NGS 101 Panel Design and Quality - Agilent · NGS 101 Panel Design and Quality . SureSelect...

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

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


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


Courtesy Geteria Onsongo


575X Average

265X Minimum

18X Average

7X Minimum

17 kb

170 bp


Silverstein Rule Bower Rule


Highest Coverage Lower Coverage

Bower Rule

Lower Coverage Highest Coverage



Silverstein Rule

Highest Coverage Lower Coverage

Lower Coverage Highest Coverage




Silverstein Rule

No Effect



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


Think about

Design

What are your metrics?

Run a Pilot!

Revise and

Improve




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



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


High-Throughput

Processing


High-Throughput

Processing

Process Design


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


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


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


Genomic Insert Adapter Adapter

Agilent Bioanalyzer


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


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


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


Sample A-

1

Sample A-

2 27 calls 1call

Blinded Proficiency

Samples

Quality Control


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)



Panel Design

Overview

>1,600 Genes

Single Capture?

Metabolism

Nephrology

Pulmonary

Cardiology

Future Design Considerations


Single Assay

Assay C

Assay B

Assay A

Performance Turnaround Functionality

Exome Capture?

Cost?

Logistics?


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


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)


Capture

Efficiency Speed Cost Complexity


HiSeq 2000 MiSeq HiSeq 2500

Cost

Speed

Length

Capacity

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

[email protected]

NGS 101 Panel Design and Quality - Agilent · NGS 101 Panel Design and Quality . SureSelect...

Documents

Transcript of NGS 101 Panel Design and Quality - Agilent · NGS 101 Panel Design and Quality . SureSelect...