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

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Presentation on theme: "Agilent Technologies SureSelect™ Target Enrichment Platform Providing focus for next-generation sequencing workflows David Willmot, PhD Sr. Applications."— Presentation transcript:

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

2 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 Institute Dr. Chad Nusbaum et al. SureSelect DNA Capture Array (on array) Developed in collaboration with Cold Spring Harbor Dr. Greg Hannon et al. Agilent 60mer Array 1-5 µg gDNA 1-5 µg gDNA, or 20 µg gDNA

3 Enabling Products for the Next-Generation Sequencing Workflow Page 3 A Choice in Agilent Target Enrichment Options for Specific Project Needs SureSelect Target Enrichment System SureSelect DNA Capture Array (244k array) ThroughputHighLow Study Sizes10-1,000s samples1-10 samples DNA Input1-3 µg1-20 µg* Capture of Target DNA Mb Custom ~1 Mb Format Kit Array plus application note/protocol or

4 SureSelect ™ Target Enrichment System: Workflow Illumina GA Kit Available End of February

5 Enabling Products for the Next-Generation Sequencing Workflow Page 5 Agilent Online Custom Solutions Flexibility in Target Enrichment 244K eArray Target Enrichment Design DNA Capture Design Custom and Catalog Designs for CGH, Gene Expression, etc researcher Manufacturing Process Development Bioinformatics SureSelect DNA Capture Array SureSelect Target Enrichment System User Sequences

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

7 SureSelect™ Target Enrichment System: Design and Order Process 1. Design & Order 2. Kit Production 3. Single Tube Kit Delivery Select custom genome partitioning set using eArray or, select catalog 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.Buffers 3.Protocol Agilent genome partitioning kit shipped to customer

8 Quality Control on the Bioanalyzer Simplified Experimental Workflow Target Enrichment

9 Enabling Products for the Next-Generation Sequencing Workflow 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

10 Agilent’s Microarray Platform Reliable inkjet printing Sensitivity of probes Flexibility of microarrays Ease of implementation Probe Fidelity can synthesize up to 200mer

11 Enabling Products for the Next-Generation Sequencing Workflow Page 11 Agilent’s Strength in Ultra-long Oligo Synthesis 3) Deblock 1) Coupling 2) Oxidation Repeat n times Depurination side reaction Manufacturing Process Development Bioinformatics

12 Agilent SureSelect™ Platform Enabling Products for the Next-Generation Sequencing Workflow Page 12 SureSelect™ Target Enrichment System: Bringing Cost-Efficiency to Next-gen Sequencing Workflows

13 Agilent SureSelect™ Platform Enabling Products for the Next-Generation Sequencing Workflow Page 13 The Cost of DNA Sequence Information: Illumina GA How much of this is useful information? What is the cost of the useful information?

14 Agilent SureSelect™ Platform Enabling Products for the Next-Generation Sequencing Workflow Page 14 SureSelect™ Target Enrichment: Cost Benefit Substantial Cost Differential

15 Enabling Products for the Next-Generation Sequencing Workflow Page 15 SureSelect™ Target Enrichment System Using Illumina GA– Sample Data from an Exon Capture Experiment Target: 9,107 exons 3 + Mb Sample: 3 µg Genomic DNA Covaris Shearing Illumina library prep End-sequencing 35bp

16 Enabling Products for the Next-Generation Sequencing Workflow Page 16 Genomic Space View (Zoomed-in View of 7 Exons) 1 st exon covered by 102 tiled baits Read depth maxes out at >269x Average read depth is 100x 2 nd exon covered by 1 bait (because it was right next to a repeat region) Read depth ~ 10x 3 rd – 5 th exons covered by 2-4 baits Read depth ~ 40x

17 SureSelect™ Target Enrichment Reproducibility Agilent Confidential

18 Agilent SureSelect™ Platform Enabling Products for the Next-Generation Sequencing Workflow Page 18 SureSelect™ Target Enrichment System: Strong Allele Balance Indicates Negligible Bias Baits are designed to wildtype sequence Long oligos lead to unbiased SNP capture and calling

19 SureSelect™ Target Enrichment System: Efficient SNP Validation and Discovery SNP IDChrBasepair Reference (reads) SNP (reads) rs rs rs rs Novel rs Allelic balance  no bias Efficient identification of SNPs Illumina readout confirmed with CE Efficiently captures mutations

20 Efficient Capture of 5 bp deletion on the X-Chromosome: Menke’s Syndrome SureSelect™ Target Enrichment Kit Efficiently Captures 5 bp Mutant Readout on Illumina GA

21 Summary The Agilent SureSelect platform Flexible custom designs with Agilent ’ s eArray portal - free of charge. Efficiently captures mutations, with 1/10 th 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 usage  DNA input  Labor  Data handling For more information See Protocol:G options

22 A QuickStart Guide for the Creation and Ordering of SureSelect Target Enrichment Oligo Sets Using eArray, a free web-based design tool

23 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

24 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 Started Access eArray The Login Name is the address used to register

25 Confirm that the Application Type is TargetEnrichment Slide 25 Getting Started Confirm Application Type If necessary, select Switch Application Type to select Target Enrichment

26 Choose between two methods for creating a SureSelect Library Slide 26 Getting Started Select Method BenefitsCaveats 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

27 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

28 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 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 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. Create Library from Bait Upload: If you have already designed the baits that will recognize your desired target regions, use this option.

29 Slide 29 Create Library by Bait Tiling Step 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. 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. 6.Submit: Select Submit when Options and Details are completed.

30 Create Library by Bait Tiling Design Strategy Slide 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) 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. 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’. 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

31 (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 Group Centered versus Justified Justified Centered 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

32 Create Library by Bait Tiling Formats 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: |chrX: |chrX: |chrX: |chrX: 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 1: Type in the genomic intervals to be targeted. The format for typing in the intervals is as follows: chrX: |chrX: |chrX: |chrX: |chrX: 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. 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.

33 Create Library by Bait Tiling Upload 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 alert when the submission is complete Slide 33

34 Create Library by Bait Tiling View 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

35 Create Library by Bait Tiling View 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

36 Create Library by Bait Tiling Continue If satisfied with the creation of this Bait Group, return to the eArray Home page and select Continue Slide 36

37 Create Library by Bait Tiling Define 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 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. 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

38 Create Library by Bait Tiling Layout Baits Slide 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 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 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. 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.

39 Create Library by Bait Tiling Save 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

40 Create Library by Bait Tiling Final 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 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 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

41 Create Library by Bait Tiling Download Library The following window results from having selected Download: Slide 41 1.Place a checkmark next to the file types desired Example of a BED file Example of TDT file 1.Place a checkmark next to the file types desired Example 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 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

42 Create Library by Bait Tiling Provide Quote Details The following window results from having selected Quote: Slide 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 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

43 Create Library by Bait Tiling Review and Submit Quote Review Quote details, if satisfied, select Submit Slide 43

44 Some Differences in Workflow for the Non-Wizard Approach

45 Create a Bait Group Design 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

46 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

47 Create a Bait Group Download 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:

48 Create 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

49 Examine Bait Group Look 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): = 342 targets were not assigned baits; these can be viewed on the right in the BaitTiling_fate file Slide 49

50 Examine Bait Group View 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

51 Examine Bait Group Identify 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.

52 For more information:


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