Exome Sequencing Platforms Research Articles

Feasibility and tissue concordance of genomic sequencing of urinary cytology in upper tract urothelial carcinoma

BackgroundThere is limited ability to accurately diagnose and clinically stage patients with upper tract urothelial carcinoma (UTUC). The most easily available and widely used urinary biomarker is urine cytology, which evaluates cellular material yet lacks sensitivity. We sought to assess the feasibility of performing next-generation sequencing (NGS) on urine cytology specimens from patients with UTUC and evaluate the genomic concordance with tissue from primary tumor. MethodsIn this retrospective study, we identified 48 patients with a diagnosis of UTUC treated at Memorial Sloan Kettering Cancer Center (MSK) between 2019 and 2022 who had banked or fresh urine samples. A convenience cohort of matching, previously sequenced tumor tissue was used when available. Urine specimens were processed and the residual material, including precipitated cell-free DNA, was sequenced using our tumor-naïve, targeted exome sequencing platform that evaluates 505 cancer-related genes (MSK-IMPACT). The primary outcome was at least 1 detectable mutation in urinary cytology specimens. The secondary outcome was concordance to matched tissue (using ANOVA or Chi-Square, as indicated). ResultsGenomic sequencing was successful for 45 (94%) of the 48 urinary cytology patient samples. The most common mutations identified were TERT (62.2%), KMT2D (46.7%), and FGFR3 (35.6%). All patients with negative urine cytology and low-grade tissue had successful cytology sequencing. Thirty-six of the 45 patients had matching tumor tissue available; concordance to matched tissue was 55% overall (131 of the total 238 oncogenic or likely oncogenic somatic mutations identified). However, in 94.4% (n = 34/36) of patients, the cytology had at least 1 shared mutation with tissue. Eleven (30.6%) patients had 100% concordance between cytology and tissue. ConclusionsSequencing urinary specimens from selective UTUC cytology is feasible in nearly all patients with UTUC. Prospective studies are underway to investigate a clinical role for this promising technology.

Prevalence of Pathogenic Germline Mutations in 13 Hereditary Cancer-Related Genes in Breast Cancer Patients in Narathiwat Province, Thailand.

BRCA1 and BRCA2 genes are known to increase breast cancer's lifetime risk. Early identification of women with this inherited risk can potentially reduce the risk of breast and/or ovarian cancer and, together with early screening, decrease the mortality rate. This study explored the frequency and distribution of genetic variants in consecutive cases of breast cancer in Narathiwat province, one of the three provinces in the southernmost Thai border. A series of 64 consecutive breast cancer patients who underwent treatment in two general hospitals in the province during the period from the year 2021 to 2022. Genotyping studies were performed using a whole exome sequencing platform. Moderate to high penetrance variants recommended by the National Comprehensive Cancer Network (NCCN) guidelines 2022 (ATM, BARD1, BRCA1, BRCA2, CDH1, CHEK2, NF1, PALB2, PTEN, RAD51C, RAD51D, STK11, TP53) were annotated and filtered for pathogenic, likely pathogenic, or high-impact variants. Pathogenic germline variants were found in 8/64 cases (12.5%), namely BRCA1 in 3 (4.7%), BRCA2 in 4 (6.3%), ATM in 1 (1.6%), and PALB2 in 1 (1.6%). One patient had two concomitant germline mutations in BRCA2 and ATM. This is the first study on the frequency of germline mutations in BRCA1/2 and other breast cancer-predisposing genes in the southernmost provinces of Thailand. At least one pathogenic germline mutation was identified in 12.5% of the study patients, which suggests that genetic testing in this population has a high potential to provide benefits.

Exome sequencing vs targeted gene panels for the evaluation of nonimmune hydrops fetalis

Next-generation sequencing is increasingly used in prenatal diagnosis. Targeted gene panels and exome sequencing are both available, but the comparative diagnostic yields of these approaches are not known. We compared the diagnostic yield of exome sequencing with the simulated application of commercial targeted gene panels in a large cohort of fetuses with nonimmune hydrops fetalis. This was a secondary analysis of a cohort study of exome sequencing for nonimmune hydrops fetalis, in which recruitment, exome sequencing, and phenotype-driven variant analysis were completed in 127 pregnancies with features of nonimmune hydrops fetalis. An Internet search was performed to identify commercial laboratories that offer targeted gene panels for the prenatal evaluation of nonimmune hydrops fetalis or for specific disorders associated with nonimmune hydrops fetalis using the terms "non-immune hydrops fetalis," "fetal non-immune hydrops," "hydrops," "cystic hygroma," "lysosomal storage disease," "metabolic disorder," "inborn error of metabolism," "RASopathy," and "Noonan." Our primary outcome was the proportion of all genetic variants identified through exome sequencing that would have been identified if a targeted gene panel had instead been used. The secondary outcomes were the proportion of genetic variants that would have been identified by type of targeted gene panel (general nonimmune hydrops fetalis, RASopathy, or metabolic) and the percent of variants of uncertain significance that would have been identified on the panels, assuming 100% analytical sensitivity and specificity of panels for variants in the included genes. Exome sequencing identified a pathogenic or likely pathogenic variant in 37 of 127 cases (29%) in a total of 29 genes. A variant of uncertain significance, strongly suspected to be associated with the phenotype, was identified in another 12 cases (9%). We identified 7 laboratories that offer 10 relevant targeted gene panels; 6 are described as RASopathy panels, 3 as nonimmune hydrops fetalis panels, and 1 as a metabolic panel. The median number of genes included on each of these panels is 22, ranging from 11 to 148. Had a nonimmune hydrops fetalis targeted gene panel been used instead of exome sequencing, 13 to 15 of the 29 genes (45%-52%) identified in our nonimmune hydrops fetalis cohort would have been sequenced, and 19 to 24 of the pathogenic variants (51%-62%) would have been detected. The yield was predicted to be the lowest with the metabolic panel (11%) and the highest with the largest nonimmune hydrops fetalis panel (62%). The largest nonimmune hydrops fetalis targeted gene panel would have had a diagnostic yield of 18% compared with 29% with exome sequencing. The exome sequencing platform used provided 30× or more coverage for all of the exons on the commercial targeted gene panels, supporting our assumption of 100% analytical sensitivity for exome sequencing. The broader coverage of exome sequencing for genetically heterogeneous disorders, such as nonimmune hydrops fetalis, made it a superior alternative to targeted gene panel testing.

2 - Genetic mutations and signatures in lung cancer – findings from a whole exome sequencing platform

2 - Genetic mutations and signatures in lung cancer – findings from a whole exome sequencing platform

Illustration of temporal evolution in patients with metastatic renal cell carcinoma (mRCC) using both circulating tumor DNA (ctDNA) and tissue-based genomic data.

347 Background: We have previously demonstrated the feasibility of ctDNA assessment in mRCC and preliminarily showed agreement between ctDNA and tissue-based genomic findings (Zengin et al ESMO 2020). Our data suggested that the degree of agreement is dependent upon the temporal separation of blood and tissue samples. We sought to further explore this temporal impact in a separate validation cohort. Methods: Patients (pts) with mRCC who underwent ctDNA genomic profiling were identified. ctDNA analysis was performed using a CLIA-certified 73-74 gene panel (Guardant360). From this cohort we identified a subset of pts who also underwent tissue-based genomic profiling using either a whole exome sequencing platform (GemExtra [TGen, Phoenix, AZ]) or a targeted next generation sequencing platform (Foundation Medicine [Cambridge, MA] or Tempus [Chicago, IL]). Only alterations covered by both assays were included for the current analysis. The difference in the proportion of alterations detected on tissue and ctDNA was compared between these cohorts and at a 6-mo landmark using the χ2 test. Results: In total, ctDNA and tissue based genomic profiling was assessed in 112 pts (M:F, 81:31); with most common histology was clear cell (85.7%). Median time between ctDNA and tissue assessments was 9.8 months (IQR 1.15-23.7). When examining paired samples in which >1 ctDNA alteration was detected, 32% (43/133) of alterations detected on tissue were also detected in ctDNA. This proportion increased to 43% (29/67) when samples collected within 6 months of each other, and was 51% (28/55) in samples collected within 3 months of each other. There was no significant difference in the frequency of shared mutations between the cohorts (P=0.09; Table). Conclusions: Our study confirms that ctDNA and tissue-based genomic profiling continue to provide consistently high levels of agreement. Notably, the percentage of samples with ≥1 ctDNA alteration detected was significantly lower in both cohorts compared to previous studies in RCC. More shared alterations were found on ctDNA when both ctDNA and tissue-based assessment were obtained at closer intervals. [Table: see text]

Abstract PS18-41: Somatic mutational landscapes of invasive ductal and lobular carcinomas in the GENIE consortium cohort: Real-world gene actionability assessment of 8,756 breast cancer patients

Abstract Background: Invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC) of the breast typically present distinct clinicopathological characteristics and responsiveness to systemic therapy. In addition, breast cancer data from The Cancer Genome Atlas (TCGA) have shown these two pathological subtypes also present distinct genomic features when analyzed using DNA copy number arrays and whole exome sequencing platforms. More recently, the AACR Project GENIE Consortium, which is a publicly accessible international cancer registry of real-world data assembled through data sharing among leading cancer centers in the world, have allowed in-depth analyses of clinical actionability using patient-level data from clinical next-generation sequencing (NGS) assays. In this study, we assessed the somatic mutational landscapes of a large cohort (n = 8,756) of invasive breast carcinomas from 19 institutions participating in the GENIE Consortium Cohort (v8.0) and examined clinical actionability of unique mutations identified in each breast cancer subtype. Method: We assessed the eighth data release of the GENIE Consortium Cohort encompassing targeted sequencing data from 7,647 IDC and 1,109 ILC cases. Clinical features and somatic mutations including single-nucleotide variants, small indels, fusions, and copy number alterations (CNAs) were retrieved from cBioportal and SAGE Bionetworks. Gene actionability was examined using both OncoKB and CiVIC publicly available knowledgebases. All patient samples were de-identified and encoded with GENIE sample codes. Results: Patients with IDC tumors were 5 years younger than patients with ILC tumors at the time sequencing data was reported (median 55 versus median 60 years old, Kruskal-Wallis, p < 10e-10). Both IDC and ILC had on average 2 mutations per tested sample. Overall, IDC and ILC tumors had median fractions of 22% and 14% of their genomes altered, respectively (Kruskal-Wallis, p < 10e-10). An initial gene enrichment analysis including 938 genes with point mutations and small indels identified CDH1 [Log-ratio (LR) 4.66, p < 1e-10], RHOA (LR 2.81, p = 1.3e-10), PTK2B (LR 2.68, p = 5.2e-4), ERBB2 (LR 1.80, p < 1e-10), TBX3 (LR 1.72, p < 1e-10), FOXA1 (LR 1.49, p = 2.5e-10) and RUNX1 (LR 1.25, p = 3.1e-9) as genes significantly enriched in ILC tumors. On the other hand, mutations in GATA3 (LR = 1.67, p < 1e-10) and TP53 (LR = 1.55, p < 1e-10) were significantly enriched in IDC tumors. A further gene enrichment analysis for copy-number alterations in 1139 genes showed amplification in PARP1 (LR 1.55 p = 2.5e-3) and deep deletions in IKZF1 (LR 2.8, p = 2.2e-3) and CDH1 (LR = 1.88, p = 1.7e-4) as the most enriched genes with CNAs in ILC. In parallel, amplifications in AURKA (LR 3.2, p = 1e-8), PPM1D (LR 3.1, p <1e-10), RAD51C (LR 2.85, p = 3.8e-8), BRIP1 (LR 2.66, p < 1e-10), ERBB2 (LR 1.65, p = 1e-10), MYC (LR 1.64, p = 1e-10), CDK12 (LR 1.55, p = 2.6e-9), and COL22A1 (LR 1.19, p = 1.6e-5), and deep deletion in CDKN2A (LR 2.1, p = 1.9e-6) were enriched in IDC tumors. Among those enriched alterations for each histological subtype, the knowledgebase CiVIC did not present curated data available for genes TBX3, FOXA1, GATA3, COL22A1, BRIP1, PPM1D, and RAD51C. OncoKB only missed genes PTK2B and COL22A1. Conclusions: Real-world genomic data from the GENIE Consortium Cohort support that breast cancer presents distinct mutational landscapes for IDC and ILC tumors. For each histological subtype, we confirmed there are different levels of enrichments for shared mutations in actionable genes. Despite the fact that publicly available knowledgebases present comprehensive curated information about commonly mutated genes in cancer, we noticed that actionability data of important cancer driver genes were missed. Citation Format: Alessandro Leal, Patricia Taranto, Poliana BG Blasi, Carlos Tadeu Garrote, Fernando Moura. Somatic mutational landscapes of invasive ductal and lobular carcinomas in the GENIE consortium cohort: Real-world gene actionability assessment of 8,756 breast cancer patients [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-41.

Germline variants discovered in lymphoma patients undergoing tumor profiling: a case series.

Clinical tumor sequencing protocols often depend on obtaining germline DNA from patients to aid in the identification of de novo variants in the tumor, and therefore come with the possibility for the incidental discovery of germline variants. Ninety-one adult patients with lymphoma were consented and enrolled in MIONCOSEQ, an IRB-approved tumor profiling protocol that utilizes an exome sequencing platform. Charts were retrospectively reviewed for germline variants from sequencing results, personal and/or family history of cancer and genetic counseling referral. After review of the 91 lymphoma cases, seven (8%) cases revealed germline variants. Only one of these, CHEK2 p.I157T, has been previously recovered as a germline variant in lymphoma. Two of the seven patients received genetic counseling, two died before genetic counseling could be arranged and three did not follow-up with a genetics provider. None of the patients had a personal or family history that would have otherwise suggested an indication for cancer genetics referral, especially notable as lymphoma is not traditionally associated with inherited cancer syndromes. Importantly, as only two of seven patients had appropriate genetic counseling for their variant, timely genetic counseling should be a critical part of all tumor profiling platforms that use non-tumor DNA.

Abstract A19: HLA allele-specific loss of heterozygosity detection using augmented exome capture approach

Abstract Loss of human leukocyte antigen (HLA) is of increasing interest as a mechanism of cancer immune evasion and biomarker for cancer immunotherapy response. Each cancer patient has six class I HLA alleles that are capable of presenting a set of tumor-specific neoantigens. However, HLA alleles are often deleted in tumors, resulting in a loss of heterozygosity (LOH). When LOH occurs, neoantigen presentation is significantly impaired, potentially facilitating tumor immune evasion. Given the biologic impact of HLA LOH, there is a need for robust algorithms that can detect allele-specific HLA LOH in tumor samples. Here, we describe a novel computational approach to detect HLA LOH from exome sequencing, demonstrate the robustness of the method, and apply the method to calculate the frequencies of HLA LOH in key cancer types. We performed exome sequencing with augmented HLA region capture on the ImmunoID NeXT platform for tumor and normal samples of 184 patients across several cancer types and identified 430 nonhomozygous HLA genes. Next, we extracted the reads mapping to a custom HLA database and mapped them on to the patient-specific HLA alleles. For each allele, we calculated two key features: 1) the tumor b-allele frequency normalized by the native b-allele frequency and 2) the allele-specific tumor to normal coverage ratio. Using these two features, along with tumor purity and ploidy values, we trained a random forest model on a subset of the HLA genes (n=300). While standard copy-number variant (CNV) tools are unable to detect LOH in the polymorphic HLA genes, they can accurately measure deletions in their flanking regions, which we used to validate the accuracy of our allele-specific HLA LOH algorithm. In our test set (n=130), we found a high concordance between our allele-specific deletion calls and the generic deletion calls (94% accuracy, 0.85 F1 Score). When we constrained our test set to samples with high tumor content (>50%, n=40), we saw even stronger concordance (98% accuracy, 0.95 F1 Score). The only discordant call was a focal deletion within an HLA gene that was detected by our algorithm but missed by the CNV tool. Next, we ran our algorithm on patient samples of different cancer indications. For non-small cell lung cancer, we found a high frequency of patients affected by LOH (35%, 9 of 26), which is similar to frequencies previously reported in the literature. Furthermore, we found a lower frequency of melanoma tumors with LOH (15%, 7 of 48). In conclusion, we developed a novel algorithm to call allele-specific HLA LOH on the ImmunoID NeXT exome sequencing platform that augments coverage in the polymorphic HLA locus and demonstrated overall robust performance. The relatively high frequency of LOH events we detected in the melanoma and lung cancer samples suggests the importance of LOH analysis to inform cancer immunotherapy biomarker studies and personalized cancer therapies that depend on neoantigen presentation. Citation Format: Rachel Marty Pyke, Charles Abbott, Simo V. Zhang, Datta Mellacheruvu, John West, Richard Chen, Sean Michael Boyle. HLA allele-specific loss of heterozygosity detection using augmented exome capture approach [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A19.

A targeted sequencing panel identifies rare damaging variants in multiple genes in the cranial neural tube defect, anencephaly.

Neural tube defects (NTDs) affecting the brain (anencephaly) are lethal before or at birth, whereas lower spinal defects (spina bifida) may lead to lifelong neurological handicap. Collectively, NTDs rank among the most common birth defects worldwide. This study focuses on anencephaly, which despite having a similar frequency to spina bifida and being the most common type of NTD observed in mouse models, has had more limited inclusion in genetic studies. A genetic influence is strongly implicated in determining risk of NTDs and a molecular diagnosis is of fundamental importance to families both in terms of understanding the origin of the condition and for managing future pregnancies. Here we used a custom panel of 191 NTD candidate genes to screen 90 patients with cranial NTDs (n = 85 anencephaly and n = 5 craniorachischisis) with a targeted exome sequencing platform. After filtering and comparing to our in‐house control exome database (N = 509), we identified 397 rare variants (minor allele frequency, MAF < 1%), 21 of which were previously unreported and predicted damaging. This included 1 frameshift (PDGFRA), 2 stop‐gained (MAT1A; NOS2) and 18 missense variations. Together with evidence for oligogenic inheritance, this study provides new information on the possible genetic causation of anencephaly.

Evaluation of Next Generation Sequencing Platforms for Whole Exome Variant Analysis

Exome analysis is potentially a cost effective approach for detecting mutations in human body. It is preferred widely over WGS (Whole Genome Sequencing) since it focuses upon the coding and functional variants of the human genome. There are several platforms involved in whole exome sequencing, but the relative study of the same sample on two widely used exome sequencing platforms is taken into consideration here. The results of this study demonstrate the systematic evaluation of two widely used sequencing platforms. However, it reported accuracy of ~98% in terms of SNPs predicted by both the platforms, infact the number of SNPs exclusively falling into exonic region were found to harmonize inspite of the initial difference in raw SNP calling step.

GE-02 * CLINICAL TARGETED EXOME-BASED SEQUENCING FOR CHILDREN DIAGNOSED WITH BRAIN TUMORS: INITIAL RESULTS OF AN INSTITUTE-WIDE PROJECT ON PERSONALIZED MEDICINE

BACKGROUND: PROFILE (DFCI#11-104) is a cancer center-wide project that aims to assess feasibility and utility of identifying genomic and potential targetable alterations in pediatric brain tumor patients. METHODS: Eligible patients/families are offered enrollment. Tumor tissues (FFPE and frozen) are collected, DNA extracted and a CLIA-certified platform is applied. First-generation platform, OncoMap, a multiplex mass spectrometry-based platform, detected mutations in 471 loci from 41 cancer genes. This has been replaced by a second-generation platform, OncoPanel, a multiplexed targeted exome-sequencing platform, which surveys 275 cancer genes to identify mutations and copy number variations, in addition to 90 introns from 30 genes to detect rearrangements. Alterations are tiered based on clinical significance and results are communicated to the treating physician. RESULTS: Between February 2013 and June 2014, 79 patients were enrolled. 25 had insufficient tumor tissue. Of the remaining 54, 33 have been profiled thus far (Oncomap = 4; Oncopanel = 29). Diagnoses include low-grade glioma (11), medulloblastoma (6), ependymoma (6), GBM (3), PNET/pineoblastoma (3), ATRT (1), AA (1), meningioma (1) and pituitary adenoma (1). Estimated mean tumor purity was 76%. We detected 401 mutations, including two GBM patients with high mutation rates (which accounted for 314 of 401 mutations detected). Of these, one had deletion of the mismatch-repair gene MSH2. Excluding these two patients, we identified 87 mutations, of which 20 have evidence for potential clinical utility (including investigational or preclinical evidence). Alterations with clinical trial relevance included gains/duplications of BRAF observed in two glioma patients; four medulloblastoma patients were noted to harbor subtype-related alterations of relevance for trial enrollment. CONCLUSIONS: We show that it is possible to perform comprehensive genomic analyses of known cancer genes in the setting of a research protocol. Significant challenges remain in determining the clinical impact of utilizing personalized genomic data and integrating these profiles into the therapeutic decisions for children with brain tumors.

Abstract 4276: T200: a high depth targeted exome sequencing platform to identify actionable alterations in FFPE solid tumor samples

Abstract Background: We developed and established a high depth targeted sequencing platform of 202 genes (all exons) to identify actionable DNA alterations in tumor samples. We aimed a minimum of 500x depth and defined actionable if the aberration had a direct link to an available clinical treatment or clinical trial. Our platform was optimized for FFPE specimens and low input DNA while the data analysis pipeline was designed to detect low frequency mutations and copy number alterations. Over 500 tumor samples and matched normal were analyzed using this platform. Methods: Libraries were made from a minimum of 170 ng of FFPE DNA (tumors) or blood DNA (matched normal). Capture of 202 genes (over 5,000 exons) was performed using Nimblegen probes to a minimum of 50x fold enrichment. Captured libraries were sequenced using Illumina HiSeq2000. Duplicate reads were removed from the raw data and the reads were mapped hg19 reference genome. We used VarScan2 for calling somatic, germline and loss of heterozygosity SNVs and short indels. Copy number variation was also reported when significant gain/loss on an exon was detected. Additionally, we used other tolls to annotate the functional consequence of the SNVs, such as CanDrA to predict if an SNV is driver or passenger event. Results: We sequenced over 500 tumor samples and their normal match (blood). The mean coverage for most of samples was over 1500x, which allowed us to detect low frequency mutations (5% or higher) with accuracy. The most common disease sites analyzed where we found genetic alterations were: breast, colon, brain, ovary, endometrial, skin, lung, prostate, head and neck, sarcoma, kidney and stomach. About 98% of all tumors analyzed had at least one somatic alteration (SNVs or copy number) and although the vast majority of the samples analyzed were derived from FFPE blocks, the number of samples that failed was less than 5%. Among the most common genes mutated are several “actionable genes” such as BRAF, EGFR and KRAS. The samples tested in this platform were also tested on ion torrent Ampliseq 46 gene panel (46 overlapping genes) in a CLIA certified laboratory at MD Anderson Cancer Center with a very high level of concordance. Conclusion: With a very low level of failure and high depth provided, our results indicate that this platform can be used for FFPE tumor samples with high level of accuracy and sensitivity, showing that this platform is reliable to be implemented in clinical settings. Citation Format: Karina Eterovic, Ken Chen, Hao Zhao, Funda Meric-Bernstam, Raja Luthra, Aldape Kenneth, Mark Routbort, Scott Kopetz, Michael Davies, John de Groot, Stacy Moulder, Yong Mao, Chacha Horombe, Lin-ya Tang, Song Ping, Zhang Qingxiu, Ezzeddine Nader, Lan Zhang, Kenna M. Shaw, John Mendelsohn, Gordon B. Mills. T200: a high depth targeted exome sequencing platform to identify actionable alterations in FFPE solid tumor samples. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4276. doi:10.1158/1538-7445.AM2014-4276

Abstract 1999: A targeted exome sequencing platform for routine clinical molecular profiling of advanced thoracic malignancies relevant to targeted therapy.

Abstract Increasing number of oncogenic mutations important to the clinical management of advanced thoracic malignancies is being identified. To translate this into routine clinical testing environment, we developed and validated a targeted exome sequencing method using next generation sequencing (NGS) technology that is designed to detect both single nucleotide variants and short insertions or deletions (indels) by employing a custom designed target selection system. In each individual assay, 197 cancer related genes, including 12 clinically relevant genes (AKT1, AKT2, BRAF, EGFR, ERBB2, HRAS, KIT, KRAS, MEK1, NRAS, PIK3CA, and PTEN) are simultaneously sequenced from 100-500ng of total DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue. To date, we have performed this assay on over 250 FFPE tissues from Non-Small-Cell Lung Cancers (NSCLC), Small-Cell Lung Cancers (SCLC), and thymic malignancies. Mutations in all 12 clinically relevant genes including EGFR L858R, KRAS codon 12 missense mutations as well as EGFR exon 19 deletions, EGFR exon 20 insertions, and ERBB2 exon 20 insertions have been identified and validated with alternate technology in approximately 30% of all samples tested. Compared with conventional capillary sequencing, which typically requires in excess of 25% mutant DNA, this assay can detect mutations with 5% - 10% mutant allele frequency. This is a robust, reliable and cost effective assay that not only is suitable for prospective molecular profiling of clinically relevant targets, but also provides a powerful tool for retrospective analysis of existing FFPE tissues from clinical trials. Citation Format: Christopher C. Lau, Keith Killian, Yuelin J. Zhu, Yonghong Wang, Marbin Pineda, Anish Thomas, Yisong Wang, Betsy Morrow, Liqiang Xi, Mark Raffeld, Giuseppe Giaccone, Paul S. Meltzer. A targeted exome sequencing platform for routine clinical molecular profiling of advanced thoracic malignancies relevant to targeted therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1999. doi:10.1158/1538-7445.AM2013-1999

Exome Sequencing by Targeted Enrichment

This unit describes methods for targeted enrichment of the exon-coding portions of the genome using Agilent SureSelect Human All Exon 50 Mb and Roche Nimblegen SeqCap EZ Exome platforms. Each platform targets and enriches a large overlapping portion of the greater human exome. The protocols here describe the biochemical procedures used to enrich exomic DNA with each platform, including recommended modifications to the manufacturers' protocols. In addition, a brief description of the sequencing protocol and estimation of the needed amount of sequencing for each platform is included. Finally, a detailed analytical pipeline for processing the subsequent data is described. These protocols focus specifically on human exome sequencing platforms, but can be applied with some modification to other organisms and targeted enrichment approaches.

Performance comparison of exome DNA sequencing technologies

Whole exome sequencing by high-throughput sequencing of target-enriched genomic DNA (exome-seq) has become common in basic and translational research as a means of interrogating the interpretable part of the human genome at relatively low cost. We present a comparison of three major commercial exome sequencing platforms from Agilent, Illumina and Nimblegen applied to the same human blood sample. Our results suggest that the Nimblegen platform, which is the only one to use high-density overlapping baits, covers fewer genomic regions than the other platforms but requires the least amount of sequencing to sensitively detect small variants. Agilent and Illumina are able to detect a greater total number of variants with additional sequencing. Illumina captures untranslated regions, which are not targeted by the Nimblegen and Agilent platforms. We also compare exome sequencing and whole genome sequencing (WGS) of the same sample, demonstrating that exome sequencing can detect additional small variants missed by WGS.