The continuous evolution of NGS technology has led to an enormous diversification in NGS applications and dramatically decreased the costs to sequence a complete human genome.
In this presentation, we will discuss the following major topics:
• Basic overview of NGS sequencing technologies
• Next-generation sequencing workflow
• Spectrum of NGS applications
• QIAGEN universal NGS solutions
Sequencing is one of the major technological advancement that has taken shape in the last two or three decade. Starting from Sanger and Maxam-Gilbert sequencing methods to the latest high-throughput methods, sequencing technologies has changed the the landscape of biological sciences.
This slide takes a look a the major sequencing methods over time.
Note: Several images included here have been sourced from GOOGLE IMAGES. The content has been extracted from several SCIENTIFIC PAPERS and WEBSITES.
PLEASE DO CONTACT THE AUTHOR DIRECTLY IF ANY COPYRIGHT ISSUE ARISES.
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.
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).
Sequencing is one of the major technological advancement that has taken shape in the last two or three decade. Starting from Sanger and Maxam-Gilbert sequencing methods to the latest high-throughput methods, sequencing technologies has changed the the landscape of biological sciences.
This slide takes a look a the major sequencing methods over time.
Note: Several images included here have been sourced from GOOGLE IMAGES. The content has been extracted from several SCIENTIFIC PAPERS and WEBSITES.
PLEASE DO CONTACT THE AUTHOR DIRECTLY IF ANY COPYRIGHT ISSUE ARISES.
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.
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).
In this lecture tried to introduce some basic methods of DNA sequencing like pyrosequencing, sequencing by ligation, sequencing by synthesis and Ion Semiconductor Sequencing
and describe them. Also introduced some new sequencing method (third generation sequencing) like SMRT (Single Molecule Real-Time Sequencing) and GridION.
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.
complete Single Nucleotide Polymorphiitsm Detection methods with Advance techniques with its applications
Single nucleotide polymorphisms are single base variations between genomes within a species.
There are at least 10 million polymorphic sites in the human genome.
SNPs can distinguish individuals from one another
Denaturing Gradient Gel Electrophoresis
Chemical Cleavage Of Mismatch
Single-stranded Conformation Polymorphism (SSCP)
MutS Protein-binding Assays
Mismatch Repair Detection (MRD)
Heteroduplex Analysis (HA)
Denaturing High Performance Liquid Chromatography (DHPLC)
UNG-Mediated T-Sequencing
RNA-Mediated Finger printing with MALDI MS Detection
Sequencing by Hybridization
Direct DNA Sequencing
Single-feature polymorphism (SFP)
Invader probe
Allele-specific oligonucleotide probes
PCR-based methods
Allele specific primers
Sequence Polymorphism-Derived (SPD) markers
Targeting induced local lesions in genomes (TILLinG)
Minisequencing primers
Allele-specific ligation probes
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.
whole genome analysis
history
needs
steps involved
human genome data
NGS
pyrosequencing
illumina
SOLiD
Ion torrent
PacBio
applications
problems
benefits
Explore the Illumina workflow, including sequencing by synthesis (SBS) technology, in 3-dimensional detail. Go from sample preparation, to cluster generation, to sequencing on a system flow cell with the proprietary SBS process.
DNA microarray:
A DNA microarray (also commonly known as gene or genome chip, DNA chip, or gene array) is a collection of microscopic DNA spots, commonly representing single genes, arrayed on a solid surface by covalent attachment to a chemical matrix. DNA arrays are different from other types of microarray only in that they either measure DNA or use DNA as part of its detection system. Qualitative or quantitative measurements with DNA microarrays utilize the selective nature of DNA-DNA or DNA-RNA hybridization under high-stringency conditions and fluorophore-based detection. DNA arrays are commonly used for expression profiling, i.e., monitoring expression levels of thousands of genes simultaneously.
QIAseq Technologies for Metagenomics and Microbiome NGS Library PrepQIAGEN
In this slide deck, learn about the innovative technologies that form the basis of QIAGEN’s portfolio of QIAseq library prep solutions for metagenomics and microbiome sequencing. Whether your research starts from single microbial cells, 16s rRNA PCR amplicons, or gDNA for whole genome analysis, QIAseq technologies offer tips and tricks for capturing the genomic diversity of your samples in the most unbiased, streamlined way possible.
Global Next Generation Sequencing (NGS) Industry By Market Size & Forecast to...DavidClark206
This research report covers end-to-end market for NGS in terms of the workflow; presequencing, NGS platforms, consumables and services, sequencing services and bioinformatics market. From an insight perspective, this research report focuses on the qualitative data, market size, share, and growth of various segments and sub-segments, competitive landscape, and company profiles.
Inquire For Discount (Single User Report Price US $4650) @ http://www.reportsnreports.com/contacts/Discount.aspx?name=257153 .
In this lecture tried to introduce some basic methods of DNA sequencing like pyrosequencing, sequencing by ligation, sequencing by synthesis and Ion Semiconductor Sequencing
and describe them. Also introduced some new sequencing method (third generation sequencing) like SMRT (Single Molecule Real-Time Sequencing) and GridION.
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.
complete Single Nucleotide Polymorphiitsm Detection methods with Advance techniques with its applications
Single nucleotide polymorphisms are single base variations between genomes within a species.
There are at least 10 million polymorphic sites in the human genome.
SNPs can distinguish individuals from one another
Denaturing Gradient Gel Electrophoresis
Chemical Cleavage Of Mismatch
Single-stranded Conformation Polymorphism (SSCP)
MutS Protein-binding Assays
Mismatch Repair Detection (MRD)
Heteroduplex Analysis (HA)
Denaturing High Performance Liquid Chromatography (DHPLC)
UNG-Mediated T-Sequencing
RNA-Mediated Finger printing with MALDI MS Detection
Sequencing by Hybridization
Direct DNA Sequencing
Single-feature polymorphism (SFP)
Invader probe
Allele-specific oligonucleotide probes
PCR-based methods
Allele specific primers
Sequence Polymorphism-Derived (SPD) markers
Targeting induced local lesions in genomes (TILLinG)
Minisequencing primers
Allele-specific ligation probes
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.
whole genome analysis
history
needs
steps involved
human genome data
NGS
pyrosequencing
illumina
SOLiD
Ion torrent
PacBio
applications
problems
benefits
Explore the Illumina workflow, including sequencing by synthesis (SBS) technology, in 3-dimensional detail. Go from sample preparation, to cluster generation, to sequencing on a system flow cell with the proprietary SBS process.
DNA microarray:
A DNA microarray (also commonly known as gene or genome chip, DNA chip, or gene array) is a collection of microscopic DNA spots, commonly representing single genes, arrayed on a solid surface by covalent attachment to a chemical matrix. DNA arrays are different from other types of microarray only in that they either measure DNA or use DNA as part of its detection system. Qualitative or quantitative measurements with DNA microarrays utilize the selective nature of DNA-DNA or DNA-RNA hybridization under high-stringency conditions and fluorophore-based detection. DNA arrays are commonly used for expression profiling, i.e., monitoring expression levels of thousands of genes simultaneously.
QIAseq Technologies for Metagenomics and Microbiome NGS Library PrepQIAGEN
In this slide deck, learn about the innovative technologies that form the basis of QIAGEN’s portfolio of QIAseq library prep solutions for metagenomics and microbiome sequencing. Whether your research starts from single microbial cells, 16s rRNA PCR amplicons, or gDNA for whole genome analysis, QIAseq technologies offer tips and tricks for capturing the genomic diversity of your samples in the most unbiased, streamlined way possible.
Global Next Generation Sequencing (NGS) Industry By Market Size & Forecast to...DavidClark206
This research report covers end-to-end market for NGS in terms of the workflow; presequencing, NGS platforms, consumables and services, sequencing services and bioinformatics market. From an insight perspective, this research report focuses on the qualitative data, market size, share, and growth of various segments and sub-segments, competitive landscape, and company profiles.
Inquire For Discount (Single User Report Price US $4650) @ http://www.reportsnreports.com/contacts/Discount.aspx?name=257153 .
Next-Generation Sequencing an Intro to Tech and Applications: NGS Tech Overvi...QIAGEN
This slidedeck provides a technical overview of DNA/RNA preprocessing, template preparation, sequencing and data analysis. It covers the applications for NGS technologies, including guidelines for how to select the technology that will best address your biological question.
Next Generation Sequencing (NGS) in food safety-Game changer or just another ...ExternalEvents
http://tiny.cc/faowgsworkshop
The use of genome sequencing technology on food safety management. Presentation from the FAO expert workshop on practical applications of Whole Genome Sequencing (WGS) for food safety management - 7-8 December 2015, Rome, Italy.
“Next-Generation Sequencing (NGS) Global Market – Forecast To 2022”Vinay Shiva Prasad
Next Generation Sequencing (NGS) has revolutionized the way the human genomes are being sequenced now a days. The time and cost to sequence has tremendously reduced . In the last five years, the sequencing cost has rapidly reduced and this is supposed to have a huge impact on the NGS market in the coming years.
The field of next-generation sequencing (NGS) has been experiencing explosive growth over the past several years and shows little sign of slowing down. The increasing capabilities and dramatically lowered costs have expanded NGS's reach beyond that of the human genome into nearly every corner of biological research. An overview of the platforms on the market today, including an assessment of their relative strengths and weaknesses, will be presented. The presentation will conclude with a peek into where the technology is going and what will be available in the future.
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.
NGS technologies - platforms and applicationsAGRF_Ltd
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. Next Gen Sequencing Platforms and Applications was presented by AGRF Next Gen Manager, Mr. Matt Tinning.
Presented: 1st August 2012
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
Innovative NGS Library Construction TechnologyQIAGEN
Next-generation sequencing (NGS) is a driving force for numerous new and exciting applications, including cancer research, stem cell research, metagenomics, population genetics, medical research and single cell analysis. While NGS technology is continuously improving, library preparation remains one of the biggest bottlenecks in the NGS workflow and includes several time-consuming steps that can result in considerable sample loss and the potential to introduce handling errors. Moreover, conducting single-cell genomic analysis using NGS methods has traditionally been challenging since the amount of genomic DNA present in a single cell is very limited.
Getting Started with NGS (Discover the Benefits of Technology and How it Oper...Tekmatic
The massively parallel sequencing technology is known as “next-generation sequencing” (NGS). It provides extremely high throughput, scalability, and speed. The term “next-generation sequencing” (NGS) refers to high-throughput technologies that can quickly ascertain the sequence of a particular nucleic acid strand, such as DNA or RNA.
Digital RNAseq Technology Introduction: Digital RNAseq Webinar Part 1QIAGEN
QIAseq RNA is a revolutionary turnkey solution for digital gene expression analysis by NGS. From 10 genes to 1000, from one sample to 100, QIAseq RNA delivers precise results on both ION and Illumina sequencing platforms. The data from QIAseq RNA is directly comparable to expression analysis derived from whole transcriptome sequencing or by qRTPCR, only better, cheaper, faster, and more flexible. This webinar will describe the principles of digital expression analysis by NGS, and review the features and benefits of the QIAseq system, options available, and the integrated data analysis package.
Course: Bioinformatics for Biomedical Research (2014).
Session: 2.1.1- Next Generation Sequencing. Technologies and Applications. Part I: NGS Introduction and Technology Overview.
Statistics and Bioinformatisc Unit (UEB) & High Technology Unit (UAT) from Vall d'Hebron Research Institute (www.vhir.org), Barcelona.
The independent study on how Cloud Computing can be used to introduce a new Next Generation Sequencing method in terms of better understanding of the limitations of existing Next Generation Sequencing Methods.
New Technologies at the Center for Bioinformatics & Functional Genomics at Mi...Andor Kiss
A review of recent molecular biology technologies at the core genomics facility at Miami University (Ohio). The aim of this talk is to introduce the facility's capabilities to faculty, graduate and undergraduate students at MiamiU.
Design and evaluation of a genomics variant analysis pipeline using GATK Spar...Paolo Missier
A paper presented at the annual Italian Database conference (SEBD): http://sisinflab.poliba.it/sebd/2018/
here is the paper: http://sisinflab.poliba.it/sebd/2018/papers/June-27-Wednesday/1-Big-Data/SEBD_2018_paper_23.pdf
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.
Digital RNAseq for Gene Expression Profiling: Digital RNAseq Webinar Part 2QIAGEN
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. In this webinar we will cover, in depth, the 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.
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.
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.
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.
Antibiotic Stewardship by Anushri Srivastava.pptxAnushriSrivastav
Stewardship is the act of taking good care of something.
Antimicrobial stewardship is a coordinated program that promotes the appropriate use of antimicrobials (including antibiotics), improves patient outcomes, reduces microbial resistance, and decreases the spread of infections caused by multidrug-resistant organisms.
WHO launched the Global Antimicrobial Resistance and Use Surveillance System (GLASS) in 2015 to fill knowledge gaps and inform strategies at all levels.
ACCORDING TO apic.org,
Antimicrobial stewardship is a coordinated program that promotes the appropriate use of antimicrobials (including antibiotics), improves patient outcomes, reduces microbial resistance, and decreases the spread of infections caused by multidrug-resistant organisms.
ACCORDING TO pewtrusts.org,
Antibiotic stewardship refers to efforts in doctors’ offices, hospitals, long term care facilities, and other health care settings to ensure that antibiotics are used only when necessary and appropriate
According to WHO,
Antimicrobial stewardship is a systematic approach to educate and support health care professionals to follow evidence-based guidelines for prescribing and administering antimicrobials
In 1996, John McGowan and Dale Gerding first applied the term antimicrobial stewardship, where they suggested a causal association between antimicrobial agent use and resistance. They also focused on the urgency of large-scale controlled trials of antimicrobial-use regulation employing sophisticated epidemiologic methods, molecular typing, and precise resistance mechanism analysis.
Antimicrobial Stewardship(AMS) refers to the optimal selection, dosing, and duration of antimicrobial treatment resulting in the best clinical outcome with minimal side effects to the patients and minimal impact on subsequent resistance.
According to the 2019 report, in the US, more than 2.8 million antibiotic-resistant infections occur each year, and more than 35000 people die. In addition to this, it also mentioned that 223,900 cases of Clostridoides difficile occurred in 2017, of which 12800 people died. The report did not include viruses or parasites
VISION
Being proactive
Supporting optimal animal and human health
Exploring ways to reduce overall use of antimicrobials
Using the drugs that prevent and treat disease by killing microscopic organisms in a responsible way
GOAL
to prevent the generation and spread of antimicrobial resistance (AMR). Doing so will preserve the effectiveness of these drugs in animals and humans for years to come.
being to preserve human and animal health and the effectiveness of antimicrobial medications.
to implement a multidisciplinary approach in assembling a stewardship team to include an infectious disease physician, a clinical pharmacist with infectious diseases training, infection preventionist, and a close collaboration with the staff in the clinical microbiology laboratory
to prevent antimicrobial overuse, misuse and abuse.
to minimize the developme
CRISPR-Cas9, a revolutionary gene-editing tool, holds immense potential to reshape medicine, agriculture, and our understanding of life. But like any powerful tool, it comes with ethical considerations.
Unveiling CRISPR: This naturally occurring bacterial defense system (crRNA & Cas9 protein) fights viruses. Scientists repurposed it for precise gene editing (correction, deletion, insertion) by targeting specific DNA sequences.
The Promise: CRISPR offers exciting possibilities:
Gene Therapy: Correcting genetic diseases like cystic fibrosis.
Agriculture: Engineering crops resistant to pests and harsh environments.
Research: Studying gene function to unlock new knowledge.
The Peril: Ethical concerns demand attention:
Off-target Effects: Unintended DNA edits can have unforeseen consequences.
Eugenics: Misusing CRISPR for designer babies raises social and ethical questions.
Equity: High costs could limit access to this potentially life-saving technology.
The Path Forward: Responsible development is crucial:
International Collaboration: Clear guidelines are needed for research and human trials.
Public Education: Open discussions ensure informed decisions about CRISPR.
Prioritize Safety and Ethics: Safety and ethical principles must be paramount.
CRISPR offers a powerful tool for a better future, but responsible development and addressing ethical concerns are essential. By prioritizing safety, fostering open dialogue, and ensuring equitable access, we can harness CRISPR's power for the benefit of all. (2998 characters)
Navigating the Health Insurance Market_ Understanding Trends and Options.pdfEnterprise Wired
From navigating policy options to staying informed about industry trends, this comprehensive guide explores everything you need to know about the health insurance market.
Leading the Way in Nephrology: Dr. David Greene's Work with Stem Cells for Ki...Dr. David Greene Arizona
As we watch Dr. Greene's continued efforts and research in Arizona, it's clear that stem cell therapy holds a promising key to unlocking new doors in the treatment of kidney disease. With each study and trial, we step closer to a world where kidney disease is no longer a life sentence but a treatable condition, thanks to pioneers like Dr. David Greene.
Telehealth Psychology Building Trust with Clients.pptxThe Harvest Clinic
Telehealth psychology is a digital approach that offers psychological services and mental health care to clients remotely, using technologies like video conferencing, phone calls, text messaging, and mobile apps for communication.
Welcome to Secret Tantric, London’s finest VIP Massage agency. Since we first opened our doors, we have provided the ultimate erotic massage experience to innumerable clients, each one searching for the very best sensual massage in London. We come by this reputation honestly with a dynamic team of the city’s most beautiful masseuses.
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.
Introduction to Next-Generation Sequencing (NGS) Technology
1. Sample to Insight
Introduction to Next-Generation Sequencing (NGS)
Wolfgang Krebs, R&D Scientist, QIAGEN
1Intro to NGS, 11.30.2016
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Overview of NGS technologies and innovative NGS library
prep methods
Part 1: Introduction to next-generation sequencing (NGS)
technology
Part 2: Innovative NGS library construction technology
Part 3: Advanced NGS library prep for challenging samples
Welcome to a 3-part series: NGS technology and applications
Intro to NGS, 11.30.2016
3. Sample to Insight
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.
Legal disclaimer
3Intro to NGS, 11.30.2016
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Illumina® with HiSeq® X in 2014… BUT data interpretation not included
Veritas Genetics in 2016 $1000 genome
Almost the $1000 genome….
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Data analysis
&
interpretation
Sequencing
Sample
extraction
Library
preparation
Genomic DNA
or RNA
Library preparation
Bring the DNA/RNA into a
format that is usable to the
sequencer
Sequencing on Illumina
or Ion Torrent™ platforms
Data analysis
The universal NGS workflow for Illumina/Ion Torrent
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1. Create DNA fragments
2. Add platform-specific adapter sequences to every fragment
3. Amplify library molecules (optional)
Library
preparation
Data analysis
&
interpretation
Sequencing
Sample
extraction
What NGS library prep accomplishes
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300 bp sample DNA insert
Adapter
ligation
point
Flowcell
What the adapters do #1: Bind library to a flowcell or bead
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Adapter
ligation
point
A C TG
A C TG
A C TG
A C TG
A C TG
Seq
primer 1
Seq primer
binding site
Bridge amplification
to form a cluster
Bind primer and sequence
What the adapters do #2: Add seq primer binding sites
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Barcode 1
Each 96-plex adapter includes two 8-base barcodes
8 barcode 1’s x 12 barcode 2’s = 96 combinations
Pool
barcoded
libraries
Single
multiplexed
NGS run
Demultiplexing
fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq
fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq
fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq
fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq
fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq
fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq
fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq
fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq
Separate FASTQ data files for each sample
Multiplexed sequencing: Multiple samples sharing one sequencing reaction to reduce
per-sample costs and control the amount of data generated per sample.
Adapter
ligation
point
What the adapters do #3: Add barcodes for multiplexing
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Adapter – A short double-stranded DNA fragment that is ligated to the sample DNA
fragments prior to sequencing.
Barcode – Unique molecular identifiers included in adapter sequences
.
Library complexity – A measure of the proportion of unique molecules within a library.
More complexity is better, and a high-complexity library is generally free of PCR
duplicates, adapter-dimers and other artifacts.
Multiplexing – Combining several different samples in one sequencing run.
Demultiplexing – The act of separating reads from an NGS run into separate piles
based on their index or barcode reads.
Key NGS vocabulary
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1st generation sequencing
• Sequence many identical molecules
• Sequencing in large gels or capillary
tubing limits scale
What came before next-generation sequencing?
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How is next-generation sequencing different?
Intro to NGS, 11.30.2016
1st generation sequencing
• Sequence many identical
molecules
• Sequencing in large gels
or capillary tubing limits
scale
2nd generation sequencing
• Sequence millions of
clonally amplified molecules
per run
• Using a reversible, stepwise
sequencing chemistry
• Immobilized on a surface
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What comes after NGS?
Intro to NGS, 11.30.2016
3rd generation sequencing
(NGS)
• Single molecule sequencing
in real-time
• Generation of very long
reads (>>5 kb)
• Faster than 2nd gen (hours
instead of days)
• Still high on error rates
1st generation sequencing
• Sequence many identical
molecules
• Sequencing in large gels
or capillary tubing limits
scale
2nd generation sequencing
• Sequence millions of
clonally amplified molecules
per run
• Using a reversible, stepwise
sequencing chemistry
• Immobilized on a surface
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Example: Illumina platforms
1. Cluster amplification 2. Sequencing
What happens on the sequencer?
Intro to NGS, 11.30.2016
Source: Cram genomics http://www.cram.com/flashcards/genomics-6416574
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• DNA fragments are flanked with adaptors (library)
• A solid surface is coated with primers complementary to the two adaptor sequences
• Isothermal amplification, with one end of each “bridge” tethered to the surface
• Clusters of DNA molecules are generated on the chip. Each cluster is originated from
a single DNA fragment, and is thus a clonal population.
• Used by Illumina
21
Bridge amplification (isothermal amplification)
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• Run time: 1–10 days
• Produces: 2–1000 Gb of sequence
• Read length: 2 x 50 bp – 2 x 250 bp
(paired-end)
• Cost: $0.05–$0.40/Mb
22
Illumina HiSeq/MiSeq®
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Image source: Nature Reviews 11. http://www.nature.com/nrg/journal/v11/n1/images/nrg2626-f2.jpg
23. Sample to Insight
Single-end reading (SE):
• Sequencer reads a fragment from only one primer binding site
Paired-end reading (PE):
• Sequencer reads both ends of the same fragment
• More sequencing information, reads can be more accurately placed (“mapped”)
• May not be required for all experiments, more expensive and time-consuming
• Required for high-order multiplexing of samples (indexes on both sides)
Single-end
reading
2nd strand
synthesis
Paired-end
reading
23
Illumina single-end vs. paired-end
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• Fragments with adaptors (the library) are PCR amplified within a water drop in oil
• One PCR primer is attached to the surface of a bead
• DNA molecules are synthesized on the beads in the water droplet. Each bead bears clonal
DNA originated from a single DNA fragment
• Beads (with attached DNA) are then deposited into the wells of sequencing chips – one well,
one bead
24
Emulsion PCR: Ion Torrent
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Image source: Shendure, J. and Hanlee, J. (2008) Next-generation DNA sequencing. Nature Biotechnology 26, 1135–45.
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• Run time: 3 h; no termination or deprotection steps
• Read length: 100–300 bp
• Throughput determined by chip size (pH meter array): 10Mb – 5 Gb
• Cost: $1–$20/Mb
25
Ion Torrent PGM/Proton
Intro to NGS, 11.30.2016
Image sources: Rothberg, J.M., et al. (2011) An integrated semiconductor device enabling non-optical genome sequencing. Nature 475, 348–52.
Tinning, M. (2012) Next gen sequencing platforms and applications. Australian Genome Research Facility slideshare. http://www.slideshare.net/AGRF_Ltd/ngs-
technologies-platforms-and-applications?qid=d1e6ef45-c2de-41e5-8fa8-c7872ced32d3&v=&b=&from_search=1
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NGS – Next-generation sequencing.
Flow cell – Name of the Illumina sample chip. Ready-to-sequence libraries are
bound to the flow cell and bridge amplified prior to sequencing. Depending on the
type of sequencer and the number of samples, different flow cell sizes are
available.
FASTQ file – default file format for sequencer output.
Alignment – the act of “matching” reads to a reference genome.
Key NGS vocabulary
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Application areas
Intro to NGS, 11.30.2016
Source: Rizzo, J.M. and Buck, M.J. (2012) Key principles and clinical applications of “next-generation” DNA sequencing. Cancer Prev Res (Phila) 5, 887–900.
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Large fraction of genome = greater costs, but greater ability to compare samples or patients across studies
Small targeted panels = easier and less expensive to complete the project, but limited ability to follow up on new
hypotheses or discover new associations
Whole
Genome
Exome
Sequencing
DNA Targeted
Panels
RNA-Seq
RNA targeted panels
• Gene expression
• Gene fusions
• Splice variants (known targets)
Four key NGS applications
Intro to NGS, 11.30.2016
Image source: Simon, R. and Roychowdhury, S. (2013) Implementing personalized cancer genomics in clinical trials. Nature 12, 358–69.
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AnalyteSample Type
Blood
Tissue
Metagenomics
Blood cells
Circulating cell-free
nucleic acids
Circulating tumor cells
(CTCs)
Exosomes
FFPE
Fresh frozen
Single cells
Stool, soil, water, etc.
NGS applications – Details
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AnalyteSample Type
Blood
Tissue
Metagenomics
Blood cells
Circulating cell-free
Nucleic acids
Circulating tumor cells
(CTCs)
Exosomes
DNA
RNA
Epigenetics
FFPE
Fresh Frozen
Single Cells
Stool, soil, water, etc.
NGS applications – Details
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AnalyteSample Type
Blood cells
Circulating cell-free
nucleic acids
Circulating tumor cells
(CTCs)
Exosomes
DNA
RNA
Epigenetics
FFPE
Fresh frozen
Single cells
Stool, soil, water, etc.
Sequencing
Targeted
Non-targeted
NGS applications – Details
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Targeted Sequencing
PCR-based target enrichment
(amplicon sequencing)
Hybrid capture target enrichment
(whole exome, targeted regions)
Lib prep: End repair, A-tailing,
adapter ligation
PCR primers, target
enrichment
Adapter
llgation
point
T
Shearing of gDNA
(enzymatic, mechanical)
Lib prep: End repair, A-tailing,
adapter ligation
Hybrid capture with target-specific
probes;
enrich for
target regions
Extracted DNA
Hybrid capture vs. amplicon sequencing
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Whole
genome
Exome
sequencing
RNA-Seq
DNA
targeted
panels
All bases,
low depth
~5–100 M reads
(variable)
RNA
targeted
panels
90 Gb
(30x human)
~25 M reads
(100x)
~2 M reads*
(1000x)
Few bases,
high depth
ChIP-
Seq
~5 M reads
(variable)
~2 M reads
(1000x)
RNA
DNA
Gb = # reads x read length
* Depends on gene number and sequencing depth
Typical coverage or sequencing depth for key NGS applications
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Capacity of different sequencers is defined by number of reads generated per run
(sequencer throughput)
Illumina
Thermo
Fisher
MiniSeq™
15–50 M
7.5 Gb
MiSeq
25–50 M
15 Gb
NextSeq™ 500
400 M
120 Gb
HiSeq 2500
4000 M
1000 Gb
HiSeq X
6000 M
1800 Gb
PGM
5 M
2 Gb
S5/S5XL
80 M
15 Gb
Low
throughput
High
throughput
Sequencers have different capacities (throughput)
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RNA targeted panels
DNA targeted panels
mRNA-Seq
ChIP-seq Exome sequencing
Whole genome(Small WGS)
The sequencer is an indicator for the NGS application
Intro to NGS, 11.30.2016
Illumina
MiniSeq™ MiSeq NextSeq™ 500 HiSeq 2500 HiSeq X
Thermo
Fisher
PGM S5/S5XL
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Coverage – The proportion of a reference genome that has reads aligned to it.
Sequencing depths – Refers to the number of reads that cover a given position in a
reference.
Whole genome sequencing – Sequencing as much of the genome as possible to either
assemble a draft genome for new organisms or to identify sequence variants, chromosomal
rearrangements or other structural variants for organisms with a reference.
Exome sequencing = hybrid capture sequencing – Sequence only a subset of the genome
containing features of interest, which are selected out of a whole genome library using hybrid
capture.
Targeted panel sequencing = amplicon panel sequencing = targeted resequencing –
Regions of interest are amplified from gDNA or cDNA via PCR, and the amplicons are
sequenced. Offers greater sequencing depth than WGS, but focus on a small set of genomic
regions.
Key NGS vocabulary
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The QIAseq portfolio covers a broad range of NGS applications
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Explore QIAseq NGS solutions: https://www.qiagen.com/products/ngs/ngs-life-sciences/
Intro to NGS, 11.30.2016
QIAseq NGS solutions
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Focus products for whole genome or exome sequencing
Intro to NGS, 11.30.2016
NGS
Library
preparation
NGS
library
preparation
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Overview of NGS technologies and innovative NGS library
prep methods
Part 1: Introduction to next-generation sequencing (NGS)
technology
Part 2: Innovative NGS library construction technology
Part 3: Advanced NGS library prep for challenging samples
Upcoming webinars
Intro to NGS, 11.30.2016
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Questions?
All our solutions from Sample to Insight on:
QIAGEN.com
Contact QIAGEN technical service
Call: 1-800-426-8157 for US
Call: +49 2103-29-12400 EU
Email:
techservice-na@QIAGEN.com
techservice-eu@QIAGEN.com
QIASeq.NGS@QIAGEN.com
QIAwebinars@QIAGEN.com
Thank you for attending!
Intro to NGS, 11.30.2016
Editor's Notes
In the past (10–15 years ago), sequencing may have been a more rarely used academic method – or a method only reserved for rich people desiring a personalized diagnostic. In 2016, the technique has become a standardized method to detect genetic diseases or cancer and has revolutionized the biomedical research and personalized healthcare. McKinsey listed next-generation sequencing (NGS) as one of the 12 technologies that will transform our lives.
HiSeq 2000 0.2 10E+9 Kb
HiSeq X System 0.9-1.8 10E+9 Kb almost 10 times more than HiSeq2000
Drastic decrease in sequencing costs per genome.
To illustrate the nature of the reductions in DNA sequencing costs, each graph also shows hypothetical data reflecting Moore's Law, which describes a long-term trend in the computer hardware industry that involves the doubling of “computer power” every two years (See: Moore's Law [wikipedia.org]). Technology improvements that keep up with Moore's Law are widely regarded to be doing exceedingly well, making it useful for comparison.
Moore’s law square.
A very high proportion of current NGS sequencers use platforms from Illumina and Ion Torrent.
Thus, I will describe a general NGS workflow applicable for these two sequencer types – which require a library preparation step, followed by performance of the sequencing reaction, itself, with sequencing-by-synthesis technology.
Due to less efficient library construction – or due to the multiple PCR cycles required for library amplification – complexity can potentially decrease. A higher complexity means more unique molecules within an NGS library, which is always preferred to a lower complexity.
[Mention our CLC Analysis software]
Makes our Sample to Insight mission possible.
Used either chemical or enzymatic methods to generate a nested set of DNA fragments.
Used electrophoretic methods to separate the fragments.
Required lots of DNA (100s of ng to 1 ug), so it typically involved cloning and/or PCR.
Limited scale and throughput.
Sequencers on the picture:
Life Technologies/Applied Biosystems; now part of Thermo Fisher Scientific, SOLID 5500
Illumina MiSeq
Ion Torrent PGM, also now part of Thermo Fisher Scientific
Roche / 454 Pyrosequencer
QIAGEN GeneReader
--Pacific Biosciences RS II System / Sequel System (pictures 1+2) with the SMRT (single molecule real-time) sequencing technology
Zero-Mode Waveguides (ZMWs) allow light to illuminate only the bottom of a well in which a DNA polymerase/template complex is immobilized. Phospholinked nucleotides allow observation of the immobilized complex as the DNA polymerase produces a completely natural DNA strand
Allows simultaneous epigenetic characterization (modified vs. unmodified bases)
--Oxford Nanopore (pictures 3+4, “Pocket-sized” minION and “Desktop” PromethION [not available, only in early access program])
The Nanopore system uses a technique that passes intact DNA polymers through a protein nanopore, sequencing in real time as the DNA translocates the pore. DNA is sequenced by measuring the change in current across the membrane when the DNA passes through the pore; different base pairs have different conductance, and thus different signals.
FFPE = Formalin-fixed paraffin-embedded
Especially for targeted panels, the read counts and sequencing depth depends not only on the type of the panel with a lower or higher number of target regions but also on the type of analysis. A very sensitive variant calling analysis with 1% detection threshold needs a higher sequencing converage in comparison to a 5% threshold analysis.