ngs 101 panel design and quality - agilent · ngs 101 panel design and quality . sureselect...
TRANSCRIPT
Adam Hauge
Development Manager
University of Minnesota Genomics Center
NGS 101
Panel Design and Quality
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
Disclaimer
Quality at the Bench
Future Considerations
Panel Design
Overview
Quality at the Bench
Future Considerations
Panel Design
Overview
University of Minnesota
Genomics Center
• Expression Analysis
• Genotyping
• Sequencing
Fairview Molecular
Diagnostic Laboratory
• Clinical Diagnostics
• Disease Monitoring
Minnesota
Supercomputing Institute
• Computing Resources
• University Outreach
Introduction
Introduction
Single Assay
Assay 3
Assay 2
Assay 1
Documentation
Training
Cost
130 Disease Conditions
600 Genes 10,000 Exons
Single Capture
Pediatrics
Neurology
Hematology
Panel Design Process
Panel Design Process
2X bait coverage Single bait
coverage
sample# unique
paired reads
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
100 % coverage at 30X 100 % coverage at 20X 100 % coverage at 10X
% on Target
30X Coverage
Reads on Target
Total Reads
Panel Design Process
Courtesy Geteria Onsongo
Panel Design Process
575X Average
265X Minimum
18X Average
7X Minimum
17 kb
170 bp
Courtesy Geteria Onsongo
Silverstein Rule Bower Rule
Panel Design Process
Highest Coverage Lower Coverage
Bower Rule
Lower Coverage Highest Coverage
Panel Design Process
Courtesy Geteria Onsongo
Silverstein Rule
Highest Coverage Lower Coverage
Lower Coverage Highest Coverage
Panel Design Process
Courtesy Geteria Onsongo
Panel Design Process
Silverstein Rule
No Effect
Courtesy Geteria Onsongo
Panel Design Process
90.00%
91.00%
92.00%
93.00%
94.00%
95.00%
96.00%
97.00%
98.00%
99.00%
100.00%
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Perc
en
t o
f E
xo
ns
Sample
Exons at 100% Coverage
10X
20X
30X
Courtesy Geteria Onsongo
Think about
Design
What are your metrics?
Run a Pilot!
Revise and
Improve
Panel Design Process
Quality at the Bench
Future Considerations
Panel Design
Overview
Quality at the Bench
Future Considerations
Panel Design
Overview
Workflow Overview
Sample Receipt
Shearing
Pre-capture
Library Creation
Hybridization and
Capture
Post-Capture
Amplification
Normalization and
Pooling
Sample Collection
and Extraction
Fairview MDL
UMGC
Quality Control
PicoGreen
PicoGreen
Bioanalyzer
PicoGreen
Bioanalyzer
QPCR
Workflow Overview
Sequencing Data Delivery
Variant Calling Report Generation
Fairview MDL
UMGC
Quality Control
Sequence Quality
Solution-based hybridization of RNA baits to target DNA within the genome
Shear DNA
Isolate and enrich target regions of interest
Add Adapters Sequence Library
SureSelect Target Enrichment
Sample Barcode
Sample A 835823885
Sample B 292058338
Sample C 242948290
Sample Handling
Quality at the Bench
Quality at the Bench
1 2 3 4
A
B
C
D
E
F
G
H
1 2 3 4
A
B
C
D
E
F
G
H
Pre-Capture Library Capture Plate
Hyb
Sample Handling
Process Design
Quality at the Bench
High-Throughput
Processing
Quality at the Bench
High-Throughput
Processing
Process Design
Quality at the Bench
Hybridization and Capture
Process Design
Assess Labware
Avoid
Evaporation
! "#$%&' () *' +, --%" . / 0#' (1
! "#$ %&' ()*+ , -. / 0001220343
5*6+7(%)+ %&' ()*+ 2. -4 0001202. 1386*77 %&' ()*+ 2. -4/ 0001220343
9%: ; %&' ()*+ 2. -4/ 00010<0/0.! "#$ +%' (6=&)>=()*+ ?-/ 000120, <2,
5*6+7(%)+ +%' (6=&)>=()*+ ?-1 000120, /10
Off-target
On-target
Quality at the Bench
1 2 3 4 5 6 7 8
Lanes Sample
Pool
1, 2 1
3, 4 2
5, 6 3
7, 8 4
Normalization and Loading
Process Design
Quality at the Bench and Beyond
Laboratory Information
Systems
Process Design
Quality at the Bench
0.00%
2.00%
4.00%
6.00%
8.00%
10.00%
12.00%
Co
eff
icie
nt
of
Vari
ati
on
Sequence Run
Sample Balance in Sequencing Quantitation
Quality Control
Normalized Sample
Input
Quality at the Bench
Genomic Insert Adapter Adapter
Agilent Bioanalyzer
Quality at the Bench
0
20
40
60
80
100
150 300 400 P
erc
en
t o
f B
ases
Insert Size (bp)
% of exon bases with 30X coverage
Sample 1
Sample 2
Genomic Insert Adapter Adapter
Agilent Bioanalyzer
Quality Control
Quality at the Bench
0
10
20
30
40
Pre-Capture
On-Target #1
On-Target #2
On-Target #3
Off-Target #1
Off-Target #2
Off-Target #3
0
10
20
30
40
Post-Capture
On-Target #1
On-Target #2
On-Target #3
Off-Target #1
Off-Target #2
Off-Target #3
1
10
100
1000
10000
100000
1000000
Fo
ld E
nri
ch
men
t
Sample
0.00
10.00
20.00
30.00
40.00
1 3 5 7 9 11 13 15 17 19 21 23
Post-Capture Amplified
On-Target #1
On-Target #2
On-Target #3
Off-Target #1
qPCR Capture Efficiency
Quality Control
Quality at the Bench and Beyond
0
5
10
15
20
25
30
35
40
R1 R2 R1 R2 R1 R2 R1 R2 R1 R2 R1 R2
L1 L2 L3 L4 L5 L6
Qu
ality
Sco
re
Lane/Read
0
10,000,000
20,000,000
30,000,000
40,000,000
50,000,000
60,000,000
70,000,000
80,000,000
Pass-F
ilte
r R
ead
s
Sample
Quality Cutoff
Sequence Output
Quality Control
Quality at the Bench and Beyond
Sample A-
1
Sample A-
2 27 calls 1call
Blinded Proficiency
Samples
Quality Control
Quality at the Bench and Beyond
Process Design
Quality Control
90.00%
92.00%
94.00%
96.00%
98.00%
100.00%
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 P
erc
en
t o
f E
xo
ns
Sample
Exons at 100% Coverage (10, 20, 30X)
90.00%
92.00%
94.00%
96.00%
98.00%
100.00%
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Pe
rce
nt
of
Ex
on
s
Sample
Exons at 100% Coverage (30X)
Quality at the Bench
Future Considerations
Panel Design
Overview
Quality at the Bench
Future Considerations
Panel Design
Overview
>1,600 Genes
Single Capture?
Metabolism
Nephrology
Pulmonary
Cardiology
Future Design Considerations
Future Design Considerations
Single Assay
Assay C
Assay B
Assay A
Performance Turnaround Functionality
Exome Capture?
Cost?
Logistics?
Future Design Considerations
0
2
4
6
8
10
12
14
16
18
Sep 2012 Oct 2012 Nov 2012 Dec 2012 Jan 2013 Feb 2013 Mar 2013 Apr 2013 May 2013 June 2013
Tu
rna
rou
nd
tim
e (
we
ek
s)
Trend for turnaround time in first year
Turnaround time (weeks)
Operational Efficiency Courtesy Matt Bower
Future Design Considerations
Workflow Overview
Sample Receipt
Shearing
Pre-capture Library Creation
Hybridization and Capture
Post-Capture Amplification
Normalization and Pooling
Sample Collection and Extraction
Test Order
Physician
Fairview MDL
UMGC
Quality Control
PicoGreen
PicoGreen Bioanalyzer
PicoGreen Bioanalyzer
QPCR
Workflow Overview
Sequencing Data Delivery
Variant Calling Report Generation
Interpretation of Report and Decision
Physician
Fairview MDL
UMGC
Quality Control
Sequence Quality
1 - 4 1 - 3 2 - 4 2 - 4
Total Turnaround (weeks)
Future Design Considerations
Capture
Efficiency Speed Cost Complexity
Future Design Considerations
HiSeq 2000 MiSeq HiSeq 2500
Cost
Speed
Length
Capacity
How can you
improve?
What are your
options?
How should you sequence?
Future Design Considerations
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