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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 Agilent’s SureSelect Platform: New Options
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, or 20 µg gDNA 1-5 µg gDNA So in 2009, Agilent will be releasing 2 new products that we believe take significant strides in addressing the workflow bottleneck of NGS. The Agilent On-Array method, developed in collaboration with Greg Hannon at CSHL, is an obvious product for Agilent since we already have the expertise in manufacturing high-quality 60mer arrays. In this method, genomic DNA is hybridized to custom arrays containing 60mer probes specific to the target regions. After a wash step to remove the nontargetted DNA, the hybridized regions are released and ready for sequencing. We will also be coming out with a completely novel method that does not use microarrays at all but continues to leverage our expertise in making high quality long oligos. For this method, while the oligos are manufactured on an array, we release the manufactured oligos, allowing the reaction to occur in-solution. This method was developed in collaboration with Chad Nusbaum’s group at the Broad Institute. Enabling Products for the Next-Generation Sequencing Workflow

3 A Choice in Agilent Target Enrichment Options for Specific Project Needs
SureSelect Target Enrichment System SureSelect DNA Capture Array (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 So we are providing 2 relatively different technologies but for a similar purpose, and each of these technologies has its own features that make it more suitable to certain study designs. The in-solution method will be very scalable, as it is performed in tubes and could be automated, while the on-array method is more difficult to scale to that degree. We are still optimizing the in-solution method, but currently it is feasible to process 100 samples at a time, with hands-on time to the customer of about 4 hours. In part because of the ease of throughput, but also because of the scalability of the product itself, we would recommend that projects with more than samples would be run using the in-solution method. As the sample #s grow, the array costs remain consistent, whereas the in-solution method can become cheaper per sample with more samples. We can make larger kit sizes, working in economy scale, and pass those savings on to the customer. On the other hand, for a project running only a few samples, it might remain more economical to take the on-array method because of the fewer steps it takes us to make those products. DNA input for the in-solution method is much lower than for the on-array method. While we’ve written 3 ug here, we’ve actually been successfully testing much lower amounts and can expect this number to decrease. The idea here is that on the array, you have to add a lot of DNA to drive the equilibrium such that most of the target is hybridized to the array. In solution, you can actually add a very large amount of the probes instead, driving the kinetics to completion but without requiring a high DNA input. Finally, the amount of DNA that you can capture is higher for the in-solution method. These are current estimates, but using the in-solution method, it is possible to combine the probes from multiple arrays for really high multiplexing or really large target regions, or of course there is flexibility and a small amount of DNA can be targeted for capture using either method. Human exome is 30Mb Enabling Products for the Next-Generation Sequencing Workflow

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

5 Agilent Online Custom Solutions Flexibility in Target Enrichment
Manufacturing Process Development Bioinformatics Agilent Online Custom Solutions Flexibility in Target Enrichment eArray Target Enrichment Design DNA Capture Design researcher Custom and Catalog Designs for CGH, Gene Expression, etc User Sequences In addition to the manufacturing benefits and the process development benefits that our GP products leverage, the benefit that will be most immediately obvious to users is the bioinformatics. Agilent has an infrastructure already well built for users to create custom oligo arrays to study the areas that most interest them, and this infrastructure is also being applied for ordering GP products. The researcher simply submits the sequences of their target regions into our online eArray site. We are extending eArray functionality to include design features for both custom capture arrays and custom in-solution kits. As I’ve mentioned, the submitted design is immediately sent to the manufacturing facility, which then produces either the array or the tube for quick delivery. Although this is a custom product, there is no setup fee since we don’t care what we’re printing on our arrays, we can print any sequence as easily as the next, and there is no major delay in receiving the product because, again, it’s just as easy for us to make a custom array as it is to make a catalog array. 244K SureSelect DNA Capture Array SureSelect Target Enrichment System Enabling Products for the Next-Generation Sequencing Workflow

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

7 3. Single Tube Kit Delivery
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 55K unique 120 mer oligos synthesized on one wafer Agilent genome partitioning kit shipped to customer Oligos released eArray Web Portal Kit Includes Biotinylated-cRNA Buffers Protocol or, select catalog oligo set Oligo IVT to RNA-biotin

8 Quality Control on the Bioanalyzer
Simplified Experimental Workflow Target Enrichment

9 Santa Clara Manufacturing Facility
Manufacturing Process Development Bioinformatics 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 Here’s a view of our manufacturing facility. The boxes here are the printers, and here is the inkjet which is printing either an A, a G, a C, or a T nucleotide, or the blocking reagent. The manufacturing facility is linked directly to our online custom array orders site, so that as soon as a customer finishes selecting the design and hits submit, the information is immediately sent to this facility so that the arrays or kit can be manufactured. Enabling Products for the Next-Generation Sequencing Workflow

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 Agilent’s Strength in Ultra-long Oligo Synthesis
Manufacturing Process Development Bioinformatics 1) Coupling Depurination side reaction 20 40 60 80 100 50 150 200 Oligo Length (mer) Full Length (%) 98% cycle yield, no depurination 99.5% cycle yield, no depurination 98% cycle yield, with depurination 99.5% cycle yield, with depurination Agilent Conventional Processes 3) Deblock 2) Oxidation Repeat n times N1 O P RO N2 Ni HO Our process development has allowed us to move from making high quality 60mers, to being able to create high quality 200mers in a process we call Oligo Library Synthesis, or OLS. The synthesis follows 3 main steps, there is the coupling step in which the nucleotide is added first to the glass slide and then to the growing oligo. Then there is the oxidation step where the phosphate is converted from P3 to P5 to prepare for the next reaction. Finally, there is a deblocking step to prep the oligo for the next nucleotide. This deblocking step, which is used in conventional methods for making long oligos as well, is known to cause some damage to the A’s in the growing oligo, but we’ve found a method to avoid this depurination step. We’ve also found that we can make the cycle yield increase by some additional modifications. When looking at the efficiency at creating long oligos, you can see that conventional processes where the cycle yield is about 98% and depurination is occurring, its very difficult to create oligos up to 100 nt in length. We found that the various steps we’ve developed to better this process work additively so that we’re now able to make long oligos far more effectively than any of the other options available. Enabling Products for the Next-Generation Sequencing Workflow

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

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

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

15 SureSelect™ Target Enrichment System Using Illumina GA– Sample Data from an Exon Capture Experiment
3 µg Genomic DNA Covaris Shearing Illumina library prep End-sequencing 35bp Target: 9,107 exons 3+ Mb Using the same target as described in the previous slide, here is an example of some results. Left graph: note that there are 2 Y-axes. The graph shows depth distribution across various read depths. The bars match the axis on the left (% per bin, or per read depth), the line represents the axis on the right (cumulative %). Enabling Products for the Next-Generation Sequencing Workflow

16 Genomic Space View (Zoomed-in View of 7 Exons)
1st exon covered by 102 tiled baits Read depth maxes out at >269x Average 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 baits Read depth ~ 40x Again, same sample as previous slide. Here you can see the targeted enrichment of 7 exons. The exons are indicated by the blue RefSeq bar. Above that is the GC percent, which doesn’t seem to affect Dna capture here. Above are bloack boxes indicating locations of the probes in the oligo library. The graph shows read depth in brown. With only a few oligos to capture the smaller exons, the read depth was still about 40, in comparison to 0 in the adjacent intronic regions. In the large exon on the left, with a large # of oligos for capture, the read depths peaked at (checking on the #). Enabling Products for the Next-Generation Sequencing Workflow

17 SureSelect™ Target Enrichment Reproducibility
Correlation of read depth across target intervals for technical replicate SureSelect captures. Replicate captures were performed on a SureSelect Target Enrichment library designed against exonic targets covering 3.3Mb with 50% probe overlap (ensuring coverage of most bases by 2 separate oligonucleotide baits). 4a) Average read depth of mapped Illumina sequencing reads across each of target intervals (median length 300 bp) for sample 1 (X axis) and sample 2 (Y axis); R2 value =0.96; 4b) Normalized read depth (average read depth normalized for the number of sequence reads) of sample 1 (X axis) vs. the ratio of normalized read depths for sample 1 and sample 2 (Y axis). Horizontal lines denote mean and standard deviation. Agilent Confidential

18 SureSelect™ Target Enrichment System: Strong Allele Balance Indicates Negligible Bias
In heterozygous SNP calls, the number of reference bases vs. non-reference bases. SNPs were called using the MAQ software and filtered for quality (minimum consensus quality = 20, maximum mapping quality = 40) and number of reads (minimum read depth = 15). Looked at 2000 SNPs and sequenced after targeting. Showed allele balance. Bait is wildtype but captured equally well the SNPs. Baits are designed to wildtype sequence Long oligos lead to unbiased SNP capture and calling Agilent SureSelect™ Platform Enabling Products for the Next-Generation Sequencing Workflow Page 18 18

19 SureSelect™ Target Enrichment System: Efficient SNP Validation and Discovery
SNP ID Chr Basepair Reference (reads) SNP (reads) rs 1  47  42 rs  52  40 rs831043 2 44 52 rs 5  41 Novel 9  36 rs944682  0  91 Allelic balance  no bias Efficient identification of SNPs Illumina readout confirmed with CE Efficiently captures mutations Sanger sequencing confirmation of example single nucleotide polymorphisms found in a SureSelect Target Enrichment captured genomic DNA sample. 7A) Shown is a summary of Illumina sequencing results for six SNPs that reflect differences between the sample (NA15510) and the reference genome. Included are the number of calls for each nucleotide in each direction (+ or -) . 7b-f) trace files from sanger sequencing of PCR products (AB3730). SNPS are highlighted in blue. Base and SNP identity calls are shown below the respective trace files.

20 SureSelect™ Target Enrichment Kit Efficiently Captures 5 bp Mutant
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 Sequencing results from captures of a 5BP deletion on the X-Chromosome: Menke’s Syndrome Menke’s syndrome is a copper transport disease associated with loss of function of the ATP7A gene . We performed captures using a Sureselect library specific for the nonrepetitive regions of the X chromosome (designed to the reference genome) on DNA derived from a Menke’s syndrome donor. This XY donor had a confirmed 5bp deletion at nucleotide 803 in exon 4 of the ATP7A (MNK) gene on the X chromosome (803_807delATCTC) resulting in a frameshift mutation after Leu The sureselect library contains two probes spanning the deletion region. Shown is an alignment of sequence reads spanning the genomic region of the reference genome corresponding to a segment of the X chromosome which encocdes the ATP7A gene. Top: reference genome with known 5BP deletion in red. Bottom, alignments of 10 Illumina single –end sequence reads to the reference genome. These data clearly demonstrate the potential for capture of small deletions with Sureselect.

21 Summary The 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 usage DNA input Labor Data handling For more information See Protocol: G Contact options 2x2x1

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

23 Summary of Steps Getting Started Create Library by Bait Tiling
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 Getting Started Access eArray
Access eArray at: Log in to eArray, if this is your first time visiting, click Request for Registration The Login Name is the address used to register Slide 24 24

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

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

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 27

28 Initiate the Wizard Select the method for Library creation in the Library Wizards quadrant, and click Next 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 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 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. Slide 28 28

29 1. Enter a name for the design job. 5. Genomic Avoid Intervals:
Create Library by Bait Tiling Step 1: Library Options and Target Details 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. 1. Enter a name for the design job. 6.Submit: Select Submit when Options and Details are completed. 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. Slide 29 29

30 Create Library by Bait Tiling Design Strategy
2. Centered versus Justified: (see next slide for visuals) 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. 1. Change the parameters: To be able to change these parameters, first remove the checkbox from the “Use Optimized Parameters” option. 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’. 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. 2X Tiling: 3X Tiling: 4X Tiling: 5X Tiling: Target Baits Slide 30 30

31 Create a Bait Group Centered versus Justified
(a) Target region is large (example of 2x tiling) 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) target region baits (b) Target region is 2 times the bait length  Design is the same for Centered and Justified (c) Target region is shorter than the bait length  Design is the same for Centered and Justified 31

32 Create Library by Bait Tiling Formats for describing Target Intervals
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 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. Slide 32 32

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 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 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 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 36

37 Create Library by Bait Tiling Define Library
A control grid is always, and automatically, included in a library 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 Slide 37 37

38 Create Library by Bait Tiling Layout Baits
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 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 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. 5. When completed, select Next Slide 38 38

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 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 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 Slide 40 40

41 Create Library by Bait Tiling Download Library
The following window results from having selected Download: 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 Slide 41 41

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

43 Create Library by Bait Tiling Review and Submit Quote
Review Quote details, if satisfied, select Submit Slide 43 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 Click on Baits Click on Bait Tiling Slide 45 45

46 Workflow Summary Create a Bait Group by tiling Baits across target intervals Examine Bait Group, determine which targets were avoided and how many baits were tiled Combine desired Bait Groups into a Library (minimum of 1) Save and Submit Library If desired, create additional Bait Groups, using additional target intervals or alternate design parameters Generate Quote Slide 46 46

47 Create a Bait Group Download the Design (II)
There are four files included in the downloaded zip: Example of a Fate file: Example of a BED file: Example of a Summary file: Example of a TDT (tab delimited table) file: Slide 47 47

48 Create Bait Group Create Bait Group
Check the status of the Bait Group creation in the Pending Jobs quadrant of the Home page Once satisfied with the Bait Group Design, select Create Bait Group While waiting for the Bait Group to be created, design additional Bait Groups, if desired Slide 48 48

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 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 Segment of chrX with baits tiled across exons (target intervals) Baits Genes RepeatMasker Zoomed in view of baits tiled across a single exon Baits Genes RepeatMasker Slide 50 50

51 Examine Bait Group Identify Targets for Potential Re-Tiling
1. Select targets not assigned baits in the Fate file. 2. Upload BED file to the UCSC browser, and zoom in on the target to identify the reason that it was provided zero baits. 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. Baits Genes RepeatMasker 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. Slide 51 51

52 For more information:

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