NGS 101 Panel Design and Quality. Adam Hauge Development Manager University of Minnesota Genomics Center
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1 NGS 101 Panel Design and Quality Adam Hauge Development Manager University of Minnesota Genomics Center
2 Disclaimer SureSelect products are research use only SureSelect has not been validated by Agilent Technologies for clinical use All claims and uses presented today have been evaluated independently of Agilent Technologies
3 Overview Panel Design Quality at the Bench Future Considerations
4 Overview Panel Design Quality at the Bench Future Considerations
5 Introduction University of Minnesota Genomics Center Expression Analysis Genotyping Sequencing Minnesota Supercomputing Institute Computing Resources University Outreach Fairview Molecular Diagnostic Laboratory Clinical Diagnostics Disease Monitoring
6 Introduction Documentation Training Cost Assay 2 Assay 1 Assay 3 Single Assay
7 Panel Design Process Pediatrics Neurology Hematology 130 Disease Conditions 600 Genes 10,000 Exons Single Capture
8 Panel Design Process 2X bait coverage Single bait coverage
9 Panel Design Process % on Target Reads on Target Total Reads sample # unique paired reads 100 % coverage at 30X 100 % coverage at 20X 100 % coverage at 10X exons genes exons genes exons genes 1 15,408, (94.6 %) (96.9 %) (98.6 %) ,332, (94.5 %) (96.8 %) (98.6 %) ,946, (93.5 %) (96.4 %) (98.4 %) ,659, (96.0 %) (97.7 %) (98.9 %) ,512, (95.6 %) (97.5 %) (98.9 %) ,917, (95.4 %) (97.4 %) (98.7 %) ,029, (97.2 %) (98.4 %) (99.1 %) ,982, (95.9 %) (97.6 %) (98.9 %) ,194, (94.3 %) (96.8 %) (98.6 %) ,160, (95.7 %) (97.3 %) (98.8 %) ,176, (95.7 %) (97.4 %) (98.9 %) ,800, (95.7 %) (97.5 %) (98.8 %) X Coverage Courtesy Geteria Onsongo
10 Panel Design Process 17 kb 575X Average 265X Minimum 170 bp 18X Average 7X Minimum Courtesy Geteria Onsongo
11 Panel Design Process Silverstein Rule Bower Rule
12 Panel Design Process Highest Coverage Lower Coverage Lower Coverage Highest Coverage Bower Rule Courtesy Geteria Onsongo
13 Panel Design Process Highest Coverage Lower Coverage Lower Coverage Highest Coverage Silverstein Rule Courtesy Geteria Onsongo
14 Panel Design Process No Effect Silverstein Rule Courtesy Geteria Onsongo
15 Percent of Exons Panel Design Process % Exons at 100% Coverage 99.00% 98.00% 97.00% 96.00% 95.00% 94.00% 93.00% 10X 20X 30X 92.00% 91.00% 90.00% Sample Courtesy Geteria Onsongo
16 Panel Design Process Think about Design What are your metrics? Run a Pilot! Revise and Improve
17 Overview Panel Design Quality at the Bench Future Considerations
18 Overview Panel Design Quality at the Bench Future Considerations
19 Workflow Overview Sample Collection and Extraction Fairview MDL UMGC Sample Receipt Shearing Pre-capture Library Creation Hybridization and Capture Post-Capture Amplification Normalization and Pooling Quality Control PicoGreen PicoGreen Bioanalyzer PicoGreen Bioanalyzer QPCR
20 Workflow Overview Variant Calling Report Generation Fairview MDL UMGC Sequencing Data Delivery Quality Control Sequence Quality
21 SureSelect Target Enrichment Shear DNA Add Adapters Sequence Library Solution-based hybridization of RNA baits to target DNA within the genome Isolate and enrich target regions of interest
22 Quality at the Bench Sample Handling Sample Barcode Sample A Sample B Sample C
23 Quality at the Bench Sample Handling Pre-Capture Library Capture Plate A A B C D Hyb B C D E E F F G G H H Process Design
24 High-Throughput Processing Quality at the Bench
25 Quality at the Bench High-Throughput Processing Process Design
26 Quality at the Bench Hybridization and Capture Off-target On-target Assess Labware Avoid Evaporation! "#$%&'() *' +, --%". / 0#'(1! "#$ %&' ()*+, -. / *6+7(%)+ %&' ()* *77 %&' ()* / %: ; %&' ()* / 00010<0/0.! "#$ +%' (6=&)>=()*+?-/ , <2, 5*6+7(%)+ +%' (6=&)>=()*+? , /10 Process Design
27 Quality at the Bench Normalization and Loading Lanes Sample Pool 1, 2 1 3, 4 2 5, 6 3 7, Process Design
28 Quality at the Bench and Beyond Laboratory Information Systems Process Design
29 Coefficient of Variation Quality at the Bench Quantitation 12.00% Sample Balance in Sequencing 10.00% 8.00% 6.00% 4.00% 2.00% 0.00% Sequence Run Normalized Sample Input Quality Control
30 Quality at the Bench Agilent Bioanalyzer Adapter Genomic Insert Adapter
31 Percent of Bases Quality at the Bench Agilent Bioanalyzer % of exon bases with 30X coverage Sample 1 Sample Insert Size (bp) Adapter Genomic Insert Adapter Quality Control
32 Fold Enrichment Quality at the Bench qpcr Capture Efficiency Post-Capture Amplified Pre-Capture On-Target #1 On-Target #2 On-Target #3 Off-Target #1 Off-Target # On-Target #1 On-Target #2 On-Target #3 Off-Target #1 0 Off-Target # Post-Capture On-Target #1 On-Target #2 On-Target #3 Off-Target #1 Off-Target #2 Off-Target # Sample Quality Control
33 Pass-Filter Reads Quality Score Quality at the Bench and Beyond Sequence Output Quality Cutoff R1 R2 R1 R2 R1 R2 R1 R2 R1 R2 R1 R2 L1 L2 L3 L4 L5 L6 Lane/Read 80,000,000 70,000,000 60,000,000 50,000,000 40,000,000 30,000,000 20,000,000 10,000,000 0 Sample Quality Control
34 Quality at the Bench and Beyond Blinded Proficiency Samples Sample A- 1 1call 27 calls Sample A- 2 Quality Control
35 Percent of Exons Percent of Exons Quality at the Bench and Beyond Quality Control Process Design % 98.00% 96.00% 94.00% 92.00% 90.00% % 98.00% 96.00% 94.00% 92.00% 90.00% Exons at 100% Coverage (10, 20, 30X) Sample Exons at 100% Coverage (30X) Sample
36 Overview Panel Design Quality at the Bench Future Considerations
37 Overview Panel Design Quality at the Bench Future Considerations
38 Future Design Considerations Metabolism Nephrology Cardiology Pulmonary >1,600 Genes Single Capture?
39 Future Design Considerations Exome Capture? Assay B Assay A Assay C Cost? Logistics? Single Assay Performance Functionality Turnaround
40 Turnaround time (weeks) Future Design Considerations 18 Trend for turnaround time in first year Sep 2012 Oct 2012 Nov 2012 Dec 2012 Jan 2013 Feb 2013 Mar 2013 Apr 2013 May 2013 June 2013 Turnaround time (weeks) Operational Efficiency Courtesy Matt Bower
41 Future Design Considerations Workflow Overview Workflow Overview Physician Test Order Physician Interpretation of Report and Decision Sample Collection and Extraction Variant Calling Report Generation Fairview MDL UMGC Fairview MDL UMGC Sample Receipt Shearing Pre-capture Library Creation Hybridization and Capture Post-Capture Amplification Normalization and Pooling Sequencing Data Delivery Quality Control PicoGreen PicoGreen Bioanalyzer PicoGreen Bioanalyzer QPCR Quality Control Sequence Quality Total Turnaround (weeks)
42 Future Design Considerations Capture Efficiency Speed Complexity Cost
43 Cost Speed Length Capacity Future Design Considerations HiSeq 2000 HiSeq 2500 MiSeq
44 Future Design Considerations How can you improve? What are your options? How should you sequence?
45 Final Thoughts SureSelect is a Great Tool Invest in Design and Run Pilots Don t Underestimate Quality Control
46 Acknowledgements University of Minnesota Genomics Center Kenneth Beckman Archana Deshpande Aaron Becker Karina Sartorio Adam Hauge Minnesota Supercomputing Institute Kevin Silverstein Getiria Onsongo Jesse Erdman Fairview Molecular Diagnostic Laboratory Bharat Thyagarajan Matt Bower Matt Schomaker Teresa Kemmer Sophia Yohe
47 Thank You! Questions? Adam Hauge
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