Agilent Technologies SureSelect™ Target Enrichment Platform Providing focus for next-generation sequencing workflows David Willmot, PhD Sr. Applications

Agilent TechnologiesSureSelect™

Target Enrichment Platform

Providing focus fornext-generation sequencing workflows

David Willmot, PhD Sr. Applications Scientist

Enabling Products for the Next-Generation Sequencing Workflow

Page 2

Agilent’s SureSelect Platform: New Options

SureSelect Target Enrichment System

(in solution)Developed in collaboration

with the Broad InstituteDr. Chad Nusbaum et al.

SureSelect DNA Capture Array

(on array)Developed in collaboration

with Cold Spring HarborDr. Greg Hannon et al.

Agilent 60mer Array

1-5 µg gDNA

1-5 µg gDNA, or20 µg gDNA


Page 3

A Choice in Agilent Target Enrichment Options for Specific Project Needs



(244k array)

Throughput High Low

Study Sizes 10-1,000s samples 1-10 samples

DNA Input 1-3 µg 1-20 µg*

Capture of Target DNA

3.3+ Mb Custom ~1 Mb

Format Kit Array plus application note/protocol

or

SureSelect™ Target Enrichment System: Workflow

SureSelect™Target Enrichment System

Illumina GA Kit Available End of February


Page 5

Agilent Online Custom SolutionsFlexibility in Target Enrichment

244K

eArrayTarget Enrichment

Design

DNA Capture Design

Custom and CatalogDesigns for CGH, Gene

Expression, etc

researcher

Manufacturing Process Development

Bioinformatics



User Sequences

Basis of SureSelect Target Enrichment System: publ. by Broad Inst. Feb. 2009 Nature Biotechnology

SureSelect™ Target Enrichment System:Design and Order Process

1. Design & Order

2. Kit Production

3. Single Tube Kit

DeliverySelect customgenome partitioning

set using eArray

or, selectcatalog oligo set

eArray Web Portal

55K unique 120 mer oligos synthesized on

one wafer

Oligos released

Oligo IVT to RNA-biotin

Kit Includes

1. Biotinylated-cRNA

2. Buffers3. Protocol

Agilent genome partitioning kit

shipped to customer

SureSelect™Target Enrichment System

Quality Control on the Bioanalyzer

• Simplified Experimental Workflow

Target Enrichment


Page 9

Santa Clara Manufacturing Facility

- Industrial manufacturing – Class 10,000 clean-room

- Wired directly into eArray, allowing direct customer access to fully customizable products

- High-performance inkjet printing enables long oligo manufacturing

Manufacturing Process

Development Bioinformatics

Agilent’s Microarray Platform

• Reliable inkjet printing

• Sensitivity of probes

• Flexibility of microarrays

• Ease of implementation

• Probe Fidelity can synthesize up to 200mer

mailto:[email protected]


Page 11

Agilent’s Strength in Ultra-long Oligo Synthesis

3) Deblock

1) Coupling

2) Oxidation

Repeat n times

Depurinationside reaction

0

20

40

60

80

100

0 50 100 150 200

Oligo Length (mer)

Fu

ll L

en

gth

(%

)

98% cycle yield, no depurination99.5% cycle yield, no depurination

98% cycle yield, with depurination99.5% cycle yield, with depurination

Agilent

Conventional Processes

N1O

OP O

ROO

N2O

OP O

ROO

NiO

OP O

ROO

HO

Manufacturing Process

Development Bioinformatics

Agilent SureSelect™ PlatformEnabling Products for the Next-Generation Sequencing WorkflowPage 12

SureSelect™ Target Enrichment System: Bringing Cost-Efficiency to Next-gen Sequencing

Workflows


The Cost of DNA Sequence Information: Illumina GA

Typical Illumina Sequence Experiment Costs

Bases/ Run 2,000,000,000Lanes/ Run 8Samples/ Run 7Bases/ Lane(Sample) 250,000,000Cost/ Run (Sequencing Chemistry/ Flow Cell) $7,000Cost/ Lane (Sample) $1,000Cost/ Megabase Read $4.00

How much of this is useful information?What is the cost of the useful information?


SureSelect™ Target Enrichment: Cost Benefit

Typical Target Experiment

Size of Interesting Area of Genome (Mb) 3

Non-targeted Experiment % On Target Reads 0.10%# of Useful Bases/ Sequencing Lane 250,000Cost per Useful Mb Sequenced $4,000

SureSelect % On Target Reads 40%# of Useful Bases/ Sequencing Lane 100,000,000Cost of Targeting Enrichment per 3.3 Mb $1,100Cost of Target Enrichment $1,100Cost per Useful Mb Sequenced $21Cost Savings with SureSelect Per Mb Targeted $3,979

SubstantialCost

Differential


Page 15

SureSelect™ Target Enrichment System Using Illumina GA– Sample Data from an Exon Capture Experiment

0 60 120

180

240

300

360

420

480

540

600

660

720

780

840

900

960

1020

1080

0%

1%

2%

3%

4%

5%

6%

7%

8%

9%

10%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Depth distribution

Read Depth

% p

er B

in

Cum

ulati

ve %

Target: 9,107 exons3+ Mb

Sample: 3 µg Genomic DNA Covaris Shearing Illumina library prep End-sequencing 35bp


Page 16

Genomic Space View (Zoomed-in View of 7 Exons)

1st exon covered by 102 tiled baitsRead depth maxes out at >269xAverage read depth is 100x

2nd exon covered by 1 bait (because it was right next to a repeat region)Read depth ~ 10x

3rd – 5th exons covered by 2-4 baitsRead depth ~ 40x

SureSelect™ Target Enrichment Reproducibility

Agilent Confidential

0 10 20 30 40 50 60 70 80 90 1000

20

40

60

80

100

120

140

160

180

R² = 0.961609744553544

Average read depth of technical replicates

Average read depth per target interval (AGT1)

Avera

ge r

ead d

epth

per

targ

et

inte

rval (A

GT7)


SureSelect™ Target Enrichment System: Strong Allele Balance Indicates Negligible Bias

number non-reference bases number reference bases0

5000

10000

15000

20000

25000

Heterozygous SNP calls

• Baits are designed to wildtype sequence• Long oligos lead to unbiased SNP capture and calling

SureSelect™ Target Enrichment System: Efficient SNP Validation and Discovery

SNP ID Chr Basepair Reference

(reads)

SNP (reads

)

rs11204545 124612607

6 47 42

rs11204546 124612608

5 52 40

rs831043 216981133

2 44 52

rs6886628 511292735

1 52 41

Novel 910338143

4 36 44

rs944682 115095921

2 0 91

GAC C A GG GC AA/TGT T C CT C AT/CGC T C T T C T A C

rs11204545 and rs11204546

GAC C A GG GC AA/TGT T C CT C AT/CGC T C T T C T A CGAC C A GG GC AA/TGT T C CT C AT/CGC T C T T C T A C

rs11204545 and rs11204546

T T G C T T G C A CA/GC T A G C T T A T C

rs6886628

T T G C T T G C A CA/GC T A G C T T A T CT T G C T T G C A CA/GC T A G C T T A T C

rs6886628

A C A C A G G A T C G T C T G G C T G C T

rs944682

A C A C A G G A T C G T C T G G C T G C TA C A C A G G A T C G T C T G G C T G C T

rs944682

G A A A G A G C C C A/C G A G A A G T G T T

Novel Chr9:103381434

G A A A G A G C C C A/C G A G A A G T G T TG A A A G A G C C C A/C G A G A A G T G T T


A A C C A C C C A C A/GG A G C A G T G T G

rs831043

A A C C A C C C A C A/GG A G C A G T G T GA A C C A C C C A C A/GG A G C A G T G T G

rs831043

GAC C A GG GC AA/TGT T C CT C AT/CGC T C T T C T A C

rs11204545 and rs11204546

GAC C A GG GC AA/TGT T C CT C AT/CGC T C T T C T A CGAC C A GG GC AA/TGT T C CT C AT/CGC T C T T C T A C

rs11204545 and rs11204546

T T G C T T G C A CA/GC T A G C T T A T C

rs6886628

T T G C T T G C A CA/GC T A G C T T A T CT T G C T T G C A CA/GC T A G C T T A T C

rs6886628

A C A C A G G A T C G T C T G G C T G C T

rs944682

A C A C A G G A T C G T C T G G C T G C TA C A C A G G A T C G T C T G G C T G C T

rs944682

G A A A G A G C C C A/C G A G A A G T G T T


G A A A G A G C C C A/C G A G A A G T G T TG A A A G A G C C C A/C G A G A A G T G T T


A A C C A C C C A C A/GG A G C A G T G T G

rs831043

A A C C A C C C A C A/GG A G C A G T G T GA A C C A C C C A C A/GG A G C A G T G T G

rs831043

• Allelic balance no bias• Efficient identification of SNPs• Illumina readout confirmed with CE• Efficiently captures mutations

Efficient Capture of 5 bp deletion on the X-Chromosome: Menke’s Syndrome

SureSelect™ Target Enrichment Kit Efficiently Captures 5 bp MutantReadout on Illumina GA

hg18_ChrX_77131408_77131467_+ : Wildtype Bait Design

CTATTGTTTATCAACCTCATCTTATCTCAGTAGAGGAAATGAAAAAGCAGATTGAAGCT

CTATTGTTTATCAACCTCATCTT AGTAGAGGAAATGAAAA ATTGTTTATCAACCTCATCTT AGTAGAGGAAATGAAAAAGTTGTTTATCAACCTCATCTT AGTAGAGGAAATGAAAAAGCGTTTATCAACCTCATCTT AGTAGAGGAAATGAAAAAGCAG

TATCAACCTCATCTT AGTAGAGGAAATGAAAAAGCAGATTATCAACCTCATCTT AGTAGAGGAAATGAAAAAGCAGATTGATCAACCTCATCTT AGTAGAGGAAATGAAAAAGCAGATTGATCAACCTCATCTT AGTAGAGGAAATGAAAAAGCAGATTGCAACCTCATCTT AGTAGAGGAAATGAAAAAGCAGATTGAA

CCTCATCTT AGTAGAGGAAATGAAAAAGCAGATTGAAGCT

hg18_ChrX_77131408_77131467_+ : Wildtype Bait Design

CTATTGTTTATCAACCTCATCTTATCTCAGTAGAGGAAATGAAAAAGCAGATTGAAGCT

CTATTGTTTATCAACCTCATCTT AGTAGAGGAAATGAAAA ATTGTTTATCAACCTCATCTT AGTAGAGGAAATGAAAAAGTTGTTTATCAACCTCATCTT AGTAGAGGAAATGAAAAAGCGTTTATCAACCTCATCTT AGTAGAGGAAATGAAAAAGCAG

TATCAACCTCATCTT AGTAGAGGAAATGAAAAAGCAGATTATCAACCTCATCTT AGTAGAGGAAATGAAAAAGCAGATTGATCAACCTCATCTT AGTAGAGGAAATGAAAAAGCAGATTGATCAACCTCATCTT AGTAGAGGAAATGAAAAAGCAGATTGCAACCTCATCTT AGTAGAGGAAATGAAAAAGCAGATTGAA

CCTCATCTT AGTAGAGGAAATGAAAAAGCAGATTGAAGCT

SummaryThe Agilent SureSelect platform• Flexible custom designs with Agilent’s eArray portal - free of charge.• Efficiently captures mutations, with 1/10th the gDNA vs. competing

products• Scalable solutions for small scale to large population studies and

automation• Array-based target enrichment application coming shortly as well as

SOLiD and 454 in-solution protocols• Target Enrichment lowers:

Reagent usageDNA inputLaborData handling

For more information http://www.opengenomics.com/SureSelect

See Protocol: G3360-90010

Contact [email protected] 1-800-227-9770 options 2x2x1

http://www.opengenomics.com/SureSelect

mailto:[email protected]

A QuickStart Guidefor the Creation and

Ordering ofSureSelect Target

Enrichment Oligo Sets

Using eArray, a free web-based design tool

Summary of Steps

• Getting Started– Access eArray– Confirm Application Type– Select Method

• Terms and Definitions• Initiate the Wizard

• Create Library by Bait Tiling– Library Options and Target Details

Design Strategy– Centered versus Justified– Formats for Describing Target

Intervals– Upload Message– View the Design– Continue to Create a Library– Define Library– Layout Baits– Save and Submit Library– Final Steps– Download Library– Provide Quote Details– Review and Submit Quote

• Access eArray at: https://earray.chem.agilent.com/earray/

• Log in to eArray, if this is your first time visiting, click Request for Registration

Slide 24

Getting StartedAccess eArray

The Login Name is the e-mail address

used to register

• Confirm that the Application Type is TargetEnrichment

Slide 25

Getting StartedConfirm Application Type

If necessary, select Switch

Application Type to select Target

Enrichment

• Choose between two methods for creating a SureSelect Library

Slide 26

Getting Started Select Method

Benefits Caveats When to Use

Following the Wizard

• Easy, stream-lined method• The Wizard takes the user from initiation of design to submission

• The user will only be able to download the library details after the library is completed• The user will not have access to a "fate" file

When a simple design is planned

Independent of the Wizard*

• User can download additional files, including a Fate file that lists the # of baits for each target interval • User can fine-tune the design, creating and combining multiple bait groups parameter settings• User can track the success of bait tiling for individual targets

• User will not be guided by a Wizard

When iterations may be desired for an optimal design

Terms and Definitions

• Bait:

– A single oligo sequence of pre-determined length (120 bp) that complements a targeted region of the genome

• Bait Group:

– Consists of a group of Baits designed to complement a single or set of targeted intervals

– May be formed from baits generated within eArray, baits uploaded into eArray, or bait search results within eArray

• Library:

– Consists of one or more Bait Groups– Represents the set of oligos that will be produced for the

kit

Slide 27

• Select the method for Library creation in the Library Wizards quadrant, and click Next

Slide 28

Initiate the Wizard

Create Library by Bait Tiling:Use this option to upload the

genomic intervals of your targets and allow eArray’s algorithm to

design baits for these targets. This is the option outlined in this tutorial.

Create Library from Existing Bait Group(s):

If you have already created the bait group(s) that will be used for this

library, use this option.

Create Library from Bait Upload:If you have already designed the

baits that will recognize your desired target regions, use this option.

Slide 29

Create Library by Bait TilingStep 1: Library Options and Target Details

1. Enter a name for the design job.

2. Design Strategy: To use the parameters previously optimized for general bait tiling, leave the checkmark

on this option. To change the parameters, uncheck this option. The next slide describes the parameter changes

that can be applied.

3. Species: Select the species for which the target intervals were designed. The Genome Build will

then automatically be populated by eArray.

4. Genomic Target Intervals: Either type in or upload the genomic intervals for the

targets to be enriched. Examples are provided on a later slide.

5. Genomic Avoid Intervals:• Choose to avoid the standard repeat masked

regions by leaving a checkmark next to this option (based on the UCSC RepeatMasker track)

• Add additional intervals to avoid with the baits by typing those intervals in or uploading them in the same format as the target intervals.

6.Submit:Select Submit when

Options and Details are completed.

Create Library by Bait TilingDesign Strategy

Slide 30

1. Change the parameters: To be able to change these parameters, first remove the checkbox from the “Use Optimized Parameters” option.

2. Centered versus Justified: (see next slide for visuals)

3. Bait Length: Currently, 120 bp is available as the only bait length option. All baits will be designed to be 120 bp in length.

4. Bait Tiling Frequency: Options include 2X, 3X, 4X, and 5X and indicate the amount of bait overlap. Tiling frequency is not enforced at

target edges. Increasing the frequency will lead to the ability to cover fewer or smaller regions in a library.

5. Allowed overlap into avoid regions: Centered baits may overlap with regions

adjacent to the target. In case the targets are

adjacent to Avoid Regions, enter the

acceptable amount of overlap with these in

bp. To ensure that there is no overlap in any Avoid Regions,

select ‘0 bp’.

Centered:Baits are centered, and

evenly distributed, across each target region. Baits

may overlap regions outside of the target.

Why use this? This is the method tested most

extensively.

Justified:Baits are first tiled across the

target. If baits extend past the target interval, all baits are shifted inward so that

there will be no overlap with adjacent but un-targeted

genomic regions.

Why use this? If it is desired to avoid having baits

to any region adjacent to targets, for example, if

sequencing cDNA.

2X Tiling: 3X Tiling: 4X Tiling: 5X Tiling:

Target

Baits

(b) Target region is 2 times the bait length

baits

(a) Target region is large (example of 2x tiling)

(c) Target region is shorter than the bait length

Create a Bait GroupCentered versus Justified

JustifiedCentered

target region

Design is the same for

Centered and Justified

Centered baits extend past interval boundaries, but have even coverage

across entire region Justified baits do not

extend past boundaries, but may have uneven

coverage (e.g., see circle where regions have both

2x and 3x tiling)

Design is the same for

Centered and Justified

Create Library by Bait TilingFormats for describing Target Intervals

Slide 32

Option 1: Type in the genomic intervals to be targeted.The format for typing in the intervals is as follows:

chrX:100003816-100003948|chrX:100004037-100004218|chrX:100004314-100004465|

chrX:100004329-100004465|chrX:100004804-100004888

Each interval should be separated by the | character. If there is a long list of intervals, it would be better to use

option 2.

Option 2: Upload a file that includes the genomic intervals to be targeted:

The format for uploading the intervals is as follows:

Each interval should be presented on a separate line, and the file should be saved as a text file.

When using this option, select Upload, Browse to find the saved file, and then select Upload File.

Create Library by Bait TilingUpload Message

• Once you’ve clicked on Submit, you will receive the above Upload Message

• You have now submitted a design for the creation of a Bait Group!

• Select Exit, and return to the Home page on eArray

• You can now monitor the status of your submission in the Library Wizards quadrant

– Click on Refresh to view the most current status

– You will receive an e-mail alert when the submission is complete

Slide 33

Create Library by Bait TilingView the Design (I)

• Once the Bait submission is complete and Baits are uploaded, return to the eArray Home page and select View Design

Slide 34

Create Library by Bait TilingView the Design (II)

• Toggle between Design Summary, Design Details, Target Fate, and Bed File to view the details of the bait design

• Only the first 100 lines are displayed

Slide 35

Create Library by Bait TilingContinue

• If satisfied with the creation of this Bait Group, return to the eArray Home page and select Continue

Slide 36

Create Library by Bait TilingDefine Library

Slide 37

Provide a name for the library:Previously in the Wizard, a Bait Group was defined and created. Now we

are creating a Library, which might consist of one or more Bait Groups. Therefore, it also requires a name.

Provide a description for the library:Here, it is possible to include a description,

keywords, and comments to help characterize this library.

When completed, select Next

A control grid is always, and

automatically, included in a library

Create Library by Bait TilingLayout Baits

Slide 38

3. Determine the # of Replicates per Bait

Group:When a Library is not full, it is possible to

create replicates of one or more Bait Groups

5. When completed, select Next

1. Describe the Control Type:

Leave this empty if the Bait Group is not

a control.

2. If desired, add additional, pre-

made Bait Groups to this library:

Select Add, Search for the Bait by name,

Add to the box on the right, and click

Done

4. Check Library Statistics:The Percentage Filled value should be less than or equal to100%. If there are a number of features still

available, or the Percentage Filled is low, it is possible to add replicates or additional, existing Bait Groups.

Create Library by Bait TilingSave and Submit Library

• Save the new Library in the format of choice– To prepare the Library for manufacturing, it is necessary

to choose Submit– When choosing Submit, click on Design check list and

complete the checklist before choosing Save• After Submitting a Library, it is ready for ordering, but you

must still generate the Quote

– The library is not ordered at this step, it is only submitted

Slide 39

Create Library by Bait TilingFinal Steps

• Once a Library has been submitted, it is available for generating a Quote

• The Library can be found in the My Libraries quadrant in the eArray home page– If the library is not listed there, select Refresh– If the library is still not listed there, it may have been saved but not submitted– The library can also be found using the Library Search feature in the Search quadrant of

eArray

Slide 40

Quote:Select this option to generate a

Quote and initiate the manufacture of this library

Download:Select this option to download the BED file (for

viewing of baits in the UCSC browser, for instance), or a TDT (tab delimited table) summarizing the

baits in this library

Create Library by Bait TilingDownload Library• The following window results from having selected Download:

Slide 41

1. Place a checkmark next to the file types desiredExample of a BED file

Example of TDT file

2. Click Download:If the files do not download, hold down the Ctrl key when clicking on Download and until the

window appears that requests whether you want to Save or Open the file

Create Library by Bait TilingProvide Quote Details

• The following window results from having selected Quote:

Slide 42

1. Determine the number of Libraries desired

2. Determine the number of reactions per library:A reaction size of 50 means that a single Library can

be used for capture with 50 DNA samples

3. Determine the sequencing technology and protocol

4. Click Next

Create Library by Bait TilingReview and Submit Quote

• Review Quote details, if satisfied, select Submit

Slide 43

Some Differences in Workflow for the

Non-Wizard Approach

Create a Bait GroupDesign Baits

• To create a Bait Group, go to the Baits page, and select Bait Tiling

• This will bring you to the page where you can define design parameters for a Bait Group

Slide 45

Click on Baits

Click on Bait Tiling

Workflow Summary

Slide 46

Create a Bait Group by tiling Baits across target

intervals

Examine Bait Group, determine which targets were avoided and how many baits were tiled

If desired, create additional Bait Groups, using additional target intervals or

alternate design parameters

Combine desired Bait Groups into a Library

(minimum of 1)

Save and Submit Library

Generate Quote

Create a Bait GroupDownload the Design (II)

There are four files included in the downloaded zip:

Slide 47

Example of a BED file:

Example of a TDT (tab delimited table) file:

Example of a Fate file:

Example of a Summary file:

Create Bait GroupCreate Bait Group

Slide 48

Once satisfied with the Bait Group Design, select Create Bait Group

Check the status of the Bait Group creation in the Pending

Jobs quadrant of the Home page

While waiting for the Bait Group to be created, design additional Bait Groups, if desired

Examine Bait GroupLook for Missed Targets• Open the BaitTiling_fate file in Excel

• Sort the file for Status to identify invalid (failed) genomic coordinates– These are targets for which the genomic coordinates could not be correctly assigned

• Sort the file for Baits Generated to identify targets for which no baits were assigned

– In this example, according to the BaitTiling_sum file (left): 8423 - 8081 = 342 targets were not assigned baits; these can be viewed on the right in the BaitTiling_fate file

Slide 49

Examine Bait GroupView Design in UCSC Browser• The BED file can be uploaded into the UCSC browser as an easy method to

visualize the probes tiled across targets of interest

Slide 50

Segment of chrX with baits tiled across exons

(target intervals)

Zoomed in view of baits

tiled across a single exon

Baits

Genes

RepeatMasker

Baits

Genes

RepeatMasker

Examine Bait GroupIdentify Targets for Potential Re-Tiling

Slide 51

Baits

Genes

RepeatMasker

1. Select targets not assigned baits in the

Fate file.

3. In this example, the exon on the right is covered by a section identified as "repeats" by the RepeatMasker track, and

design parameters had specified to avoid these regions. Therefore, this target received zero baits.

If the target is still desired, it is possible to create a new, additional Bait Group for this target with new design parameters that allow repeat regions. Both Bait

Groups can later be included in the final library.

2. Upload BED file to the UCSC browser, and zoom in on the target to identify the reason that it was provided

zero baits.

For more information:

• www.agilent.com/chem/eArray

• www.OpenGenomics.com/sureselect

• [email protected]

• 1-800-229-9770

http://www.opengenomics.com/sureselect

http://www.agilent.com/chem/eArray

Documents

Agilent Technologies SureSelect™ Target Enrichment Platform Providing focus for next-generation sequencing workflows David Willmot, PhD Sr. Applications