I. Introduc+ons II. Transcriptomics 101
Sep 25, 2020
I.Introduc+ons
II.Transcriptomics101
WorkshopLearningObjec+ves
1. Whattypesofresearchques+onsthattranscriptomicscanbeusedtoaddress
2. Keythingstoconsiderinexperimentaldesign&analysisapproaches
3. ConceptualunderstandingofstepsanddecisionsinvolvedinRNA-Seqdataanalysis
4. BecomecomfortableworkingwithRNA-Seqdatainkeyworkflowsteps
5. Knowaboutresourcesandwheretogoforhelp/futurelearning(includingbuildingcontactscommunityherewithotherpar6cipants!)
Schedule&CodeofConduct
Logis+cs• S"ckyNotes
• Etherpad
• Personalnotetakingsugges"ons
• Interac"veEnvironment
JohnMcCrowComputa"onalStaffScien"stAllenLab,JCVI/[email protected]
Luke Thompson Research Associate
NOAA SWFSC & AOML
Genomics
Metagenomics Amplicon sequencing
Transcriptomics
Bioinformatics Data science
Environmental sequencing Fisheries biology
Ecosystem assessment
Tessa Pierce!
Nonmodel Transcriptomics - Environmental Stressors - Time Series Analysis
TIME%
@saltyscientist
LisaKomoroskeDept.ofEnvironmentalConserva"onSWFSCMarineMammal&[email protected]://Lmkomooroske.com
LisaKomoroske
LisaJohnson(Cohen)TitusBrown–LabforDataIntensiveBiologyAndrewWhitehead–EnvironmentalGenomics
UCDavis@monsterbashseq
10
Brennan,2017
Keelingetal.2014PMID:24959919
Whitehead2010
StudentIntroduc+ons
• Whatorganisms/systemsareyoustudying?
• Whyareyouinterestedintranscriptomics?
DNA RNA Proteins
Transcriptomics101• WhatisTranscriptomics?
• Whenwoulditbeagoodtool?
• Whattypeofdataisgenerated?
Transcription RNA splicing & editing
Translation
the code the messages the effectors
GeneralWorkflow
GeneralWorkflow
FASTQ format @K00180:331:HHVHJBBXX:1:1101:32471:1033 1:N:0:GTGGCC NCAAGGCGGTGGACGTCCCCAAAACCAACCGAGGAAGCTTTGGGATAGCA + #<-A-FJFA7FAJFJA<JJAJJJJFJJJJFFA7FFJF<FA<FFJFFJJJJ
Sequence
Qualityscores(samelengthassequence)
Header
Illuminaformatincludesreadpairandindex
FASTA format >K00180:331:HHVHJBBXX:1:1101:32471:1033 1:N:0:GTGGCC CAAGGCGGTGGACGTCCCCAAAACCAACCGAGGAAGCTTTGGGATAGCA
Sequence
Header
NoqualityscoresTrimmedforhigh-qualitybasesonly(Q30)
WhatisTranscriptomics?•SequencingofcellularmRNAtranscripts(ordepletedtotalRNA-prokaryotes)•Powerfulmeasureoffunc"onalresponsetochangingenvironmentalcondi"ons,celltypes,
orpopula"ons,etc.•Small"ssuerequirements&mul"plexingcapacity•Non-omicsapproaches:RT-qPCR,RNaseprotec"onassay,Northernblot•Otherapplica"ons(e.g.,variantcalling)
WhyuseTranscriptomics?•Wanttoknowaboutmanygenes,don’tknowwhichgenesarepresentorareinteres"ng•Wanttoknowaboutoverallbiologicalprocessesand/orpathwaysaffected•Genomesarenotcomprisedofjustsingle-copysingle-func"onproteins,especiallyinmore
complexeukaryotes•Evenwhenmeasurementofashortlistofgenesisdesired,valueinknowingthecontextof
othergenes.