IGV Hands-on Exercise IGV Basics · 1. Launch IGV 2. Select reference genome. • Click on Human...
Transcript of IGV Hands-on Exercise IGV Basics · 1. Launch IGV 2. Select reference genome. • Click on Human...
1.LaunchIGV
2.Selectreferencegenome.• ClickonHumanhg19 inthegenome
drop-downmenuintheupperleftcorner.
3. LoaddatafromtheIGVhostedserver.• SelectFile>LoadfromServer…
• OpentheTutorialsmenu(UseonMac,and+onWindows)and clickontheUIBasicscheckbox.
Fourtracksareloaded:ENCODEprojectChIP-seq datarepresentinghistonemodifications.Eachtrackisdisplayedasabarchartofsignalintensities.
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MakesureyouonlyopentheTutorialsmenu.Donot checktheboxnexttoTutorials.ThatwillselecteverythingunderTutorials, butweonlywantUIBasics forthisexercise.
IGVBasics- page1
ThistypeofdataisperfectforaUIbasicsexercisebecausethetracksarevisuallysimple– butthenavigationbasicsarethesamenomatterwhattypeofdatayouload.
IfthisisthefirsttimeyourunIGV,theremaybeonlyone entryinthemenu.Moreaboutthatlater...
IfyouonlyseeHumanhg18inthemenu,it’soktoselectthatinstead
IGVHands-onExercise IGVBasics
4. Navigate acrossdifferentgenomiclociandatdifferentzoomlevels,fromwholegenomeviewanddowntobase-pairresolution.
4a. Startatwholegenomeview:• SelectAllfromthechromosomedrop-downmenu–OR– ClicktheHomebutton.
4b. Zoomintoviewonewholechromosome:• SelectChr1fromthechromosomedrop-downmenu–OR– Clickthe1inthegenomeruler.
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Rulerdisplaysallthechromosomes
Genomerulernowhasmoredetailsandacytoband viewofthechromosome
Atthiszoomedoutview,thegenetrackdisplaysgenedensity
Genetrackstartsshowingindividualgenes,butstilltoo
manytosee
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4c. Zoominfurther:• Clickanddragtozoominonaregionsweptoutintheruler
• Double-clickinthedatatracktozoominonapointofinterest.[Alt-clicktozoomout]
Rulermeasurementsandaredboxonthecytoband diagramshowwhereyouareinthechromosome
4d. Movearoundwithinthechromosome:• Jump toanotherregioninthesamechromosome(nochangeinzoomlevel):
Clickanywhereinthecytoband diagram.
• Scroll acrossgenomecoordinates:Clickanywhereinthedatapanelanddragleft &right.
4e. Navigatetospecificlocusorgeneonanychromosome• TypeintothesearchboxintheIGVtoolbarandclickGo:
eitheralocusingenomiccoordinates(e.g.chr1:144,874-969,268) oragenename(e.g.NRAS)
IGVBasics-page3
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4f. Zoomintobase-pairresolution:• Keepzoominginasbefore,orclickononeoftherightmostticksonthe
“railroadtrack”zoomwidgetintheupperrightcorner.
5. Optionsforviewingthereferencesequencetrack• Clickanywhereonthesequencetoshow/hidea3-frametranslation
• Bydefault,thesequencefortheforwardstrandisshown.Clickonthearrowtoreversethestrand.
6.OptionsforviewingthegenetrackandotherannotationtracksIGVusesaUCSCbrowserstylegenerepresentation:
• Expand thetrackusingtheright-clickpopupmenu
Referencesequence
5’ UTR
Exons
Intron 3’ UTR
Featuresaredrawninasingleline,bydefault
UseSquishedforanevenmorecompactview
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1.Clearoutthedatafromthepreviousexercise:SelectFile>NewSession
2.SelectGenomes >LoadGenomefromFile
andbrowsetotheigvData workshopfolder,andthentothegenomesubfolderandopenchr1.fasta
3. NoteintheIGVwindow:thereisnogenetrack,andnocytoband ideograminthegenomeruler.
IGVhostedgenomespackageeverythingtogether, butyouloadedonlytheFASTAfilewiththesequence.Youcanzoominandoutasbefore,andenterthenumericvalueofalocus,butyoucannotfindagenelocusbyenteringthenameinthesearchbox.
4. LoadageneannotationtrackSelectFile >LoadfromFileandopenrefSeq_chr1.bedfromtheigvData /genomefolder.
Nowyoucanjumptoalocusbyenteringthenameofageneonchr1inthesearchbox,e.g.CAP9
Notpartofthisexercise:YoucanusetheUCSCTableBrowser togetafileofgeneannotations.
5.Thecytoband cannotbeloadedseparatelyintothegenomeruler.
Thechr1.fasta filecontainschromosome1
fromHumanhg18.
http://genome.ucsc.edu/cgi-bin/hgTables
Note: thisistheFilemenu,not theGenomemenu
Note: thisistheGenomemenu,not theFilemenu
IGVHands-onExercise Loadgenomefromfile
ENDOFEXERCISE
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IGVHands-onExercise
ViewingSNPS:page1
1.ReferenceGenome
Firstmakesurethereferencegenomeissettotheoneyouloadedinthepreviousexercise:Selectchr1.fasta fromthegenomedropdownmenu
3.Navigatetofirst putativeSNPlocus
Typesnp1inthesearchboxandclickGo
4.Optional
a) Modifytheinfopopupbehaviorifyoudon’twanttheyellowinfowindowvisibleallthetimeè Clickontheyellowballoonicon,andselectShowdetailsonclick
b) Foralargerdatapanel,click&dragthewindowdivider
Note:Donotloadthe.bai file
ViewingSNPs
2.Loaddata
ClickFile>LoadfromFile
NavigatetotheworkshopigvData folder,andthenthesnps subfolder.Openthefollowingfiles
igvData /snps /snp_calls.bedigvData /snps /NA12878.SLX.sample.bam
IGVautomaticallyfindstheindexfile– aslongasitisnamedcorrectlyandisinthesamefolderasthe.bamfile
5.Sortthemismatchedalignedreadsbybase
First,clickanddragtopositionthemismatchedbasesbetweenthecenterguidelines
Right-click(onMac:control-click)anywhereinthealignedreads,andselectSortalignmentsby>base
6.Seetheallelecountsandfrequencies
Mouseoverthered/bluebarinthecoveragetrack(Orclick onthebar,ifyouchangedtheinformationpopupbehaviortodisplayonclickonly)
Observe thedistributionofmismatchesatthatlocus.Observe thelackothermismatchesintheregion.
è ThisappearstobeaheterozygousSNP.
7.Gotothelocusofthesecond putativeSNP
Typesnp2 inthesearchbox andclickGo
Observe themismatchedbasesandtheirapparentlowqualities.(Mismatchedbasesaredrawninafaintercolor
ifthebasecallisoflowquality)
8.Disableshadingbyquality
Click&dragtopositionthesnp2 locus(withthe5blueCs)betweentheverticalcenterguidelines.
Right-click(onMac:control-click)anywhereinthealignedreads,andclickShadebasebyquality
Observe themismatchedbases.
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9.Sortandcolorthealignedreadsbyreadstrand
Right-click(onMac:control-click)anywhereinthealignedreads,andselectSort alignmentsby>readstrand
Right-click(onMac:control-click)anywhereinthealignedreads,andselectColor alignmentsby>readstrand
Observewherethemismatchesare.
Note:Weknowthatthissequencingwasnotwithastrand-preservinglibrary,sotheexpectedstranddistributionis50-50.
è Thisislikelyafalsepositive.
ENDOFEXERCISE
ViewingSNPS:page3
IGVHands-onExercise Pre-computecoveragetrack
Thisexerciseassumesthefollowinghavealreadybeenloadedinthepreviousexercises:
a) Referencegenomechr1.fasta(fromexercise“Loadgenomefromfile”)
b)BAMfilefromworkshopfolder:igvData /snps /NA12878.SLX.sample.bam(fromexercise“ViewingSNPs”)
1.First,zoomallthewayoutbyclickingontheleftmosttickontherailroadtrackinthezoomtool.
Observethereisnodatainthecoveragetrack
2.Launchigvtools:clickTools>Runigvtools
3.RuntheCounttool
>SelectCountfromtheCommanddropdownmenu>SettheInputFiletotheNA12878.SLX.sample.bam fileintheworkshopfolderigvData /snps.TheOutputFile willautomaticallybesettothesamefolder,andsamename+.tdf suffix
>Keepthedefaultsforallothervalues.>ClickRun
Waituntilyousee“Done”intheMessagesarea,andclickClose.
Pre-computingcoveragedata:page1
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4.Associatethenew.tdf filewiththecoveragetrack
>Right-click(command-clickonMac)anywhereonthecoveragetrack intheIGVwindow...
>...andselectLoadpre-computedcoveragedata.
>BrowsetotheworkshopfolderigvData/snpsandselectthefileNA12878.SLX.sample.bam.tdf
2.Observethe3spikesinthecoveragetrack.
The.bamfilefortheexercisewasstrippeddownandonlyhasdatainthese3regions.
Ifyouzoominontheleftmostspike,youwillseethesnp1locusfromtheViewingSNPsexercise.
Pre-computingcoveragedata:page2
1.SetpreferencesforviewingRNA-seq data
ClickView>Preferences
SelectAlignmentstab
CheckSpliceJunctionTrack
2.Loaddata
SelectHumanhg19fromthegenomedropdownmenu
ClickFile>LoadfromServer
OpentheTutorialsmenu(UseonMac,and+onWindows)andclickonRNA-Seq (BodyMap)andthenclickonOK
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MakesureyouonlyopentheTutorialsmenu.Donot checktheboxnexttoTutorials.ThatwillselecteverythingunderTutorials, butweonlywantRNA-Seq forthisexercise.
ViewingRNA-Seq data:page1
IGVHands-onExercise ViewingRNA-Seq Data
3.JumptogeneSLC25A3
TypeSLC25A3 inthesearchboxandclickGo
4.Expandgenetracktoseeisoforms
Right-clickovertheRefSeq Genestrack,andselect Squished
Coverage
Junctions
5.Zoominonfirst3exons
Clickanddraginrulerregionoverareashown
ViewingRNA-Seq data:page2
6.Noteevidenceofalternativesplicing.
ObservewhichisoformsintheRefSeq trackareexpressedineachtissue.
7.Zoombackouttoviewwholegene
Clickthebackbuttoninthecommandbartozoomouttopreviousview
8.OpenSashimiplot
Right-clickoverjunctiontrackoralignmentsandselect“SashimiPlot”
Verifybothheart andliver arechecked,andclickOK
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9.ExamineSashimiplot
Note:
• Arcsrepresentreadsspanningexonjunctions
• Peaksrepresentexoncoverage
10.Filteroutlow-countsplicingevents
Rightclickoverred(heart)trackandselectSetJunctionCoverageMin.Enter50 andclickOK.
Repeatforblue(liver)track.
11.Comparewithnon-filteredview
12.Zoominon5’end
Click“+”button2times
Click-and-dragtrackstotherighttobringthefirst3exonsinview.
13.Observethealternativesplicingofthe3rd exon
ViewingRNA-Seqdata:page4 ENDOFEXERCISE
1.Loaddata
ClickFile>OpenSession
Genotypes
Thennavigatetotheworkshopfolder /igvData /vcfandopenvcf_session.xml
2.Observethedifferentdatapanels
Variantsites
Sampleinformation
Hoveroverthevariantsitesandthegenotypestoseethedetails.(Ifyouchangedthepopupbehaviorwiththetool,youmayhavetoclick toseethedetails)
Observe howthesamevaluesinthesampleinformationpanelareassignedthesamecolor.Tryclickingonthesampleinformationcolumnheaderstosortbyattribute.
Sampleinfocolumnheaders
3.HighlightinterestingeventingeneAPOL1
- TypeAPOL1:S342GinthesearchboxandclickGo
- Right-clickovergenotypesandselectDisplayMode:Squished
Viewingvariants(VCFfile)-page1
IGVHands-onExercise Viewingvariants(VCFfile)
4.ObservedifferencesbetweengroupsUsethescrollbartoscrolldownandseeallthegroups.
Notethatthevariantsatthislocusarenotpresentinsomepopulationgroupsandareprevalentinothers.
Scrollbar
3.(continued)HighlightinterestingeventingeneAPOL1
- Right-clickovergenotypesandselectGroupBy,thenselectattributesuper_pop.
Viewingvariants(VCFfile)-page2
ENDOFEXERCISE