Mutation CallingIGV Exercises

• Run IGV – Web search IGV (Integrative Genomics Viewer)– Go to Download page – may need to provide email– Launch with JavaWebStart – 1.2 GB will work

• Set genome (top left) to Human hg19• Copy Exome.bam Exome.bam.bai and

Exome.bam.tdf to your computer.• Use File / Load from File to open the .bam file

Setup

Exercise #1

• Find BRCA2 gene. • Double click to zoom in and see the reads • Can you find a mutation in BRCA2 that will

affect the protein sequence of the gene? • If so, what is the codon change and protein

impact?

#1 BRCA2

No VariantPoor CoverageLack of supporting Reads

#1 BRCA2

VariantBut No Coding Impact

#1 BRCA2

Gene: BRCA2gTa->gCaProtein: Val(V) to Ala(A)Missense

Exercise #2

• Go to the genomic position:

chr 10 124066731

• How would you characterize this mutation? • What is its protein impact?

#2 Chr 10 124066731

Gene: BTBD16ctT_>ctGHetrozygousProtein L to LSynonymous

Zygosity call in cancer is complicated

K562

Exercise #3

• Go to the genomic position:

chr 2 136575534

• What would the impact of this mutation be?

#3 Chr 2 136575534

NegativeStrandGene

#3 Chr 2 136575534

NegativeStrandGene

Gene: LCTMissense MutationAtc->GtcProtein I to V

Exercise #4• Go to the genomic position:

chr 2 272256

• If T was mutated to G what would the impact be?• If a G was inserted at the position what would the

impact be?• If TGA was deleted at the position what would the

impact be?

#4 chr2 272256 T to G

Multiple Effects on different transcripts:Gene: ACP1NM_070099 NM_001040649gTg->gGg Tga->GgaV to G Stop to G

Exercise #5

• Go to the genomic position:

chr 13 32953452• If the 'G' that shows up on one read represented a

real mutation, what would the impact on the protein be?

#5 Chr 13 32953452

NegativeStrandGene

If the G represented a realVariant, impact:SpliceSite mutation with potentialto cause exon skip.

Exercise #6

• Go to the genomic position:

chr 2 11094125

• Is there any reason to have reduced confidence in a mutation call here?

#6 Chr 2 11094125

Low Complexity RegionMakes Mutation Calling Less Reliable