Deciphering DNA sequences is essential for virtually all branches of biological research. With the
advent of capillary electrophoresis (CE)-based Sanger sequencing, scientists gained the ability to
elucidate genetic information from any given biological system. This technology has become widely
adopted in laboratories around the world, yet has always been hampered by inherent limitations in
throughput, scalability, speed, and resolution that often preclude scientists from obtaining the essential
information they need for their course of study. To overcome these barriers, an entirely new technology
was required—Next-Generation Sequencing (NGS), a fundamentally different approach to sequencing
that triggered numerous ground-breaking discoveries and ignited a revolution in genomic science.
It contains information about- DNA Sequencing; History and Era sequencing; Next Generation Sequencing- Introduction, Workflow, Illumina/Solexa sequencing, Roche/454 sequencing, Ion Torrent sequencing, ABI-SOLiD sequencing; Comparison between NGS & Sangers and NGS Platforms; Advantages and Applications of NGS; Future Applications of NGS.
Sanger sequencing is one of the DNA sequencing methods used to identify and determine the sequence (Nucleotide) of DNA .This is an enzymatic method of sequencing developed by Fred Sanger.
A real-time polymerase chain reaction is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR, i.e. in real-time, and not at its end, as in conventional PCR.
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It contains information about- DNA Sequencing; History and Era sequencing; Next Generation Sequencing- Introduction, Workflow, Illumina/Solexa sequencing, Roche/454 sequencing, Ion Torrent sequencing, ABI-SOLiD sequencing; Comparison between NGS & Sangers and NGS Platforms; Advantages and Applications of NGS; Future Applications of NGS.
Sanger sequencing is one of the DNA sequencing methods used to identify and determine the sequence (Nucleotide) of DNA .This is an enzymatic method of sequencing developed by Fred Sanger.
A real-time polymerase chain reaction is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR, i.e. in real-time, and not at its end, as in conventional PCR.
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STS stands for sequence tagged site which is short DNA sequence, generally between 100 and 500 bp in length, that is easily recognizable and occurs only once in the chromosome or genome being studied.
Sanger sequencing is a method of DNA sequencing based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication.
SNP (Single Nucleotide Polymorphic), SNP mapping, SNP profile, SNP types, SNP analysis by gel electropherosis and by mass spectrometry, SNP effects, single strand conformation polymorphism, SNP advantages and disadvantages and application of SNP profile in drug choice
Next Generation Sequencing (NGS) Is A Modern And Cost Effective Sequencing Technology Which Enables Scientists To Sequence Nucleic Acids At Much Faster Rate. In This Presentation, You Will Learn About What is NGS, Idea Behind NGS, Methodology And Protocol, Widely Adapted NGS Protocols, Applications And References For Further Study.
Next Generation Sequencing and its Applications in Medical Research - Frances...Sri Ambati
The so-called “next-generation” sequencing (NGS) technologies allows us, in a short time and in parallel, to sequence massive amounts of DNA, overcoming the limitations of the original Sanger sequencing methods used to sequence the first human genome. NGS technologies have had an enormous impact on biomedical research within a short time frame. This talk will give an overview of these applications with specific examples from Mendelian genomics and cancer research. #h2ony
STS stands for sequence tagged site which is short DNA sequence, generally between 100 and 500 bp in length, that is easily recognizable and occurs only once in the chromosome or genome being studied.
Sanger sequencing is a method of DNA sequencing based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication.
SNP (Single Nucleotide Polymorphic), SNP mapping, SNP profile, SNP types, SNP analysis by gel electropherosis and by mass spectrometry, SNP effects, single strand conformation polymorphism, SNP advantages and disadvantages and application of SNP profile in drug choice
Next Generation Sequencing (NGS) Is A Modern And Cost Effective Sequencing Technology Which Enables Scientists To Sequence Nucleic Acids At Much Faster Rate. In This Presentation, You Will Learn About What is NGS, Idea Behind NGS, Methodology And Protocol, Widely Adapted NGS Protocols, Applications And References For Further Study.
Next Generation Sequencing and its Applications in Medical Research - Frances...Sri Ambati
The so-called “next-generation” sequencing (NGS) technologies allows us, in a short time and in parallel, to sequence massive amounts of DNA, overcoming the limitations of the original Sanger sequencing methods used to sequence the first human genome. NGS technologies have had an enormous impact on biomedical research within a short time frame. This talk will give an overview of these applications with specific examples from Mendelian genomics and cancer research. #h2ony
Sequencing is one of the major technological advancement that has taken shape in the last two or three decade. Starting from Sanger and Maxam-Gilbert sequencing methods to the latest high-throughput methods, sequencing technologies has changed the the landscape of biological sciences.
This slide takes a look a the major sequencing methods over time.
Note: Several images included here have been sourced from GOOGLE IMAGES. The content has been extracted from several SCIENTIFIC PAPERS and WEBSITES.
PLEASE DO CONTACT THE AUTHOR DIRECTLY IF ANY COPYRIGHT ISSUE ARISES.
Nucleic acid techniques in diagnostic microbiologymohit kumar
in this presentation, you learn about the microbiological techniques which help in the molecular diagnosis of any single pathogens. with this, you aware of some commercially available kits for polymerase chain reaction both for realtime as well as conventional PCR and genome extraction kits.
Molecular marker technology in studies on plant genetic diversityChanakya P
A molecular marker is a molecule contained within a sample taken from an organism (biological markers) or other matter. It can be used to reveal certain characteristics about the respective source. DNA, for example, is a molecular marker containing information about genetic disorders, genealogy and the evolutionary history of life. Specific regions of the DNA (genetic markers) are used to diagnose the autosomal recessive genetic disorder cystic fibrosis, taxonomic affinity (phylogenetics) and identity (DNA Barcoding). Further, life forms are known to shed unique chemicals, including DNA, into the environment as evidence of their presence in a particular location.Other biological markers, like proteins, are used in diagnostic tests for complex neurodegenerative disorders, such as Alzheimer's disease. Non-biological molecular markers are also used, for example, in environmental studies.
Lecture ON Polymerase Chain Reaction.
The polymerase chain reaction (PCR) is a powerful core molecular biology technique - Sometimes called "molecular photocopying. • Developed by Kary Mullis in 1985.
• It is an efficient and rapid in vitro method for enzymatic amplification of specific DNA or RNA sequences from nucleic acids of various sources. •
It generates microgram (µg) quantities of DNA copies (up to billion copies) of the desired DNA (or RNA) segment.
A simple PCR reaction consists of
i. A DNA preparation containing the desired segment to be amplified.
ii. A set of synthetic oligonucleotide primers that flank the target DNA
sequence, of about 20 bases long, specific, i.e., complementary.
iii. A thermostable DNA polymerase e.g., Taq isolated from the
bacterium Thermus acquaticus, Pfu – Pyrococcus furiosus and Vent
from Thermococcus litoralis. Pfu and Vent are more efficient than
Taq polymerase.
iv. Four deoxynucleoside triphosphate (dNTPs): TTP – thymidine
triphosphate, dCTP – deoxycyctidine triphosphate, dATP –
deoxyadenosine triphosphate and dGTP – deoxyguanosine
triphosphate
Key Trends Shaping the Future of Infrastructure.pdfCheryl Hung
Keynote at DIGIT West Expo, Glasgow on 29 May 2024.
Cheryl Hung, ochery.com
Sr Director, Infrastructure Ecosystem, Arm.
The key trends across hardware, cloud and open-source; exploring how these areas are likely to mature and develop over the short and long-term, and then considering how organisations can position themselves to adapt and thrive.
GDG Cloud Southlake #33: Boule & Rebala: Effective AppSec in SDLC using Deplo...James Anderson
Effective Application Security in Software Delivery lifecycle using Deployment Firewall and DBOM
The modern software delivery process (or the CI/CD process) includes many tools, distributed teams, open-source code, and cloud platforms. Constant focus on speed to release software to market, along with the traditional slow and manual security checks has caused gaps in continuous security as an important piece in the software supply chain. Today organizations feel more susceptible to external and internal cyber threats due to the vast attack surface in their applications supply chain and the lack of end-to-end governance and risk management.
The software team must secure its software delivery process to avoid vulnerability and security breaches. This needs to be achieved with existing tool chains and without extensive rework of the delivery processes. This talk will present strategies and techniques for providing visibility into the true risk of the existing vulnerabilities, preventing the introduction of security issues in the software, resolving vulnerabilities in production environments quickly, and capturing the deployment bill of materials (DBOM).
Speakers:
Bob Boule
Robert Boule is a technology enthusiast with PASSION for technology and making things work along with a knack for helping others understand how things work. He comes with around 20 years of solution engineering experience in application security, software continuous delivery, and SaaS platforms. He is known for his dynamic presentations in CI/CD and application security integrated in software delivery lifecycle.
Gopinath Rebala
Gopinath Rebala is the CTO of OpsMx, where he has overall responsibility for the machine learning and data processing architectures for Secure Software Delivery. Gopi also has a strong connection with our customers, leading design and architecture for strategic implementations. Gopi is a frequent speaker and well-known leader in continuous delivery and integrating security into software delivery.
Accelerate your Kubernetes clusters with Varnish CachingThijs Feryn
A presentation about the usage and availability of Varnish on Kubernetes. This talk explores the capabilities of Varnish caching and shows how to use the Varnish Helm chart to deploy it to Kubernetes.
This presentation was delivered at K8SUG Singapore. See https://feryn.eu/presentations/accelerate-your-kubernetes-clusters-with-varnish-caching-k8sug-singapore-28-2024 for more details.
State of ICS and IoT Cyber Threat Landscape Report 2024 previewPrayukth K V
The IoT and OT threat landscape report has been prepared by the Threat Research Team at Sectrio using data from Sectrio, cyber threat intelligence farming facilities spread across over 85 cities around the world. In addition, Sectrio also runs AI-based advanced threat and payload engagement facilities that serve as sinks to attract and engage sophisticated threat actors, and newer malware including new variants and latent threats that are at an earlier stage of development.
The latest edition of the OT/ICS and IoT security Threat Landscape Report 2024 also covers:
State of global ICS asset and network exposure
Sectoral targets and attacks as well as the cost of ransom
Global APT activity, AI usage, actor and tactic profiles, and implications
Rise in volumes of AI-powered cyberattacks
Major cyber events in 2024
Malware and malicious payload trends
Cyberattack types and targets
Vulnerability exploit attempts on CVEs
Attacks on counties – USA
Expansion of bot farms – how, where, and why
In-depth analysis of the cyber threat landscape across North America, South America, Europe, APAC, and the Middle East
Why are attacks on smart factories rising?
Cyber risk predictions
Axis of attacks – Europe
Systemic attacks in the Middle East
Download the full report from here:
https://sectrio.com/resources/ot-threat-landscape-reports/sectrio-releases-ot-ics-and-iot-security-threat-landscape-report-2024/
Neuro-symbolic is not enough, we need neuro-*semantic*Frank van Harmelen
Neuro-symbolic (NeSy) AI is on the rise. However, simply machine learning on just any symbolic structure is not sufficient to really harvest the gains of NeSy. These will only be gained when the symbolic structures have an actual semantics. I give an operational definition of semantics as “predictable inference”.
All of this illustrated with link prediction over knowledge graphs, but the argument is general.
Dev Dives: Train smarter, not harder – active learning and UiPath LLMs for do...UiPathCommunity
💥 Speed, accuracy, and scaling – discover the superpowers of GenAI in action with UiPath Document Understanding and Communications Mining™:
See how to accelerate model training and optimize model performance with active learning
Learn about the latest enhancements to out-of-the-box document processing – with little to no training required
Get an exclusive demo of the new family of UiPath LLMs – GenAI models specialized for processing different types of documents and messages
This is a hands-on session specifically designed for automation developers and AI enthusiasts seeking to enhance their knowledge in leveraging the latest intelligent document processing capabilities offered by UiPath.
Speakers:
👨🏫 Andras Palfi, Senior Product Manager, UiPath
👩🏫 Lenka Dulovicova, Product Program Manager, UiPath
Smart TV Buyer Insights Survey 2024 by 91mobiles.pdf91mobiles
91mobiles recently conducted a Smart TV Buyer Insights Survey in which we asked over 3,000 respondents about the TV they own, aspects they look at on a new TV, and their TV buying preferences.
Software Delivery At the Speed of AI: Inflectra Invests In AI-Powered QualityInflectra
In this insightful webinar, Inflectra explores how artificial intelligence (AI) is transforming software development and testing. Discover how AI-powered tools are revolutionizing every stage of the software development lifecycle (SDLC), from design and prototyping to testing, deployment, and monitoring.
Learn about:
• The Future of Testing: How AI is shifting testing towards verification, analysis, and higher-level skills, while reducing repetitive tasks.
• Test Automation: How AI-powered test case generation, optimization, and self-healing tests are making testing more efficient and effective.
• Visual Testing: Explore the emerging capabilities of AI in visual testing and how it's set to revolutionize UI verification.
• Inflectra's AI Solutions: See demonstrations of Inflectra's cutting-edge AI tools like the ChatGPT plugin and Azure Open AI platform, designed to streamline your testing process.
Whether you're a developer, tester, or QA professional, this webinar will give you valuable insights into how AI is shaping the future of software delivery.
When stars align: studies in data quality, knowledge graphs, and machine lear...
NEXT GENERATION SEQUENCING
1. What is sequencing??
• Deciphering the code hidden in biological
sequences like DNA, polypeptides etc.
• Method and technologies that enables us to
determine the order of nucleotides and amino
acids in DNA and Polypeptide respectively.
2. Traditional methods of Sequencing and
its limitations
• Maxam-Gilbert Method
Use of radioactive labels.
Sanger Method
It utilize the fluorescent dye for labeling.
separation of extended fragments of DNA with the
addition of di-deoxynucleotides (lack a 3’-OH group)
Thus, chain termination.
Limitation
Slow
High cost per run.
3. Automated Sanger method
1. Bacterial cloning or PCR
template purification
2. labelling of DNA
fragments using the chain
termination method with
energy transfer
3. dye-labelled di-de
oxynucleotides and a DNA
polymerase
4. capillary electrophoresis
5. fluorescence detection that
provides four-colour plots
to reveal the DNA
sequence.
4. NEXT GENERATION SEQUENCING
•Also known as
▫High throughput sequencing or
▫ultra-deep sequencing or
▫massively parallel sequencing.
5. What is next generation sequencing ??
• Automated Sanger method (1st generation)
• Technologies developed after that are known as
next generation sequencing.
• NGS enables the sequencing of biological codes
at a very rapid pace with low cost per operation.
• This is the primary advantage over conventional
methods.
• For example Billions of short reads can be
sequenced in one operation.
6. Major Platforms for NGS
•454 ( By Roche)
•SOLiD (By Applied Biosystems)
•Solexa (By Illumina)
7. • Above mentioned platform varies in
strategies, application and type of
data generated.
• However, all technologies are
common in
▫ That they generate sequences on an
unprecedented scale
▫ DNA cloning is not required
▫ and very low operation cost.
8. What NGS Consists of
Next generation technologies for
sequencing is combination of strategies
for
• template preparation
• sequencing and imaging
• genome alignment
• assembly methods
9. Template preparation
As even most sensitive imaging technique
is not able to detect single
molecule, amplification of templates is
inevitable.
• Clonally amplified templates
▫ By emulsion PCR (emPCR) e.g. 454 and
SOLiD
▫ Solid phase amplification e.g. illumina
• Single-molecule templates
10. Template preparation: Traditional vs. NGS
• Immobilization of templates fragments over
bead /glass plate allows billions of the
sequencing reaction run simultaneously.
11. sequencing and imaging
• Sequencing
▫ cyclic reversible termination(CRT) e.g.
illumina/solexa
▫ single-nucleotide addition (SNA) e.g.
454/roche
▫ real-time sequencing: R&D going on
(pacific Bioscience)
▫ Sequencing by ligation (SBL) e.g. SOLiD
• Imaging
▫ measuring bioluminescent signals
▫ four-colour imaging of single molecular
events e.g. illumina/solexa.
12. Genome alignment and assembly
After NGS reads have been generated, they are
aligned to either
• a known reference sequence
or
• assembled de novo
13. 454 (Pyrosequencing)
• DNA is
fragmented, joined to
adapters at either end of
the fragmented DNA
• amplified in an emulsion
PCR (includes agarose
bead with complimentary
adaptors to fragmented
DNA)
• PCR amplified allowing
up to 1 million identical
fragments around one
bead and finally dropped
into a PicoTitreTube
(PTT)
14. PCR amplification
Pico Titre Tube
• Adapter containing the
universal priming site
are ligated to target
ends
• Same primer can be
used for amplification
15. • In Pico titre tube reaction
of fluorescence occurs
with the addition of
nucleotides
Nucleotide addition
18. SOLiD
(support oligonucleotide ligation detection)
• Sequencing by Oligo/Ligation
and Detection.
• Steps
▫ Library Preparation
two types of libraries
sequencing-fragment or
mate-paired are prepared.
▫ Emulsion PCR/Bead
Enrichment
amplification of template
fragments is done in same
manner as 454.
▫ Bead Deposition
Deposit 3’ modified beads
onto a glass slide.
19. Sequencing by Ligation
• Primers hybridize to the P1
adapter sequence on the
templated beads
• The method uses two-base-
encoded probes(4
probes), which has the primary
advantage of improved
accuracy.
• Multiple cycles of
ligation, detection and cleavage
are performed.
• Extension product is removed
and the template is reset with a
primer complementary to the
n-1 position for a second round
of ligation cycles.
20.
21. Illumina
• Breaking up DNA
• Adding adaptors, but
in this case attach not
to a bead but to a
slide
• Fold-back PCR is
then used to amplify
the fragmented DNA
into a cluster
23. NGS and Bioinformatics
• Alignment of sequence reads to a
reference
BLAST doesn’t blast here
Short read aligners side-lines BLAST
Software
• Bowtie
• MAQ
• BWA
24. • Above strategy works if reference genome exist.
• de novo assembly from paired or unpaired reads
• base-calling and/or polymorphism detection
• structural variant detection
• genome browsing.
25. Application of NGS
• Variants discovery in targeted region or whole
genome by re-sequencing
• Reassembling genome of lower organism by de
novo method.
• Cost-effective sequencing of complex samples at
remarkable scale and speed.
• Sequencing entire transcriptome.
• In Meta genomics : Sequencing genome of entire
biological communities
• Replacing ChIP-on-chip with ChIP-seq in case of
multicellular eukaryotes.
• Personalized genome for personalized medicine
26. • Further Readings
1. Branton, D. et al. The potential and challenges
of nanopore sequencing. Nature Biotech. 26
1146–1153 (2008).
2. Wang, Z., Gerstein, M. & Snyder, M. RNA-Seq: a
revolutionary tool for transcriptomics. Nature
Rev. Genet. 10, 57–63 (2009).
3. Petrosino, J. F., Highlander, S., Luna, R.
A., Gibbs, R. A. & Versalovic, J. Metagenomic
pyrosequencing and microbial identification.
Clin. Chem. 55, 856–866 (2009).
4. Park, P. J. ChIP–seq: advantages and challenges
of a maturing technology. Nature Rev. Genet.
10, 669–680 (2009).