abel gonzalez-perez research associate biomedical...

Abel Gonzalez-PerezResearch Associate

Biomedical Genomics Lab

0. A word about detection...

0. A word about detection

1. Annotation of SNVs and small indels. Vocabularies and tools

2. Functional Impact of SNVs. Approaches and tools

3. Somatic mutations in cancer

NGS technologies compared to Sanger sequencing

B&FG 3eTable 9-1Page 382

FASTQ format

The FASTQ format stores DNA sequence data as well as associated Phred quality scores of each base.

DNA read

Base quality score

Next-generation sequence data: visualizing of short reads aligned to a reference genome

reference genome

reads

variant

B&FG 3eFig.9-13Page 403

home/bioinformatics$ samtools view 030c_S7.bam | lessM01121:5:000000000-A2DTN:1:2111:20172:15571 163 chrM480 60 148M2S = 524 195 AATCTCATCAATACAACCCTCGCCCATCCTACCCAGCACACACACACCGCTGCTAACCCCATACCCCGAACCAACCAAACCCCAAAGACACCCCCCACAGTTTATGTAGCTTACCTCCTCAAAGCAATAACCTGAAAATGTTTAGACGGG BBBBBFFB5@FFGGGFGEGGGEGAAACGHFHFEGGAGFFHAEFDGG?E?EGGGFGHFGHF?FFCHFH00E@EGFGGEEE1FFEEEHBGEFFFGGGG@</01BG212222>F21@F11FGFG1@1?GC<G11?1?FGDGGF=GHFFFHC.-RG:Z:Sample7 XC:i:148 XT:A:U NM:i:3 SM:i:37AM:i:37 X0:i:1 X1:i:0 XM:i:3 XO:i:0 XG:i:0 MD:Z:19C109C0A17

(1)Thequerynameof theread isgiven(M01121…)

(2)Thef l agvalueis163(thisequals1+2+32+128)

(3)Thereferencesequencename,chrM,refersto themitochondrial genome

(4)Position480 istheleft-mostcoordinatepositionof thisread

(5)ThePhred-scaledmappingqualityis60(anerrorrateof1in10 )

(6)TheCIGARstring(148M2S)shows148matchesand2soft-clipped(unaligned)bases

(7)An=signshowsthatthematereferencematchesthereferencename

(8)The1-based leftposition is524

(9)Theinsert sizeis195 bases

(10)ThesequencebeginsAATCT andendsACGGG(itslength is150bases)

(11)Eachbaseisassignedaqualityscore(fromBBBBBendingFHC.-)

(12)Thisreadhasadditional,optionalf i el dst

h

ataccompanytheMiSeqanalysi

Anatomy of a Sequence Alignment/Map (SAM) file

B&FG 3eTable 9.6Page 411

Variant Call Format (VCF) file summarizes variation

A VCF file includes the following information:

B&FG 3eFig.9-17Page 412

Variant Call Format (VCF) file summarizes variation

SNP Insertion

Deletion Replacement

Alignment VCFrepresentation1234 POS REF ALTACGT 2 C TATGT

Alignment VCFrepresentation12345 POS REF ALTAC-GT 2 C CTACTGT

Alignment VCFrepresentation1234 POS REF ALTACGT 1 ACG AA--T

Alignment VCFrepresentation1234 POS REF ALTACGT 1 ACG ATA-TT

1. Annotation of SNVs and small indels. Vocabularies and tools.


Genomic variant

Effect on transcript

Phenotypic effect


Genomic variant


Phenotypic effect

Functional Impact

Consequence type


Genomic variant


Phenotypic effect

Association studies


Genomic variant


Phenotypic effect

Functional Impact

Consequence type

Annotation

Assessment


Taken from Ensembl Variation Database


The Ensembl Variant Effect Predictor

Genomic coordinates/ change of the variant

Map to overlapping genomic features(transcripts, reg. regions, etc)

VEP standalone PERL script using Ensembl APIs

VEP web tool


The Ensembl Variant Effect Predictor


The Ensembl Variant Effect Predictor(Example)


Predicting functional impact of nsSNVs


Predicting functional impact of nsSNVs:three exemplary tools

http://sift.jcvi.org/

SIFT



Query sequence + aminoacid change

Psi-Blast +Motif

Closely related sequences

MSA

Closely related sequences

PSSM

Ng and Henikoff, 2001. Genome Research

SIFT



http://genetics.bwh.harvard.edu/pph2/PolyPhen2





Adzhubei et al, 2010. Nature Methods

PolyPhen2


SNVs catalogs

1000 Genomes Project:genetic variants with frequencies of at least 1% in the populations studied

~2,504 individuals so far

http://www.1000genomes.org/


SNVs catalogs

http://www.ncbi.nlm.nih.gov/projects/SNP/

dbSNPCentral repository of SNVs without allele frequency limitation


SNVs catalogs

http://hapmap.ncbi.nlm.nih.gov/

HAPMAPProject to characterize haplotypes (close co-ocurring variants)

in human populations


SNVs catalogs

https://www.gwascentral.org/

GWAS Centralcentralized compilation of summary level findings from genetic

association studies, both large and small


Navigating variants in Ensembl http://www.ensembl.org

SMARCA4: genomic region

SMARCA4: transcripts


Navigating variants in Ensembl

3. A word about somatic mutations in cancer

Mike Stratton. EMBO Molecular Medicine (2013)

Mutagenic processes

(internal and external)

Mechanisms of DNA repair

Mutational processes

Mutagenic processes

(internal and external)

Mechanisms of DNA repair

This interaction affects he genome at different levels of resolution



UV light signature

Tobacco smoke

Mutation burden and mutationsignature affecting the whole

genome


Sabarinathan et al. Nature. 2016

More mutations in transcription factor binding sites of melanocytes

Schuster-Böckler and Lehner et al. Nature 2012

More mutations in closed chromatin regions

More mutations in regions lowly expressed genes

Lawrence et al. Nature. 2013

Fewer mutations in exons than expectedin POLE-mutant tumorsFrigola, Sabarinathan et al. Nature Genetics. 2017

3. Interpreting somatic mutations in cancer

Understand the genomic mechanisms that fuel the tumor phenotypes

Drivers of cancer

Hanahan and Weinberg. Cell 2011

Driver versus passenger events

Needle in a haystack

Which mutations in driver genes are drivers?

How deleterious are cancer driver mutations?


Deleteriousness

Driv

erne

ss

Yes

No

Yes No

● Highly deleterious mutations affecting cancer genes

● Highly deleterious mutations affecting non-cancer genes

● Lowly deleterious mutations that contribute to tumor phenotype?

● Lowly deleterious mutations affecting non-cancer genes

Pas

seng

er

Carter et al., 2009. Cancer Research


It depends on the mode of action of the gene!!

OncodriveFM

OncodriveCLUST

MuSiC-SMG

F

C

RUsing complementary signals help obtaining a more comprehensive list of cancer drivers

Tamborero et al., Scientific Reports 2013

How about other types of variants?

Copy Number Alterations

Other genomic rearrangements

Repositories of known oncogenic variants

PTEN*

Temsirolimus

Vemurafenib(V600E) (R130G)

PI3K

AKT

mTOR

BRAF*

(R175H)

TP53*

rAd-p53

Strategies to target cancer drivers

IndirectGene Therapy

Direct

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