rna seq data therapy 12jan2018 - university of connecticut · analysis rna fastq fastq sam/bam...
TRANSCRIPT
![Page 1: RNA seq Data Therapy 12Jan2018 - University of Connecticut · analysis RNA fastq fastq SAM/BAM fasta GFF/GTF RNA-seqvs Microarray • Higher sensitivity and dynamic range • Lower](https://reader034.vdocument.in/reader034/viewer/2022050409/5f85c8cc6b292f30c646bf78/html5/thumbnails/1.jpg)
RNA-Seq Analysis
![Page 2: RNA seq Data Therapy 12Jan2018 - University of Connecticut · analysis RNA fastq fastq SAM/BAM fasta GFF/GTF RNA-seqvs Microarray • Higher sensitivity and dynamic range • Lower](https://reader034.vdocument.in/reader034/viewer/2022050409/5f85c8cc6b292f30c646bf78/html5/thumbnails/2.jpg)
ExperimentalDesign
Sequencing
DataQualityControl
Readmapping
ReferenceGenome
ReferenceTranscriptome
DifferentialExpressionanalysis
RNA
fastq
fastq
SAM/BAM
fasta
GFF/GTF
QualityControlchecks• Reproducibility• Reliability
![Page 3: RNA seq Data Therapy 12Jan2018 - University of Connecticut · analysis RNA fastq fastq SAM/BAM fasta GFF/GTF RNA-seqvs Microarray • Higher sensitivity and dynamic range • Lower](https://reader034.vdocument.in/reader034/viewer/2022050409/5f85c8cc6b292f30c646bf78/html5/thumbnails/3.jpg)
Introduction
ExperimentalDesign
Sequencing
DataQualityControl
Readmapping
ReferenceGenome
ReferenceTranscriptome
DifferentialExpressionanalysis
RNA
fastq
fastq
SAM/BAM
fasta
GFF/GTF
![Page 4: RNA seq Data Therapy 12Jan2018 - University of Connecticut · analysis RNA fastq fastq SAM/BAM fasta GFF/GTF RNA-seqvs Microarray • Higher sensitivity and dynamic range • Lower](https://reader034.vdocument.in/reader034/viewer/2022050409/5f85c8cc6b292f30c646bf78/html5/thumbnails/4.jpg)
Introduction
ExperimentalDesign
Sequencing
DataQualityControl
Readmapping
ReferenceGenome
ReferenceTranscriptome
DifferentialExpressionanalysis
RNA
fastq
fastq
SAM/BAM
fasta
GFF/GTF
RNA-seq vsMicroarray
• Highersensitivityanddynamicrange• Lowertechnicalvariation• Availableforallspecies• Noveltranscriptidentification• Alternatesplicing• Allelespecificexpression• Fusiongenes• HigherInformaticsCost
![Page 5: RNA seq Data Therapy 12Jan2018 - University of Connecticut · analysis RNA fastq fastq SAM/BAM fasta GFF/GTF RNA-seqvs Microarray • Higher sensitivity and dynamic range • Lower](https://reader034.vdocument.in/reader034/viewer/2022050409/5f85c8cc6b292f30c646bf78/html5/thumbnails/5.jpg)
Introduction
ExperimentalDesign
Sequencing
DataQualityControl
Readmapping
ReferenceGenome
ReferenceTranscriptome
DifferentialExpressionanalysis
RNA
fastq
fastq
SAM/BAM
fasta
GFF/GTF
Reproducibility,LinearityandSensitivity
<2%ofgenome
Mortazavi etal.,NatureMethods5,621- 628(2008)
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RNAisolation
TotalRNA
Poly(A)selection
rRNA depletion SizeselectionClinicalSamples(tissuebiopsies)BacterialRNAsamples
SmallncRNA
Introduction
ExperimentalDesign
Sequencing
DataQualityControl
Readmapping
ReferenceGenome
ReferenceTranscriptome
DifferentialExpressionanalysis
RNA
fastq
fastq
SAM/BAM
fasta
GFF/GTF
![Page 7: RNA seq Data Therapy 12Jan2018 - University of Connecticut · analysis RNA fastq fastq SAM/BAM fasta GFF/GTF RNA-seqvs Microarray • Higher sensitivity and dynamic range • Lower](https://reader034.vdocument.in/reader034/viewer/2022050409/5f85c8cc6b292f30c646bf78/html5/thumbnails/7.jpg)
Introduction
ExperimentalDesign
Sequencing
DataQualityControl
Readmapping
ReferenceGenome
ReferenceTranscriptome
DifferentialExpressionanalysis
RNA
fastq
fastq
SAM/BAM
fasta
GFF/GTF
![Page 8: RNA seq Data Therapy 12Jan2018 - University of Connecticut · analysis RNA fastq fastq SAM/BAM fasta GFF/GTF RNA-seqvs Microarray • Higher sensitivity and dynamic range • Lower](https://reader034.vdocument.in/reader034/viewer/2022050409/5f85c8cc6b292f30c646bf78/html5/thumbnails/8.jpg)
Introduction
ExperimentalDesign
Sequencing
DataQualityControl
Readmapping
ReferenceGenome
ReferenceTranscriptome
DifferentialExpressionanalysis
RNA
fastq
fastq
SAM/BAM
fasta
GFF/GTF
GeneA GeneB GeneC
Sample1 6 1 4
GeneA GeneB GeneC Total
Sample1 3 1 1 5
Sample2 6 3 6 15
#ofreads
GeneA GeneB GeneC
Sample1 3 1 1Readsperkbofexon
GeneA GeneB GeneC Total
Sample1 0.6 0.2 0.2 5
Sample2 0.4 0.2 0.4 15
Readsperkbofexon
Readsperkbofexonpermillionmappedreads- RPKM
![Page 9: RNA seq Data Therapy 12Jan2018 - University of Connecticut · analysis RNA fastq fastq SAM/BAM fasta GFF/GTF RNA-seqvs Microarray • Higher sensitivity and dynamic range • Lower](https://reader034.vdocument.in/reader034/viewer/2022050409/5f85c8cc6b292f30c646bf78/html5/thumbnails/9.jpg)
Introduction
ExperimentalDesign
Sequencing
DataQualityControl
Readmapping
ReferenceGenome
ReferenceTranscriptome
DifferentialExpressionanalysis
RNA
fastq
fastq
SAM/BAM
fasta
GFF/GTF
![Page 10: RNA seq Data Therapy 12Jan2018 - University of Connecticut · analysis RNA fastq fastq SAM/BAM fasta GFF/GTF RNA-seqvs Microarray • Higher sensitivity and dynamic range • Lower](https://reader034.vdocument.in/reader034/viewer/2022050409/5f85c8cc6b292f30c646bf78/html5/thumbnails/10.jpg)
Introduction
ExperimentalDesign
Sequencing
DataQualityControl
Readmapping
ReferenceGenome
ReferenceTranscriptome
DifferentialExpressionanalysis
RNA
fastq
fastq
SAM/BAM
fasta
GFF/GTF
ExperimentalDesign
Whataremygoals?• Transcriptassembly• DifferentialExpressionanalysis• Identifynew/raretranscripts
WhatareCharacteristicsofmysystem?• Largeandcomplexgenome• Intronsandhighdegreeofalternativesplicing• Noreferencegenomeortranscriptome.
![Page 11: RNA seq Data Therapy 12Jan2018 - University of Connecticut · analysis RNA fastq fastq SAM/BAM fasta GFF/GTF RNA-seqvs Microarray • Higher sensitivity and dynamic range • Lower](https://reader034.vdocument.in/reader034/viewer/2022050409/5f85c8cc6b292f30c646bf78/html5/thumbnails/11.jpg)
Introduction
ExperimentalDesign
Sequencing
DataQualityControl
Readmapping
ReferenceGenome
ReferenceTranscriptome
DifferentialExpressionanalysis
RNA
fastq
fastq
SAM/BAM
fasta
GFF/GTF
ExperimentalDesign
• BiologicalComparison(s)• PairedEndvsSingleend• Readdepth• Readlength• Replicates
![Page 12: RNA seq Data Therapy 12Jan2018 - University of Connecticut · analysis RNA fastq fastq SAM/BAM fasta GFF/GTF RNA-seqvs Microarray • Higher sensitivity and dynamic range • Lower](https://reader034.vdocument.in/reader034/viewer/2022050409/5f85c8cc6b292f30c646bf78/html5/thumbnails/12.jpg)
Introduction
ExperimentalDesign
Sequencing
DataQualityControl
Readmapping
ReferenceGenome
ReferenceTranscriptome
DifferentialExpressionanalysis
RNA
fastq
fastq
SAM/BAM
fasta
GFF/GTF
ExperimentalDesign
SimpleDesign- Pairwisecomparison
TwoGroups
ComplexdesignControl Experimental
treatment
CancerSubtypeA
- +drug
CancerSubtypeB
- +drug
Consultastatistician
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Introduction
ExperimentalDesign
Sequencing
DataQualityControl
Readmapping
ReferenceGenome
ReferenceTranscriptome
DifferentialExpressionanalysis
RNA
fastq
fastq
SAM/BAM
fasta
GFF/GTF
ExperimentalDesign
ReaddepthandreadlengthSmallgenomewithnoalternatesplicing(yeast)Annotatedtranscriptome
10millionreadspersample,50bpsingle-endreads
Mammaliangenomes(Largetranscriptome,alternativesplicing,geneduplication)
30millionreadspersample,
Transcriptomeassembly(100Xcoverageoftranscriptome)50-200millionreadspersample,100bppairedendreads
Natureofsamples.• Whatistheexpectedpurityofyoursample?• Istherecontaminationorheterogeneityexpected?
Ifyes,thenHighcoveragetodetectvariantsatlowerfrequencyduetoimpurityorbecausetheycomefromminor(butpossiblystillinteresting)subpopulationsofyoursample.
![Page 14: RNA seq Data Therapy 12Jan2018 - University of Connecticut · analysis RNA fastq fastq SAM/BAM fasta GFF/GTF RNA-seqvs Microarray • Higher sensitivity and dynamic range • Lower](https://reader034.vdocument.in/reader034/viewer/2022050409/5f85c8cc6b292f30c646bf78/html5/thumbnails/14.jpg)
Introduction
ExperimentalDesign
Sequencing
DataQualityControl
Readmapping
ReferenceGenome
ReferenceTranscriptome
DifferentialExpressionanalysis
RNA
fastq
fastq
SAM/BAM
fasta
GFF/GTF
ExperimentalDesign
Replicates:Factorsdeterminingnumberofreplicates:• Variabilityinmeasurements(TechnicalnoiseandBiologicalvariation)• Statisticalpoweranalysis
TechnicalReplicatesNotNeeded:HighreproducibilityatsequencingstepErrorpronestepsRNAfragmentation,cDNAsynthesis,adapterligation,PCRamplification,bar-coding,laneloadingSpikeIns:Qualitycontrolandlibrary-sizenormalisationMinimizebatcheffectsRandomizesamplesatlibrarypreparationandsequencingruns
BiologicalReplicatesNotrequiredfortranscriptionassemblyEssentialfordifferentialexpressionanalysisComplexdesigns:
• 3+forcelllines• 5+forinbredlines• 20+forhumansamples
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Introduction
ExperimentalDesign
Sequencing
DataQualityControl
Readmapping
ReferenceGenome
ReferenceTranscriptome
DifferentialExpressionanalysis
RNA
fastq
fastq
SAM/BAM
fasta
GFF/GTF
ExperimentalDesign
Scotty:http://scotty.genetics.utah.edu/
![Page 16: RNA seq Data Therapy 12Jan2018 - University of Connecticut · analysis RNA fastq fastq SAM/BAM fasta GFF/GTF RNA-seqvs Microarray • Higher sensitivity and dynamic range • Lower](https://reader034.vdocument.in/reader034/viewer/2022050409/5f85c8cc6b292f30c646bf78/html5/thumbnails/16.jpg)
Introduction
ExperimentalDesign
Sequencing
DataQualityControl
Readmapping
ReferenceGenome
ReferenceTranscriptome
DifferentialExpressionanalysis
RNA
fastq
fastq
SAM/BAM
fasta
GFF/GTF
![Page 17: RNA seq Data Therapy 12Jan2018 - University of Connecticut · analysis RNA fastq fastq SAM/BAM fasta GFF/GTF RNA-seqvs Microarray • Higher sensitivity and dynamic range • Lower](https://reader034.vdocument.in/reader034/viewer/2022050409/5f85c8cc6b292f30c646bf78/html5/thumbnails/17.jpg)
Introduction
ExperimentalDesign
Sequencing
DataQualityControl
Readmapping
ReferenceGenome
ReferenceTranscriptome
DifferentialExpressionanalysis
RNA
fastq
fastq
SAM/BAM
fasta
GFF/GTF
Sequencing
Illuminasequencingbysynthesis
SOLID“Color-Space”readsLowerrorrate
454pyrosequencingLongerreads,lowthroughput
Pacific-Bioscience(pacBio)/OxfordNanoporeLongerread(Recoveryoffulllengthtranscripts)
![Page 18: RNA seq Data Therapy 12Jan2018 - University of Connecticut · analysis RNA fastq fastq SAM/BAM fasta GFF/GTF RNA-seqvs Microarray • Higher sensitivity and dynamic range • Lower](https://reader034.vdocument.in/reader034/viewer/2022050409/5f85c8cc6b292f30c646bf78/html5/thumbnails/18.jpg)
Introduction
ExperimentalDesign
Sequencing
Readmapping
ReferenceGenome
ReferenceTranscriptome
DifferentialExpressionanalysis
Transcriptomeassembly
RNA
fastq
fastq
SAM/BAM
fasta
GFF/GTF
DataQualityControl
![Page 19: RNA seq Data Therapy 12Jan2018 - University of Connecticut · analysis RNA fastq fastq SAM/BAM fasta GFF/GTF RNA-seqvs Microarray • Higher sensitivity and dynamic range • Lower](https://reader034.vdocument.in/reader034/viewer/2022050409/5f85c8cc6b292f30c646bf78/html5/thumbnails/19.jpg)
Introduction
ExperimentalDesign
Sequencing
Readmapping
ReferenceGenome
ReferenceTranscriptome
DifferentialExpressionanalysis
Transcriptomeassembly
RNA
fastq
fastq
SAM/BAM
fasta
GFF/GTF
DataQualityControl
SequenceDataFormat.FASTQ
QualityScorePhred +33
ReadIDSequence
MachineID
QCFilterflagY=badN=goodReadpair#1
Readpair#2
Sample1_R1.fastqSample1_R2.fastq
R1
R2
SampleID/Barcode
![Page 20: RNA seq Data Therapy 12Jan2018 - University of Connecticut · analysis RNA fastq fastq SAM/BAM fasta GFF/GTF RNA-seqvs Microarray • Higher sensitivity and dynamic range • Lower](https://reader034.vdocument.in/reader034/viewer/2022050409/5f85c8cc6b292f30c646bf78/html5/thumbnails/20.jpg)
Introduction
ExperimentalDesign
Sequencing
Readmapping
ReferenceGenome
ReferenceTranscriptome
DifferentialExpressionanalysis
RNA
fastq
fastq
SAM/BAM
fasta
GFF/GTF
DataQualityControl
DataQualityAssessment
Evaluaterawreadlibraryquality• Sequencequality• GCcontentforbiases• AdapterContamination• K-mer overrepresentation• Duplicatereads• PCRartifacts
Software/Tools• FASTQC(Commandline)
Illuminareadfiles• NGSQC
SupportreadsfromanyplatformSupportqualitybasedreadtrimmingandfiltering
• SAMSat (Commandline)AlsoworkwithBamalignmentfiles
![Page 21: RNA seq Data Therapy 12Jan2018 - University of Connecticut · analysis RNA fastq fastq SAM/BAM fasta GFF/GTF RNA-seqvs Microarray • Higher sensitivity and dynamic range • Lower](https://reader034.vdocument.in/reader034/viewer/2022050409/5f85c8cc6b292f30c646bf78/html5/thumbnails/21.jpg)
Introduction
ExperimentalDesign
Sequencing
Readmapping
ReferenceGenome
ReferenceTranscriptome
DifferentialExpressionanalysis
RNA
fastq
fastq
SAM/BAM
fasta
GFF/GTF
DataQualityControl
DataQualityAssessment
![Page 22: RNA seq Data Therapy 12Jan2018 - University of Connecticut · analysis RNA fastq fastq SAM/BAM fasta GFF/GTF RNA-seqvs Microarray • Higher sensitivity and dynamic range • Lower](https://reader034.vdocument.in/reader034/viewer/2022050409/5f85c8cc6b292f30c646bf78/html5/thumbnails/22.jpg)
Introduction
ExperimentalDesign
Sequencing
Readmapping
ReferenceGenome
ReferenceTranscriptome
DifferentialExpressionanalysis
RNA
fastq
fastq
SAM/BAM
fasta
GFF/GTF
DataQualityControl
DataQualityAssessment
Sequencequality:Qualityscoresoverbases
Good
BadTrimmingrequired
Software/ToolsFastX-ToolkitTrimmomaticSickleCutadapt
Phred 30=1error/1000basesPhred 20=1error/100bases
![Page 23: RNA seq Data Therapy 12Jan2018 - University of Connecticut · analysis RNA fastq fastq SAM/BAM fasta GFF/GTF RNA-seqvs Microarray • Higher sensitivity and dynamic range • Lower](https://reader034.vdocument.in/reader034/viewer/2022050409/5f85c8cc6b292f30c646bf78/html5/thumbnails/23.jpg)
Introduction
ExperimentalDesign
Sequencing
Readmapping
ReferenceGenome
ReferenceTranscriptome
DifferentialExpressionanalysis
RNA
fastq
fastq
SAM/BAM
fasta
GFF/GTF
DataQualityControl
DataQualityAssessment
GCDistribution:AcceptablelevelsdependonSourceofsample
Good
Bad
![Page 24: RNA seq Data Therapy 12Jan2018 - University of Connecticut · analysis RNA fastq fastq SAM/BAM fasta GFF/GTF RNA-seqvs Microarray • Higher sensitivity and dynamic range • Lower](https://reader034.vdocument.in/reader034/viewer/2022050409/5f85c8cc6b292f30c646bf78/html5/thumbnails/24.jpg)
Introduction
ExperimentalDesign
Sequencing
Readmapping
ReferenceGenome
ReferenceTranscriptome
DifferentialExpressionanalysis
RNA
fastq
fastq
SAM/BAM
fasta
GFF/GTF
DataQualityControl
DataQualityAssessment
![Page 25: RNA seq Data Therapy 12Jan2018 - University of Connecticut · analysis RNA fastq fastq SAM/BAM fasta GFF/GTF RNA-seqvs Microarray • Higher sensitivity and dynamic range • Lower](https://reader034.vdocument.in/reader034/viewer/2022050409/5f85c8cc6b292f30c646bf78/html5/thumbnails/25.jpg)
Introduction
ExperimentalDesign
Sequencing
Readmapping
ReferenceGenome
ReferenceTranscriptome
DifferentialExpressionanalysis
RNA
fastq
fastq
SAM/BAM
fasta
GFF/GTF
DataQualityControl
DataQualityAssessment
![Page 26: RNA seq Data Therapy 12Jan2018 - University of Connecticut · analysis RNA fastq fastq SAM/BAM fasta GFF/GTF RNA-seqvs Microarray • Higher sensitivity and dynamic range • Lower](https://reader034.vdocument.in/reader034/viewer/2022050409/5f85c8cc6b292f30c646bf78/html5/thumbnails/26.jpg)
Introduction
ExperimentalDesign
Sequencing
Readmapping
ReferenceGenome
ReferenceTranscriptome
DifferentialExpressionanalysis
RNA
fastq
fastq
SAM/BAM
fasta
GFF/GTF
DataQualityControl
DataQualityAssessment
Recommendations:
• Generatequalityplotsforallreadlibraries• Trimand/orfilterdataifneeded
Alwaystrimandfilterfordenovotranscriptomeassembly
• Regeneratequalityplotsaftertrimmingandfilteringtodetermineeffectiveness
• AcceptableduplicationK-mer orGCcontentlevelsareexperimentandorganismspecificbutthevaluesshouldbehomogeneousforsamplesinthesameexperiment.
• Outlierswith>30%disagreementshouldbediscarded.
![Page 27: RNA seq Data Therapy 12Jan2018 - University of Connecticut · analysis RNA fastq fastq SAM/BAM fasta GFF/GTF RNA-seqvs Microarray • Higher sensitivity and dynamic range • Lower](https://reader034.vdocument.in/reader034/viewer/2022050409/5f85c8cc6b292f30c646bf78/html5/thumbnails/27.jpg)
Introduction
ExperimentalDesign
Sequencing
DataQualityControl
Readmapping
ReferenceGenome
ReferenceTranscriptome
DifferentialExpressionanalysis
RNA
fastq
fastq
SAM/BAM
fasta
GFF/GTF
![Page 28: RNA seq Data Therapy 12Jan2018 - University of Connecticut · analysis RNA fastq fastq SAM/BAM fasta GFF/GTF RNA-seqvs Microarray • Higher sensitivity and dynamic range • Lower](https://reader034.vdocument.in/reader034/viewer/2022050409/5f85c8cc6b292f30c646bf78/html5/thumbnails/28.jpg)
Introduction
ExperimentalDesign
Sequencing
DataQualityControl
Readmapping
ReferenceGenome
ReferenceTranscriptome
DifferentialExpressionanalysis
RNA
fastq
fastq
SAM/BAM
fasta
GFF/GTF
ReadMapping
(1) Withreferencegenome(with/withouttranscriptome).(2) Withreferencetranscriptome.(3) Referencefreeassembly.
![Page 29: RNA seq Data Therapy 12Jan2018 - University of Connecticut · analysis RNA fastq fastq SAM/BAM fasta GFF/GTF RNA-seqvs Microarray • Higher sensitivity and dynamic range • Lower](https://reader034.vdocument.in/reader034/viewer/2022050409/5f85c8cc6b292f30c646bf78/html5/thumbnails/29.jpg)
Introduction
ExperimentalDesign
Sequencing
DataQualityControl
Readmapping
ReferenceGenome
ReferenceTranscriptome
DifferentialExpressionanalysis
RNA
fastq
fastq
SAM/BAM
fasta
GFF/GTF
RNA-seq:AssemblyvsMapping
RNA-seqreads
ReferencebasedRNA-seq
Ref:GenomeorTranscriptome
DenovoRNA-seqcontig 1 contig 2
![Page 30: RNA seq Data Therapy 12Jan2018 - University of Connecticut · analysis RNA fastq fastq SAM/BAM fasta GFF/GTF RNA-seqvs Microarray • Higher sensitivity and dynamic range • Lower](https://reader034.vdocument.in/reader034/viewer/2022050409/5f85c8cc6b292f30c646bf78/html5/thumbnails/30.jpg)
Introduction
ExperimentalDesign
Sequencing
DataQualityControl
Readmapping
ReferenceGenome
ReferenceTranscriptome
DifferentialExpressionanalysis
RNA
fastq
fastq
SAM/BAM
fasta
GFF/GTF
Mappingwithreferencegenome
Shortreadsfastq
Mappingtogenome
Gappedmapper TopHatSTAR,HISAT2
Cufflinks,htseq-count,subread
Transcriptidentification&counting
WithGFF WithoutGFF
Transcriptdiscovery&counting
Functionalannotation
Blast2GOHomologybased
![Page 31: RNA seq Data Therapy 12Jan2018 - University of Connecticut · analysis RNA fastq fastq SAM/BAM fasta GFF/GTF RNA-seqvs Microarray • Higher sensitivity and dynamic range • Lower](https://reader034.vdocument.in/reader034/viewer/2022050409/5f85c8cc6b292f30c646bf78/html5/thumbnails/31.jpg)
Introduction
ExperimentalDesign
Sequencing
DataQualityControl
Readmapping
ReferenceGenome
ReferenceTranscriptome
DifferentialExpressionanalysis
RNA
fastq
fastq
SAM/BAM
fasta
GFF/GTF
MappingwithreferenceTranscriptome
Shortreadsfastq
MappingtoTranscriptome
Ungapped mapper Bowtie2
RSEMKallistoexpress
Transcriptidentification&counting
![Page 32: RNA seq Data Therapy 12Jan2018 - University of Connecticut · analysis RNA fastq fastq SAM/BAM fasta GFF/GTF RNA-seqvs Microarray • Higher sensitivity and dynamic range • Lower](https://reader034.vdocument.in/reader034/viewer/2022050409/5f85c8cc6b292f30c646bf78/html5/thumbnails/32.jpg)
Introduction
ExperimentalDesign
Sequencing
DataQualityControl
Readmapping
ReferenceGenome
ReferenceTranscriptome
DifferentialExpressionanalysis
RNA
fastq
fastq
SAM/BAM
fasta
GFF/GTF
Mappingwithoutreference
Shortreadsfastq
Assemblyintotranscripts
DeBrujin graphs TrinityOasesVelvet
Mapreadsback
Ungapped mapper Bowtie
Htseq-countRSEM,express
Functionalannotation
GTF-based
Counting
Blast2GOHomologybased
![Page 33: RNA seq Data Therapy 12Jan2018 - University of Connecticut · analysis RNA fastq fastq SAM/BAM fasta GFF/GTF RNA-seqvs Microarray • Higher sensitivity and dynamic range • Lower](https://reader034.vdocument.in/reader034/viewer/2022050409/5f85c8cc6b292f30c646bf78/html5/thumbnails/33.jpg)
Introduction
ExperimentalDesign
Sequencing
DataQualityControl
Readmapping
ReferenceGenome
ReferenceTranscriptome
DifferentialExpressionanalysis
RNA
fastq
fastq
SAM/BAM
fasta
GFF/GTF
Alignmenttools
![Page 34: RNA seq Data Therapy 12Jan2018 - University of Connecticut · analysis RNA fastq fastq SAM/BAM fasta GFF/GTF RNA-seqvs Microarray • Higher sensitivity and dynamic range • Lower](https://reader034.vdocument.in/reader034/viewer/2022050409/5f85c8cc6b292f30c646bf78/html5/thumbnails/34.jpg)
Introduction
ExperimentalDesign
Sequencing
DataQualityControl
Readmapping
ReferenceGenome
ReferenceTranscriptome
DifferentialExpressionanalysis
RNA
fastq
fastq
SAM/BAM
fasta
GFF/GTF
Alignmenttools
Conesaetal,GenomeBiology201617:13
![Page 35: RNA seq Data Therapy 12Jan2018 - University of Connecticut · analysis RNA fastq fastq SAM/BAM fasta GFF/GTF RNA-seqvs Microarray • Higher sensitivity and dynamic range • Lower](https://reader034.vdocument.in/reader034/viewer/2022050409/5f85c8cc6b292f30c646bf78/html5/thumbnails/35.jpg)
`
Introduction
ExperimentalDesign
Sequencing
DataQualityControl
Readmapping
ReferenceGenome
ReferenceTranscriptome
DifferentialExpressionanalysis
RNA
fastq
fastq
SAM/BAM
fasta
GFF/GTF
ReadMapping
![Page 36: RNA seq Data Therapy 12Jan2018 - University of Connecticut · analysis RNA fastq fastq SAM/BAM fasta GFF/GTF RNA-seqvs Microarray • Higher sensitivity and dynamic range • Lower](https://reader034.vdocument.in/reader034/viewer/2022050409/5f85c8cc6b292f30c646bf78/html5/thumbnails/36.jpg)
Introduction
ExperimentalDesign
Sequencing
DataQualityControl
Readmapping
ReferenceGenome
ReferenceTranscriptome
DifferentialExpressionanalysis
RNA
fastq
fastq
SAM/BAM
fasta
GFF/GTF
![Page 37: RNA seq Data Therapy 12Jan2018 - University of Connecticut · analysis RNA fastq fastq SAM/BAM fasta GFF/GTF RNA-seqvs Microarray • Higher sensitivity and dynamic range • Lower](https://reader034.vdocument.in/reader034/viewer/2022050409/5f85c8cc6b292f30c646bf78/html5/thumbnails/37.jpg)
StatisticalpropertiesofedgeR (exact)asafunctionoflog2(FC)threshold,T,andthenumberofreplicates,n
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Introduction
ExperimentalDesign
Sequencing
DataQualityControl
Readmapping
ReferenceGenome
ReferenceTranscriptome
DifferentialExpressionanalysis
RNA
fastq
fastq
SAM/BAM
fasta
GFF/GTF
DifferentialexpressionCommontoolsfordifferentialexpressionanalysis
![Page 39: RNA seq Data Therapy 12Jan2018 - University of Connecticut · analysis RNA fastq fastq SAM/BAM fasta GFF/GTF RNA-seqvs Microarray • Higher sensitivity and dynamic range • Lower](https://reader034.vdocument.in/reader034/viewer/2022050409/5f85c8cc6b292f30c646bf78/html5/thumbnails/39.jpg)
Decisiontreeforsoftwareselection
Introduction
ExperimentalDesign
Sequencing
DataQualityControl
Readmapping
ReferenceGenome
ReferenceTranscriptome
DifferentialExpressionanalysis
RNA
fastq
fastq
SAM/BAM
fasta
GFF/GTF
![Page 40: RNA seq Data Therapy 12Jan2018 - University of Connecticut · analysis RNA fastq fastq SAM/BAM fasta GFF/GTF RNA-seqvs Microarray • Higher sensitivity and dynamic range • Lower](https://reader034.vdocument.in/reader034/viewer/2022050409/5f85c8cc6b292f30c646bf78/html5/thumbnails/40.jpg)
Sources:
Conesaetal,GenomeBiology201617:13
Schurch etal,https://arxiv.org/abs/1505.02017
ZengAndMortazavi NatImmunol 13(9),802-7
Mortazavi etalNatureMethods volume5, pages621–628 (2008)