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/SIOjmccrow@jcvi.org

Luke Thompson Research Associate

NOAA SWFSC & AOML

Genomics

Metagenomics Amplicon sequencing

Transcriptomics

Bioinformatics Data science

Environmental sequencing Fisheries biology

Ecosystem assessment

Luke.thompson@noaa.gov

Tessa Pierce!

Nonmodel Transcriptomics - Environmental Stressors - Time Series Analysis

TIME%

ntpierce@ucdavis.edu

@saltyscientist

LisaKomoroskeDept.ofEnvironmentalConserva"onSWFSCMarineMammal&TurtleDivisionLisa.komoroske@noaa.govhQps://Lmkomooroske.com

LisaKomoroske

LisaJohnson(Cohen)TitusBrown–LabforDataIntensiveBiologyAndrewWhitehead–EnvironmentalGenomics

UCDavis@monsterbashseq

ljcohen@ucdavis.edu

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.