tutorial - salk institute for biological studies

SUMMERTutorial07-29-2020SalkIGC

Systeminformationbox:Usedforinstructionsandwarningmessages.

Datauploadpanel

Dataanalysistabs

OverviewofSUMMERwebserver

downloadsampledataset

Torunatestdataset:

1. Checkthisbox(ontopleftcorner)

2.Clickthisbutton(bottomleftcorner)

Torunanewanalysis:

1.choosetousetranscriptomeorproteomedata.Note:ifyoudon’thaveeither,leavetheselectionasis,skiptheuploadoftranscriptomicsdata,click“Runanalyses”.

3.uploadthemetabolomicsdata(must).Chooseiftheinputtablehasaheaderlineandwhetherthetableisdelimitedbycomma,semicolon,ortab.

4.uploadthetranscriptomicsorproteomicsdata(optional).

Apaneltouploadproteomicsdatawillappearif“Proteome”isselectedinstep1.

5.Don’tforgettocheckthisboxformicroarraydata.Datawillbequantile-normalizeduponthisselection.

2.choosespecies.

Torunanewanalysis(continued):

6.setupexperimentdesignformetabolomicsdata.First,choosethecolumnheadersfromdrop-downmenuthatcontainKEGGmetaboliteIDs.Then,choosethecolumnheadersthatcontainreplicatesforeachcondition

7.setupexperimentdesignfortranscriptomics/proteomicsdata.

8.click“Runanalyses”tostarttheanalysis.

Runanalyses

Afterclicking“Runanalyses”,gototheinformationbox.Itwillshowhowmanymetabolites,transcripts/proteinsaremappedandfiltered.Replicatenumbersareallowedtobedifferentformetabolomicsandtranscriptomics/proteomicsfornow.

Nowitisreadytoactuallyruntheanalyses.Note:Pathwayanalysisandnetworkgraphcanonlyberunafterdifferentialexpressionanalysisisperformed.

PrincipleComponentAnalysis(PCA)

Thisbuttonwillappeariftheuploadeddatalooksgood.ClicktorunPCA.

ClickPCAtab.

PrincipleComponentAnalysis(PCA)

Afterclickingthe“RunPCA”button,thePCAplotswillbegeneratedtovisualizesampleseparations.Detailswillbeshownuponmousehovering.

Ittakesabouthalfaminutetorendertheplots,pleasebepatient.

PCApanelswillbedisplayedintheorderofmetabolite(top),gene(middle),andprotein(bottom).

Plotscanbedownloaded

DifferentialExpression(DE)Analysis

ClickDifferentialExpressiontab.

Thisbuttonwillappeariftheuploadeddatalooksgood.Note:bootstraptakesabout2-5minstorundependingondatasize.

DifferentialExpression(DE)Analysis

DownloadbuttontodownloadtheDEresults.

• ThedefaultDEcutoffisabsolutelogFC>0.5andadjustedp-val <0.05formetabolites,absolutelogFC>0.5andrankingscore<0.05forreactions.

• ThedefaultDEcutoffisusedforthisbarplot,andthepathwayenrichmentanalysisonthenextslide.

• ThedefaultDEcutoffisnotadjustableatthismoment.

PathwayAnalysisClickPathwayAnalysistab.

ThisbuttonwillappeariftheDEanalysisisperformed.

Currently,onlyOver-representationAnalysisissupported.ChooseDEcutoffforthepathwayenrichmentresult.

PathwayAnalysis

Downloadbuttontodownloadthepathwayresults.

Barplots ofsignificantlyenrichedKEGGmetabolicpathwaytermsforDEmetabolitesandDEreactions.

PathwayAnalysis

ChooseKEGGmetabolicpathwaysfromdrop-downmenustogenerateanetworkgraphforthatpathway.Allmeasuredmetaboliteswillbeshownregardlessofitsstatisticalsignificance.

Downloadbuttontodownloadthepathwaynetworkgraphindifferentformats.

PathwayviewofNetworkGraph

NetworkGraph

ClickNetworkGraphtab.

ThisbuttonwillappearaftertheDEanalysisisperformed.

ChoosethestatisticalsignificancecutofftoconstructanetworkofDEmetabolitesandDEreactionsplustheirassociatedpartners.

NetworkGraph

GlobalviewofNetworkGraph

Canbemagnifiedbymousescrolling.

Choosefromdrop-downmenuthemetabolite/reactionneedstobehighlighted.

Downloadbuttontodownloadthenetworkgraphindifferentformats.

Square=reaction,circle=metabolite.red=up-regulation,blue=down-regulation.Networknodesizerepresentsitsrelativeconnectivitywithothers.Soonemetabolitenodecanbetinyifitonlyconnectswithonereaction.