This document provides instructions for using GeneRead DNAseq Gene Panels, which are targeted exon enrichment panels for next-generation sequencing. The key points are:
- GeneRead panels use multiplex PCR to amplify targeted regions of interest from genomic DNA for sequencing.
- The process involves PCR amplification of DNA samples using primer mixes, pooling and purification of products, and library preparation for sequencing on platforms like Illumina or Ion Torrent.
- Detailed protocols are provided for PCR setup, pooling and purification, library construction, and data analysis. Important considerations like DNA quality controls and recommended reagents are also outlined.
Advancing Microbiome Research: From challenging samples to insight with Confi...QIAGEN
Microbiome research encompasses sample types as diverse as the human gut, Antarctic soil, ocean water and acidic hot spring biofilms. These samples are challenging because they are difficult to lyse, with some microbes containing a tough extracellular matrix. Incomplete lysis of a microbial community results in an inaccurate representation of the microbial content of the sample. Additionally, PCR inhibitors present in these samples, especially humic acids, polysaccharides, polyphenolics, lipids and heavy metals result in inaccurate quantification of nucleic acids that may inhibit downstream applications such as qPCR and NGS.
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.
Quality Control of RNA Samples - For Gene-Expression Results you Can Rely onQIAGEN
By their very nature RNA molecules, especially mRNA and regulator RNA, are labile and can be highly unstable and sensitive to heat, UV and RNase contamination. The quality, relevance and scientific impact of gene expression results directly depends on the ability to extract RNA without losing any fraction of interest, while preserving the integrity of the biological information it carries. RNA quality control is thus critical to ensure high-quality results and for turning these results into actionable insights with confidence.
In this webinar, we will introduce you to the main parameters influencing RNA-based assays and their respective impact on downstream applications, discuss how to monitor them and cover the advantages of automation for quality control along complex workflows.
Optimal RNAlater® incubation and removal conditions prior to isolation of tot...QIAGEN
RNA is highly sensitive to degradation. Handling methods and prolonged storage of cells can greatly affect the quality of the RNA that can be later isolated. Contamination with RNases is the most significant problem, especially as they are so ubiquitous in the environment. They can degrade RNA to the point where results of downstream analyses become meaningless.
Submerging cells in RNAlater, an RNA stabilization reagent, helps to stabilize the RNA within the cells and prevent degradation, supporting accurate downstream gene expression analyses. However, to avoid any interference from any RNAlater components in isolation and analyses, cells must be pelleted and the reagent must be removed. The separation of cells from excess RNAlater via centrifugation is impeded due to the higher density of the reagent compared to standard culture medium. This means it requires higher centrifugal forces, which might damage cells due to increased shearing forces, leading to reduced RNA yield. The aim of this study was to establish the optimal conditions for the recovery of cells from RNAlater after RNA stabilization for maximum RNA yield and integrity.
Canvax Catalog 2017 - Accelerating your Molecular Biology DiscoveriesJesús C. Morales
Canvax Biotech is a leading spanish manufacturer and supplier of of 450+ high quality, cost-effective and easy-to-use Highly Innovative Molecular Biology solutions, services, kits and R&D Reagents that accelerates Scientists Success since 2001.
What we manufacture?
>> DNA Cloning >> Mammalian & Bacterial Expression vectors >> GPCR cDNA ORF Clones >> PCR Essentials
>> Cell based assay and molecule detection kits >> DNA & RNA Purification Kits >> Recombinant proteins
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.
Advancing Microbiome Research: From challenging samples to insight with Confi...QIAGEN
Microbiome research encompasses sample types as diverse as the human gut, Antarctic soil, ocean water and acidic hot spring biofilms. These samples are challenging because they are difficult to lyse, with some microbes containing a tough extracellular matrix. Incomplete lysis of a microbial community results in an inaccurate representation of the microbial content of the sample. Additionally, PCR inhibitors present in these samples, especially humic acids, polysaccharides, polyphenolics, lipids and heavy metals result in inaccurate quantification of nucleic acids that may inhibit downstream applications such as qPCR and NGS.
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.
Quality Control of RNA Samples - For Gene-Expression Results you Can Rely onQIAGEN
By their very nature RNA molecules, especially mRNA and regulator RNA, are labile and can be highly unstable and sensitive to heat, UV and RNase contamination. The quality, relevance and scientific impact of gene expression results directly depends on the ability to extract RNA without losing any fraction of interest, while preserving the integrity of the biological information it carries. RNA quality control is thus critical to ensure high-quality results and for turning these results into actionable insights with confidence.
In this webinar, we will introduce you to the main parameters influencing RNA-based assays and their respective impact on downstream applications, discuss how to monitor them and cover the advantages of automation for quality control along complex workflows.
Optimal RNAlater® incubation and removal conditions prior to isolation of tot...QIAGEN
RNA is highly sensitive to degradation. Handling methods and prolonged storage of cells can greatly affect the quality of the RNA that can be later isolated. Contamination with RNases is the most significant problem, especially as they are so ubiquitous in the environment. They can degrade RNA to the point where results of downstream analyses become meaningless.
Submerging cells in RNAlater, an RNA stabilization reagent, helps to stabilize the RNA within the cells and prevent degradation, supporting accurate downstream gene expression analyses. However, to avoid any interference from any RNAlater components in isolation and analyses, cells must be pelleted and the reagent must be removed. The separation of cells from excess RNAlater via centrifugation is impeded due to the higher density of the reagent compared to standard culture medium. This means it requires higher centrifugal forces, which might damage cells due to increased shearing forces, leading to reduced RNA yield. The aim of this study was to establish the optimal conditions for the recovery of cells from RNAlater after RNA stabilization for maximum RNA yield and integrity.
Canvax Catalog 2017 - Accelerating your Molecular Biology DiscoveriesJesús C. Morales
Canvax Biotech is a leading spanish manufacturer and supplier of of 450+ high quality, cost-effective and easy-to-use Highly Innovative Molecular Biology solutions, services, kits and R&D Reagents that accelerates Scientists Success since 2001.
What we manufacture?
>> DNA Cloning >> Mammalian & Bacterial Expression vectors >> GPCR cDNA ORF Clones >> PCR Essentials
>> Cell based assay and molecule detection kits >> DNA & RNA Purification Kits >> Recombinant proteins
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.
Nucleic Acid Quantification from FFPE Samples – Are You Doing it Right?QIAGEN
Formalin-fixation and paraffin-embedding is a standard method for long-term preservation of tissue biopsies and these stored samples are a valuable tool for studying diseases such as cancer, especially when they are histologically and pathologically well characterized, and follow-up clinical data is available. The quality of nucleic acids extracted from FFPE samples is influenced by a number of factors, including how the samples were handled before, during and after fixation and embedding. Moreover, there are several difficulties when purifying nucleic acids from FFPE samples as the chemicals and temperature used during the process can degrade the DNA.
In this webinar, we will discuss the variability in quantity and purity of DNA purified from FFPE material. We will show data from different quantification and quality control methods, demonstrate the impact of inaccurate quantification on downstream results and discuss how to overcome these challenges.
5 Tips for Successful qRT-PCR Results InfographicQIAGEN
Market research shows that 66.6% of researchers use qRT-PCR for gene-expression profiling. Clearly, this is a very effective and popular technique for detecting RNA expression levels, but it’s still prone to pitfalls that can sometimes lead to disappointing results.
To help improve your experiments, we’ve made a new infographic with some interesting facts about qRT-PCR and 5 tips to help with your gene expression studies. This will cover everything from experimental design to data analysis.
RNase H2-dependent PCR (rhPCR) is a powerful method for increasing PCR specificity and eliminating primer-dimers by using blocked primers and a thermostable RNase H2 from Pyrococcus abyssi (P. abyssi). Primers will only support extension and replication after the blocked portion is cleaved. Cleavage by the RNase H2 enzyme occurs only when primers are bound to their complementary target sequence, thus providing increased specificity. Also, the thermostability of P. abyssi RNase H2 provides a “hot start” capability to the reaction. In this presentation, Dr Joseph Dobosy (senior research scientist in the molecular genetics research division of IDT) gives a detailed explanation of the rhPCR mechanism, offer tips on how to design assays using this powerful technology, and discuss examples of applications that benefit from rhPCR.
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.
RNA integrity and quality - Standardize RNA quality controlQIAGEN
RNA integrity and quality are critical to obtain meaningful and reliable downstream data. This slidedeck details the important considerations and critical factors in RNA preparation. It also highlights the need for quality control analysis and common methods for RNA integrity and quality assessment.
The importance of controls and novel solutions for successful real-time qPCRQIAGEN
The increasing demand for streamlined, monitored and ultrafast qPCR procedures requires high-performance, real-time quantitative RT and PCR chemistries. Particularly, procedures utilizing generic kits for gene expression analysis should include in-process safety measures to avoid variables and control accuracy of procedures and results. This slidedeck presents innovative solutions for one-step and two-step RT-PCR that significantly enhance performance and reliability in qRT-PCR. The new QuantiNova kit family offers a combination of various integrated safety features to remove variables and prevent artifacts. Internal control RNA, removal of genomic DNA, room temperature set-up capability for RT-PCR and a built-in visual pipetting control verify accurate procedures, ensuring reliable gene expression profiling.
This slidedeck explains the principles of the technologies and shows data demonstrating performance in qRT-PCR. Find out how you can verify accurate performance in qRT-PCR and improve your results!
Enabling RNA-Seq With Limited RNA Using Whole Transcriptome AmplificationQIAGEN
RNA-Seq was developed to perform transcriptome profiling and provides a highly precise measurement of expression levels of transcripts and their isoforms. Normally, RNA-Seq analysis requires at least 500 ng –1 μg of total RNA. When working with small biopsies, single cells (such as circulating tumor cells), or other limited material, whole transcriptome amplification (WTA) is normally required. Various WTA methods overcome limited RNA availability and enable transcriptome analysis from limited material or even single cells. In standard PCR-based WTA procedures, however, bias from uneven coverage of cDNA regions with high GC or AT content or amplification errors can lead to the loss of transcripts and wrong variant calling. Here, we compare a standard RNA-Seq library preparation method and the REPLI-g RNA library protocol. The REPLI-g procedure is a PCR-free protocol to efficiently generate RNA-Seq libraries from small amounts of RNA or a single cell in 6.5–7 hours. The REPLI-g protocol uses whole transcriptome amplification based on multiple displacement amplification (MDA), combined with an efficient library adaptor ligation procedure, to prepare RNA-Seq libraries from small RNA amounts. The procedure demonstrates high fidelity, minimal bias and retention of sample‘s transcriptional profile. Compared to standard RNA-Seq library prep, the REPLI-g protocol demonstrates similar reproducibility and sensitivity in transcript detection.
PCR - From Setup to Cleanup: A Beginner`s Guide with Useful Tips and Tricks -...QIAGEN
This End-Point PCR Beginner´s Guide will not only give you a comprehensive overview of tools and techniques to help you to get the most out of your samples, but also give you information on dedicated solutions and complete workflows on multiplex PCR and PCR fragment analysis.
Improving RNA yield and quality is easy with the right protocols. This trouble shooting guide presents methods to overcome common difficulties in RNA purification. Prevent RNA degradation, improve RNA yields, reduce genomic DNA contamination, and more.
Real-Time quantitative PCR (qPCR) is a mainstream method that is used in research and diagnostic applications for quantification of gene expression. IDT has developed a robust and affordable qPCR master mix for use with probe-based qPCR in single and multiplex assays. In this presentation, we explore a variety of applications of PrimeTime® Gene Expression Master Mix. We cover the use of PrimeTime master mix with probe based assays from IDT. We also look at the use of PrimeTime master mix in multiplex applications without the loss of sensitivity that is commonly observed. Finally, we demonstrate the unmatched stability of PrimeTime master mix under ambient temperatures, saving your research money and minimizing on shipping delays.
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.
Addressing the Pre-PCR Analytical Variability of FFPE SamplesCandy Smellie
Despite technical advances, assessing the accuracy of pre-PCR steps, which include DNA extraction from formalin-fixed paraffin-embedded (FFPE) tissues, DNA quantitation and DNA quality control, remain a key challenge in external quality assurance.
In the webinar we will discuss the latest results from recent studies and look at ways that the accuracy of pre-PCR workflows can be improved.
New Progress in Pyrosequencing for DNA MethylationQIAGEN
Pyrosequencing is a highly flexible technology that lets you rapidly analyze short- to medium-length sequences fast and quantitatively with high accuracy. The real-time, high-resolution sequence output makes the technology highly suitable for applications including complex mutation analysis, microbial identification and DNA methylation quantification.
The main bottleneck in Pyrosequencing has been limited sequence length, which is critical for some applications. Our new technology, software, and chemistry overcome this bottleneck and give sequence reads that are typically twice as long as those from previous PyroMark systems. The new PyroMark Q24 Advanced system also reduces background noise, improving quantification even at sites distant from the sequencing start. The new system is ideal for applications requiring analysis of longer sequences, such as DNA methylation analysis in epigenetic research, frequency determination in mutation analysis, and various de novo sequencing applications.
In this presentation, we will discuss the following applications and technology improvements:
• DNA methylation analysis at single base resolution at CpG and CpN sites
• Improved quantification of sequence variations at any sequence position
• Easy and improved base calling functionality
To assess the effect of formalin on genomic DNA and assay performance for som...Candy Smellie
What is the impact of assay failure in your laboratory and how do you monitor for it?
Application of Companion Diagnostics - driving better treatment for cancer patients
PCR Array Data Analysis Tutorial: qPCR Technology Webinar Series Part 3QIAGEN
Using actual PCR Array data, this slidedeck presents an easy-to-use and free web-based data analysis tool to calculate fold-differences in gene expression from your raw real-time PCR threshold cycles. Learn how you can look at your results in different formats, including heat map, scatter, volcano, clustergram and multigroup plot.
RNA Integrity and Quality – Standardize RNA Quality Control QIAGEN
RNA integrity and quality are critical to obtain meaningful and reliable downstream data. This slidedeck details the challenges and considerations of handling RNA samples, the need for quality control analysis and common methods for RNA integrity and quality assessment. The QIAxcel Advanced System will be introduced to automate the process of RNA sample integrity analysis and obtain objective quality measurement. Application data will be presented.
Nucleic Acid Quantification from FFPE Samples – Are You Doing it Right?QIAGEN
Formalin-fixation and paraffin-embedding is a standard method for long-term preservation of tissue biopsies and these stored samples are a valuable tool for studying diseases such as cancer, especially when they are histologically and pathologically well characterized, and follow-up clinical data is available. The quality of nucleic acids extracted from FFPE samples is influenced by a number of factors, including how the samples were handled before, during and after fixation and embedding. Moreover, there are several difficulties when purifying nucleic acids from FFPE samples as the chemicals and temperature used during the process can degrade the DNA.
In this webinar, we will discuss the variability in quantity and purity of DNA purified from FFPE material. We will show data from different quantification and quality control methods, demonstrate the impact of inaccurate quantification on downstream results and discuss how to overcome these challenges.
5 Tips for Successful qRT-PCR Results InfographicQIAGEN
Market research shows that 66.6% of researchers use qRT-PCR for gene-expression profiling. Clearly, this is a very effective and popular technique for detecting RNA expression levels, but it’s still prone to pitfalls that can sometimes lead to disappointing results.
To help improve your experiments, we’ve made a new infographic with some interesting facts about qRT-PCR and 5 tips to help with your gene expression studies. This will cover everything from experimental design to data analysis.
RNase H2-dependent PCR (rhPCR) is a powerful method for increasing PCR specificity and eliminating primer-dimers by using blocked primers and a thermostable RNase H2 from Pyrococcus abyssi (P. abyssi). Primers will only support extension and replication after the blocked portion is cleaved. Cleavage by the RNase H2 enzyme occurs only when primers are bound to their complementary target sequence, thus providing increased specificity. Also, the thermostability of P. abyssi RNase H2 provides a “hot start” capability to the reaction. In this presentation, Dr Joseph Dobosy (senior research scientist in the molecular genetics research division of IDT) gives a detailed explanation of the rhPCR mechanism, offer tips on how to design assays using this powerful technology, and discuss examples of applications that benefit from rhPCR.
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.
RNA integrity and quality - Standardize RNA quality controlQIAGEN
RNA integrity and quality are critical to obtain meaningful and reliable downstream data. This slidedeck details the important considerations and critical factors in RNA preparation. It also highlights the need for quality control analysis and common methods for RNA integrity and quality assessment.
The importance of controls and novel solutions for successful real-time qPCRQIAGEN
The increasing demand for streamlined, monitored and ultrafast qPCR procedures requires high-performance, real-time quantitative RT and PCR chemistries. Particularly, procedures utilizing generic kits for gene expression analysis should include in-process safety measures to avoid variables and control accuracy of procedures and results. This slidedeck presents innovative solutions for one-step and two-step RT-PCR that significantly enhance performance and reliability in qRT-PCR. The new QuantiNova kit family offers a combination of various integrated safety features to remove variables and prevent artifacts. Internal control RNA, removal of genomic DNA, room temperature set-up capability for RT-PCR and a built-in visual pipetting control verify accurate procedures, ensuring reliable gene expression profiling.
This slidedeck explains the principles of the technologies and shows data demonstrating performance in qRT-PCR. Find out how you can verify accurate performance in qRT-PCR and improve your results!
Enabling RNA-Seq With Limited RNA Using Whole Transcriptome AmplificationQIAGEN
RNA-Seq was developed to perform transcriptome profiling and provides a highly precise measurement of expression levels of transcripts and their isoforms. Normally, RNA-Seq analysis requires at least 500 ng –1 μg of total RNA. When working with small biopsies, single cells (such as circulating tumor cells), or other limited material, whole transcriptome amplification (WTA) is normally required. Various WTA methods overcome limited RNA availability and enable transcriptome analysis from limited material or even single cells. In standard PCR-based WTA procedures, however, bias from uneven coverage of cDNA regions with high GC or AT content or amplification errors can lead to the loss of transcripts and wrong variant calling. Here, we compare a standard RNA-Seq library preparation method and the REPLI-g RNA library protocol. The REPLI-g procedure is a PCR-free protocol to efficiently generate RNA-Seq libraries from small amounts of RNA or a single cell in 6.5–7 hours. The REPLI-g protocol uses whole transcriptome amplification based on multiple displacement amplification (MDA), combined with an efficient library adaptor ligation procedure, to prepare RNA-Seq libraries from small RNA amounts. The procedure demonstrates high fidelity, minimal bias and retention of sample‘s transcriptional profile. Compared to standard RNA-Seq library prep, the REPLI-g protocol demonstrates similar reproducibility and sensitivity in transcript detection.
PCR - From Setup to Cleanup: A Beginner`s Guide with Useful Tips and Tricks -...QIAGEN
This End-Point PCR Beginner´s Guide will not only give you a comprehensive overview of tools and techniques to help you to get the most out of your samples, but also give you information on dedicated solutions and complete workflows on multiplex PCR and PCR fragment analysis.
Improving RNA yield and quality is easy with the right protocols. This trouble shooting guide presents methods to overcome common difficulties in RNA purification. Prevent RNA degradation, improve RNA yields, reduce genomic DNA contamination, and more.
Real-Time quantitative PCR (qPCR) is a mainstream method that is used in research and diagnostic applications for quantification of gene expression. IDT has developed a robust and affordable qPCR master mix for use with probe-based qPCR in single and multiplex assays. In this presentation, we explore a variety of applications of PrimeTime® Gene Expression Master Mix. We cover the use of PrimeTime master mix with probe based assays from IDT. We also look at the use of PrimeTime master mix in multiplex applications without the loss of sensitivity that is commonly observed. Finally, we demonstrate the unmatched stability of PrimeTime master mix under ambient temperatures, saving your research money and minimizing on shipping delays.
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.
Addressing the Pre-PCR Analytical Variability of FFPE SamplesCandy Smellie
Despite technical advances, assessing the accuracy of pre-PCR steps, which include DNA extraction from formalin-fixed paraffin-embedded (FFPE) tissues, DNA quantitation and DNA quality control, remain a key challenge in external quality assurance.
In the webinar we will discuss the latest results from recent studies and look at ways that the accuracy of pre-PCR workflows can be improved.
New Progress in Pyrosequencing for DNA MethylationQIAGEN
Pyrosequencing is a highly flexible technology that lets you rapidly analyze short- to medium-length sequences fast and quantitatively with high accuracy. The real-time, high-resolution sequence output makes the technology highly suitable for applications including complex mutation analysis, microbial identification and DNA methylation quantification.
The main bottleneck in Pyrosequencing has been limited sequence length, which is critical for some applications. Our new technology, software, and chemistry overcome this bottleneck and give sequence reads that are typically twice as long as those from previous PyroMark systems. The new PyroMark Q24 Advanced system also reduces background noise, improving quantification even at sites distant from the sequencing start. The new system is ideal for applications requiring analysis of longer sequences, such as DNA methylation analysis in epigenetic research, frequency determination in mutation analysis, and various de novo sequencing applications.
In this presentation, we will discuss the following applications and technology improvements:
• DNA methylation analysis at single base resolution at CpG and CpN sites
• Improved quantification of sequence variations at any sequence position
• Easy and improved base calling functionality
To assess the effect of formalin on genomic DNA and assay performance for som...Candy Smellie
What is the impact of assay failure in your laboratory and how do you monitor for it?
Application of Companion Diagnostics - driving better treatment for cancer patients
PCR Array Data Analysis Tutorial: qPCR Technology Webinar Series Part 3QIAGEN
Using actual PCR Array data, this slidedeck presents an easy-to-use and free web-based data analysis tool to calculate fold-differences in gene expression from your raw real-time PCR threshold cycles. Learn how you can look at your results in different formats, including heat map, scatter, volcano, clustergram and multigroup plot.
RNA Integrity and Quality – Standardize RNA Quality Control QIAGEN
RNA integrity and quality are critical to obtain meaningful and reliable downstream data. This slidedeck details the challenges and considerations of handling RNA samples, the need for quality control analysis and common methods for RNA integrity and quality assessment. The QIAxcel Advanced System will be introduced to automate the process of RNA sample integrity analysis and obtain objective quality measurement. Application data will be presented.
Comparison of Different NGS Library Construction Methods for Single-Cell Sequ...QIAGEN
Recent advances in whole genome amplification (WGA), whole transcriptome amplification (WTA) technologies and next-generation sequencing (NGS) have enabled whole genome or transcriptome sequencing at the single-cell level. Single-cell sequencing studies have yielded new insights into the heterogeneity of the genome and transcriptome in individual cells. Such heterogeneity at the single-cell level has been shown to be closely related to cellular function, differentiation, development, and diseases. A critical element of the single-cell sequencing workflow is sequencing library construction following WGA or WTA. An efficient library construction method is required to convert a high percentage of the DNA fragments to an adaptor-ligated sequencing library and to ensure high sequence complexity of the library. Furthermore, uniform representation of all genomic regions in a sequencing library is essential for retaining all important sequence information.
Here we compared 2 library construction methods following a REPLI-g MDA-mediated WGA or WTA:
• A ligation-based library construction method using a GeneRead Library Prep Kit (QIAGEN)
• A ‘tagmentation’-based method using Nextera DNA Sample Prep Kit (Illumina), which simultaneously fragments and tags DNA.
Our results demonstrated that the Nextera library construction method can be directly used with the REPLI-g-amplified DNA following MDA reaction, without the need for DNA purification. This could be beneficial if working with a high number of samples or if the complete workflow of single WGA/WTA and library construction should be automated. However, compared with the tagmentation method, the ligation-based library construction method is more flexible with regard to the input DNA amount and delivers sequencing libraries with higher complexity and less bias. This is critical for sensitive applications, such as identification of genomic variants or comprehensive profiling of transcriptomes.
Advanced miRNA Expression Analysis: miRNA and its Role in Human Disease Webin...QIAGEN
miRNAs are small functional RNAs, which regulate gene expression post-transcriptionally. The miScript miRNA PCR Array System is a sensitive and reliable technology for detection of mature miRNAs in any laboratory. In this slideshow, the challenges of miRNA data analysis and solutions that the miScript miRNA PCR Arrays provide for researchers interested in identifying miRNA from cells, tissues and FFPE samples are described. You will also learn how to use our GeneGlobe Data Analysis Center to identify miRNAs that may be important in your favorite biological pathway or disease.
Molecular Mechanisms of Neurodegeneration: Neurodegenerative Disorders Webin...QIAGEN
Common molecular mechanisms and pathways leading to neurodegeneration, such as Alzheimer’s Disease, Parkinson’s Disease, Huntington’s Disease or Multiple Sclerosis, are presented in this slideshow. Learn more about research and therapeutic strategies as well as how these discoveries and tools can be used to facilitate your neurodegeneration research.
Real-time quantitative PCR (qPCR) is a preferred platform for high throughput gene expression profiling, where large numbers of samples are characterized for hundreds of expression markers. Technically, the qPCR measurements are performed in the same way as when classical qPCR is used to analyze only a few targets per sample, but the higher throughput introduces additional sources of potential confounding variation that must be controlled for. In this presentation, Dr Kubista describes how high throughput qPCR profiling studies are designed. He covers assay optimization and validation, sample quality testing, and how to merge multi-plate measurements into a common analysis. Dr Kubista also discusses how to cost-effectively measure and compensate for background due to genomic DNA.
Setting up a qPCR experiment is so simple that it actually becomes dangerous. Without appropriate controls and data normalization, results can be misleading at best. This presentation addresses selection and validation of suitable reference genes as well as the use of the global mean normalization method to obtain accurate data. Also discussed are tools for data generation and analysis.
Multicopy reference assay (MRef) — a superior normalizer of sample input in D...QIAGEN
Copy number variations (CNVs) and alterations (CNAs) are a source of genetic diversity in humans and are often pathogenic. Numerous CNVs and CNAs are being identified with various genome analysis platforms, including array comparative genomic hybridization (aCGH), single nucleotide polymorphism (SNP) genotyping platforms, and next-generation sequencing. Independent verification of copy number changes is a critical step. Quantitative real-time PCR (qPCR) is a classic method to verify microarray copy number findings. Traditional copy number assays that use qPCR typically rely on a putative single-copy gene reference assay (e.g., RNase P or TERT) to normalize the DNA input for downstream ΔΔCT-based copy number calculation for comparison to a reference genome. When applied to cancer samples, these single-copy reference assays may no longer be a reliable indicator of DNA input due to the presence of complex chromosome composition (both in chromosome number and structure). To meet the need for an accurate DNA input normalizer, especially for heterogeneous tumor samples, QIAGEN developed a multicopy reference (MRef) assay for real-time PCR copy number analysis. This assay, in conjunction with QIAGEN’s greater than 10 million genomewide copy number assays and pathway- and disease-focused copy number PCR arrays (Figure 1), provides a successful solution for copy number analysis. This article will address the assay design considerations, development, and performance of this multicopy reference (MRef) assay.
Toll-like Receptors in Inflammation: Host Defense Webinar Series Part 2QIAGEN
Toll-like receptors (TLRs) have been implicated in both innate and adaptive immunity-induced inflammation, thereby playing critical roles in providing the host with short- and long-term protection against infections. This slidedeck provides an overview of the roles that TLRs play in the regulation of inflammation and solutions for studying these roles. An overview of TLR-mediated inflammation, the key signaling players involved in TLR-mediated inflammation, and the contribution of TLR-mediated inflammation to various physiological processes are also presented.
Functional Analysis of miRNA: miRNA and its Role in Human Disease Webinar Ser...QIAGEN
This slideshow highlights the use of miRNA mimics, inhibitors and target protectors to increase, decrease and adjust the cellular concentration of miRNA and disrupt specific miRNA–mRNA interactions. A ready-to-use screening tool for identifying miRNA targets and info on how to predict mRNA targets using miRNA expression data are also highlighted.
Guide to Molecular Cloning - Download the GuideQIAGEN
Molecular cloning can be sometimes tricky with significant challenges involved. Overcome the challenges with the essential knowledge and tips for successful cloning.
Introduction to Real Time PCR (Q-PCR/qPCR/qrt-PCR): qPCR Technology Webinar S...QIAGEN
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.
Host-pathogen Interactions, Molecular Basis and Host Defense: Pathogen Detect...QIAGEN
Host–pathogen interactions are strikingly complex during infection. This slidedeck provides an overview of the molecular basis of these intricate interactions: the impact of microbiota on innate and adaptive immunity, metabolism, and insulin resistance and host defense mechanisms. Various research tools will be introduced to simplify and streamline each step of studying the host response, enabling detection of pathogens, analysis of gene expression and regulation, epigenetic modification, genotyping and signal transduction pathway activation.
qPCR assays using intercalating dyes, such as SYBR® Green dye, are an economical and effective tool for measuring gene expression. To interpret intercalating dye assays, users need to know how to analyze melt curves, and understand the benefits and limitations of melt curve analysis. In this presentation, Nick Downey, PhD, covers melt curve basics and shares examples of multiple peaks due to suboptimal sample prep, primer dimers, and asymmetric GC content of amplicons. He demonstrates troubleshooting strategies. Experienced and novice users will benefit from an overview of uMeltSM software, developed by the Wittwer lab at the University of Utah, that can predict the melt profile of your assay before you run your experiment.
Sequential Automation of RNA and DNA preps on the same QIAcube instrumentQIAGEN
Automation of QIAGEN spin-column kits on the QIAcube saves valuable time and ensures standardized results. Since the same QIAcube may be used by multiple researchers for different applications, cross-contamination between samples and preparation technologies must be avoided (e.g., when nucleases are used). The unique instrument design and features minimize contamination between sequential preps, allowing both RNA and DNA preps to be performed on the same instrument. To show the process safety and robustness, we performed alternating automated RNA preps (requiring a DNase step) and DNA plasmid preps (requiring an RNase step). The preps were sequentially performed on the same QIAcube instrument using the RNeasy® Mini Kit and the QIAprep® Spin Miniprep Kit, respectively.
Independently, we performed a series of manually processed preps to compare with the automated preps. RNA and DNA quality and yields were similar between the two methods, showing the absence of carryover of nucleases.
High data quality and accuracy are recognized characteristics of Sanger re-sequencing projects and are primary reasons that next generation sequencing projects compliment their results by capillary electrophoresis data validation. We have developed an on-line tool called Primer Designer™ to streamline the NGS-to-Sanger sequencing workflow by taking the laborious task of PCR primer design out of the hands of the researcher by providing pre-designed assays for the human exome. The primer design tool has been created to enable scientists using next generation sequencing to quickly confirm variants discovered in their work by providing the means to quickly search, order and receive suitable pre-designed PCR primers for Sanger sequencing. Using the Primer Designer™ tool to design M13-tailed and non-tailed PCR primers for Sanger sequencing we will demonstrate validation of 28-variants across 24-amplicons and 19-genes using the BDD, BDTv1.1 and BDTv3.1 sequencing chemistries on the 3500xl Genetic Analyzer capillary electrophoresis platform.
Next generation sequencing & microarray-- Genotypic TechnologyGenotypic Technology
Greetings from Genotypic Technology, Bangalore (www.genotypic.co.in). We are a 13 year old genomics and bioinformatics company ( 65+ employees, Service. Products and R & D) based in Bangalore, India, primarily working on applications of Microarrays and Next Generation Sequencing in Human Health and Disease, including in Molecular Diagnostics, Prognosis, Therapeutics, Vaccine Research, Basic Science Research, Veterinary Science, Agriculture, Industrial Biotechnology, Microbial Genetics and more.
Our major strength is in customized genomics solutions, particularly in your field, we can develop panel of markers for specific diseases, optimize, validate and help commercialize on open platforms or specific instrument platforms- in microarrays and sequencing based methods/ assays. We can also use genomic markers to aid in treatment of specific disease using personalized medicine approaches. All this can be done on a comprehensive end-to-end manner in our company as we have a very good blend of molecular biology and bioinformatics with totally 6 Ph.Ds. We work closely with Agilent's R &D as their partner.
Identity testing by NGS as a means of risk mitigation for viral gene therapiesMerck Life Sciences
Watch the presentation of this webinar here: https://bit.ly/3RijkHC
Detailed description:
Imagine you’ve just completed a manufacturing run for your viral vector. Identity testing is performed to confirm the vector sequence. But when the results come back the data reveals unexpected sequence variants! With an appropriate risk mitigation testing strategy, this situation can be prevented.
The situation described above is not hypothetical, and happens more that you think, costing valuable time and resources.
Investigatory testing has shown that sequence variants present in starting materials (e.g. plasmids) are likely to make their way to the final product. Adequate identification of low-level variants with an appropriately sensitive method is critical in ensuring the quality of the final product. A risk-based testing strategy, in the context of identity, for viral vector manufacturing will be presented, focusing on key testing points. NGS assays for identity and variant detection will be highlighted due to their extremely sensitive nature compared to traditional approaches.
In this webinar, we'll explore:
• Regulatory requirements for identity testing
• NGS applications for identity testing as compared to traditional methods
• A case study on the impact of not establishing a proper risk-based testing strategy
Presented by: Bradley Hasson, Director of Lab Operations for NGS Services
Identity testing by NGS as a means of risk mitigation for viral gene therapiesMilliporeSigma
Watch the presentation of this webinar here: https://bit.ly/3RijkHC
Detailed description:
Imagine you’ve just completed a manufacturing run for your viral vector. Identity testing is performed to confirm the vector sequence. But when the results come back the data reveals unexpected sequence variants! With an appropriate risk mitigation testing strategy, this situation can be prevented.
The situation described above is not hypothetical, and happens more that you think, costing valuable time and resources.
Investigatory testing has shown that sequence variants present in starting materials (e.g. plasmids) are likely to make their way to the final product. Adequate identification of low-level variants with an appropriately sensitive method is critical in ensuring the quality of the final product. A risk-based testing strategy, in the context of identity, for viral vector manufacturing will be presented, focusing on key testing points. NGS assays for identity and variant detection will be highlighted due to their extremely sensitive nature compared to traditional approaches.
In this webinar, we'll explore:
• Regulatory requirements for identity testing
• NGS applications for identity testing as compared to traditional methods
• A case study on the impact of not establishing a proper risk-based testing strategy
Presented by: Bradley Hasson, Director of Lab Operations for NGS Services
1. November 2012
GeneRead DNAseq Gene Panel
Handbook
For targeted exon enrichment for nextgeneration sequencing
Sample & Assay Technologies
2. QIAGEN Sample and Assay Technologies
QIAGEN is the leading provider of innovative sample and assay technologies,
enabling the isolation and detection of contents of any biological sample. Our
advanced, high-quality products and services ensure success from sample to
result.
QIAGEN sets standards in:
Purification of DNA, RNA, and proteins
Nucleic acid and protein assays
microRNA research and RNAi
Automation of sample and assay technologies
Our mission is to enable you to achieve outstanding success and
breakthroughs. For more information, visit www.qiagen.com.
3. Contents
Kit Contents
4
Shipping and Storage
5
Product Use Limitations
5
Product Warranty and Satisfaction Guarantee
5
Technical Assistance
6
Safety Information
6
Quality Control
7
Introduction
8
Principle and procedure
8
Equipment and Reagents to Be Supplied by User
11
Important Notes
13
DNA preparation and quality control
13
DNA quantification and quality control
14
Protocols
PCR Setup
15
Sample Pooling and Purification
17
Troubleshooting Guide
19
References
19
Appendix A: Library Construction using the NEBNext Fast DNA Library
Prep Set for Ion Torrent
20
Appendix B: NEBNext DNA Library Prep Master Mix Set for Illumina with
NEBNext Singleplex Oligos for Illumina (E7350) or NEBNext Multiplex
Oligos for Illumina (Index Primers 1–12) (E7335)
25
Appendix C: Library Quantification and Quality Control
29
Appendix D: Data Analysis using QIAGEN Web Portal
29
Ordering Information
30
GeneRead DNAseq Gene Panel Handbook 11/2012
3
4. Kit Contents
GeneRead DNAseq Gene Panel Primer Mixes
180941*
Tubes with enough primers for 12 or 96 samples, depending
on pack size
4
Handbook
1
* Gene panel tubes are labeled A1, B1, C1, and D1.
GeneRead DNAseq Gene Panel High-Content Primer
Mixes
180942†
Tubes with enough primers for 12 or 96 samples, depending
on pack size
Handbook
†
8
1
Gene panel tubes are labeled A1, B1, C1, D1, A2, B2, C2, and D2.
GeneRead DNAseq Gene Panel Mix-n-Match Primer
Mixes
180944‡
Tubes with laboratory-verified primers for 24 samples
Handbook
‡
4
1
Selection limited to 124 genes.
GeneRead DNAseq Gene Panel Custom Primer Mixes
180946
Tubes with primers for any gene or genes in the human
genome for 500 samples
Handbook
4
4
1
GeneRead DNAseq Gene Panel Handbook 11/2012
5. GeneRead Panel Mastermix*
(0.6 ml)
(4.8 ml)
Catalog no.
180962
180964
GeneRead Panel Mastermix,
containing:
HotStart DNA Taq
Polymerase
Sufficient reagents Sufficient reagents for
384 PCR
for 48 PCR
amplification
amplification
reactions
reactions
PCR Buffer
dNTP mix (dATP, dCTP,
dGTP, dTTP)
DNase-free water
Shipping and Storage
GeneRead DNAseq Gene Panel Kits are shipped frozen or at ambient
temperature and should be stored at –20°C immediately upon arrival. When
stored properly at –20°C, all reagents are stable for up to 6 months after
delivery.
GeneRead Panel Mastermixes are shipped on cold packs. For long-term
storage, keep tubes at –20°C. If the entire volume will not be used at once, we
recommend dividing into aliquots and storing at –20°C. Avoid repeated
freezing and thawing. If stored under these conditions, GeneRead Panel
Mastermixes are stable for 6 months after receipt.
Product Use Limitations
GeneRead DNAseq Gene Panels and GeneRead Panel Mastermix are intended
for molecular biology applications. These products are not intended for the
diagnosis, prevention, or treatment of a disease.
All due care and attention should be exercised in the handling of the products.
We recommend all users of QIAGEN products to adhere to the NIH guidelines
that have been developed for recombinant DNA experiments, or to other
applicable guidelines.
Product Warranty and Satisfaction Guarantee
QIAGEN guarantees the performance of all products in the manner described
in our product literature. The purchaser must determine the suitability of the
product for its particular use. Should any product fail to perform satisfactorily
due to any reason other than misuse, QIAGEN will replace it free of charge or
refund the purchase price. We reserve the right to change, alter, or modify any
GeneRead DNAseq Gene Panel Handbook 11/2012
5
6. product to enhance its performance and design. If a QIAGEN product does not
meet your expectations, simply call your local Technical Service Department or
distributor. We will credit your account or exchange the product — as you wish.
Separate conditions apply to QIAGEN scientific instruments, service products,
and to products shipped on dry ice. Please inquire for more information.
A copy of QIAGEN terms and conditions can be obtained on request, and is
also provided on the back of our invoices. If you have questions about product
specifications or performance, please call QIAGEN Technical Services or your
local distributor (see back cover or visit www.qiagen.com).
Technical Assistance
At QIAGEN, we pride ourselves on the quality and availability of our technical
support. Our Technical Service Departments are staffed by experienced
scientists with extensive practical and theoretical expertise in sample and assay
technologies and the use of QIAGEN products. If you have any questions or
experience any difficulties regarding GeneRead DNAseq Gene Panels or
GeneRead Panel Mastermix, or QIAGEN products in general, please do not
hesitate to contact us.
QIAGEN customers are a major source of information regarding advanced or
specialized uses of our products. This information is helpful to other scientists as
well as to the researchers at QIAGEN. We therefore encourage you to contact
us if you have any suggestions about product performance or new applications
and techniques.
For technical assistance and more information, please see our Technical
Support Center at www.qiagen.com/Support or call one of the QIAGEN
Technical Service Departments or local distributors (see back cover or visit
www.qiagen.com).
Safety Information
When working with chemicals, always wear a suitable lab coat, disposable
gloves, and protective goggles. For more information, please consult the
appropriate safety data sheets (SDSs). These are available online in convenient
and compact PDF format at www.qiagen.com/safety where you can find, view,
and print the SDS for each QIAGEN kit and kit component.
24-hour emergency information
Emergency medical information in English, French, and German can be
obtained 24 hours a day from:
Poison Information Center Mainz, Germany
Tel: +49-6131-19240
6
GeneRead DNAseq Gene Panel Handbook 11/2012
7. Quality Control
In accordance with QIAGEN’s Quality Management System, each lot of
GeneRead DNAseq Gene Panels and GeneRead Panel Mastermix is tested
against predetermined specifications to ensure consistent product quality.
GeneRead DNAseq Gene Panel Handbook 11/2012
7
8. Introduction
DNA resequencing is a useful tool to detect genetic variations, including
somatic mutations, SNPs, and small insertions and deletions. Targeted
enrichment technology enables next-generation sequencing (NGS) platform
users to sequence specific regions of interest instead of the entire genome.
GeneRead DNAseq Gene Panels use multiplex PCR-based target enrichment
technology in combination with a sophisticated primer design and separation
algorithm to enable amplification and enrichment of any gene or targeted
region in the human genome in order to detect genetic variation using nextgeneration sequencing (Figure 1). GeneRead DNAseq Gene Panels are
designed to analyze a panel of genes related to a disease state and can be
used with any major next-generation sequencing platforms. The targeted
enrichment process is essential for the efficient utilization of medium-throughput
sequencers such as Life Technologies®’ Ion Torrent™ PGM Sequencer and
Illumina®’s MiSeq® Personal Sequencer.
GeneRead DNAseq Gene Panels have been optimized in combination with
GeneRead Panel Mastermix to provide superior sensitivity and linear multiplex
amplification. The simplicity of the PCR method makes these panels accessible
for routine use in every research laboratory.
Principle and procedure
GeneRead DNAseq Gene Panels are provided as sets of four tubes, each
containing primer mix, with up to 1400 primer pairs. The number of 4-tube sets
included is determined by the number of genes in a panel. GeneRead DNAseq
Gene Panels can enrich selected genes using as little as 80 ng genomic DNA in
two hours (Figure 2). Briefly, add genomic DNA to primer mix and PCR
mastermix and put them into a regular thermocycler for PCR amplification. After
the reaction is complete, pool the product for the same DNA sample and purify
the enriched DNA. The purified DNA then is ready for NGS library construction
and sequencing using the NGS platform of your choice. The sequencing results
can be analyzed on our server at http://ngsdataanalysis.sabiosciences.com,
and genetic variations can be detected (Figure 3).
8
GeneRead DNAseq Gene Panel Handbook 11/2012
9. Figure 1. Multiplex PCR-based target enrichment scheme. GeneRead DNAseq Gene
Panels use multiplex PCR-based target enrichment technology in combination with a
sophisticated primer design and separation algorithm to maximize design coverage and
minimize nonspecific amplification.
Figure 2. GeneRead DNAseq Gene Panel procedure.
GeneRead DNAseq Gene Panel Handbook 11/2012
9
10. Figure 3. Overview of the NGS workflow with GeneRead DNAseq Gene Panels. The
procedure involves DNA extraction (QIAGEN QIAamp DNA Mini Kit or QIAmp DNA FFPE
Tissue Kit is recommended), followed by target enrichment with GeneRead DNAseq Gene
Panels, NGS library construction, sequencing, and data analysis using the QIAGEN NGS Data
Analysis Web Portal.
10
GeneRead DNAseq Gene Panel Handbook 11/2012
11. Equipment and Reagents to Be Supplied by User
When working with chemicals, always wear a suitable lab coat, disposable
gloves, and protective goggles. For more information, consult the appropriate
material safety data sheets (MSDSs), available from the product supplier.
In addition to the GeneRead DNAseq Gene Panel and GeneRead Panel
Mastermix, the following supplies are required:
For genomic DNA isolation:
See page 13 for specific recommendations.
For target enrichment:
High-quality, nuclease-free water. Do not use DEPC-treated water.
Agencourt® AMPure® XP Kit
70% ethanol
Low TE
Magnetic rack for 1.5 ml tubes
1.5 ml LoBind tubes
0.2 ml PCR tubes, 96-well reaction plates, or PCR strips and caps
Thermal cycler
Multichannel pipettor
Single-channel pipettor
DNase-free pipet tips and tubes
For NGS library construction for Ion Torrent™ PGM (optional):
NEBNext Fast DNA Library Prep Set for Ion Torrent
Agencourt AMPure XP Kit
80% ethanol
QIAGEN’s GeneRead DNAseq Library Quant Array for Ion Torrent PGM
Thermal cycler
A real-time PCR machine compatible with 96-well plates
For NGS library construction for Illumina MiSeq/HiSeq (optional):
NEBNext® DNA Library Prep Master Mix Set for Illumina
NEBNext Multiplex Oligos for Illumina (index primers 1–12, for multiplex
sequencing)
GeneRead DNAseq Gene Panel Handbook 11/2012
11
12.
NEBNext Singleplex Oligos for Illumina (for singleplex sequencing)
Agencourt AMPure XP Kit
80% ethanol
QIAGEN’s GeneRead DNAseq Library Quant Array for Illumina
Thermal cycler
A real-time PCR machine compatible with 96-well plates
12
GeneRead DNAseq Gene Panel Handbook 11/2012
13. Important Notes
DNA preparation and quality control
High-quality DNA is essential for obtaining good sequencing results
The most important prerequisite for any DNA sequence analysis experiment is
consistent, high-quality DNA from every experimental sample. Therefore,
sample handling and DNA isolation procedures are critical to the success of the
experiment. Residual traces of proteins, salts, or other contaminants will either
degrade the DNA or decrease the efficiency of, if not block completely, the
enzyme activities necessary for optimal whole genome amplification and realtime PCR performance.
Recommended genomic DNA preparation method
The QIAGEN QIAamp DNA Mini Kit (cat. no. 51304) and QIAamp DNA FFPE
Tissue Kit (cat. no. 56404) are highly recommended for the preparation of
genomic DNA samples from fresh tissues and FFPE tissue samples. Ensure that
samples have been treated for the removal of RNA, as RNA contamination will
cause inaccuracies in DNA concentration measurements. Do not omit the
recommended RNase treatment step to remove RNA. If genomic DNA samples
need to be harvested from biological samples for which kits are not available,
please contact Technical Support representatives for suggestions.
For best results, all DNA samples should be resuspended in DNase-free water
or alternatively in DNase-free 10 mM Tris buffer pH 8.0. Do not use DEPCtreated water.
Recommended library quantification method
The QIAGEN GeneRead DNAseq Library Quant Array (cat. no. 180601) is
highly recommended for the quantification of the prepared library. Each
GeneRead DNAseq Gene Panel contains a set of spike-in controls, and the
GeneRead DNAseq Library Quant Array provides predispensed primer assays
to measure those controls, for determination of the quality of the prepared
sample library. Additionally, the GeneRead DNAseq Library Quant Array
contains five predispensed, sequential 10-fold dilutions of Illumina or Ion
Torrent DNA Standard mixed with a PCR primer assay in triplicate, and PCR
primer assays in the remaining wells of a 96-well, 384-well, or 100-well PCR
plate. The predispensed, serially diluted DNA standards and PCR primer assay
are a convenient method for quantification of library input. In total, the
GeneRead DNAseq Library Quant Array determines quantity as well as quality
of the prepared library.
GeneRead DNAseq Gene Panel Handbook 11/2012
13
14. DNA quantification and quality control
For best results, all DNA samples should also demonstrate consistent quality
according to the following criteria:
Concentration and purity determined by UV spectrophotometry
The concentration and purity of DNA should be determined by measuring the
absorbance in a spectrophotometer. Prepare dilutions and measure absorbance
in 10 mM Tris·Cl,* pH 8.0. The spectral properties of nucleic acids are highly
dependent on pH.
A260:A230 ratio should be greater than 1.7
A260:A280 ratio should be greater than 1.8
Concentration determined by A260 should be >2.5 µg/ml DNA
DNA integrity
For best results, the genomic DNA should be greater than 2 kb in length with
some fragments greater than 10 kb. This can be checked by running a fraction
of each DNA sample on a 1% agarose gel.
* When working with chemicals, always wear a suitable lab coat, disposable gloves, and
protective goggles. For more information, please consult the appropriate material safety data
sheets (MSDSs), available from the product supplier.
14
GeneRead DNAseq Gene Panel Handbook 11/2012
15. Protocol: PCR Setup
Procedure
1. Dilute DNA sample to 4 ng/µl. For each sample, 80 ng (20 µl) DNA
is required for the 4-pool panel, or 160 ng (40 µl) for the 8-pool
panel.
2. Determine the number of reactions needed. For a 4-pool panel, 4
reactions for each sample are required. For an 8-pool panel, 8
reactions for each sample are required. Prepare PCR strips or PCR
plate according to the number of reactions. Label with sample names
and pool numbers.
3. Aliquot 5 µl each DNA sample into each well.
4. Prepare the PCR reaction mix on ice according to Table 1. For each
sample, 4 or 8 PCR reaction mixes will be needed. Mix gently by
pipetting up and down.
Table 1. Preparation of PCR reaction mix for each primer mix pool
Component
Per 1 sample
Per n samples
GeneRead Panel
Mastermix
11 µl
11 x n µl
Primer mix pool x*
5.5 µl
5.5 x n µl
16.5 µl
16.5 x n µl
Total volume
* The number of primer mix pools is determined by the panel size.
5. Aliquot 15 µl of each PCR reaction mix, and put it into the well with
DNA samples accordingly. Mix gently by pipetting up and down.
6. Seal the wells with PCR tube caps. Place strips or plate in
thermocycler and set up reaction parameters according to Table 2.
GeneRead DNAseq Gene Panel Handbook 11/2012
15
16. Table 2. PCR program
Cycle
Temperature
Time
1
95°C
10 min
20
95°C
15 s
60°C
2 min
1
72°C
10 min
1
4°C
∞
7. After the reaction is complete, place the reactions on ice and proceed
with sample pooling and purification.
Note: If the samples are to be stored prior to purification, transfer them to
a –20°C freezer.
16
GeneRead DNAseq Gene Panel Handbook 11/2012
17. Protocol: Sample Pooling and Purification
Procedure
1. Combine all 4 or 8 reactions from the same sample into one well.
Mix thoroughly. The volume of each sample should be approximately
80 µl for a 4-pool panel or 160 µl for an 8-pool panel.
2. Transfer 25 µl from each sample to a 1.5 ml Lobind tube for
purification. Store the rest at –20°C.
3. For each sample, add 45 µl (1.8x volume) of AMPure XP Reagent to
the sample and mix by pipetting up and down.
4. Incubate for 10 minutes at room temperature.
5. Pulse-spin the tube and place in a magnetic rack for 5 minutes or
until the beads have collected to the wall of the tube and the solution
is clear.
6. Carefully remove and discard the supernatant without disturbing the
beads.
7. Keep the tube on the magnet and add 500 µl freshly prepared 70%
ethanol.
8. While keeping the tube in the magnetic rack, rotate the tube 180
degrees. When the beads have collected to the opposite side of the
tube (near the magnet), turn the tube another 180 degrees. Rotate
the tube two more times, each time waiting until the beads collect to
the opposite side of the tube.
9. Allow the solution to become clear, and carefully remove and
discard the supernatant.
10. Repeat steps 7–9.
11. Pulse-spin the tube, return it to the magnet, and remove any residual
ethanol with a pipet.
12. Keeping the tube in the magnetic rack, with the cap open, air-dry the
beads for 5 minutes at room temperature.
13. Resuspend the beads in 25 µl low TE. Mix well by vortexing.
14. Pulse-spin the tube, return to the magnet, and collect the
supernatant into a new Lobind tube.
15. Proceed to library construction according to the sequencing platform
of your choice. Refer to Appendix A for recommended library
construction protocol for sequencing with Ion Torrent PGM. Refer to
GeneRead DNAseq Gene Panel Handbook 11/2012
17
18. Appendix B for recommended library construction protocol for
sequencing with Illumina MiSeq/HiSeq.
Note: If reactions are to be stored prior to library construction, transfer
them to a –20°C freezer.
18
GeneRead DNAseq Gene Panel Handbook 11/2012
19. Troubleshooting Guide
For technical support, please call us at 1-888-503-3187 or 1-301-682-9200.
For more information, see also the Frequently Asked Questions page at our
Technical Support Center:
www.SABiosciences.com/support_faq.php?target=PCR. The scientists in
QIAGEN Technical Services are always happy to answer any questions you may
have about either the information and protocols in this handbook or sample
and assay technologies (for contact information, see back cover or visit
www.qiagen.com).
References
QIAGEN maintains a large, up-to-date online database of scientific
publications utilizing QIAGEN products. Comprehensive search options allow
you to find the articles you need, either by a simple keyword search or by
specifying the application, research area, title, etc.
For a complete list of references, visit the reference database online at
www.SABiosciences.com/support_publication.php#pcrarray or contact QIAGEN
Technical Services or your local distributor.
GeneRead DNAseq Gene Panel Handbook 11/2012
19
20. Appendix A: Library Construction using the NEBNext
Fast DNA Library Prep Set for Ion Torrent
Procedure
End repair of DNA
A1.
Add the components in Table 3 to a 0.2 ml PCR tube on ice.
Table 3. DNA end-repair reaction components
Component
Volume
PCR-enriched DNA from previous step
25 µl
NEBNext End Repair Reaction Buffer
6 µl
NEBNext Repair Enzyme Mix
3 µl
DNase-free water
26 µl
Total
60 µl
A2.
Mix the components by pipetting up and down several times.
A3.
Incubate in a thermal cycler for 20 minutes at 25°C, followed by 10
minutes at 70°C.
Pulse-spin the microfuge tube and return to ice.
A4.
Preparation of adaptor-ligated DNA
A5.
20
Add the reagents in Table 4 to the PCR tube.
GeneRead DNAseq Gene Panel Handbook 11/2012
21. Table 4. Reagents for preparation of adaptor-ligated DNA
Component
Volume
DNase-free water
14 µl
T4 DNA Ligase Buffer (10x)
10 µl
NEBNext DNA Library Adaptors
for Ion Torrent
10 µl
T4 DNA Ligase
6 µl
Total
40 µl
A6.
The total volume in the microfuge tube should be 100 µl. Mix the
contents by pipetting up and down several times.
A7.
Incubate in a thermal cycler for 15 minutes at 16°C.
A8.
Transfer the entire volume to a 1.5 ml Lobind tube.
Cleanup of adaptor-ligated DNA
A1.
Add 160 µl (1.6x volume) AMPure XP Reagent to the sample and
mix by pipetting up and down.
A2.
Incubate for 5 minutes at room temperature.
A3.
Pulse-spin the tube and place in a magnetic rack for 5 minutes or
until the beads have collected to the wall of the tube and the
solution is clear.
A4.
Carefully remove and discard the supernatant without disturbing
the beads.
A5.
Keep the tube on the magnet and add 400 µl freshly prepared
80% ethanol.
A6.
While keeping the tube in the magnetic rack, rotate the tube 180
degrees. When the beads have collected to the opposite side of the
tube (near the magnet), turn the tube another 180 degrees. Rotate
the tube two more times, each time waiting until the beads collect
to the opposite side of the tube.
A7.
Allow the solution to become clear, and carefully remove and
discard the supernatant.
A8.
Repeat previous three steps (A5–A7).
A9.
Pulse-spin the tube, return to the magnet, and remove any
residual ethanol with a pipet.
GeneRead DNAseq Gene Panel Handbook 11/2012
21
22. A10. Keeping the tube in the magnetic rack, with the cap open, air-dry
the beads for 5 minutes at room temperature.
A11. Resuspend the beads in 150 µl sterile water. Mix well by vortexing.
A12. Pulse-spin the tube, return to the magnet, and collect the
supernatant.
Size selection
A1.
Add 105 μl (0.7x) AMPure XP beads to 150 μl DNA solution. Mix
well on a vortex mixer or by pipetting up and down at least 10
times.
A2.
Incubate for 5 minutes at room temperature.
A3.
Pulse-spin the tube and place tube on magnetic rack to separate
beads from supernatant. After the solution is clear (about 5
minutes), carefully transfer the supernatant to a new tube. Discard
the beads which contain the large fragments.
Note: Do not the discard the supernatant!
A4.
Add 120 μl (0.8x) AMPure XP beads to the supernatant, mix well
and incubate for 5 minutes at room temperature.
A5.
Pulse-spin the tube and place tube on magnetic rack and wait until
solution is clear (about 5 minutes). Carefully remove and discard
supernatant. Be careful not to disturb the beads, which contain the
DNA target.
Note: Do not discard beads.
A6.
Add 400 μl fresh 80% ethanol to the tube while on magnetic rack.
Incubate at room temperature for 30 seconds, and then carefully
remove and discard the supernatant.
A7.
Repeat step A6 once.
A8.
Briefly spin the tube, and place on magnetic rack. Completely
remove residual ethanol and dry beads for 10 minutes while tube
is on rack with lid open.
A9.
Elute DNA target beads into 25 μl 0.1x TE buffer. Mix well by
vortexing. Spin down briefly and place tube on rack until solution
is clear.
A10. Transfer the supernatant to a clean PCR tube and proceed to PCR
amplification.
22
GeneRead DNAseq Gene Panel Handbook 11/2012
23. PCR amplification of adaptor-ligated DNA
A1.
Mix the components in Table 5 in a 0.2 ml PCR tube.
Table 5. Reaction components for PCR amplification
Component
Volume
Adaptor-ligated DNA
25 µl
Primers
4 µl
DNase-free water
21 µl
OneTaq® Hot Start 2x Master Mix
50 µl
Total
A2.
100 µl
Set up the cycler using the cycling conditions in Table 6.
Table 6. Cycling conditions for amplification of adaptor-ligated DNA
Step
Temperature
Time
Nick translation
68°C
20 min
Initial denaturation
94°C
30 sec
5 cycles
94°C
30 sec
58°C
30 sec
68°C
1 min
4°C
∞
Hold
A3.
Transfer the entire volume to a 1.5 ml Lobind tube.
Cleanup of adaptor-ligated DNA
A1.
Add 140 μl (1.4X volume) of AMPure XP Reagent to the sample
and mix by pipetting up and down.
A2.
Incubate for 5 minutes at room temperature.
A3.
Pulse-spin the tube and place in a magnetic rack for 5 minutes or
until the beads have collected to the wall of the tube and the
solution is clear.
GeneRead DNAseq Gene Panel Handbook 11/2012
23
24. A4.
Carefully remove and discard the supernatant without disturbing
the beads.
A5.
Keep the tube on the magnet and add 400 μl freshly prepared
80% ethanol.
A6.
While keeping the tube in the magnetic rack, rotate the tube 180
degrees. When the beads have collected to the opposite side of the
tube (near the magnet), turn the tube another 180 degrees. Rotate
the tube two more times, each time waiting until the beads collect
to the opposite side of the tube.
A7.
Allow the solution to become clear, and carefully remove and
discard the supernatant.
A8.
Repeat previous three steps (A5–A7).
A9.
Pulse-spin the tube, return to the magnet, and remove any
residual ethanol with a pipet.
A10. Keeping the tube in the magnetic rack, with the cap open, air-dry
the beads for 5 minutes at room temperature.
A11. Resuspend the beads in 22 μl 0.1x TE buffer. Mix well by vortexing.
A12. Pulse-spin the tube, return to the magnet, and collect the
supernatant.
Library quantification using GeneRead DNAseq Library Quant Array
The library can be stored in a –20°C freezer prior to quantification.
24
GeneRead DNAseq Gene Panel Handbook 11/2012
25. Appendix B: NEBNext DNA Library Prep Master Mix
Set for Illumina with NEBNext Singleplex Oligos for
Illumina (E7350) or NEBNext Multiplex Oligos for
Illumina (Index Primers 1–12) (E7335)
Procedure
Adaptor ligation of PCR product
B1. Add the components from Table 7 to a 0.2 ml PCR tube.
Table 7. Reagents for adaptor ligation of PCR product
Component
Volume
Purified PCR product
25 µl
Quick Ligation Reaction Buffer (5X)
10 µl
NEBNext Adaptor
2 µl
Quick T4 DNA Ligase
2 µl
DNase-free water
11 µl
Total
50 µl
B2.
Incubate in a thermal cycler for 15 minutes at 20°C.
B3.
Add 3 µl USER™ enzyme mix by pipetting up and down and
incubate at 37°C for 15 minutes.
B4.
Transfer the entire volume to a 1.5 ml Lobind tube.
Cleanup using AMPure XP Beads
B1.
Add 90 μl (1.8X volume) of AMPure XP Reagent to the sample and
mix by pipetting up and down.
B2.
Incubate for 5 minutes at room temperature.
B3.
Pulse-spin the tube and place in a magnetic rack for 5 minutes or
until the beads have collected to the wall of the tube and the
solution is clear.
B4.
Carefully remove and discard the supernatant without disturbing
the beads.
GeneRead DNAseq Gene Panel Handbook 11/2012
25
26. B5.
Keep the tube on the magnet and add 400 μl freshly prepared
80% ethanol.
B6.
While keeping the tube in the magnetic rack, rotate the tube 180
degrees. When the beads have collected to the opposite side of the
tube (near the magnet), turn the tube another 180 degrees. Rotate
the tube two more times, each time waiting until the beads collect
to the opposite side of the tube.
B7.
Allow the solution to become clear, and carefully remove and
discard the supernatant.
B8.
Repeat previous three steps (B5–B7).
B9.
Pulse-spin the tube, return to the magnet, and remove any
residual ethanol with a pipet.
B10. Keeping the tube in the magnetic rack, with the cap open, air dry
the beads for 5 minutes at room temperature.
B11. Resuspend the beads in 150 μl sterile water. Mix well by vortexing.
B12. Pulse-spin the tube, return to the magnet, and collect the
supernatant.
Size selection
B1.
Add 120 µl (0.8x) AMPure XP beads to 150 μl DNA solution. Mix
well on a vortex mixer or by pipetting up and down at least 10
times.
B2.
Incubate for 5 minutes at room temperature.
B3.
Pulse-spin the tube and place tube on magnetic rack to separate
beads from supernatant. After the solution is clear (about 5
minutes), carefully transfer the supernatant to a new tube. Discard
the beads which contain the large fragments.
Note: Do not the discard the supernatant.
B4.
Add 90 µl (0.6x) AMPure XP beads to the supernatant, mix well
and incubate for 5 minutes at room temperature.
B5.
Pulse-spin the tube and place tube on magnetic rack and wait until
solution is clear (about 5 minutes). Carefully remove and discard
supernatant. Be careful not to disturb the beads which contain the
DNA target.
Note: Do not discard the beads.
B6.
26
Add 400 µl fresh 80% ethanol to the tube while on magnetic rack.
Incubate at room temperature for 30 seconds, and then carefully
remove and discard the supernatant.
GeneRead DNAseq Gene Panel Handbook 11/2012
27. B7.
Repeat step B6 once.
B8.
Briefly spin the tube, and place on magnetic rack. Completely
remove residual ethanol and dry beads for 10 minutes while tube
is on rack with lid open.
B9.
Elute DNA target beads into 23 µl 0.1x TE buffer. Mix well by
vortexing. Spin down briefly and place tube on rack until solution
is clear.
B10. Transfer the supernatant to a clean PCR tube and proceed to PCR
amplification.
PCR amplification of adaptor-ligated DNA
B1.
Add the reagents in Table 8 to a 0.2 ml PCR tube.
Table 8. Reagents for PCR amplification of adaptor-ligated DNA
Component
Volume
Adaptor-ligated DNA
23 µl
Universal PCR primer (25 µM)
1 µl
Index primer (1)* (25 µM)
1 µl
Phusion® High-Fidelity PCR
Master Mix with HF Buffer, 2X
25 µl
Total
50 µl
* If NEBNext Multiplex Oligos for Illumina (Index Primers 1-12) are used, for
each reaction, only one of the 12 PCR primer indices is used during the PCR
step.
B2.
Set up the cycler using the cycling conditions in Table 9.
GeneRead DNAseq Gene Panel Handbook 11/2012
27
28. Table 9. Cycling conditions for PCR amplification of adaptor-ligated DNA
Step
Temperature
Time
Initial denaturation
98°C
30 sec
12 cycles
98°C
10 sec
65°C
30 sec
72°C
30 sec
Index primer (1)* (25 µM)
98°C
5 min
Phusion High-Fidelity PCR Master
Mix with HF Buffer, 2X
98°C
∞
B3.
Transfer the entire volume to a 1.5 ml Lobind tube.
Cleanup using AMPure XP Beads
B1.
Add 60 µl (1.2x volume) AMPure XP Reagent to the sample and
mix by pipetting up and down.
B2.
Incubate for 5 minutes at room temperature.
B3.
Pulse-spin the tube and place in a magnetic rack for 5 minutes or
until the beads have collected to the wall of the tube and the
solution is clear.
B4.
Carefully remove and discard the supernatant without disturbing
the beads.
B5.
Keep the tube on the magnet and add 400 µl freshly prepared
80% ethanol.
B6.
While keeping the tube in the magnetic rack, rotate the tube 180
degrees. When the beads have collected to the opposite side of the
tube (near the magnet), turn the tube another 180 degrees. Rotate
the tube two more times, each time waiting until the beads collect
to the opposite side of the tube.
B7.
Allow the solution to become clear, and carefully remove and
discard the supernatant.
B8.
Repeat previous three steps (B5–B7).
B9.
Pulse-spin the tube, return to the magnet, and remove any
residual ethanol with a pipet.
B10. Keeping the tube in the magnetic rack, with the cap open, air-dry
the beads for 5 minutes at room temperature.
28
GeneRead DNAseq Gene Panel Handbook 11/2012
29. B11. Resuspend the beads in 22 µl 0.1x TE Buffer. Mix well on a vortex
mixer or by pipetting up and down, and put the tube in the
magnetic stand until the solution is clear.
B12. Transfer supernatant to a clean 1.5 ml LoBind tube.
Library quantification using GeneRead DNAseq Library Quant Array
The library may be stored in a –20°C freezer prior to quantification.
Appendix C: Library Quantification and Quality
Control
Quality control for the target enrichment and library construction process can
be performed using QIAGEN’s GeneRead DNAseq Library Quant Array. With
this array, the correct dilution of the library can also be determined for
sequencing. Please refer to the corresponding user manual for library
quantification and QC.
Appendix D: Data Analysis using QIAGEN Web
Portal
After sequencing, results can be analyzed using QIAGEN’s Next-Generation
Sequencing Data Analysis Web Portal. Our data analysis server will perform
reads trimming (removing primer sequences), mapping to reference genome,
and variants identification. Please refer to the corresponding document for data
analysis.
GeneRead DNAseq Gene Panel Handbook 11/2012
29
30. Ordering Information
Product
Contents
Cat. no.
GeneRead DNAseq
Gene Panels
Sets of 4 tubes containing wet-bench
verified primer sets for targeted exon
enrichment of a pathway-focused panel
of genes
180941
GeneRead DNAseq
Gene Panels: HighContent
Sets of 8 tubes containing wet-bench
verified primer sets for exon enrichment
of a pathway-focused panel of genes
180942
GeneRead Custom
DNAseq Gene Panels
Tubes containing primer sets for
targeted exon enrichment of a
customized panel of genes
180946
GeneRead DNAseq
Mix’n’Match Gene
Panels
Tubes containing wet-bench verified
primer sets for targeted exon
enrichment of a custom panel of genes
180944
GeneRead Panel
Mastermix
Mastermix for use with the GeneRead
DNAseq Gene Panel System
Varies
Related products
GeneRead DNAseq
Library Quant Array
Reagents for NGS sample library
quantification following targeted exon
enrichment with the GeneRead DNAseq
Gene Panel System
180601
GeneRead Library
Quant Array
Reagents for NGS sample library
quantification
180611
GeneRead Library
Quant Kit
Reagents for NGS sample library
quantification
180612
GeneRead qPCR SYBR
Green Mastermix
Mastermix for use with the GeneRead
Library Quant Arrays and Kit
Varies
QIAamp DNA Mini Kit
(50)
For 50 DNA preps: 50 QIAamp Mini
Spin Columns, QIAGEN Proteinase K,
Collection Tubes (2 ml), reagents and
buffers
51304
30
GeneRead DNAseq Gene Panel Handbook 11/2012
31. Product
Contents
QIAamp DNA FFPE
Tissue Kit (50)
For 50 DNA preps: 50 QIAamp
MinElute Columns, Proteinase K,
Collection Tubes (2 ml), buffers
Cat. no.
56404
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.
GeneRead DNAseq Gene Panel Handbook 11/2012
31