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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