1. Be familiar with next generation molecular diagnostic techniques that can provide guidance in clinical decision making
2. Identify the utility of these diagnostic approaches with some examples
3. Be aware of the challenges that exist in implementing these tools as part of the routine clinical decision making process, especially in resource limited settings
Developing a Rapid Clinical Sequencing System to Classify Meningioma: Meet th...QIAGEN
Meningioma’s display a broad spectrum of clinical, histological and cytogenetic features even within the same WHO grade often posing a challenge for classification and prognostic stratification. In this webinar, we will describe our experience of using targeted amplicon sequencing to develop rapid clinical sequencing system to identify and confirm the meningioma genotype in just two weeks. In addition the details of the three meningioma categories and the genes involved will be discussed.
This document discusses next-generation sequencing (NGS) and its role in cancer research. It begins with an introduction to NGS technology and applications. The speaker then discusses how NGS can help with cancer research by identifying new cancer genes and mutations, assisting with diagnosis and targeted therapies, and enabling patient stratification. The need for NGS is explained by discussing cancer genetics, statistics on cancer incidence, and the need to identify driver mutations and develop personalized treatments. Qiagen offers an end-to-end NGS workflow and solutions including sample preparation, target enrichment, library preparation, sequencing, and data analysis. Their targeted gene panels and analysis software provide a streamlined sample-to-result experience for cancer research.
Next Generation Sequencing and its Applications in Medical Research - Frances...Sri Ambati
The so-called “next-generation” sequencing (NGS) technologies allows us, in a short time and in parallel, to sequence massive amounts of DNA, overcoming the limitations of the original Sanger sequencing methods used to sequence the first human genome. NGS technologies have had an enormous impact on biomedical research within a short time frame. This talk will give an overview of these applications with specific examples from Mendelian genomics and cancer research. #h2ony
overview on Next generation sequencing in breast csncerSeham Al-Shehri
Next-generation sequencing (NGS) and its application to breast cancer research is discussed. NGS allows for comprehensive profiling of microRNA (miRNA) expression, which can provide insights into tumorigenesis pathways. The document outlines the NGS process, including template preparation, sequencing/imaging, and complex data analysis using statistical methods and software. Key applications are identifying miRNA involvement in cancer driver genes and exploring miRNA expression patterns to discover potential diagnostic or prognostic biomarkers for breast cancer subtypes.
Next generation sequencing (NGS) can simultaneously detect aneuploidy and gene defects in single cells. It involves fragmenting DNA, sequencing short fragments, and using bioinformatics to map the fragments to the human genome. NGS can detect chromosome abnormalities and mutations with the same resolution as array comparative genomic hybridization. It also allows detection of mitochondrial DNA abnormalities and simultaneous analysis of aneuploidy and gene defects. In the future, whole gene sequencing may be possible. NGS is being used successfully for preimplantation genetic diagnosis and was used to produce the first baby born from NGS analysis.
This document summarizes information from a student's assignment on plant genome sequencing techniques. It discusses early phenotypic selection methods and their limitations. It then summarizes different sequencing strategies used for important crop plants like rice, poplar, and Arabidopsis. These include BAC-by-BAC, whole genome shotgun, and various next-generation sequencing platforms. The document also summarizes applications of sequencing including identifying genes related to rice yield and flowering time and using sequencing to improve potato and maize varieties.
This year's 3rd Annual TCGC: The Clinical Genome Conference, held June 10-12, 2014 in San Francisco, is a three-day event that weaves together the science of sequencing and the business of implementing genomics in the clinic. It uniquely illustrates the mutual influence of those areas and the need to therefore consider the needs, challenges and opportunities of both - from next-generation sequencing and variant interpretation to insurance reimbursement and electronic health records - throughout the entire research process.Learn more at http://www.clinicalgenomeconference.com
Next generation sequencing (NGS) involves chemical assays other than traditional Sanger sequencing to sequence DNA. The basic steps of NGS are: 1) library preparation where DNA is fragmented and adapters are ligated, 2) clonal amplification where fragments are amplified, and 3) sequencing reactions using techniques like pyrosequencing or reversible dye terminators to determine the sequence. The sequenced fragments are then analyzed bioinformatically to map reads and assemble sequences.
Developing a Rapid Clinical Sequencing System to Classify Meningioma: Meet th...QIAGEN
Meningioma’s display a broad spectrum of clinical, histological and cytogenetic features even within the same WHO grade often posing a challenge for classification and prognostic stratification. In this webinar, we will describe our experience of using targeted amplicon sequencing to develop rapid clinical sequencing system to identify and confirm the meningioma genotype in just two weeks. In addition the details of the three meningioma categories and the genes involved will be discussed.
This document discusses next-generation sequencing (NGS) and its role in cancer research. It begins with an introduction to NGS technology and applications. The speaker then discusses how NGS can help with cancer research by identifying new cancer genes and mutations, assisting with diagnosis and targeted therapies, and enabling patient stratification. The need for NGS is explained by discussing cancer genetics, statistics on cancer incidence, and the need to identify driver mutations and develop personalized treatments. Qiagen offers an end-to-end NGS workflow and solutions including sample preparation, target enrichment, library preparation, sequencing, and data analysis. Their targeted gene panels and analysis software provide a streamlined sample-to-result experience for cancer research.
Next Generation Sequencing and its Applications in Medical Research - Frances...Sri Ambati
The so-called “next-generation” sequencing (NGS) technologies allows us, in a short time and in parallel, to sequence massive amounts of DNA, overcoming the limitations of the original Sanger sequencing methods used to sequence the first human genome. NGS technologies have had an enormous impact on biomedical research within a short time frame. This talk will give an overview of these applications with specific examples from Mendelian genomics and cancer research. #h2ony
overview on Next generation sequencing in breast csncerSeham Al-Shehri
Next-generation sequencing (NGS) and its application to breast cancer research is discussed. NGS allows for comprehensive profiling of microRNA (miRNA) expression, which can provide insights into tumorigenesis pathways. The document outlines the NGS process, including template preparation, sequencing/imaging, and complex data analysis using statistical methods and software. Key applications are identifying miRNA involvement in cancer driver genes and exploring miRNA expression patterns to discover potential diagnostic or prognostic biomarkers for breast cancer subtypes.
Next generation sequencing (NGS) can simultaneously detect aneuploidy and gene defects in single cells. It involves fragmenting DNA, sequencing short fragments, and using bioinformatics to map the fragments to the human genome. NGS can detect chromosome abnormalities and mutations with the same resolution as array comparative genomic hybridization. It also allows detection of mitochondrial DNA abnormalities and simultaneous analysis of aneuploidy and gene defects. In the future, whole gene sequencing may be possible. NGS is being used successfully for preimplantation genetic diagnosis and was used to produce the first baby born from NGS analysis.
This document summarizes information from a student's assignment on plant genome sequencing techniques. It discusses early phenotypic selection methods and their limitations. It then summarizes different sequencing strategies used for important crop plants like rice, poplar, and Arabidopsis. These include BAC-by-BAC, whole genome shotgun, and various next-generation sequencing platforms. The document also summarizes applications of sequencing including identifying genes related to rice yield and flowering time and using sequencing to improve potato and maize varieties.
This year's 3rd Annual TCGC: The Clinical Genome Conference, held June 10-12, 2014 in San Francisco, is a three-day event that weaves together the science of sequencing and the business of implementing genomics in the clinic. It uniquely illustrates the mutual influence of those areas and the need to therefore consider the needs, challenges and opportunities of both - from next-generation sequencing and variant interpretation to insurance reimbursement and electronic health records - throughout the entire research process.Learn more at http://www.clinicalgenomeconference.com
Next generation sequencing (NGS) involves chemical assays other than traditional Sanger sequencing to sequence DNA. The basic steps of NGS are: 1) library preparation where DNA is fragmented and adapters are ligated, 2) clonal amplification where fragments are amplified, and 3) sequencing reactions using techniques like pyrosequencing or reversible dye terminators to determine the sequence. The sequenced fragments are then analyzed bioinformatically to map reads and assemble sequences.
This document provides an overview of a study that aims to validate somatic mutations in cervical cancer reported by next-generation sequencing (NGS) using Sanger sequencing. The study involved selecting mutations from NGS data, designing primers, performing PCR and purification on tumor and blood samples, conducting Sanger sequencing, and analyzing the results. 16 out of 27 mutations selected were validated as somatic or germline by Sanger sequencing, for a validation rate of around 60%. Several of the validated mutations occurred in important cancer-related genes. The validated mutations were also found to be present in cervical cancer cell lines.
Presentation from the ECDC expert consultation on Whole Genome Sequencing organised by the European Centre of Disease Prevention and Control - Stockholm, 19 November 2015
The Molecular Diagnostics Laboratory processes around 20,000 specimens annually for various testing categories including infectious diseases, hematological malignancies, solid tumor malignancies, and inherited disorders. Next generation sequencing is proposed as a solution to provide more comprehensive genetic testing by simultaneously evaluating variations in multiple genes from a single sample. While next generation sequencing offers advantages over traditional testing methods, there are also technical and clinical challenges to address in implementation, including optimizing detection of structural variations and interpretation of variants with unclear clinical significance.
Dr. Douglas Marthaler - Use of Next Generation Sequencing for Whole Genome An...John Blue
Use of Next Generation Sequencing for Whole Genome Analysis of Pathogens - Dr. Douglas Marthaler, Veterinary Diagnostic Laboratory, College of Veterinary Medicine, University of Minnesota, from the 2016 Allen D. Leman Swine Conference, September 17-20, 2016, St. Paul, Minnesota, USA.
More presentations at http://www.swinecast.com/2016-leman-swine-conference-material
The document describes Phase II of the ABRF Next Generation Sequencing Study which aims to establish reference data sets for evaluating DNA sequencing performance across multiple platforms and laboratories. Phase II will sequence various human and bacterial genomic samples to assess accuracy, coverage, and limits of detection using different platforms and library preparation methods. A collaboration with NIST Genome in a Bottle will provide standardized samples to the participating laboratories. The study aims to provide a resource for ongoing method development and evaluation of sequencing performance.
GTC group 8 - Next Generation SequencingYanqi Chan
DNA sequencing is the process of determining the precise order of nucleotides within a DNA molecule. Discuss the application of next generation sequencing in cancer treatment.
This document provides an overview of next generation sequencing (NGS) for cancer genomics. It discusses how NGS allows sequencing of whole genomes or targeted regions to identify genetic changes between normal and tumor DNA. Analysis involves aligning DNA reads to reference genomes, detecting mutations, and categorizing them as silent, non-silent, oncogenic drivers, or signatures of mutational processes. RNA sequencing can also profile gene expression and detect fusion genes. The data requires specialized bioinformatics analysis to interpret genetic changes driving cancer.
Ernesto Picardi – Bioinformatica e genomica comparata: nuove strategie sperim...eventi-ITBbari
Bioinformatica e genomica comparata: nuove strategie sperimentali e computazionali per la produzione e analisi di dati NGS finalizzati a sviluppare processi e prodotti innovativi per la salute dell’uomo, l’ambiente e l’agroalimentare.
Aug2013 Heidi Rehm integrating large scale sequencing into clinical practiceGenomeInABottle
This document discusses integrating large scale genomic sequencing into clinical practice. It describes how the Laboratory for Molecular Medicine at Partners Healthcare has transitioned from testing a few genes to now offering over 150 tests using next generation sequencing technologies. It also discusses the evolution of testing panels, moving from targeted panels to exome and genome sequencing. Validation of sequencing methods and challenges of variant interpretation are discussed.
Translational Genomics and Prostate Cancer: Meet the NGS Experts Series Part 2QIAGEN
Advanced prostate cancer is highly heterogeneous but this inter-patient heterogeneity has until recently not been understood. We have through an international research effort dissected the molecular landscape of advanced castration resistant prostate, elucidating key molecular targets in this group of diseases. We have also shown that PARP inhibitors have antitumor activity against a significant proportion of these cancers, mainly in men whose cancers harbor DNA repair defects.
Next-Generation Sequencing Clinical Research Milestones InfographicQIAGEN
DNA mutations have been implicated in several diseases, particularly cancer. NGS presents an ideal technology to efficiently profile the multitude of mutations in a high throughput manner. In 2001 the first draft of human genome was published. Since then many major milestones have been reached. Do you know when PIK3CA was identified in colon cancer? When was Olaparib for ovarian cancer treatment? This infographic traces the major clinical research milestones starting from the first draft of the human genome.
Course: Bioinformatics for Biomedical Research (2014).
Session: 2.1.3- Next Generation Sequencing. Technologies and Applications. Part III: NGS Applications II.
Statistics and Bioinformatisc Unit (UEB) & High Technology Unit (UAT) from Vall d'Hebron Research Institute (www.vhir.org), Barcelona.
Clinical Validation of an NGS-based (CE-IVD) Kit for Targeted Detection of Ge...Thermo Fisher Scientific
In recent years, advances in next-generation sequencing (NGS) technologies have enabled faster and cheaper methods for uncovering the genetic basis of disease. For cancer, NGS based screening for known tumour subtypes can inform diagnosis and allow the clinician to tailor a specific therapy based on testing outcome. Here we present the validation of one such NGS based kit approved for CE-IVD* use to screen for specific chromosomal translocations in non-small cell lung cancer (NSCLC) samples by targeting specific breakpoints in known fusion transcripts.
The kit tested (Oncomine™ Solid Tumour Fusion Transcript Kit) included a single primer poolcontaining amplicon designs to simultaneously screen for over 75 specific rearrangements involving the receptor tyrosine kinase (RTK) genes ALK, RET and ROS1 as well as NTRK1. The panel was compatible with formalin-fixed paraffin-embedded (FFPE) lung tumour samples and achieved high sensitivity down to 10 ng of RNA input. In addition, amplicon assays designed at the 5’ and 3’ ends the RTK genes provide non-specific evidence that a translocation exists in a sample by comparing expression imbalance between the two ends. Validation testing was carried out at three external clinical laboratories (CLIA, CAP, INAB). In addition to positive and negative control samples, each site contributed FFPE lung tumour samples for which ALK fusion status was known prior to NGS library preparation carried out using the Ion AmpliSeq workflow. For site-specific samples (n=144, 16 samples per sequencing run), high concordance, sensitivity and specificity were measured at 97.2%, 90.5% and 98.4%, respectively.
Bioinformatics tools are essential for analyzing next-generation sequencing (NGS) data. The summary describes the typical stages of NGS data analysis:
1. Primary analysis involves demultiplexing, base calling and quality control to produce fastq files.
2. Secondary analysis maps reads to a reference genome to produce SAM/BAM files and calls variants to produce VCF files.
3. Tertiary analysis annotates and filters variants to prioritize those relevant to disease.
Development of a Multi-Variant Frequency Ladder™ for Next Generation Sequenci...Thermo Fisher Scientific
Increasing adoption of NGS has shed light on the need for more
standardized controls to evaluate and optimize system performance.
Samples containing mutations of interest are difficult to source and cell
line pooling experiments to determine limit of detection require significant
investments of time and money. To simultaneously evaluate variant
calling performance in >200 unique amplicons across 50 genes targeted
by NGS tests, AcroMetrix® has developed a proprietary
genomic/synthetic DNA material containing over 550 mutations as a
mixture of SNV’s indels and MNP’s. The limit of detection was then
determined for >400 variants using multiple platforms. Tumor samples
were diluted with matched normal samples to mimic a range of
frequencies. Linearity between the material and diluted tumor tissue
samples were compared. Overall, highly multiplex controls with tunable
frequencies allow for much more extensive, yet streamlined, assay
evaluation and facilitate implementation and impart confidence to NGS
testing.
The document provides information about a short course on next generation sequencing and analysis of sequence variants. It includes an agenda with sessions on introduction to NGS applications in medical research, data analysis pipelines, interpretation of variants, and tools for predicting pathogenicity. It also provides background on the organizing institutions, the CNAG sequencing center and its projects, and an overview of bioinformatics analysis pipelines and resources.
Supporting Genomics in the Practice of Medicine by Heidi RehmKnome_Inc
View the webinar at http://www.knome.com/webinar-supporting-genomics-practice-medicine. In this presentation, Dr. Heidi Rehm, Chief Laboratory Director of the Laboratory for Molecular Medicine at Partners Healthcare and one of the Principal Investigators on ClinGen, elucidates the challenges of genomics in medicine and outlined the path to integrating large scale sequencing into clinical practice.
Utilization of NGS to Identify Clinically-Relevant Mutations in cfDNA: Meet t...QIAGEN
Pancreatic cancer is a uniquely lethal malignancy characterized by frequent mutations in KRAS, CDKN2A, SMAD4, TP53 and many others. We have shown that KRAS mutation can be detected in cell-free, circulating tumor DNA (ctDNA) isolated from the plasma in a subset of patients and is associated with poor prognosis. The ability to simultaneously detect multiple pancreatic cancer-specific mutations in ctDNA would open a new avenue for detection of clinically-relevant mutations. In this study, we performed ultra-deep sequencing of ctDNA from advanced pancreatic cancer patients prior to treatment with Gemcitabine and Erlotinib following target enrichment. Somatic, non-synonymous variants were identified in 29 different genes at allele frequencies typically less than 0.5%. Updated results of ultra-deep NGS analysis will be presented.
Molecular diagnostics market & forecast (by application, technology, countrie...Renub Research
Renub Research (http://renub.com/report/molecular-diagnostics-market-forecast-by-application-technology-countries-companies-clinical-trials-to-2017-global-analysis-114) has announced the addition of the "Molecular Diagnostics Market & Forecast (By Application, Technology, Countries, Companies & Clinical Trials) to 2017: Global Analysis" report to its offering
Quantifiler™ Trio: Decision-support to help streamline Sexual Assault sample ...Thermo Fisher Scientific
Quantifiler™ Trio:
Decision-support to help streamline
Sexual Assault sample processing. Sheri Olson, Thermo Fisher Scientific
Bode West Meeting
March 2015, Coronado, CA
This document provides an overview of a study that aims to validate somatic mutations in cervical cancer reported by next-generation sequencing (NGS) using Sanger sequencing. The study involved selecting mutations from NGS data, designing primers, performing PCR and purification on tumor and blood samples, conducting Sanger sequencing, and analyzing the results. 16 out of 27 mutations selected were validated as somatic or germline by Sanger sequencing, for a validation rate of around 60%. Several of the validated mutations occurred in important cancer-related genes. The validated mutations were also found to be present in cervical cancer cell lines.
Presentation from the ECDC expert consultation on Whole Genome Sequencing organised by the European Centre of Disease Prevention and Control - Stockholm, 19 November 2015
The Molecular Diagnostics Laboratory processes around 20,000 specimens annually for various testing categories including infectious diseases, hematological malignancies, solid tumor malignancies, and inherited disorders. Next generation sequencing is proposed as a solution to provide more comprehensive genetic testing by simultaneously evaluating variations in multiple genes from a single sample. While next generation sequencing offers advantages over traditional testing methods, there are also technical and clinical challenges to address in implementation, including optimizing detection of structural variations and interpretation of variants with unclear clinical significance.
Dr. Douglas Marthaler - Use of Next Generation Sequencing for Whole Genome An...John Blue
Use of Next Generation Sequencing for Whole Genome Analysis of Pathogens - Dr. Douglas Marthaler, Veterinary Diagnostic Laboratory, College of Veterinary Medicine, University of Minnesota, from the 2016 Allen D. Leman Swine Conference, September 17-20, 2016, St. Paul, Minnesota, USA.
More presentations at http://www.swinecast.com/2016-leman-swine-conference-material
The document describes Phase II of the ABRF Next Generation Sequencing Study which aims to establish reference data sets for evaluating DNA sequencing performance across multiple platforms and laboratories. Phase II will sequence various human and bacterial genomic samples to assess accuracy, coverage, and limits of detection using different platforms and library preparation methods. A collaboration with NIST Genome in a Bottle will provide standardized samples to the participating laboratories. The study aims to provide a resource for ongoing method development and evaluation of sequencing performance.
GTC group 8 - Next Generation SequencingYanqi Chan
DNA sequencing is the process of determining the precise order of nucleotides within a DNA molecule. Discuss the application of next generation sequencing in cancer treatment.
This document provides an overview of next generation sequencing (NGS) for cancer genomics. It discusses how NGS allows sequencing of whole genomes or targeted regions to identify genetic changes between normal and tumor DNA. Analysis involves aligning DNA reads to reference genomes, detecting mutations, and categorizing them as silent, non-silent, oncogenic drivers, or signatures of mutational processes. RNA sequencing can also profile gene expression and detect fusion genes. The data requires specialized bioinformatics analysis to interpret genetic changes driving cancer.
Ernesto Picardi – Bioinformatica e genomica comparata: nuove strategie sperim...eventi-ITBbari
Bioinformatica e genomica comparata: nuove strategie sperimentali e computazionali per la produzione e analisi di dati NGS finalizzati a sviluppare processi e prodotti innovativi per la salute dell’uomo, l’ambiente e l’agroalimentare.
Aug2013 Heidi Rehm integrating large scale sequencing into clinical practiceGenomeInABottle
This document discusses integrating large scale genomic sequencing into clinical practice. It describes how the Laboratory for Molecular Medicine at Partners Healthcare has transitioned from testing a few genes to now offering over 150 tests using next generation sequencing technologies. It also discusses the evolution of testing panels, moving from targeted panels to exome and genome sequencing. Validation of sequencing methods and challenges of variant interpretation are discussed.
Translational Genomics and Prostate Cancer: Meet the NGS Experts Series Part 2QIAGEN
Advanced prostate cancer is highly heterogeneous but this inter-patient heterogeneity has until recently not been understood. We have through an international research effort dissected the molecular landscape of advanced castration resistant prostate, elucidating key molecular targets in this group of diseases. We have also shown that PARP inhibitors have antitumor activity against a significant proportion of these cancers, mainly in men whose cancers harbor DNA repair defects.
Next-Generation Sequencing Clinical Research Milestones InfographicQIAGEN
DNA mutations have been implicated in several diseases, particularly cancer. NGS presents an ideal technology to efficiently profile the multitude of mutations in a high throughput manner. In 2001 the first draft of human genome was published. Since then many major milestones have been reached. Do you know when PIK3CA was identified in colon cancer? When was Olaparib for ovarian cancer treatment? This infographic traces the major clinical research milestones starting from the first draft of the human genome.
Course: Bioinformatics for Biomedical Research (2014).
Session: 2.1.3- Next Generation Sequencing. Technologies and Applications. Part III: NGS Applications II.
Statistics and Bioinformatisc Unit (UEB) & High Technology Unit (UAT) from Vall d'Hebron Research Institute (www.vhir.org), Barcelona.
Clinical Validation of an NGS-based (CE-IVD) Kit for Targeted Detection of Ge...Thermo Fisher Scientific
In recent years, advances in next-generation sequencing (NGS) technologies have enabled faster and cheaper methods for uncovering the genetic basis of disease. For cancer, NGS based screening for known tumour subtypes can inform diagnosis and allow the clinician to tailor a specific therapy based on testing outcome. Here we present the validation of one such NGS based kit approved for CE-IVD* use to screen for specific chromosomal translocations in non-small cell lung cancer (NSCLC) samples by targeting specific breakpoints in known fusion transcripts.
The kit tested (Oncomine™ Solid Tumour Fusion Transcript Kit) included a single primer poolcontaining amplicon designs to simultaneously screen for over 75 specific rearrangements involving the receptor tyrosine kinase (RTK) genes ALK, RET and ROS1 as well as NTRK1. The panel was compatible with formalin-fixed paraffin-embedded (FFPE) lung tumour samples and achieved high sensitivity down to 10 ng of RNA input. In addition, amplicon assays designed at the 5’ and 3’ ends the RTK genes provide non-specific evidence that a translocation exists in a sample by comparing expression imbalance between the two ends. Validation testing was carried out at three external clinical laboratories (CLIA, CAP, INAB). In addition to positive and negative control samples, each site contributed FFPE lung tumour samples for which ALK fusion status was known prior to NGS library preparation carried out using the Ion AmpliSeq workflow. For site-specific samples (n=144, 16 samples per sequencing run), high concordance, sensitivity and specificity were measured at 97.2%, 90.5% and 98.4%, respectively.
Bioinformatics tools are essential for analyzing next-generation sequencing (NGS) data. The summary describes the typical stages of NGS data analysis:
1. Primary analysis involves demultiplexing, base calling and quality control to produce fastq files.
2. Secondary analysis maps reads to a reference genome to produce SAM/BAM files and calls variants to produce VCF files.
3. Tertiary analysis annotates and filters variants to prioritize those relevant to disease.
Development of a Multi-Variant Frequency Ladder™ for Next Generation Sequenci...Thermo Fisher Scientific
Increasing adoption of NGS has shed light on the need for more
standardized controls to evaluate and optimize system performance.
Samples containing mutations of interest are difficult to source and cell
line pooling experiments to determine limit of detection require significant
investments of time and money. To simultaneously evaluate variant
calling performance in >200 unique amplicons across 50 genes targeted
by NGS tests, AcroMetrix® has developed a proprietary
genomic/synthetic DNA material containing over 550 mutations as a
mixture of SNV’s indels and MNP’s. The limit of detection was then
determined for >400 variants using multiple platforms. Tumor samples
were diluted with matched normal samples to mimic a range of
frequencies. Linearity between the material and diluted tumor tissue
samples were compared. Overall, highly multiplex controls with tunable
frequencies allow for much more extensive, yet streamlined, assay
evaluation and facilitate implementation and impart confidence to NGS
testing.
The document provides information about a short course on next generation sequencing and analysis of sequence variants. It includes an agenda with sessions on introduction to NGS applications in medical research, data analysis pipelines, interpretation of variants, and tools for predicting pathogenicity. It also provides background on the organizing institutions, the CNAG sequencing center and its projects, and an overview of bioinformatics analysis pipelines and resources.
Supporting Genomics in the Practice of Medicine by Heidi RehmKnome_Inc
View the webinar at http://www.knome.com/webinar-supporting-genomics-practice-medicine. In this presentation, Dr. Heidi Rehm, Chief Laboratory Director of the Laboratory for Molecular Medicine at Partners Healthcare and one of the Principal Investigators on ClinGen, elucidates the challenges of genomics in medicine and outlined the path to integrating large scale sequencing into clinical practice.
Utilization of NGS to Identify Clinically-Relevant Mutations in cfDNA: Meet t...QIAGEN
Pancreatic cancer is a uniquely lethal malignancy characterized by frequent mutations in KRAS, CDKN2A, SMAD4, TP53 and many others. We have shown that KRAS mutation can be detected in cell-free, circulating tumor DNA (ctDNA) isolated from the plasma in a subset of patients and is associated with poor prognosis. The ability to simultaneously detect multiple pancreatic cancer-specific mutations in ctDNA would open a new avenue for detection of clinically-relevant mutations. In this study, we performed ultra-deep sequencing of ctDNA from advanced pancreatic cancer patients prior to treatment with Gemcitabine and Erlotinib following target enrichment. Somatic, non-synonymous variants were identified in 29 different genes at allele frequencies typically less than 0.5%. Updated results of ultra-deep NGS analysis will be presented.
Molecular diagnostics market & forecast (by application, technology, countrie...Renub Research
Renub Research (http://renub.com/report/molecular-diagnostics-market-forecast-by-application-technology-countries-companies-clinical-trials-to-2017-global-analysis-114) has announced the addition of the "Molecular Diagnostics Market & Forecast (By Application, Technology, Countries, Companies & Clinical Trials) to 2017: Global Analysis" report to its offering
Quantifiler™ Trio: Decision-support to help streamline Sexual Assault sample ...Thermo Fisher Scientific
Quantifiler™ Trio:
Decision-support to help streamline
Sexual Assault sample processing. Sheri Olson, Thermo Fisher Scientific
Bode West Meeting
March 2015, Coronado, CA
Aula ministrada durante a disciplina de Biologia Molecular de Microrganismos, no Programa de Pós-Graduação em Microbiologia da Universidade Federal de Minas Gerais.
Chronic myeloid leukemia (CML) is a type of leukemia originating from abnormal bone marrow stem cells that are consistently associated with the BCR-ABL1 fusion gene. It accounts for 10-15% of myeloid leukemias and is more prevalent in males. CML progresses through three phases: chronic phase, accelerated phase, and blast crisis phase. Treatment involves tyrosine kinase inhibitors like imatinib, with the goal of controlling the disease long-term. Upfront hematopoietic stem cell transplantation may provide a cure but also carries risks of toxicity and mortality. Ongoing research is evaluating whether newer tyrosine kinase inhibitors or stopping imatinib after deep responses could improve outcomes while reducing costs and side
Molecular diagnostics of colorectal cancerAddisu Alemu
This document provides an outline for a presentation on colorectal cancer (CRC). It discusses the etiology, types, molecular pathogenesis and diagnosis of CRC. Key points include: CRC accounts for 9% of cancer deaths worldwide and arises through genetic and epigenetic changes. The main types are sporadic, inherited syndromes like Lynch syndrome, and familial CRC. Molecularly, CRC arises through chromosomal instability, mismatch repair defects, and epigenetic silencing. Important genes mutated include APC, KRAS, BRAF, p53 and DCC. Familial adenomatous polyposis and Lynch syndrome are inherited CRC syndromes associated with APC and mismatch repair gene mutations respectively.
1) O documento discute o diagnóstico molecular de vírus respiratórios, comparando diferentes métodos como cultura celular, testes rápidos, imunofluorescência e PCR.
2) É destacada a importância de detectar novos vírus como o metapneumovírus e bocavírus humano usando técnicas moleculares como PCR em tempo real.
3) A detecção frequente de coinfecções virais e a interação entre vírus e bactérias respiratórias são abordadas.
O documento resume os principais métodos de diagnóstico de infecções virais, incluindo métodos diretos como microscopia eletrônica, isolamento viral e detecção de ácidos nucleicos, e métodos indiretos como detecção de anticorpos por soroneutralização, inibição da hemaglutinação e ELISA indireto.
O documento discute teoria e aplicações da PCR quantitativa (qPCR). Resume os principais tópicos da qPCR, incluindo ensaios químicos disponíveis, conceitos importantes como curvas padrão e de dissociação, e fatores que afetam a eficiência. Também aborda cálculos como quantificação absoluta e relativa usando métodos como Livak.
Aula04 tecnicas diagnostico virologia parte 2 finalHugo Sousa
O documento discute várias técnicas de diagnóstico virológico, incluindo métodos de amplificação como PCR em tempo real, extração de ácidos nucleicos, e aplicações destas técnicas para detecção e quantificação de vírus.
Diagnóstico de Doenças Infecciosas e ParasitáriasMessias Miranda
1) O documento apresenta informações sobre diagnóstico laboratorial de doenças infecciosas e parasitárias, abordando exames de sangue, fezes e amostras biológicas.
2) São descritos métodos como hemograma, cultura bacteriana, identificação de parasitas em fezes e testes sorológicos para detecção de vírus.
3) Também são apresentados detalhes sobre técnicas moleculares como PCR e testes imunológicos como ELISA para diagnóstico.
The observer witnessed an interaction between herself and a toddler named Evelyn. Evelyn pulled the observer's hair twice while she was reading a story to the children. When confronted by the observer, Evelyn cried and falsely claimed the observer hurt her arm. The early childhood educator was called over and had Evelyn apologize to the observer. The observer accepted the apology but emphasized the importance of always telling the truth.
Introdução de tecnicas de diagnostico molecular Safia Naser
1. O documento discute técnicas de diagnóstico molecular, incluindo detecção de agentes infecciosos e alterações genéticas que auxiliam no diagnóstico e prognóstico de doenças.
2. São descritas várias técnicas moleculares como PCR, Southern Blot, RFLP e sequenciamento que permitem avaliar a estrutura do DNA para diagnóstico.
3. O diagnóstico molecular é um campo emergente que oferece vantagens como alta sensibilidade, rapidez e especificidade no
Este documento discute a genética forense, incluindo seu uso na identificação criminal por meio de DNA desde 1985. Detalha amostras biológicas de interesse forense, como sangue e saliva, e os passos para obter perfis de DNA, incluindo isolamento, corte e separação de fragmentos. Também menciona bancos de dados criminais como CODIS usado pelo FBI para comparar perfis genéticos.
O documento resume a história da descoberta do DNA, desde as primeiras observações de Mendel sobre a hereditariedade até a elucidação da estrutura em dupla hélice por Watson e Crick em 1953. Destaca os principais estudos de Miescher, Griffith, Avery e Chargaff que levaram à compreensão do papel do DNA na transmissão de características genéticas.
The children were playing outside when a disagreement arose over which game to play. John did not want Athene to play with them and told Bavyansh not to play with her. The teacher used active listening to have each child express their perspective. This allowed John to understand that Athene can choose her own games and friends. The children then separated amicably to play different games, coming back together later to play catching. The teacher reflected that the solution went well but could improve by better understanding each child's temperament.
O documento discute a genética forense e seu uso na justiça criminal, mencionando o primeiro caso de identificação criminal usando DNA em 1985 na Inglaterra e os métodos atuais de análise de DNA, incluindo STRs e bancos de dados como CODIS.
Este documento resume los principales aspectos de la genética forense, incluyendo el análisis de polimorfismos de ADN genómico y mitocondrial para aplicaciones como la paternidad, criminalística e identificación. Describe técnicas como la digestión con enzimas de restricción, reacción en cadena de la polimerasa y electroforesis para analizar muestras de ADN pequeñas o degradadas.
Intel introduced Light Peak in 2009, an optical cable interface that could transfer data at 10Gbps. In 2011, they launched "Copper Peak" which used copper cables instead of fiber optics. Also in 2011, Apple updated the MacBook Pro with Thunderbolt, a strictly copper-based port that could transfer data at 10Gbps and replaced USB and other ports.
Molecular diagnostic techniques involve manipulating and analyzing DNA, RNA, and proteins. These techniques are used in areas like neoplastic disorders, infectious diseases, and inherited conditions. Common techniques include amplification methods like PCR, blotting techniques such as Southern blot and Western blot, and hybridization methods like in situ hybridization and microarrays. Molecular diagnostics provides advantages like automation, speed, reliability, and sensitivity, though limitations include potential errors and sensitivity to inhibitors. These techniques have revolutionized fields like medicine, agriculture, forensics, and more by enabling abundant production of DNA and detection of genetic disorders.
DNA microarray technology allows for the high-throughput analysis of differential gene expression. The document discusses DNA microarray approaches, including spotted arrays and oligonucleotide chips. Key steps in a microarray experiment are described such as sample preparation, hybridization, and data analysis. Examples are given of microarray studies examining gene expression changes related to atherosclerosis, endothelial function, and macrophage response to oxidized LDL. Challenges in microarray design, analysis, and validation of results are also discussed.
DNA microarray is a technique that allows high-throughput analysis of gene expression. It involves depositing DNA fragments onto a glass slide and using fluorescent probes made from sample RNA to detect expression levels of thousands of genes simultaneously. The document discusses the basic principles and steps of DNA microarray, including sample preparation, hybridization, image analysis and data normalization. It also compares different microarray fabrication technologies and platforms, and discusses quality control considerations and limitations of the technique.
DNA microarray is a technique that allows high-throughput analysis of gene expression. It involves depositing DNA fragments onto a glass slide and using fluorescent probes made from sample RNA to detect expression levels of thousands of genes simultaneously. The document discusses the basic principles and steps of DNA microarray, including sample preparation, hybridization, imaging, and data analysis. It also compares different microarray fabrication technologies and highlights some challenges in the field, such as lack of standardization and high rates of false positives.
High Sensitivity Detection of Tumor Gene Mutations-v3Michael Powell
This document summarizes a new method called QClamp PCR that can highly sensitively detect tumor gene mutations. QClamp uses synthetic XNA probes to selectively block amplification of wild-type DNA sequences, improving the sensitivity of PCR to detect mutations present in less than 0.1% of samples. This level of sensitivity allows detection of mutations from biopsy, tissue, or blood samples. The document discusses applications of QClamp PCR for detection of EGFR mutations in lung cancer patients and enriching samples for DNA sequencing. QClamp represents an improvement over other methods by reducing the excess of wild-type DNA that creates high background signals.
This document discusses the use of DNA microarrays in researching vulnerable plaque. DNA microarrays allow high-throughput analysis of gene expression and have opened doors to exploring unknown molecular mechanisms. The author's research group is conducting genomic and proteomic experiments on human atherosclerotic plaques to shed light on the molecular mechanisms involved in atherosclerosis development and vulnerability. Proteomic analysis provides insights not available through genomics alone. Understanding these molecular processes could lead to better understanding of vulnerable plaque development and complications.
This document discusses the use of DNA microarrays in studying vulnerable atherosclerotic plaques. It provides background on atherosclerosis and plaque rupture. DNA microarrays allow high-throughput analysis of gene and protein expression, which can provide insights into molecular mechanisms underlying plaque vulnerability. One study used microarrays to analyze gene expression differences between ruptured and stable plaques, identifying perilipin as upregulated in ruptured plaques. However, microarray analysis of atherosclerosis is still in its early stages with many technical challenges to address.
This document discusses the use of DNA microarrays in researching vulnerable plaque. DNA microarrays allow high-throughput analysis of gene expression and have opened doors to exploring unknown molecular mechanisms. The author's research group is conducting genomic and proteomic experiments on human atherosclerotic plaques to shed light on the molecular mechanisms involved in atherosclerosis development and vulnerability. They are examining differential gene and protein expression between ruptured and stable plaques using various techniques including laser capture microdissection. The goal is to gain a better understanding of the molecular processes leading to vulnerable plaques and their complications.
The document provides an overview of genomics and molecular profiling techniques. It discusses:
- The lab for bioinformatics and computational genomics which has 10 "genome hackers" and 42 scientists.
- An introduction to personalized medicine and biomarkers.
- First generation molecular profiling techniques like gene sequencing, microarrays, PCR.
- Next generation sequencing techniques like Roche 454, Illumina, SOLID which allow high throughput sequencing.
- Next generation applications like RNA sequencing, exome sequencing, epigenetic profiling.
- The role of bioinformatics in analyzing large genomic and molecular profiling data.
This document discusses next generation molecular profiling technologies. It begins with an overview of first generation molecular profiling techniques like gene sequencing, microarrays, and fluorescence in situ hybridization (FISH). It then describes some key advantages of next generation sequencing technologies like their ability to generate more sequence data at lower cost. Examples of second generation DNA and RNA profiling methods are provided, including exome sequencing and RNA-sequencing. The document also briefly discusses emerging areas like third generation sequencing and next generation protein profiling using mass spectrometry. Epigenetic profiling using techniques like methyl-binding domain sequencing is summarized in the section on next generation epigenetic profiling.
NSA Diagnostic Laboratory has been operating since 1958, founded by Prof. Nasseh Amin. NSA is considered as one of the most advanced labs in Egypt. Maintaining personalized services for its stakeholders, as well as the main role of the lab "Diagnosis"
NSA Diagnostic Laboratory operates through two different segments.
Firstly, a group of stand-alone labs located at prime locations all over Egypt, with the latest and up to date equipments.
Secondly, being the backbone of well reputed hospitals and some polyclinics where NSA is the lab that is responsible for all medical testing there, serving all our patients with class A quality.
Our main focus is delivering quality care and with Cost-value return. NSA plays a key role in improving the health of many Egyptians, by providing access to quality service for more than 200,000 patients annually.
This document discusses the use of DNA microarrays in vulnerable plaque research. It provides background on atherosclerosis and identifies DNA microarrays as a tool that can be used to investigate the molecular mechanisms underlying plaque vulnerability. The document outlines the basic steps in performing a DNA microarray experiment and discusses considerations for experimental design, data analysis, and quality control. It summarizes several studies that have used microarrays or related techniques to examine gene expression in atherosclerosis. The author's research group plans to use genomic and proteomic approaches like microarrays and laser capture microdissection to study gene and protein expression differences between stable and ruptured plaques.
This document discusses the use of DNA microarrays in vulnerable plaque research. It provides background on atherosclerosis and describes how DNA microarrays allow high-throughput analysis of gene expression. The document outlines the basic steps of a microarray experiment and issues around data analysis and quality control. It summarizes several studies that used microarrays or related techniques to analyze gene expression in atherosclerosis and vulnerable plaques. The author's research group aims to shed light on molecular mechanisms in atherosclerosis by examining differential gene and protein expression between stable and ruptured plaques using microarrays, proteomics, and other techniques.
This document discusses the use of DNA microarrays in vulnerable plaque research. It provides background on atherosclerosis and describes how DNA microarrays allow high-throughput analysis of gene expression. The document outlines the basic steps of a microarray experiment and issues around data analysis and quality control. It summarizes several studies that used microarrays or related techniques to analyze gene expression in atherosclerosis and vulnerable plaques. The author's research group aims to shed light on molecular mechanisms in atherosclerosis by examining differential gene and protein expression between stable and ruptured plaques using microarrays, proteomics, and other approaches.
This document discusses the use of DNA microarrays in researching vulnerable atherosclerotic plaques. It provides background on atherosclerosis and plaque rupture. DNA microarrays allow high-throughput analysis of gene and protein expression, which can provide insights into molecular mechanisms of plaque vulnerability. However, DNA microarray technology for studying atherosclerosis is still in its infancy due to lack of data. The document outlines the basic steps of a microarray experiment and issues to consider, such as sample preparation, data normalization, and quality controls. It also reviews a study that used suppression subtractive hybridization followed by macroarray to identify genes potentially involved in plaque rupture.
This document discusses the use of DNA microarrays in vulnerable plaque research. It provides background on atherosclerosis and identifies DNA microarrays as a tool that can be used to investigate the molecular mechanisms underlying plaque vulnerability. The document outlines the basic steps of a DNA microarray experiment and discusses considerations for experimental design, data analysis, and validation of results. It also summarizes several studies that have used DNA microarrays or related techniques to examine gene expression in atherosclerosis.
Molecular techniques for pathology research - MDX .pdfsabyabby
This document discusses molecular techniques used in pathology research such as PCR, microarrays, next generation sequencing, immunohistochemistry, ELISA, and Western blotting. It provides details on each technique including the basic principles, applications in research, and examples of uses in studies of gene expression, cancer, bone disease, and growth retardation. The learning outcomes are to understand these techniques and their uses in basic and clinical research.
Exploring the neuroblastoma epigenome: perspectives for improved prognosisMaté Ongenaert
EXPLORING THE NEUROBLASTOMA EPIGENOME: PERSPECTIVES FOR THE DISCOVERY OF PROGNOSISTIC BIOMARKERS
M. Ongenaert, A. Decock, J. Vandesompele, F. Speleman
Center for Medical Genetics, Ghent University, Ghent, Belgium (mate.ongenaert@ugent.be)
Neuroblastoma (NB) is a childhood tumor originating from sympathetic nervous system cells. Although recently new insights into genes involved in NB have emerged, the molecular basis of neuroblastoma development and progression still remains poorly understood. The best-characterized genetic alterations include amplification of the proto-oncogene MYCN, ALK activating mutations or amplification, gain of chromosome arm 17q and losses of 1p, 3p, and 11q. Epigenetic alterations have been described as well: caspase-8 (CASP8) and RAS-association domain family 1 isoform A (RASSF1A) DNA-methylation are important events for the development and progression of neuroblastoma. In total, there are about 75 genes described as epigenetically affected in NB cell lines and/or NB primary samples.
Most of these methylation markers are found using ‘candidate gene’ approaches and the methylation frequencies are usually very low. In order to find novel methylation markers that can be used for improved prognosis, we applied a whole-genome methylation screen. This technique relies on capturing with the MBD2 protein, containing a methyl-binding domain (MBD), with a very high affinity towards methylated genomic regions. In an initial phase, MBD2-seq was performed on 8 NB cell lines (where we also had micro-array data of, before and after treatment with DAC). As these results are promising, we will explore the complete methylomes of 45 primary NB tumors.
Based on an integrative analysis (re-expression results, expression micro-arrays, MBD2-sequencing on cell lines), 48 MSP (Methylation Specific PCR) assays were tested on 89 primary neuroblastoma patients of different risk categories. The results of this validation study demonstrate the power of epigenetic biomarkers as several assays are informative for prognosis and survival.
Microarrays allow researchers to examine gene expression patterns across thousands of genes simultaneously. A microarray contains probes for known genes that are used to detect complementary mRNA in a biological sample. Microarrays can be used to study gene expression differences between normal and diseased tissues, classify tumor subtypes, and diagnose cancers. They also show promise for personalized cancer treatment by predicting patient prognosis and response to therapy.
Dna Based Molecular Diagnosis(CMH)Final (1).pptxRajKumarSingha5
The document discusses Abu Sufian, the DNA analyst at the 1st molecular diagnostic lab in Bangladesh. It provides information on DNA, RNA, molecular diagnostic techniques like PCR, sequencing, and applications in genetic disease diagnosis, oncology, microbiology, and more. The lab offers diagnostic tests for conditions like thalassemia, aneuploidy, microdeletions and performs transplant matching.
Similar to Clinical molecular diagnostics for drug guidance (20)
This particular slides consist of- what is Pneumothorax,what are it's causes and it's effect on body, risk factors, symptoms,complications, diagnosis and role of physiotherapy in it.
This slide is very helpful for physiotherapy students and also for other medical and healthcare students.
Here is a summary of Pneumothorax:
Pneumothorax, also known as a collapsed lung, is a condition that occurs when air leaks into the space between the lung and chest wall. This air buildup puts pressure on the lung, preventing it from expanding fully when you breathe. A pneumothorax can cause a complete or partial collapse of the lung.
Mental Health and well-being Presentation. Exploring innovative approaches and strategies for enhancing mental well-being. Discover cutting-edge research, effective strategies, and practical methods for fostering mental well-being.
As Mumbai's premier kidney transplant and donation center, L H Hiranandani Hospital Powai is not just a medical facility; it's a beacon of hope where cutting-edge science meets compassionate care, transforming lives and redefining the standards of kidney health in India.
Emotional and Behavioural Problems in Children - Counselling and Family Thera...PsychoTech Services
A proprietary approach developed by bringing together the best of learning theories from Psychology, design principles from the world of visualization, and pedagogical methods from over a decade of training experience, that enables you to: Learn better, faster!
English Drug and Alcohol Commissioners June 2024.pptxMatSouthwell1
Presentation made by Mat Southwell to the Harm Reduction Working Group of the English Drug and Alcohol Commissioners. Discuss stimulants, OAMT, NSP coverage and community-led approach to DCRs. Focussing on active drug user perspectives and interests
Sectional dentures for microstomia patients.pptxSatvikaPrasad
Microstomia, characterized by an abnormally small oral aperture, presents significant challenges in prosthodontic treatment, including limited access for examination, difficulties in impression making, and challenges with prosthesis insertion and removal. To manage these issues, customized impression techniques using sectional trays and elastomeric materials are employed. Prostheses may be designed in segments or with flexible materials to facilitate handling. Minimally invasive procedures and the use of digital technologies can enhance patient comfort. Education and training for patients on prosthesis care and maintenance are crucial for compliance. Regular follow-up and a multidisciplinary approach, involving collaboration with other specialists, ensure comprehensive care and improved quality of life for microstomia patients.
Hypertension and it's role of physiotherapy in it.Vishal kr Thakur
This particular slides consist of- what is hypertension,what are it's causes and it's effect on body, risk factors, symptoms,complications, diagnosis and role of physiotherapy in it.
This slide is very helpful for physiotherapy students and also for other medical and healthcare students.
Here is summary of hypertension -
Hypertension, also known as high blood pressure, is a serious medical condition that occurs when blood pressure in the body's arteries is consistently too high. Blood pressure is the force of blood pushing against the walls of blood vessels as the heart pumps it. Hypertension can increase the risk of heart disease, brain disease, kidney disease, and premature death.
At Malayali Kerala Spa Ajman, Full Service includes individualized care for every client. We specifically design each massage session for the individual needs of the client. Our therapists are always willing to adjust the treatments based on the client's instruction and feedback. This guarantees that every client receives the treatment they expect.
By offering a variety of massage services, our Ajman Spa Massage Center can tackle physical, mental, and emotional illnesses. In addition, efficient identification of specific health conditions and designing treatment plans accordingly can significantly enhance the quality of massaging.
At Malayali Kerala Spa Ajman, we firmly believe that everyone should have the option to experience top-quality massage services regularly. To achieve that goal we offer cheap massage services in Ajman.
If you are interested in experiencing transformative massage treatment at Malayali Kerala Spa Ajman, you can use our Ajman Massage Center WhatsApp Number to schedule your next massage session.
Contact @ +971 529818279
Visit @ https://malayalikeralaspaajman.com/
R3 Stem Cell Therapy: A New Hope for Women with Ovarian FailureR3 Stem Cell
Discover the groundbreaking advancements in stem cell therapy by R3 Stem Cell, offering new hope for women with ovarian failure. This innovative treatment aims to restore ovarian function, improve fertility, and enhance overall well-being, revolutionizing reproductive health for women worldwide.
The Importance of Black Women Understanding the Chemicals in Their Personal C...bkling
Certain chemicals, such as phthalates and parabens, can disrupt the body's hormones and have significant effects on health. According to data, hormone-related health issues such as uterine fibroids, infertility, early puberty and more aggressive forms of breast and endometrial cancers disproportionately affect Black women. Our guest speaker, Jasmine A. McDonald, PhD, an Assistant Professor in the Department of Epidemiology at Columbia University in New York City, discusses the scientific reasons why Black women should pay attention to specific chemicals in their personal care products, like hair care, and ways to minimize their exposure.
Digital Health in India_Health Informatics Trained Manpower _DrDevTaneja_15.0...DrDevTaneja1
Digital India will need a big trained army of Health Informatics educated & trained manpower in India.
Presently, generalist IT manpower does most of the work in the healthcare industry in India. Academic Health Informatics education is not readily available at school & health university level or IT education institutions in India.
We look into the evolution of health informatics and its applications in the healthcare industry.
HIMMS TIGER resources are available to assist Health Informatics education.
Indian Health universities, IT Education institutions, and the healthcare industry must proactively collaborate to start health informatics courses on a big scale. An advocacy push from various stakeholders is also needed for this goal.
Health informatics has huge employment potential and provides a big business opportunity for the healthcare industry. A big pool of trained health informatics manpower can lead to product & service innovations on a global scale in India.
Digital Health in India_Health Informatics Trained Manpower _DrDevTaneja_15.0...
Clinical molecular diagnostics for drug guidance
1. Utility of Molecular Diagnostics in
clinical decision making
Nikhil Phadke, PhD
GenePath Dx, Pune, India
This presentation is the property of
2. This presentation is the property of
Learning Objectives
1. Be familiar with next generation molecular diagnostic
techniques that can provide guidance in clinical
decision making
2. Identify the utility of these diagnostic approaches
with some examples
3. Be aware of the challenges that exist in
implementing these tools as part of the routine
clinical decision making process, especially in
resource limited settings
4. This presentation is the property of
mt
16.6 Kb
37
1013 cells
Our DNA
Haploid Human Genome
~ 3.09 * 109 bp
Distributed in 23
chromosomes & mitochondria
Coding regions ~ 2%
(85% of all known mutations)
Total Genes 25,000 - 45,000
(Histone 500bp – Dystrophin 2.2 Mbp)
5. This presentation is the property of
Where can things go wrong?
What do we need to detect?
Analogy (using plain English)
♀THE DOG BIT THE CAT | THE CAT ATE THE RAT
♂THE DOG BIT THE CAT | THE CAT ATE THE RAT
Presence and quantification of foreign nucleic acids (e.g.
infectious agents like viruses and bacteria)
COW EGG PIG ASS
Single nucleotide polymorphisms (SNPs) (e.g. Sickle Cell
Anemia)
THA DOG BIT THE CAT
THE DOG BIT THE BAT
Insertions / Deletions (InDels) & Nucleotide Repeat
Polymorphisms – can cause frameshifts (e.g. Fragile X,
Huntingtons Disease)
THE EDO GBI TTH ECA T
THD OGB ITT HEC AT
Copy number variations (CNVs) e.g. (Deletion,
Duplications such as Her2 amplification in some Breast
Cancers)
THE DOG BIT THE CAT THE DOG BIT THE CAT
THE DOG DOG BIT THE CAT
OR
THE BIT THE CAT
Inversions (e.g. Factor VIII Haemophilia) THE GOD BIT THE CAT
TAC EHT TIB GOD EHT
Translocations (e.g. chronic myelogenous leukemia - CML
Philadelphia chromosome 22 & 9 translocation causes
BCR-ABL fusion)
THE DOG BIT THE CAT ATE THE RAT
THE CAT ATE THE DOG BIT
Loss of Heterozygosity (LoH) (e.g. retinoblatoma) /
Uniparental Disomy (e.g. Prader Willi Syndrome)
THE DOG BIT THE CAT
THE DOG BIT THE CAT
THE DOG BIT THE CAT
6. How do we detect these anomalies?
This presentation is the property of
Analysis scope
Single/ Few
Defined
Variations
Multiple/
Unknown
Variations
Cytogenetics Molecular Genetics
FISH
Karyotyping
(& Spectral)
(digital) PCR
SNaPshot
Sanger
Sequencing
MLPA
dHPLC/ HRM
Microarrays
Next Generation
Sequencing
Low resolution High resolution
M-FISH
7. The evolution of sequencing technologies
1970s 80s and 90s 2000s 2010s
This presentation is the property of
8. Traditional Capillary/Sanger Sequencing
Chromatogram
+ -
Shorter Longer
fragments fragments
This presentation is the property of
Nucleotides dA dT dG dC,
ddA ddT ddG ddC
Polymerase
Sequence X-X-X-T-T-G-S-T-A-T
Complementary Template Y-Y-Y-A-A-C-W-A-T-A
Primer X-X-X
X-X-X-T
Synthesized X-X-X-T-T
labeled DNA X-X-X-T-T-G
fragments X-X-X-T-T-G-C
X-X-X-T-T-G-G
X-X-X-T-T-G-C-T
X-X-X-T-T-G-C-T-A
X-X-X-T-T-G-C-T-A-T
X-X-X-T
X-X-X-T-T
X-X-X-T-T-G
X-X-X-T-T-G-C
X-X-X-T-T-G-G
X-X-X-
T-T-G-C-T
X-X-X-T-T-G-C-
T-A
X-X-X-
T-T-G-C-T-A-T
Capillary Electrophoretic
Separation
Sequencing Reaction
9. Pros and Cons of Capillary Sequencing
- Slow, limited throughput
- Expensive (per base)
- Limited sensitivity
- Not ideal for multiplexing
- Not ideal for multigenic or
large gene analyses
This presentation is the property of
Gold standard
Relatively long reads (700 -
1000 nucleotides)
Nobel Prize for Fred Sanger
in 1980
Used for Human Genome
Project (3000 scientists, multiple
centers, 6 countries, over 13
years, cost US$ ~ 3B)
10. Next (2nd) generation sequencing (NGS)
This presentation is the property of
Library Preparation
Data Capture &
Data Analysis
Fragmentation /
Targeted Amplification
Addition of Priming &
Multiplexing adapters
Massively
Parallel Sequencing
by Synthesis
Clonal amplification
(Emulsion/Bridge PCR)
11. Single molecule (3rd gen) sequencing
Single Molecule Real Time Nanopore Sequencing
This presentation is the property of
Sequencing
dA dT
dG dC
Zero-mode
Waveguide
Labeled
nucleotides
Anchored
Polymerase
Detection of incorporated
polymerase at base of ZMV
DNA
unwound,
ratcheted
through
protein
graphene
nanopore
Nucleotide detected
based on conductivity
12. Clinical applications of NGS
• Sequencing: de novo genome, resequencing, exome,
targeted, transcriptome profiling, metagenome analysis
• Large genes / multiple genes / multiple samples
• Ultra-deep sequencing for rare mutations and circulating
nucleic acids (e.g. plasma tumor DNA and maternal DNA for
non-invasive prenatal diagnosis)
This presentation is the property of
13. Digital PCR – An evolution of PCR
Conventional PCR Real-time PCR Digital PCR
Real time detection in
closed system
High Low
This presentation is the property of
Temp
Thermal cycling
Post PCR gel
electrophoretic analysis
Fluorescence
Cycles
Negative
End-point detection
of droplets
+ -
+
-
+
+
+
+
+
+
-
+
dA dT dG dC Polymerase + Primers dA dT dG dC
+ (Probes) +
Nucleotides + Target;
Individual Target DNA
physically separated
Polymerase + Primers
+ (Probes) +
Nucleotides + Target
in a
Homogenous system
15. Molecular diagnostics across medical disciplines
This presentation is the property of
Pathogen
identification
e.g. MTB
Pathogen
quantification
e.g. HIV load
Drug
resistance
status e.g.
MRSA
Predictive testing
e.g. BRCA1/2
Companion
diagnostics e.g.
EGFR, HER2
Disease
monitoring e.g.
BCR-ABL
Prognostic testing
e.g. ‘OncotypeDx’
e.g. Neonatal
Diabetes,
Congenital
Adrenal
Hyperplasia and
Prader Willi
Syndrome
Transplantation
e.g. HLA typing
and chimerism
monitoring
Blood disorders
e.g.
Thalassemia
and DVT
e.g. Trisomy 21
testing by
amniocentesis,
CVS, or NIPD
Infectious
Diseases
Endocrinology
Hematology
Identity
Prenatal
Testing
Oncology
16. The utility of BRCA testing
1. Modify surveillance options
2. Suggest risk reduction measures
3. Provide treatment guidance
4. Understand risk to other family members
5. Potential for pre-implantation diagnostics (PIGD)
This presentation is the property of
17. BRCA Testing in India in 2012 (Sanger Seq)
58 year old female diagnosed with Breast Cancer with
family history of HBOC; common Indian mutations
tested
This presentation is the property of
BRCA1 Exon 2 185del AG
BRCA1 Exon 8 632 dupT
BRCA1 Exon 12 IVS17+1, G>A, 5154delC
BRCA2 Exon 9 IVS8-2, A>G(c.682-2 A>G)
BRCA2 Exon 25 W3127X
All negative. Results inconclusive
18. BRCA Testing in India in 2014 (NGS Panel)
High penetrance breast cancer susceptibility genes
BRCA1, BRCA2, TP53, PTEN, STK11, CDH1
Moderate susceptibility breast cancer genes:
ATM, CHEK2, PALB2, BRIP1, NBN, RAD51C, RAD51D
This presentation is the property of
Other genes in the panel:
MEN1, CDC73, DICER1, MUTYH, NF2, MSH2, BMPR1A, APC, KIT, RET, SMAD4,
RB1, VHL, EPCAM, ALK, CDK4, AIP, MSH6, MET, MLH1, SDHAF2, SDHB, SDHD,
SDHC, SUFU, WT1, TSC2, TSC1, PMS2.
Pathogenic frameshift BRCA1 Mutation Detected c.933_933delT
19. Genetic testing in Neonatal Diabetes
• ATP-sensitive potassium channel (KATP) consists of two
subunits - SUR1 (ABCC8 – 4000bp) and Kir6.2 (KCNJ11 –
84000bp).
• Multiple mutations in these genes are implicated in Neonatal
Diabetes (NDM); for some mutations, normal activity can be
restored with Sulfonylurea drugs
K+ K+ K+
This presentation is the property of
Kir6.2 (KCNJ11)
Membrane
Glucose ATP
SIR1 (ABCC8)
Sulfonylurea
Normal Insulin Decreased Insulin (hyperglycemia) Normalized Insulin
20. Sanger Sequencing of KCNJ11 & ABCC8
Loss of
function/inactivatin
g G111R
causing recessively
inherited congenital
hyperinsulinism
(inherited from
asymptomatic
father)
Gain of function
R826W causing
This presentation is the property of
transient
neonatal
diabetes
(inherited from
asymptomatic
mother)
• One month old baby with NDM
(very high sugar)
• Initially managed with 2 insulin
injections per day
• Sanger Sequencing
• Detected two missense mutations
in ABCC8 gene G111R and
R826W
• Switched to oral sulfonylurea
(glibenclamide). Responded well
21. NGS Panel for Neonatal Diabetes
• Sanger sequencing of ABCC8 and KCNJ11 is laborious,
expensive (~ $2000) and requires long turn-around times
• Cases of suspected NDM with no detected mutations in
This presentation is the property of
ABCC8 and KCNJ11
• Development of NGS panel with several additional genes
involved in neonatal diabetes/hyperinsulinism
ABCC8 KCNJ11 GLUD1 GCK HADH INS HNF1A HNF4A HNF1B INS PDX1
PTF1A NEUROD1 NEUROG3 RFX6 EIF2AK3 FOXP3 GLIS3 SLC19A2 SLC2A2
IER3IP1 ZFP57 WFS1 GATA6 GATA4 CEL PAX4 BLK KLF11 LMNA PPARG
• Panel has allowed for relatively rapid and cheaper testing for
NDM and detection of new mutations in ABCC8, EIF2AK3,
GATA6, GCK, PDX1, SLC19A2
22. EGFR mutations in Lung Cancer
• Epidermal growth factor receptor (EGFR) can be mutated in
certain Non Small Cell Lung Cancers (NSCLC) and other
cancers
• Tyrosine kinase inhibitors (TKIs) (Gefitinib and Erlotinib) can
act on some mutated forms of EGFR
• Mutations usually detected from formalin fixed paraffin
embedded (FFPE) blocks by Sanger Sequencing or PCR
based methods
• Sanger sequencing has limited (~ 20%) sensitivity
• PCR methods need you to know the mutations a priori
This presentation is the property of
23. EGFR mutations by NGS
• Ultra-deep sequencing of EGFR from FFPE samples can
detect mutations at sensitivities up to 0.1%
• Somatic mutation NGS panels can be used to detect
mutations not just in the hotspots of certain genes, but over
entire sequences of multiple genes (e.g. KRAS, NRAS,
BRAF)
• Research projects underway to determine the clinical
relevance of EGFR mutations detected from circulating tumor
DNA in plasma
This presentation is the property of
24. BCR-ABL1 Fusions in CML
• Chronic Myelogenous Leukemia characterized by reciprocal
translocation of chromosomes 9 & 22 resulting in the
Philadelphia chromosome
• Fusion of Abelson (ABL1) proto-oncogene on chr 9 with the
breakpoint cluster region gene (BCR) on chr 22 , generating
the constitutively active BCR-ABL1 fusion oncogene
• Responds to tyrosine kinase inhibitor (TKI) – Imatinib
• Need to monitor minimal residual disease (MRD)
This presentation is the property of
25. BCR-ABL1 Testing in CML by RQ-PCR
Reverse transcribe RNA to DNA using Random Hexamers + TaqMan DNA quantitation
14
12
10
8
6
4
2
BCR-ABL ABL
This presentation is the property of
Tube Ct* Calculated Conc
(copies/uL)
Tube Ct* Calculated Conc
(copies/uL)
Standard 101 copies 32.97 9.8 Standard 101 copies 34.60 11
Standard 102 copies 29.44 104.8 Standard 102 copies 31.29 97
Standard 103 copies 26.12 977 Standard 103 copies 27.84 1017
Patient Major BCR-ABL 33.13 6.7 Patient ABL 26.45 5240
Major BCR-ABL/ABL Ratio is 0.13%
0
0 10 20 30 40
Log of target concentration
26. Digital PCR for BCR-ABL1 in CML
This presentation is the property of
dA dT dG dC
Polymerase + Nucleotides +
Primers + Probes
+ -
+
-
+
+
+
+
+
+
-
+
+ -
+
-
+
+
+
+
+
+
-
+
• Droplet digital RT-PCR
• Two color droplet detection of
BCR-ABL1 fusion gene and
ABL1 as reference gene
• Absolute count as against
relative quantitation
• No need for a standard curve
• Lower limit of detection (LOD)
+
+ +
+
+
+
+
+
+
+
+ +
+
+
+
+
+
+
28. Accessibility and Cost (India vs. US)
• US Population – 316 Mn,
17% rural
• Per capita income (PPP) - $
44800
• NGS machines: ~1000
• Physicians/ 1000 people:
2.45
• Public health expenditure (%
of GDP) – 8.3%
• Total health expenditure (per
capita) - $8233
• Established insurance system
This presentation is the property of
• India Population – 1.25 Bn,
68% rural
• Per capita income (PPP) –
$ 5350
• NGS machines: ~100 (80% in
research)
• Physicians/ 1000 people: 0.7
• Public health expenditure (%
of GDP) – 4.0%
• Total health expenditure (per
capita) - $126
• Out of pocket payment
Sources: WHO (2010), World Bank (2013)
29. Awareness and Education
• Limited exposure to genetics and molecular biology
during medical training
• Mandatory CMEs only recently introduced
• Limited ability to select appropriate tests and interpret
test results for optimal patient management
• Usage of molecular diagnostics guided decision making
is limited to very few conditions (e.g. Imatinib in CML,
Traustuzumab in Breast Cancer, Sulphonylurea in NDM)
This presentation is the property of
30. This presentation is the property of
Other Challenges
• Complex data interpretation & data storage
requirements
• Variants of unknown significance (VUS)
• Limited databases for ethnicities
• Incidental findings
• Ethical considerations
31. Questions that need to be answered
• Is the test relevant? Could it’s results alter the course of
treatment?
• What are the criteria for testing?
This presentation is the property of
• Are the results actionable?
• Will the results be reliable?
• Will the results be available in time?
• Will the results be interpretable?
• Is the test affordable? Is the follow-up treatment affordable? Is
the test more expensive that the treatment?
32. This presentation is the property of
Some solutions
• Increased availability of economical tests
• Availability and selection of relevant and actionable tests
• Multiplexing of tests and samples to reduce costs
• Reporting of only relevant and actionable data from panels
• Quick turn around time
• Clear communication with the clinicians
The basics
Examples to illustrate
Challenges and questions raised
Some take home messages
Fragmentation / targeted amplification
Addition of Priming and Multiplexing Adapters / Barcodes (ligation or PCR)
Clonal amplification of each fragment (Emulsion PCR or Bridge PCR)
Physical separation of each clonally amplified fragment through droplets captured in picotiter plates or on the surface of a flow cell.
Real-time sequencing by sequential addition of one nucleotide at a time, or through the use of flurescently labeled reversibly terminated nucleotides
Data acquisition through a cascade reaction that either produces an H+ ion (nanofabricated pH meter) or a bioluminescence event (luciferase) or through fluorescence monitoring of the flow cell
Conversion into nucleotide base calls. Assembly of fragment data to final form.
Polymerase anchored at base of Zero-Mode Waveguide.
All nucleotides fluorescently labeled
Detection of single nucleotide being incorporated by polymerase in real-time
Large arrays of ZMWs
5) DNA unwound and single stranded DNA ratcheted through protein nanopores embedded in artificial substrate
6) DNA bases detected as strand is stalled based on conductivity across the nanopore
7) Large arrays of nanopores
Microreactors in digital PCR. Much like the emulsion PCR sample prep for NGS.
From Qualitative results to relative quantification to absolute quantification (Poisson statistics)
1) Frequency of screening. Type of screening (e.g. alternating mammogram & MRI), Age of initial screening note mammo slightly less sensitive for BRCA1 due to higher breast density
2) e.g. Salpingo-oophorectomy after childbearing, bilateral mastectomy, chemoprevention like anti-estrogen therapy for BRCA2 mutation carriers
3) e.g. avoidance of radiation for TP53 mutation carriers, gene-specific treatment options like PARP inhibitors and platinum salts for BRCA1 carriers.
4) Evaluation of risk and testing of other family members
5) Evaluate the option on pre-implanatation genetic testing
Tumor Supressor genes.
Large genes. BRCA1 (81kb/24 exons/1863amino acids), BRCA2 (84kb/26 exons/3418 amino acids).
Nearly 800 clinically relevant mutations reported.
Laborious and expensive to sequence. ~ $3500/sample.
Patent related complications.
Cheaper and quicker way to sequence BRCA1 & 2
Several additional genes included in the panel (the one’s in bold – PALB2 and MEN1 were recently reported as important in NEJM articles)
Sulphonylurea Receptor (SUR1) and Inwardly Rectifying potassium channel (Kir6.2) - 4 subunits of each form the Katp channels. In pancreatic beta cells high glucose levels lead to increased ATP production, Channels sense the intracellular ATP levels and close. This causes increased membrane potential which opens voltage dependent calcium channels which trigger insulin exocytosis,
Activating mutations can lead to neonatal diabetes
FFPE DNA extraction is hard, and sometimes poor quality DNA
Tumor tissue is heterogenous mass along with normal tissue. Low level mutations can be easily missed
3) Sanger has low sensitivity, and PCR based methods need prior knowledge. Looking for all mutations by PCR can be laborious and expensive.
Often get patients who simply cannot afford the tests.
In many of those cases, even if the cost of the test were removed from the equation, the patient would not be able to afford the cost and burden of treatment
In some cases (especially infectious diseases) the cost of the test overwhelms the cost of the treatment, and the accessibility to the treatment, so it becomes easier to try a variety of treatments rather than test guided therapy
Large storage requirements. Need computaional horsepower, and fast internet connectivity. Also need data management and interpretation expertise
How do you handle large numbers of variants of unknown significance. Up to 1200 VUS in a single exome sequencing
Most data has been generated on Caucasian populations. Limited data available for Indians and other ethnic groups (further complicated by heterogenous populations). This exacerbates the VUS problem further.
How do you report incidental finding i.e. something that you were not looking for in the first case?
How do you manage the data, it’s privacy. How do you deal with findings that might impact other family members?
From the perspective of the clinician
Is the test affordable? Is it more expensive than the treatment? Is the treatment affordable?
Is the test relevant?Are the results actionable?Will the results be reliable?Will the results be available in time?From the perspective of the diagnosticianIs there sufficient awareness about the need for the test?Do the clinicians have the knowledge and ability to interpret the results of the tests?
Availability of relevant tests include the ability of data for the ethnicities that are being tested