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
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
AGRF in conjunction with EMBL Australia recently organised a workshop at Monash University Clayton. This workshop was targeted at beginners and biologists who are new to analysing Next-Gen Sequencing data. The workshop also aimed to provide users with a snapshot of bioinformatics and data analysis tips on how to begin to analyse project data. An introduction to RNA-seq data analysis was presented by AGRF Senior Bioinformatician Dr. Sonika Tyagi.
Presented: 1st August 2012
Deciphering DNA sequences is essential for virtually all branches of biological research. With the
advent of capillary electrophoresis (CE)-based Sanger sequencing, scientists gained the ability to
elucidate genetic information from any given biological system. This technology has become widely
adopted in laboratories around the world, yet has always been hampered by inherent limitations in
throughput, scalability, speed, and resolution that often preclude scientists from obtaining the essential
information they need for their course of study. To overcome these barriers, an entirely new technology
was required—Next-Generation Sequencing (NGS), a fundamentally different approach to sequencing
that triggered numerous ground-breaking discoveries and ignited a revolution in genomic science.
NGS Targeted Enrichment Technology in Cancer Research: NGS Tech Overview Webi...QIAGEN
This slidedeck discusses the most biologically efficient, cost-effective method for successful NGS. The GeneRead DNA QuantiMIZE Kits enable determination of the optimum conditions for targeted enrichment of DNA isolated from biological samples, while the GeneRead DNAseq Panels V2 allow you to quickly and reliably deep sequence your genes of interest. Applications in translational and clinical research are highlighted.
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
AGRF in conjunction with EMBL Australia recently organised a workshop at Monash University Clayton. This workshop was targeted at beginners and biologists who are new to analysing Next-Gen Sequencing data. The workshop also aimed to provide users with a snapshot of bioinformatics and data analysis tips on how to begin to analyse project data. An introduction to RNA-seq data analysis was presented by AGRF Senior Bioinformatician Dr. Sonika Tyagi.
Presented: 1st August 2012
Deciphering DNA sequences is essential for virtually all branches of biological research. With the
advent of capillary electrophoresis (CE)-based Sanger sequencing, scientists gained the ability to
elucidate genetic information from any given biological system. This technology has become widely
adopted in laboratories around the world, yet has always been hampered by inherent limitations in
throughput, scalability, speed, and resolution that often preclude scientists from obtaining the essential
information they need for their course of study. To overcome these barriers, an entirely new technology
was required—Next-Generation Sequencing (NGS), a fundamentally different approach to sequencing
that triggered numerous ground-breaking discoveries and ignited a revolution in genomic science.
NGS Targeted Enrichment Technology in Cancer Research: NGS Tech Overview Webi...QIAGEN
This slidedeck discusses the most biologically efficient, cost-effective method for successful NGS. The GeneRead DNA QuantiMIZE Kits enable determination of the optimum conditions for targeted enrichment of DNA isolated from biological samples, while the GeneRead DNAseq Panels V2 allow you to quickly and reliably deep sequence your genes of interest. Applications in translational and clinical research are highlighted.
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.
CRISPR (clustered regularly interspaced short palindromic repeats) is a family of DNA sequences found within the genomes of prokaryotic organisms such as bacteria and archaea. These sequences are derived from DNA fragments of bacteriophages that have previously infected the prokaryote and are used to detect and destroy DNA from similar phages during subsequent infections. Hence these sequences play a key role in the antiviral defense system of prokaryotes.
Cas9 (CRISPR-associated protein 9) is an enzyme that uses CRISPR sequences as a guide to recognize and cleave specific strands of DNA that are complementary to the CRISPR sequence. Cas9 enzymes together with CRISPR sequences form the basis of a technology known as CRISPR-Cas9 that can be used to edit genes within organisms.This editing process has a wide variety of applications including basic biological research, development of biotechnology products, and treatment of diseases.
The CRISPR-Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as those present within plasmids and phages that provides a form of acquired immunity. RNA harboring the spacer sequence helps Cas (CRISPR-associated) proteins recognize and cut foreign pathogenic DNA. Other RNA-guided Cas proteins cut foreign RNA. CRISPR are found in approximately 50% of sequenced bacterial genomes and nearly 90% of sequenced archaea.
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
Introduction to real-Time Quantitative PCR (qPCR) - Download the slidesQIAGEN
This slidedeck introduces the concepts of real-time PCR and how to conduct a real-time PCR assay. The topics that are covered include an overview of real-time PCR chemistries, protocols, quantification methods, real-time PCR applications and factors for success.
Have you considered that protein over-expression or inefficient mRNA knockdown may be masking physiological effects in your assays? Increasingly scientists are moving to endogenous gene-editing to characterise the function of their genes of interest.
Dr Chris Thorne from Cambridge Biotech Horizon Discovery discusses the ground breaking gene-editing technology CRISPR. The simplicity of experimental design has led to rapid adoption of the technology across the scientific community. However, challenges remain.
This Slidedeck focuses specifically on implementing CRISPR experiments, and explore a number of key considerations crucial to maximising chances of targeting success, whether your goal is to generate a knock-out or a knock-in. Chris also takes a look at some of the alternative uses of CRISPR, including sgRNA genome wide synthetic lethality screens.
The slides aim to support those researchers either planning to or already using CRISPR gene-editing in their lab. Horizon Discovery have also recently launched a program aimed specifically at academic cell biologists to promote the adoption of CRISPR by offering FREE CRISPR Reagents for knock-out cell line generation - more information available here. http://www.horizondiscovery.com/what-we-do/discovery-toolbox/genassist-crispr--raav-genome-editing-tools
A workshop is intended for those who are interested in and are in the planning stages of conducting an RNA-Seq experiment. Topics to be discussed will include:
* Experimental Design of RNA-Seq experiment
* Sample preparation, best practices
* High throughput sequencing basics and choices
* Cost estimation
* Differential Gene Expression Analysis
* Data cleanup and quality assurance
* Mapping your data
* Assigning reads to genes and counting
* Analysis of differentially expressed genes
* Downstream analysis/visualizations and tables
The next generation of crispr–cas technologies and Applicationsiqraakbar8
The prokaryote-derived CRISPR–Cas genome editing systems have transformed our ability to manipulate, detect, image and annotate specific DNA and RNA sequences in living cells of diverse species. The ease of use and robustness of this technology have revolutionized genome editing for research ranging from fundamental science to translational medicine. Initial successes have inspired efforts to discover new systems for targeting and manipulating nucleic acids, including those from Cas9, Cas12, Cascade and Cas13 orthologues.
This slidedeck details two comprehensive informatics solutions — the Biomedical Genomics Workbench and Ingenuity Knowledge Base Variant Analysis platforms. We show the intuitive user interface of CLC Cancer Research Workbench and demonstrate how the rich biological content from Ingenuity Knowledge Base helps you rapidly identify critical variants in your samples.
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.
CRISPR (clustered regularly interspaced short palindromic repeats) is a family of DNA sequences found within the genomes of prokaryotic organisms such as bacteria and archaea. These sequences are derived from DNA fragments of bacteriophages that have previously infected the prokaryote and are used to detect and destroy DNA from similar phages during subsequent infections. Hence these sequences play a key role in the antiviral defense system of prokaryotes.
Cas9 (CRISPR-associated protein 9) is an enzyme that uses CRISPR sequences as a guide to recognize and cleave specific strands of DNA that are complementary to the CRISPR sequence. Cas9 enzymes together with CRISPR sequences form the basis of a technology known as CRISPR-Cas9 that can be used to edit genes within organisms.This editing process has a wide variety of applications including basic biological research, development of biotechnology products, and treatment of diseases.
The CRISPR-Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as those present within plasmids and phages that provides a form of acquired immunity. RNA harboring the spacer sequence helps Cas (CRISPR-associated) proteins recognize and cut foreign pathogenic DNA. Other RNA-guided Cas proteins cut foreign RNA. CRISPR are found in approximately 50% of sequenced bacterial genomes and nearly 90% of sequenced archaea.
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
Introduction to real-Time Quantitative PCR (qPCR) - Download the slidesQIAGEN
This slidedeck introduces the concepts of real-time PCR and how to conduct a real-time PCR assay. The topics that are covered include an overview of real-time PCR chemistries, protocols, quantification methods, real-time PCR applications and factors for success.
Have you considered that protein over-expression or inefficient mRNA knockdown may be masking physiological effects in your assays? Increasingly scientists are moving to endogenous gene-editing to characterise the function of their genes of interest.
Dr Chris Thorne from Cambridge Biotech Horizon Discovery discusses the ground breaking gene-editing technology CRISPR. The simplicity of experimental design has led to rapid adoption of the technology across the scientific community. However, challenges remain.
This Slidedeck focuses specifically on implementing CRISPR experiments, and explore a number of key considerations crucial to maximising chances of targeting success, whether your goal is to generate a knock-out or a knock-in. Chris also takes a look at some of the alternative uses of CRISPR, including sgRNA genome wide synthetic lethality screens.
The slides aim to support those researchers either planning to or already using CRISPR gene-editing in their lab. Horizon Discovery have also recently launched a program aimed specifically at academic cell biologists to promote the adoption of CRISPR by offering FREE CRISPR Reagents for knock-out cell line generation - more information available here. http://www.horizondiscovery.com/what-we-do/discovery-toolbox/genassist-crispr--raav-genome-editing-tools
A workshop is intended for those who are interested in and are in the planning stages of conducting an RNA-Seq experiment. Topics to be discussed will include:
* Experimental Design of RNA-Seq experiment
* Sample preparation, best practices
* High throughput sequencing basics and choices
* Cost estimation
* Differential Gene Expression Analysis
* Data cleanup and quality assurance
* Mapping your data
* Assigning reads to genes and counting
* Analysis of differentially expressed genes
* Downstream analysis/visualizations and tables
The next generation of crispr–cas technologies and Applicationsiqraakbar8
The prokaryote-derived CRISPR–Cas genome editing systems have transformed our ability to manipulate, detect, image and annotate specific DNA and RNA sequences in living cells of diverse species. The ease of use and robustness of this technology have revolutionized genome editing for research ranging from fundamental science to translational medicine. Initial successes have inspired efforts to discover new systems for targeting and manipulating nucleic acids, including those from Cas9, Cas12, Cascade and Cas13 orthologues.
This slidedeck details two comprehensive informatics solutions — the Biomedical Genomics Workbench and Ingenuity Knowledge Base Variant Analysis platforms. We show the intuitive user interface of CLC Cancer Research Workbench and demonstrate how the rich biological content from Ingenuity Knowledge Base helps you rapidly identify critical variants in your samples.
Step by Step, from Liquid Biopsy to a Genomic Biomarker: Liquid Biopsy Series...QIAGEN
Liquid biopsies enable us to monitor the evolution of genetic aberrations in primary tumors as they shed the tumor cells into the circulation. The limitation is the ability to detect these low frequency genetic aberrations in a consistent manner to understand short- and long-term implications and how this information will be used in the clinic. This slidedeck will cover the challenges and solutions associated with multiple steps as one starts with liquid biopsy and move towards finding a new biomarker.
Targeted RNAseq for Gene Expression Using Unique Molecular Indexes (UMIs): In...QIAGEN
Traditional RNA sequencing (RNA-Seq) is a powerful tool for expression profiling, but is hindered by PCR amplification bias and inaccuracy at low expressing genes. QIAseq RNA is a flexible and precise tool developed for mitigating these complications, allowing digital gene expression analysis. This in-depth webinar will cover sample requirements, experimental design, NGS platform-specific challenges and workflow for gene enrichment, library prep and sequencing. The applications of QIASeq RNA Panels in cancer research, stem cell differentiation and elucidating the effects small molecules on signaling pathways will be highlighted.
DNA sequence analysis and genotyping of biological samples using innovative instrumentation, such as next-generation sequencing (NGS) platforms, is often limited by the small amount of sample available. The REPLI-g Single Cell Kit is specially designed to uniformly amplify genomic DNA from single cells (1 to <1000 bacterial or tumor cells) or purified genomic DNA with complete genome coverage. Additional protocols are also available for use with fresh or dried blood or fresh or frozen tissue. Dedicated buffers and reagents undergo a unique, controlled decontamination procedure to avoid amplification of contaminating DNA, ensuring highly reliable results every time. Accurate amplification of genomes with negligible sequence bias and no genomic drop-outs is achieved with innovative Multiple Displacement Amplification (MDA) technology. In contrast to PCR-based WGA technologies, high fidelity rates are increased up to 1000-fold, avoiding costly false positive or negative results.
Analyzing Fusion Genes Using Next-Generation SequencingQIAGEN
Fusion genes are hybrid genes formed by the fusion of two separate genes. Translocation, interstitial deletion and chromosomal inversions are some of the genetic events that can lead to the formation of fusion genes. The occurrence of fusion genes and its implications in cancer have already been known, but the emergence of NGS technology – especially RNA sequencing – offers the potential to detect novel gene fusions. You can learn more about fusion genes and applying NGS to detect them at our upcoming webinar, presented by Raed Samara, Ph.D., QIAGEN’s Global Product Manager for NGS technologies.
In this webinar, Dr. Raed Samara will cover:
1. Fusion genes: what they are and a historical perspective
2. Fusion gene detection: the current status
3. RNA sequencing vs. digital RNA sequencing
4. How to detect and accurately quantify novel fusion genes in your sample
Advanced NGS Library Prep for Challenging SamplesQIAGEN
Rapidly developing next-generation sequencing (NGS) technologies provide highly sensitive methods for discovering and characterizing the genetic information of a variety of samples. However, DNA samples are often limited in quantity, as well as compromised in quality. Such samples are not suitable for standard NGS library construction methods, which commonly require hundreds of nanograms of high-quality DNA. Examples of such challenging samples include circulating DNA, laser capture microdissection (LCM) samples, formalin-fixed paraffin-embedded (FFPE) samples, ancient DNA and chromatin immunoprecipitation (ChIP) samples.
This webinar discusses the measures that should be taken into consideration while sequencing such challenging samples. It also presents methods that can be used to optimize library construction to efficiently convert small amounts of DNA samples into high-quality sequencing libraries.
Speaker: Benedict C. S. Cross, PhD, Team leader (Discovery Screening), Horizon Discovery
CRISPR–Cas9 mediated genome editing provides a highly efficient way to probe gene function. Using this technology, thousands of genes can be knocked out and their function assessed in a single experiment. We have conducted over 150 of these complex and powerful screens and will use our experience to guide you through the process of screen design, performance and analysis.
We'll be discussing:
• How to use CRISPR screening for target ID and validation, understanding drug MOA and patient stratification
• The screen design, quality control and how to evaluate success of your screening program
• Horizon’s latest developments to the platform
• Horizon’s novel approaches to target validation screening
The clinical application development and validation of cell free dna assays -...Candy Smellie
What is the impact of assay failure in your laboratory and how do you monitor for it?
In cancer patients, cell-free DNA carries tumour-related genetic alterations that are relevant to cancer development, disease progression and response to therapy.
Cell-free DNA detection allows:
Early detection
Frequent sampling
Monitoring of disease progression
Measure response to therapy
Detection of resistance mutation
Non-invasive diagnostic tool development
Analysis and Interpretation of Cell-free DNAQIAGEN
Identification and monitoring of cancer mutations from cell free DNA-Seq data is a key application in liquid biopsy. In this part of the webinar we will show how mutations can be best identified from this type of data and how they can be interpreted. Furthermore, potential challenges when analyzing this type of data will be discussed together with relevant strategies.
Using methylation patterns to determine origin of biological material and ageQIAGEN
In this QIAGEN sponsored webinar, our guest speakers from the San Francisco Police Department (SFPD) Crime Lab and Florida International University (FIU) discuss their research on the potential of epigenetic methylation as a procedure for body fluid identification and age estimation from DNA left at crime scenes. Several approaches have been studied, including an analysis of methyl array data and an initial validation of procedures such as pyrosequencing and real-time PCR. The presentation focuses on a number of tissue-specific epigenetic markers for body fluid and age determination with a promise of future integration of these markers into the forensic lab due to the simplicity of analysis and the ease of application.
Learn more about the Pyrosequencing technology and our solutions at
https://www.qiagen.com/resources/technologies/pyrosequencing-resource-center/
Take lung cancer research to a new molecular dimensionQIAGEN
Circulating Tumor Cells (CTCs) can provide researchers with important new discoveries on the mechanism of cancer. Find out more about the latest technology that provides researchers the necessary tools to conduct CTC research in lung cancer.
Circulating Tumor Cells (CTCs) can provide researchers with important new discoveries on the mechanism of cancer. Find out more about the latest technology that provides researchers the necessary tools to conduct CTC research in AR-V7 related prostate cancer.
Learn about the power of LNA (Locked Nucleic Acid) technology and QIAGEN's LNA enhanced product portfolio for RNA and DNA research. Download the slide deck!
Take your RNA research to the next level with QIAGEN LNA tools!QIAGEN
Download the flyer!
Experience truly exceptional RNA research with QIAGEN's next-generation, LNA®-enhanced tools. LNA (Locked Nucleic Acid) oligos bind with much higher affinity and specificity to RNA targets than standard DNA and RNA oligos – This enables specific and sensitive detection of small RNAs and discrimination between highly similar
sequences.
An Approach to De-convolution of Mixtures in Touch DNA Samples. Download now!QIAGEN
7th QIAGEN Investigator Forum - Lisbon, March 8, 2018 . An Approach to De-convolution of Mixtures in Touch DNA Samples. Presenter: Lisa Dierig, Institute of Legal Medicine, Ulm
Assessment of Y chromosome degradation level using the Investigator® Quantipl...QIAGEN
Assessment of Y chromosome degradation level using the Investigator® Quantiplex® Pro RGQ Kit, presented by Dr. Tomasz Kupiec, Head of the Forensic Genetics Section, Institute of Forensic Research, Krakow, Poland on June 14, 2018.
ICMP MPS SNP Panel for Missing Persons - Michelle Peck et al.QIAGEN
Optimization and Performance of a Very Large MGS SNP Panel for Missing Persons, by Michelle Peck et al., International Commission on Mission Persons. Presented May 3, 2018, at the QIAGEN Investigator Forum, San Antonio, TX.
Exploring the Temperate Leaf Microbiome: From Natural Forests to Controlled E...QIAGEN
The aerial surfaces of plants, the phyllosphere, harbors a diverse community of microorganisms. The increasing awareness of the potential roles of phyllosphere microbial communities calls for a greater understanding of their structure and dynamics in natural and urban ecosystems. To do so, we characterized the community structure and assembly dynamics of leaf bacterial communities in natural temperate forests of Quebec by comparing the relative influence of host species identity, site, and time on phyllosphere bacterial community structure. Second, we tested the value of characterizing a tree’s complete phyllosphere microbial community through a single sample by measuring the intra-individual, inter-individual and interspecific variation in leaf bacterial communities. Third, we quantified the relationships among phyllosphere bacterial diversity, plant species richness, plant functional diversity and identity, and plant community productivity in a biodiversity-ecosystem function experiment with trees. Finally, we compared tree leaf bacterial communities in natural and urban environments, as well as along a gradient of increasing anthropogenic pressures. The work presented here thus offers an original assessment of the dynamics at play in the tree phyllosphere.
Cancer Research & the Challenges of FFPE Samples – An IntroductionQIAGEN
A cascade of complex genetic and epigenetic changes regulate tumor formation and progression. Gene expression analyses can shed light on these changes at a molecular level and identify the key genes and associated pathways involved in cancer. Often the samples used in cancer research are FFPE samples, which pose a significant challenge in terms of nucleic acid quality. The quality of nucleic acids extracted from FFPE samples depends on a number of factors, including how the samples were handled before, during and after fixation and embedding.
Dr. Vishwadeepak Tripathi describes the variability of sample purification from FFPE samples – in particular, samples to be used in cancer research. What are the challenges and solutions, and what quality control approach can ensure credible results? This webinar will focus on sample purification and the quality control of FFPE samples and compare different automated purification procedures.
The Microbiome of Research Animals : Implications for Reproducibility, Transl...QIAGEN
The human gut microbiota (GM) has emerged as a key factor in susceptibility to, as well as a potential biomarker of, several diseases and conditions. Similarly, researchers now appreciate that the GM of laboratory animals could affect the reproducibility and translatability of many disease models, including a complete loss of phenotype. While associations between characteristics of the GM and differential disease model phenotypes are of concern, they can also be viewed as sources of discovery related to disease pathogenesis. As such, there is considerable interest in factors that inadvertently influence the composition of the GM and methods of manipulating the GM prospectively to investigate such associations and standardize or optimize disease models. The webinar will present data on variables capable of influencing the GM of laboratory rodents citing several examples and animal models, considerations related to manipulation of the GM in mice and rats, and recent data supporting the use of “dirty” mice in biomedical research.
Building a large-scale missing persons ID SNP panel - Download the studyQIAGEN
In this webinar, we will take a look at a large-scale SNP-based forensic identification panel for DNA analysis with massively parallel sequencing (MPS). The panel was specifically designed for the challenges of identifying missing persons; where DNA is frequently highly degraded, and relationship tests may involve reference samples from across several generations and in a deficient pedigree.
Rapid DNA isolation from diverse plant material for use in Next Generation Se...QIAGEN
Isolation of DNA from plant material is often a tedious process which involves significant hands on time and leads to varying results due to the diverse nature of the material. Different parts of the plants as well as the plants themselves differ in both consistency of material and presence of inhibitory substances, making dependable isolation of DNA difficult.
Here, we developed a method for the efficient extraction of DNA from different plant types, including strawberry leaf, pine needle, grape leaf, and cotton and coffee seeds (workflow at right). A novel bead beating method and lysis chemistry led to more efficient sample lysis with minimal hands-on time and significantly increased DNA yield compared to conventional methods. Through the use of multiple technologies to improve removal of secondary metabolites, such as polyphenols, complex polysaccharides, alkaloids and tannins that may inhibit downstream applications, the isolated DNA was of high quality and purity.
The resulting DNA is suitable for immediate use in downstream reactions, including PCR, qPCR and Next Generation Sequencing based applications. Using this method we were further able to design a workflow that included DNA isolation, library preparation and bioinformatics analyses for the efficient detection of plant pathogens isolated from infected samples. With this, our protocol is a substantial improvement within workflows used for plant microbiome and plant pathology studies as well as in plant breeding and engineering.
Rapid extraction of high yield, high quality DNA from tissue samples - Downlo...QIAGEN
Genetic and genomic analysis from tissue samples requires the extraction of high quality DNA. Mechanical disruption methods such as bead milling provide high yield from tissue samples, but cause damage to the nucleic acids. Purely enzymatic methods such as proteinase K digestion can extract nucleic acid without damage, but require long incubation times, often proceeding overnight, and without approaching the yields achieved by mechanical disruption techniques. Thus a method is needed which can provide a rapid extraction of high yield, high quality DNA from tissue samples. See the new method.
Critical Factors for Successful Real-Time PCR: Multiplex PCRQIAGEN
Multiplex end-point PCR is a powerful tool for genotyping and many other applications. QIAGEN’s multiplex PCR chemistry is optimized for reliable amplification of many different templates with high variability in copy numbers. Thus it enables very quick establishment of a new lab routine and instant success for your multiplex PCR strategy.
There is a set of critical factors which we recommend to be regarded for planning and performing this kind of PCR. These will be discussed in detail in the webinar. Additionally, our multiplex PCR chemistry has recently been gaining increasing popularity among scientists who are utilizing it for their next-generation sequencing workflows.
Practical hints and new solutions for successful real-time PCR studies QIAGEN
Part 1: Practical hints and new solutions for successful real-time PCR studies
In this webinar we will cover the following topics which are critical steps for efficient and precise gene expression studies using real-time PCR technology:
- Effect of RNA integrity on real-time PCR results – tips to achieve a true RNA profiling suitable for real-time PCR studies
- Improved methods for cDNA synthesis, optimized for real-time PCR
- Real-time PCR analysis
o Real-time PCR essentials and background information on different quantification strategies
o SYBR Green real-time PCR – factors influencing specificity
o Introduction to probe technology
o New, fast and efficient real-time PCR solutions
Part 2: Critical Factors for Successful Multiplex Real-Time PCR
Multiplex real-time PCR is a powerful tool for gene expression analysis, viral load monitoring, genotyping, and many other applications. The ability to amplify and detect several genomic DNA, cDNA, or RNA targets in the same reaction offers many benefits:
• Conservation of precious samples – more quantification data per sample
• Increased throughput – more targets analyzed per run on a cycler
• Reliable results – no well-to-well variability due to co-amplification of internal control
• Reduced costs – save time and reagents
The QuantiFast Multiplex PCR and RT-PCR kits are optimized for reliable amplification of many different templates despite a high variability in abundance. Thus they enable successful amplification of multiple targets on the first attempt without optimization.
This webinar explains the principles of the QIAGEN multiplex technologies and shows data demonstrating the exceptional multiplex real-time PCR performance of the QuantiFast Multiplex kits.
Overcome the challenges of Nucleic acid isolation from PCR inhibitor-rich mic...QIAGEN
This presentation will focus on nucleic acid extraction tools developed by QIAGEN that facilitate accurate non-biased community analysis and eliminate common amplification problems via the depletion of endogenous polymerase inhibitors using our patented Inhibitor Removal Technology.
RotorGene Q A Rapid, Automatable real-time PCR Instrument for Genotyping and...QIAGEN
QIAGEN has developed a selection of robust, novel chemistries to prevent PCR crosstalk. We can successfully measure target abundance and fold change in real-time assays, and perform sub-genotyping using a fast, high-throughput and powerful High-Resolution Melting (HRM) statistical analysis program. In this presentation, we will demonstrate these features and benefits with examples.
Reproducibility, Quality Control and Importance of AutomationQIAGEN
In this webinar, we will introduce you to the key sample quality parameters, discuss their respective impact on downstream applications and how to monitor them, and present the advantages of automating quality control along complex workflows.
Automated Nucleic Acid Purification from Diverse Sample types using dedicated...QIAGEN
This webinar will focus on the automation of QIAGEN’s new line of DNA and RNA sample prep kits for the microbiome. We will show how automation on the QIAcube enables efficient and reliable use of these samples for sensitive downstream applications such as qPCR and NGS. In addition, you will learn how to successfully use the CLC Microbial Genomics Module for metagenome sequencing and identification of microbial composition and diversity.
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For those battling kidney disease and exploring treatment options, understanding when to consider a kidney transplant is crucial. This guide aims to provide valuable insights into the circumstances under which a kidney transplant at the renowned Hiranandani Hospital may be the most appropriate course of action. By addressing the key indicators and factors involved, we hope to empower patients and their families to make informed decisions about their kidney care journey.
India Diagnostic Labs Market: Dynamics, Key Players, and Industry Projections...Kumar Satyam
According to the TechSci Research report titled “India Diagnostic Labs Market Industry Size, Share, Trends, Competition, Opportunity, and Forecast, 2019-2029,” the India Diagnostic Labs Market was valued at USD 16,471.21 million in 2023 and is projected to grow at an impressive compound annual growth rate (CAGR) of 11.55% through 2029. This significant growth can be attributed to various factors, including collaborations and partnerships among leading companies, the expansion of diagnostic chains, and increasing accessibility to diagnostic services across the country. This comprehensive report delves into the market dynamics, recent trends, drivers, competitive landscape, and benefits of the research report, providing a detailed analysis of the India Diagnostic Labs Market.
Collaborations and Partnerships
Collaborations and partnerships among leading companies play a pivotal role in driving the growth of the India Diagnostic Labs Market. These strategic alliances allow companies to merge their expertise, strengthen their market positions, and offer innovative solutions. By combining resources, companies can enhance their research and development capabilities, expand their product portfolios, and improve their distribution networks. These collaborations also facilitate the sharing of technological advancements and best practices, contributing to the overall growth of the market.
Expansion of Diagnostic Chains
The expansion of diagnostic chains is a driving force behind the growing demand for diagnostic lab services. Diagnostic chains often establish multiple laboratories and diagnostic centers in various cities and regions, including urban and rural areas. This expanded network makes diagnostic services more accessible to a larger portion of the population, addressing healthcare disparities and reaching underserved populations. The presence of diagnostic chain facilities in multiple locations within a city or region provides convenience for patients, reducing travel time and effort. A broader network of labs often leads to reduced waiting times for appointments and sample collection, ensuring that patients receive timely and efficient diagnostic services.
Rising Prevalence of Chronic Diseases
The increasing prevalence of chronic diseases is a significant driver for the demand for diagnostic lab services. Chronic conditions such as diabetes, cardiovascular diseases, and cancer require regular monitoring and diagnostic testing for effective management. The rise in chronic diseases necessitates the use of advanced diagnostic tools and technologies, driving the growth of the diagnostic labs market. Additionally, early diagnosis and timely intervention are crucial for managing chronic diseases, further boosting the demand for diagnostic lab services.
CHAPTER 1 SEMESTER V - ROLE OF PEADIATRIC NURSE.pdfSachin Sharma
Pediatric nurses play a vital role in the health and well-being of children. Their responsibilities are wide-ranging, and their objectives can be categorized into several key areas:
1. Direct Patient Care:
Objective: Provide comprehensive and compassionate care to infants, children, and adolescents in various healthcare settings (hospitals, clinics, etc.).
This includes tasks like:
Monitoring vital signs and physical condition.
Administering medications and treatments.
Performing procedures as directed by doctors.
Assisting with daily living activities (bathing, feeding).
Providing emotional support and pain management.
2. Health Promotion and Education:
Objective: Promote healthy behaviors and educate children, families, and communities about preventive healthcare.
This includes tasks like:
Administering vaccinations.
Providing education on nutrition, hygiene, and development.
Offering breastfeeding and childbirth support.
Counseling families on safety and injury prevention.
3. Collaboration and Advocacy:
Objective: Collaborate effectively with doctors, social workers, therapists, and other healthcare professionals to ensure coordinated care for children.
Objective: Advocate for the rights and best interests of their patients, especially when children cannot speak for themselves.
This includes tasks like:
Communicating effectively with healthcare teams.
Identifying and addressing potential risks to child welfare.
Educating families about their child's condition and treatment options.
4. Professional Development and Research:
Objective: Stay up-to-date on the latest advancements in pediatric healthcare through continuing education and research.
Objective: Contribute to improving the quality of care for children by participating in research initiatives.
This includes tasks like:
Attending workshops and conferences on pediatric nursing.
Participating in clinical trials related to child health.
Implementing evidence-based practices into their daily routines.
By fulfilling these objectives, pediatric nurses play a crucial role in ensuring the optimal health and well-being of children throughout all stages of their development.
Deep Leg Vein Thrombosis (DVT): Meaning, Causes, Symptoms, Treatment, and Mor...The Lifesciences Magazine
Deep Leg Vein Thrombosis occurs when a blood clot forms in one or more of the deep veins in the legs. These clots can impede blood flow, leading to severe complications.
India Clinical Trials Market: Industry Size and Growth Trends [2030] Analyzed...Kumar Satyam
According to TechSci Research report, "India Clinical Trials Market- By Region, Competition, Forecast & Opportunities, 2030F," the India Clinical Trials Market was valued at USD 2.05 billion in 2024 and is projected to grow at a compound annual growth rate (CAGR) of 8.64% through 2030. The market is driven by a variety of factors, making India an attractive destination for pharmaceutical companies and researchers. India's vast and diverse patient population, cost-effective operational environment, and a large pool of skilled medical professionals contribute significantly to the market's growth. Additionally, increasing government support in streamlining regulations and the growing prevalence of lifestyle diseases further propel the clinical trials market.
Growing Prevalence of Lifestyle Diseases
The rising incidence of lifestyle diseases such as diabetes, cardiovascular diseases, and cancer is a major trend driving the clinical trials market in India. These conditions necessitate the development and testing of new treatment methods, creating a robust demand for clinical trials. The increasing burden of these diseases highlights the need for innovative therapies and underscores the importance of India as a key player in global clinical research.
COVID-19 PCR tests remain a critical component of safe and responsible travel in 2024. They ensure compliance with international travel regulations, help detect and control the spread of new variants, protect vulnerable populations, and provide peace of mind. As we continue to navigate the complexities of global travel during the pandemic, PCR testing stands as a key measure to keep everyone safe and healthy. Whether you are planning a business trip, a family vacation, or an international adventure, incorporating PCR testing into your travel plans is a prudent and necessary step. Visit us at https://www.globaltravelclinics.com/
How many patients does case series should have In comparison to case reports.pdfpubrica101
Pubrica’s team of researchers and writers create scientific and medical research articles, which may be important resources for authors and practitioners. Pubrica medical writers assist you in creating and revising the introduction by alerting the reader to gaps in the chosen study subject. Our professionals understand the order in which the hypothesis topic is followed by the broad subject, the issue, and the backdrop.
https://pubrica.com/academy/case-study-or-series/how-many-patients-does-case-series-should-have-in-comparison-to-case-reports/
PET CT beginners Guide covers some of the underrepresented topics in PET CTMiadAlsulami
This lecture briefly covers some of the underrepresented topics in Molecular imaging with cases , such as:
- Primary pleural tumors and pleural metastases.
- Distinguishing between MPM and Talc Pleurodesis.
- Urological tumors.
- The role of FDG PET in NET.
Digital DNA-seq Technology: Targeted Enrichment for Cancer Research
1. Sample to Insight
Mutational analysis using QIAGEN’s QIAseq® panels and Sample
to Insight® NGS solutions
Raed Samara, PhD
Global Product Manager, QIAGEN
1QIAseq Targeted NGS for Cancer Research, 10.10.2016
2. Sample to Insight
2
Legal disclaimer
QIAseq Targeted NGS for Cancer Research, 10.10.2016
• QIAGEN products shown here are intended for molecular biology
applications. These products are not intended for the diagnosis,
prevention or treatment of a disease.
• For up-to-date licensing information and product-specific
disclaimers, see the respective QIAGEN kit handbook or user
manual. QIAGEN kit handbooks and user manuals are available
at www.QIAGEN.com or can be requested from QIAGEN
Technical Services or your local distributor.
3. Sample to Insight
3
Precision medicine: Right drug, right patient, right time and dose
QIAseq Targeted NGS for Cancer Research, 10.10.2016
“One size fits all” does not work
4. Sample to Insight
Mutations
AGCTCGTTGCTCAGCTC
Reference genome
AGCTCGTTGCTCAGCGTTC
Insertion
AGCTC---GCTCAGCTC
Deletion
Indels Copy number variations
T
G
CA
T
G
A
C
RS 4
DNA variants for precision medicine
QIAseq Targeted NGS for Cancer Research, 10.10.2016
5. Sample to Insight
RS 5
Actionable DNA variants for precision medicine
QIAseq Targeted NGS for Cancer Research, 10.10.2016
Mutations
AGCTCGTTGCTCAGCTC
Reference genome
AGCTCGTTGCTCAGCGTTC
Insertion
AGCTC---GCTCAGCTC
Deletion
Indels Copy number variations
Only a handful of mutations are actionable
Actionable DNA
Variant
BRAF V600E
EGFR E746-750
+ Kinase domain
mutation
HER2
Disease Melanoma Lung adenocarcinomas IDC-Breast cancer
Therapy Vemurafenib (PLX4032) Erlotinib / Gefitinib Trastuzumab
6. Sample to Insight
RS 6
Actionable DNA variants for precision medicine
QIAseq Targeted NGS for Cancer Research, 10.10.2016
How many?
7. Sample to Insight
EGFR
(L858R)
KRAS
(G12C)
+
Response rates of
>70% in patients with
non-small cell lung
cancer treated with
either erlotinib or
gefitinib
Poor response rate in
patients with non-small
cell lung cancer
treated with either
erlotinib or gefitinib
KRAS
25%
EGFR
23%
EML4-ALK
6%
BRAF
3%PIKC3A
3%
MET
2%
ERBB2
1%
MAP2K1
0.4%
NRAS
0.2%
Unknown
37%
RS 7
Precision medicine for lung cancer
QIAseq Targeted NGS for Cancer Research, 10.10.2016
Current lung cancer biomarker landscape
• How many mutations to test for?
• How to test for these mutations
◦ Sequential testing
◦ Parallel testing
Adapted from: Govindan, R. et al. (2012). Genomic landscape of non-small cell lung cancer in smokers and never-smokers. Cell 150, 1121–34.
8. Sample to Insight
Attribute /
Parameter
Information
level
Cost per
sample
Coverage
achieved
DNA input
No. of samples
multiplexed
Whole
Genome
Sequencing
3 x 109 bps
$5000
30x
1 µg
1
Whole
Exome
Sequencing
5 x 107 bps
$2000
100x
100–200 ng
2
Targeted
DNA
Sequencing
6 x 104 bps
$200
1000x
10 ng
96
Benefits of Targeted
DNA Sequencing:
More relevant data
More cost effective
Detect low-frequency
mutations
Lower DNA
requirements
Higher multiplexing
capabilities
RS 8
Actionable DNA variants for precision medicine
QIAseq Targeted NGS for Cancer Research, 10.10.2016
Targeted DNA sequencing delivers accurate information required for precision medicine
Clinical utility requires targeted analysis
9. Sample to Insight
• Well-defined content
• Small target size
• More reads per sample
RS 9
Why choose targeted DNA sequencing?
QIAseq Targeted NGS for Cancer Research, 10.10.2016
Targeted DNA sequencing limits the genes or targets to be sequenced
Features Benefits
• Examine variants that matter
• Multiplex many samples to save money
• Detect low frequency variants
10. Sample to Insight
Sample Insight
The principle of targeted enrichment is to simultaneously sequence millions of small
DNA fragments that represent the region of interest
gDNA
Variants Report:
KRAS G12D
EGFR T790M
IDH1 R132H
KRAS
EGFR
IDH1
RS 10
Sample
isolation
Library
construction
& targeted
enrichment
NGS run
Data
analysis
Interpretation
Targeted DNA Sequencing (TDS)
QIAseq Targeted NGS for Cancer Research, 10.10.2016
Shrink the genome
11. Sample to Insight
RS 11
Why choose PCR-based targeted enrichment?
QIAseq Targeted NGS for Cancer Research, 10.10.2016
• Offers specificity that beats capture-based
approaches
Features Benefits
• Lets you use sequencing capacity on regions
targeted by the panel, with minimal off-target
sequencing
• Lets you achieve more uniform enrichment for
more sequencing efficiency
It delivers unmatched specificity and uniformity (compared to capture-based methods)
12. Sample to Insight
12
Sample Insight
• Turnaround
time, and
limited
amounts of
DNA
• Uniformity of
enrichment
• Coverage of
GC-rich
regions
• Platform-
dependent
challenges
• Data
processing
& variant
calling
• Isolation of
high-quality
DNA samples
• Quantification
of amplifiable
(not total)
amounts of
DNA
• Clinical &
biological
interpretation
of data
RS
Sample
QC Library QC
Variant
confirmation
Sample
isolation
Library
construction
& targeted
enrichment
NGS run
Data
analysis
Interpretation
Sample to Insight: Integrated universal targeted NGS workflow
QIAseq Targeted NGS for Cancer Research, 10.10.2016
To overcome NGS challenges
13. Sample to Insight
13
Inability to
detect low-
frequency
mutations
Inefficient
enrichment and
sequencing of
GC-rich
regions
PCR and sequencing errors
• Limits sensitivity and accuracy of calling low-frequency variants
o Doesn’t let you confidently call variants down to 1% variant allele
frequency (VAF)
Suboptimal
uniformity of
enrichment and
sequencing
Suboptimal, GC-rich region-incompatible PCR chemistry
• Limits comprehensiveness of panel coverage
o Doesn’t let you efficiently sequence clinically-relevant genes such as
CEBPA or CCND1 – or clinically-relevant regions such as TERT
promoter
Conventional PCR protocols and two-primer amplicon design
• Increases variability in coverage across targeted genomic regions
o Causes you to over-sequence to accommodate the under-
sequenced
o Doesn’t let you call variants in low-depth regions
Mainly due to inferior PCR amplification approaches
Challenges of current DNA targeted sequencing approaches
QIAseq Targeted NGS for Cancer Research, 10.10.2016
14. Sample to Insight
14
DNA
dsDNA
PCR amplification & sequencing
PCR and sequencing errors
The necessary evil: PCR amplification
QIAseq Targeted NGS for Cancer Research, 10.10.2016
PCR amplification is required for target enrichment, but…
15. Sample to Insight
15
5 reads OR library fragments that look exactly the same.
Cannot tell whether they represent:
1. 5 unique DNA molecules, or
2. Quintuplets of the same DNA molecule (PCR duplicates)
Conventional targeted
DNA sequencing
EGFR exon 21
Quantification based on non-unique reads does
not reflect quantities of original DNA molecules
Challenges of conventional targeted DNA sequencing
QIAseq Targeted NGS for Cancer Research, 10.10.2016
PCR duplicates limit accurate quantification
16. Sample to Insight
16
Conventional targeted
DNA sequencing
EGFR exon 21
*
Variant calling based on non-unique reads does not
reflect the mutational status of original DNA molecules
Challenges of conventional targeted DNA sequencing
QIAseq Targeted NGS for Cancer Research, 10.10.2016
A mutation is seen in 1 out of 5 reads that map to EGFR exon
21. Cannot accurately tell whether the mutation is:
1. A PCR or sequencing error (artifact) / false positives, or
2. A true low-frequency mutations
PCR and sequencing errors (artifacts) limit variant calling accuracy
17. Sample to Insight
17
• Proprietary PCR chemistry to enrich even
GC-rich regions
• Primers based on single primer extension
(SPE) approach for enhanced uniformity
Panel box (kit)
QIAGEN’s solutions to overcome challenges of targeted NGS
QIAseq Targeted NGS for Cancer Research, 10.10.2016
QIAseq targeted DNA panels
• Molecularly-barcoded library adapters to
incorporate unique molecular indices
(UMIs).
Index box (kit)
18. Sample to Insight
18
With the QIAseq targeted DNA panels, variant detection is done by analyzing unique
DNA molecules instead of total reads
Overcoming current challenges
QIAseq Targeted NGS for Cancer Research, 10.10.2016
Current approach Challenges
How QIAseq targeted DNA
panels overcome challenges of
current approaches
• Conventional targeted DNA
sequencing for variant
detection
• PCR and sequencing errors • UMIs that enable digital
sequencing to correct for PCR
and sequencing errors
• Inefficient sequencing of GC-
rich regions
• Proprietary chemistry to
efficiently sequence GC-rich
regions
• Suboptimal uniformity of
enrichment and sequencing
• SPE-based primer design to
increase uniformity
For optimal variant detection
19. Sample to Insight
19
TATCGTACAGAT
(12 nucleotides long)
Incorporate this random barcode (signature)
into the original DNA molecules before
amplification to preserve their uniqueness
What is a UMI (molecular barcode)?
QIAseq Targeted NGS for Cancer Research, 10.10.2016
Tag (barcode) to identify unique DNA molecules
20. Sample to Insight
20
DNA
dsDNA
TATCGTACAGAT
Molecularly-barcoded adapter Incorporate this random
barcode (signature) into
the original DNA
molecules before
amplification to preserve
their uniqueness
PCR amplification & sequencing
Correct for PCR duplicates & errors
How are UMIs incorporated?
QIAseq Targeted NGS for Cancer Research, 10.10.2016
Ligate molecularly-barcoded adapters to unique DNA molecules before amplification
21. Sample to Insight
21
5 unique DNA molecules
since 5 molecular barcodes are detected
Quintuplets of the same DNA molecule (PCR duplicates)
since 1 molecular barcode is detected
UMI
Digital sequencing
with UMIs
UMIs before any
amplification
Achieve accurate quantification with molecular barcodes
QIAseq Targeted NGS for Cancer Research, 10.10.2016
Count and analyze single original molecules (not total reads) = digital sequencing
5 reads OR library fragments that look exactly the same.
Cannot tell whether they represent:
1. 5 unique DNA molecules, or
2. Quintuplets of the same DNA molecule (PCR duplicates)
Conventional targeted
DNA sequencing
EGFR exon 21
22. Sample to Insight
22
False variant is present in some fragments
carrying the same UMI
True variant is present in all fragments
carrying the same UMI
UMI
UMIs before any
amplification
* *****
Digital sequencing
with UMIs
Achieve accurate variant calling with molecular barcodes
QIAseq Targeted NGS for Cancer Research, 10.10.2016
Conventional targeted
DNA sequencing
EGFR exon 21
*A mutation is seen in 1 out of 5 reads that map to EGFR exon
21. Cannot accurately tell whether the mutation is:
1. A PCR or sequencing error (artifact) / false positives, or
2. A true low-frequency mutations
Count and analyze single original molecules (not total reads) = digital sequencing
23. Sample to Insight
23
Sample
isolation
Library
construction
& targeted
enrichment
NGS run
Data
analysis
InterpretationSample Insight
Panels and
molecularly-
barcoded
adapters
Barcode-aware
variant calling
pipeline
QIAseq targeted DNA panels: Sample to Insight
QIAseq Targeted NGS for Cancer Research, 10.10.2016
Panels, molecularly-barcoded adapters and data analysis algorithms
24. Sample to Insight
24
Specifications of QIAseq targeted DNA panels
QIAseq Targeted NGS for Cancer Research, 10.10.2016
DNA input As little as 20 ng DNA
Primer multiplexing level 11,500 / 9600 primers (Catalog / Custom DNA)
Number of primer pools 1
Enrichment technology SPE-based with molecularly-barcoded adapters
Amplicon size Average 150 bp
Sample multiplexing level 384 (Illumina), 96 (Ion Torrent)
Total workflow time 8–9 hours
Number of libraries per sample 1
Sequencer compatibility Illumina and Ion Torrent platforms
Variant allele frequency called 1%
25. Sample to Insight
End repair and A tailing
Adapter ligation / library construction (incorporation
of adapters, molecular barcodes and sample
indexes)
5’
5’
5’
5’
MB
MB
Adapter
5’
IL-F
RSP
Add GSPs and UP*
5’
IL-U
SIP
Add indexes and UP*
Universal PCR amplification
Sample indexing and amplification
Sequencing-ready library
MB: Molecular barcode
RSP: Region-specific primer
FP: Forward primer
UP: Universal primer
SIP: Sample index primer
A
A
5’MB
Target enrichment by SPE
5’
*Preceded by bead cleanup
Lib quant*
Enzyme-based random DNA fragmentation
DNA
5’
5’
1.5Days
QIAseq Targeted DNA Panel: Workflow (Illumina®)
QIAseq Targeted NGS for Cancer Research, 10.10.2016 25
26. Sample to Insight
QIAseq Targeted DNA Panel: Workflow (Ion Torrent™)
QIAseq Targeted NGS for Cancer Research, 10.10.2016 26
End repair and A tailing
Adapter ligation / library construction (incorporation
of adapters, molecular barcodes and sample
indexes)
5’
5’
5’
5’
MB
MB
Adapter
5’
LT-F
RSP
Add GSPs and UP*
5’
LT-U
P1
Add indexes and UP*
Universal PCR amplification
Sample indexing and amplification
Sequencing-ready library
MB: Molecular barcode
RSP: Region-specific primer
FP: Forward primer
UP: Universal primer
P1: P1 primer
A
A
5’MB
Target enrichment by SPE
5’
*Preceded by bead cleanup
Lib quant*
Enzyme-based random DNA fragmentation
DNA
5’
5’
1.5Days
27. Sample to Insight
27
1. Exonic regions of genes plus 10 bases to cover intron / exon junctions
2. Mix of type of coverage 1 (for tumor suppressor genes) and HotSpots for oncogenes
3. SNPs
4. Full chromosome
QIAseq targeted DNA panels
QIAseq Targeted NGS for Cancer Research, 10.10.2016
Panel
Variant (Cat)
number
Number of
genes
Number of
primers
Type of
coverage
Breast cancer panel DHS-001Z 93 4831 1
Colorectal cancer panel DHS-002Z 71 2929 1
Myeloid Neoplasms panel DHS-003Z 141 5887 1
Lung cancer panel DHS-005Z 72 4149 1
Actionable solid tumor panel DHS-101Z 23 651 2
BRCA1 and BRCA2 panel DHS-102Z 2 223 1
BRCA1 and BRCA2 Plus panel DHS-103Z 6 348 1
Pharmacogenomics panel DHS-104Z 39 146 3
Mitochondria panel DHS-105Z Chromosome M 222 4
Inherited diseases panel DHS-3011Z 298 11,579 1
Comprehensive cancer panel DHS-3501Z 275 11,311 1
List of panels
Types of coverage:
28. Sample to Insight
28
QIAseq targeted DNA panels
QIAseq Targeted NGS for Cancer Research, 10.10.2016
List of panels
Panel
Variant (Cat)
number
Panel size
(bases)
Specificity
(reads with
primers, %)
Uniformity
(0.2x mean
baseMT, %)
Breast cancer panel DHS-001Z 370,942 96.47 99.84
Colorectal cancer panel DHS-002Z 215,328 90.39 99.79
Myeloid Neoplasms panel DHS-003Z 436,672 95.31 99.71
Lung cancer panel DHS-005Z 318,059 97.3 99.91
Actionable solid tumor panel DHS-101Z 15,160 90.48 99.85
BRCA1 and BRCA2 panel DHS-102Z 16,405 99.59 100
BRCA1 and BRCA2 Plus panel DHS-103Z 25,590 99.46 99.92
Pharmacogenomics panel DHS-104Z 3313 93.43 99.34
Mitochondria panel DHS-105Z 16,570 99.72 99.08
Inherited diseases panel DHS-3011Z 838,627 97.29 99.21
Comprehensive cancer panel DHS-3501Z 836,670 97.42 199.76
Uniformity and specificity are defined based on NA12878 tests
29. Sample to Insight
Extended and Custom
What is the list of your targets?
RS 29
Extended
panels
Custom panels
• Extend the contents of an existing cataloged panel
• Turnaround time = 14 days
• Bioinformatically target any gene(s) or genomic region(s) within the
human genome
• Turnaround time = 14 days
Customized panels
QIAseq Targeted NGS for Cancer Research, 10.10.2016
30. Sample to Insight
30
CEBPA
GC content
Coverage
GC content
Coverage
CCND1
The proprietary
PCR chemistry
used in the
QIAseq targeted
DNA panels
enables efficient
coverage of
regions high in
GC content
Comprehensive coverage of GC-rich regions
QIAseq Targeted NGS for Cancer Research, 10.10.2016
31. Sample to Insight
31
830 kb region was enriched from 20 ng of NA12878 DNA with
Comprehensive Cancer Panel. Library was constructed for sequencing on a
MiSeq, with 2600x read depth. The panel achieved a uniformity of 99.5% at
0.2x of mean coverage, and 98% at 0.3x of mean coverage.
Unmatched uniformity
QIAseq Targeted NGS for Cancer Research, 10.10.2016
32. Sample to Insight
32
Benefits of QIAseq targeted DNA panels
QIAseq Targeted NGS for Cancer Research, 10.10.2016
Feature Benefit
Low DNA input (as low as 20 ng DNA) Preserve sample
High primer multiplexing capability (up to 10,000
primers)
Detect a large number of DNA variants
Single pool of primers Easier sample handling
SPE-based target enrichment Flexibility in primer design
Small amplicons (average size 150 bp)
Generate relatively small library fragments to maintain
compatibility with fragmented DNA (FFPE and ctDNA samples)
High sample multiplexing (up to 384 samples) Increased sample throughput to decrease sequencing costs
Automation-friendly workflow Streamline operations for high throughput
Molecular barcode-aware variant caller Confidently call low-frequency mutations
Suite of complementary data analysis tools Save resources
Affordable per-sample cost Save $$$
All required reagents (including beads) in 2 kits Simplified logistics & ordering
33. Sample to Insight
33
This online GenomeWeb seminar focused on the design of a large cohort study for assessing
breast cancer risk and how using an innovative digital sequencing approach is able to solve
the previously unmet challenges of this type of NGS study design.
Fergus J. Couch, PhD
Professor and Chair Division of Experimental Pathology,
Department of Laboratory Medicine and Pathology, Mayo
Clinic
https://genomeweb.webex.com/genomeweb/lsr.php?RCID=30c8e3fe698b7feca1ed79ac117fbed0
Application: Large cohort study for breast cancer risk
QIAseq Targeted NGS for Cancer Research, 10.10.2016
Sequencing 60,000 samples
34. Sample to Insight
RS 34
Sample to Insight: Integrated universal targeted NGS workflow
QIAseq Targeted NGS for Cancer Research, 10.10.2016
To overcome NGS challenges
Sample Insight
• Turnaround
time, and
limited
amounts of
DNA
• Uniformity of
enrichment
• Coverage of
GC-rich
regions
• Platform-
dependent
challenges
• Data
processing
& variant
calling
• Isolation of
high-quality
DNA samples
• Quantification
of amplifiable
(not total)
amounts of
DNA
• Clinical &
biological
interpretation
of data
Sample
QC Library QC
Variant
confirmation
Sample
isolation
Library
construction
& targeted
enrichment
NGS run
Data
analysis
Interpretation
35. Sample to Insight
35
Barcode-aware variant caller has been developed
Caller is available on the cloud
In conjunction with molecular barcodes incorporated in the workflow, the caller can
confidently call low-frequency variants (down to 1% variant allele frequency, “VAF”)
Variant caller will do the following:
• Mapping
• Alignment
• Molecular barcode counting
• Variant / calling
• Variant / annotation – variants based on public databases
Data analysis with barcode-aware variant caller – overview
QIAseq Targeted NGS for Cancer Research, 10.10.2016
For QIAseq targeted DNA panels
36. Sample to Insight
Data analysis with barcode-aware variant caller – overview
FASTQ or BAM files are uploaded into cloud-based data analysis portal
The following inputs are needed (by customer):
• Set up file
• Panel used
• File lanes
o 1-lane (MiSeq/HiSeq/NextSeq concatenated)
o 4-lane (NextSeq individual lane files)
Inputs
37. Sample to Insight
37
Data analysis with barcode-aware variant caller – overview
QIAseq Targeted NGS for Cancer Research, 10.10.2016
Outputs
Summary file
• Stats
o Specificity
o Uniformity
o Molecular barcode counts
• Variants
o Frequency
o Annotations
VCF
38. Sample to Insight
Actionable
solid tumor
Disease-
specific Comprehensive
Detection
Discovery
Multiplexing
Target size
Custom & extended
PanelsApplicationsSpecifications
Clinical research Translational & discovery research
Whole Exome Seq
Whole Genome Seq
Targeted DNA sequencing: robust detection, limited discovery
RS 38
Why choose targeted DNA sequencing?
QIAseq Targeted NGS for Cancer Research, 10.10.2016
Detect known variants & discover novel variants
39. Sample to Insight
Thank you for attending today’s webinar!
Contact QIAGEN
Call: 1-800-426-8157
Email:
techservice-na@qiagen.com
BRCsupport@QIAGEN.com
QIASeq.NGS@QIGAEN.COM
QIAWebinars@QIAGEN.COM
Questions?
39
Thank you for attending
QIAseq Targeted NGS for Cancer Research, 10.10.2016
Editor's Notes
The concept of one-size-fits all for disease management is not effective. With this approach, some patients show positive effects, while others show no effects or adverse effects.
The concept of precision medicine – delivering the right drug, to the right patient, at the right time and dose – is more effective. It builds on the idea of utilizing genetic information for disease management.
There are several DNA variants that can be used for precision medicine. Three are shown here.
Out of all the DNA variants, only a handful are actionable – which means we can use the information from these variants for disease management. For example, the presence of the V600E mutation in BRAF is seen in a large number of melanoma patients. While this variant causes the development and progression of the disease, its presence is favorable for melanoma patients because the variant (mutation) can be targeted by an approved therapy (vemurafenib).
How many actionable mutations are there? Only a handful.
Shown here is a typical process of filtering variants to build a list of actionable mutations.
Starting with millions of common variants, we end up with 10s–100s of actionable variants.
As an example, in lung cancer we see the following mutations accumulate in lung cancer patients.
We can use the information on the type of mutations for disease management. For instance, patients who harbor the EGFR L858R mutation respond very well to tyrosine kinase inhibitors – while among patients who harbor an additional mutation in KRAS, patient response to the same class of drugs drops.
There are three main sequencing levels which can be used to detect actionable mutations. The table here shows how these three levels differ on several parameters.
Targeted DNA sequencing and analysis is extensively used in clinical research.
What are the benefits of targeted DNA sequencing?
What to include in a targeted panel? There are many approaches. One approach, highlighted by Dr. Carl Morrison, is to include genes for which actionable information exists. In other words, examine targets upon which you can act.
With targeted DNA sequencing, one shrinks the genome to a number of targets that are crucial to the disease being examined. In this example, the user is interested in only the three genes shown.
The reason we chose a PCR approach for target enrichment is to take advantage of the high specificity and uniformity that PCR provides, as shown in this report. Here, the authors compared different enrichment approaches and show that the specificity and uniformity of PCR surpasses those of hybridization approaches.
QIAGEN has built a Sample to Insight workflow to overcome some of the main challenges associated with NGS.
A variant identified in a sample represents one of two events: a true variant or a false variant. False variants can be introduced at any step during the workflow, including sequencing reactions. This results in the inability to accurately and confidently call rare variants (those present at 1% of the sample). Due to PCR duplicates generated in amplification steps, all DNA fragments look exactly the same – and there is no way to tell whether a specific DNA fragment is a unique DNA molecule or a duplicate of a DNA molecule. With molecular barcodes, since each unique DNA molecule is barcoded before any amplification takes place, unique DNA molecules are identified by their unique barcodes – and PCR duplicates carrying the same barcode are removed, thereby increasing the sensitivity of the panel.
We also offer 2 approaches to build your own unique panel that fits your own needs and requirements.
The Mix-n-Match approach gives you access to our 570 wet bench-tested gene designs, which is a very unique offering in the market.
In case your genes of interest are not part of Mix-n-Match, you can use our Custom panels to build a fully customized panel.
The QIAseq DNA panels use a proprietary buffer mixture to efficiently sequence both regular and GC-rich regions within the genome in a single reaction. Two examples are shown here: CEBPA and CCND1. Complete coverage of exonic regions within those two genes is achieved.