Bioinformatics tools for NGS data analysis
- 1. Bioinformatics tools for NGS data analysis Kalfakakou Despoina Bioinformatician, PhD candidate Molecular Diagnostics Laboratory @ NCSR Demokritos
- 2. History of Sequencing 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. Apply DNA fragments to flow cell Sequencing procedure Cluster generation by solid phase PCR (Bridge Amplification) Sequencing by Synthesis
- 10. Step 1 Apply DNA fragments to flow cell Sequencing procedure
- 11. Step 2 Cluster generation by solid phase PCR (Bridge Amplification) Sequencing procedure
- 12. Step 3 Sequencing procedure Sequencing by Synthesis
- 14. NGS data analysis Overview Primary Analysis De-multiplexing Base Calling & Quality Control (Fastq File) Secondary Analysis Sequence Alignment aka Read Mapping (SAM/BAM File) Variant Calling (VCF File) Tertiary Analysis Variant Annotation Variant Filtering
- 15. NGS data analysis Overview Primary Analysis De-multiplexing
- 16. Conceptual overview of sample multiplexing Picture source: http://www.illumina.com/technology/next-generation-sequencing/multiplexing-sequencing-assay.html
- 17. NGS data analysis Overview Primary Analysis Base Calling & Quality Control (Fastq File)
- 18. Fastq Generation Sequence Identifier Sequence Quality Score per base See more at: https://en.wikipedia.org/wiki/FASTQ_format
- 19. Phred Score Q = -10 log10 Perr Q=0: Perr =1 Q=40: Perr =0.0001
- 22. NGS data analysis Overview Secondary Analysis Sequence Alignment aka Read Mapping (SAM/BAM File)
- 23. Short Sequence Aligners for NGS data DNA RNA Methylation miRNA Source: https://www.ebi.ac.u k/~nf/hts_mappers/
- 24. 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!
- 25. Map to Reference Genome
- 26. Map to Reference Genome Reference Genome
- 27. Map to Reference Genome Reads mapped to this area
- 28. Map to Reference Genome MiSeq Coverage: ~100-700 reads
- 29. NGS data analysis Overview Secondary Analysis Variant Calling (VCF File)
- 31. VCF File example
- 32. NGS data analysis Overview Tertiary Analysis Variant Annotation
- 33. 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 HapMap gnomAD 1000 Genome Pathogenicity Prediction PolyPhen MutationTaster SIFT AlignGVGD Predicted Genomic Features ESEFinder HSF MicroT Consortia ENIGMA BCAC OCAC COMPLEXO
- 34. NGS data analysis Overview Tertiary Analysis Variant Filtering
- 35. Variant Filtering Variant Quality: ▪ Coverage at position ▪ Allele Balance ▪ Base/Mapping qualities ▪ Passed filters ▪ Known artefacts Variant Prioritization: ▪ Minor Allele Frequency ▪ Functional Impact ▪ Mode of Inheritance ▪ Previous report of clinical significance ▪ Segregation in family
- 37. 7 incidents <40 years (5 incidents <35 years) BRCA1c.843_846delCTCA p.S282TfsX15
- 38. 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 Koulis Yannoukakos Reena Konstantopoulou Florentia Fostira Vivi Apostolou Andromachi Vagena Myrto Papamentzelopoulou Angeliki Delimitsou Despoina Kalfakakou Eugenia Giannakopoulou Vicky Dellatola
- 39. Total Count: 339 C: 178, 52% T: 161, 48% BRCA1 c.5497G>A p.V1833M
- 40. Total Count: 189 WT: 189 Del: 165 BRCA1c.843_846delCTCA p.S282TfsX15
- 41. CanVaS Cancer Variation reSource • The first database of Greek cancer patient genetic variation. • Data from DNA sequencing of >10000 patients analyzed at Molecular Diagnostics Laboratory. Sanger Sequencing TruSight cancer panel Panel testing in collaboration with other Organizations
- 42. 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.
Editor's Notes
- #7: Targeted sequencing: Reduces cost, DNA input, length of workflows Increases sequencing accuracy
- #8: Τα γονιδιάκά πάνελ στοχεύουν σε 5 έως περισσότερα από 100 γονίδια μεταλλάξεις στα οποία φέρουν πάνω από 2 φορές μεγαλύτερο διά βίου κίνδυνο ανάπτυξης καρκίνου. Η συχνότητα των μεταλλάξεων στον πληθυσμό είναι κάτω του μισό τοις εκατό. Επίσης στοχεύουν κάποιες περιοχές του γονιδιώματος,που έχουν ανιχνευθεί από Μελέτες συσχέτισης ολόκληρου του γονιδιώματος, μεταλλάξεις στις οποίες δεν ανεβάζουν πολύ τον κίνδυνο ανάπτυξης καρκίνου αλλά έχει παρατηρηθεί ότι λειτουργούν πολλαπλασιαστικά μαζί με τα μεταλλαγμένα γονίδια.
- #40: Conflicting interpretations MDL Classification: Probably pathogenic XX families found to carry this mutation
- #41: E10