How to use earray to create custom content for the SureSelect Target Enrichment platform. Page 1

Transcription

1 How to use earray to create custom content for the SureSelect Target Enrichment platform Page 1

2 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 2

3 earray Navigation Application Views- Users can select between applications, where each view shows only functionality for the current application Page 3

4 earray Navigation Content Bar- Provides access to functionality surrounding the basic concepts in earray Page 4

5 earray Navigation Search Allows you to search for Libraries, Baits, or Bait Groups in your account Page 5

6 earray Navigation Design Wizards Takes you through the most common scenarios for creating a custom library design, from start to finish Page 6

7 earray Navigation Pending Jobs Allows you to monitor your design progress Page 7

8 earray Navigation My Libraries where you can order or download your library contents Page 8

9 Important Considerations before designing your custom library What chromosomal regions do you want covered? Do you have a list of genomic coordinates? Is there a list of specific genes/ regions that you want covered? Do you only want exons? Would you like to include UTRs? If whole gene region including introns, you can use the Interval Finder function on earray If exons only, use Exon Interval Finder function on earray note that all annotated exons (including 5 and 3 UTRs) are included when this is used (see next slide) If not interested in UTRs, manually delete these regions from results of Exon Interval Finder. Alternatively, UTRless intervals could be obtained by manually going through regions on UCSC Do you want promoter regions? Look up relevant publications for identified promoter/enhancer regions and include this to your target region list Do you want to test for splicing mutations? To interrogate splice donor/acceptor sites, add 2bp at both ends of the exons To interrogate branch site mutations, add 10bp flanking sequence Do you want to specifically avoid regions? Note that during bait tiling, by default, earray avoids regions recognized by RepeatMasker and Tandem Repeats Finder. If there are additional regions that you want avoided in addition to this, you would have to specify these within the Avoid Custom Interval field Page 9

10 Exon Interval Finder Search Search by gene symbol, accession or cytoband List of species that earray supports Page 10

11 Interval Finder Search Use this search if you want to tile both exons and introns Page 11

12 Genomic Interval Search Search by genomic target intervals List of species earray supports Page 12

13 Bait Tiling Design 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. If using exon or interval finders, they show up automatically 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. Page 13

14 Bait Tiling Design Design Strategy 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. 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. 4. Bait Tiling Frequency: Options include 1X, 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. 1X Tiling 2X Tiling 3X Tiling 4X Tiling 5X Tiling Target Baits Slide 14

15 Bait Tiling Design Centered versus Justified Centered Justified (a) Target region is large (example of 2x tiling) target region baits 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 (eg, see circle where regions have both 2x and 3x tiling) (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 Page 15

16 Create a Bait Group When bait tiling is complete, click on View Design to view the result or Create Bait Group to save it in your workspace Page 17

17 Create a Bait Group Download the Design 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: Page 18

18 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. Page 19

19 Looking for regions with high GC using UCSC Genome Browser Look for regions of high GC (>65%) by using the GC Percent track on the UCSC Genome Browser. Create a separate target interval file for regions with high GC since both tiling frequency and number of replicates will be increased for these regions compared to others to allow for increased capture efficiency Once bait tiling is done for these regions, upload the BED file for the baits as a custom track to examine design more closely and verify that bait representation is sufficient Page 20

20 What to do if earray returns zero baits for your regions of interests Check if your genomic interval input format is correct In most cases, your missed interval(s) may fall into the repeat masked regions If the target is still desired, it is possible to create a new bait tiling design again but turn off Avoid Standard Repeat Masked Regions option BLAT your bait sequence to check for Homology is a good idea - Page 21

21 UCSC Genome Bioinformatics-BLAT Search Tool Page 22

22 BLAT Search Result Baits with high secondary homology score should be taken out of the design Keep the bait if homology score is between or less BAD Good Page 23

23 Create library When you have your bait group(s) ready, the next step is to create library Page 24

24 Create library Step 1: Select Species Select species from the drop-down window Design wizard contains 4-easy step to follow When complete, select Next Page 25

25 Create library Step 2: Define Library Provide a name for the library A control grid is always, and automatically, included in a library When complete, select Next Provide a description for the library: Here, it is possible to include a description, keywords, and comments to help characterize this library. Page 26

26 Create library Step 3: Layout Baits 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 If desired, add additional, premade 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. When completed, select Next Page 27

27 Create library Step 4: Create 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 Page 28

28 Request Quote Page 29

29 Request Quote: Library Design Information Page 30

30 For more information: options x3x4x2 Page 31