Presentation for the NCSR Demokritos INRaSTES researchers. How can bioinformatics help understand the basic mechanisms of hereditary cancer?

1. Bioinformatics as a tool
for understanding
carcinogenesis
Kalfakakou Despoina
Bioinformatician, PhD candidate
Molecular Diagnostics Laboratory @ NCSR Demokritos
Supervisor: Dr I. Konstantopoulou
NCSR Demokritos
Industrial PhD Scholarship Program
Supported by Stavros Niarchos Foundation

2. History of Sequencing Technology
1977
Sanger
Sequencing
1990
Launching of
Human Genome
Project (HGP)
HGP
completed
2003
2007
NGS
Introduced
Dramatic
drop of
sequencing
cost and
time
2008
Routine
Whole
Genome
Sequencing
20??

3. New era in sequencing technology
Graph sources:
http://www.futuretimeline.net/21stcentury/2015.htm#.WFkM4X0qxrI
https://www.genome.gov/sequencingcostsdata/

4. Add a Slide Title - 3

5. Add a Slide Title - 3
For each person the
file containing all of
their raw data from a
WGS experiment is
240GB

6. WGS VS WES VS Targeted Sequencing

7. Germline Multiplex Sequencing Panels
in Cancer Genetics
▪ 5 to more than
100 genes
▪ Several SNPs
>2 times greater risk of developing cancer
Carrier Frequency: <0.5%
~1-1.5 times greater risk of developing cancer
Carrier Frequency: >1%

8. Breast Cancer Associated
Ovarian Cancer Associated
CRC/Polyposis Associated
Thyroid Cancer Associated
BRCA1APC ATM BRCA2BMPR1A BRIP1
CDH1 CHEK2
EPCAM ERCC4 FANCA FANCB FANCC FANCD2
FANCE FANCF FANCG FANCI FANCL FANCM FH
METMEN1 MLH1 MSH2 MSH6 MUTYH
PALB2 PMS2 PTEN RAD51C RAD51D
RET SDHB SDHC SDHD SLX4 SMAD4 STK11
TP53 VHL
Renal Cancer Associated
Fanconi Anemia Associated
HD Gastric Cancer Associated
Other cancers/syndromes

9. Sequencing procedure
Image source:
https://sites.google.com/site/geneexpressionprofiling/home/transcriptome-sequencing

11. NGS data analysis Overview
Primary Analysis
De-multiplexing
Base Calling & Quality Control (Fastq File)
Secondary Analysis
Read Mapping (SAM/BAM File)
Variant Calling (VCF File)
Tertiary Analysis
Variant Annotation
Variant Filtering

12. NGS data analysis Overview
Primary Analysis
De-multiplexing

13. Conceptual overview of sample
multiplexing
Picture source:
http://www.illumina.com/technology/next-generation-sequencing/multiplexing-sequencing-assay.html

14. NGS data analysis Overview
Primary Analysis
Base Calling & Quality Control (Fastq File)

15. Fastq Generation
Sequence Identifier
Sequence
Quality Score
per base
See more at: https://en.wikipedia.org/wiki/FASTQ_format

16. Phred Score
Q = -10 log10 Perr
Q = 0: Perr = 1
Q = 40: Perr = 0.0001

17. NGS data analysis Overview
Secondary Analysis
Read Mapping (SAM/BAM File)

18. Read Mapping is like
throwing 400 average
city phone books into
a paper shredder…
…and then sorting
through all the shreds
and matching where
they go to recreate
all 400 phone books!

19. Map to Reference Genome

20. Map to Reference Genome
Reference Genome

21. Map to Reference Genome
Reads mapped to this area

22. Map to Reference Genome
MiSeq Coverage: ~100-700 reads

23. NGS data analysis Overview
Secondary Analysis
Variant Calling (VCF File)

24. VCF File Example
Variant Calling
• Locate suspicious of variation regions
comparing to reference genome.
• Local realignment in order to increase
sensitivity.

25. NGS data analysis Overview
Tertiary Analysis
Variant Annotation

26. Variant Annotation
Gene/Variant –
Phenotype
Association DBs
OMIM
ClinVar
LOVD
Gene/Disease Specific DBs
IARC TP53 DB
InSiGHT DBs (MMR genes)
BRCA Share (BR1/2 DBs)
BIC (NIH BrCa DB)
Variant ID and
Frequency
dbSNP
ExAC
gnomAD
1000 Genome
Pathogenicity
Prediction
PolyPhen
MutationTaster
SIFT
AlignGVGD
Predicted Genomic
Features
ESEFinder
HSF
MicroT
Consortia
ENIGMA
BCAC
OCAC
COMPLEXO

27. NGS data analysis Overview
Tertiary Analysis
Variant Filtering

28. Variant Filtering
Variant Quality:
▪ Base qualities
▪ Coverage at position
▪ Strand Bias
▪ Allele Balance
▪ Known artefacts
Variant Prioritization:
▪ Minor Allele Frequency
▪ Functional Impact
▪ Mode of Inheritance
▪ Previous report of
clinical significance
▪ Segregation in family

29. Total Count: 189
WT: 189
Del: 165
BRCA1 c.843_846delCTCA
p.S282TfsX15

30. 7 incidents <40 years (5 incidents <35 years)
BRCA1 c.843_846delCTCA
p.S282TfsX15

31. Total Count: 339
C: 178, 52%
T: 161, 48%
BRCA1 c.5497G>A
p.V1833M

32. BRCA1 c.5497G>A
p.V1833M

33. Total Count: 966
C: 876, 91%
T: 90, 9%
TP53 c.646G>A
p.V216M

34. Data
Integration
Targets and
Biomarkers
Discovery
Pathway
Analysis
Mathematical
Modeling
Cancer
Prognosis -
Patient
Management
Clinical Data
Bioinformatics
Bioinformatics in cancer research

35. CanVaS
Cancer Variation reSource
• The first database of Greek cancer patient
genetic variation.
• Data from DNA sequencing of >5000 patients
analyzed at Molecular Diagnostics Laboratory.
 Sanger Sequencing
 TruSight cancer panel
 Panel testing in collaboration
with other Organizations

36. CanVaS
Cancer Variation reSource
• Variants in 100 known cancer susceptibility
genes.
• >3000 variants in coding regions.
• >3800 variants in non-coding regions.
Will provide:
 MAF for the Greek population.
 Possible genotype-phenotype association.
 Impact on clinical significance.
 Links to other public databases.
 Data available for download.

37. Cooperations - Funding
ΗeLLENIC COOPERATIVE ONCOLOGY GROUP
Panhellenic Association of Women with Breast Cancer
“Alma Zois”
θΑΛΗΣ – Molecular Subtyping of Triple Negative Breast
Cancer
AΡΙΣΤΕΙΑ Extreme Phenotypes in BReast-Ovarian CAncer:
Whole exome analysis in very early onset (17-
35yrs) cases
COOPERATION II
"Detection of New Breast and ovarian Cancer predisposing
Alleles using whole genome next generation
sequencing". Duration 2012-2015. Principal
Investigator: I. Ragoussis. (Total budget:
800.000, MDL: 300.000)
Participation in international consortia
Consortium of Investigators of Modifiers of BRCA1 and
BRCA2 (CIMBA). Coordinated by Dr Georgia Chenevix-
Trench, Queensland Institute of Medical Research,
Brisbane, Australia.
Breast Cancer Association Consortium. Coordinated by
Prof. Doug Easton, Cambridge University, UK.
Evidence-based Network for the Interpretation of
Germline Mutant Alleles – ENIGMA. Coordinated by
Prof. A. Monteiro, Univ. S. Florida, Tampa, USA.
http://enigmaconsortium.org/
Ovarian Cancer Association Consortium – OCAC.
Coordinated by Prof. Paul Pharoah, Cambridge
University
Collaborative Oncological Gene-environment Study – COGS.
Coordinated by Prof. Per Hall, Karolinska
Institute, Sweden http://cogseu.org
Collaborative genomics for human health and cooperation
in the Mediterranean region. Coordinated by Prof.
Mary-Claire King, University of Washington,
Seattle
Molecular Diagnostics
Laboratory
D. Yannoukakos
I. Konstantopoulou
F. Fostira
P. Apostolou
I. Konstanta
A. Vagena
M. Papamentzelopoulou
A. Delimitsou
D. Kalfakakou
I. Koudouna
E. Giannakopoulou