Data on file.For Research Use Only. Not for use in diagnostic procedures.

SINGLE-TUBE LIBRARYCONSTRUCTION

SeqCap EZ Prime Choice ProbesDiscover more, sequence less

Features• Fully customizable designs for human and non-human designs up to 200 Mb

• Improved coverage of GC-rich regions

• Compatible with our HyperCap products and workflow

Benefits• Create economical custom panels for a wide range of target sizes

• Less sequencing costs for challenging regions (e.g., first exons)

• Convenient one-stop shop for ordering, service, and support for a complete sample prep solution

SeqCap EZ Prime Choice Probes are Roche Sequencing Solution’s newest generation of DNA probes used for NGS Target Enrichment. Made using a new manufacturing process that retains the excellent enrichment properties of SeqCap EZ Choice, SeqCap Prime Choice Probes also improve the coverage of GC-rich regions.

TARGETEDSEQUENCING

2Data on file.For Research Use Only. Not for use in diagnostic procedures.

Good enrichment starts with good probe design

• The Roche design algorithm selects probes where others may not, providing you more coverage of the target area to better enable your variant discovery research

• Design your own custom SeqCap EZ Prime Choice panel via our NimbleDesign software or work with our live design team

Figure 1. SeqCap EZ Prime Choice Probes provide superior design coverage over Supplier A. Design comparison between SeqCap EZ Prime Choice Probes and Supplier A for a 500 Kb target on chr11 (61 – 61.5 Mb). SeqCap EZ Prime Choice Probes target 20% more of the target region (362 Kb vs. 256 Kb), as illustrated by an increase in red areas, denoting more coverage of the target regions. Note: The SeqCap EZ Prime Choice design was created using Roche’s most stringent design parameters which picks only unique probes. Supplier A’s design was created using Supplier A’s online design tool using its most stringent design parameters.

chr11(q12.2) 11p15.4 15.2 p15.1 p14.3 14.1 11p13 11p12 p11.2 q12.1 q13.4 11q14.1 q14.3 q21 q22.1 11q22.3 11q23.3 24.2

chr11(q12.2) 11p15.4 15.2 p15.1 p14.3 14.1 11p13 11p12 p11.2 q12.1 q13.4 11q14.1 q14.3 q21 q22.1 11q22.3 11q23.3 24.2

Supplier A’s probe coverage SeqCap EZ Prime Choice Probes’ coverage

61,08

9,000

61,09

0,000

61,06

7,000

61, 0

68,00

061

,069,0

0061

,069,0

0061

,070,0

0061

,071,0

0061

,072,0

0061

,073,0

0061

,074,0

0061

,075,0

0061

,076,0

0061

,077,0

0061

,078,0

0061

,079,0

0061

,080,0

0061

,081,0

0061

,082,0

0061

,083,0

0061

,084,0

0061

,085,0

0061

,086,0

0061

,087,0

0061

,088,0

00

Supplier A

Target Region

SeqCap EZ Prime Choice

SeqCap EZ Prime Choice Probes: compatibility with the streamlined HyperCap WorkflowThe HyperCap Workflow integrates KAPA HyperPrep and HyperPlus Kits with our new SeqCap EZ Prime Choice Probes, enabling a complete sample prep solution that can be tailored to different applications, sample types, and starting DNA amounts. This workflow streamlines hands-on time and costs while maintaining excellent sequence data quality.

• Reduce workflow time to 1.5 days—from extracted DNA to sequencing-ready libraries

• HyperCap products include our new Roche Universal Blocking Oligo (UBO) and KAPA Dual-Indexed (96) Adapter Kit

Set up Hybridization

Figure 2. KAPA HyperPrep and HyperPlus Kits reduce library construction time by half. (A) Workflow of various library preparation kits. *Covaris shearing time is dependent on instrument, make, model, and batch size. Time estimate for the KAPA Library Preparation Kit and KAPA HyperPrep Kit do not include Covaris shearing. (B) Workflow of the SeqCap EZ target enrichment protocols.

KAPA Library Preparation Kit protocol

in SeqCap EZ workflow v5.1

KAPA HyperPrep Kit protocol in SeqCap

EZ HyperCap workflow

KAPA HyperPlus

Kit protocol in SeqCap

EZ HyperCap workflow

Supplier A library prep kit

Covaris Shearing* Covaris Shearing* Covaris Shearing*EnzymaticFragmentation

Total Time: ~6 hrs*

End Repair

Size Selection

Adapter Ligation

A-tailing

PCR

Bead Cleanup

Bead Cleanup

Bead Cleanup

Bead Cleanup

Total Time: ~6 hrs*

End Repair

Size Selection

Adapter Ligation

A-tailing

PCR

Bead Cleanup

Bead Cleanup

Bead Cleanup

Bead Cleanup

Total Time: ~3 hrs

Adapter Ligation

Sing

le T

ube

End Repairand A-tailing

Bead Cleanup

Bead Cleanup

Size Selection

PCR

Total Time: ~3 hrs

Adapter Ligation

Sing

le T

ube

End Repairand A-tailing

Bead Cleanup

Bead Cleanup

Size Selection

PCR

Supplier A workflow

SeqCap EZ workflow v5.1

SeqCap EZ HyperCap workflow

Day

1D

ay 2

Probe Preparation

Set up Hybridization

Sequencing Day

3D

ay 1

Day

2

Probe Preparation

Set up Hybridization

Sequencing

Day

3D

ay 1

Day

2

Library Preparation

Set up Hybridization

Sequencing

BA

3Data on file.For Research Use Only. Not for use in diagnostic procedures.

Input Amount

Conv

ersion

(%)

Figure 4. SeqCap EZ Prime Choice Probes resulted in more functional coverage of full genes. NGS libraries starting with100 ng of NA 12891 were 12-plexed and captured on the 4,000 gene human genetic disease content made with both SeqCap EZ Prime Choice and SeqCap EZ Choice Probes. The HyperCap Workflow v1.0 was used; first exon and full gene coverage at ≥20x depth was analyzed. The values shown for first exon and full gene are representative of an average of the 12 samples.

…and of first exons and full genesImproved coverage of first exons can lead to more complete coverage of full genes which can reduce the amount of Sanger Sequencing necessary to fill in uncovered genes, saving valuable time and resources.

• SeqCap EZ Prime Choice Probes’ improved GC-rich coverage enables 384 more first exons on average, covered at 20x vs. the same panel made with the SeqCap EZ Choice Probes

• Obtain on average 573 more genes fully covered ≥20x compared to SeqCap EZ Choice Probes

Coverage bin

SeqCap EZ ChoiceD_100%

SeqCap EZ Prime Choice

3000

2500

2000

1500

1000

500

0

Genes in bin

1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12

Conv

ersio

n (%

)

3500

3400

3300

3200

3100

3000

2900

2800

2700SeqCap EZ Choice SeqCap EZ Prime Choice

Figure 3. Significantly improved mean coverage in GC-rich regions. Three panels of varying size were created on both SeqCap EZ Choice and SeqCap EZ Prime Choice Probes for comparison. 1. SeqCap EZ Human Oncology Panel (200 Kb) 2. SeqCap EZ Human Oncology Panel (2.75 Mb) 3. SeqCap EZ Inherited Disease Panel (11 Mb, approx. 4,000 genes). All panels were processed using the HyperCap Workflow v1.0 starting with 100 ng of Coriell Sample NA 12891. Additionally, 100 ng of formalin treated Horizon TruQ sample was run on both versions of the 2.75 Mb SeqCap EZ Human Oncology Panel. In all cases, the SeqCap EZ Prime Choice Panels significantly improved normalized mean coverage vs. the SeqCap EZ Choice Panels in GC-rich regions.

SeqCap EZ Primes Choice Probes offer improved coverage…

…in GC-rich regions• SeqCap EZ Prime Choice Probes deliver consistently higher coverage in GC-rich regions (>65%) across varying design

sizes and sample types vs. SeqCap EZ Choice Probes

• On average, the coverage of GC-rich regions is 46% higher vs. SeqCap EZ Choice Probes

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

OncologyPanel(3Mb) OncologyPanel(200Kb) Inherited DiseasePanel(11Mb) OncologyPanel(3Mb)- FormalinTreatedDNA

Average

NOrm

alize

dMeanCoverage

SeqCapEZChoicevsEZPrimeChoiceNormalizedCoverageofHigh-GCRegions

EZChoice

EZPrimeChoice

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0

Nor

mal

ized

mea

n co

vera

ge

SeqCap EZ Choice Probes vs SeqCap EZ Prime Choice Probes; normalized coverage of GC-rich regions

EZ Choice EZ Prime Choice

Oncology Panel (3 Mb)

Oncology Panel (200 Kb)

Inherited Disease Panel (11 Mb)

Oncology Panel (3 Mb) - Formalin

Treated DNA

Average

Published by:

Roche Sequencing Solutions, Inc.4300 Hacienda DrivePleasanton, CA 94588

sequencing.roche.com

Data on file.For Research Use Only. Not for use in diagnostic procedures.HYPERCAP, KAPA, NIMBLEDESIGN, and SEQCAP are trademarks of Roche. All other product names and trademarks are the property of their respective owners. ©2018 Roche Sequencing Solutions, Inc. All rights reserved. SEQ100193 A038 03/18

SeqCap EZ Prime Choice ProbesRoche Cat. No. Description Pack Size08247447001

Target up to 7 Mb human (HG18 or HG19) genomic regions

4 rxn

08247455001 12 rxn

08247463001 24 rxn

08247471001 48 rxn

08247480001 96 rxn

08247498001 384 rxn

08247501001 960 rxn

SeqCap EZ Prime Choice XL ProbesRoche Cat. No. Description Pack Size08247510001

Target >7 – 200 Mb human (HG19 or HG38) genomic regions

4 rxn

08247528001 12 rxn

08247536001 24 rxn

08247544001 48 rxn

08247552001 96 rxn

08247579001 384 rxn08247587001 960 rxn

SeqCap EZ Prime Developer ProbesRoche Cat. No. Description Pack Size08247595001

Capture up to 200 Mb custom regions from non-human genomes

4 rxn

08247609001 12 rxn

08247617001 24 rxn08247625001 48 rxn08247633001 96 rxn08247641001 384 rxn08247650001 960 rxn

Ordering Information