Utilization of the MiSeq in a clinical lab. Tony Krentz, PhD PreventionGenetics

Transcription

1 Utilization of the MiSeq in a clinical lab Tony Krentz, PhD PreventionGenetics

2 PreventionGenetics Founded in 2004 in Marshfield, Wisconsin by James Weber ~90 employees Largest test menu in US Vision: Disease Prevention through Genetic Testing

3 PreventionGenetics Marshfield

4 Our Services DNA Banking DNA Banking is our only direct-to-consumer service Clinical DNA Testing Will only take test orders from physicians, genetic counselors, and other healthcare professionals

5 DNA Banking Schedule a blood draw with your physician or lab One time fee of $98 Guarantee to store DNA for at least 20 years

6 Our Services DNA Banking DNA Banking is our only direct-to-consumer service Clinical DNA Testing Will only take test orders from physicians, genetic counselors, and other healthcare professionals

7 Clinical DNA Testing Analytical Platforms Sanger dideoxy sequencing Gene-centric acgh for CNV Next-Gen Sequencing for disease specific panels

8 Next-Gen Sequencing Focus on disease-specific panels Utilize MiSeq sequencer 2x150bp reads

9 Benefits of MiSeq Sequencer 1. Extremely easy to use 2. Fast TAT 2x150bp takes ~26hrs 3. Ability to multiplex 4. Reagent costs per run ~$ Relatively affordable compared to other sequencers on the market

10 Our Next-Gen Strategy 1. Use biotinylated oligos to capture coding exons plus 20bp flanking intronic sequence and any documented-causative variants located in introns, regulatory regions, etc 2. Backfill any dropout or low coverage regions with Sanger 3. Confirm any pathogenic, VUS with Sanger 4. Use STRP test for any mononucleotide repeats >8

11 Next-Gen Library Prep Validation Process 1. Ran 2 HapMap samples and compared results to Sanger sequencing results produced in-house for ~100 genes 2. Ran 2 HapMap samples and compared results to external sources 3. Step 1 repeated by 3 different technicians on different days 4. Ran >50 past patients and compared our NGS results to Sanger results 5. Step 1 is repeated at least every 6 months for internal PT and anytime a step is modified in the workflow

12 Next-Gen Panels Bardet-Biedl Syndrome Catecholaminergic Polymorphic Ventricular Tachycardia Chromosomal Instability Syndromes Congenital Muscular Dystrophy Dystroglycan-related Congenital Muscular Dystrophy Fanconi Anemia Hereditary Breast and Ovarian Cancer Heterotaxy/Situs Inversus And Kartagener's Syndrome Hypertrophic Cardiomyopathy Immotile Cilia Syndrome/Primary Ciliary Dyskinesia (PCD) Joubert and Meckel-Gruber Syndromes Marfan Syndrome and Related Aortopathies Multiple Epiphyseal Dysplasia Nephronophthisis and Senior-Loken Syndrome Noonan Spectrum Disorders/Rasopathies Pan-Ciliopathy Stickler Syndrome

13 Next-Gen Panels Bardet-Biedl Syndrome Catecholaminergic Polymorphic Ventricular Tachycardia Chromosomal Instability Syndromes Congenital Muscular Dystrophy Dystroglycan-related Congenital Muscular Dystrophy Fanconi Anemia Hereditary Breast and Ovarian Cancer Heterotaxy/Situs Inversus And Kartagener's Syndrome Hypertrophic Cardiomyopathy Immotile Cilia Syndrome/Primary Ciliary Dyskinesia (PCD) Joubert and Meckel-Gruber Syndromes Marfan Syndrome and Related Aortopathies Multiple Epiphyseal Dysplasia Nephronophthisis and Senior-Loken Syndrome Noonan Spectrum Disorders/Rasopathies Pan-Ciliopathy Stickler Syndrome

14 Most Cells Have Cilia

15 Organ Systems Affected ALMS, Alström syndrome; BBS, Bardet-Biedl syndrome; CORS, cerebello-oculo-renal syndrome; EVC, Ellis-van Creveld syndrome; JATD, Jeune asphyxiating thoracic dystrophy; JBTS, Joubert syndrome; LCA, Leber congenital amaurosis; MKS, Meckel syndrome; NPHP, nephronophthisis; OFD1, oral-facialdigital syndrome type 1; PCD, primary ciliary dyskinesia; PKD, polycystic kidney disease Lee and Gleeson Genome Medicine :59

16 Ciliopathies Bardet-Biedl Syndrome (17 genes) Joubert and Meckel-Gruber Syndromes (25 genes) Nephronophthisis and Senior-Loken Syndrome (14 genes) Immotile Cilia Syndrome/Primary Ciliary Dyskinesia (PCD) (17 genes) Heterotaxy/Situs Inversus And Kartagener's Syndrome (19 genes) Pan-Ciliopathy

17 Ciliopathy Capture Strategy Nephronophthisis and Senior-Loken syndrome DNAH11 DNAH5 DNAI1 DNAI2 HEATR2 RSPH9 DNAL1 CCDC103 CCDC39 RSPH4A DNAAF1 DNAAF2 CCDC40 DNAAF3 LRRC6 RPGR INVS CCDC114 ANKS6 OFD1 ARL13B NPHP3 AHI1 TMEM138 GLIS2 TCTN2 INPP5E NPHP4 NPHP1 TMEM231 TCTN1 IQCB1 ZNF423 TCTN3 CEP164 CEP41 TMEM237 RPGRIP1L TMEM216 NEK8 CC2D2A B9D1 CEP290 C5ORF42 B9D2 TTC21B TTC21B SDCCAG8 MKS1 TMEM67 WDPCP KIF7 BBS1 ARL6 BBS4 BBS2 BBS9 BBS7 BBS10 BBS5 MKKS BBS12 TRIM32 CDCC28B BBS8 Primary Ciliary Dyskinesia Meckel-Gruber and Joubert syndrome Bardet-Biedl syndrome

18 Bardet-Biedl Syndrome Genetically heterogeneous; Autosomal recessive Primary features of obesity, polydactyly, pigmentary retinopathy, hypogonadism, renal anomalies and intellectual disabilities Secondary features include diabetes, hypertension and congenital heart defects Forsythe and Beales (2013) EJHG

19 Forsythe and Beales (2013) EJHG Function of BBS proteins

20 Clinical Sensitivity of BBS NGS TRIM32 TTC21B UNKNOWN BBS1 BBS5 WDPCP ARL6 TTC8 SDCCAG8 CEP290 BBS7 BBS4 MKS1 BBS10 BBS12 MKKS BBS9 BBS2

21 Bardet-Biedl Coverage % 99.50% % of bases above 99.00% >20x >50x >100x 98.50% 98.00% Sample #

22 Bardet-Biedl Case BBS5: c.143-1g>c BBS5: c.1004delg (p.gly335aspfs*5) BBS5: c.143-1g>c and c.1004delg (p.gly335aspfs*5) 14 yo polydactyly of hands, obesity, DD 10yo 6mo CHD, polydactyly of hands and feet

23 Clinical Sensitivity of BBS NGS TRIM32 WDPCP SDCCAG8 TTC21B BBS5 ARL6 TTC8 CEP290 BBS7 BBS4 UNKNOWN MKS1 BBS12 BBS1 BBS10 MKKS BBS9 BBS2

24 Pan-Ciliopathy Case Proband Baby Girl born with Nephronophthisis and Situs Inversus--died shortly after birth INVS/NPHP2 negative Nephronophthisis and Senior-Loken syndrome Bardet-Biedl syndrome NPHP4 CEP164 NEK8 NPHP3 DNAH11 DNAI2 HEATR2 DNAH5 DNAI1 RSPH9 DNAL1 CCDC103 CCDC39 RSPH4A DNAAF1 DNAAF2 CCDC40 DNAAF3 LRRC6 RPGR GLIS2 IQCB1 CCDC114 OFD1 ANKS6 ARL13B AHI1 TMEM138 TCTN2 INPP5E NPHP1 TMEM231 TCTN1 ZNF423 TCTN3 CEP41 TMEM237 RPGRIP1L TMEM216 CC2D2A B9D1 INVS CEP290 C5ORF42 B9D2 TTC21B TTC21B MKS1 SDCCAG8 TMEM67 WDPCP KIF7 BBS1 ARL6 BBS4 BBS2 BBS9 BBS7 BBS10 BBS5 MKKS BBS12 TRIM32 CDCC28B BBS8 Primary Ciliary Dyskinesia Meckel-Gruber and Joubert syndrome

25 Pan-Ciliopathy Case Proband Baby Girl born with Nephronophthisis and Situs Inversus--died shortly after birth INVS/NPHP2 negative Sequenced 60+ genes with the Pan-Ciliopathy NGS panel BBS10: Ser303Argfs*3 TMEM67: Lys819Argfs*7 BBS10: Cys91Trp BBS10: Cys91Trp BBS10: Cys91Trp and Ser303Argfs*3 both doc.causative (Stoetzel et al. 2006) TMEM67: Lys819Argfs*7 -undocumented

26 Summary We are using MiSeq to sequence diseasespecific NGS panels Use a combination of Sanger sequencing and NGS to cover every base Providing a comprehensive, cost effective test for our clients

27 Questions and Discussion