This document provides an overview of DNA sequencing technologies. It begins by defining DNA sequencing as reading the exact nucleotide sequence of a genome. Several sequencing methods are described, including Maxam-Gilbert chemical degradation, Sanger chain termination, Illumina sequencing-by-synthesis using fluorescence, SOLiD ligation-based sequencing using fluorescent probes, Ion Torrent semiconductor sequencing detecting pH changes, nanopore sequencing detecting electrical currents, and single molecule real-time sequencing. The principles, detection methods, and applications of these techniques are outlined. The document emphasizes understanding the core concepts of each method and highlights their comparisons.
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/
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/
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.
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.
Original Next Gen Seq Methods set of slides prepared for Technorazz Vibes 2016. There is also a shorter version.
This starts with an introduction to qPCR followed by an introduction to Library Complexity. Microarrays are discussed as well along with a very short introduction to FISH. Finally discussion of Next gen seq methods is done where generation of sequencers are discussed and a short discussion of the ILLUMINA protocol. Finally comparison of ILLUMINA amongst other 3rd gen sequencer, description of the standard pipeline and the omics technologies that have risen from this seq data.
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.
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.
This lecture covers some nice stories about the origins of the words "genome" and the derived word "genomics". the lecture also introduces viral, bacterial, and eukaryotic genomes.
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.
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.
Original Next Gen Seq Methods set of slides prepared for Technorazz Vibes 2016. There is also a shorter version.
This starts with an introduction to qPCR followed by an introduction to Library Complexity. Microarrays are discussed as well along with a very short introduction to FISH. Finally discussion of Next gen seq methods is done where generation of sequencers are discussed and a short discussion of the ILLUMINA protocol. Finally comparison of ILLUMINA amongst other 3rd gen sequencer, description of the standard pipeline and the omics technologies that have risen from this seq data.
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.
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.
This lecture covers some nice stories about the origins of the words "genome" and the derived word "genomics". the lecture also introduces viral, bacterial, and eukaryotic genomes.
This lecture covers key findings to the development of genomics as a field. This first part covers briefly Mendel to knowing that DNA is the genetic material by Hershey and Chase
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.
Detailed explanation about gene sequencing methods
Sequencing the gene is an important step toward understanding the gene.
A gene sequence contains some clues about where genes are.
Gene sequencing give us understanding how the genome as a whole works-how genes work together to direct the growth, development and maintenance of an entire organism.
It help scientists to study the part of genome outside the genes-regulatory regions
Microarray -types, DNA chip, Principle and application of microarray, Preparation of DNA Chip, Affymetrix chip, microarray in genomics and proteomics, advantages and limitations of microarray
The material of a talk that I prepared to give in the online camel conference of Oman. Unfortunately, I had a death in the family the day before the conference and the material was presented by my friend Dr. Mohammed Alabri from Oman. The material is in Arabic and focused for camel breeders.
The material of a two days workshop that I gave at Sultan Qaboos University in Oman about the importance of livestock biobanks and how to establish an organized one. The workshop was given in Arabic.
A presentation as a webinar for the Winn Feline Foundation that focuses on recent findings related to the signatures of selection in the domestic cat genome
This was my presentation at the Plant and Animal Genome Conference 2019 in San Diego. My talk was a presentation of the thesis project of my student Mona Abdi. The focus of the presentation and project was the genomic signatures of selection in the domestic cat breeds.
This is a my lecture about camels title "Journey around the camel". The lecture was in Arabic and is related to a book under preparation with the same title. This part of the journey around the camel introduces the major camel breeds in the Arabian Peninsula and their external phenotypes and groupings.
This pdf is about the Schizophrenia.
For more details visit on YouTube; @SELF-EXPLANATORY;
https://www.youtube.com/channel/UCAiarMZDNhe1A3Rnpr_WkzA/videos
Thanks...!
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.
(May 29th, 2024) Advancements in Intravital Microscopy- Insights for Preclini...Scintica Instrumentation
Intravital microscopy (IVM) is a powerful tool utilized to study cellular behavior over time and space in vivo. Much of our understanding of cell biology has been accomplished using various in vitro and ex vivo methods; however, these studies do not necessarily reflect the natural dynamics of biological processes. Unlike traditional cell culture or fixed tissue imaging, IVM allows for the ultra-fast high-resolution imaging of cellular processes over time and space and were studied in its natural environment. Real-time visualization of biological processes in the context of an intact organism helps maintain physiological relevance and provide insights into the progression of disease, response to treatments or developmental processes.
In this webinar we give an overview of advanced applications of the IVM system in preclinical research. IVIM technology is a provider of all-in-one intravital microscopy systems and solutions optimized for in vivo imaging of live animal models at sub-micron resolution. The system’s unique features and user-friendly software enables researchers to probe fast dynamic biological processes such as immune cell tracking, cell-cell interaction as well as vascularization and tumor metastasis with exceptional detail. This webinar will also give an overview of IVM being utilized in drug development, offering a view into the intricate interaction between drugs/nanoparticles and tissues in vivo and allows for the evaluation of therapeutic intervention in a variety of tissues and organs. This interdisciplinary collaboration continues to drive the advancements of novel therapeutic strategies.
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.
Slide 1: Title Slide
Extrachromosomal Inheritance
Slide 2: Introduction to Extrachromosomal Inheritance
Definition: Extrachromosomal inheritance refers to the transmission of genetic material that is not found within the nucleus.
Key Components: Involves genes located in mitochondria, chloroplasts, and plasmids.
Slide 3: Mitochondrial Inheritance
Mitochondria: Organelles responsible for energy production.
Mitochondrial DNA (mtDNA): Circular DNA molecule found in mitochondria.
Inheritance Pattern: Maternally inherited, meaning it is passed from mothers to all their offspring.
Diseases: Examples include Leber’s hereditary optic neuropathy (LHON) and mitochondrial myopathy.
Slide 4: Chloroplast Inheritance
Chloroplasts: Organelles responsible for photosynthesis in plants.
Chloroplast DNA (cpDNA): Circular DNA molecule found in chloroplasts.
Inheritance Pattern: Often maternally inherited in most plants, but can vary in some species.
Examples: Variegation in plants, where leaf color patterns are determined by chloroplast DNA.
Slide 5: Plasmid Inheritance
Plasmids: Small, circular DNA molecules found in bacteria and some eukaryotes.
Features: Can carry antibiotic resistance genes and can be transferred between cells through processes like conjugation.
Significance: Important in biotechnology for gene cloning and genetic engineering.
Slide 6: Mechanisms of Extrachromosomal Inheritance
Non-Mendelian Patterns: Do not follow Mendel’s laws of inheritance.
Cytoplasmic Segregation: During cell division, organelles like mitochondria and chloroplasts are randomly distributed to daughter cells.
Heteroplasmy: Presence of more than one type of organellar genome within a cell, leading to variation in expression.
Slide 7: Examples of Extrachromosomal Inheritance
Four O’clock Plant (Mirabilis jalapa): Shows variegated leaves due to different cpDNA in leaf cells.
Petite Mutants in Yeast: Result from mutations in mitochondrial DNA affecting respiration.
Slide 8: Importance of Extrachromosomal Inheritance
Evolution: Provides insight into the evolution of eukaryotic cells.
Medicine: Understanding mitochondrial inheritance helps in diagnosing and treating mitochondrial diseases.
Agriculture: Chloroplast inheritance can be used in plant breeding and genetic modification.
Slide 9: Recent Research and Advances
Gene Editing: Techniques like CRISPR-Cas9 are being used to edit mitochondrial and chloroplast DNA.
Therapies: Development of mitochondrial replacement therapy (MRT) for preventing mitochondrial diseases.
Slide 10: Conclusion
Summary: Extrachromosomal inheritance involves the transmission of genetic material outside the nucleus and plays a crucial role in genetics, medicine, and biotechnology.
Future Directions: Continued research and technological advancements hold promise for new treatments and applications.
Slide 11: Questions and Discussion
Invite Audience: Open the floor for any questions or further discussion on the topic.
Seminar of U.V. Spectroscopy by SAMIR PANDASAMIR PANDA
Spectroscopy is a branch of science dealing the study of interaction of electromagnetic radiation with matter.
Ultraviolet-visible spectroscopy refers to absorption spectroscopy or reflect spectroscopy in the UV-VIS spectral region.
Ultraviolet-visible spectroscopy is an analytical method that can measure the amount of light received by the analyte.
Introduction:
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is an important biological process for modulating eukaryotic gene expression.
It is highly conserved process of posttranscriptional gene silencing by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.
dsRNA-induced gene silencing (RNAi) is reported in a wide range of eukaryotes ranging from worms, insects, mammals and plants.
This process mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What are small ncRNAs?
micro RNA (miRNA)
short interfering RNA (siRNA)
Properties of small non-coding RNA:
Involved in silencing mRNA transcripts.
Called “small” because they are usually only about 21-24 nucleotides long.
Synthesized by first cutting up longer precursor sequences (like the 61nt one that Lee discovered).
Silence an mRNA by base pairing with some sequence on the mRNA.
Discovery of siRNA?
The first small RNA:
In 1993 Rosalind Lee (Victor Ambros lab) was studying a non- coding gene in C. elegans, lin-4, that was involved in silencing of another gene, lin-14, at the appropriate time in the
development of the worm C. elegans.
Two small transcripts of lin-4 (22nt and 61nt) were found to be complementary to a sequence in the 3' UTR of lin-14.
Because lin-4 encoded no protein, she deduced that it must be these transcripts that are causing the silencing by RNA-RNA interactions.
Types of RNAi ( non coding RNA)
MiRNA
Length (23-25 nt)
Trans acting
Binds with target MRNA in mismatch
Translation inhibition
Si RNA
Length 21 nt.
Cis acting
Bind with target Mrna in perfect complementary sequence
Piwi-RNA
Length ; 25 to 36 nt.
Expressed in Germ Cells
Regulates trnasposomes activity
MECHANISM OF RNAI:
First the double-stranded RNA teams up with a protein complex named Dicer, which cuts the long RNA into short pieces.
Then another protein complex called RISC (RNA-induced silencing complex) discards one of the two RNA strands.
The RISC-docked, single-stranded RNA then pairs with the homologous mRNA and destroys it.
THE RISC COMPLEX:
RISC is large(>500kD) RNA multi- protein Binding complex which triggers MRNA degradation in response to MRNA
Unwinding of double stranded Si RNA by ATP independent Helicase
Active component of RISC is Ago proteins( ENDONUCLEASE) which cleave target MRNA.
DICER: endonuclease (RNase Family III)
Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)
One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute
ARGONAUTE PROTEIN :
1.PAZ(PIWI/Argonaute/ Zwille)- Recognition of target MRNA
2.PIWI (p-element induced wimpy Testis)- breaks Phosphodiester bond of mRNA.)RNAse H activity.
MiRNA:
The Double-stranded RNAs are naturally produced in eukaryotic cells during development, and they have a key role in regulating gene expression .
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.
This presentation explores a brief idea about the structural and functional attributes of nucleotides, the structure and function of genetic materials along with the impact of UV rays and pH upon them.
2. AIMS
• Introduce DNA sequencing technologies.
• Highlight method of sequencing.
• Highlight sequence detection.
3.
4. What is DNA sequencing?
It is reading the letters of the book.
It is reading the exact nucleotide sequence of the
genome.
5. Sequencing methods available now!
1. Maxam and Gilbert chemical
degradation method (extinct).
2. Sanger sequencing (dideoxy
or chain termination method).
3. Illumina sequencing.
4. SOLiD sequencing.
5. Pyrosequencing.
6. Ion Torrent method.
7. Single molecule sequencing.
6. Why DNA sequencing?
• DNA sequencing can be considered the ultimate
characterization of gene(s) or fragment(s) of DNA.
• DNA Sequencing is used for:
• Mapping genomes
• Determining gene structure and thus function
• Detecting polymorphism (single nucleotide
polymorphism SNP)
• Analyzing genetic variation
• Predicting the possible product(s) of DNA
fragments
• Many purposes depending on the questions one is
asking
7. In 1977, Allan Maxam and Walter Gilbert
developed chemical sequencing method
Maxam-Gilbert Sequencing
8. 1.Base modification.
2.Removal of modified base from its sugar.
3.Breaking the phosphodiester bond.
4.Analyzing the fragments using gel
electrophoresis.
Maxam-Gilbert Sequencing
11. Sanger sequencing
1.Fredrick Sanger has
developed a sequencing
method and received a
Noble prize for it.
2.Sanger sequencing method
is also called Chain
Termination Method and
Dideoxy sequencing
method.
12. • Employs:
• specific primers
• dNTPs
• ddNTPs
• DNA polymerase
• DNA template
Sanger sequencing
17. 3’5’5’5’
G5’
G A5’
AG A5’
AG A T5’
AG TA TAG TA T5’
C C5’
AG CTA TAG CTA T T5’
C
Sanger sequencing procedure
AG TA TGAATTC3’ 5’
5’
G
5’
G A
5’
AG A
5’
AG A T
5’
AG TA T
AG TA T5’
C
C5’
AG CTA T
AG CTA T T5’
C
18. A T G C
+
3’
5’
• Analysis using high
resolution
polyacrylamide gel
electrophoresis.
• Fragments are
detected using
radioactive markers
and autoradiography.
A
C
T
G
G
T
C
A
A
T
C
G
A
T
C
G
T
A
Sanger sequencing procedure
-
20. • Each dideoxy nucleotide is attached to a
florescent marker.
• At the end of each cycle, a laser beam can detect
the florescent marker and thus record the position
of the nucleotide.
Sanger sequencing - Automated
24. Next-generation sequencing must die!
http://www.acgt.me/blog/2014/3/7/next-generation-
sequencing-must-die
“NGS technologies have already been around
for almost 20 years. It doesn't strike me as
particularly helpful to keep on labeling all of
these different technologies with the same
name.” Keith Bradnam.
Current sequencing technologies
25. • Stop using NGS to describe sequencing technology.
• Refer to the technology by its method (e.g.
sequencing-by-synthesis).
• Refer to the technology by the company that provides
it (e.g. Oxford Nanopore).
• Use the term “current sequencing technologies” if
people can know what current is.
What is “current” to you today?
Current sequencing technologies
http://www.acgt.me/blog/2014/3/7/next-generation-
sequencing-must-die
What should we call them?
26. Pyrosequencing
• Method developed by Pål Nyrén and Mustafa
Ronaghi in 1996.
• It is a DNA sequencing method by synthesis.
• The method detects incorporation of
nucleotides by the pyrophosphate by product.
33. ABi SOLiD Sequencing
• SOLiD stands for (Sequencing by Oligonucleotide
Ligation and Detection).
• Sequencing by ligation.
• Ligation of four fluorescent labeled di-base
probes.
• Different universal primers (different lengths) are
used.
• The overlap of all ligation rounds will determine
the DNA sequence.
http://www.appliedbiosystems.com/absite/us/en/home/applications-technologies/solid-
next-generation-sequencing/next-generation-systems/solid-sequencing-chemistry.html
36. Ion Torrent sequencing
• A semi-conductor sequencing technology.
• Small chips contain millions of wells/sensors.
• Does not use fluorescent light or camera imaging.
37. Principle
• Incorporation of correct nucleotide causes
changes in pH.
• Why?
• Ion sensor that set under the well can detects
measures the changes of the pH.
• The chemical signals and converts it to voltage.
• This process is repeated every 15 seconds with
different nucleotides washing over the chip.
40. • No Synthesis or ligation.
• Realtime sequencing.
• Uses nanopores.
• Changes in electrical current passing through the
nanopore is the method of nucleotide detection
and identification.
• Each nucleotide has a specific electrical current
disruption signature.
Nanopore sequencing
41. Principle
1. Double stranded DNA
attaches to a helicase.
2. Single strand DNA passes
through the nanopore and
changes in flow of electric
current is detected.
3. Each base has a different
electrical signal.
42. Fast - Realtime - Accurate - Small device - Cheap
The future?
43.
44. Which one to choose?
http://www.molecularecologist.com/next-gen-fieldguide-2014/
I suggest you look at this website for nice
comparisons between sequencing technologies
45. Which one to choose?
http://www.molecularecologist.com/next-gen-fieldguide-2014/
46. Expectations
• For each of the sequencing methods/
technology, you know:
• The general principle.
• Sequencing by?
• Sequence detection by?
48. Disclaimer
Figures, photos, and graphs in my lectures are
collected using google searches. I do not claim to have
personally produced the material (except for some). I
do cite only articles or books used. I thank all owners of
the visual aid that I use and apologize for not citing
each individual item. If anybody finds the inclusion of
their material in my lectures a violation of their copy
rights, please contact me via email.
hhalhaddad@gmail.com
