NGS 101 Panel Design and Quality Adam Hauge Development Manager University of Minnesota Genomics Center
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
Overview Panel Design Quality at the Bench Future Considerations
Overview Panel Design Quality at the Bench Future Considerations
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
Introduction Documentation Training Cost Assay 2 Assay 1 Assay 3 Single Assay
Panel Design Process Pediatrics Neurology Hematology 130 Disease Conditions 600 Genes 10,000 Exons Single Capture
Panel Design Process 2X bait coverage Single bait coverage
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 15,408,360 9525 (94.6 %) 308 9755 (96.9 %) 392 9929 (98.6 %) 475 2 19,332,263 9517 (94.5 %) 317 9748 (96.8 %) 391 9926 (98.6 %) 473 3 14,946,525 9413 (93.5 %) 287 9705 (96.4 %) 371 9909 (98.4 %) 462 4 20,659,002 9664 (96.0 %) 352 9836 (97.7 %) 430 9953 (98.9 %) 489 5 17,512,596 9620 (95.6 %) 344 9815 (97.5 %) 413 9953 (98.9 %) 487 6 18,917,409 9606 (95.4 %) 351 9802 (97.4 %) 411 9940 (98.7 %) 487 7 24,029,918 9788 (97.2 %) 408 9906 (98.4 %) 461 9975 (99.1 %) 499 8 18,982,380 9653 (95.9 %) 355 9824 (97.6 %) 425 9952 (98.9 %) 490 9 15,194,449 9498 (94.3 %) 310 9745 (96.8 %) 379 9929 (98.6 %) 474 10 18,160,028 9632 (95.7 %) 349 9800 (97.3 %) 410 9946 (98.8 %) 484 11 18,176,710 9636 (95.7 %) 350 9801 (97.4 %) 409 9959 (98.9 %) 489 12 17,800,123 9630 (95.7 %) 349 9820 (97.5 %) 420 9946 (98.8 %) 479 30X Coverage Courtesy Geteria Onsongo
Panel Design Process 17 kb 575X Average 265X Minimum 170 bp 18X Average 7X Minimum Courtesy Geteria Onsongo
Panel Design Process Silverstein Rule Bower Rule
Panel Design Process Highest Coverage Lower Coverage Lower Coverage Highest Coverage Bower Rule Courtesy Geteria Onsongo
Panel Design Process Highest Coverage Lower Coverage Lower Coverage Highest Coverage Silverstein Rule Courtesy Geteria Onsongo
Panel Design Process No Effect Silverstein Rule Courtesy Geteria Onsongo
Percent of Exons Panel Design Process 100.00% 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% 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Sample Courtesy Geteria Onsongo
Panel Design Process Think about Design What are your metrics? Run a Pilot! Revise and Improve
Overview Panel Design Quality at the Bench Future Considerations
Overview Panel Design Quality at the Bench Future Considerations
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
Workflow Overview Variant Calling Report Generation Fairview MDL UMGC Sequencing Data Delivery Quality Control Sequence Quality
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
Quality at the Bench Sample Handling Sample Barcode Sample A 835823885 Sample B 292058338 Sample C 242948290
Quality at the Bench Sample Handling Pre-Capture Library Capture Plate 1 2 3 4 1 2 3 4 A A B C D Hyb B C D E E F F G G H H Process Design
High-Throughput Processing Quality at the Bench
Quality at the Bench High-Throughput Processing Process Design
Quality at the Bench Hybridization and Capture Off-target On-target Assess Labware Avoid Evaporation! "#$%&'() *' +, --%". / 0#'(1! "#$ %&' ()*+, -. / 0001220343 5*6+7(%)+ %&' ()*+ 2. -4 0001202. 13 86*77 %&' ()*+ 2. -4/ 0001220343 9%: ; %&' ()*+ 2. -4/ 00010<0/0.! "#$ +%' (6=&)>=()*+?-/ 000120, <2, 5*6+7(%)+ +%' (6=&)>=()*+?-1 000120, /10 Process Design
Quality at the Bench Normalization and Loading Lanes Sample Pool 1, 2 1 3, 4 2 5, 6 3 7, 8 4 1 2 3 4 5 6 7 8 Process Design
Quality at the Bench and Beyond Laboratory Information Systems Process Design
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
Quality at the Bench Agilent Bioanalyzer Adapter Genomic Insert Adapter
Percent of Bases Quality at the Bench Agilent Bioanalyzer % of exon bases with 30X coverage 100 80 60 40 20 Sample 1 Sample 2 0 150 300 400 Insert Size (bp) Adapter Genomic Insert Adapter Quality Control
Fold Enrichment Quality at the Bench qpcr Capture Efficiency 40.00 Post-Capture Amplified 40 30 20 10 Pre-Capture On-Target #1 On-Target #2 On-Target #3 Off-Target #1 Off-Target #2 30.00 20.00 10.00 0.00 1 3 5 7 9 11 13 15 17 19 21 23 1000000 On-Target #1 On-Target #2 On-Target #3 Off-Target #1 0 Off-Target #3 100000 10000 40 30 20 10 0 Post-Capture On-Target #1 On-Target #2 On-Target #3 Off-Target #1 Off-Target #2 Off-Target #3 1000 100 10 1 Sample Quality Control
Pass-Filter Reads Quality Score Quality at the Bench and Beyond Sequence Output Quality Cutoff 40 35 30 25 20 15 10 5 0 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
Quality at the Bench and Beyond Blinded Proficiency Samples Sample A- 1 1call 27 calls Sample A- 2 Quality Control
Percent of Exons Percent of Exons Quality at the Bench and Beyond Quality Control Process Design 100.00% 98.00% 96.00% 94.00% 92.00% 90.00% 100.00% 98.00% 96.00% 94.00% 92.00% 90.00% Exons at 100% Coverage (10, 20, 30X) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Sample Exons at 100% Coverage (30X) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Sample
Overview Panel Design Quality at the Bench Future Considerations
Overview Panel Design Quality at the Bench Future Considerations
Future Design Considerations Metabolism Nephrology Cardiology Pulmonary >1,600 Genes Single Capture?
Future Design Considerations Exome Capture? Assay B Assay A Assay C Cost? Logistics? Single Assay Performance Functionality Turnaround
Turnaround time (weeks) Future Design Considerations 18 Trend for turnaround time in first year 16 14 12 10 8 6 4 2 0 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
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 1-4 1-3 2-4 2-4 Total Turnaround (weeks)
Future Design Considerations Capture Efficiency Speed Complexity Cost
Cost Speed Length Capacity Future Design Considerations HiSeq 2000 HiSeq 2500 MiSeq
Future Design Considerations How can you improve? What are your options? How should you sequence?
Final Thoughts SureSelect is a Great Tool Invest in Design and Run Pilots Don t Underestimate Quality Control
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
Thank You! Questions? Adam Hauge haug0296@umn.edu