suredesign v.1.2.1.15 laura pellegrino phd...designed for ffpe compatibility june 30, 2014...

1

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

June 30, 2014

Confidentiality Label

2

www.agilent.com/genomics/suredesign

Replaces eArray for DNA NGS/CGH custom design

BASIC DESIGN

June 30, 2014

Confidentiality Label

3

June 30, 2014

Confidentiality Label

4

Step 1: Define Design

June 30, 2014

Confidentiality Label

5

Haloplex

Step 2: Define Targets

June 30, 2014

Confidentiality Label

6

Step 3: Review Targets

June 30, 2014

Confidentiality Label

7

Direct links to UCSC Genome Browser

Step 4: Enter Parameters (only with Advantge Option)

June 30, 2014

Confidentiality Label

8

Step 5: Select probes

Step 6: Finalize

June 30, 2014

Confidentiality Label

9

June 30, 2014

Confidentiality Label

10

PDF Reports

Ways to Find Existing SureDesign Content

Expanded Search Capability

• Global search across applications, workgroups, and collaborations

• Search by design creator

June 30, 2014

Confidentiality Label

11

ADVANCED HALO DESIGN

June 30, 2014

Confidentiality Label

12

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

June 30, 2014

Confidentiality Label

13

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

June 30, 2014

Confidentiality Label

14

Read Length

June 30, 2014

Confidentiality Label

15

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

June 30, 2014

Confidentiality Label

16

Pseudogene designs

- Probe Stringency alteration

17

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

18

Balanced probe selection – 100% coverage of MYH6

Maximize specificity probe selection – 96.6% coverage of MYH6

SureDesign – FFPE Compatibility

19

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

June 30, 2014

Confidentiality Label

22

#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

23

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

June 30, 2014

Confidentiality Label

24

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

June 30, 2014

Confidentiality Label

33

www.agilent.com/genomics/suredesign