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

IGV Hands-on Exercise Viewing SNPS: page 1 1. Reference Genome First make sure the reference genome is set to the one you loaded in the previous exercise: Select chr1.fasta from the genome dropdown menu 3. Navigate to first putative SNP locus Type snp1 in the search box and click Go 4. Optional a) Modify the info popup behavior if you don’t want the yellow info window visible all the time. Click on the yellow balloon icon, and select Show details on click b) For a larger data panel, click & drag the window divider Note: Do not load the .bai file Viewing SNPs 2. Load data Click File > Load from File Navigate to the folder that contains the workshop igvData folder, and then the snps subfolder. Open the following files igvData / snps / snp_calls.bed igvData / snps / NA12878.SLX.sample.bam IGV automatically finds the index file – as long as it is named correctly and is in the same folder as the .bam file

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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

Page 2: IGV Exercise 3 SNPs · IGV Hands-on Exercise Viewing SNPS: page 1 1. Reference Genome First make sure the reference genome is set to the one you loaded in the previous exercise: Select

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.

ViewingSNPS:page2

Page 3: IGV Exercise 3 SNPs · IGV Hands-on Exercise Viewing SNPS: page 1 1. Reference Genome First make sure the reference genome is set to the one you loaded in the previous exercise: Select

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

ViewingSNPS:page3

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