SlideShare a Scribd company logo
Next-Generation Sequencing and its
Applications in RNA-Seq

                        Perdacher Martin




                  1
Overview

• Next-Generation Sequencing
 • technologies
 • applications
• RNA-Seq

                   2
Next-Generation Sequencing
           technologies


• Ion semiconductor sequencing
• Pyrosequencing
• Sequencing by ligation
• Cyclic reversible termination

                    3
Ion semiconductor sequencing




             4
Pyrosequencing


• pyrophosphate (PPi) is translated into light
  emission in two enzymatic steps
  • ATP-sulfurylase converts PPi in ATP
  • luciferase converts Luciferin in
    oxiluciferin and emitted light (ATP is the
    fuel for this reaction).


                      5
Sequencing by ligation




          6
Cyclic reversible termination
• incorporation of modified nucleotides
• fluorescence imaging
• washing of fluorescent dye and removing of
  terminating group




                      7
Overview

• Next-Generation Sequencing
 • technologies
 • applications
• RNA-Seq

                   8
Anwendungen von Next-Generation
           Sequencing
                             Transcriptome Sequencing:


• Transcriptome sequencing
                             RNA Sequenzierung
                                  coding
                                  non-coding (rRNA, tRNA, miRNA,
                                  siRNA, piRNA)



• Genomic sequencing         Genomic Sequencing
                                 de novo
                                 resequencing (copy number variation,



• Epigenetic applications
                                 SNP point mutations)

                             Epigenetic
                                  Chip Seq



• Additional applications
                                  Methylation profiling

                             Additional:
                                  Clip Seq, RIP-Seq, HITS Clip, - RNA
                                  interaktion mit RNA-bindenden Proteinen




                    9
Overview

• Next-Generation Sequencing
 • technologies
 • applications
• RNA-Seq

                   10
RNA-Seq
• biological background
• workflow of RNA-Seq
• transcriptome reconstruction
• differential gene expression

                    11
background biology of RNA-Seq




              12
RNA-Seq

• biological background
• workflow of RNA-Seq
• transcriptome reconstruction
• differential gene expression

                    13
Workflow of RNA-Seq

• sample preparation        ribo minus and strand specific libs
                            (paired end)




• seqeuncing
• read mapping
                            Fragmentation of cDNA
                            Purification

                            Adapter ligation
                            Size-based purification of ligation
                            products using

                            PCR of ligation products


                            Purification and sequencing of the
                                fragments




                       14
Workflow of RNA-Seq

• sample preparation        ribo minus and strand specific libs
                            (paired end)




• seqeuncing
• read mapping
                            Fragmentation of cDNA
                            Purification

                            Adapter ligation
                            Size-based purification of ligation
                            products using

                            PCR of ligation products


                            Purification and sequencing of the
                                fragments




                       14
Read mapping in RNA-Seq




           15
Read mapping
                               unspliced alignment match PERFECTLY the



•
                               genome

    unspliced read alignment   seed : after mapping, the seed is extended with
                               smith waterman method



    • seed methods
                               Burrow-Wheeler: transform genome into
                               efficient data structure




    • Burrows-Wheeler transform methods
• spliced aligners
 • exon first
 • seed and extend
                      16
Read mapping (spliced alignment
                                        methods)
2 steps:
     unspliced read aligner                                   break reads into shorter
                                                              (k-mer-)seeds.
     unmapped reads are
     split into fragments                                     seed-regions are evaluated
     and aligned                                              with sensitive alignment
     independently                                            methods (smith
                                                              waterman)

                                                              much slower method




                                                           pseudogene is dysfunctional
                                                           and does not contain
                                                           introns. in the first step of
                                                           exon-first approaches reads
                                                           are aligned to the genome
                                                           based on unspliced read
                                                           mappers. here it is detected
                                                           as gene and not as
                                                           pseudogene.




                                             17
RNA-Seq

• biological background
• workflow of RNA-Seq
• transcriptome reconstruction
• differential gene expression

                18
Transcriptome reconstruction
                               exon identification: is used for very


• genome guided reconstruction
                               short reads (36 bp)

                               first map reads to the genome. the
                               unmapped reads are tested for all


 • exon identification
                               possibilities of exon-exon junctions

                               not able to identify full transcript
                               structures



 • genome-guided assembly
• genome independent reconstruction


                   19
Transcriptome reconstruction
                               exon identification: is used for very


• genome guided reconstruction
                               short reads (36 bp)

                               first map reads to the genome. the
                               unmapped reads are tested for all


 • exon identification
                               possibilities of exon-exon junctions

                               not able to identify full transcript
                               structures



 • genome-guided assembly
• genome independent reconstruction


                   19
Transcriptome reconstruction
                                                           break reads into k-mers.
align reads to the genome                                  build de-brujin graph.
and use spliced reads to
build transcript graph                                     alignment to the genome
(node: read fragment,                                      is because of annotation
edge: link bet ween                                        purpose
fragments) one paht
through the graph
represents an isoform of
the transcript)




                                         20
RNA-Seq

• biological background
• workflow of RNA-Seq
• transcriptome reconstruction
• differential gene expression

                 21
Differential gene expression for RNA-Seq


 • comparing the count of reads for one gene
   in different experiments (conditions)
 • the problem is that one read cannot
   assigned uniquely to one gene, because of
   the overlap of genes and their different
   isoforms



                      22
Differential gene expression for RNA-Seq




                   23
Summary
• Next-Generation Sequencing, (technologies
  and applications)
• RNA-Seq
  • biology
  • workflow of RNA-Seq (read mapping)
  • transcriptome reconstruction
  • differential gene expression
                      24

More Related Content

What's hot

Tag-based transcript sequencing: Comparison of SAGE and CAGE
Tag-based transcript sequencing: Comparison of SAGE and CAGETag-based transcript sequencing: Comparison of SAGE and CAGE
Tag-based transcript sequencing: Comparison of SAGE and CAGE
Matthias Harbers
 
RNASeq - Analysis Pipeline for Differential Expression
RNASeq - Analysis Pipeline for Differential ExpressionRNASeq - Analysis Pipeline for Differential Expression
RNASeq - Analysis Pipeline for Differential Expression
Jatinder Singh
 
Next generation sequencing
Next generation sequencingNext generation sequencing
Next generation sequencing
PALANIANANTH.S
 
Next generation sequencing
Next generation sequencingNext generation sequencing
Next generation sequencing
Swathi Prabakar
 
RNA sequencing: advances and opportunities
RNA sequencing: advances and opportunities RNA sequencing: advances and opportunities
RNA sequencing: advances and opportunities
Paolo Dametto
 
Introduction to next generation sequencing
Introduction to next generation sequencingIntroduction to next generation sequencing
Introduction to next generation sequencing
VHIR Vall d’Hebron Institut de Recerca
 
Oxford nanopore sequencing
Oxford nanopore sequencingOxford nanopore sequencing
Oxford nanopore sequencing
Sangeetha80717
 
1.Role of next generation sequencing in plant pathology
1.Role of next generation sequencing in plant pathology1.Role of next generation sequencing in plant pathology
1.Role of next generation sequencing in plant pathology
*noT yeT workinG! !M stilL studyinG*
 
Next generation-sequencing.ppt-converted
Next generation-sequencing.ppt-convertedNext generation-sequencing.ppt-converted
Next generation-sequencing.ppt-converted
Shweta Tiwari
 
next generation sequencing (recent collection2018)
next generation sequencing (recent collection2018) next generation sequencing (recent collection2018)
next generation sequencing (recent collection2018)
Newborn Screening KW
 
Next generation sequencing
Next generation sequencingNext generation sequencing
Next generation sequencing
Tapish Goel
 
Next generation sequencing technologies for crop improvement
Next generation sequencing technologies for crop improvementNext generation sequencing technologies for crop improvement
Next generation sequencing technologies for crop improvement
anjaligoud
 
New generation Sequencing
New generation Sequencing New generation Sequencing
New generation Sequencing
Vijay Raj Yanamala
 
Next generation sequencing
Next generation sequencingNext generation sequencing
Next generation sequencing
ehsan sepahi
 
Next generation sequencing
Next generation sequencingNext generation sequencing
Next generation sequencing
Uzma Jabeen
 
Sequenced taged sites (sts)
Sequenced taged sites (sts)Sequenced taged sites (sts)
Sequenced taged sites (sts)
DHANRAJ GIRIMAL
 
Why Transcriptome? Why RNA-Seq? ENCODE answers….
Why Transcriptome? Why RNA-Seq?  ENCODE answers….Why Transcriptome? Why RNA-Seq?  ENCODE answers….
Why Transcriptome? Why RNA-Seq? ENCODE answers….
Mohammad Hossein Banabazi
 
Sequence assembly
Sequence assemblySequence assembly
Sequence assembly
Ramya P
 
New generation sequencing equipments
New generation sequencing equipmentsNew generation sequencing equipments
New generation sequencing equipmentsKalaivani P
 
Introduction to NGS
Introduction to NGSIntroduction to NGS
Introduction to NGS
cursoNGS
 

What's hot (20)

Tag-based transcript sequencing: Comparison of SAGE and CAGE
Tag-based transcript sequencing: Comparison of SAGE and CAGETag-based transcript sequencing: Comparison of SAGE and CAGE
Tag-based transcript sequencing: Comparison of SAGE and CAGE
 
RNASeq - Analysis Pipeline for Differential Expression
RNASeq - Analysis Pipeline for Differential ExpressionRNASeq - Analysis Pipeline for Differential Expression
RNASeq - Analysis Pipeline for Differential Expression
 
Next generation sequencing
Next generation sequencingNext generation sequencing
Next generation sequencing
 
Next generation sequencing
Next generation sequencingNext generation sequencing
Next generation sequencing
 
RNA sequencing: advances and opportunities
RNA sequencing: advances and opportunities RNA sequencing: advances and opportunities
RNA sequencing: advances and opportunities
 
Introduction to next generation sequencing
Introduction to next generation sequencingIntroduction to next generation sequencing
Introduction to next generation sequencing
 
Oxford nanopore sequencing
Oxford nanopore sequencingOxford nanopore sequencing
Oxford nanopore sequencing
 
1.Role of next generation sequencing in plant pathology
1.Role of next generation sequencing in plant pathology1.Role of next generation sequencing in plant pathology
1.Role of next generation sequencing in plant pathology
 
Next generation-sequencing.ppt-converted
Next generation-sequencing.ppt-convertedNext generation-sequencing.ppt-converted
Next generation-sequencing.ppt-converted
 
next generation sequencing (recent collection2018)
next generation sequencing (recent collection2018) next generation sequencing (recent collection2018)
next generation sequencing (recent collection2018)
 
Next generation sequencing
Next generation sequencingNext generation sequencing
Next generation sequencing
 
Next generation sequencing technologies for crop improvement
Next generation sequencing technologies for crop improvementNext generation sequencing technologies for crop improvement
Next generation sequencing technologies for crop improvement
 
New generation Sequencing
New generation Sequencing New generation Sequencing
New generation Sequencing
 
Next generation sequencing
Next generation sequencingNext generation sequencing
Next generation sequencing
 
Next generation sequencing
Next generation sequencingNext generation sequencing
Next generation sequencing
 
Sequenced taged sites (sts)
Sequenced taged sites (sts)Sequenced taged sites (sts)
Sequenced taged sites (sts)
 
Why Transcriptome? Why RNA-Seq? ENCODE answers….
Why Transcriptome? Why RNA-Seq?  ENCODE answers….Why Transcriptome? Why RNA-Seq?  ENCODE answers….
Why Transcriptome? Why RNA-Seq? ENCODE answers….
 
Sequence assembly
Sequence assemblySequence assembly
Sequence assembly
 
New generation sequencing equipments
New generation sequencing equipmentsNew generation sequencing equipments
New generation sequencing equipments
 
Introduction to NGS
Introduction to NGSIntroduction to NGS
Introduction to NGS
 

Viewers also liked

An introduction to RNA-seq data analysis
An introduction to RNA-seq data analysisAn introduction to RNA-seq data analysis
An introduction to RNA-seq data analysis
AGRF_Ltd
 
DNA sequencing methods
DNA sequencing methodsDNA sequencing methods
DNA sequencing methods
Abo algasim Goma
 
Introduction to RNA-seq and RNA-seq Data Analysis (UEB-UAT Bioinformatics Cou...
Introduction to RNA-seq and RNA-seq Data Analysis (UEB-UAT Bioinformatics Cou...Introduction to RNA-seq and RNA-seq Data Analysis (UEB-UAT Bioinformatics Cou...
Introduction to RNA-seq and RNA-seq Data Analysis (UEB-UAT Bioinformatics Cou...
VHIR Vall d’Hebron Institut de Recerca
 
RNA-seq: general concept, goal and experimental design - part 1
RNA-seq: general concept, goal and experimental design - part 1RNA-seq: general concept, goal and experimental design - part 1
RNA-seq: general concept, goal and experimental design - part 1
BITS
 
RNA Sequencing for Full Length Transcript Discovery
RNA Sequencing for Full Length Transcript DiscoveryRNA Sequencing for Full Length Transcript Discovery
RNA Sequencing for Full Length Transcript Discovery
Anne Deslattes Mays
 
RNA-Seq To Identify Novel Markers For Research on Neural Tissue Differentiation
RNA-Seq To Identify Novel Markers For Research on Neural Tissue DifferentiationRNA-Seq To Identify Novel Markers For Research on Neural Tissue Differentiation
RNA-Seq To Identify Novel Markers For Research on Neural Tissue Differentiation
Thermo Fisher Scientific
 
Next generation sequencing methods (final edit)
Next generation sequencing methods (final edit)Next generation sequencing methods (final edit)
Next generation sequencing methods (final edit)
Mrinal Vashisth
 
2012 august 16 systems biology rna seq v2
2012 august 16 systems biology rna seq v22012 august 16 systems biology rna seq v2
2012 august 16 systems biology rna seq v2Anne Deslattes Mays
 
Catalyzing Plant Science Research with RNA-seq
Catalyzing Plant Science Research with RNA-seqCatalyzing Plant Science Research with RNA-seq
Catalyzing Plant Science Research with RNA-seq
Manjappa Ganiger
 
Domagoj šegregur, croatia, fishes as bioindicators of water pollution of river
Domagoj šegregur, croatia,  fishes as bioindicators of water pollution of riverDomagoj šegregur, croatia,  fishes as bioindicators of water pollution of river
Domagoj šegregur, croatia, fishes as bioindicators of water pollution of riverMohd Subri
 
Dna sequencing
Dna sequencingDna sequencing
Dna sequencing
dibya ranjan
 
Examining gene expression and methylation with next gen sequencing
Examining gene expression and methylation with next gen sequencingExamining gene expression and methylation with next gen sequencing
Examining gene expression and methylation with next gen sequencing
Stephen Turner
 
Genetic testing in breast cancer
Genetic testing in breast cancerGenetic testing in breast cancer
Genetic testing in breast cancer
Vibhay Pareek
 
Table comparing Illumina s new sequencers
Table comparing Illumina s new sequencersTable comparing Illumina s new sequencers
Table comparing Illumina s new sequencers
abizarl
 
Guide to Molecular Cloning - Download the Guide
Guide to Molecular Cloning  - Download the GuideGuide to Molecular Cloning  - Download the Guide
Guide to Molecular Cloning - Download the Guide
QIAGEN
 
Reproductive biology
Reproductive biologyReproductive biology
Reproductive biology
sanchu yadav
 
Recent lipid metabolism
Recent lipid metabolismRecent lipid metabolism
Recent lipid metabolism
Alhassan Ali
 

Viewers also liked (20)

An introduction to RNA-seq data analysis
An introduction to RNA-seq data analysisAn introduction to RNA-seq data analysis
An introduction to RNA-seq data analysis
 
DNA sequencing methods
DNA sequencing methodsDNA sequencing methods
DNA sequencing methods
 
Introduction to RNA-seq and RNA-seq Data Analysis (UEB-UAT Bioinformatics Cou...
Introduction to RNA-seq and RNA-seq Data Analysis (UEB-UAT Bioinformatics Cou...Introduction to RNA-seq and RNA-seq Data Analysis (UEB-UAT Bioinformatics Cou...
Introduction to RNA-seq and RNA-seq Data Analysis (UEB-UAT Bioinformatics Cou...
 
RNA-seq: general concept, goal and experimental design - part 1
RNA-seq: general concept, goal and experimental design - part 1RNA-seq: general concept, goal and experimental design - part 1
RNA-seq: general concept, goal and experimental design - part 1
 
Introduction to statistics iii
Introduction to statistics iiiIntroduction to statistics iii
Introduction to statistics iii
 
RNA Sequencing for Full Length Transcript Discovery
RNA Sequencing for Full Length Transcript DiscoveryRNA Sequencing for Full Length Transcript Discovery
RNA Sequencing for Full Length Transcript Discovery
 
RNA-Seq To Identify Novel Markers For Research on Neural Tissue Differentiation
RNA-Seq To Identify Novel Markers For Research on Neural Tissue DifferentiationRNA-Seq To Identify Novel Markers For Research on Neural Tissue Differentiation
RNA-Seq To Identify Novel Markers For Research on Neural Tissue Differentiation
 
Next generation sequencing methods (final edit)
Next generation sequencing methods (final edit)Next generation sequencing methods (final edit)
Next generation sequencing methods (final edit)
 
2012 august 16 systems biology rna seq v2
2012 august 16 systems biology rna seq v22012 august 16 systems biology rna seq v2
2012 august 16 systems biology rna seq v2
 
Catalyzing Plant Science Research with RNA-seq
Catalyzing Plant Science Research with RNA-seqCatalyzing Plant Science Research with RNA-seq
Catalyzing Plant Science Research with RNA-seq
 
Domagoj šegregur, croatia, fishes as bioindicators of water pollution of river
Domagoj šegregur, croatia,  fishes as bioindicators of water pollution of riverDomagoj šegregur, croatia,  fishes as bioindicators of water pollution of river
Domagoj šegregur, croatia, fishes as bioindicators of water pollution of river
 
Rna seq
Rna seqRna seq
Rna seq
 
Dna sequencing
Dna sequencingDna sequencing
Dna sequencing
 
Examining gene expression and methylation with next gen sequencing
Examining gene expression and methylation with next gen sequencingExamining gene expression and methylation with next gen sequencing
Examining gene expression and methylation with next gen sequencing
 
Genetic testing in breast cancer
Genetic testing in breast cancerGenetic testing in breast cancer
Genetic testing in breast cancer
 
Table comparing Illumina s new sequencers
Table comparing Illumina s new sequencersTable comparing Illumina s new sequencers
Table comparing Illumina s new sequencers
 
Guide to Molecular Cloning - Download the Guide
Guide to Molecular Cloning  - Download the GuideGuide to Molecular Cloning  - Download the Guide
Guide to Molecular Cloning - Download the Guide
 
Reproductive biology
Reproductive biologyReproductive biology
Reproductive biology
 
Recent lipid metabolism
Recent lipid metabolismRecent lipid metabolism
Recent lipid metabolism
 
Dna repair
Dna repairDna repair
Dna repair
 

Similar to Next-Generation Sequencing and its Applications in RNA-Seq

20110524zurichngs 1st pub
20110524zurichngs 1st pub20110524zurichngs 1st pub
20110524zurichngs 1st pubsesejun
 
Experimentos de nubes científicas: Medical Genome Project
Experimentos de nubes científicas: Medical Genome ProjectExperimentos de nubes científicas: Medical Genome Project
Experimentos de nubes científicas: Medical Genome Project
Fundación Ramón Areces
 
Introduction to Apollo for i5k
Introduction to Apollo for i5kIntroduction to Apollo for i5k
Introduction to Apollo for i5k
Monica Munoz-Torres
 
Automated Solutions for working with DNA/RNA
Automated Solutions for working with DNA/RNAAutomated Solutions for working with DNA/RNA
Automated Solutions for working with DNA/RNALuc Van Laer
 
Approaches to cDNA Cloning and Analysis
Approaches to cDNA Cloning and AnalysisApproaches to cDNA Cloning and Analysis
Approaches to cDNA Cloning and Analysis
Matthias Harbers
 
Next-generation sequencing course, part 1: technologies
Next-generation sequencing course, part 1: technologiesNext-generation sequencing course, part 1: technologies
Next-generation sequencing course, part 1: technologiesJan Aerts
 
Functional genomics
Functional genomicsFunctional genomics
Functional genomics
ajay301
 
Genomics lecture 3
Genomics lecture 3Genomics lecture 3
Genomics lecture 3
iainj88
 
R na sequencing and its methods
R na sequencing and its methodsR na sequencing and its methods
R na sequencing and its methods
Sabaiftikhar10
 
Functional genomics
Functional genomicsFunctional genomics
Functional genomics
Sreenivasa Reddy Thalla
 
Genotyping by sequencing
Genotyping by sequencingGenotyping by sequencing
Genotyping by sequencing
Bhavya Sree
 
New Molecular Approaches to Identify 21st Century Microbes - Dr Melissa Mille...
New Molecular Approaches to Identify 21st Century Microbes - Dr Melissa Mille...New Molecular Approaches to Identify 21st Century Microbes - Dr Melissa Mille...
New Molecular Approaches to Identify 21st Century Microbes - Dr Melissa Mille...
Eastern Pennsylvania Branch ASM
 
Sts
StsSts
Processing Raw scRNA-Seq Sequencing Data
Processing Raw scRNA-Seq Sequencing DataProcessing Raw scRNA-Seq Sequencing Data
Processing Raw scRNA-Seq Sequencing Data
Alireza Doustmohammadi
 
PCR applications.pptx
PCR applications.pptxPCR applications.pptx
PCR applications.pptx
B. BHASKAR
 
nextgenerationsequencing-170606100132.pdf
nextgenerationsequencing-170606100132.pdfnextgenerationsequencing-170606100132.pdf
nextgenerationsequencing-170606100132.pdf
AkhileshPathak33
 
Next generation sequencing
Next generation sequencingNext generation sequencing
Next generation sequencing
ARUNDHATI MEHTA
 
Construction and screening of genomic library
Construction and screening of genomic libraryConstruction and screening of genomic library
Construction and screening of genomic library
DrMayankChaudhary
 

Similar to Next-Generation Sequencing and its Applications in RNA-Seq (20)

20110524zurichngs 1st pub
20110524zurichngs 1st pub20110524zurichngs 1st pub
20110524zurichngs 1st pub
 
Experimentos de nubes científicas: Medical Genome Project
Experimentos de nubes científicas: Medical Genome ProjectExperimentos de nubes científicas: Medical Genome Project
Experimentos de nubes científicas: Medical Genome Project
 
Introduction to Apollo for i5k
Introduction to Apollo for i5kIntroduction to Apollo for i5k
Introduction to Apollo for i5k
 
Automated Solutions for working with DNA/RNA
Automated Solutions for working with DNA/RNAAutomated Solutions for working with DNA/RNA
Automated Solutions for working with DNA/RNA
 
Approaches to cDNA Cloning and Analysis
Approaches to cDNA Cloning and AnalysisApproaches to cDNA Cloning and Analysis
Approaches to cDNA Cloning and Analysis
 
Next-generation sequencing course, part 1: technologies
Next-generation sequencing course, part 1: technologiesNext-generation sequencing course, part 1: technologies
Next-generation sequencing course, part 1: technologies
 
Functional genomics
Functional genomicsFunctional genomics
Functional genomics
 
Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2Biotech autumn2012-02-ngs2
Biotech autumn2012-02-ngs2
 
Genomics lecture 3
Genomics lecture 3Genomics lecture 3
Genomics lecture 3
 
R na sequencing and its methods
R na sequencing and its methodsR na sequencing and its methods
R na sequencing and its methods
 
Functional genomics
Functional genomicsFunctional genomics
Functional genomics
 
Genotyping by sequencing
Genotyping by sequencingGenotyping by sequencing
Genotyping by sequencing
 
New Molecular Approaches to Identify 21st Century Microbes - Dr Melissa Mille...
New Molecular Approaches to Identify 21st Century Microbes - Dr Melissa Mille...New Molecular Approaches to Identify 21st Century Microbes - Dr Melissa Mille...
New Molecular Approaches to Identify 21st Century Microbes - Dr Melissa Mille...
 
Sts
StsSts
Sts
 
ChipSeq Data Analysis
ChipSeq Data AnalysisChipSeq Data Analysis
ChipSeq Data Analysis
 
Processing Raw scRNA-Seq Sequencing Data
Processing Raw scRNA-Seq Sequencing DataProcessing Raw scRNA-Seq Sequencing Data
Processing Raw scRNA-Seq Sequencing Data
 
PCR applications.pptx
PCR applications.pptxPCR applications.pptx
PCR applications.pptx
 
nextgenerationsequencing-170606100132.pdf
nextgenerationsequencing-170606100132.pdfnextgenerationsequencing-170606100132.pdf
nextgenerationsequencing-170606100132.pdf
 
Next generation sequencing
Next generation sequencingNext generation sequencing
Next generation sequencing
 
Construction and screening of genomic library
Construction and screening of genomic libraryConstruction and screening of genomic library
Construction and screening of genomic library
 

Recently uploaded

Builder.ai Founder Sachin Dev Duggal's Strategic Approach to Create an Innova...
Builder.ai Founder Sachin Dev Duggal's Strategic Approach to Create an Innova...Builder.ai Founder Sachin Dev Duggal's Strategic Approach to Create an Innova...
Builder.ai Founder Sachin Dev Duggal's Strategic Approach to Create an Innova...
Ramesh Iyer
 
"Impact of front-end architecture on development cost", Viktor Turskyi
"Impact of front-end architecture on development cost", Viktor Turskyi"Impact of front-end architecture on development cost", Viktor Turskyi
"Impact of front-end architecture on development cost", Viktor Turskyi
Fwdays
 
UiPath Test Automation using UiPath Test Suite series, part 3
UiPath Test Automation using UiPath Test Suite series, part 3UiPath Test Automation using UiPath Test Suite series, part 3
UiPath Test Automation using UiPath Test Suite series, part 3
DianaGray10
 
Empowering NextGen Mobility via Large Action Model Infrastructure (LAMI): pav...
Empowering NextGen Mobility via Large Action Model Infrastructure (LAMI): pav...Empowering NextGen Mobility via Large Action Model Infrastructure (LAMI): pav...
Empowering NextGen Mobility via Large Action Model Infrastructure (LAMI): pav...
Thierry Lestable
 
GDG Cloud Southlake #33: Boule & Rebala: Effective AppSec in SDLC using Deplo...
GDG Cloud Southlake #33: Boule & Rebala: Effective AppSec in SDLC using Deplo...GDG Cloud Southlake #33: Boule & Rebala: Effective AppSec in SDLC using Deplo...
GDG Cloud Southlake #33: Boule & Rebala: Effective AppSec in SDLC using Deplo...
James Anderson
 
ODC, Data Fabric and Architecture User Group
ODC, Data Fabric and Architecture User GroupODC, Data Fabric and Architecture User Group
ODC, Data Fabric and Architecture User Group
CatarinaPereira64715
 
FIDO Alliance Osaka Seminar: Overview.pdf
FIDO Alliance Osaka Seminar: Overview.pdfFIDO Alliance Osaka Seminar: Overview.pdf
FIDO Alliance Osaka Seminar: Overview.pdf
FIDO Alliance
 
When stars align: studies in data quality, knowledge graphs, and machine lear...
When stars align: studies in data quality, knowledge graphs, and machine lear...When stars align: studies in data quality, knowledge graphs, and machine lear...
When stars align: studies in data quality, knowledge graphs, and machine lear...
Elena Simperl
 
The Future of Platform Engineering
The Future of Platform EngineeringThe Future of Platform Engineering
The Future of Platform Engineering
Jemma Hussein Allen
 
IOS-PENTESTING-BEGINNERS-PRACTICAL-GUIDE-.pptx
IOS-PENTESTING-BEGINNERS-PRACTICAL-GUIDE-.pptxIOS-PENTESTING-BEGINNERS-PRACTICAL-GUIDE-.pptx
IOS-PENTESTING-BEGINNERS-PRACTICAL-GUIDE-.pptx
Abida Shariff
 
Assuring Contact Center Experiences for Your Customers With ThousandEyes
Assuring Contact Center Experiences for Your Customers With ThousandEyesAssuring Contact Center Experiences for Your Customers With ThousandEyes
Assuring Contact Center Experiences for Your Customers With ThousandEyes
ThousandEyes
 
Search and Society: Reimagining Information Access for Radical Futures
Search and Society: Reimagining Information Access for Radical FuturesSearch and Society: Reimagining Information Access for Radical Futures
Search and Society: Reimagining Information Access for Radical Futures
Bhaskar Mitra
 
PHP Frameworks: I want to break free (IPC Berlin 2024)
PHP Frameworks: I want to break free (IPC Berlin 2024)PHP Frameworks: I want to break free (IPC Berlin 2024)
PHP Frameworks: I want to break free (IPC Berlin 2024)
Ralf Eggert
 
De-mystifying Zero to One: Design Informed Techniques for Greenfield Innovati...
De-mystifying Zero to One: Design Informed Techniques for Greenfield Innovati...De-mystifying Zero to One: Design Informed Techniques for Greenfield Innovati...
De-mystifying Zero to One: Design Informed Techniques for Greenfield Innovati...
Product School
 
Key Trends Shaping the Future of Infrastructure.pdf
Key Trends Shaping the Future of Infrastructure.pdfKey Trends Shaping the Future of Infrastructure.pdf
Key Trends Shaping the Future of Infrastructure.pdf
Cheryl Hung
 
FIDO Alliance Osaka Seminar: Passkeys and the Road Ahead.pdf
FIDO Alliance Osaka Seminar: Passkeys and the Road Ahead.pdfFIDO Alliance Osaka Seminar: Passkeys and the Road Ahead.pdf
FIDO Alliance Osaka Seminar: Passkeys and the Road Ahead.pdf
FIDO Alliance
 
Neuro-symbolic is not enough, we need neuro-*semantic*
Neuro-symbolic is not enough, we need neuro-*semantic*Neuro-symbolic is not enough, we need neuro-*semantic*
Neuro-symbolic is not enough, we need neuro-*semantic*
Frank van Harmelen
 
DevOps and Testing slides at DASA Connect
DevOps and Testing slides at DASA ConnectDevOps and Testing slides at DASA Connect
DevOps and Testing slides at DASA Connect
Kari Kakkonen
 
From Siloed Products to Connected Ecosystem: Building a Sustainable and Scala...
From Siloed Products to Connected Ecosystem: Building a Sustainable and Scala...From Siloed Products to Connected Ecosystem: Building a Sustainable and Scala...
From Siloed Products to Connected Ecosystem: Building a Sustainable and Scala...
Product School
 
Smart TV Buyer Insights Survey 2024 by 91mobiles.pdf
Smart TV Buyer Insights Survey 2024 by 91mobiles.pdfSmart TV Buyer Insights Survey 2024 by 91mobiles.pdf
Smart TV Buyer Insights Survey 2024 by 91mobiles.pdf
91mobiles
 

Recently uploaded (20)

Builder.ai Founder Sachin Dev Duggal's Strategic Approach to Create an Innova...
Builder.ai Founder Sachin Dev Duggal's Strategic Approach to Create an Innova...Builder.ai Founder Sachin Dev Duggal's Strategic Approach to Create an Innova...
Builder.ai Founder Sachin Dev Duggal's Strategic Approach to Create an Innova...
 
"Impact of front-end architecture on development cost", Viktor Turskyi
"Impact of front-end architecture on development cost", Viktor Turskyi"Impact of front-end architecture on development cost", Viktor Turskyi
"Impact of front-end architecture on development cost", Viktor Turskyi
 
UiPath Test Automation using UiPath Test Suite series, part 3
UiPath Test Automation using UiPath Test Suite series, part 3UiPath Test Automation using UiPath Test Suite series, part 3
UiPath Test Automation using UiPath Test Suite series, part 3
 
Empowering NextGen Mobility via Large Action Model Infrastructure (LAMI): pav...
Empowering NextGen Mobility via Large Action Model Infrastructure (LAMI): pav...Empowering NextGen Mobility via Large Action Model Infrastructure (LAMI): pav...
Empowering NextGen Mobility via Large Action Model Infrastructure (LAMI): pav...
 
GDG Cloud Southlake #33: Boule & Rebala: Effective AppSec in SDLC using Deplo...
GDG Cloud Southlake #33: Boule & Rebala: Effective AppSec in SDLC using Deplo...GDG Cloud Southlake #33: Boule & Rebala: Effective AppSec in SDLC using Deplo...
GDG Cloud Southlake #33: Boule & Rebala: Effective AppSec in SDLC using Deplo...
 
ODC, Data Fabric and Architecture User Group
ODC, Data Fabric and Architecture User GroupODC, Data Fabric and Architecture User Group
ODC, Data Fabric and Architecture User Group
 
FIDO Alliance Osaka Seminar: Overview.pdf
FIDO Alliance Osaka Seminar: Overview.pdfFIDO Alliance Osaka Seminar: Overview.pdf
FIDO Alliance Osaka Seminar: Overview.pdf
 
When stars align: studies in data quality, knowledge graphs, and machine lear...
When stars align: studies in data quality, knowledge graphs, and machine lear...When stars align: studies in data quality, knowledge graphs, and machine lear...
When stars align: studies in data quality, knowledge graphs, and machine lear...
 
The Future of Platform Engineering
The Future of Platform EngineeringThe Future of Platform Engineering
The Future of Platform Engineering
 
IOS-PENTESTING-BEGINNERS-PRACTICAL-GUIDE-.pptx
IOS-PENTESTING-BEGINNERS-PRACTICAL-GUIDE-.pptxIOS-PENTESTING-BEGINNERS-PRACTICAL-GUIDE-.pptx
IOS-PENTESTING-BEGINNERS-PRACTICAL-GUIDE-.pptx
 
Assuring Contact Center Experiences for Your Customers With ThousandEyes
Assuring Contact Center Experiences for Your Customers With ThousandEyesAssuring Contact Center Experiences for Your Customers With ThousandEyes
Assuring Contact Center Experiences for Your Customers With ThousandEyes
 
Search and Society: Reimagining Information Access for Radical Futures
Search and Society: Reimagining Information Access for Radical FuturesSearch and Society: Reimagining Information Access for Radical Futures
Search and Society: Reimagining Information Access for Radical Futures
 
PHP Frameworks: I want to break free (IPC Berlin 2024)
PHP Frameworks: I want to break free (IPC Berlin 2024)PHP Frameworks: I want to break free (IPC Berlin 2024)
PHP Frameworks: I want to break free (IPC Berlin 2024)
 
De-mystifying Zero to One: Design Informed Techniques for Greenfield Innovati...
De-mystifying Zero to One: Design Informed Techniques for Greenfield Innovati...De-mystifying Zero to One: Design Informed Techniques for Greenfield Innovati...
De-mystifying Zero to One: Design Informed Techniques for Greenfield Innovati...
 
Key Trends Shaping the Future of Infrastructure.pdf
Key Trends Shaping the Future of Infrastructure.pdfKey Trends Shaping the Future of Infrastructure.pdf
Key Trends Shaping the Future of Infrastructure.pdf
 
FIDO Alliance Osaka Seminar: Passkeys and the Road Ahead.pdf
FIDO Alliance Osaka Seminar: Passkeys and the Road Ahead.pdfFIDO Alliance Osaka Seminar: Passkeys and the Road Ahead.pdf
FIDO Alliance Osaka Seminar: Passkeys and the Road Ahead.pdf
 
Neuro-symbolic is not enough, we need neuro-*semantic*
Neuro-symbolic is not enough, we need neuro-*semantic*Neuro-symbolic is not enough, we need neuro-*semantic*
Neuro-symbolic is not enough, we need neuro-*semantic*
 
DevOps and Testing slides at DASA Connect
DevOps and Testing slides at DASA ConnectDevOps and Testing slides at DASA Connect
DevOps and Testing slides at DASA Connect
 
From Siloed Products to Connected Ecosystem: Building a Sustainable and Scala...
From Siloed Products to Connected Ecosystem: Building a Sustainable and Scala...From Siloed Products to Connected Ecosystem: Building a Sustainable and Scala...
From Siloed Products to Connected Ecosystem: Building a Sustainable and Scala...
 
Smart TV Buyer Insights Survey 2024 by 91mobiles.pdf
Smart TV Buyer Insights Survey 2024 by 91mobiles.pdfSmart TV Buyer Insights Survey 2024 by 91mobiles.pdf
Smart TV Buyer Insights Survey 2024 by 91mobiles.pdf
 

Next-Generation Sequencing and its Applications in RNA-Seq

  • 1. Next-Generation Sequencing and its Applications in RNA-Seq Perdacher Martin 1
  • 2. Overview • Next-Generation Sequencing • technologies • applications • RNA-Seq 2
  • 3. Next-Generation Sequencing technologies • Ion semiconductor sequencing • Pyrosequencing • Sequencing by ligation • Cyclic reversible termination 3
  • 5. Pyrosequencing • pyrophosphate (PPi) is translated into light emission in two enzymatic steps • ATP-sulfurylase converts PPi in ATP • luciferase converts Luciferin in oxiluciferin and emitted light (ATP is the fuel for this reaction). 5
  • 7. Cyclic reversible termination • incorporation of modified nucleotides • fluorescence imaging • washing of fluorescent dye and removing of terminating group 7
  • 8. Overview • Next-Generation Sequencing • technologies • applications • RNA-Seq 8
  • 9. Anwendungen von Next-Generation Sequencing Transcriptome Sequencing: • Transcriptome sequencing RNA Sequenzierung coding non-coding (rRNA, tRNA, miRNA, siRNA, piRNA) • Genomic sequencing Genomic Sequencing de novo resequencing (copy number variation, • Epigenetic applications SNP point mutations) Epigenetic Chip Seq • Additional applications Methylation profiling Additional: Clip Seq, RIP-Seq, HITS Clip, - RNA interaktion mit RNA-bindenden Proteinen 9
  • 10. Overview • Next-Generation Sequencing • technologies • applications • RNA-Seq 10
  • 11. RNA-Seq • biological background • workflow of RNA-Seq • transcriptome reconstruction • differential gene expression 11
  • 12. background biology of RNA-Seq 12
  • 13. RNA-Seq • biological background • workflow of RNA-Seq • transcriptome reconstruction • differential gene expression 13
  • 14. Workflow of RNA-Seq • sample preparation ribo minus and strand specific libs (paired end) • seqeuncing • read mapping Fragmentation of cDNA Purification Adapter ligation Size-based purification of ligation products using PCR of ligation products Purification and sequencing of the fragments 14
  • 15. Workflow of RNA-Seq • sample preparation ribo minus and strand specific libs (paired end) • seqeuncing • read mapping Fragmentation of cDNA Purification Adapter ligation Size-based purification of ligation products using PCR of ligation products Purification and sequencing of the fragments 14
  • 16. Read mapping in RNA-Seq 15
  • 17. Read mapping unspliced alignment match PERFECTLY the • genome unspliced read alignment seed : after mapping, the seed is extended with smith waterman method • seed methods Burrow-Wheeler: transform genome into efficient data structure • Burrows-Wheeler transform methods • spliced aligners • exon first • seed and extend 16
  • 18. Read mapping (spliced alignment methods) 2 steps: unspliced read aligner break reads into shorter (k-mer-)seeds. unmapped reads are split into fragments seed-regions are evaluated and aligned with sensitive alignment independently methods (smith waterman) much slower method pseudogene is dysfunctional and does not contain introns. in the first step of exon-first approaches reads are aligned to the genome based on unspliced read mappers. here it is detected as gene and not as pseudogene. 17
  • 19. RNA-Seq • biological background • workflow of RNA-Seq • transcriptome reconstruction • differential gene expression 18
  • 20. Transcriptome reconstruction exon identification: is used for very • genome guided reconstruction short reads (36 bp) first map reads to the genome. the unmapped reads are tested for all • exon identification possibilities of exon-exon junctions not able to identify full transcript structures • genome-guided assembly • genome independent reconstruction 19
  • 21. Transcriptome reconstruction exon identification: is used for very • genome guided reconstruction short reads (36 bp) first map reads to the genome. the unmapped reads are tested for all • exon identification possibilities of exon-exon junctions not able to identify full transcript structures • genome-guided assembly • genome independent reconstruction 19
  • 22. Transcriptome reconstruction break reads into k-mers. align reads to the genome build de-brujin graph. and use spliced reads to build transcript graph alignment to the genome (node: read fragment, is because of annotation edge: link bet ween purpose fragments) one paht through the graph represents an isoform of the transcript) 20
  • 23. RNA-Seq • biological background • workflow of RNA-Seq • transcriptome reconstruction • differential gene expression 21
  • 24. Differential gene expression for RNA-Seq • comparing the count of reads for one gene in different experiments (conditions) • the problem is that one read cannot assigned uniquely to one gene, because of the overlap of genes and their different isoforms 22
  • 26. Summary • Next-Generation Sequencing, (technologies and applications) • RNA-Seq • biology • workflow of RNA-Seq (read mapping) • transcriptome reconstruction • differential gene expression 24

Editor's Notes

  1. \n
  2. \n
  3. \n
  4. \n
  5. \n
  6. \n
  7. \n
  8. \n
  9. \n
  10. \n
  11. \n
  12. \n
  13. \n
  14. \n
  15. \n
  16. \n
  17. \n
  18. \n
  19. \n
  20. \n
  21. \n
  22. \n
  23. \n
  24. \n