SureDesign v.1.2.1.15 Laura Pellegrino PhD...Designed for FFPE compatibility June 30, 2014...
Transcript of SureDesign v.1.2.1.15 Laura Pellegrino PhD...Designed for FFPE compatibility June 30, 2014...
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SureDesign v.1.2.1.15
Laura Pellegrino PhD
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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
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BASIC DESIGN
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Step 1: Define Design
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Haloplex
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Step 2: Define Targets
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Step 3: Review Targets
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Direct links to UCSC Genome Browser
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Step 4: Enter Parameters (only with Advantge Option)
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Step 5: Select probes
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Step 6: Finalize
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PDF Reports
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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
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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.
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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
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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
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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
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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
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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
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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
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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
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Questions and Live Demo
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www.agilent.com/genomics/suredesign