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https://www.readbyqxmd.com/read/28075488/generating-exome-enriched-sequencing-libraries-from-formalin-fixed-paraffin-embedded-tissue-dna-for-next-generation-sequencing
#1
Beth A Marosy, Brian D Craig, Kurt N Hetrick, P Dane Witmer, Hua Ling, Sean M Griffith, Benjamin Myers, Elaine A Ostrander, Janet L Stanford, Lawrence C Brody, Kimberly F Doheny
This unit describes a technique for generating exome-enriched sequencing libraries using DNA extracted from formalin-fixed paraffin-embedded (FFPE) samples. Utilizing commercially available kits, we present a low-input FFPE workflow starting with 50 ng of DNA. This procedure includes a repair step to address damage caused by FFPE preservation that improves sequence quality. Subsequently, libraries undergo an in-solution-targeted selection for exons, followed by sequencing using the Illumina next-generation short-read sequencing platform...
January 11, 2017: Current Protocols in Human Genetics
https://www.readbyqxmd.com/read/28074285/t-cell-receptor-repertoire-usage-in-cancer-as-a-surrogate-marker-for-immune-responses
#2
REVIEW
David Schrama, Cathrin Ritter, Jürgen C Becker
Characterizing the interaction of cancer cells with the host adaptive immune system is critical for understanding tumor immunology and the modus operandi of immunotherapeutic interventions to treat cancer. As the key cellular effectors of adaptive immunity, T cells are endowed with specialized receptors (the T cell receptor; TCR), to recognize and to eliminate cancer cells. The diversity of the TCR repertoire results from specialized genetic diversification mechanisms that generate an incredible variability allowing recognizing extensive collections of antigens...
January 10, 2017: Seminars in Immunopathology
https://www.readbyqxmd.com/read/28049581/validation-of-a-next-generation-sequencing-panel-for-detection-of-hotspot-cancer-mutations-in-a-clinical-laboratory
#3
Reza Shahsiah, Jenefer DeKoning, Saeed Samie, Seyed Ziaeddin Latifzadeh, Zahra Mehdizadeh Kashi
Recent advances in sequencing technologies have enabled us to scrutinize the versatile underlying mechanisms of cancer more precisely. However, adopting these new sophisticated technologies is challenging for clinical labs as it involves complex workflows, and requires validation for diagnostic purposes. The aim of this work is towards the analytical validation of a next generation sequencing (NGS) panel for cancer hotspot mutation analysis. Characterized formalin-fixed paraffin-embedded (FFPE) samples including biopsy specimens and cell-lines were examined by NGS methods utilizing the Ion Torrent™ Oncomine™ Focus DNA Assay and the PGM™ platform...
December 16, 2016: Pathology, Research and Practice
https://www.readbyqxmd.com/read/27995571/evaluating-the-feasibility-of-dna-methylation-analyses-using-long-term-archived-brain-formalin-fixed-paraffin-embedded-samples
#4
Stine T Bak, Nicklas H Staunstrup, Anna Starnawska, Tina F Daugaard, Jens R Nyengaard, Mette Nyegaard, Anders Børglum, Ole Mors, Karl-Anton Dorph-Petersen, Anders L Nielsen
We here characterize the usability of archival formalin-fixed paraffin-embedded (FFPE) brain tissue as a resource for genetic and DNA methylation analyses with potential relevance for brain-manifested diseases. We analyzed FFPE samples from The Brain Collection, Aarhus University Hospital Risskov, Denmark (AUBC), constituting 9479 formalin-fixated brains making it one of the largest collections worldwide. DNA extracted from brain FFPE tissue blocks was interrogated for quality and usability in genetic and DNA methylation analyses by different molecular techniques...
December 19, 2016: Molecular Neurobiology
https://www.readbyqxmd.com/read/27939411/identification-of-novel-mutations-in-japanese-ovarian-clear-cell-carcinoma-patients-using-optimized-targeted-ngs-for-clinical-diagnosis
#5
Yoshiaki Maru, Naotake Tanaka, Miki Ohira, Makiko Itami, Yoshitaka Hippo, Hiroki Nagase
OBJECTIVE: Ovarian clear cell carcinoma (OCCC) is an aggressive ovarian cancer with a higher frequency in Japan and often becomes chemorefractory disease. Reliable genetic diagnosis is essential to affirm the success of precision medicine for OCCC treatment. The aim of this study is, therefore, to identify novel mutations in OCCCs and develop a feasible clinical next generation sequencing (NGS) approach using formalin-fixed paraffin-embedded (FFPE) rather than preferable but not always available fresh frozen (FF) samples...
December 8, 2016: Gynecologic Oncology
https://www.readbyqxmd.com/read/27930669/development-of-rna-fish-assay-for-detection-of-oncogenic-fgfr3-tacc3-fusion-genes-in-ffpe-samples
#6
Masahiro Kurobe, Takahiro Kojima, Kouichi Nishimura, Shuya Kandori, Takashi Kawahara, Takayuki Yoshino, Satoshi Ueno, Yuichi Iizumi, Koji Mitsuzuka, Yoichi Arai, Hiroshi Tsuruta, Tomonori Habuchi, Takashi Kobayashi, Yoshiyuki Matsui, Osamu Ogawa, Mikio Sugimoto, Yoshiyuki Kakehi, Yoshiyuki Nagumo, Masakazu Tsutsumi, Takehiro Oikawa, Koji Kikuchi, Hiroyuki Nishiyama
INTRODUCTION AND OBJECTIVES: Oncogenic FGFR3-TACC3 fusions and FGFR3 mutations are target candidates for small molecule inhibitors in bladder cancer (BC). Because FGFR3 and TACC3 genes are located very closely on chromosome 4p16.3, detection of the fusion by DNA-FISH (fluorescent in situ hybridization) is not a feasible option. In this study, we developed a novel RNA-FISH assay using branched DNA probe to detect FGFR3-TACC3 fusions in formaldehyde-fixed paraffin-embedded (FFPE) human BC samples...
2016: PloS One
https://www.readbyqxmd.com/read/27911273/robust-detection-of-immune-transcripts-in-ffpe-samples-using-targeted-rna-sequencing
#7
Benjamin E Paluch, Sean T Glenn, Jeffrey M Conroy, Antonios Papanicolau-Sengos, Wiam Bshara, Angela R Omilian, Elizabeth Brese, Mary Nesline, Blake Burgher, Jonathan Andreas, Kunle Odunsi, Kevin Eng, Ji He, Maochun Qin, Mark Gardner, Lorenzo Galluzzi, Carl D Morrison
Current criteria for identifying cancer patients suitable for immunotherapy with immune checkpoint blockers (ICBs) are subjective and prone to misinterpretation, as they mainly rely on the visual assessment of CD274 (best known as PD-L1) expression levels by immunohistochemistry (IHC). To address this issue, we developed a RNA sequencing (RNAseq)-based approach that specifically measures the abundance of immune transcripts in formalin-fixed paraffin embedded (FFPE) specimens. Besides exhibiting superior sensitivity as compared to whole transcriptome RNAseq, our assay requires little starting material, implying that it is compatible with RNA degradation normally caused by formalin...
November 29, 2016: Oncotarget
https://www.readbyqxmd.com/read/27910019/fluorescence-in-situ-hybridization-on-tissue-sections
#8
Alvin S T Lim, Tse Hui Lim
Formalin-fixed paraffin-embedded (FFPE) tissues are typically the specimens available for FISH analysis of solid tissues, particularly of tumor specimens. Occasionally, tissue cores constructed as tissue microarrays from several patients are presented for simultaneous evaluation. FFPE sections can also be prepared from cell blocks derived from cell suspensions. The interphase fluorescence in situ hybridization assay employs specific nucleic acid sequences (probes) that target complementary sequences of interest to detect gains or losses of genes/gene loci or a fusion gene within the tissue...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27895798/exploring-the-impact-of-egfr-t790m-neighboring-snps-on-arms-based-t790m-mutation-assay
#9
Sanpeng Xu, Yaqi Duan, Liping Lou, Fengjuan Tang, Juan Shou, Guoping Wang
The present study aimed to explore the influence of T790M neighboring single nucleotide polymorphism (SNP) on the sensitivity of amplification refractory mutation system (ARMS)-based T790M mutation assay. Three ARMS-quantitative polymerase chain reaction (qPCR) systems (system 1 had a forward ARMS primer without rs1050171, system 2 included a forward ARMS primer with rs1050171 and system 3 contained the above two forward ARMS primers) were used to detect the T790M mutation in two series plasmid samples and genomic DNA (gDNA) of the cell line H1975...
November 2016: Oncology Letters
https://www.readbyqxmd.com/read/27858973/detection-and-genotyping-of-human-papillomaviruses-from-archival-formalin-fixed-tissue-samples
#10
Koenraad Van Doorslaer, Zigui Chen, Alison A McBride
Pathology departments routinely process and store formalin-fixed, paraffin-embedded (FFPE) tissue samples for clinical diagnosis. These collections often contain decades' worth of samples and represent a treasure trove of specimens that can be analyzed for retrospective epidemiological studies, diagnostics, and pathogen discovery. Accurate amplification and sequencing of DNA from these samples is critical for the usability of these FFPE samples. Here we present a collection of protocols that describe extraction of DNA from FFPE tissues, PCR amplification of human papillomavirus DNA, and subsequent genotyping of the infecting virus...
November 18, 2016: Current Protocols in Microbiology
https://www.readbyqxmd.com/read/27846907/copy-number-analysis-by-low-coverage-whole-genome-sequencing-using-ultra-low-input-dna-from-formalin-fixed-paraffin-embedded-tumor-tissue
#11
Tanjina Kader, David L Goode, Stephen Q Wong, Jacquie Connaughton, Simone M Rowley, Lisa Devereux, David Byrne, Stephen B Fox, Gisela Mir Arnau, Richard W Tothill, Ian G Campbell, Kylie L Gorringe
Unlocking clinically translatable genomic information, including copy number alterations (CNA), from formalin-fixed paraffin-embedded (FFPE) tissue is challenging due to low yields and degraded DNA. We describe a robust, cost-effective low-coverage whole genome sequencing (LC WGS) method for CNA detection using 5 ng of FFPE-derived DNA. CN profiles using 100 ng or 5 ng input DNA were highly concordant and comparable with molecular inversion probe (MIP) array profiles. LC WGS improved CN profiles of samples that performed poorly using MIP arrays...
November 15, 2016: Genome Medicine
https://www.readbyqxmd.com/read/27834959/k-ras-oncogene-mutation-in-pterygium
#12
B T Ozturk, M S Yıldırım, A Zamani, B Bozkurt
PurposePterygium is claimed to be a benign proliferation triggered by prolonged exposure to ultraviolet radiation. The frequency of K-ras oncogene mutation, which is among the initial mutations in tumorigenesis, is evaluated in this study.Patients and methodsIn this prospective randomized clinical, trial pterygium tissues and normal conjunctiva tissue specimens are obtained from the superotemporal quadrant of patients who underwent primary pterygium excision with autograft transplantation. DNA extraction from tissues was performed using the QIAamp DNA FFPE tissue kit...
November 11, 2016: Eye
https://www.readbyqxmd.com/read/27826616/development-and-validation-of-an-ultra-high-sensitive-next-generation-sequencing-assay-for-molecular-diagnosis-of-clinical-oncology
#13
Jiao Liang, Yaoguang She, Jiaqi Zhu, Longgang Wei, Lanying Zhang, Lianju Gao, Yan Wang, Jing Xing, Yang Guo, Xuehong Meng, Peiyu Li
Dramatic improvements in the understanding of oncogenes have spurred the development of molecular target therapies, which created an exigent need for comprehensive and rapid clinical genotyping. Next-generation sequencing (NGS) assay with increased performance and decreased cost is becoming more widely used in clinical diagnosis. However, the optimization and validation of NGS assay remain a challenge, especially for the detection of somatic variants at low mutant allele fraction (MAF). In the present study, we developed and validated the Novogene Comprehensive Panel (NCP) based on targeted capture for NGS analysis...
November 2016: International Journal of Oncology
https://www.readbyqxmd.com/read/27801728/egfr-mutational-profiling-in-non-small-cell-lung-cancer-the-clinical-performance-of-a-sensitive-reverse-hybridization-assay
#14
Gernot Kriegshäuser, Dietmar Enko, Michael Novy, Anita Reitmayr, Almute Loidl, Gabriele Halwachs-Baumann, Christian Oberkanins
In patients with non-small cell lung cancer (NSCLC), epidermal growth factor receptor (EGFR) mutations have been associated with the tumor response to targeted therapy with EGFR tyrosine kinase inhibitors. Although labor intensive and not very sensitive (ie, an analytical sensitivity of 20%), direct sequencing is widely used for mutation detection. This study aimed at evaluating the potential of a test strip-based reverse-hybridization assay (EGFR StripAssay), designed for the simultaneous detection of 16 mutations in exons 18 to 21 of the EGFR gene, to sensitively identify EGFR mutation in DNA from NSCLC tissue samples...
October 31, 2016: Applied Immunohistochemistry & Molecular Morphology: AIMM
https://www.readbyqxmd.com/read/27793035/performance-of-multiplicom-s-brca-mastr-dx-kit-on-the-detection-of-brca1-and-brca2-mutations-in-fresh-frozen-ovarian-and-breast-tumor-samples
#15
Cindy Badoer, Céline Garrec, Dirk Goossens, Gillian Ellison, John Mills, Mélina Dzial, Hakim El Housni, Sarah Berwouts, Paola Concolino, Virginie Guibert-Le Guevellou, Capucine Delnatte, Jurgen Del Favero, Ettore Capoluongo, Stéphane Bézieau
Next-generation sequencing (NGS) has enabled new approaches for detection of mutations in the BRCA1 and BRCA2 genes responsible for hereditary breast and ovarian cancer (HBOC). The search for germline mutations in the BRCA1 and BRCA2 genes is of importance with respect to oncogenetic and surgical (bilateral mastectomy, ovariectomy) counselling. Testing tumor material for BRCA mutations is of increasing importance for therapeutic decision making as the poly ADP ribose polymerase (PARP) inhibitor, olaparib, is now available to treat patients with specific forms of ovarian cancer and BRCA mutations...
October 25, 2016: Oncotarget
https://www.readbyqxmd.com/read/27792260/targeted-next-generation-sequencing-enables-reliable-detection-of-her2-erbb2-status-in-breast-cancer-and-provides-ancillary-information-of-clinical-relevance
#16
Nicole Pfarr, Roland Penzel, Volker Endris, Clemens Lier, Christa Flechtenmacher, Anna-Lena Volckmar, Martina Kirchner, Jan Budczies, Jonas Leichsenring, Esther Herpel, Aurelia Noske, Wilko Weichert, Andreas Schneeweiss, Peter Schirmacher, Hans-Peter Sinn, Albrecht Stenzinger
HER2-positive breast cancers are a heterogeneous group of tumors, which share amplification and overexpression of HER2. In routine diagnostics, the HER2 (ERBB2) status is currently assessed by immunohistochemistry (IHC) and in-situ hybridization (ISH). Data on targeted next-generation sequencing (NGS) approaches that could be used to determine the HER2 status are sparse. Employing two breast cancer-related gene panels, we performed targeted NGS of 41 FFPE breast cancers for which full pathological work-up including ISH and IHC results were available...
October 28, 2016: Genes, Chromosomes & Cancer
https://www.readbyqxmd.com/read/27791984/droplet-digital-pcr-is-a-powerful-technique-to-demonstrate-frequent-fgfr1-duplication-in-dysembryoplastic-neuroepithelial-tumors
#17
Frédéric Fina, Doriane Barets, Carole Colin, Corinne Bouvier, Laëtitia Padovani, Isabelle Nanni-Metellus, L'Houcine Ouafik, Didier Scavarda, Andrey Korshunov, David T W Jones, Dominique Figarella-Branger
Dysembryoplastic neuroepithelial tumors (DNT) share V600E mutation in the BRAF gene with other low grade neuroepithelial tumors (LGNTs). FGFR1 internal tandem duplication of the tyrosine-kinase domain (FGFR1-ITD), another genetic alteration that also leads to MAP kinase pathway alteration, has been previously reported in LGNTs by whole-genome sequencing. In the present study we searched for FGFR1-ITD by droplet digital PCR (DDPCR™) and for FGFR1 point mutations by HRM-sequencing in a series of formalin-fixed paraffin-embedded (FFPE) LGNTs including 12 DNT, 2 oligodendrogliomas lacking IDH mutation and 1p/19q co- deletion (pediatric-type oligodendrogliomas; PTOs), 3 pediatric diffuse astrocytomas (PDAs), 14 gangliogliomas (GGs) and 5 pilocytic astrocytomas (PAs)...
October 25, 2016: Oncotarget
https://www.readbyqxmd.com/read/27790419/quantification-of-dna-extracted-from-formalin-fixed-paraffin-embeded-tissue-comparison-of-three-techniques-effect-on-pcr-efficiency
#18
Dushyant Kumar, Manoj Kumar Panigrahi, Moushumi Suryavanshi, Anurag Mehta, Kandarpa Kumar Saikia
INTRODUCTION: Mutation detection from Formalin Fixed Paraffin-Embedding (FFPE) tissue in molecular lab became a necessary tool for defining potential targeted drug. Accurate quantification of DNA extracted from FFPE tissue is necessary for downstream applications like Polymerase Chain Reaction (PCR), sequencing etc. AIM: To check and define which method for FFPE DNA quantification is suitable for downstream processes. MATERIALS AND METHODS: In this experimental experience study Biorad Smartspec Plus spectrophotomery, Qubit Fluorometer, and Qiagen Rotorgene qPCR was used to compare 20 FFPE DNA quantification in Rajiv Gandhi Cancer Institute and Research Centre, in 2015 and quantified amount of DNA used for PCR reaction...
September 2016: Journal of Clinical and Diagnostic Research: JCDR
https://www.readbyqxmd.com/read/27777300/front-end-genomics-using-an-alternative-approach-for-the-recovery-of-high-quality-dna-from-core-needle-biopsies
#19
Wilfrido D Mojica, Tieying Hou, Don Sykes, Rama Dey-Rao
AIMS: Determine whether a simple prewash step will provide adequate amounts of high-quality DNA from core needle biopsies for molecular sequencing studies. METHODS: The quantitative and qualitative metrics of DNA recovered from core needle biopsies processed either by 1) formalin fixation and paraffin embedding (FFPE), 2) cells recovered after the core needle biopsy was washed, and 3) frozen sections of the core needle biopsy tissue were evaluated and compared to one another...
October 24, 2016: Journal of Clinical Pathology
https://www.readbyqxmd.com/read/27774772/touch-imprint-cytology-with-massively-parallel-sequencing-tic-seq-a-simple-and-rapid-method-to-snapshot-genetic-alterations-in-tumors
#20
Kenji Amemiya, Yosuke Hirotsu, Taichiro Goto, Hiroshi Nakagomi, Hitoshi Mochizuki, Toshio Oyama, Masao Omata
Identifying genetic alterations in tumors is critical for molecular targeting of therapy. In the clinical setting, formalin-fixed paraffin-embedded (FFPE) tissue is usually employed for genetic analysis. However, DNA extracted from FFPE tissue is often not suitable for analysis because of its low levels and poor quality. Additionally, FFPE sample preparation is time-consuming. To provide early treatment for cancer patients, a more rapid and robust method is required for precision medicine. We present a simple method for genetic analysis, called touch imprint cytology combined with massively paralleled sequencing (touch imprint cytology [TIC]-seq), to detect somatic mutations in tumors...
December 2016: Cancer Medicine
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