This document summarizes a study that aimed to identify prognostic serum microRNA profiles in neuroblastoma. The study performed microRNA profiling on serum samples from different neuroblastoma patient risk groups. Ten microRNAs were found to best discriminate between survival groups. A validation set of over 120 patients was then profiled for these 10 microRNAs, finding they could separate high-risk deceased from low-risk survivor patients. This suggests serum microRNA profiling may help identify ultra-high risk neuroblastoma patients. The document also discusses microRNA biomarker potential in general and optimization of microRNA sequencing workflows from serum samples.
This document discusses next generation sequencing technologies. It provides details on several massively parallel sequencing platforms and describes their advantages over traditional Sanger sequencing such as higher throughput, lower costs, and ability to process millions of reads in parallel. It then outlines several applications of next generation sequencing like mutation discovery, transcriptome analysis, metagenomics, epigenetics research and discovery of non-coding RNAs.
This document discusses the use of 16S ribosomal RNA (rRNA) gene sequencing for bacterial identification and phylogenetic analysis. It explains that the 16S rRNA gene is highly conserved, making it useful for comparing distantly related organisms. The document outlines the process of 16S rRNA gene sequencing, including PCR amplification using conserved primer regions and sequencing of variable regions. It also discusses various methods that have been developed using 16S rRNA, such as TRFLP profiling and ribotyping, to study microbial communities.
RNA sequencing: advances and opportunities Paolo Dametto
This document summarizes recent advances in transcriptome analysis technologies. It discusses limitations of microarray-based approaches and how next-generation sequencing-based RNA-seq provides more comprehensive transcriptome profiling. RNA-seq can detect thousands of new transcript variants and isoforms. It also describes direct RNA sequencing without cDNA conversion, revealing polyadenylation profiles with single-molecule resolution. Comprehensive polyadenylation maps in human and yeast showed previously unannotated sites and alternative polyadenylation, providing insights into regulatory mechanisms.
This document discusses bladder cancer and different types (T1NP and T1P), which are differentiated by whether the disease progresses to become muscle-invasive or metastatic. RNA sequencing was used to analyze gene expression differences between the two types. The sequencing was done using the Illumina Genome Analyzer II, and the resulting reads were analyzed using CASAVA, TopHat, and Cufflinks software. CASAVA aligns reads and detects variants, TopHat identifies splice junctions, and Cufflinks assembles transcripts and tests for differential expression. Biomarkers identified through this analysis may help predict cancer progression using archived samples.
Original Next Gen Seq Methods set of slides prepared for Technorazz Vibes 2016. There is also a shorter version.
This starts with an introduction to qPCR followed by an introduction to Library Complexity. Microarrays are discussed as well along with a very short introduction to FISH. Finally discussion of Next gen seq methods is done where generation of sequencers are discussed and a short discussion of the ILLUMINA protocol. Finally comparison of ILLUMINA amongst other 3rd gen sequencer, description of the standard pipeline and the omics technologies that have risen from this seq data.
The document provides an overview of the history and techniques of transcriptome analysis. It discusses how RNA was separated from DNA with the formulation of the central dogma in 1958. Key developments include the discoveries of messenger RNA, transfer RNA, and ribosomal RNA in the 1960s. The document outlines techniques such as serial analysis of gene expression (SAGE) and RNA sequencing (RNA-seq) that allow comprehensive analysis of gene expression patterns. It provides details on the basic steps and advantages of SAGE and describes how next generation sequencing revolutionized transcriptome analysis through massive parallel sequencing.
Lynx Therapeutics' Massively Parallel Signature Sequencing (MPSS) is an early high-throughput DNA sequencing technique. It works by attaching cDNA from an mRNA sample to beads, determining short sequence signatures from many beads in parallel, and using the signatures to count the number of individual mRNA molecules from each gene. This digital gene expression data allows MPSS to accurately quantify genes expressed at low levels by analyzing transcripts from virtually all genes simultaneously. The technique involves converting mRNA to cDNA, attaching oligonucleotide tags, PCR amplification on beads, and using fluorescent probes to determine short sequences in increments of four nucleotides from millions of beads in parallel.
This document discusses next generation sequencing technologies. It provides details on several massively parallel sequencing platforms and describes their advantages over traditional Sanger sequencing such as higher throughput, lower costs, and ability to process millions of reads in parallel. It then outlines several applications of next generation sequencing like mutation discovery, transcriptome analysis, metagenomics, epigenetics research and discovery of non-coding RNAs.
This document discusses the use of 16S ribosomal RNA (rRNA) gene sequencing for bacterial identification and phylogenetic analysis. It explains that the 16S rRNA gene is highly conserved, making it useful for comparing distantly related organisms. The document outlines the process of 16S rRNA gene sequencing, including PCR amplification using conserved primer regions and sequencing of variable regions. It also discusses various methods that have been developed using 16S rRNA, such as TRFLP profiling and ribotyping, to study microbial communities.
RNA sequencing: advances and opportunities Paolo Dametto
This document summarizes recent advances in transcriptome analysis technologies. It discusses limitations of microarray-based approaches and how next-generation sequencing-based RNA-seq provides more comprehensive transcriptome profiling. RNA-seq can detect thousands of new transcript variants and isoforms. It also describes direct RNA sequencing without cDNA conversion, revealing polyadenylation profiles with single-molecule resolution. Comprehensive polyadenylation maps in human and yeast showed previously unannotated sites and alternative polyadenylation, providing insights into regulatory mechanisms.
This document discusses bladder cancer and different types (T1NP and T1P), which are differentiated by whether the disease progresses to become muscle-invasive or metastatic. RNA sequencing was used to analyze gene expression differences between the two types. The sequencing was done using the Illumina Genome Analyzer II, and the resulting reads were analyzed using CASAVA, TopHat, and Cufflinks software. CASAVA aligns reads and detects variants, TopHat identifies splice junctions, and Cufflinks assembles transcripts and tests for differential expression. Biomarkers identified through this analysis may help predict cancer progression using archived samples.
Original Next Gen Seq Methods set of slides prepared for Technorazz Vibes 2016. There is also a shorter version.
This starts with an introduction to qPCR followed by an introduction to Library Complexity. Microarrays are discussed as well along with a very short introduction to FISH. Finally discussion of Next gen seq methods is done where generation of sequencers are discussed and a short discussion of the ILLUMINA protocol. Finally comparison of ILLUMINA amongst other 3rd gen sequencer, description of the standard pipeline and the omics technologies that have risen from this seq data.
The document provides an overview of the history and techniques of transcriptome analysis. It discusses how RNA was separated from DNA with the formulation of the central dogma in 1958. Key developments include the discoveries of messenger RNA, transfer RNA, and ribosomal RNA in the 1960s. The document outlines techniques such as serial analysis of gene expression (SAGE) and RNA sequencing (RNA-seq) that allow comprehensive analysis of gene expression patterns. It provides details on the basic steps and advantages of SAGE and describes how next generation sequencing revolutionized transcriptome analysis through massive parallel sequencing.
Lynx Therapeutics' Massively Parallel Signature Sequencing (MPSS) is an early high-throughput DNA sequencing technique. It works by attaching cDNA from an mRNA sample to beads, determining short sequence signatures from many beads in parallel, and using the signatures to count the number of individual mRNA molecules from each gene. This digital gene expression data allows MPSS to accurately quantify genes expressed at low levels by analyzing transcripts from virtually all genes simultaneously. The technique involves converting mRNA to cDNA, attaching oligonucleotide tags, PCR amplification on beads, and using fluorescent probes to determine short sequences in increments of four nucleotides from millions of beads in parallel.
Next Generation Sequencing Technologies and Their Applications in Ornamental ...Ravindra Kumar
This document summarizes research on DNA sequencing and genome sequencing techniques. It discusses early Sanger sequencing and the development of next-generation sequencing platforms like Roche 454, Illumina, Ion Torrent, and SOLiD. The document also presents two case studies, one on sequencing the carnation genome and another on obtaining the rose transcriptome to identify genes related to traits of interest. Overall, the document provides a high-level overview of the evolution of DNA sequencing technologies and their applications in sequencing plant genomes and transcriptomes.
2016. daisuke tsugama. next generation sequencing (ngs) for plant researchFOODCROPS
This document provides an overview of next-generation sequencing (NGS) for plant research. It discusses the main NGS platforms, data analysis procedures including assembly, mapping, and applications such as RNA-seq, genome sequencing, RAD-seq, MutMap, and QTL-seq. The document aims to explain what NGS is, typical analysis workflows, and how NGS can be applied to questions in plant research.
Ribosomes are complex structures found in all living cells which functions in protein synthesis machinery. Basically ribosome’s consists of two subunits, each of which is composed of protein and a type of RNA, known as ribosomal RNA (rRNA). Prokaryotic ribosomes consist of 30S subunit (small sub unit) and 50S subunit (large sub unit) which together make up the complete 70S ribosome, where S stands for Svedberg unit non-SI unit for sedimentation rate. 30S subunit is composed of 16S ribosomal RNA and 21 polynucleotide chains while 50S subunit is composed of two rRNA species, the 5S and 23S rRNAs. The presence of hyper variable regions in the 16S rRNA gene provides a species specific signature sequence which is useful for bacterial identification process. 16S Ribosomal RNA sequencing is widely used in microbiology studies to identify the diversities in prokaryotic organisms as well as other organisms and thereby studying the phylogenetic relationships between them. The advantages of using ribosomal RNA in molecular techniques are as follows
Ribosomes and ribosomal RNA are present in all cells.
RNA genes are highly conserved in nature.
Culturing of microbial cells is absent in the sequencing techniques.
Next Generation Sequencing & Transcriptome AnalysisBastian Greshake
This document discusses next generation sequencing methods like 454 sequencing, Illumina sequencing, and SOLiD sequencing. It then describes how the large amounts of sequencing data generated can be used for transcriptome analysis to study gene expression, identify new genes, and analyze non-model organisms. Finally, it outlines some of the common bioinformatics tools used for assembling sequencing reads, detecting SNPs, finding homologous genes, identifying open reading frames, and annotating genes.
Next-generation DNA sequencing technologies have significantly impacted genetics research. Three major platforms - Roche/454, Illumina Genome Analyzer, and Applied Biosystems SOLiD - utilize massively parallel sequencing to generate large amounts of sequence data. Roche/454 uses emulsion PCR to amplify DNA fragments on beads and pyrosequencing to determine sequences. Illumina performs bridge amplification on a flow cell to generate DNA clusters then sequences by synthesis. Applied Biosystems SOLiD uses ligation-based sequencing. These new methods have enabled genome-wide studies and applications such as ancient DNA sequencing and metagenomics that were previously difficult or impossible.
Role of transcriptomics in gene expression studies andSarla Rao
Transcriptomics provides a global view of gene expression by analyzing the transcriptome, which includes mRNA and various non-coding RNA transcripts. It can be used to study gene expression changes during development, in different diseases and in response to drugs. RNA-sequencing is now widely used for transcriptome analysis and offers advantages over microarrays. Transcriptomics has revealed non-coding RNAs like miRNAs play important regulatory roles. Differential miRNA expression is observed in cancers and miRNAs can influence cancer metastasis. They also show promise as biomarkers for cancer diagnosis and potential therapeutics.
MPSS is a technique for analyzing gene expression that involves sequencing cDNA fragments cloned onto microbeads. It allows for the simultaneous sequencing of over 1 million cDNA clones. MPSS generates 17-base signature sequences that uniquely identify mRNA transcripts. Gene expression levels are quantified by counting the number of signatures for each gene. MPSS provides a more in-depth analysis of gene expression compared to other methods as it can detect genes expressed at very low levels and does not require prior knowledge of gene sequences.
This document provides an overview of DNA sequencing technologies. It begins with a brief history of DNA sequencing, including the discovery of DNA's structure and Sanger sequencing. The document then focuses on next generation sequencing technologies, describing several platforms such as 454 sequencing, Illumina sequencing, Ion Torrent sequencing, and Pacific Biosciences sequencing. It also discusses third generation sequencing and compares the sequencing approaches, workflows, and applications of various sequencing technologies. In conclusion, the document notes the progress and future directions of sequencing, including increased clinical applications and reduced costs.
Digital DNA-seq Technology: Targeted Enrichment for Cancer ResearchQIAGEN
Targeted DNA sequencing has become a powerful approach by achieving high coverage of the region of interest while keeping the cost of sequencing and complexity of data interpretation manageable. However, existing PCR-based target enrichment approaches introduce errors due to PCR amplification bias and artifacts, which significantly affects quantification accuracy and limit the ability to confidently detect low-frequency DNA variants. This webinar introduces a new digital sequencing approach that is based on the use of unique molecular indices (UMIs) - QIAseq Targeted DNA Panels. With UMIs, each unique DNA molecule is barcoded before any amplification takes place to correct for PCR errors. Detailed workflow and applications in cancer research will be presented. Join us and learn about this exciting novel digital DNAseq technology
This document discusses wet-lab considerations for Illumina sequencing data analysis. It describes the typical Illumina sequencing workflow including library preparation, cluster formation, sequencing, and data analysis. It provides details on DNA and RNA input requirements, library construction steps like fragmentation and adapter ligation, and quality control methods. The document also discusses newer sequencing technologies like Pacific Biosciences and Oxford Nanopore sequencing.
Next Generation Sequencing (NGS) Is A Modern And Cost Effective Sequencing Technology Which Enables Scientists To Sequence Nucleic Acids At Much Faster Rate. In This Presentation, You Will Learn About What is NGS, Idea Behind NGS, Methodology And Protocol, Widely Adapted NGS Protocols, Applications And References For Further Study.
This document summarizes three main next generation sequencing technologies: Roche/454FLX pyrosequencing, Illumina/Solexa sequencing by synthesis, and Applied Biosystems SOLiD sequencing by ligation. Pyrosequencing works by detecting pyrophosphate released during DNA polymerization, producing light signals to determine the sequence. Roche/454FLX amplifies DNA fragments on beads in emulsions and sequences in picotiter plates. Illumina attaches DNA fragments to a flow cell for bridge amplification and sequencing by synthesis. Applied Biosystems SOLiD performs sequencing by ligation, determining sequences through sequential ligation of oligos.
Next generation sequencing (NGS) allows for high-throughput sequencing at low cost by processing millions of reactions in parallel. This overcomes limitations of earlier Sanger sequencing methods, which were expensive, time-consuming, and difficult to automate. NGS is now commonly used in plant pathology and mycology to understand fungal genomes and disease mechanisms. It provides insights to improve disease management and natural product development.
Next generation-sequencing.ppt-convertedShweta Tiwari
The advance version, sequences the whole genome efficiently with high speed and high throughput sequencing at reduce cost is termed as Next Generation Sequencing (NGS) or massively parallel sequencing (MPS).
QIAseq Targeted DNA, RNA and Fusion Gene PanelsQIAGEN
Tumor heterogeneity has been known for a while but quantifying heterogeneity is still a challenge. NGS is the method of choice in the analysis of tumor heterogeneity, however, there are some inherent challenges associated with it. These include false positives, gaps in the gene due to overrepresentation and incomplete representation of low-frequency transcripts – all contributing to an inaccurate picture. Conventional library prep strategies for NGS are based on PCR, which introduces sequence-based bias and amplification noise, leading to these inaccuracies. In this webinar, we will cover
1. Principles of UMI and the new QIAseq product porfolio
2. How UMI along with SPE (single primer extension) allows for increased uniformity across difficult-to-sequence regions, removal of library construction bias, improved data analysis and sequencing optimization
3. How data generated from using UMI and SPE is directly comparable to analysis derived from whole transcriptome and exome sequencing
4. Application of UMI and SPE in the discovery of novel gene fusions and in the analysis of gene expression and genetic variation
Bioo Scientific - Reduced Bias Small RNA Library Prep with Gel-Free or Low-In...Bioo Scientific
microRNAs (miRNAs) may provide useful markers for the development of disease diagnostic and prognostic assays. NGS brings sensitivity, specificity, and the ability to maximize data acquisition and minimize costs of miRNA sequencing by using multiplex strategies to allow many samples to be sequenced simultaneously with small RNA analysis. However, small RNA sequencing has typically suffered from three major drawbacks: severe bias, such that sequencing data does not reflect original miRNA abundances, the need to gel purify final libraries, and lack of low-input protocols. The NEXTflex™ Small RNA-Seq Kit v3 addresses these drawbacks by using two strategies: randomized adapters to reduce ligation-associated bias, and a dual approach to adapter-dimer reduction, thereby allowing gel-free or low-input small RNA library preparation.
The document describes an RNA-seq analysis workflow that includes:
1. Preprocessing raw reads including quality control, filtering, and alignment to a reference genome using tools like FastQC, Bowtie2, and TopHat.
2. Assembling transcripts and estimating abundance using Cufflinks and HTseq-count.
3. Identifying differentially expressed genes between samples using DESeq and Cuffdiff.
4. Providing gene annotations and visualizing results using tools like GO, KEGG, and CummeRbund.
The workflow follows a typical reference-based analysis approach and uses various open source tools for read mapping, assembly, quantification, and differential expression.
Whole genome sequencing analysis involves aligning sequencing reads, calling variants like SNPs, indels and structural variants, and annotating the variants. The pipeline includes alignment with tools like BWA, variant calling with GATK for SNPs/indels and Lumpy/Manta for structural variants, and annotation with Ensembl VEP. WGS can detect more types of variants than exome sequencing and has increasing diagnostic utility for diseases.
How to Become a Thought Leader in Your NicheLeslie Samuel
Are bloggers thought leaders? Here are some tips on how you can become one. Provide great value, put awesome content out there on a regular basis, and help others.
Este documento proporciona instrucciones para crear una base de datos en Access. Explica cómo crear tablas, agregar campos, establecer relaciones entre tablas, insertar registros, y diseñar formularios, informes, consultas y un panel de control. El objetivo es enseñar a los usuarios a organizar y visualizar datos en Access de una manera fácil de usar y comprensible.
Next Generation Sequencing Technologies and Their Applications in Ornamental ...Ravindra Kumar
This document summarizes research on DNA sequencing and genome sequencing techniques. It discusses early Sanger sequencing and the development of next-generation sequencing platforms like Roche 454, Illumina, Ion Torrent, and SOLiD. The document also presents two case studies, one on sequencing the carnation genome and another on obtaining the rose transcriptome to identify genes related to traits of interest. Overall, the document provides a high-level overview of the evolution of DNA sequencing technologies and their applications in sequencing plant genomes and transcriptomes.
2016. daisuke tsugama. next generation sequencing (ngs) for plant researchFOODCROPS
This document provides an overview of next-generation sequencing (NGS) for plant research. It discusses the main NGS platforms, data analysis procedures including assembly, mapping, and applications such as RNA-seq, genome sequencing, RAD-seq, MutMap, and QTL-seq. The document aims to explain what NGS is, typical analysis workflows, and how NGS can be applied to questions in plant research.
Ribosomes are complex structures found in all living cells which functions in protein synthesis machinery. Basically ribosome’s consists of two subunits, each of which is composed of protein and a type of RNA, known as ribosomal RNA (rRNA). Prokaryotic ribosomes consist of 30S subunit (small sub unit) and 50S subunit (large sub unit) which together make up the complete 70S ribosome, where S stands for Svedberg unit non-SI unit for sedimentation rate. 30S subunit is composed of 16S ribosomal RNA and 21 polynucleotide chains while 50S subunit is composed of two rRNA species, the 5S and 23S rRNAs. The presence of hyper variable regions in the 16S rRNA gene provides a species specific signature sequence which is useful for bacterial identification process. 16S Ribosomal RNA sequencing is widely used in microbiology studies to identify the diversities in prokaryotic organisms as well as other organisms and thereby studying the phylogenetic relationships between them. The advantages of using ribosomal RNA in molecular techniques are as follows
Ribosomes and ribosomal RNA are present in all cells.
RNA genes are highly conserved in nature.
Culturing of microbial cells is absent in the sequencing techniques.
Next Generation Sequencing & Transcriptome AnalysisBastian Greshake
This document discusses next generation sequencing methods like 454 sequencing, Illumina sequencing, and SOLiD sequencing. It then describes how the large amounts of sequencing data generated can be used for transcriptome analysis to study gene expression, identify new genes, and analyze non-model organisms. Finally, it outlines some of the common bioinformatics tools used for assembling sequencing reads, detecting SNPs, finding homologous genes, identifying open reading frames, and annotating genes.
Next-generation DNA sequencing technologies have significantly impacted genetics research. Three major platforms - Roche/454, Illumina Genome Analyzer, and Applied Biosystems SOLiD - utilize massively parallel sequencing to generate large amounts of sequence data. Roche/454 uses emulsion PCR to amplify DNA fragments on beads and pyrosequencing to determine sequences. Illumina performs bridge amplification on a flow cell to generate DNA clusters then sequences by synthesis. Applied Biosystems SOLiD uses ligation-based sequencing. These new methods have enabled genome-wide studies and applications such as ancient DNA sequencing and metagenomics that were previously difficult or impossible.
Role of transcriptomics in gene expression studies andSarla Rao
Transcriptomics provides a global view of gene expression by analyzing the transcriptome, which includes mRNA and various non-coding RNA transcripts. It can be used to study gene expression changes during development, in different diseases and in response to drugs. RNA-sequencing is now widely used for transcriptome analysis and offers advantages over microarrays. Transcriptomics has revealed non-coding RNAs like miRNAs play important regulatory roles. Differential miRNA expression is observed in cancers and miRNAs can influence cancer metastasis. They also show promise as biomarkers for cancer diagnosis and potential therapeutics.
MPSS is a technique for analyzing gene expression that involves sequencing cDNA fragments cloned onto microbeads. It allows for the simultaneous sequencing of over 1 million cDNA clones. MPSS generates 17-base signature sequences that uniquely identify mRNA transcripts. Gene expression levels are quantified by counting the number of signatures for each gene. MPSS provides a more in-depth analysis of gene expression compared to other methods as it can detect genes expressed at very low levels and does not require prior knowledge of gene sequences.
This document provides an overview of DNA sequencing technologies. It begins with a brief history of DNA sequencing, including the discovery of DNA's structure and Sanger sequencing. The document then focuses on next generation sequencing technologies, describing several platforms such as 454 sequencing, Illumina sequencing, Ion Torrent sequencing, and Pacific Biosciences sequencing. It also discusses third generation sequencing and compares the sequencing approaches, workflows, and applications of various sequencing technologies. In conclusion, the document notes the progress and future directions of sequencing, including increased clinical applications and reduced costs.
Digital DNA-seq Technology: Targeted Enrichment for Cancer ResearchQIAGEN
Targeted DNA sequencing has become a powerful approach by achieving high coverage of the region of interest while keeping the cost of sequencing and complexity of data interpretation manageable. However, existing PCR-based target enrichment approaches introduce errors due to PCR amplification bias and artifacts, which significantly affects quantification accuracy and limit the ability to confidently detect low-frequency DNA variants. This webinar introduces a new digital sequencing approach that is based on the use of unique molecular indices (UMIs) - QIAseq Targeted DNA Panels. With UMIs, each unique DNA molecule is barcoded before any amplification takes place to correct for PCR errors. Detailed workflow and applications in cancer research will be presented. Join us and learn about this exciting novel digital DNAseq technology
This document discusses wet-lab considerations for Illumina sequencing data analysis. It describes the typical Illumina sequencing workflow including library preparation, cluster formation, sequencing, and data analysis. It provides details on DNA and RNA input requirements, library construction steps like fragmentation and adapter ligation, and quality control methods. The document also discusses newer sequencing technologies like Pacific Biosciences and Oxford Nanopore sequencing.
Next Generation Sequencing (NGS) Is A Modern And Cost Effective Sequencing Technology Which Enables Scientists To Sequence Nucleic Acids At Much Faster Rate. In This Presentation, You Will Learn About What is NGS, Idea Behind NGS, Methodology And Protocol, Widely Adapted NGS Protocols, Applications And References For Further Study.
This document summarizes three main next generation sequencing technologies: Roche/454FLX pyrosequencing, Illumina/Solexa sequencing by synthesis, and Applied Biosystems SOLiD sequencing by ligation. Pyrosequencing works by detecting pyrophosphate released during DNA polymerization, producing light signals to determine the sequence. Roche/454FLX amplifies DNA fragments on beads in emulsions and sequences in picotiter plates. Illumina attaches DNA fragments to a flow cell for bridge amplification and sequencing by synthesis. Applied Biosystems SOLiD performs sequencing by ligation, determining sequences through sequential ligation of oligos.
Next generation sequencing (NGS) allows for high-throughput sequencing at low cost by processing millions of reactions in parallel. This overcomes limitations of earlier Sanger sequencing methods, which were expensive, time-consuming, and difficult to automate. NGS is now commonly used in plant pathology and mycology to understand fungal genomes and disease mechanisms. It provides insights to improve disease management and natural product development.
Next generation-sequencing.ppt-convertedShweta Tiwari
The advance version, sequences the whole genome efficiently with high speed and high throughput sequencing at reduce cost is termed as Next Generation Sequencing (NGS) or massively parallel sequencing (MPS).
QIAseq Targeted DNA, RNA and Fusion Gene PanelsQIAGEN
Tumor heterogeneity has been known for a while but quantifying heterogeneity is still a challenge. NGS is the method of choice in the analysis of tumor heterogeneity, however, there are some inherent challenges associated with it. These include false positives, gaps in the gene due to overrepresentation and incomplete representation of low-frequency transcripts – all contributing to an inaccurate picture. Conventional library prep strategies for NGS are based on PCR, which introduces sequence-based bias and amplification noise, leading to these inaccuracies. In this webinar, we will cover
1. Principles of UMI and the new QIAseq product porfolio
2. How UMI along with SPE (single primer extension) allows for increased uniformity across difficult-to-sequence regions, removal of library construction bias, improved data analysis and sequencing optimization
3. How data generated from using UMI and SPE is directly comparable to analysis derived from whole transcriptome and exome sequencing
4. Application of UMI and SPE in the discovery of novel gene fusions and in the analysis of gene expression and genetic variation
Bioo Scientific - Reduced Bias Small RNA Library Prep with Gel-Free or Low-In...Bioo Scientific
microRNAs (miRNAs) may provide useful markers for the development of disease diagnostic and prognostic assays. NGS brings sensitivity, specificity, and the ability to maximize data acquisition and minimize costs of miRNA sequencing by using multiplex strategies to allow many samples to be sequenced simultaneously with small RNA analysis. However, small RNA sequencing has typically suffered from three major drawbacks: severe bias, such that sequencing data does not reflect original miRNA abundances, the need to gel purify final libraries, and lack of low-input protocols. The NEXTflex™ Small RNA-Seq Kit v3 addresses these drawbacks by using two strategies: randomized adapters to reduce ligation-associated bias, and a dual approach to adapter-dimer reduction, thereby allowing gel-free or low-input small RNA library preparation.
The document describes an RNA-seq analysis workflow that includes:
1. Preprocessing raw reads including quality control, filtering, and alignment to a reference genome using tools like FastQC, Bowtie2, and TopHat.
2. Assembling transcripts and estimating abundance using Cufflinks and HTseq-count.
3. Identifying differentially expressed genes between samples using DESeq and Cuffdiff.
4. Providing gene annotations and visualizing results using tools like GO, KEGG, and CummeRbund.
The workflow follows a typical reference-based analysis approach and uses various open source tools for read mapping, assembly, quantification, and differential expression.
Whole genome sequencing analysis involves aligning sequencing reads, calling variants like SNPs, indels and structural variants, and annotating the variants. The pipeline includes alignment with tools like BWA, variant calling with GATK for SNPs/indels and Lumpy/Manta for structural variants, and annotation with Ensembl VEP. WGS can detect more types of variants than exome sequencing and has increasing diagnostic utility for diseases.
How to Become a Thought Leader in Your NicheLeslie Samuel
Are bloggers thought leaders? Here are some tips on how you can become one. Provide great value, put awesome content out there on a regular basis, and help others.
Este documento proporciona instrucciones para crear una base de datos en Access. Explica cómo crear tablas, agregar campos, establecer relaciones entre tablas, insertar registros, y diseñar formularios, informes, consultas y un panel de control. El objetivo es enseñar a los usuarios a organizar y visualizar datos en Access de una manera fácil de usar y comprensible.
This study profiled circulating microRNAs (miRNAs) in human plasma and serum samples using deep sequencing of small RNA libraries. The researchers detected placental-specific miRNAs in maternal and newborn circulation, quantifying their relative abundance. They also found that sequence variations in placental miRNA profiles could be traced to the specific placenta of origin. This deep sequencing approach provides a comprehensive characterization of the miRNA content in circulation and establishes its potential for biomarker discovery and noninvasive detection of diseases originating from solid tissues like tumors or the placenta during pregnancy.
Georgetown Innovation Center for Biomedical Informatics Symposium Precision ...Warren Kibbe
The document discusses opportunities and challenges with precision oncology and big data. It describes how big data from sources like mobile devices, social media, next generation sequencing, imaging, and electronic health records can be leveraged. Key challenges include needing synoptic and semantic EHR data to support precision medicine, and handling and analyzing large amounts of patient-derived data from various sources. Examples provided of current solutions include mobile apps to collect patient-reported outcomes and integrating natural language processing with EHRs. The document also describes several projects and tools developed at Northwestern University for mobile computing and context awareness in healthcare, such as Mobilyze for depression treatment and Purple Robot for sensor data collection.
3 Things Every Sales Team Needs to Be Thinking About in 2017Drift
Thinking about your sales team's goals for 2017? Drift's VP of Sales shares 3 things you can do to improve conversion rates and drive more revenue.
Read the full story on the Drift blog here: http://blog.drift.com/sales-team-tips
Pistoia Alliance US Conference 2015 - 1.5.4 New data - Nikolaus SchultzPistoia Alliance
The document provides an overview of cancer genomics data and tools for analyzing it. It discusses how next-generation sequencing is identifying genetic alterations in cancer at an increasing scale through projects like TCGA. The cBioPortal is highlighted as a tool that provides intuitive access to these complex cancer genomics datasets and helps identify patterns across data to provide clinical insights. It has become widely used and its code is now fully open source to further collaborative cancer research.
Please share this webinar with anyone who may be interested!
Watch all our webinars: https://www.youtube.com/playlist?list=PL4dDQscmFYu_ezxuxnAE61hx4JlqAKXpR
Cancer care is increasingly tailored to individual patients, who can undergo genetic or biomarker testing soon after diagnosis, to determine which treatments have the best chance of shrinking or eliminating tumours.
In this webinar, a pathologist and clinical oncologist discuss:
● how they are using these new tests,
● how they communicate results and treatment options to patients and caregivers, and
● how patients can be better informed on the kinds of tests that are in development or in use across Canada
View the video: https://youtu.be/_Wai_uMQKEQ
Follow our social media accounts:
Twitter - https://twitter.com/survivornetca
Facebook - https://www.facebook.com/CanadianSurvivorNet
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10 Things You Need To Know About Women In MENAMohammed Minawi
The document summarizes key insights from a large research study on women in the Middle East and North Africa region. Some of the main findings are: 1) Women play a major role in household purchase decisions across many categories like food, personal care, and appliances. 2) However, most women do not generate their own income and rely on allowances from male relatives. 3) As a result, value for money is a major consideration in purchase decisions. 4) Women are heavily connected through internet and social media but traditional media remains an important source of information and online shopping is still limited.
This Presentation presents how Data science can bring manifold benefits to Retail Broking. Machine Learning & Text Analytics can impact your business in many positive ways- gives you that competitive edge and gains you customer satisfaction & loyalty
1) A preposition is a word that explains a spatial or temporal relationship and is used before nouns. A prepositional phrase begins with a preposition and ends with a noun or clause, which is the object of the preposition.
2) A prepositional phrase functions like an adjective or adverb by answering questions like "which one?" or "how, when, or where?".
3) A prepositional phrase will never contain the subject of the sentence. The subject is always outside of the prepositional phrase.
Sales Playbook Rapid Scale strategies video simulationJ Bentley Radcliff
The document introduces ACE, a just-in-time microlearning solution from RapidScale Strategies that provides simulated sales training. It ensures salespeople demonstrate knowledge and confidence with customers, provides management confidence in sales execution, and builds trust with customers. The platform allows virtualization of subject matter experts and resources for on-demand training. It includes a virtual customer simulation to practice messaging and a virtual sales kit. The mobile-first solution improves sales performance by delivering just-in-time content and intelligence anywhere. It consolidates marketing and education into easy to digest modules accessible before customer interactions. The platform facilitates virtualization of all resources to simplify complex portfolios and build acumen around key messages for any seller.
Este documento resume una página web creada en la plataforma Wix que presenta fotografías tomadas en Colombia a lo largo de varios años. La plataforma Wix requiere de experiencia para ser manejada fácilmente, pero resulta una herramienta útil una vez dominada. El objetivo de la página es apreciar las pequeñas cosas que a menudo pasan desapercibidas y encontrar perfección en momentos sencillos capturados en fotografías.
La abdicación de la izquierda dani rodrikPAD Ancash
El autor argumenta que la izquierda ha fallado en presentar una alternativa clara a la globalización y al capitalismo del siglo XXI. Mientras que los políticos de derecha han explotado el resentimiento hacia la inmigración y la globalización, la izquierda no ha propuesto ideas económicas sólidas más allá de políticas de bienestar. Además, tecnócratas de izquierda promovieron la globalización financiera en lugar de desarrollar una alternativa. Sin embargo, ahora existen propuestas académicas sobre reformas banc
These are some of the photographs I've taken for the School of Human Ecology in promotion for events, lectures and guests or to exhibit the beautiful architecture of the building.
O documento discute conceitos de organização e estrutura organizacional, incluindo definições de organização formal e informal. Também aborda a história do trabalho e diferentes regimes de relações de produção, como escravidão e feudalismo.
1) O documento discute os princípios básicos da mecânica dos fluidos, incluindo a conservação da massa, quantidade de movimento e energia.
2) Apresenta o Teorema do Transporte de Reynolds (TTR), que relaciona a taxa de variação de propriedades em um sistema com fluxos através dos limites de um volume de controle.
3) Explica a aplicação do TTR para derivar equações de conservação para a massa, quantidade de movimento, energia e entropia.
microRNA for Clinical Research and Tumor AnalysisBioGenex
The discovery of microRNAs [miRNAs] has been one of the defining developments in cancer biology over the past decade. miRNAs are short, single stranded 20-22 nucleotide long, non-coding RNAs that regulate gene expression and have fundamental roles in Cancer growth and metastasis. miRNAs exert their function via base pairing with complementary mRNA molecules, resulting in gene silencing via transcriptional repression or target degradation. BioGenex solved the inherent difficulties in visualizing miRNAs in spatial context by using a propriety technology to synthesize modified, high-affinity oligonucleotides, labelling miRNA probes with multiple reporter molecules and developing a fully-integrated miRNA-ISH workflow solution allowing high throughput analysis of miRNA in the spatial context.
The document discusses various topics related to molecular profiling and personalized medicine. It describes first generation molecular profiling techniques like gene sequencing, microarrays, and PCR. It then covers next generation sequencing technologies like Roche 454, Illumina, and ABI SOLID. It also discusses second generation techniques for DNA and RNA profiling including exome sequencing, ChIP-seq, and RNA-seq. Finally, it briefly mentions third generation sequencing and epigenetic profiling.
Applications of transcriptomice s in modern biotechnology 2Pakeeza Rubab
Transcriptomics is the study of transcriptomes, which are the complete set of RNA transcripts produced in a cell or tissue under a specific set of conditions. Next-generation sequencing techniques like Illumina sequencing have enabled comprehensive analysis of transcriptomes. Transcriptomics has many applications in biotechnology including agriculture, stem cell research, disease studies, and assessing chemical safety. It can be used to discover gene functions, biomarkers, and responses to environmental changes. Common transcriptomics techniques are real-time PCR, microarrays, and next-generation sequencing which provide information on RNA expression levels.
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.
Total RNA Discovery for RNA Biomarker Development WebinarQIAGEN
Precision medicine offers to transform patient care by targeting treatment to those with most to gain. To date the most significant advances have been at the level of DNA, for example, the use of somatic DNA alterations as diagnostic indicators of disease and for prediction of pharmacodynamic response. Development of RNA expression signatures as biomarkers has been more problematic. While RNA expression analysis has yielded valuable insights into the biological mechanisms of disease, RNA is a more unstable molecule than DNA, and more easily damaged or degraded during sample collection and isolation. In addition, RNA levels are inherently dynamic and gene expression signatures are extraordinarily complex. Recently, much progress has been made in identifying key changes in gene expression in cancer and other diseases, as well as identifying expression signatures in circulating nucleic acid that have the potential to be developed into diagnostic and prognostic indicators.
miRNA-Target Site SNPs as Predictors for Cancer Risk and Treatment ResponseDavid W. Salzman
This document discusses the generation of isogenic cell lines containing different variants of a microRNA target site in the KRAS 3' untranslated region (UTR). Specifically, it describes a workflow to use zinc finger nucleases and homologous recombination to insert either the common "T" allele or rare "G" allele of rs61764370 into the KRAS 3'UTR of cell lines that originally only contained the "T" allele. This will allow study of the effects of the variants on KRAS expression and cell phenotypes. Sequencing is used to verify insertion of the correct allele and establish homozygous cell lines for further experimentation.
This document discusses microRNAs (miRNAs) and methods for studying their function and regulation of genes. It describes:
1) What miRNAs are, how they work by incorporating into the RISC complex and repressing target mRNAs through translational repression or degradation.
2) Techniques for manipulating miRNAs in cell lines using reporter assays, mimics, inhibitors and target protectors to study their effects on genes.
3) How to screen for miRNAs that regulate a target gene using ready-made cDNA panels and quantitative PCR. Several examples are provided of identifying miRNAs that regulate important cancer genes.
This document discusses RNA interference (RNAi) techniques such as silencing RNA and CRISPR/Cas9. It explains that double-stranded RNA is cut by the enzyme Dicer into short interfering RNAs (siRNAs) that can degrade mRNA strands in a highly specific process. RNAi is involved in regulating 30% of the human genome and acts as a defense mechanism against viruses and transposons. The document also discusses selecting effective siRNAs, considerations for species variation and secondary RNA structures, and strategies for gene knockdown screening using shRNA and CRISPR/Cas9.
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.
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.
microRNA discovery and biomarker development in clinical samplesexiqon
The webinar discussed microRNA discovery and biomarker development in clinical samples using LNATM technology. It covered how LNATM probes can overcome challenges in analyzing microRNAs due to their short length and sequence variations. The webinar also presented a case study using LNATM PCR to detect microRNAs in blood plasma as potential biomarkers for early detection of colorectal cancer. Finally, it discussed challenges in normalizing microRNA qPCR data from serum/plasma samples.
Genomics and Proteomics provides an overview of genomics and proteomics methods and their applications in medicine. It discusses how the fields of genomics and molecular biology emerged through key advances in DNA structure, recombinant DNA technology, PCR, and automated DNA sequencing. The document also reviews epigenetics, genomic and proteomic techniques including blotting, PCR, microarrays, and mass spectrometry. Applications of these methods in clinical settings are described such as genomic tests for disease diagnosis, prognosis, and personalized medicine. Proteomics uses mass spectrometry to discover biomarkers for diseases like cancer.
This document summarizes a scientific article about overcoming limitations in sample material for miRNA biomarker discovery. It discusses how miRNAs have potential as diagnostic, prognostic, and theranostic biomarkers but valuable sample types like laser-captured samples or circulating tumor cells often have insufficient amounts of material for full miRNA profiling. The article presents an integrated PCR-based system that reduces the sample amount needed for full miRNA profiling by several orders of magnitude, enabling detailed analysis of even the smallest samples. This advance opens up new possibilities for biomarker development using hard-to-obtain sample types.
The document describes RT2 miRNA PCR Arrays from SABiosciences for analyzing microRNA (miRNA) expression. Key points:
Relative Detection
(% perfect match)
1) The system allows simultaneous detection of over 700 miRNAs representing most functional human miRNAs.
hsa-miR-10a
hsa-miR-10b
100
0.01
2) It uses a universal polyadenylation and reverse transcription approach followed by SYBR Green-based real-time PCR, offering improved sensitivity, specificity, and reproducibility over other methods.
hsa-miR-10a
hsa-miR-10b
0.01
Transcriptomics is the study of RNA in cells and tissues. The transcriptome refers to the complete set of transcripts in a cell under a specific condition. Understanding the transcriptome reveals the functional elements of the genome and molecular constituents of cells. Techniques for studying the transcriptome include microarray analysis and RNA sequencing. Microarrays measure gene expression levels using fluorescently-labeled cDNA hybridized to probes on an array. RNA sequencing determines expression levels by sequencing individual cDNAs produced from target RNA. Transcriptomics provides insights into development, disease, and varying gene expression under different environmental conditions.
This document describes miScript miRNA PCR Arrays, which allow for the simultaneous detection of genome-wide or pathway-focused microRNA (miRNA) expression. It provides an overview of miRNA biology and research, details the miScript miRNA PCR Array system workflow from isolation to data analysis, and discusses applications in cancer research, development, differentiation, and genome-wide discovery. The system offers validated miRNA assays, controls, and optimized reagents to enable reproducible and reliable miRNA expression profiling from RNA samples.
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.
This document provides an overview of RNA-seq analysis using the T-BioInfo platform. It describes analyzing RNA-seq data from breast cancer patient-derived xenograft models to identify differences between cancer subtypes and mouse models. The analysis includes mapping reads, quantifying gene and isoform expression, normalizing data, performing PCA, and identifying biomarker genes for breast cancer subtypes using factor regression analysis. The goal is to gain insights into cancer biology and identify diagnostic or therapeutic targets.
Extending miRQC’s dynamic range: amplifying the view of Limiting RNA samples ...QIAGEN
The original microRNA quality control (miRQC) study provided an in-depth analysis of commercially available microRNA (miRNA) quantification platforms. Specifically, twelve different
microarray, real-time PCR and small RNA sequencing platforms were assessed for reproducibility, sensitivity, accuracy, specificity and concordance of differential expression using a variety of sample types. Overall, each platform exhibited specific strengths and weaknesses, leading to the
final suggestion that a platform should be chosen on the basis of the experimental setting and the specific research questions. With this suggestion in mind, and the fact that liquid miRNA biopsies are an area of intense interest, we sought to expand the original miRQC study. For our “miRQC extension,” we benchmarked the QIAGEN miScript® PCR System with and without preamplification, and included a specific focus on routinely used biofluids. Concurrently, we benchmarked the miScript PCR System against another SYBR® Green miRNA detection platform. Overall, QIAGEN miScript demonstrated strong reproducibility and accuracy as well as superior detection rate and sensitivity in biofluids. Collectively, QIAGEN miScript provides the leading solution for novel miRNA discoveries.
Similar to Exploiting microRNAs for precision oncology (20)
Osteoporosis - Definition , Evaluation and Management .pdfJim Jacob Roy
Osteoporosis is an increasing cause of morbidity among the elderly.
In this document , a brief outline of osteoporosis is given , including the risk factors of osteoporosis fractures , the indications for testing bone mineral density and the management of osteoporosis
Histololgy of Female Reproductive System.pptxAyeshaZaid1
Dive into an in-depth exploration of the histological structure of female reproductive system with this comprehensive lecture. Presented by Dr. Ayesha Irfan, Assistant Professor of Anatomy, this presentation covers the Gross anatomy and functional histology of the female reproductive organs. Ideal for students, educators, and anyone interested in medical science, this lecture provides clear explanations, detailed diagrams, and valuable insights into female reproductive system. Enhance your knowledge and understanding of this essential aspect of human biology.
One health condition that is becoming more common day by day is diabetes.
According to research conducted by the National Family Health Survey of India, diabetic cases show a projection which might increase to 10.4% by 2030.
These lecture slides, by Dr Sidra Arshad, offer a simplified look into the mechanisms involved in the regulation of respiration:
Learning objectives:
1. Describe the organisation of respiratory center
2. Describe the nervous control of inspiration and respiratory rhythm
3. Describe the functions of the dorsal and respiratory groups of neurons
4. Describe the influences of the Pneumotaxic and Apneustic centers
5. Explain the role of Hering-Breur inflation reflex in regulation of inspiration
6. Explain the role of central chemoreceptors in regulation of respiration
7. Explain the role of peripheral chemoreceptors in regulation of respiration
8. Explain the regulation of respiration during exercise
9. Integrate the respiratory regulatory mechanisms
10. Describe the Cheyne-Stokes breathing
Study Resources:
1. Chapter 42, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 36, Ganong’s Review of Medical Physiology, 26th edition
3. Chapter 13, Human Physiology by Lauralee Sherwood, 9th edition
Our backs are like superheroes, holding us up and helping us move around. But sometimes, even superheroes can get hurt. That’s where slip discs come in.
These lecture slides, by Dr Sidra Arshad, offer a quick overview of the physiological basis of a normal electrocardiogram.
Learning objectives:
1. Define an electrocardiogram (ECG) and electrocardiography
2. Describe how dipoles generated by the heart produce the waveforms of the ECG
3. Describe the components of a normal electrocardiogram of a typical bipolar lead (limb II)
4. Differentiate between intervals and segments
5. Enlist some common indications for obtaining an ECG
6. Describe the flow of current around the heart during the cardiac cycle
7. Discuss the placement and polarity of the leads of electrocardiograph
8. Describe the normal electrocardiograms recorded from the limb leads and explain the physiological basis of the different records that are obtained
9. Define mean electrical vector (axis) of the heart and give the normal range
10. Define the mean QRS vector
11. Describe the axes of leads (hexagonal reference system)
12. Comprehend the vectorial analysis of the normal ECG
13. Determine the mean electrical axis of the ventricular QRS and appreciate the mean axis deviation
14. Explain the concepts of current of injury, J point, and their significance
Study Resources:
1. Chapter 11, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 9, Human Physiology - From Cells to Systems, Lauralee Sherwood, 9th edition
3. Chapter 29, Ganong’s Review of Medical Physiology, 26th edition
4. Electrocardiogram, StatPearls - https://www.ncbi.nlm.nih.gov/books/NBK549803/
5. ECG in Medical Practice by ABM Abdullah, 4th edition
6. Chapter 3, Cardiology Explained, https://www.ncbi.nlm.nih.gov/books/NBK2214/
7. ECG Basics, http://www.nataliescasebook.com/tag/e-c-g-basics
Adhd Medication Shortage Uk - trinexpharmacy.comreignlana06
The UK is currently facing a Adhd Medication Shortage Uk, which has left many patients and their families grappling with uncertainty and frustration. ADHD, or Attention Deficit Hyperactivity Disorder, is a chronic condition that requires consistent medication to manage effectively. This shortage has highlighted the critical role these medications play in the daily lives of those affected by ADHD. Contact : +1 (747) 209 – 3649 E-mail : sales@trinexpharmacy.com
1. Exploiting microRNAs for precision
oncology
March 6, 2017
Jo Vandesompele, Cancer Research Institute Ghent
2. PDF version of presentation and most
references are available on
https://goo.gl/70kyab
3. • more effective and less toxic treatments for durable
responses
– combination therapies
– companion diagnostic tests > the right drug for the right
patient
• better laboratory tests
– early diagnosis
– monitoring of treatment effectivity
– early detection of relapse or recurrence
Unmet needs in oncology
4. • easy to obtain
• low risk for the patient
• serial profiling > longitudinal studies
• reflects entire tumor load
• full of biomarker potential
– cell-free nucleic acids
– circulating tumor cells
– extracellular vesicles
– tumor educated platelets
Liquid biopsies are the holy grail of
precision oncology
6. Active secretion and passive release
of RNA into circulation
Wan et al., Nature Reviews Cancer, 2017
7. • dynamic nature (time, location and condition specific)
• diverse
– different types: messenger, micro, long non-coding,
transfer, ribosomal, piwi, sn(o)RNA, etc.
– varying abundance levels: 1 copy/cell > 100,000
copies/cell
– structural differences: splicing, isoforms, fusion,
mutations
• measurement technologies are state-of-the-art
– RNA sequencing (discovery)
– quantitative and digital PCR (verification, validation,
clinical-grade test)
– sensitive, high-throughput, large dynamic range
RNA has great biomarker potential
8. The majority of human genes do not
code for proteins
protein coding mRNA
non-coding miRNA
long non-coding RNA
21000
63000
2500
• ncRNA have exquisite condition specific expression patterns
• attractive intellectual property landscape
10. MicroRNAs play a role in all the
hallmarks of cancer
Bertoli et al., Theranostics, 2015
11. • miRs undergo (epi)genetic alterations
– deletion (e.g. miR-15/16 in CLL)
– amplification (e.g. miR-17-92)
– mutation, methylation, etc.
– sponge titration (lncRNAs)
• miRNA biogenesis pathway alterations
– mutations in Drosha, Dicer, …
• mRNA target genes
– create new miR target recognition sites
– disrupt miR binding sites
– alternative splicing / differential UTR usage
MicroRNAs are genetically altered in
cancer
12. • high degree of homology between family members
• small differences in expression level among conditions
• low abundance (e.g. in body fluids)
• isomiR sequence variants
MicroRNA quantification challenges
13. Keeping track of microRNA
annotation changes
• www.mirbasetracker.org (Van Peer et al., Database, 2014)
• e.g. hsa-miR-422b
14. • comparison of 11 commercial microRNA gene expression
technologies (qPCR, microarrays, sequencing)
• novel objective and robust performance metrics
• framework for platform comparison, incl. set of
standardized samples
• Mestdagh et al., Nature Methods, 2014
miRNA quality control study
15.
16. • each platform has its own strengths and weaknesses
• selection of an optimal platform in part depends on the
application and goals of the study
– low input amount studies (e.g. serum/plasma profiling)
– discovery vs. validation
– isomiRs
• recommendation to combine 2 different technologies
for discovery and validation
• other things to consider: cost, throughput, sample input
amount, content size, ease of use, …
• TruSeq small RNA sequencing + miScript qPCR
miRQC conclusions
17. Q F
AAAAAAAAA
TTTTTTTTTT
TTTTTTTTTT
stem-loop RT universal RT
mature miRNA mature miRNA
reverse transcription
quantitative PCR
F primer
R primerprobe
reverse transcription
quantitative PCR
F primer
R primer
A BTruSeq small RNA seq miScript qPCR
• 10 cycle multiplex preamp
• lower adaptor concentration
• more PCR cycles
• Pippin lib size selection
• qPCR lib quant
18. • RNA input, library prep kit, library purification, read
depth, data processing, donor status (healthy vs.
diseased), body fluid type (platelet level in plasma)
• 500 – 800 miRNAs per 200 µl serum sample (<100
miRQC) with high reproducibility
miRNA seq on human serum
5 10 15
5
10
15
sample 9
normalized read count replicate 1
normalizedreadcountreplicate2
R = 0.963
5 10 15
5
10
15
sample 15
normalized read count replicate 1
normalizedreadcountreplicate2
R = 0.968
A
numberofdetectedmiRNAs
acrossallsamples
0
200
400
600
800
1000
1200
1400
2014−006−001
2014−006−002
2014−006−004
2014−006−006
2014−006−012
2014−006−019
B
numberofdetectedmiRNAspersample
0
200
400
600
800
2014−006−008
2014−006−009
2014−006−013
2014−006−015
2014−006−017
C
numberofdetectedmiRNAspersample
0
200
400
600
800
15M 25M
Sample1
Sample2
Sample3
Sample4
Sample5
Sample6
Sample1
Sample2
Sample3
Sample4
Sample5
data courtesy of Biogazelle
19. • optimization of the library prep workflow results in more
efficient detection of miRNAs
miRNA seq on human serum
serum 1 serum 2
miRNAreadsrelativetoSTDprotocol
0
20
40
60
80
100
120
140
serum 1 serum 2
miRNAsdetectedrelativetoSTDprotocol
0
20
40
60
80
100
120
standard protocol
optimized protocol
30% more miRNA reads 15% more miRNAs detected
data courtesy of Biogazelle
21. miSTAR has better overall performance
and equal/better precisionAreaUnderCurve
miSTAR
22. Case 1: prognostic serum microRNA
profiling in neuroblastoma
Cian Will Joep Max
low risk low risk high risk high risk
• most frequent extracranial solid tumor in children
• aim: identify ultra-high-risk patients to make them eligible
for new experimental drugs
23. • full miRNome miScript qPCR profiling (n=2405) of 5
pooled serum samples from 3 different risk groups
– low risk survivors
– high risk survivors
– high risk deceased
Experiment design
24. • full miRNome miScript qPCR profiling (n=2405) of 5
pooled serum samples from 3 different risk groups
– low risk survivors
– high risk survivors
– high risk deceased
• selection of 781 miRs expressed in the pools
• individual qPCR profiling of 781 miRs on 200 µl serum
– SIOPEN cohort of +120 high/low risk patients
• modified global mean normalization (D’haene et al.,
Methods Mol Biol, 2012)
Experiment design
26. • idasanutlin is a selective MDM2 inhibitor,
releasing TP53 from negative control
• before going to clinical phases in human during
drug development, preclinical work in animal
models is needed (safety, efficacy, biomarkers)
• goals
– identify liquid biopsy tumor markers for disease
monitoring
– identify on target drug efficacy markers
Case 2: serum miR analysis in a
preclinical model of NB
Table of Contents (TOC)
N
H
Cl
Cl
NH
O
F
CN
F
OHO
O
RG7388
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
Isadanutlin*
(RG7388)*
27. • jugular vein puncture with a lancet (100 µl blood)
Verification of miRNA seq on ½ RNA
from 50 µl of murine serum
28. • optimized TruSeq small RNA sequencing results in
massive amount of 5’ tRNA halves
Verification of miRNA seq on ½ RNA
from 50 µl of murine serum
RNA fragment size
22 nt
30 nt
readcount
29. • regulated process under stress and in cancer
tRNAs as source of small non-coding
RNAs with various functions
Anderson and Ivanov, 2014
30. Probe based removal of unwanted
small RNA fragments miRNA
5’ tRNA halves
5’ bioƟnylated
DNA probe
magneƟc
streptavidin beads
+ magneƟc field
purified RNA
miR
Beads RNa
purified RNA
u
control
beads
RNase H
0
20
40
60
80
100
120
tRNA-gly tRNA-his tRNA-val tRNA-glu
relaƟveabundance(%)
control
beads
RNase H
B
31. Probe based removal of unwanted
small RNA fragments
probes: 0 16
avg miRNAs: 169 570
tRNA %: 53.44% 3.88%
miRNA %: 1.12% 28.33%
• 25x enrichment of miRNA, 14x depletion of 5’ tRFs
• Van Goethem et al., Scientific Reports, 2016
32. Experiment design
day 7
engraftment
day 21
start treatment
day 35
end treatment
2 w
106 SH-SY5Y
cells
day 1 day 18 day 35day 22
idasanutlin
temsirolimus
2 w
33. 56 miR indicators of tumor load0
2
0 2 4 6log2(cou
0
2
4
6
0 2 4 6
hsa miR 105 5p
hsa miR 1180 3p
hsa miR 125b 2 3p
hsa miR 1269a
hsa miR 1269b
hsa miR 1271 5p
hsa miR 1301 3p
hsa miR 1307 3p
hsa miR 1307 5p
hsa miR 1468 5p
hsa miR 151a 3p
hsa miR 16 2 3p
hsa miR 182 5p
hsa miR 191 3p
hsa miR 197 3p
hsa miR 199b 5p
hsa miR 28 3p
hsa miR 301b 3p
hsa miR 330 3p
hsa miR 339 3p
hsa miR 345 5p
hsa miR 3605 3p
hsa miR 361 3p
hsa miR 3615
hsa miR 3909
hsa miR 424 3p
hsa miR 432 5p
hsa miR 4326
hsa miR 450b 5p
hsa miR 454 5p
hsa miR 483 3p
hsa miR 483 5p
hsa miR 500a 3p
hsa miR 501 3p
hsa miR 505 3p
hsa miR 561 5p
hsa miR 576 5p
hsa miR 589 3p
hsa miR 589 5p
hsa miR 598 3p
hsa miR 6511b 3p
hsa miR 654 3p/mmu miR 654 3p
-6
10
15
25
days
0.0 2.5 5.0
log2 fold change
signifcantly differentialy expressed
no
yes
24
56
4
B
log2 (count before engraftment
)
log2(countafterengraftment
)
log2 (countnot-engrafted
)
DESeq2
• 53 are human specific, 3 are conserved between
human and mouse
• 5p and 3p arms of the same pre-miR are present
• gradual increase of these 56 miRs in tumor-bearing vs.
non-engrafted over 4 time points
0
2
4
6
0 2 4 6
log2(countafterengraftment
)
0
2
4
6
0 2 4 6
hsa miR 105 5p
hsa miR 1180 3p
hsa miR 125b 2 3p
hsa miR 1269a
hsa miR 1269b
hsa miR 1271 5p
hsa miR 1301 3p
hsa miR 1307 3p
hsa miR 1307 5p
hsa miR 1468 5p
hsa miR 151a 3p
hsa miR 16 2 3p
hsa miR 182 5p
hsa miR 191 3p
hsa miR 197 3p
hsa miR 199b 5p
hsa miR 28 3p
hsa miR 301b 3p
hsa miR 330 3p
hsa miR 339 3p
hsa miR 345 5p
hsa miR 3605 3p
hsa miR 361 3p
hsa miR 3615
hsa miR 3909
hsa miR 424 3p
hsa miR 432 5p
hsa miR 4326
hsa miR 450b 5p
hsa miR 454 5p
hsa miR 483 3p
hsa miR 483 5p
hsa miR 500a 3p
hsa miR 501 3p
hsa miR 505 3p
hsa miR 561 5p
hsa miR 576 5p
hsa miR 589 3p
hsa miR 589 5p
hsa miR 598 3p
hsa miR 6511b 3p
hsa miR 654 3p/mmu miR 654 3p
hsa miR 660 5p
hsa miR 675 3p
hsa miR 675 5p
hsa miR 767 5p/mmu miR 767
hsa miR 767 5p
hsa miR 769 5p
hsa miR 7706
hsa miR 873 3p
hsa miR 887 3p
hsa miR 92b 3p/mmu miR 92b 3p
hsa miR 941
-6
10
15
25
days
24
56
4
A
C
B
D
log2 (count before engraftment
)
log2(countafterengraftment
)
log2 (countnot-engrafted
)
34. 56 serum miRs are proportional to
tumor volume
tumorweight(g)
log2meanexpressoin
log2 mean
expression
tumor weight (g)cumulative proportion
of serum miRs
in vivo luciferase imaging endpoints
35. 56 serum miRs are proportional to
tumor volume
tumorweight(g)
logluciferasesignal
log2 mean expression log2 mean expression
36. Serum tumor load miRs are high
abundant in tumor
0.0
2.5
5.0
7.5
10.0
hsa−miR−92b−3p
hsa−miR−151a−3p
hsa−miR−28−3p
hsa−miR−500a−3p
hsa−miR−769−5phsa−miR−941hsa−miR−887−3p
hsa−miR−345−5phsa−miR−301b−3phsa−miR−125b−2−3phsa−miR−1307−5p
hsa−miR−767−5phsa−miR−589−5p
hsa−miR−1307−3p
hsa−miR−197−3phsa−miR−7706hsa−miR−21−3phsa−miR−660−5p
hsa−miR−873−3p
hsa−miR−589−3p
hsa−miR−598−3phsa−miR−483−5p
hsa−miR−135a−5phsa−miR−450b−5p
hsa−miR−339−3phsa−miR−873−5p
hsa−miR−3615
hsa−miR−483−3p
hsa−miR−105−5phsa−miR−191−3p
hsa−miR−330−3p
hsa−miR−1468−5p
hsa−miR−4326
hsa−miR−3648
hsa−miR−129−2−3p
hsa−miR−675−3p
hsa−miR−499a−5phsa−miR−455−5p
log(Count)
Differentially expressed in serum NO YES
miRNA Expression in cell_line
logcounts
tumor miRs ordered according to abundance
serum tumor load miR
37. 20 out of 56 miRs are higher
expressed in human HR NB
hsa−miR−1269a hsa−miR−1307−3p hsa−miR−16−2−3p hsa−miR−191−3p
hsa−miR−199b−5p hsa−miR−330−3p hsa−miR−339−3p hsa−miR−345−5p
hsa−miR−3605−3p hsa−miR−424−3p hsa−miR−432−5p hsa−miR−454−5p
hsa−miR−4741 hsa−miR−483−3p hsa−miR−483−5p hsa−miR−500a−3p
hsa−miR−501−3p hsa−miR−675−5p hsa−miR−769−5p hsa−miR−92b−3p
0
2
4
0
2
4
6
0
2
4
6
8
0
2
4
0
2
4
0
1
2
3
4
5
0
2
4
6
0.0
2.5
5.0
7.5
0
2
4
6
0
1
2
3
4
5
0
2
4
0
1
2
3
0
1
2
3
4
5
0.0
2.5
5.0
7.5
10.0
0.0
2.5
5.0
7.5
10.0
0
1
2
3
4
0
2
4
6
0
2
4
6
0
1
2
3
4
0
1
2
3
4
NBHR
NBHR
H
S
N
R
NBHR
NBHR
H
S
N
R
NBHR
NBHR
H
S
N
R
NBHR
NBHR
H
S
N
R
log2(relativeexpression)
HR neuroblastoma
n=5
healthy children
n=5
HR neuroblastoma
n=5
rabdomyosarcoma
n=5
nephroblastoma
n=5
sarcoma
n=5
38. 24 idasanutlin induced human miRs
hsa miR 802/mmu miR 802 5p vehicle
idasanutlin
vehicle
idasanutlin
vehicle
idasanutlin
vehicle
idasanutlin2 0 2
rescaled log2 (count)
1 2 3
hsa miR 134 5p/mmu miR 134 5p
4
1 2 3 4
hsa miR 34a 5p/mmu miR 34a 5p
1 2 3 4
1dayaŌertreatment
10daysaŌertreatment
A B
hsa miR 485 3p/mmu miR 485 3p
hsa miR 143 5p/mmu miR 143 5p
hsa miR 4492
hsa miR 216a 5p/mmu miR 216a 5p
hsa miR 636/mmu miR 5126
hsa miR 146b 5p/mmu miR 146b 5p
hsa miR 378a 3p/mmu miR 378b
hsa miR 365b 5p/mmu miR 365 2 5p
hsa miR 6087
hsa miR 490 5p/mmu miR 490 5p
hsa miR 10a 5p/mmu miR 10a 5p
hsa miR 668 3p/mmu miR 668 3p
hsa miR 212 3p/mmu miR 212 3p
hsa miR 29c 3p/mmu miR 29c 3p
hsa miR 188 5p/mmu miR 188 5p
hsa miR 136 3p/mmu miR 136 3p
hsa miR 143 3p/mmu miR 143 3p
hsa miR 145 3p/mmu miR 145a 3p
hsa miR 145 5p/mmu miR 145a 5p
hsa miR 490 3p/mmu miR 490 3p
-6 10
20 1 3
1 day 11 days
0 0 0
1 day 11 days
idasanutlin
temsirolimus
15 25
miR-143/145 cluster
miR-34a
1dayaftertreatment
10days1dayaftertreatment
treatment vs.
control
+
before and
after
engraftment
39. miR-34a-5p & 212-3p are circulating
biomarkers for TP53 activation
6
7
−6 11 15 25
day
log2(count)
no yescontrol
hsa−miR−34a−5p/mmu−miR−34a−5p
tumortreatment idasanutlin
A
B
6
7
−6 11 15 25
day
log2(count)
hsa−miR−212−3p/mmu−miR−212−3p
7.5
8.0
8.5
9.0
control idasanutlin
log2(count)
hsa−miR−34a−5p/mmu−miR−34a−5p
5
6
7
control idasanutlin
log2(count)
treatment
control
idasanutlin
hsa−miR−212−3p/mmu−miR−212−3p
C
D
6
7
−6 11 15 25
day
log2(count)
no yescontrol
hsa−miR−34a−5p/mmu−miR−34a−5p
tumortreatment idasanutlin
A
B
6
7
−6 11 15 25
day
log2(count)
hsa−miR−212−3p/mmu−miR−212−3p
7.5
8.0
8.5
9.0
control idasanutlin
log2(count)
hsa−miR−34a−5p/mmu−miR−34a−5p
5
6
7
control idasanutlin
log2(count)
treatment
control
idasanutlin
hsa−miR−212−3p/mmu−miR−212−3p
C
D
tumorendpointserum
40. • tools available to study miRNAs
– miRBase Tracker, miSTAR
– miRQC, global mean normalization, tRNA depletion
• circulating miRNAs are promising biomarkers in
neuroblastoma
– outcome prediction in high-risk group
– tumor load assessment > patient monitoring / diagnosis
– target engagement in the tumor
Conclusions