Research Use Only

SureSelect:

Leading the Pack in

Target Enrichment

For Research Use Only. Not for use in diagnostic procedures

Maria Celeste Ramirez, PhD

Product Manager, SureSelect

Margherita Corioni, PhD R&D Scientist

SureSelect: The Game Changer

For Research Use Only. Not for use in diagnostic procedures

Hybridization

Baits:

- cRNA probes

- Long (120bp)

- Biotinylated

Capture

Wash / Elution / Amp

SureSelect Target Enrichment System Workflow

gDNA Library prep

Hyb set-up

For Research Use Only. Not for use in diagnostic procedures

• Catalog or custom libraries (SureDesign)

• Library QC (Bioanalyzer and Tapestation)

• Data analysis (GeneSpring NGS)

• Manual or automation (Bravo) protocols

• Exomes or custom (up to 24Mb) captures

• Standard (3ug) or Low-input (as low as 100ng)

• Post-capture or pre-capture pooling

• For sequencing on Illumina or Ion Proton systems

• Long 120-mer baits enables superior sensitivity for

SNP and InDel identification

• Preserves allelic balance

Key Benefits

For Research Use Only. Not for use in diagnostic procedures

• SureSelect Exome and custom target enrichment

kits have been used in > 350 publications on

Mendelian and complex disorders

PROVEN Technology

HIGH

Performance

FLEXIBLE Solution

COMPLETE Solution

SureSelectXT and SureSelectXT2

Low Input solutions

Low input SureSelect: Agenda

Low Input SureSelect

Changes to current protocol

Performance

Low input SureSelect: Agenda

Low Input SureSelect

Changes to current protocol

Performance

SureSelectXT and SureSelect XT2 NGS Target Enrichment Workflow

Standard protocols:

SureSelect XT 3ug input

SureSelect XT2 1ug input

Low input protocols:

SureSelect XT low input 3ug input

SureSelect XT2 low input 1ug input

SureSelectXT and SureSelectXT2 Library Prep workflow

For

Standard

or

Low input

The overall workflow for low input XT and XT2 Library Prep system

is the same as current Library Prep

DNA fragmentation

End-repair

dA-tailing

Adapter ligation

PCR

Library QC

~ 4 hours

workflow

Low input SureSelect: Agenda

Low Input SureSelect

Changes to current protocol

Performance

SureSelectXT Library Prep: workflow for 200ng input

AMPure purification 3ug input

No AMPure purification

Adaptor dilution

Shear

End-repair

dA-tailing

Ligation

PCR

200ng input

Shear

End-repair

dA-tailing

Ligation

PCR Amplify all adaptor-ligated library

SureSelectXT 200ng input: DNA shear

Shear

End-repair

dA-tailing

Ligation

PCR

Average DNA fragment

size 120-150bp

Dilute the

adaptor

before

ligation!!

Shear

End-repair

dA-tailing

Ligation

PCR

SureSelectXT 200ng input: adaptor ligation

Amplify all adaptor-ligated library

10 cycles of amplification

QC pre-capture:

Shear

End-repair

dA-tailing

Ligation

PCR

Average DNA fragment

size 225-275bp

SureSelectXT 200ng input: adaptor-ligated library PCR

SureSelectXT2 Library Prep: workflow for 100ng input

AMPure purification 1ug input

Inactivation of dA-tailing

Adaptor dilution

Amplify all adaptor-ligated

library

Shear

End-repair

dA-tailing

Ligation

PCR

100ng input

Shear

End-repair

dA-tailing

Ligation

PCR

Shear

End-repair

dA-tailing

Ligation

PCR

SureSelectXT2 100ng input: DNA shear

Average DNA fragment

size 150-200bp

Shear

End-repair

dA-tailing

Ligation

PCR

SureSelectXT2 100ng input: dA tailing

Dilute the

adaptor

before

ligation!!

Shear

End-repair

dA-tailing

Ligation

PCR

SureSelectXT2 100ng input: adaptor ligation

Amplify all adaptor-ligated library

8 cycles of amplification

QC pre-capture:

Shear

End-repair

dA-tailing

Ligation

PCR

Average DNA fragment

size 250-275bp

SureSelectXT2 100ng input: adaptor-ligated library PCR

Low input SureSelect: Agenda

Low Input SureSelect

Changes to current protocol

Performance

Low input SureSelect: Target Enrichment

SureSelectXT and SureSelectXT2 compatible with all

the existing baits

Human Exome Other

All Exon 50Mb DNA Kinome

All Exon V4 Mouse All Exon

All Exon V5 Custom 1kb-24Mb

All Exon V5 + UTRs

All Exon V5 + lncRNA

SureSelectXT 200ng input: sequencing performance

Human All Exon V5

Human All Exon V5

0.00%

10.00%

20.00%

30.00%

40.00%

50.00%

60.00%

70.00%

80.00%

90.00%

100.00%

Duplicates/ on-target

Percent duplicate reads:

Percentage reads in targeted regions:

Percentage reads in regions +/- 100bp:

0.00%

10.00%

20.00%

30.00%

40.00%

50.00%

60.00%

70.00%

80.00%

90.00%

100.00%

Coverage

...at least 1 read:

...at least 5 reads:

...at least 10 reads:

...at least 20 reads:

SureSelectXT 200ng input: sequencing performance

Human All Exon V5

dbSNP 135

3μg 200ng

Overlapping sites 41829 42238

Sites in

concordance

41785 42190

Concordance rate 99.89% 99.89%

SureSelectXT 200ng input: sequencing performance

Kinome

0.00%

10.00%

20.00%

30.00%

40.00%

50.00%

60.00%

70.00%

80.00%

90.00%

100.00%

200ng XT 200ng XT 200ng XT 200ng XT 3ug XT

Duplicates/ on-target

Percent duplicate reads:

Percentage reads in targeted regions:

Percentage reads in regions +/- 100bp:

75.00%

80.00%

85.00%

90.00%

95.00%

100.00%

200ng XT 200ng XT 200ng XT 200ng XT 3ug XT

Coverage

...at least 1 read:

...at least 5 reads:

...at least 10 reads:

...at least 20 reads:

SureSelectXT 200ng input: sequencing performance

Kinome

0.00%

10.00%

20.00%

30.00%

40.00%

50.00%

60.00%

70.00%

80.00%

90.00%

100.00%

200ng XT 200ng XT 200ng XT 200ng XT 3ug XT

Duplicates/ on-target

Percent duplicate reads:

Percentage reads in targeted regions:

Percentage reads in regions +/- 100bp:

75.00%

80.00%

85.00%

90.00%

95.00%

100.00%

200ng XT 200ng XT 200ng XT 200ng XT 3ug XT

Coverage

...at least 1 read:

...at least 5 reads:

...at least 10 reads:

...at least 20 reads:

SureSelectXT 200ng input: sequencing performance

Kinome

dbSNP 135

3μg 200ng

Overlapping sites 2259 2663

Sites in

concordance

2259 2661

Concordance rate 100% 99.92%

SureSelectXT 200ng input: sequencing performance

1Mb custom design

0.00%

10.00%

20.00%

30.00%

40.00%

50.00%

60.00%

70.00%

80.00%

90.00%

100.00%

200ng XT 200ng XT 200ng XT 200ng XT

Coverage

...at least 1 read:

...at least 5 reads:

...at least 10 reads:

...at least 20 reads:

0.00%

10.00%

20.00%

30.00%

40.00%

50.00%

60.00%

70.00%

80.00%

90.00%

100.00%

200ng XT 200ng XT 200ng XT 200ng XT

Duplicates/ on-target

Percent duplicate reads:

Percentage reads in targeted regions:

Percentage reads in regions +/- 100bp:

SureSelectXT 200ng input: sequencing performance

Human All Exome V5

0.00%

10.00%

20.00%

30.00%

40.00%

50.00%

60.00%

70.00%

80.00%

90.00%

100.00%

100ng XT2

100ng XT2

100ng XT2

100ng XT2

1ug XT2

1ug XT2

Duplicate/On-target (40 million reads/library- Exome V5)

% duplicate reads

% reads in targeted regions

% reads in targeted regions +/- 100bp

75.00%

80.00%

85.00%

90.00%

95.00%

100.00%

100ng XT2

100ng XT2

100ng XT2

100ng XT2

1ug XT2 1ug XT2

Coverage (40 million reads/library-Exome V5)

1X

5X

10X

20X

SureSelectXT2 100ng input: sequencing performance

Human All Exome V5

dbSNP 135

1μg 100ng

Overlapping sites 41460 41397

Sites in

concordance

41422 41360

Concordance rate 97.82% 97.81%

SureSelectXT2 100ng input: sequencing performance

DNA kinome

0.00% 10.00% 20.00% 30.00% 40.00% 50.00% 60.00% 70.00% 80.00% 90.00%

100.00%

100ng XT2

100ng XT2

100ng XT2

100ng XT2

1ug XT2

1ug XT2

Duplicate/On-target (3 million reads/library- Kinome bait design)

% duplicate reads

% reads in targeted regions

% reads in targeted regions +/- 100bp

75.00%

80.00%

85.00%

90.00%

95.00%

100.00%

100ng XT2

100ng XT2

100ng XT2

100ng XT2

1ug XT2 1ug XT2

Coverage (3 million reads/library-Kinome bait design)

1X

5X

10X

20X

SureSelectXT2 100ng input: sequencing performance

DNA kinome

dbSNP 135

1μg 100ng

Overlapping sites 2296 2294

Sites in

concordance

2295 2293

Concordance rate 99.95% 99.95%

SureSelectXT2 100ng input: sequencing performance

1Mb Custom Design

0.00%

10.00%

20.00%

30.00%

40.00%

50.00%

60.00%

70.00%

80.00%

90.00%

100.00%

100ng XT2

100ng XT2

100ng XT2

100ng XT2

1ug XT2

1ug XT2

Duplicate/On-target (1 million reads/library- 1Mb bait design)

% duplicate reads

% reads in targeted regions

% reads in targeted regions +/- 100bp

75.00%

80.00%

85.00%

90.00%

95.00%

100.00%

100ng XT2

100ng XT2

100ng XT2

100ng XT2

1ug XT2 1ug XT2

Coverage (1 million reads/library-1Mb bait design)

1X

5X

10X

20X

SureSelectXT2 100ng input: sequencing performance

SureSelectXT – 200ng input

SureSelectXT2 – 100ng input

No kit changes, some few protocol changes

Compatible with all existing baits

Proven sequencing performance: high sensitivity, specificity

and coverage. Greater than 99% SNP concordance rate.

SureSelectXT and SureSelectXT2 low input