Targeted DNA- and RNA-based next-generation sequencing for identifying MET exon 14 alterations in pulmonary sarcomatoid carcinoma.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e20591-e20591
Author(s):  
Jianming Ying ◽  
Lianju Gao ◽  
Yan Li ◽  
Bing Liu ◽  
Lamei Deng ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Splice Site ◽  
Point Mutations ◽  
Sarcomatoid Carcinoma ◽  
Exon Skipping ◽  
Performance Comparison ◽  
Next Generation ◽  
Dna And Rna ◽  
Pulmonary Sarcomatoid Carcinoma ◽  
Generation Sequencing

e20591 Background:Highly diverse somatic splice site alteration at MET exon 14 ( METex14) result in exon skipping, which is supposed to be a therapeutic target in NSCLC. Here we report detection of METex14 alterations using targeted DNA- and RNA-based Next-Generation Sequencing (NGS) in pulmonary sarcomatoid carcinoma (PSC) with a high frequency of METex14 skipping. Methods: Tumor specimens were collected from 100 Chinese PSC patients. DNA and RNA were subject to targeted NGS, allowing the detection of somatic splice site alterations and intragenic METex14 skipping respectively. Then, somatic mutations that lead to METex14 skipping were recognized. Meanwhile, cross-platform performance comparison for detecting MET exon 14 skipping was achieved. Moreover, Sanger sequencing was also performed on the METex14-positive specimens. Results: So far, we have detected genetic aberrations in 34 FFPE samples. For RNA-based NGS, METex14 skipping was identified in 8 (23.5%) of 34 patient cases. The population frequency was accordant to the reported percent of 22% (PMID: 26215952). And 5 (62.5%) of 8 METex14-positive patients were detected somatic splice site mutations by DNA-based NGS, including 4 cases with known point mutations at the 3’ splice region and one case with a novel deletion (chr7: 116412027- 116412042) at MET exon14 region, which is newly discovered. No somatic mutation was found at the splice junctions of METex14 in the remaining 3 samples using DNA-based NGS. Conclusions: Mutational events of MET leading to exon 14 skipping are frequent occurred in Chinese PSC patients. Our study also suggests that RNA-based NGS could identify more METex14 skipping, and thus provide more accurate results than DNA-based NGS.

scholarly journals Detection and Functional Verification of Noncanonical Splice Site Mutations in Hereditary Deafness

2021 ◽  
Vol 12 ◽  
Author(s):  
Penghui Chen ◽  
Longhao Wang ◽  
Yongchuan Chai ◽  
Hao Wu ◽  
Tao Yang
Keyword(s):  
Next Generation Sequencing ◽  
Splice Site ◽  
Negative Impact ◽  
Intron Retention ◽  
Exon Skipping ◽  
Next Generation Sequencing Data ◽  
Functional Verification ◽  
Next Generation ◽  
Hereditary Deafness ◽  
Generation Sequencing

Splice site mutations contribute to a significant portion of the genetic causes for mendelian disorders including deafness. By next-generation sequencing of 4 multiplex, autosomal dominant families and 2 simplex, autosomal recessive families with hereditary deafness, we identified a variety of candidate pathogenic variants in noncanonical splice sites of known deafness genes, which include c.1616+3A > T and c.580G > A in EYA4, c.322-57_322-8del in PAX3, c.991-15_991-13del in DFNA5, c.6087-3T > G in PTPRQ and c.164+5G > A in USH1G. All six variants were predicted to affect the RNA splicing by at least one of the computational tools Human Splicing Finder, NNSPLICE and NetGene2. Phenotypic segregation of the variants was confirmed in all families and is consistent with previously reported genotype-phenotype correlations of the corresponding genes. Minigene analysis showed that those splicing site variants likely have various negative impact including exon-skipping (c.1616+3A > T and c.580G > A in EYA4, c.991-15_991-13del in DFNA5), intron retention (c.322-57_322-8del in PAX3), exon skipping and intron retention (c.6087-3T > G in PTPRQ) and shortening of exon (c.164+5G > A in USH1G). Our study showed that the cryptic, noncanonical splice site mutations may play an important role in the molecular etiology of hereditary deafness, whose diagnosis can be facilitated by modified filtering criteria for the next-generation sequencing data, functional verification, as well as segregation, bioinformatics, and genotype-phenotype correlation analysis.

The applications of next-generation sequencing (NGS) in drug development for cancer therapy

2020 ◽  
Vol 16 ◽  
Author(s):  
Pelin Telkoparan-Akillilar ◽  
Dilek Cevik
Keyword(s):  
Next Generation Sequencing ◽  
Drug Discovery ◽  
Cancer Therapy ◽  
Target Identification ◽  
Rapid Development ◽  
Next Generation ◽  
Biomedical Sciences ◽  
Dna And Rna ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Background: Numerous sequencing techniques have been progressed since the 1960s with the rapid development of molecular biology studies focusing on DNA and RNA. Methods: a great number of articles, book chapters, websites are reviewed, and the studies covering NGS history, technology and applications to cancer therapy are included in the present article. Results: High throughput next-generation sequencing (NGS) technologies offer many advantages over classical Sanger sequencing with decreasing cost per base and increasing sequencing efficiency. NGS technologies are combined with bioinformatics software to sequence genomes to be used in diagnostics, transcriptomics, epidemiologic and clinical trials in biomedical sciences. The NGS technology has also been successfully used in drug discovery for the treatment of different cancer types. Conclusion: This review focuses on current and potential applications of NGS in various stages of drug discovery process, from target identification through to personalized medicine.

scholarly journals Development and Optimization of Metagenomic Next-Generation Sequencing Methods for Cerebrospinal Fluid Diagnostics

2018 ◽  
Vol 56 (9) ◽  
Author(s):  
Patricia J. Simner ◽  
Heather B. Miller ◽  
Florian P. Breitwieser ◽  
Gabriel Pinilla Monsalve ◽  
Carlos A. Pardo ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Next Generation Sequencing ◽  
Sample Pretreatment ◽  
Standard Of Care ◽  
Nucleic Acid Amplification ◽  
Next Generation ◽  
Bead Beating ◽  
Dna And Rna ◽  
Positive Threshold ◽  
Generation Sequencing

ABSTRACT The purpose of this study was to develop and optimize different processing, extraction, amplification, and sequencing methods for metagenomic next-generation sequencing (mNGS) of cerebrospinal fluid (CSF) specimens. We applied mNGS to 10 CSF samples with known standard-of-care testing (SoC) results (8 positive and 2 negative). Each sample was subjected to nine different methods by varying the sample processing protocols (supernatant, pellet, neat CSF), sample pretreatment (with or without bead beating), and the requirement of nucleic acid amplification steps using DNA sequencing (DNASeq) (with or without whole-genome amplification [WGA]) and RNA sequencing (RNASeq) methods. Negative extraction controls (NECs) were used for each method variation (4/CSF sample). Host depletion (HD) was performed on a subset of samples. We correctly determined the pathogen in 7 of 8 positive samples by mNGS compared to SoC. The two negative samples were correctly interpreted as negative. The processing protocol applied to neat CSF specimens was found to be the most successful technique for all pathogen types. While bead beating introduced bias, we found it increased the detection yield of certain organism groups. WGA prior to DNASeq was beneficial for defining pathogens at the positive threshold, and a combined DNA and RNA approach yielded results with a higher confidence when detected by both methods. HD was required for detection of a low-level-positive enterovirus sample. We demonstrate that NECs are required for interpretation of these complex results and that it is important to understand the common contaminants introduced during mNGS. Optimizing mNGS requires the use of a combination of techniques to achieve the most sensitive, agnostic approach that nonetheless may be less sensitive than SoC tools.

scholarly journals Performance Comparison of Different Analytic Methods in Proficiency Testing for Mutations in the BRAF, EGFR, and KRAS Genes: A Study of the College of American Pathologists Molecular Oncology Committee

2019 ◽  
Vol 143 (10) ◽  
pp. 1203-1211 ◽  
Author(s):  
Joel T. Moncur ◽  
Angela N. Bartley ◽  
Julia A. Bridge ◽  
Suzanne Kamel-Reid ◽  
Alexander J. Lazar ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Proficiency Testing ◽  
Critical Issue ◽  
Performance Comparison ◽  
Test Method ◽  
Next Generation ◽  
Molecular Oncology ◽  
Assay Methods ◽  
Commercial Kits ◽  
Generation Sequencing

Context.— The performance of laboratory testing has recently come under increased scrutiny as part of important and ongoing debates on regulation and reimbursement. To address this critical issue, this study compares the performance of assay methods, using either commercial kits or assays designed and implemented by single laboratories (“home brews”), including next-generation sequencing methods, on proficiency testing provided by the College of American Pathologists Molecular Oncology Committee. Objective.— To compare the performance of different assay methods on College of American Pathologists proficiency testing for variant analysis of 3 common oncology analytes: BRAF, EGFR, and KRAS. Design.— There were 6897 total responses across 35 different proficiency testing samples interrogating 13 different variants as well as wild-type sequences for BRAF, EGFR, and KRAS. Performance was analyzed by test method, kit manufacturer, variants tested, and preanalytic and postanalytic practices. Results.— Of 26 reported commercial kits, 23 achieved greater than 95% accuracy. Laboratory-developed tests with no kit specified demonstrated 96.8% or greater accuracy across all 3 analytes (1123 [96.8%] acceptable of 1160 total responses for BRAF; 848 [97.5%] acceptable of 870 total responses for EGFR; 942 [97.0%] acceptable of 971 total responses for KRAS). Next-generation sequencing platforms (summed across all analytes and 2 platforms) demonstrated 99.4% accuracy for these analytes (165 [99.4%] acceptable of 166 total next-generation sequencing responses). Slight differences in performance were noted among select commercial assays, dependent upon the particular design and specificity of the assay. Wide differences were noted in the lower limits of neoplastic cellularity laboratories accepted for testing. Conclusions.— These data demonstrate the high degree of accuracy and comparable performance across all laboratories, regardless of methodology. However, care must be taken in understanding the diagnostic specificity and reported analytic sensitivity of individual methods.

scholarly journals Towards Improving Embryo Prioritization: Parallel Next Generation Sequencing of DNA and RNA from a Single Trophectoderm Biopsy

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Noga Fuchs Weizman ◽  
Brandon A. Wyse ◽  
Ran Antes ◽  
Zenon Ibarrientos ◽  
Mugundhine Sangaralingam ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Trophectoderm Biopsy ◽  
Next Generation ◽  
Dna And Rna ◽  
Generation Sequencing

Next-Generation Sequencing Technology Reveals a Characteristic Pattern of Molecular Mutations in 75% of Chronic Myelomonocytic Leukemia (CMML) by Detecting Frequent Alterations in TET2, RUNX1, CBL, and RAS.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 417-417 ◽  
Author(s):  
Alexander Kohlmann ◽  
Vera Grossmann ◽  
Claudia Haferlach ◽  
Beray Kazak ◽  
Sonja Schindela ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Mutation Screening ◽  
Point Mutations ◽  
Chronic Myelomonocytic Leukemia ◽  
Melting Curve Analysis ◽  
Next Generation ◽  
Monosomy 7 ◽  
Equity Ownership ◽  
Myelomonocytic Leukemia ◽  
Generation Sequencing

Abstract Abstract 417 Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic malignancy that is characterized by features of both a myeloproliferative neoplasm and a myelodysplastic syndrome. Here, we analyzed 81 CMML cases (45 CMML-1, 36 CMML-2). In chromosome banding analysis 59/76 (77.6%) patients showed a normal karyotype (data not availabel in 5 cases). Recurrent chromosome aberrations were trisomy 8 (n=6; 7.9%), monosomy 7 (n=3; 3.9%), and loss of the Y-chromosome (n=5; 6.6%). Fluorescence in situ hybridization (FISH) detected the deletion of one allele of the TET2 gene in 4/71 cases (5.6%). Thus, the majority of cases can not be genetically characterized by these techniques. Therefore, we applied next-generation sequencing (NGS) technology to investigate 7 candidate genes, represented by 43 PCR-products, at known mutational hotspot regions, i.e. CBL (exons 8 and 9), JAK2 (exons 12 and 14), MPL (exon 10), NRAS (exons 2 and 3), and KRAS (exons 2 and 3). In addition, complete coding regions were analyzed for RUNX1 (beta isoform) and TET2. NGS was performed using 454 FLX amplicon chemistry (Roche Diagnostics Corporation, Branford, CT). The median number of base pairs sequenced per patient was 9.24 Mb. For each target gene a median of 911 reads was generated (coverage range: 736-fold to 1606-fold). This approach allowed a high-sensitive detection of molecular mutations, e.g. detecting the JAK2 V617F mutation down to 1.16% of reads. In total, 146 variances were detected by this comprehensive molecular mutation screening (GS Amplicon Variant Analyzer software version 2.0.01). In 80.4% of variances consistent results were obtained after confirming NGS mutations with melting curve analysis and conventional sequencing. In the remaining discrepant variances (19.6%) NGS deep-sequencing outperformed conventional methods due to the higher sensitivity of the platform. After excluding 19 polymorphisms or silent mutations 127 distinct mutations in 61/81 patients (75.3%) were detected: CBL: n=21 point mutations and one deletion (18 bp) found in 20 cases (24%); JAK2: n=8 mutations (V617F) found in 8 cases (9.8%); MPL: no mutations found; NRAS: n=23 mutations found in 18 cases (22.2%); KRAS: n=12 mutations found in 10 cases (12.3%); RUNX1: n=6 point mutations and one deletion (14 bp) found in 7 cases (8.6%); and TET2: n=49 point mutations and 6 deletions (2-19 bp; 5/6 out-of-frame) found in 41 cases (50.6%). Furthermore, in 21 TET2-mutated cases 11 mutations previously described in the literature were detectable, whereas 28 cases carried novel mutations (n=28). In the cohort of TET2-mutated cases 17/41 (41.3%) patients harbored TET2 abnormalities as sole aberration. Interestingly, CBL mutations were found to be significantly associated with TET2 mutations (Fisher's exact test, p=0.008). In 17 of 20 (85.0%) CBL-mutated cases TET2 abnormalities were concomitantly observed. In contrast, no significant associations were found between any of the point mutations or deletions and the karyotype. There were also no associations observed between molecular aberrations and the diagnostic categories CMML-1 and CMML-2. With respect to clinical data a trend for better outcome was seen for patients that carried either or both TET2 and CBL mutations (median OS 130.4 vs. 17.3 months, alive at 2 yrs: 72.0% vs. 43.9%; p=0.13). In conclusion, 75.3% of CMMLs harbored at least one molecular aberration. In median 2 mutations per case were observed. Compared to limited data from the literature we detected not only a higher frequency of CBL mutations, but also add data on novel TET2 mutations. In particular, comprehensive NGS screening here for the first time has demonstrated its strength to further genetically characterize and delineate prognostic groups within this type of hematological malignancy. Disclosures: Kohlmann: MLL Munich Leukemia Laboratory: Employment. Grossmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Kazak:MLL Munich Leukemia Laboratory: Employment. Schindela:MLL Munich Leukemia Laboratory: Employment. Weiss:MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Sign in / Sign up

Export Citation Format

Share Document