igv exercise 3 snps · igv hands-on exercise viewing snps: page 1 1. reference genome first make...

IGVHands-onExercise

ViewingSNPS:page1

1.ReferenceGenome

Firstmakesurethereferencegenomeissettotheoneyouloadedinthepreviousexercise:

Selectchr1.fasta fromthegenomedropdownmenu

3.Navigatetofirst putativeSNPlocus

Typesnp1inthesearchboxandclickGo

4.Optional

a) Modifytheinfopopupbehaviorifyoudon’twanttheyellowinfowindowvisibleallthetime.Clickontheyellowballoonicon,andselectShowdetailsonclick

b) Foralargerdatapanel,click&dragthewindowdivider

Note:Donotloadthe.bai file

ViewingSNPs

2.Loaddata

ClickFile>LoadfromFile

NavigatetothefolderthatcontainstheworkshopigvData 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.

àThisappearstobeaheterozygousSNP.

7.Gotothelocusofthesecond putativeSNP

Typesnp2 inthesearchbox andclickGo

Observe themismatchedbasesandtheirapparentlowqualities.(Mismatchedbasesaredrawninafaintercolorifthebasecallisoflowquality)

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.

à Thisislikelyafalsepositive.

ENDOFEXERCISE

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