ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) is a technique used in molecular biology to study chromatin accessibility. The key part of the ATAC-seq procedure is the action of the transposase Tn5 on the genomic DNA of the sample.
This presentation is explains about the genome sequencing, its traditional method and modern method. This basically focus on Next Generation Sequencing and its types.
The next generation sequencing platform of roche 454creativebiogene1
454 is totally different from Solexa and Hiseq of Illumina. The disadvantage of 454 is that it is unable to accurately measure the homopolymer length. For this unavoidable reason, 454 technology will introduce insertion and deletion sequencing errors to the results.
Introduction to Next-Generation Sequencing (NGS) TechnologyQIAGEN
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
This presentation is explains about the genome sequencing, its traditional method and modern method. This basically focus on Next Generation Sequencing and its types.
The next generation sequencing platform of roche 454creativebiogene1
454 is totally different from Solexa and Hiseq of Illumina. The disadvantage of 454 is that it is unable to accurately measure the homopolymer length. For this unavoidable reason, 454 technology will introduce insertion and deletion sequencing errors to the results.
Introduction to Next-Generation Sequencing (NGS) TechnologyQIAGEN
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
This presentation gives an easy introduction to ChIP-seq analyses and is part of a bioinformatics workshop. The accompanying websites are available at http://sschmeier.github.io/bioinf-workshop/#!galaxy-chipseq/
Course: Bioinformatics for Biomedical Research (2014).
Session: 4.1- Introduction to RNA-seq and RNA-seq Data Analysis.
Statistics and Bioinformatisc Unit (UEB) & High Technology Unit (UAT) from Vall d'Hebron Research Institute (www.vhir.org), Barcelona.
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.
A workshop is intended for those who are interested in and are in the planning stages of conducting an RNA-Seq experiment. Topics to be discussed will include:
* Experimental Design of RNA-Seq experiment
* Sample preparation, best practices
* High throughput sequencing basics and choices
* Cost estimation
* Differential Gene Expression Analysis
* Data cleanup and quality assurance
* Mapping your data
* Assigning reads to genes and counting
* Analysis of differentially expressed genes
* Downstream analysis/visualizations and tables
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.
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.
INTRODUCTION TO REAL TIME PCR IS GIVEN, basic principle of realtime pcr, along with the process of operating this, diagrammatic representation of the process, advantages and disadvantages o f reatimem pcr, applications of the same is also there
This presentation gives an easy introduction to ChIP-seq analyses and is part of a bioinformatics workshop. The accompanying websites are available at http://sschmeier.github.io/bioinf-workshop/#!galaxy-chipseq/
Course: Bioinformatics for Biomedical Research (2014).
Session: 4.1- Introduction to RNA-seq and RNA-seq Data Analysis.
Statistics and Bioinformatisc Unit (UEB) & High Technology Unit (UAT) from Vall d'Hebron Research Institute (www.vhir.org), Barcelona.
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.
A workshop is intended for those who are interested in and are in the planning stages of conducting an RNA-Seq experiment. Topics to be discussed will include:
* Experimental Design of RNA-Seq experiment
* Sample preparation, best practices
* High throughput sequencing basics and choices
* Cost estimation
* Differential Gene Expression Analysis
* Data cleanup and quality assurance
* Mapping your data
* Assigning reads to genes and counting
* Analysis of differentially expressed genes
* Downstream analysis/visualizations and tables
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.
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.
INTRODUCTION TO REAL TIME PCR IS GIVEN, basic principle of realtime pcr, along with the process of operating this, diagrammatic representation of the process, advantages and disadvantages o f reatimem pcr, applications of the same is also there
Some sample sources present special challenges in RNA isolation or contain substances that cause problems in RNA analysis. These guides to RNA isolation have tips for a whole range of sample types, including guidance on the best kits for each.
Struggling with low editing efficiency or delivery problems? IDT has developed a simple and affordable CRISPR-Cas9 solution that outperforms other methods. In this presentation we present the advantages of using a Cas9:tracrRNA:crRNA ribonucleoprotein (RNP) complex in genome editing experiments, and explain why it is the most efficient driver for genome editing compared to alternative methods, such as expression plasmids or the use of sgRNAs. We also review RNP delivery using cationic lipids and electroporation, and provide tips for optimized transfection in your system.
Cloning and expression of the Nodamura virus RNA-dependent RNA polymerase
Poster presentation at Society for the Advancement of Chicanos and Native Americans in Science (SACNAS) National Conference, October 2012, Seatltle, WA
Technical Guide to Qiagen PCR Arrays - Download the GuideQIAGEN
Total RNA discovery with RT2 and miScript PCR Arrays : Explore the RNA universe - Whatever your destination within the RNA universe, QIAGEN will help you get there. The miRNeasy kits deliver pure, high-quality total RNA from a broad range of samples. The RT2 and miScript PCR arrays are a complete solution both for focused analysis of gene and microRNA expression and for validation of microarray and RNA sequencing experiments. Together with the powerful analytics tools of GeneGlobe® and QIAGEN Ingenuity® Pathway Analysis, these products give you a smooth path from your sample to high-quality results.
Circulating cell free DNA is a potential tumor marker in a non-invasive blood test for the treatment and evaluation of cancer and recurrence monitoring. As circulating tumor DNA is often present at low frequencies within circulating cell free DNA, targeted sequencing on the Ion Torrent™ platform is an optimal tool or mutation detection with very little sample input required. Here, we demonstrate a complete workflow from isolation through molecular characterization of circulating tumor DNA. We have optimized a protocol using magnetic beads to isolate circulating cell free DNA. This protocol is easily automated to process up to 192 samples a day. It is also easily scalable for any input volume and can elute in volumes down to 15 μL resulting in no loss of low frequency alleles. We demonstrate comparable performance between this bead based isolation and column based isolation. We have completed molecular characterization of circulating cell free DNA using the multiplexing capabilities of AmpliSeq™ and the Ion PGM™. With the Ion AmpliSeq™ Cancer Hotspot Panel v2, we performed targeted sequencing of 50 genes of interest, covering 2800 COSMIC mutations. We demonstrate good reproducibility of amplicon representation as well as allelic frequencies. Through saturation studies and subsampling, we have determined the limit of detection of hotspots circulating cell free DNA on the Ion PGM™ to be below 1%. We further demonstrate proof of principle of this workflow on circulating cell free DNA and matched FFPE samples. Our results verify the accuracy and ease of our workflow. This protocol, from isolation through targeted sequencing, will not only result in a simple sample preparation for circulating cell free DNA but also facilitate rapid mutation detection to advance cancer research.
A next generation sequencing based sample-to-result pharmacogenomics research...Thermo Fisher Scientific
Pharmacogenomics (PGx) is the study of genetic variations in terms of their response to drugs. Variations in gene sequence or copy numbers may result in complete loss of function, partial decrease or increase in enzyme activity, or an altered affinity for substrates, which may in turn significantly impact drug efficacy. PGx studies are becoming increasingly important for precision medicine. We have developed a next generation sequencing (NGS) PGx research solution with increased flexibility on the assay targets and combined detection of SNP/INDEL genotyping and CNV using Ion AmpliSeq™ technology for low to medium throughput laboratories. With this highly multiplexed PGx research panel we can profile a set of 136 genetic markers in 40 known PGx related genes (Table 1) and determine CYP2D6 copy number variation (CNV, Figure 1) in a single reaction using Ion Torrent™ semiconductor sequencing.
CAP Trapper Technologies and Applications, CAP Analysis of Gene Expression (C...Laura Berry
Presented at the NGS Tech and Applications Congress: USA. To find out more, visit:
www.global-engage.com
Masayoshi Itoh is a Senior Scientist at the RIKEN Center for Life Science Technologies (CLST) and Coordinator of the RIKEN Preventive Medicine and Diagnostics Innovation Program (PMI). In this presentation Masayoshi introduces CAP trapper technologies and presents the findings of the FANTOM5 project.
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.
Codons Optimization by Creative BiogeneDonglin Bao
Codon optimization is one of the key steps in achieving the high-level expression of target gene. There are some key factors for consideration including transcription efficiency, translation efficiency, gene synthesis and protein folding. Creative Biogene can optimize almost perfectly these key factors in codon optimization.
Principle and method of RNAi-Creative BiogeneDonglin Bao
In recent years, studies have shown that mRNA will occur specific degradation if dsRNA which is composed of sense RNA and antisense RNA from mRNA is transferred into the cell, eventually it leads to target genes silence. The post-transcriptional gene silencing (PTGS) is described as RNAi.
Codon optimization is one of the key steps in achieving the high level expression of target gene. There are some key factors for consideration including transcription efficiency, translation efficiency, gene synthesis and protein folding.
RT-PCR (reverse transcription-polymerase chain reaction) is a variant of the polymerase chain reaction (PCR) which are now widely used. Traditionally RT-PCR involves two steps: the RT reaction and PCR amplification. RNA is first reverse transcribed into cDNA using a reverse transcriptase as described here, the resulting cDNA is used as templates for subsequent PCR amplification using primers specific for one or more genes. RT-PCR can be used to quantify mRNA levels from much smaller samples. In fact, this technique is sensitive enough to enable quantitation of RNA from a single cell.
Gene overexpression is the process which leads to the abundant target protein expression subsequently. The process may be in the cell where the gene is originally located or in other expression systems. The fundamental principle is to add regulatory elements to the upstream of the target gene through artificial construction, so that genes can be transcribed and translated efficiently under controlled conditions.
CRISPR system is very simple, consisted of a Cas9 protein and a single guided RNA. With the guidance of sgRNA, Cas9 could cause a double stranded breaks in the target site.
Nutraceutical market, scope and growth: Herbal drug technologyLokesh Patil
As consumer awareness of health and wellness rises, the nutraceutical market—which includes goods like functional meals, drinks, and dietary supplements that provide health advantages beyond basic nutrition—is growing significantly. As healthcare expenses rise, the population ages, and people want natural and preventative health solutions more and more, this industry is increasing quickly. Further driving market expansion are product formulation innovations and the use of cutting-edge technology for customized nutrition. With its worldwide reach, the nutraceutical industry is expected to keep growing and provide significant chances for research and investment in a number of categories, including vitamins, minerals, probiotics, and herbal supplements.
THE IMPORTANCE OF MARTIAN ATMOSPHERE SAMPLE RETURN.Sérgio Sacani
The return of a sample of near-surface atmosphere from Mars would facilitate answers to several first-order science questions surrounding the formation and evolution of the planet. One of the important aspects of terrestrial planet formation in general is the role that primary atmospheres played in influencing the chemistry and structure of the planets and their antecedents. Studies of the martian atmosphere can be used to investigate the role of a primary atmosphere in its history. Atmosphere samples would also inform our understanding of the near-surface chemistry of the planet, and ultimately the prospects for life. High-precision isotopic analyses of constituent gases are needed to address these questions, requiring that the analyses are made on returned samples rather than in situ.
Richard's entangled aventures in wonderlandRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
Cancer cell metabolism: special Reference to Lactate PathwayAADYARAJPANDEY1
Normal Cell Metabolism:
Cellular respiration describes the series of steps that cells use to break down sugar and other chemicals to get the energy we need to function.
Energy is stored in the bonds of glucose and when glucose is broken down, much of that energy is released.
Cell utilize energy in the form of ATP.
The first step of respiration is called glycolysis. In a series of steps, glycolysis breaks glucose into two smaller molecules - a chemical called pyruvate. A small amount of ATP is formed during this process.
Most healthy cells continue the breakdown in a second process, called the Kreb's cycle. The Kreb's cycle allows cells to “burn” the pyruvates made in glycolysis to get more ATP.
The last step in the breakdown of glucose is called oxidative phosphorylation (Ox-Phos).
It takes place in specialized cell structures called mitochondria. This process produces a large amount of ATP. Importantly, cells need oxygen to complete oxidative phosphorylation.
If a cell completes only glycolysis, only 2 molecules of ATP are made per glucose. However, if the cell completes the entire respiration process (glycolysis - Kreb's - oxidative phosphorylation), about 36 molecules of ATP are created, giving it much more energy to use.
IN CANCER CELL:
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
introduction to WARBERG PHENOMENA:
WARBURG EFFECT Usually, cancer cells are highly glycolytic (glucose addiction) and take up more glucose than do normal cells from outside.
Otto Heinrich Warburg (; 8 October 1883 – 1 August 1970) In 1931 was awarded the Nobel Prize in Physiology for his "discovery of the nature and mode of action of the respiratory enzyme.
WARNBURG EFFECT : cancer cells under aerobic (well-oxygenated) conditions to metabolize glucose to lactate (aerobic glycolysis) is known as the Warburg effect. Warburg made the observation that tumor slices consume glucose and secrete lactate at a higher rate than normal tissues.
Multi-source connectivity as the driver of solar wind variability in the heli...Sérgio Sacani
The ambient solar wind that flls the heliosphere originates from multiple
sources in the solar corona and is highly structured. It is often described
as high-speed, relatively homogeneous, plasma streams from coronal
holes and slow-speed, highly variable, streams whose source regions are
under debate. A key goal of ESA/NASA’s Solar Orbiter mission is to identify
solar wind sources and understand what drives the complexity seen in the
heliosphere. By combining magnetic feld modelling and spectroscopic
techniques with high-resolution observations and measurements, we show
that the solar wind variability detected in situ by Solar Orbiter in March
2022 is driven by spatio-temporal changes in the magnetic connectivity to
multiple sources in the solar atmosphere. The magnetic feld footpoints
connected to the spacecraft moved from the boundaries of a coronal hole
to one active region (12961) and then across to another region (12957). This
is refected in the in situ measurements, which show the transition from fast
to highly Alfvénic then to slow solar wind that is disrupted by the arrival of
a coronal mass ejection. Our results describe solar wind variability at 0.5 au
but are applicable to near-Earth observatories.
Professional air quality monitoring systems provide immediate, on-site data for analysis, compliance, and decision-making.
Monitor common gases, weather parameters, particulates.
2. ATAC-seq is a technique used in molecular biology to study chromatin
accessibility. It identifies regions of open chromatin using a hyperactive
prokaryotic Tn5-transposase, which preferentially inserts into accessible
chromatin and tags the sites with sequencing adaptors. An ATAC-seq
experiment will typically produce millions of next generation sequencing
reads that can be successfully mapped to the reference genome. And it
also can be used to infer nucleosome positions. ATAC-seq provides a
first look at cell-to-cell variability in chromatin organization irregularities
in gene expression by gathering data on hundreds or thousands of single
cells in parallel. In addition, ATAC-seq will also be a useful diagnostic
tool.
A Brief Introduction of ATAC-seq
4. Cell preparation
1. 50,000 cells are counted and harvested, then cells are centrifuged 5
min at 500g, 4 .℃
2. Remove and discard supernatant. Wash cells with 50 uL cold PBS
buffer and centrifuge 5 min at 500g, 4 .℃
3. Remove and discard supernatant. Resuspend the cell pellet with 50
uL cold lysis buffer centrifuge 10 min at 500g, 4 .℃
4. Discard the supernatant, the Lyse cells will be generate a crude
nuclei. Next, they need immediately continue to transposition reaction.
5. The joint that connects with a ME sequence 1.0-1.5 uL
10×TPS buffer 2 uL
Robust Tn5 Transposase 1 uL
TE buffer Up to 20 uL
6. Incubate the mixture at 25 for 30 min.℃
7. The transposome can be immediately used to
transposition reaction, or stored at -20 if necessary.℃
Construction of Transposome
5. To make the reaction system, combine the following:
6. Tagmentation and purification
8. To make the transposition reaction mix, combine the following:
DNA to be fragmented ( from step 4) 100 ng
5×LM buffer 6 uL
Transposome 4 uL
Nuclease-free HO Up to 30 uL
9. Incubate the transposition reaction mix at 37 for 2 hours or at℃
56 for 10-15 min.℃
10. Purify the fragmented and tagged DNA using a Creative Biogene
PCR Purification Kit. Stored purified DNA at -20 if necessary.℃
7. PCR amplification and purification
11. Amplify transposed DNA fragments using a Creative Biogene PCR
amplification Kit.
12. Purify the PCR amplified library using a Creative Biogene PCR Purification
Kit.Library sequencing
Sequence the library and correlate reads with open and closed chromatin.
When sequencing ATAC-seq libraries, sequencing primers and reagents must
be used. For nucleosome mapping, paired-end sequencing is preferred. Foe
inferring differences in open chromatin with human samples,we generally
use>50,000,000 mapped reads, and for transcription factor foot-printing, we
use>200,000,000 mapped reads. The mitochondrial read fraction can vary
between 10% and >50%, depending on cell type.
8. Sample demands are greatly reduced.
ATAC-seq is compatible with a number of methods for cell separation and
isolation. The demand for cells ranged from 500 to 50,000. ATAC-seq provides
a great convenience.
Lower sequencing depth.
ATAC-seq data sets can be bioinformatically separated into reads that are shorter
than the canonical length generally protected by a nuclesome. And compared
MNase-seq sequencing, the sequencing effort of ATAC-seq has been reduced
several times. However, signals of ATAC-seq are very good.
Simple operation.
Genome-wide mapping of insertion ends by high-throughput sequensing allows
for multi-dimensional assays of the regulatory landscape of chromatin with a
relatively simple protocol.
The Features of ATAC-seq
9. At the start of the experiment, cells must be at healthy living
condition to ensure obtain more complete information in subsequent
studies;
Cells should be lysed under mild conditions to ensure that the
structure of the chromosomes is not destroyed;
After the treatment of Tn5 transposase, purify the fragmented is
necessary, otherwise it can produce false positives.
Some Notes
10. Creative Biogene has years of enzyme production experience, and all
enzymes are tested in standard functional assays and certified free of
nucleases and proteases. Creative Biogene's modification enzymes, including
DNA and RNA polymerases, phosphatases, kinases, ligases, nucleases,
methyltransferases, DNA repair proteins, recombinases etc, are used for a
variety of molecular biology applications. And we also provide robust Tn5
transposase for accelerating your research, especially tagmentation-based
sequencing library construction.
About Us
If you have any questions, please feel free to contact us. We look forward
to working with you in the future.
