FONS Nové sekvenační technologie vklinickédiagnostice?

Transcription

1 FONS 2010 Nové sekvenační technologie vklinickédiagnostice?

2 Sekvenování amplikonů Sequence capture Celogenomové sekvenování

3 FONS 2010 Sekvenování amplikonů

4 Amplicon sequencing - amplicon sequencing enables the identification and quantification of known and novel sequence variations within complex mixtures of PCR products - reads are compared to a user defined reference sequence to identify and quantify known variants and facilitate the discovery of novel variants. - detection of rare sequence variants in a complex sample (e.g. cancer studies) - population studies to find rare alleles - investigation of diversity in environmental samples - investigation of diversity in clinical research studies

5 Comparison Sanger and NGS Sequenced Based Typing Resolution of Ambiguity T T C T Sanger Sequencing T C T T Population sequencing = mixed reads sensitivity ~ 15% T T C T T T NGS Sequencing T T T T T C Clonal sequencing = 1 DNA = 1 read sensitivity < 1%

6 HIV resistance testing 3 mutations are frequently observed in multidrug resistant HIV viruses: M46I/L, V82A/F/S/T, and L90M - The combination confers resistance to all protease inhibitors currently in use If all three were found on a single major species before treatment, protease inhibitors would have minimal effect However, if they were on distinct species, they could be suppressed by different inhibitors

7 HIV resistance testing

8 Effective Detection of Low Frequency Drug Resistance Mutations in HIV at a 1% level Detection of low frequency mutations conferring resistance to HIV drugs Mutations -Type -Frequency C984A 2.7% D177E T990G 5.8% no Δ C993A 2.3% no Δ C993T 2.2% no Δ A995G 1.0% Y181C T996C 0.9% no Δ C1002T 0.5% no Δ T1012C 0.4% no Δ D177E: common polymorphism (20% of clade B viruses) Reference Sequence

9 HLA Region (Human Leukocyte Antigen) (Chromosome 6p) DP DM TAP DQ DR TNF B C A 150 kb 50 kb 200 kb 50 kb 850 kb 250 kb 100 kb 1270 kb B1 A1 B A 1 2 B1 A1 B1 B3 A1 B4 B A B Green = serotypes class II loci class I loci The HLA region is the most polymorphic region of the human genome 4,478 alleles at 12 loci as of May 2010

10 Molecular Targets of HLA Typing SJ Lee, et al. Blood 2007

11 HLA Disease Associations (selected examples) Autoimmunity / Inflammation IDDM (type 1 diabetes) MS IBD (UC and CD) RA, juvenile RA Pemphigus Vulgaris MG Ankylosing Spondylitis Psoriasis Celiac Disease Unknown Narcolepsy Cancer Cervical Carcinoma NP Carcinoma HD Infectious Disease Malaria Tuberculosis Leprosy HCV HIV Drug Allergic Hypersensitivity Abacavir carbamizine

12 HLA sequencing CLASS I class II class I exons 2, 3 and 4 from HLA-A exons 2, 3 and 4 from HLA-B exons 2, 3 and 4 from HLA-C blue exons 8 primer pairs & red exons 6 primer pairs MIDs MIDs CLASS II Medium High exon 2 from DPB1 Resolution Resolution exon 2 from DQA1 exons 2 and 3 from DQB1 exon 2 from DRB1

13 Conexio ATF Genotype of HLA-B

14 Amplicon Sequencing Major research applications will include the discovery and verification of causative mutations for mono or multi genetic disorders somatic mutations in cancer Technology of choice to substitute Sanger Sequencing in Clinical Research Predisposition testing like syndroms (blindness, deafness), breast cancer (BRCA etc) Patient stratification: EGFR, KRAS, BRAF, PIK3CA, ERBB2, NRAS, HRAS, PTEN, KIT, PDGFRA Technology of choice for Diagnostic Sequencing HIV, HLA etc

15 FONS 2010 Sequence capture

16 SEQUENCE CAPTURE

17 Key Driver is Complexity Reduction Sequencing Sample Preparation Sequencing Sample Preparation? Capillary Sequencing PCR Next-Gen Sequencing PCR Complexity reduction by enrichment of smaller genomic portion will exploit the full potential of next-generation sequencing instruments

18 NimbleGen Sequence Capture Exon1 Exon2 Exon3 Exon4 Exon5 Exon6 Fragment & Add Linkers Genomic DNA hybridize Wash Background DNA Probes Target DNA Elute Amplification Target DNA Array NGS Sequencing

19 Agilent Sequence Capture

20 Sequence Capture Targeting the 11p12 Diabetes Locus NimbleGen Sequence Capture array: 385,000 probes targeted ~3 Mb 72% of the region covered by probes Repetitive regions

21 Benefit of Sequence Capture Time Saved (vs. LR-PCR) Cost Saved (vs. parallel PCR) Long Range PCR $30,000 $25,000 PCR cost / sample (10 sample project) PCR cost / sample (100 sample project) Work Days Needed Sequence Capture Cost / Sample $20,000 $15,000 $10,000 Sequence capture cost / sample 100 $5, Size of Region (Kb) $ # of Exons SeqCap offers customer with significant savings in time and cost.

22

23

24

25 FONS 2010 Celogenomové sekvenování

26 Proc. Natl. Acad. Sci. U. S. A. 105,

27 Proc. Natl. Acad. Sci. U. S. A. 105, The z-score of a potentially aneuploid chromosome is expected to be higher for pregnancies with an aneuploid fetus (cases E H shown in green) than for those with a euploid fetus (cases A D shown in blue).

28 Stephen R Quake Stephen Quake, D.Phil. Professor of Bioengineering Co-Chair, Department of Bioengineering Investigator, Howard Hughes Medical Institute D. Phil., Physics, University of Oxford, MS, Mathematics, Stanford University, BS, Mathematics, Stanford University, 1991

29 Clinical assessment incorporating a personal genome Lancet 2010; vol. 375:

30 Clinical characteristics of the patient

31 Patient pedigree

32 Approach to variants

33 Clinical risk incorporating genetic-risk estimates for major diseases Pre-test probabilities or disease prevalence (in white men in the patient s age range) - backs of the arrowheads show pre-test probabilities -arrows point in the direction of change in probability - blue lines show lowered posttest probabilities - red lines increased post-test probabilities n=number of independent single nucleotide polymorphisms used in calculation of post-test probability for that disorder

34 Contribution of individual alleles to overall risk of prostate cancer

35 Contribution of individual alleles to overall risk of Alzheimer s disease

36 Findings & Interpretation analysis of 2 6 million single nucleotide polymorphisms and 752 copy number variations increased genetic risk for myocardial infarction, type 2 diabetes, and some cancers we discovered rare variants in three genes that are clinically associated with sudden cardiac death TMEM43, DSP, and MYBPC3 a variant in LPA was consistent with a family history of coronary artery disease the patient had a heterozygous null mutation in CYP2C19 suggesting probable clopidogrel resistance, several variants associated with a positive response to lipid-lowering therapy, and variants in CYP4F2 and VKORC1 that suggest he might have a low initial dosing requirement for warfarin many variants of uncertain importance were reported Although challenges remain, our results suggest that whole-genome sequencing can yield useful and clinically relevant information for individual patients

37 Lancet 2010; vol. 376: 869

38 NATURE, 2010, vol. 464:

39 Lancet 2010, vol. 375:

40

41

42

43 FONS 2010 Děkuji za pozornost. Dotazy?