IGV Hands-on Exercise IGV Basics · 1. Launch IGV 2. Select reference genome. • Click on Human...
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1. Launch IGV 2. Select reference genome. • Click on Human hg19 in the genome drop-down menu in the upper left corner. 3. Load data from the IGV hosted server. • Select File > Load from Server… • Open the Tutorials menu (Use on Mac, and + on Windows) and click on the UI Basics checkbox. Four tracks are loaded: ENCODE project ChIP-seq data representing histone modifications. Each track is displayed as a bar chart of signal intensities. ✔ Make sure you only open the Tutorials menu. Do not check the box next to Tutorials. That will select everything under Tutorials, but we only want UI Basics for this exercise. IGV Basics - page 1 This type of data is perfect for a UI basics exercise because the tracks are visually simple – but the navigation basics are the same no matter what type of data you load. If this is the first time you run IGV, there may be only one entry in the menu. More about that later... If you only see Human hg18 in the menu, it’s ok to select that instead IGV Hands-on Exercise IGV Basics
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.
✔
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
ENDOFEXERCISE
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
ENDOFEXERCISE
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
✔
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
