Alternatív splicing változások elemzése rákgenom projektekben
Sebestyén Endre - Computational Genomics, Universitat Pompeu Fabra, Dr. Aiguader 88, E08003 Barcelona, Spain
Az elmúlt évtizedben a rákkutatást jelentősen elősegítette a különféle genomszekvenáló módszerek szédületes fejlődése, és az egyszerre előállítható adatmennyiség. Az e módszerekre alapuló tanulmányok nagy része azonban csak a DNS mutációk katalogizálásával foglalkozik, kevés figyelmet fordítva más változásokra. Az alternatív splicing mechanizmusa, amely lehetővé teszi, hogy egy génről több különböző mRNS változat, majd fehérje is képződjön, valószínűleg fontos szerepet játszik a rák kialakulásában, azonban átfogóan eddig nem vizsgálták. Munkánk során 9 ráktípushoz tartozó mintegy 4000 mintában kerestünk alternatív splicing változásokat, és elemeztük ezek kapcsolatát a DNS-ben fellépő mutációkkal. Több mint 250, mutációktól általában független, adott rákra jellemző, vagy több ráktípusban is előforduló konzisztens változást írtunk le, amelyek lehetséges jövőbeli terápiás célpontok, esetleg diagnosztikai módszerek fejlesztésében is felhasználhatók.
Budapest Science Meetup, 2014. szeptember 11.
Alternatív splicing változások elemzése rákgenom projektekben
Sebestyén Endre - Computational Genomics, Universitat Pompeu Fabra, Dr. Aiguader 88, E08003 Barcelona, Spain
Az elmúlt évtizedben a rákkutatást jelentősen elősegítette a különféle genomszekvenáló módszerek szédületes fejlődése, és az egyszerre előállítható adatmennyiség. Az e módszerekre alapuló tanulmányok nagy része azonban csak a DNS mutációk katalogizálásával foglalkozik, kevés figyelmet fordítva más változásokra. Az alternatív splicing mechanizmusa, amely lehetővé teszi, hogy egy génről több különböző mRNS változat, majd fehérje is képződjön, valószínűleg fontos szerepet játszik a rák kialakulásában, azonban átfogóan eddig nem vizsgálták. Munkánk során 9 ráktípushoz tartozó mintegy 4000 mintában kerestünk alternatív splicing változásokat, és elemeztük ezek kapcsolatát a DNS-ben fellépő mutációkkal. Több mint 250, mutációktól általában független, adott rákra jellemző, vagy több ráktípusban is előforduló konzisztens változást írtunk le, amelyek lehetséges jövőbeli terápiás célpontok, esetleg diagnosztikai módszerek fejlesztésében is felhasználhatók.
Budapest Science Meetup, 2014. szeptember 11.
This document describes various in vitro models and methods that can be used to study hepatotoxicity, including hepatocyte cell cultures, assays to measure cell viability and metabolic activity (trypan blue dye exclusion test, MTT assay), staining to visualize lipid accumulation (Oil Red O), and techniques to examine gene and protein expression changes (RT-PCR, western blotting). Specifically, it discusses using these methods to establish models of non-alcoholic fatty liver disease (NAFLD) by treating hepatocyte cultures with fatty acids like palmitic and oleic acid, and models of drug-induced hepatotoxicity by treating with acetaminophen or amiodarone. Key readouts include lipid accumulation, apoptosis levels
This document summarizes various liver diseases and their etiologies. It discusses alcoholic liver disease, drug-induced liver injury, viral hepatitis infections from hepatitis B, C, and D viruses, autoimmune disorders like autoimmune hepatitis and primary biliary cirrhosis, genetic disorders, non-alcoholic fatty liver disease, cirrhosis, and hepatocellular carcinoma. The liver's important functions are outlined. Causes, pathogenesis, clinical features, diagnosis, and treatment approaches are described for each disease.
An introduction to experimental epidemiology improvemed
This document provides an overview of experimental epidemiology methods. It discusses the key features and types of experimental epidemiology studies, including controlled field trials and community trials. Controlled field trials involve dividing healthy subjects into an exposed group that receives an active substance (like a vaccine) and an unexposed control group that receives a placebo. Community trials involve entire exposed and unexposed communities. Randomized controlled trials, which assign individual subjects randomly to intervention or control groups, are described as the most common experimental method but are covered in more depth separately. Overall, the document outlines the design and purpose of various experimental epidemiology study types.
Genotyping methods of nosocomial infections pathogenimprovemed
Nosocomial infections afflict around 2 million patients in the US each year, resulting in around 88,000 deaths and $4.5 billion in excess healthcare costs. Understanding the distribution and relatedness of pathogens that cause these infections is important for designing effective control methods. Historically, phenotypic characterization was used, but increasingly molecular or genotyping techniques are being used, including pulsed-field gel electrophoresis, multilocus sequence typing, and polymerase chain reaction-based methods. Studies have shown that integrating molecular typing into infection control programs can significantly reduce infection rates and healthcare costs.
Use of MALDI-TOF in the diagnosis of infectious diseasesimprovemed
MALDI-TOF MS has revolutionized clinical microbiology by drastically improving the time needed to identify bacterial cultures from over 24 hours to just a few minutes. Whereas the entire process from sampling to results previously took 2-3 days or more, new methods like MALDI-TOF MS and molecular technology have reduced this to just a few hours or one day. MALDI-TOF MS is a powerful, cost-effective, and easy to implement technique that provides rapid and reliable identification of bacteria and yeast from clinical samples at the genus and species level through analysis of their protein mass spectral signatures.
1. Molecular microbiology methods like PCR and hybridization have revolutionized clinical diagnostics by enabling fast and direct detection of pathogens from clinical samples.
2. PCR in particular has become a mainstay technique, allowing amplification of specific DNA sequences from small amounts of input DNA. Variations like real-time PCR, multiplex PCR, and broad-range PCR further expanded diagnostic capabilities.
3. Emerging technologies like DNA microarrays promise even greater multiplexing, with the ability to simultaneously genotype large genomic regions or measure expression of many genes, positioning them as promising future molecular diagnostic tools.
This document provides information about setting up and conducting experiments with isolated organs and tissue rings, including:
1. Describing the mechanical setup for a four-channel system bath for isolated organs.
2. Explaining the preparation of Krebs-Hanseleit solution and common drugs used.
3. Outlining typical experiment protocols, including stabilizing tissues, pre-contraction testing, and assessing endothelial function.
4. Noting that each experiment begins by preparing Krebs-Hanseleit solution and activating the system before surgery and setting rings in wells.
This document describes the components, work principles, and experimental protocols for using a pressure myograph system to study isolated blood vessels. The system allows measuring vessel diameter in response to drugs and stimuli while maintaining constant temperature. Experiments involve isolating small arteries from rats and attaching them to glass micropipettes in a chamber filled with physiological salt solution. Vessel diameter is recorded under varying pressures and drug exposures to study endothelial function and vasoactive mechanisms. Statistical analysis of diameter changes under different conditions uses repeated measures ANOVA to compare responses between experimental groups.
Notes for Measuring blood flow and reactivity of the blood vessels in the ski...improvemed
This document describes the laser Doppler flowmetry (LDF) method for measuring blood flow in the microcirculation of skin. Specifically, it discusses post-occlusive reactive hyperemia (PORH) testing using LDF to assess microvascular reactivity by inducing a brief occlusion of blood vessels. It also covers iontophoresis of acetylcholine and sodium nitroprusside combined with LDF to evaluate endothelium-dependent and independent vasodilation respectively. Standardization of methods like occlusion duration and probe placement is important for reproducibility. LDF provides a general index of microvascular function rather than direct flow measurements.
Notes for STAINING AND ANALYSIS of HISTOLOGICAL PREPARATIONSimprovemed
This document provides an overview of histological staining techniques. It discusses how histological preparations are stained using interactions between dyes, solvents, and tissue components. Different staining methods result in different colors that highlight various structures. A classic example is hematoxylin and eosin staining, where hematoxylin stains acidic components blue and eosin stains basic components pink. Specialized staining techniques also exist, such as immunohistochemistry. Proper staining selection depends on the tissue and research goals. Histological preparations are then analyzed under a microscope to study cell and tissue morphology.
Notes for Fixation of tissues and organs for educational and scientific purposesimprovemed
Fixation of tissues and organs is done to preserve them for scientific and educational purposes. Various chemical fixatives are used including formaldehyde, alcohols, and acids. Formaldehyde cross-links proteins to harden the tissue while maintaining the original structure. Several fixation protocols are used for different purposes, balancing preservation of color and long-term durability. Key steps include diffusion or injection of fixatives, followed by storage in preservative solutions. Proper fixation and storage are necessary to prevent degradation over time.
The document summarizes the process of preparing tissue samples for histological analysis, including fixation, dehydration, infiltration/embedding, sectioning, staining, and examination. Key steps involve fixing tissues to prevent degradation, dehydrating using increasing alcohol concentrations, infiltrating with paraffin wax or resin for structural support during sectioning, precisely cutting thin sections, mounting them to glass slides, staining, and examining under a microscope. The quality of prepared samples depends on carefully following each step of the preparation process.
Notes for The principle and performance of capillary electrophoresisimprovemed
This document provides an overview of capillary electrophoresis (CE). It begins by introducing CE and its advantages over other separation techniques. It then describes the basic theory behind CE, including electrophoretic mobility, electroosmotic flow, and how samples migrate through the capillary when an electric field is applied. The document details the key components of a CE instrument and various CE separation techniques such as capillary zone electrophoresis, micellar electrokinetic chromatography, and capillary isoelectric focusing. It focuses on the principles and applications of CE.
Notes for The principle and performance of liquid chromatography–mass spectro...improvemed
This document provides an overview of liquid chromatography-mass spectrometry (LC-MS). It describes the basic components and functioning of an LC-MS system, including the liquid chromatograph and mass spectrometer connected by an interface. The document discusses various ionization sources like electrospray ionization and atmospheric pressure chemical ionization, as well as mass analyzers like quadrupoles and time-of-flight analyzers. It also covers detectors used in LC-MS like electron multipliers and photomultipliers. Overall, the document serves as a technical introduction to the principles and components of LC-MS.
This document provides an overview of basic cell culture techniques. It discusses the history of cell culture, defining primary and secondary cell cultures. It describes different types of cell lines and how cells grow as monolayers or in suspension. The document outlines the key equipment needed for a cell culture laboratory, including biosafety cabinets, CO2 incubators, centrifuges, microscopes, and supplies. It emphasizes the importance of aseptic technique to prevent microbial contamination when working with cell cultures.
This document discusses systems biology and its goals of understanding how biological molecules interact and systems function as a whole. It covers:
1) Systems biology uses large datasets from "omics" experiments and computational models to understand complex biological interactions beyond individual molecules.
2) Pioneering work used microarrays to measure thousands of genes in serum-stimulated cells, finding over 500 changed in proliferation.
3) The field aims to discover emergent system properties and functions not evident from separate parts, like switches that change cell behavior.
Systems biology for Medicine' is 'Experimental methods and the big datasetsimprovemed
This document discusses experimental methods used in systems biology to generate large datasets, including microarrays, sequencing-based methods, mass spectrometry, and liquid chromatography. It explains that systems biology studies must be quantitative and enable computational modeling. Key methods covered are microarrays, RNA-seq, ChIP-seq, whole-genome sequencing, whole-exome sequencing, proteomics using mass spectrometry, and combining liquid chromatography with mass spectrometry for lipidomics, metabolomics and glycomics. Sources of variation are also discussed for genomic and proteomic studies.
Systems biology for medical students/Systems medicineimprovemed
Systems biology takes a holistic approach to studying biological systems by considering all the interactions within a system and how they generate complex behaviors. Lecture 1 introduces key concepts in systems biology like how increasing levels of biological organization give rise to new system properties like robustness. Lecture 2 discusses experimental methods like genomics, proteomics, and metabolomics that generate large data sets for systems analysis. Lecture 3 covers mathematical and statistical tools for analyzing these data sets, such as using differential equations to model signaling networks. Lecture 4 provides examples of medical applications of systems biology in finding diagnostic markers, personalizing therapy, and predicting disease interactions from human disease networks, with the future of medicine taking a more predictive, preventive, and personalized approach
The document discusses several use cases for applying data mining and machine learning techniques in healthcare and biomedical research. Three examples are:
1) Early diagnosis of cancers like lung cancer and breast cancer through predictive modeling of patient data to detect cancers at earlier stages when survival rates are higher.
2) Predicting patient responses to drug therapies for cancers like breast cancer by combining different types of molecular profiling data using techniques like support vector machines and random forests.
3) Using imaging data and temporal analysis of metrics like medication purchases to better understand and predict chronic diseases like diabetes and associated health complications.
This document describes various in vitro models and methods that can be used to study hepatotoxicity, including hepatocyte cell cultures, assays to measure cell viability and metabolic activity (trypan blue dye exclusion test, MTT assay), staining to visualize lipid accumulation (Oil Red O), and techniques to examine gene and protein expression changes (RT-PCR, western blotting). Specifically, it discusses using these methods to establish models of non-alcoholic fatty liver disease (NAFLD) by treating hepatocyte cultures with fatty acids like palmitic and oleic acid, and models of drug-induced hepatotoxicity by treating with acetaminophen or amiodarone. Key readouts include lipid accumulation, apoptosis levels
This document summarizes various liver diseases and their etiologies. It discusses alcoholic liver disease, drug-induced liver injury, viral hepatitis infections from hepatitis B, C, and D viruses, autoimmune disorders like autoimmune hepatitis and primary biliary cirrhosis, genetic disorders, non-alcoholic fatty liver disease, cirrhosis, and hepatocellular carcinoma. The liver's important functions are outlined. Causes, pathogenesis, clinical features, diagnosis, and treatment approaches are described for each disease.
An introduction to experimental epidemiology improvemed
This document provides an overview of experimental epidemiology methods. It discusses the key features and types of experimental epidemiology studies, including controlled field trials and community trials. Controlled field trials involve dividing healthy subjects into an exposed group that receives an active substance (like a vaccine) and an unexposed control group that receives a placebo. Community trials involve entire exposed and unexposed communities. Randomized controlled trials, which assign individual subjects randomly to intervention or control groups, are described as the most common experimental method but are covered in more depth separately. Overall, the document outlines the design and purpose of various experimental epidemiology study types.
Genotyping methods of nosocomial infections pathogenimprovemed
Nosocomial infections afflict around 2 million patients in the US each year, resulting in around 88,000 deaths and $4.5 billion in excess healthcare costs. Understanding the distribution and relatedness of pathogens that cause these infections is important for designing effective control methods. Historically, phenotypic characterization was used, but increasingly molecular or genotyping techniques are being used, including pulsed-field gel electrophoresis, multilocus sequence typing, and polymerase chain reaction-based methods. Studies have shown that integrating molecular typing into infection control programs can significantly reduce infection rates and healthcare costs.
Use of MALDI-TOF in the diagnosis of infectious diseasesimprovemed
MALDI-TOF MS has revolutionized clinical microbiology by drastically improving the time needed to identify bacterial cultures from over 24 hours to just a few minutes. Whereas the entire process from sampling to results previously took 2-3 days or more, new methods like MALDI-TOF MS and molecular technology have reduced this to just a few hours or one day. MALDI-TOF MS is a powerful, cost-effective, and easy to implement technique that provides rapid and reliable identification of bacteria and yeast from clinical samples at the genus and species level through analysis of their protein mass spectral signatures.
1. Molecular microbiology methods like PCR and hybridization have revolutionized clinical diagnostics by enabling fast and direct detection of pathogens from clinical samples.
2. PCR in particular has become a mainstay technique, allowing amplification of specific DNA sequences from small amounts of input DNA. Variations like real-time PCR, multiplex PCR, and broad-range PCR further expanded diagnostic capabilities.
3. Emerging technologies like DNA microarrays promise even greater multiplexing, with the ability to simultaneously genotype large genomic regions or measure expression of many genes, positioning them as promising future molecular diagnostic tools.
This document provides information about setting up and conducting experiments with isolated organs and tissue rings, including:
1. Describing the mechanical setup for a four-channel system bath for isolated organs.
2. Explaining the preparation of Krebs-Hanseleit solution and common drugs used.
3. Outlining typical experiment protocols, including stabilizing tissues, pre-contraction testing, and assessing endothelial function.
4. Noting that each experiment begins by preparing Krebs-Hanseleit solution and activating the system before surgery and setting rings in wells.
This document describes the components, work principles, and experimental protocols for using a pressure myograph system to study isolated blood vessels. The system allows measuring vessel diameter in response to drugs and stimuli while maintaining constant temperature. Experiments involve isolating small arteries from rats and attaching them to glass micropipettes in a chamber filled with physiological salt solution. Vessel diameter is recorded under varying pressures and drug exposures to study endothelial function and vasoactive mechanisms. Statistical analysis of diameter changes under different conditions uses repeated measures ANOVA to compare responses between experimental groups.
Notes for Measuring blood flow and reactivity of the blood vessels in the ski...improvemed
This document describes the laser Doppler flowmetry (LDF) method for measuring blood flow in the microcirculation of skin. Specifically, it discusses post-occlusive reactive hyperemia (PORH) testing using LDF to assess microvascular reactivity by inducing a brief occlusion of blood vessels. It also covers iontophoresis of acetylcholine and sodium nitroprusside combined with LDF to evaluate endothelium-dependent and independent vasodilation respectively. Standardization of methods like occlusion duration and probe placement is important for reproducibility. LDF provides a general index of microvascular function rather than direct flow measurements.
Notes for STAINING AND ANALYSIS of HISTOLOGICAL PREPARATIONSimprovemed
This document provides an overview of histological staining techniques. It discusses how histological preparations are stained using interactions between dyes, solvents, and tissue components. Different staining methods result in different colors that highlight various structures. A classic example is hematoxylin and eosin staining, where hematoxylin stains acidic components blue and eosin stains basic components pink. Specialized staining techniques also exist, such as immunohistochemistry. Proper staining selection depends on the tissue and research goals. Histological preparations are then analyzed under a microscope to study cell and tissue morphology.
Notes for Fixation of tissues and organs for educational and scientific purposesimprovemed
Fixation of tissues and organs is done to preserve them for scientific and educational purposes. Various chemical fixatives are used including formaldehyde, alcohols, and acids. Formaldehyde cross-links proteins to harden the tissue while maintaining the original structure. Several fixation protocols are used for different purposes, balancing preservation of color and long-term durability. Key steps include diffusion or injection of fixatives, followed by storage in preservative solutions. Proper fixation and storage are necessary to prevent degradation over time.
The document summarizes the process of preparing tissue samples for histological analysis, including fixation, dehydration, infiltration/embedding, sectioning, staining, and examination. Key steps involve fixing tissues to prevent degradation, dehydrating using increasing alcohol concentrations, infiltrating with paraffin wax or resin for structural support during sectioning, precisely cutting thin sections, mounting them to glass slides, staining, and examining under a microscope. The quality of prepared samples depends on carefully following each step of the preparation process.
Notes for The principle and performance of capillary electrophoresisimprovemed
This document provides an overview of capillary electrophoresis (CE). It begins by introducing CE and its advantages over other separation techniques. It then describes the basic theory behind CE, including electrophoretic mobility, electroosmotic flow, and how samples migrate through the capillary when an electric field is applied. The document details the key components of a CE instrument and various CE separation techniques such as capillary zone electrophoresis, micellar electrokinetic chromatography, and capillary isoelectric focusing. It focuses on the principles and applications of CE.
Notes for The principle and performance of liquid chromatography–mass spectro...improvemed
This document provides an overview of liquid chromatography-mass spectrometry (LC-MS). It describes the basic components and functioning of an LC-MS system, including the liquid chromatograph and mass spectrometer connected by an interface. The document discusses various ionization sources like electrospray ionization and atmospheric pressure chemical ionization, as well as mass analyzers like quadrupoles and time-of-flight analyzers. It also covers detectors used in LC-MS like electron multipliers and photomultipliers. Overall, the document serves as a technical introduction to the principles and components of LC-MS.
This document provides an overview of basic cell culture techniques. It discusses the history of cell culture, defining primary and secondary cell cultures. It describes different types of cell lines and how cells grow as monolayers or in suspension. The document outlines the key equipment needed for a cell culture laboratory, including biosafety cabinets, CO2 incubators, centrifuges, microscopes, and supplies. It emphasizes the importance of aseptic technique to prevent microbial contamination when working with cell cultures.
This document discusses systems biology and its goals of understanding how biological molecules interact and systems function as a whole. It covers:
1) Systems biology uses large datasets from "omics" experiments and computational models to understand complex biological interactions beyond individual molecules.
2) Pioneering work used microarrays to measure thousands of genes in serum-stimulated cells, finding over 500 changed in proliferation.
3) The field aims to discover emergent system properties and functions not evident from separate parts, like switches that change cell behavior.
Systems biology for Medicine' is 'Experimental methods and the big datasetsimprovemed
This document discusses experimental methods used in systems biology to generate large datasets, including microarrays, sequencing-based methods, mass spectrometry, and liquid chromatography. It explains that systems biology studies must be quantitative and enable computational modeling. Key methods covered are microarrays, RNA-seq, ChIP-seq, whole-genome sequencing, whole-exome sequencing, proteomics using mass spectrometry, and combining liquid chromatography with mass spectrometry for lipidomics, metabolomics and glycomics. Sources of variation are also discussed for genomic and proteomic studies.
Systems biology for medical students/Systems medicineimprovemed
Systems biology takes a holistic approach to studying biological systems by considering all the interactions within a system and how they generate complex behaviors. Lecture 1 introduces key concepts in systems biology like how increasing levels of biological organization give rise to new system properties like robustness. Lecture 2 discusses experimental methods like genomics, proteomics, and metabolomics that generate large data sets for systems analysis. Lecture 3 covers mathematical and statistical tools for analyzing these data sets, such as using differential equations to model signaling networks. Lecture 4 provides examples of medical applications of systems biology in finding diagnostic markers, personalizing therapy, and predicting disease interactions from human disease networks, with the future of medicine taking a more predictive, preventive, and personalized approach
The document discusses several use cases for applying data mining and machine learning techniques in healthcare and biomedical research. Three examples are:
1) Early diagnosis of cancers like lung cancer and breast cancer through predictive modeling of patient data to detect cancers at earlier stages when survival rates are higher.
2) Predicting patient responses to drug therapies for cancers like breast cancer by combining different types of molecular profiling data using techniques like support vector machines and random forests.
3) Using imaging data and temporal analysis of metrics like medication purchases to better understand and predict chronic diseases like diabetes and associated health complications.
1. Improved Medical Education in Basic
Sciences
for Better Medical Practicing
ImproveMEd
Rendszerbiológia orvostudományhoz
III. Hogyan elemezzük a nagy adatkészleteket?
2. A rendszerbiológiai tanulmányok
gyakran kezdenek expressziós
profillal (a gyógyszerrel kezelt, illetve
a nem kezelt sejtek, a normális
versus rákos sejtek, a különböző
fejlődési szakaszokban lévő sejtek) ...
microarray vagy RNAseq
használatával... microarray
használata költséghatékony
megközelítés ...
És ezt kapjuk
3. A microarray 10 000 foltot tartalmazhat. Tegyük fel, hogy
minden pont egy gén - hogyan szervezünk foltokat /
géneket az eredmény extraktálása érdekében?
A lézer szkenner egy fluoreszcens címkét mér, majd egy
másikat, amit ráhelyez és így tovább. Minden címkét
kétszer szkenneli.
a fluoreszcens jel intenzitása = a kötött DNS mennyisége
Minden pont helyettesíthető egy számmal, amely relatív
változást jelent a „normál” szinttől.
N = R / G… ..1 egyenlő expressziót jelent mindkét mintában
R = vörös fluoreszcencia (tumor)
4. A színeket számokká alakítják, mert a számok könnyebben
kezelhetők!
Minden pont helyettesíthető egy olyan számmal, amely relatív
változást jelent a "normál" szintekről.
R = piros fluoreszcencia (tumor)
G = zöld fluoreszcencia (normál sejt)
N = R/G
N=1 egyenlő expresszió mindkét mintában
N›1 indukció
N‹1 repressuió
http://www.hhmi.org/biointeractive/how-analyze-dna-microarray-
data
http://www.hhmi.org/biointeractive/scanning-lifes-matrix-genes-
proteins-and-small-molecules
Több mintát hasonlíthatunk össze ...
vagy egyet időben követünk- humán
fibroblasztok szérummal stimulálva és
24 órán keresztül követve (Iyer et al.,
1999)
And organize genes so that
induced one are clustered at
one end-opposite from
repressed one…
Az adatok ilyen bemutatását Heat Map (Heat Map)
nevezzük
5. A nagy adatokból származó ismeretek
kibontásához statisztikai módszerekre
van szükségünk!
Gyakran használt - R statisztikai csomag
LIMMA
A klaszterek azonosításához
használhatunk - klaszterelemzést!
Eredeti számok logaritmizáltak (2-es
vagy 10-es bázissal), és a hasonlósági
pontszámok kiszámításánál a
microarray platformot kísérő
számítógépes program segítségével.
Az adatok vizuális megjelenítéséhez a
számokat színre cseréljük, de ezúttal a
zöld az repressziót, a vörös pedig az
indukciót jelenti.
6. Az adatok bemutatásának másik
módja a Volcano plot (a GWS
tanulmányok esetében gyakori).
Az adatokat a "szórvány-plot" -on
mutatjuk be, hogy gyorsan
megtalálhassuk a
legérdekesebbeket, pl. génjelölt
bizonyos betegségben.
Két statisztikai vizsgálatot
kombinál: egy p értéket egy
ANOVA modelltől a változás
nagyságával.
Az adatok gyors felismerése
(gének, stb.), Amelyek
nagymértékű, statisztikailag
szignifikáns változásokat mutatnak.
A p>0.05 &
p<0.05
közötti határ
A két mintában az azonos paraméterek közötti különbség "fold change"-
ként jelenik meg.
A szürke változások kisebbek, mint 2x.
http://genomicsclass.github.io/book/pages/using_limma.html
Statisztikai
szignifikancia
Érdekes adatok
7. Mind a Heat Map, mind a Volcano Plot (és a mögöttük álló
statisztikai elemzés) az első lépés a megfigyelt fenotípus
mögötti gének / fehérjék azonosítása és rangsorolása felé. A
megfigyelt mechanizmusokért vagy potenciális terápiás
célokért felelős gének listáját a különböző bioinformatikai
eszközök lehet feldolgozni.
The gene list can be fed into: Gene Ontology, géncsoport dúsulás
vizsgálata,
Transzkripciós faktor analízis…
A létrehozott listáknak az egyedi nómenklatúrát kell használniuk ahhoz, hogy kölcsönösen összehasonlíthatók legyenek.
8. Gene Ontology – http://geneontology.org/
Bioinformatikai eszköz, amely alkalmas arra, hogy a
megfelelő nevet hozzárendelje a szekvenciához és
összekapcsolja a molekuláris változásokat a
sejtfolyamatokkal
A gének és a fehérjék a legtöbb élő szervezetben
megmaradnak, és közös funkciók vannak. A gén szerepe az
egyik szervezetben segíthet a másikban betöltött
szerepének megvilágításában. A Gene Ontológia
Consortium foglalkozik a génnómenklatúrával.
A készleteket az alábbiak szerint szervezzük:
Biológiai folyamat
Molekuláris funkció
Celluláris rekesz
The Gene Ontology Consortium, Nature, 2000.
Biológiai folyamatok, például: sejtnövekedés,
proliferáció, transzláció vagy cAMP szintézis ...
11. géncsoport dúsulás vizsgálata– GSEA
Analitikai módszer a génkészletek megtalálására és
értelmezésére.
Olyan géneket keres, amelyek együtt változnak
meghatározza az azonos jelátviteli útvonalon részt vevő
fehérjék szintjét
ugyanazon biológiai folyamatban részt vevő molekulákat
keresi
Ingyenes szoftvercsomag 1,325 biológiailag definiált
géncsoport kezdeti adatbázisával.
http://software.broadinstitute.org/gsea/index.jsp
Subramanian et al. (2005) PNAS 102:15545
1. Szortírozza a géneket egy kritérium, pl. expressziós szint
szerint
2. Hasonlítsa össze a listát egyes már létező listákkal, és
rendelje hozzá az egyes géneket az "erichrichment score" -
hez - a túlreprezentált vagy túlzottan csökkentett gének a
Kolmogorov-Smirnov típusú statisztikák szerint
3. A Max. Enrichment Score (MES) egy létező gén
relevancia-mutatója egy új adatkészlethez, amelyet most
vizsgálnak
12. Transzkripciós faktor analízis
Az expresszálódás szintjét megváltoztató géneket
ugyanaz a transzkripciós faktor szabályozhatja.
A géneket az omics adatok és az előzetes ismeretek
kombinálásával azonosítják.
A ChEA adatbázis jelenleg 159 transzkripciós faktort
kapcsol össze több mint 30 000 génnel - összesen
361 299 interakcióval -, amelyek 157 publikációból
származnak.
TRANSFAC, PAINT, JASPAR - egyéb adatbázisok a ChIP
számára
Kináz dúsítás elemzése (KEA)
Web alapú parancssori szoftver, amely összeköti az
emlős fehérjék listáját a protein kinázokkal, amelyek
valószínűleg foszforilizálják őket. Az adatbázis 436
kinázot és 14 374 interakciót tartalmaz 3469
publikációból.
http://amp.pharm.mssm.edu/Enrichr/
https://www.ncbi.nlm.nih.gov/pmc/articl
es/PMC2944209/
14. A kromatin immunprecipitáció
egy választott módszer a
fehérjékkel kölcsönhatásban
lévő összes szekvencia
megtalálására. Az összes ChIP-
seq kísérletből származó adatok
ugyanabban az adatbázisban
(ChEA) táplálhatók
...https://galaxyproject.org/tutorials/chip/
15. Expression2Kinases –X2K
A szoftver, amely egyesíti a különböző
adatbázisokat és eszközöket.
INPUT: a különbözőképpen expresszált gének
listája
OUTPUT: protein kinázok, transzkripciós faktorok
és proteinkomplexek, amelyek a bejuttatott gének
feltételezett szabályozói.
Ilyen szoftverek felhasználásával hipotetikus
szabályozási útvonalakat építhetünk fel, és protein-
interakciós hálózatokat hozhatunk létre.
Az eredményeket kísérleti bizonyítékokkal is alá kell
támasztani!
The work-flow of X2K
Chen et al. (2012) Bioinformatics 28:105
16. Amit igazán akarunk az, hogy a listát hálózattá alakítsuk át -
gyakran használják a sejtösszetevők közötti kölcsönhatások
kimutatására
Euler, 1700s, Seven Bridges of Konigsberg
Csomópont
molekula
Él interakció
17. A rendszerbiológiához kapcsolódó hálózatok típusai
1. Sejt jelátviteli hálózatok
- rák jelátviteli hálózat
doi:10.1038/psp.2013.38
2. Protein-protein interakciós hálózatok
- Dystrophin fehérje-fehérje kereszteződések
http://parendogen677s10.weebly.com/protein-protein-interactions.html
3. Génszabályozó hálózatok
- A Drosophila szem fejlődése
- http://dev.biologists.org/content/140/1/82
18. Genes2Networks
Lists2Networks
Kombinálja a kísérleti adatokat (mRNS
expressziós mikroarray, genom-wide ChI-X,
RNAi screen, proteomika és
foszfoproteomika) minden ismert
kölcsönhatás (előzetes biológiai tudás)
http://www.lists2networks.org
19.
20. További szoftverek léteznek a hálózatok vizualizálásához és
elemzéséhez:
Pajek (Vladimir Batagelj & Andrej Mrvar, Ljubljana,
Slovenia)
http://vlado.fmf.uni-
lj.si/pub/networks/doc/gd.01/Pajek2.png
http://vlado.fmf.uni-lj.si/pub/networks/doc/pajek.pdf
Cytoscape (Trey Ideker, Shannon et al.,2003.))
http://www.cytoscape.org/
SNAVI (Ma’ayan et al. 2009)
yEd…..
Az útvonalak, alhálózatok, klaszterek, a hálózati sajátosságok
azonosítása ...
21. A molekuláris adatokat tovább lehetne
integrálni a strukturális adatokkal a 3D
modellek (makromolekuláris komplexek,
virtuális sejtek) előállítása érdekében.
Patwardhan és mtsai. 2017, DOI: 10,7554 /
eLife.25835
(plazmodiummal fertőzött eritrociták)
22. 1. A statisztikai elemzés kritikus fontosságú a nagy adathalmazokról
szerzett tudásbővítés során. A statisztikai analízis a vizsgálat
szempontjából releváns gének / fehérjék / RNS-ek listáját állítja
elő.
2. A gének listáját a bioinformatikai eszközökbe lehet bevinni, és az
előzetes ismeretekkel kombinálva új elméleti utakat,
alhálózatokat, szabályozási mechanizmust találhatunk ...
3. A kísérleti nagy adathalmazok és a korábbi ismeretek (több
adatbázis) integrálása lehetővé teszi a fiziológiás funkciók,
patofiziológia vagy farmakokinetika sokrétű megértését.
4. A számítással előállított jóslatokat kísérletileg bizonyítani kell.