p.R168H | 0.37-0.46 | 0.29-0.33 | 0.31 | 0.32 | 0.01 | 4.40% | 5 of 5 | 100.0% (47.8%, 100.0%) | | JAK3 Exon 4 | c.366G>T | p.K122N | 0.56-0.66 | 0.30-0.35 | 0.32 | 0.33 | 0.02 | 5.71% | 5 of 5 | 100.0% (47.8%, 100.0%) | | PNRC1 Exon 1 | c.169C>A | p.L57I | 1.38-1.84 | 0.30-0.33 | 0.32 | 0.31 | 0.01 | 3.23% | 5 of 5 | 100.0% (47.8%, 100.0%) | | RPTOR Exon 22 | c.2621C>T | p.A874V | 0.97-1.33 | 0.28-0.33 | 0.31 | 0.30 | 0.02 | 5.53% | 5 of 5 | 100.0% (47.8%, 100.0%) | 34 {34} | Gene/ Exon | Coding Change | Protein Change | NC Range | MAF Range | MAF Mean | MAF Median | MAF (SD) | MAF (%CV) | Positive/ Total Calls | Positive Call Rate (two-sided 95% CI) | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | BRCA1 Exon 10 | c.3784T>C | p.S1262P | 0.19-0.49 | 0.26-0.37 | 0.32 | 0.32 | 0.04 | 12.74% | 5 of 5 | 100.0%(47.8%, 100.0%) | | SMARCD 1 Exon 6 | c.699C>A | p.F233L | 0.18-0.25 | 0.24-0.30 | 0.28 | 0.28 | 0.02 | 7.81% | 5 of 5 | 100.0%(47.8%, 100.0%) | | MDM4 Exon 4 | c.266G>A | p.S89N | 0.46-0.55 | 0.25-0.30 | 0.27 | 0.28 | 0.02 | 6.50% | 5 of 5 | 100.0%(47.8%, 100.0%) | | APC Exon 8 | c.694C>T | p.R232* | 0.47-0.53 | 0.31-0.36 | 0.33 | 0.33 | 0.02 | 5.25% | 5 of 5 | 100.0%(47.8%, 100.0%) | | PIK3CG Exon 2 | c.1426C>T | p.R476C | 1.68-1.89 | 0.30-0.34 | 0.31 | 0.32 | 0.01 | 4.77% | 5 of 5 | 100.0%(47.8%, 100.0%) | | KMT2B Exon 28 | c.5943G>T | p.E1981D | 1.40-1.65 | 0.31-0.33 | 0.32 | 0.32 | 0.01 | 1.87% | 5 of 5 | 100.0%(47.8%, 100.0%) | | NOTCH2 Exon 7 | c.1175C>T | p.T392I | 1.04-1.14 | 0.24-0.34 | 0.31 | 0.32 | 0.04 | 11.57% | 5 of 5 | 100.0%(47.8%, 100.0%) | | SETD2 Exon 3 | c.4305G>T | p.E1435D | 0.49-0.60 | 0.26-0.32 | 0.28 | 0.28 | 0.02 | 7.34% | 5 of 5 | 100.0%(47.8%, 100.0%) | | BAP1 Exon 6 | c.437+3G>C | None | 0.80-1.66 | 0.32-0.34 | 0.32 | 0.32 | 0.01 | 2.32% | 5 of 5 | 100.0%(47.8%, 100.0%) | | RICTOR Exon 22 | c.2054T>A | p.L685H | 0.20-0.31 | 0.28-0.39 | 0.33 | 0.29 | 0.05 | 14.93% | 5 of 5 | 100.0%(47.8%, 100.0%) | | ESR1 Exon 6 | c.767G>A | p.R256Q | 1.30-1.46 | 0.30-0.35 | 0.32 | 0.32 | 0.02 | 4.69% | 5 of 5 | 100.0%(47.8%, 100.0%) | | TGFBR1 Exon 2 | c.166G>A | p.V56I | 0.55-0.66 | 0.33-0.34 | 0.33 | 0.33 | 0.00 | 1.28% | 5 of 5 | 100.0%(47.8%, 100.0%) | | STK11 Exon 1 | c.83G>A | p.R28H | 1.70-2.00 | 0.31-0.34 | 0.33 | 0.34 | 0.01 | 4.07% | 5 of 5 | 100.0%(47.8%, 100.0%) | | ARID5B Exon 1 | c.10A>G | p.N4D | 0.51-0.78 | 0.26-0.36 | 0.32 | 0.33 | 0.03 | 9.90% | 5 of 5 | 100.0%(47.8%, 100.0%) | | MPL Exon 12 | c.1697G>A | p.S566N | 1.28-1.48 | 0.30-0.36 | 0.32 | 0.32 | 0.02 | 6.55% | 5 of 5 | 100.0%(47.8%, 100.0%) | 35 {35} | Gene/ Exon | Coding Change | Protein Change | NC Range | MAF Range | MAF Mean | MAF Median | MAF (SD) | MAF (%CV) | Positive/ Total Calls | Positive Call Rate (two-sided 95% CI) | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | STAG2 Exon 30 | c.3197G>A | p.R1066Q | 0.41-0.44 | 0.27-0.35 | 0.31 | 0.32 | 0.03 | 9.60% | 5 of 5 | 100.0% (47.8%, 100.0%) | | MSH2 Exon 4 | c.687dupA | p.A230Sfs *2 | 0.34-0.46 | 0.29-0.34 | 0.32 | 0.32 | 0.02 | 6.35% | 5 of 5 | 100.0% (47.8%, 100.0%) | | NBN Exon 3 | c.200C>T | p.T67I | 0.23-0.34 | 0.27-0.45 | 0.32 | 0.28 | 0.07 | 21.98% | 5 of 5 | 100.0% (47.8%, 100.0%) | | EPHA5 Exon 18 | c.3103G>A | p.V1035 M | 0.18-0.23 | 0.25-0.33 | 0.30 | 0.31 | 0.03 | 9.81% | 5 of 5 | 100.0% (47.8%, 100.0%) | | AXIN1 Exon 10 | c.2350G>A | p.V784I | 0.79-1.34 | 0.32-0.35 | 0.33 | 0.34 | 0.01 | 2.94% | 5 of 5 | 100.0% (47.8%, 100.0%) | | PAK1 Exon 13 | c.1313G>A | p.R438Q | 0.47-0.52 | 0.27-0.30 | 0.28 | 0.28 | 0.01 | 3.89% | 5 of 5 | 100.0% (47.8%, 100.0%) | | PDGFRA Exon 18 | c.2532G>A | p.M844I | 0.46-0.56 | 0.27-0.35 | 0.31 | 0.31 | 0.03 | 9.09% | 5 of 5 | 100.0% (47.8%, 100.0%) | | RECQL Exon 12 | c.1217-1G>T | None | 0.20-0.27 | 0.26-0.32 | 0.28 | 0.27 | 0.02 | 8.79% | 5 of 5 | 100.0% (47.8%, 100.0%) | | NCOR1 Exon 12 | c.1297G>A | p.D433N | 0.46-0.50 | 0.34-0.36 | 0.35 | 0.34 | 0.01 | 2.39% | 5 of 5 | 100.0% (47.8%, 100.0%) | | PIK3CA Exon 2 | c.241G>A | p.E81K | 0.26-0.30 | 0.27-0.32 | 0.29 | 0.29 | 0.02 | 5.74% | 5 of 5 | 100.0% (47.8%, 100.0%) | | EP300 Exon 30 | c.4897C>A | p.L1633M | 1.49-1.64 | 0.30-0.33 | 0.31 | 0.31 | 0.01 | 3.48% | 5 of 5 | 100.0% (47.8%, 100.0%) | | SMARCA 4 Exon 29 | c.4047G>T | p.K1349N | 0.81-1.02 | 0.33-0.36 | 0.34 | 0.33 | 0.01 | 3.52% | 5 of 5 | 100.0% (47.8%, 100.0%) | | CIC Exon 4 | c.494C>T | p.P165L | 1.60-1.80 | 0.30-0.35 | 0.32 | 0.31 | 0.02 | 5.05% | 5 of 5 | 100.0% (47.8%, 100.0%) | | SYK Exon 4 | c.625C>T | p.H209Y | 1.63-1.81 | 0.31-0.35 | 0.32 | 0.32 | 0.02 | 4.80% | 5 of 5 | 100.0% (47.8%, 100.0%) | | SMO Exon 10 | c.1798G>A | p.V600M | 0.76-0.91 | 0.31-0.37 | 0.35 | 0.35 | 0.02 | 5.97% | 5 of 5 | 100.0% (47.8%, 100.0%) | 36 {36} | Gene/ Exon | Coding Change | Protein Change | NC Range | MAF Range | MAF Mean | MAF Median | MAF (SD) | MAF (%CV) | Positive/ Total Calls | Positive Call Rate (two-sided 95% CI) | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | CREBBP Exon 31 | c.5597G>A | p.R1866H | 1.96-2.33 | 0.33-0.34 | 0.33 | 0.33 | 0.00 | 1.37% | 5 of 5 | 100.0% (47.8%, 100.0%) | | ASXL2 Exon 8 | c.799G>A | p.A267T | 0.78-0.86 | 0.30-0.34 | 0.32 | 0.32 | 0.02 | 6.00% | 5 of 5 | 100.0% (47.8%, 100.0%) | | FLT4 Exon 3 | c.266C>T | p.A89V | 1.15-1.34 | 0.14-0.18 | 0.16 | 0.16 | 0.01 | 8.97% | 5 of 5 | 100.0% (47.8%, 100.0%) | | FLT3 Exon 2 | c.143G>A | p.G48E | 0.32-0.40 | 0.26-0.30 | 0.28 | 0.29 | 0.02 | 6.24% | 5 of 5 | 100.0% (47.8%, 100.0%) | | CIC Exon 10 | c.2153C>A | p.P718H | 1.62-1.93 | 0.32-0.35 | 0.33 | 0.33 | 0.01 | 3.52% | 5 of 5 | 100.0% (47.8%, 100.0%) | | MSH6 Exon 4 | c.3104G>A | p.R1035Q | 0.37-0.46 | 0.26-0.36 | 0.31 | 0.32 | 0.03 | 10.45% | 5 of 5 | 100.0% (47.8%, 100.0%) | | INSR Exon 10 | c.2195T>C | p.F732S | 0.50-0.53 | 0.29-0.35 | 0.32 | 0.31 | 0.02 | 5.93% | 5 of 5 | 100.0% (47.8%, 100.0%) | | SDHA Exon 5 | c.501G>T | p.K167N | 1.50-1.68 | 0.22-0.25 | 0.23 | 0.22 | 0.01 | 5.07% | 5 of 5 | 100.0% (47.8%, 100.0%) | | SH2B3 Exon 2 | c.446G>A | p.R149H | 0.90-1.05 | 0.23-0.26 | 0.25 | 0.25 | 0.01 | 5.29% | 5 of 5 | 100.0% (47.8%, 100.0%) | | PTPRS Exon 14 | c.2044C>A | p.L682M | 0.72-1.24 | 0.31-0.35 | 0.34 | 0.34 | 0.02 | 4.51% | 5 of 5 | 100.0% (47.8%, 100.0%) | | APC Exon 17 | c.5002G>A | p.E1668K | 0.52-0.63 | 0.05-0.12 | 0.07 | 0.06 | 0.02 | 32.23% | 5 of 5 | 100.0% (47.8%, 100.0%) | | ASXL2 Exon 6 | c.545G>A | p.C182Y | 0.94-1.14 | 0.30-0.33 | 0.31 | 0.30 | 0.01 | 4.10% | 5 of 5 | 100.0% (47.8%, 100.0%) | | SETD2 Exon 12 | c.6034C>T | p.L2012F | 0.64-0.71 | 0.30-0.36 | 0.32 | 0.30 | 0.03 | 9.70% | 5 of 5 | 100.0% (47.8%, 100.0%) | | MSH6 Exon 4 | c.2039C>T | p.A680V | 0.37-0.46 | 0.26-0.39 | 0.33 | 0.34 | 0.04 | 13.47% | 5 of 5 | 100.0% (47.8%, 100.0%) | | KDM6A Exon 24 | c.3493G>A | p.V1165I | 0.24-0.28 | 0.31-0.37 | 0.35 | 0.35 | 0.02 | 6.09% | 5 of 5 | 100.0% (47.8%, 100.0%) | 37 {37} | Gene/ Exon | Coding Change | Protein Change | NC Range | MAF Range | MAF Mean | MAF Median | MAF (SD) | MAF (%CV) | Positive/ Total Calls | Positive…
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