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SureDesign v.1.2.1.15

Laura Pellegrino PhD

SureDesign

Design with Confidence

• Modify existing catalog and custom designs

• Direct link to UCSC browser to review target regions

• Easily modify design settings (FFPE, stringency, etc.)

• Download .BED and design report

• Basic and advanced design options

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www.agilent.com/genomics/suredesign

Replaces eArray for DNA NGS/CGH custom design

BASIC DESIGN

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Step 1: Define Design

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Haloplex

Step 2: Define Targets

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Step 3: Review Targets

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Direct links to UCSC Genome Browser

Step 4: Enter Parameters (only with Advantge Option)

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Step 5: Select probes

Step 6: Finalize

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PDF Reports

Ways to Find Existing SureDesign Content

Expanded Search Capability

• Global search across applications, workgroups, and collaborations

• Search by design creator

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ADVANCED HALO DESIGN

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Halo Advanced Design Wizard (Ion and Illumina)

Define Design

• Library name, sequencing platform, read length, etc

Add / Review Content - LOOP

• Create one or more probe groups

– By Tiling

• Access to additional design parameters

– By Selecting Existing Probes

• Entire design

• Subset of a design

• List of amplicon IDs from a design

Finalize

• Design is available for quoting and ordering

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Read Length Parameter

• For Halo Illumina libraries only

• In Simple and Advanced wizards

• Choice of 100bp, 150bp or 250bp

• Probe selection algorithm is the

same for 100bp, 150bp and 250

read lengths

• Different coverage calculation

– to account for longer coverage

contribution per amplicon

needed for sequencing

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Read Length

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100bp 250bp

Add / Review Content – By Tiling

Same as tiling in HaloPlex simple design workflow

With access to additional design parameters

• Stringency

– Maximize Specificity, Balanced, or Maximize Coverage

• Optimize for Fragmented Samples

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Pseudogene designs

- Probe Stringency alteration

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1. Maximize Specificity - Requires probe with only one unique hit in the

genome

2. Balanced – Allows selection of probes with 2 hits in the genome.

3. Maximize Coverage – Allows up to 5 hits in the genome.

Pseudogene Designs

- Probe Stringency Alteration

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Balanced probe selection – 100% coverage of MYH6

Maximize specificity probe selection – 96.6% coverage of MYH6

SureDesign – FFPE Compatibility

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For FFPE optimized designs

1. The design will include shorter fragments (50-400bp)

2. Both target polarities will be selected

Target Region Target Region

Normally fragments in the

range of 100-400bp are

used

For FFPE samples shorter

fragments (50-400bp) are

included

Standard design

Designed for FFPE compatibility

Exon Exon

FFPE design

One probe is targeting the sense and one the anti-sense strand of the target DNA

Sense

Strand

AntiSense

Strand

DNA TARGET FRAGMENT

Probes are designed for both target fragment polarities

• Improves capture efficiency

• Eliminate strand specific FFPE artifacts

Designed for FFPE compatibility

Designed for FFPE compatibility

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#chrom chromStart chromEnd name score strand

chr17 7565227 7565285 AM_175245_000003 1000 –

chr17 7565317 7565419 AM_175245_000002 1000 –

chr17 7565330 7565382 AM_175245_000001 1000 –

chr17 7569302 7569549 AM_175245_000008 1000 –

chr17 7569302 7569549 AM_175245_000007 1000 –

chr17 7569374 7569604 AM_175245_000004 1000 –

chr17 7569419 7569771 AM_175245_000006 1000 –

chr17 7569515 7569618 AM_175245_000005 1000 –

#chrom chromStart chromEnd name score strand

chr17 7565227 7565285 AM_719573_000005 1000 +

chr17 7565227 7565285 AM_719573_000004 1000 –

chr17 7565317 7565419 AM_719573_000001 1000 –

chr17 7565318 7565420 AM_719573_000003 1000 +

chr17 7565326 7565382 AM_719573_000002 1000 +

chr17 7569300 7569549 AM_719573_000012 1000 +

chr17 7569300 7569549 AM_719573_000011 1000 +

chr17 7569302 7569549 AM_719573_000014 1000 –

Standard FFPE

SureDesign – Ion Torrent vs Illumina

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Polarities

Illumina: Probes are selected on one of the two

polarities of the region targeted

(PE sequencing)

Ion Torrent: Probes are selected on both polarities

(SE sequencing) Pseudo PE Sequencing

Capacities

Illumina: Up to 5 MB

Ion Torrent: Up to 2.5 MB

Add / Review Content – Selecting Existing Probes

Options are to choose:

• One design / probegroup

• Subset of one design / probegroup

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Exon 1 Exon 2

• All HaloPlex exome probes overlapping with defined targets will be selected

• Exome probes do not exist for non-coding regions, none will be selected for

these regions

SureDesign

Use amplicons from other probe groups (ex: HaloPlex Exome)

1. Define target regions

2. Use all exome probes for coding regions

3. Combine with custom design for non coding regions

Questions and Live Demo

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www.agilent.com/genomics/suredesign