Advanced Custom NGS Panel Design with … Custom NGS Panel Design with SureDesign Webinar Creating...

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Advanced Custom NGS Panel Design with SureDesign Webinar

Creating Complex NGS

Designs

June 1, 2017

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Topics for today’s presentation

For Research Use Only. Not for

use in diagnostic procedures.

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Enabling advanced features

Login to SureDesign



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Advanced settings changes workflow

Basic settings Advanced settings



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Probegroups: when and how to efficiently use

• A probegroup is a collection of baits that were designed

using the same parameters

• Use a probegroup as a sub-design (e.g., all of the genes

related to inflammation that might be included in an

infectious disease design as well as in a cancer design)

• Probegroups can be used to create baits according to design

parameters like stringency and balancing

• Probegroups can be repeated for optimization



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Design approaches: tiling and probe reuse from other design(s)

Tiling

• Simple steps to design

• May need tweaking for long and short

regions as tiling algorithm does not

consider region length

Reuse probes (catalog or custom)

• More steps in SureDesign

• Use performance metrics from

previous runs of your design

• Agilent catalog designs empirically

optimized

• Likely increase bait counts

considerably



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Tiling design considerations (long regions)

Long tiled regions may get covered “too well” in the middle (about 700 bp)

4X tiling 760 reads at peak

Staggered baits result in fewer reads; more even coverage

Remove some of the baits in the center of the region



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Tiling design considerations (short regions)

Similarly, tiling short regions may not provide enough coverage (110 bp)

Need to add more copies of each bait in shorter regions

16 replicates of each bait to get 56X coverage



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• Identify “missed” regions by downloading the design and examining the AllTracks.bed file

• Because fragments can extend outside the bait, small regions outside the baits will likely be captured. Don’t be concerned with gaps smaller than 40 bp

• Relax the masking OR increase the extension into masked areas by creating a new probegroup

• Reuse probes from an Agilent catalog design to see if gap can be closed (may need to expand area slightly). Choose catalog design based on compatibility with your research goals

Optimizing SureSelect designs (design gaps)

Be very careful using Least Stringent or No Masking



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• If you have an existing design and sequencing performance

metrics, you may selectively increase the bait replication

factor (individually or probegroup replicates) or selectively

delete baits from an existing design (see previous tiling slides)

• You may modify the Boosting for all baits in a probegroup

Max Performance and Balanced will increase replication values for individual baits

based on GC content (up to 16 or 4, respectively) based on GC content

• The same bait may reside in multiple probegroups

• Adjusting a bait’s location slightly may improve performance

• Easily create a probegroup from an existing design

Optimizing SureSelect designs (performance)



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Optimizing HaloPlex and HaloPlexHS designs

• SureDesign allows few optimizations for HaloPlex designs

within the tool. Most optimizations would have to be

performed by Agilent personnel using tools outside of

SureDesign.

• Use the FFPE design option to add probes to both strands

(Illumina).

• If amplicons are captured to a lesser degree than desired,

you can create a probegroup that contains the baits for those

amplicons and increase the number of replicates for the

probegroup.



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Design visualization

UCSC View link at design level shows

Covered.bed file for design

Or download AllTracks.bed file to view

Targets, Covered, and Missed regions

and upload into your favorite genome

browser



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While on the subject of design files…..simple or catalog designs

Target, Covered, Missing

Union of all regions covered by baits

Covered.bed ± 100 bp

Chromosomal coordinates of targets

List of design targets

Use Covered.bed for on-target metrics but use Padded.bed file for SNP calling



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Design files….complex designs with probegroups

Top level files for design; must create

your own padded.bed file from

Covered.bed file

One set of files per probegroup.

If designed hierarchically (use targets and

then try to close gaps for same targets), you

will have to look at the last probegroup’s

AllTracks.bed file for gaps.



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Collaborations and sharing designs

• Your custom designs are private; even Agilent field

employees cannot see the details nor can anyone else order

them

• If you wish to share designs with a coworker, collaborator, or

an Agilent employee, you must create a collaboration and

invite members to join using their SureDesign login id (email)

• Once Collaboration is created, you must move the design

over to the Collaboration space or create it in that

collaboration



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Creating a collaboration in SureDesign

2) Click on Collaborations Tab

1) From Home Screen, Find Designs>SureSelect DNA

First time message

3) In left panel, select Collaborations>Manage Collaborations

4) Select Add at bottom of screen 5) On next screen, enter Name and

Save, Invite using SureDesign login

ids, and Close



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Designs can be created in collaboration space

Your default folder or

workspace

All probegroups must reside in the same workspace or collaboration



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Tips & Tricks (specifying Targets)

• Targets can be a mixture of gene names, genomic coordinates, SNP IDs, Entrez IDs

• Targets can be cut and pasted into the window

• Targets may be in a file that gets Uploaded

• After selecting Next, SureDesign translates Targets into Regions which you can immediately download and modify in Excel if you want to do something like expand upstream by 100 bases and downstream by 30 bases



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Tips & Tricks (specifying targets)

Choose databases

wisely.

Different levels of

curation for each

database.

If UTR specified

with all databases

selected, the UTR

regions will

expand

tremendously.

Coding Regions + UTRs must

be selected for noncoding

RNAs (antisense tx, lncRNA,

microRNAs)

Allow Synonyms should only be

used if your target cannot be

found. Otherwise, you may have

multiple genes for same target.

Perfect use for probegroups



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Tips & Tricks (SureDesign extras)

Some SureSelect and HaloPlex catalog design

files contain sample data for download HaloPlex designs also contain the

QC BioAnalyzer trace

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Further information

Register for SureDesign account at link provided in chat

(designing or quote requests)

3 ways to create custom designs

1) Design it yourself using SureDesign

2) Consult your local Field Application Scientist

3) Contact Agilent Design Services through your account manager

Software questions about SureDesign: contact

[email protected]



Thank you for your attention

Advanced Custom NGS Panel Design with … Custom NGS Panel Design with SureDesign Webinar Creating...

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