LastUpdated:9/7/16

TutorialbyHansVasquez-GrossUCDavis-DubcovskyLab

Tutorial:HowtousetheWheatTILLINGdatabase

1. Visithttp://dubcovskylab.ucdavis.edu/wheat_blasttogototheBLASTpageorclickonthe‘WheatBLAST’buttononthehomepage.

2. A)Pasteinyournucleotidesequence,B)selectthe‘TILLINGGenomicReference(4xand6x)’database,andthenC)hitthe“Basicsearch”button.

3. Wait5-20secondswhiletheBLASTsearchruns.Thetimeisdependentonnumberofquerysequencesandthelengthofthequery.

A)

B)

C)

LastUpdated:9/7/16

TutorialbyHansVasquez-GrossUCDavis-DubcovskyLab

4. Whenfinished,youwillbepresentedwithatableofBLASThitresults.Here,A)youcanviewtheBLASTHTMLoutput,B)downloadtabularBLASTTSVoutput,C)checktheboxforcontigsequenceyouwanttodownload,D)clickonScorestoviewtheBLASTHTMLalignmentforaspecifichit,andE)clickalinktoviewtheannotationwithpossiblemutantlinesinthesameregion.Fromthispage,youcanalsodownloadfullcontigsequencesbasedonyourresults.

5. Afterclickingonavisualizationlink,allowJBrowsetoloadthesequenceandannotationdata.Thismaytakeanywherefrom5secondsto3minutesdependingonconnectionspeed.Oncefinishedloading,youwillhaveyourhitregionhighlightedinyellow,withyourBLASTHSPsdepictedbygreenbarsintheBlastHSPResultstrack.BelowtheBlastHSPResultstrack,wecanseetheHighConfidenceMutantstrackwhereSingleNucleotideVariants(SNVs)arecolor-codeddependingontheseverityofthevarianteffect.SNVsthataremostseverearecoloredinRED(stop_gained,stop_lost,splice_sitechanges),mediumseveritySNVsarecoloredinPURPLE(missense/aminoacidchange),theleastsevereSNVsarecoloredinGREEN(synonymous/noaminoacidchange),andlastlySNVsarecoloredinBLUEforintron_variantsorwhentheeffectisunknown(I.E.inthecaseofallUCWchromosomeassembledcontigsoranythingthatisnotannotatedinIWGSC).

A)

B)

C) D) E)

LastUpdated:9/7/16

TutorialbyHansVasquez-GrossUCDavis-DubcovskyLab

6. WhenclickingonaparticularSNV,aninformationwindowwillbedisplayedwithdetails.

7. Inordertodownloadnucleotidesequencedataforonlyaparticularregion,youcanusetheReferenceSequencetracktoexportthesequenceinFASTAformat.First,zoomintotheregionofinterestsothatitisdisplayedinthevisualizationwindow.Tozoom,holdthe<SHIFT>keyanduseyourmousetoclickanddragovertheexactregionyouwant.YoushouldseeaREDlinewiththepositionalstartinformationafterpressingSHIFT.

LastUpdated:9/7/16

TutorialbyHansVasquez-GrossUCDavis-DubcovskyLab

8. Then,clickonthereferencetrackdownarrowandchoose‘Savetrackdata’.Thistrackmaybeatthebottomoftheinterface.

9. Youwillthenbepresentedwithadialogwindowwhereyoucanchoosefromacoupleofoptions:Highlightedregion,visibleregion,orwholereferencesequence.Inthiscase,sincewearetryingtograbonlyaparticularsub-regionofthehighlightedregion,wewillchoose‘Visibleregion’andthenclickthe‘Save’button

10. TodownloadSNPdatainTSVformat(canbeopenedinexcel),right-clickonaSNPfordifferentexportoptions.

LastUpdated:9/7/16

TutorialbyHansVasquez-GrossUCDavis-DubcovskyLab

Alternatedownloadoption:

• DownloadingmutationdatacanalsobesearchedbasedonIDsfromtheSampleSearchpage:http://dubcovskylab.ucdavis.edu/wheat-tilling/sample-search

• Thefirst3options(LineName,GeneName,orScaffoldName)willsearchautomaticallyandpresentyouwithalistofoptions.Clickingononeinthedropdownboxwillautomaticallydownloadthemutationdataforthesearchterm.PleasenotethesearchisCASESENSITVE.

• Additionally,thebottomboxcanbeusedtopasteinmixedIDlisttoautomaticallygenerateonefileforallthesearchedmutationdata.