SCICLONE SURESELECT XT NGS WORKSTATION USER S GUIDE: AGILENT TARGET ENRICHMENT ON THE

Transcription

1 USER S GUIDE: AGILENT SURESELECT XT TARGET ENRICHMENT ON THE SCICLONE NGS WORKSTATION Agilent SureSelect XT Target Enrichment System for Illumina Paired-End Sequencing Libraries with Multiplexing (Protocol v1.2 May 2011) Sciclone NGS Workstation with Maestro 6.0 Sciclone NGSx Workstation with Maestro 6.2

2 Table of Contents Introduction 3 Maestro SureSelect XT Overview 3-6 Required Materials and Reagents 7-8 Reagents 7 General Laboratory Equipment and Supplies 7 Sciclone NGS Accessories 7 Consumables 8 Running the Maestro SureSelect XT Workflow 8 Sample Preparation 8 SureSelectXT Initial SPRI Cleanup Application (Day 1) 8 Quality Control of Initial SPRI Samples (Optional) 10 SureSelectXT Library Prep Application (Day 2) 10 Quality Control and Quantification of Amplified Libraries 13 SureSelectXT Pre-Capture Normalization Application (Day 3) 14 SureSelectXT Hyb Setup Application (Day 3) 15 SureSelectXT Target Selection Application (Day 4) 17 Quality Control and Quantification of Captured Libraries 20 Appendix A: Step-by-Step Guide to the SureSelectXT Initial SPRI Cleanup Application 21 Appendix B: Step-by-Step Guide to the SureSelectXT Library Prep Application 21 Appendix C: Step-by-Step Guide to the SureSelectXT Normalization Application 22 Appendix D: Step-by-Step Guide to the SureSelectXT Hyb Setup Application 22 Appendix E: Step-by-Step Guide to the SureSelectXT Target Selection Application 22 2

3 Introduction Preparation of DNA samples for cluster generation and sequencing on the Illumina platform requires a series of manipulations to efficiently ligate appropriate indexed adapters onto DNA fragments to produce paired-end libraries. Selection of specific regions of the genome for sequencing requires additional steps including hybridization with capture probes, isolation of captured sequences, and enrichment of captured libraries. Automating the process has the advantage of avoiding sample tracking errors and reducing sample-tosample variability while dramatically increasing throughput. The Maestro-based SureSelect XT Workflow from PerkinElmer provides a pre-programmed solution for the SureSelect XT Target Enrichment System on the PerkinElmer Sciclone NGS Workstation. Overview of the Maestro Workflow The Maestro Workflow for SureSelect XT sample preparation is a validated process for library preparation from fragmented DNA samples that follows the steps outlined in Figure 1. Samples are processed in 96-well PCR plates, and the number of samples to process (1 to 12 columns of 8 samples each) is selected at the start of each run. Pre-set tip-tracking utilities written into the Maestro applications guide the instrument to pick up appropriate numbers of tips, and refill/replace tip boxes as needed. INHECO temperature blocks installed on the deck of the Sciclone NGS Workstation allow for appropriate 4 C or heated storage of reagents and controlled incubation temperatures for reactions and wash steps. Reaction mixes are pre-arrayed prior to addition to sample to ensure equal incubation times across the sample plate. Easy-to-follow user interfaces guide the reagent and deck setup process and prompt the user for any necessary interventions. Figure 1. Overview of the Maestro Workflow for the SureSelect XT protocol. 3

4 The Maestro Workflow for the SureSelect XT DNA Sample Preparation Consists of 5 Independent Maestro Applications Run Time (Including Run Time Set-up Setup Thermocycler (including Maestro Application Time thermocycler Maestro Application Time steps) Steps) 1 SureSelectXT Initial SPRI Cleanup SureSelectXT Initial SPRI Cleanup 10 min 10 min 1 hour 1 hour 2 SureSelectXT Library Prep SureSelectXT Library Prep 1 hour 1 hour 6 hours 6 hours 3 SureSelectXT PreCapture Normalization 10 min SureSelectXT PreCapture Normalization 10 min up to 1.5 hours up to 1.5 hours SureSelectXT 4 SureSelectXT Hyb Hyb Setup Setup min min 15 min 15 min SureSelectXT 5 SureSelectXT Target Target Selection Selection min min 7 hours 7 hours In the SureSelectXT Initial SPRI Cleanup, 130 µl sheared DNA samples are split into two aliquots for the AMPure XP bead cleanup to accommodate the necessary sample/bead volumes in standard Hard-Shell PCR plates. After elution in 15 µl each, the split samples are pooled back together for a total sample volume of 30 µl. The SureSelectXT Initial SPRI Cleanup application processes a maximum of 48 samples (6 columns); it is implemented twice if more than 48 samples are being processed. In the SureSelectXT Library Prep procedure, the volume of the End Repair reaction is reduced from the Agilent - recommended volume of 100 µl. Instead, the End Repair reaction is set up in a total volume of 50 µl (30 µl sample plus 20 µl End-Repair Mix) to allow for post End-Repair SPRI Cleanup (with 90 µl AMPure XP beads) in a Bio- Rad 96-well PCR plate. A single aliquot of AMPure XP beads is used per sample for SPRI Cleanups following the End-Repair, A-Tailing, and Ligation steps. The DNA in the sample is driven on and off the beads via changes in PEG and NaCl concentrations. This strategy increases yield by limiting the number of times samples are transferred to new wells/plates. For the library prep, use of Elution Buffer (Qiagen EB), rather than water, is recommended for all pre-pcr elution steps to ensure proper buffering during resuspension. When the post-ligation SPRI Cleanup is complete, the samples are split into two aliquots: 15 µl is added directly to PCR master mix for enrichment and further processing and 15 µl is transferred to a clean PCR plate and stored at -20 C for future use. A Post-PCR SPRI Cleanup step is integrated into the SureSelectXT Library Prep application. This step requires fresh AMPure XP beads and elution in water rather than EB. The full library preparation (End-Repair through Post-PCR SPRI Cleanup) can be completed for up to 96 samples in about 6 hours. The quality, size distribution, and concentration of library in each sample must be checked prior to continuing to the next steps. In the SureSelectXT PreCapture Normalization procedure, samples are diluted with water to a concentration of 18.9 ng/µl in 26.4 µl (total 500 ng DNA). This is undertaken one sample at a time and requires up to 1.5 hours for a 96 sample plate. The Maestro application simplifies sample tracking by importing concentration data directly from a Microsoft Excel spreadsheet and automatically calculating the proper sample and water volumes for each well. The excess volume in the normalized sample is reduced appropriately by evaporation during the high temperature denaturing step of hybridization setup. For SureSelectXT Hyb Setup, hybridization buffer and capture library are mixed by the user at room temperature, then arrayed into a 96-well plate and heated to 65 C on the Sciclone. Samples are mixed with blocking solution, denatured on the deck at 95 C, then transferred to the 65 C plate containing the capture library. The user is required to promptly seal and transfer the plate to a nearby thermocycler for the overnight incubation. In the SureSelectXT Target Selection application, wash steps are carried out in 96-well PCR plates with 150 µl wash buffer per well, rather than the Agilent recommended 200 and 500 µl per sample. Additional wash cycles are used to compensate for reduced wash volumes. At the completion of the wash steps, samples (with streptavidin beads) are resuspended in 50 µl 10 mm Tris ph 8.0. The SureSelect XT Elution Buffer and SureSelect XT Neutralization Buffer are not used. Half the resuspended beads/sample (25 µl) is transferred to a plate containing PCR Master Mix for PCR enrichment followed by a post-pcr SPRI Cleanup. The remaining 25 µl resuspended beads/sample (25 µl) is stored at -20 C for future use. The SureSelectXT Target Selection application, including the PCR and Post-PCR SPRI Cleanup steps, can process up to 96 libraries in 7 hours. 4

5 Flowcharts for Individual Steps of the Maestro Workflow for the SureSelect XT Protocol Figure 2. SureSelectXT Initial SPRI Cleanup Application (Day 1). Figure 3. SureSelectXT Library Prep Application (Day 2). 5

6 Flowcharts for Individual Steps of the Maestro Workflow of the SureSelect XT Workflow Figure 4. SureSelectXT Pre-Capture Normalization Application (Day 3). Figure 5. SureSelectXT Hyb Setup Application (Daay 3). Figure 6. SureSelectXT Target Selection Application (Day 4). 6

7 Required Materials and Reagents Reagents Reagent Sciclone NGS Workstation Accessories Accessory Vendor and Part No. Agilent SureSelect XT Human All Exon Kit or Agilent SureSelectXT Custom Target Enrichment System Kit Agilent Herculase II Fusion DNA Polymerase* Agilent Dynabeads MyOne Streptavidin T1 Nuclease-Free Water Ampure XP Beads (200 rxn) (400 rxn) Invitrogen (2 ml) (10 ml) (100 ml) PerkinElmer Part No. Agencourt 96-ring Magnet (2) CLS Spacer Assembly for Agencourt 96-ring Magnet CLS (qty 4) and CLS (1) INHECO Deepwell plate adapter (Corning 1 ml) CLS INHECO Deepwell plate adapter (12-row Pyramid) CLS INHECO 384-well plate adapter CLS INHECO 96-well adapters (3) CLS INHECO 96-well adapter/shaker CLS Various Beckman Coulter A63880 (5 ml) A63881 (60 ml) A63882 (450 ml) 100% EtOH Sigma E % PEG 8000 Sigma M NaCl Sigma S mm Tris ph 8.0 Various Elution Buffer (EB) Qiagen General Laboratory Equipment and Supplies Equipment Microfuge Vortexer Supplier 200 µl Multichannel pipettor and appropriate barrier tips Various 1000 µl Multichannel pipettor and appropriate barrier tips Various Covaris S2 or E210 System and appropriate tubes Microplate centrifuge Thermocycler** Various Various Various Various Bio-Rad Laboratories (MJ Research) DNA Engine PTC-200, or equivalent MicroAmp Clear Adhesive Plate Seals Applied Biosystems LabChip GX or Agilent Bioanalyzer with appropriate chips and reagents ** The standard sample plates used in this application are fully skirted Bio-Rad Hard-Shell 96-PCR plates. Please check if your thermocycler is compatible with this plate type. If it is not, please contact your PerkinElmer Field Application Scientist to discuss modifications to the application to support semi-skirted PCR plates. Various 7

8 Consumables PerkinElmer No. Used Consumable Description Part No. Vendor and Part No. per Run PCR Plates 96-well PCR Plate, Bio-Rad Hard-Shell, Full Skirt CLS Bio-Rad HSP Boxed Tips Pipette Tip 150 µl, Art, Box, Sterile Racks CLS PerkinElmer 58 (96 samples) Deepwell Plates Deepwell-96 POS, Square 2.0 ml Well, Polypro, CLS Seahorse Bioscience Seahorse Bioscience Reservoir- 12-Column Pyramid Bottom, 290 ml CLS Seahorse Bioscience Deepwell Seahorse Bioscience Lids 946 Lid Universal, Robotic Friendly, Polystyrene CLS Seahorse Bioscience well Plates Microplate 384-well, Round Bottom, CLS Corning, Inc Polypropylene (Pkg. 10) Deepwell Plates Microplate-96 well, 1 ml Well Volume, Polypropylene CLS Corning, Inc (1 ml) Corning Running the Maestro SureSelect XT Workflow Please read and familiarize yourself with all steps described in this section prior to beginning the run. A 4-day process is suggested for running the full set of Maestro SureSelect XT applications, but the process may be modified according to the laboratory schedule. Approximate times for setting up and running each application are indicated in the chart on Page 4. Be sure to plan properly for the 24 hour hybridization step to allow enough time to complete the subsequent Target Selection application during the following working day. SureSelectXT Initial SPRI Cleanup application (Day 1) This application serves to concentrate the fragmented DNA in order to reduce the volume of sample presented for the library prep application. Because each sample is split into two aliquots, the maximum sample number for each run of the SureSelectXT Initial SPRI Cleanup application is 48 samples. All subsequent applications in the workflow can process up to 96 samples. Sample preparation Genomic DNA should be fragmented to an average size of bp on a Covaris S2 or E210 instrument as described in the Agilent SureSelect XT Target Enrichment System for Illumina Paired-Ended Sequencing Library with Multiplexing Protocol v1.2 May Samples should be presented for the Maestro SureSelectXT Initial SPRI Cleanup run as up to 3 µg sheared genomic DNA in 130 µl TE in a Bio-Rad Hard-Shell 96-well PCR plate. Note: AMPure XP beads should be warmed at room temperature for about 30 min before use. They may be taken out of 4 C storage before beginning. If necessary, boot up the system by first starting the Sciclone NGS Workstation and the INHECO units, then starting the PC controller. 1. Modify the SS Initial SPRI worksheet in the SureSelectXT Workbook to specify the number of samples to run The workbook must be located in the filepath: C:\Program Data\CaliperLS\Maestro\Workbooks and must have the name SureSelectXT Workbook.xls. If the file is moved or the name is changed, Maestro will not be able to find the information necessary to begin the run. 8

9 The sample number must be set by indicating the number of columns to run in the worksheet titled SS Initial SPRI. The Maestro application only processes full columns of 8 samples each. 3. Prepare sample plate Up to 48 samples may be processed (as sets of 8 samples per column) in the SureSelectXT Initial SPRI application. Transfer 130 µl sheared DNA sample (containing up to 3 µg) to a Bio-Rad 96-well PCR plate. Fill columns 1-6 in order from left to right. Inspect the plate to ensure that air has not been trapped in the wells. If necessary, spin briefly to bring liquid to the bottom of the wells. If >48 samples are to be processed, prepare a second plate containing up to 48 additional samples. After processing the first set of 48 samples, all steps of the SureSelectXT Initial SPRI application must be repeated with the second plate. 4. Prepare reagents for the run Figure 7. SS Initial SPRI worksheet. After modifying the entry for the number of columns to process, the spreadsheet will update the appropriate volumes/ wells to fill on the reagent plates. Save the modified spreadsheet with its original name in its original file path. Print the SS Initial SPRI spreadsheet to use as a guide while setting up reagents. 2. Start the Maestro SureSelectXT Initial SPRI Cleanup run Launch the Maestro software and open the SureSelectXT Initial SPRI Cleanup Application. Ensure that the INHECO units have the correct adapters for the run: Position A3 Position A4 Position D2 Position D4 96-well Plate Adapter 96-well Plate Adapter 96-well Plate Adapter 96-well Plate Shaker Adapter Start the run by selecting the play button. If running in Edit mode, be sure to start the Main Method. Verify that the INHECO units for position A4 is set to 4 C and is cooling. Verify that the INHECO units for positions A3, D2, and D4 are set to 22 C. Note: when the run is started, the instrument will complete all initialization steps for the hardware and the specific application. The run will automatically pause and prompt the user to set up the deck and confirm proper setup prior to beginning the SPRI Cleanup steps. Ensure AMPure XP beads are at room temperature and thoroughly resuspended in storage solution. Using a multichannel pipettor, aliquot 90 µl AMPure XP beads per well into a Bio-Rad Hard-Shell PCR plate as shown in the SS Initial SPRI spreadsheet. Inspect the plate to ensure that air has not been trapped in the wells. If necessary, spin gently to bring liquid to the bottom of the wells. Store the bead plate at room temperature. Note: Two columns of AMPure XP beads will be used for each column of samples to be processed in the run. Make 50 ml fresh 80% EtOH solution by diluting 40 ml 100% Ethanol with 10 ml nuclease-free molecular biology grade water. Pour into a Seahorse Deepwell reservoir, cover with lid, and store at room temperature. Pour 20 ml Qiagen Elution Buffer (EB), or 10 mm Tris ph 8.0 into a Seahorse Deepwell reservoir and store at room temperature. If running a limited number of samples, a 12-column Seahorse Deepwell reservoir may be used instead. For every column to be run, fill 2 columns of the reservoir with 3 ml EB. 5. Set up the Sciclone NGS Workstation deck Confirm that the Maestro Sciclone NGS Workstation software has correctly read the workbook and is set to run the correct number of columns. Step through the pictures, placing the indicated consumables/prepared plates in the indicated locations. Take care to note whether or not a lid is needed at each location. Place new tip boxes in the indicated locations. 9

10 6. Run the SureSelectXT Initial SPRI Cleanup Steps Confirm that the deck setup matches the final picture in the setup window. Selecting Finished will prompt the application to begin the run. The Maestro SureSelectXT Initial SPRI application will automatically proceed through the steps indicated in the flowchart on Page 5 and Appendix A. While the application is running, the green light at the top of the instrument will blink. If there is a problem with the run, the light will change to yellow and an alarm will sound to indicate that user intervention is necessary. The application may be set up so that an is sent if an error occurs or when additional tip boxes are needed on the deck. After the final elution of sample from beads, the split samples will be re-pooled into single wells with a total volume of 30 µl. If properly sheared and size selected with the Initial SPRI application, the DNA samples will contain a majority of fragments in the bp (±10%) range. Note that the Initial SPRI Step will select against fragments smaller than 100 bp, so it is likely that the profile of the smear will shift as shown below. 7. Application complete When the Initial SPRI is complete, the application will pause and show a message indicating that the sample plate should be removed from the deck. The samples may be stored at -20 C, checked for quality/concentration, or passed immediately into Library Preparation steps. If processing > 48 samples through the SureSelect XT procedure, repeat steps 1-7 with the second plate of samples. Quality control of Initial SPRI samples (optional) For high throughput projects, a LabChip GX Automated Electrophoresis System is recommended for assessing sample quality and concentration. Samples, diluted into water in a 96-well PCR plate, may be analyzed sequentially by the LabChip GX System in a single run at a rate of about 1 sample per minute. To set up the LabChip GX System, follow the instructions in the LabChip GX System user guides, making sure to allow 30 minutes for the chip and reagents to equilibrate to room temperature prior to preparing the chip. DNA 1K assay: dilute sample 1:10 in a Bio-Rad 96-well PCR plate. DNA High Sensitivity assay: dilute sample 1:25 in a Bio-Rad 96-well PCR plate. Ensure the diluted sample is well mixed by pipetting up and down at least 3 times. Centrifuge the plate at 1000 xg for 1 min to eliminate bubbles and pellet any beads prior to beginning the LabChip GX System run. For lower throughput projects, the Agilent Bioanalyzer can be used in place of the LabChip GX System. Use the Agilent Bioanalyzer DNA 1000 kit for sheared DNA QC. Allow 30 minutes for reagents to equilibrate to room temperature, and prepare the chip according to the Agilent protocol. Figure 8. Representative data tracings showing LabChipGX analysis of 1:25 dilutions of a sheared total human DNA sample before (blue) and after (red) the Initial SPRI Cleanup Step. SureSelectXT Library Prep Application (Day 2) Please read and familiarize yourself with all steps described in this section prior to beginning the run. The Library Preparation application includes a thermocycler step and a post-pcr SPRI Cleanup. It is recommended to run the entire application prior to storing samples. If time allows, quantification of the library may be carried out on the same day as the library prep. Sample preparation Genomic DNA should be fragmented to an average size of bp on a Covaris S2 or E210 instrument as described in the Agilent SureSelect XT Target Enrichment System for Illumina Paired-Ended Sequencing libraries with multiplexing v1.2 May Samples should be purified and concentrated with the Maestro SureSelectXT Initial SPRI Cleanup application (or manual cleanup according to the Agilent protocol) and presented in 30 µl EB Buffer or 10 mm Tris ph 8.0 in a Bio-Rad 96-well PCR plate for the Maestro SureSelectXT Library Prep run. Note: AMPure XP beads should be warmed at room temperature for about 30 min before use. They may be taken out of 4 C storage before beginning. Do not thaw SureSelect XT reagents until steps 1 and 2 have been completed. If necessary, boot up the system by first starting the Sciclone NGS Workstation and the INHECO units, then starting the PC controller. 10

11 1. Modify the SS XT Lib Prep spreadsheet in the SureSelectXT Workbook to specify the number of samples to run The workbook must be located in the filepath: C:\ProgramData\CaliperLS\Maestro\Workbooks and must have the name SureSelectXT Workbook. If the file is moved or the name is changed, Maestro will not be able to find the information necessary to begin the run. The sample number must be set by indicating the number of columns to run in the worksheet titled SS XT Lib Prep. The Maestro application only processes full columns of 8 samples each. Figure 9. SS XT Lib Prep worksheet. After modifying entries for the number of columns to process, the spreadsheet will update the recipes for the reagent mixes and the appropriate volumes for the reagent plates. Save the modified spreadsheet with its original name in its original file path. Print the SS XT Lib Prep spreadsheet to use as a guide while setting up reagents. 2. Start the Maestro SureSelectXT Library Prep run Launch the Maestro software and open the SureSelectXT Library Prep Application. Ensure that the INHECO units have the correct adapters for the run: Position A3 Position A4 Position D2 Position D4 96-well PCR Plate Adapter 384-well Plate Adapter 96-well PCR Plate Adapter 96-well PCR Plate Shaker Adapter Start the run by selecting the play button. If running in Edit mode, be sure to start the Main Method. Verify that the INHECO units for positions A3 and A4 are set to 4 C and are cooling. Verify that the INHECO units for positions D2 is set to 22 C and D4 is set to 20 C. Note: when the run is started, the instrument will complete all initialization steps for the hardware and the specific application. The run will automatically pause and prompt the user to set up the deck and confirm proper setup prior to beginning the library preparation steps. Starting the application prior to thawing and diluting reagents ensures that the cold blocks are pre-chilled and ready for on-deck reagent storage. 3. Thaw the SureSelect XT library preparation reagents and place on ice Using the SS XT Lib Prep spreadsheet as a guide, thaw the reagents from the SureSelect XT Library Preparation Kit necessary to make the End Repair Mix, A-Tailing Mix, and Adapter Ligation Mix. Do not thaw the reagents for the PCR Master Mix at this time. Note: care should be taken to avoid freeze-thaw cycles with all SureSelect XT reagents. Reagents should be aliquoted appropriately if planning to use a kit for more than 2 independent runs. 4. Prepare the PEG, Elution Buffer, AMPure XP bead plates, and 80% EtOH reservoir Make fresh 20% PEG/2.5 M NaCl solution according to the recipe in the SS XT Library Prep spreadsheet. Using a multichannel pipettor, aliquot 190 µl 20% PEG/2.5 M NaCl solution per well into a Bio-Rad 96-well PCR plate for each column of samples to be run. Cover the plate with a lid and store at room temperature. The Elution Buffer (EB Buffer from Qiagen ), should be placed in a Seahorse Deepwell 12-column reservoir. Starting with column 1, add 3 ml buffer for each column of samples to be run. For higher throughput runs, an open Seahorse Deepwell reservoir with 50 ml Elution Buffer may be used instead. Note: water is not recommended for elution during the library preparation steps, as the lack of buffering can interfere with proper performance. Water is only used as elution buffer in the Post-PCR SPRI Cleanup just prior to sample normalization and hyb setup. Maestro will prompt the user to exchange the Elution Buffer for water prior to starting the Post-PCR SPRI Cleanup step. 11

12 Thoroughly resuspend AMPure XP beads (warmed to room temperature) by inverting/rotating the bottle. Using a multichannel pipettor, aliquot 95 µl AMPure XP beads per well into a Bio-Rad 96-well PCR plate for each column of samples to be run (see SS XT Library Prep spreadsheet). Store at room temperature. Note: a second plate of AMPure XP beads, with 90 µl beads per well, will be necessary for the Post-PCR SPRI Cleanup step at the end of Library Prep. AMPure XP beads for this plate may be left at room temperature at this time and aliquoted into a Bio-Rad 96-well PCR plate during the amplification step. Make 100 ml fresh 80% EtOH solution by diluting 80 ml 100% Ethanol with 20 ml nuclease-free molecular biology grade water. Pour into a Seahorse Deepwell reservoir, cover with a lid and store at room temperature. 5. Aliquot the adapter mixes Transfer the appropriate volumes of adapter mix into the Adapter Oligo Plate as indicated in the SS XT Library Prep spreadsheet. Cover the plate with a lid and store on ice or at 4 C. 6. Make the reaction mixes and aliquot into the Master Mix Plate The following mixes should be prepared at this step according to the recipes on the SS XT Library Prep spreadsheet: End-Repair Mix, A-Tailing Mix, Ligation Mix. Care should be taken to pipet accurately, as the reaction mixes have minimal overage volumes. Keep the reaction mixes on ice. Note: to avoid loss of polymerase activity, the PCR Mix is not prepared at this time. The application will prompt for placement of the PCR mix on the deck at the appropriate time during the run. Prepare the Master Mix plate as specified on the spreadsheet. Keep the plate on ice, pipette carefully into the bottom of the wells, and avoid trapping air or creating bubbles. Cover the plate with a lid and store on ice or at 4 C. 7. Set Up the Sciclone NGS Workstation Deck Inspect all prepared reagent plates to ensure that air has not been trapped in the wells. If necessary, spin the plates briefly to bring reagents to the bottom of the wells. Confirm that the Maestro software has correctly read the workbook and is set to run the correct number of columns. Step through the pictures, placing the indicated consumables/prepared plates in the indicated locations. Take care to note whether or not a lid is needed at each location. Place new tip boxes in the indicated locations. Note: when the SureSelectXT Library Prep application is started, the variables used for tip tracking are reset. The run must be started with new, full tip boxes in the indicated positions, as Maestro will not retain tip tracking information from the previous run. 8. Run the Library Preparation Steps Confirm that the deck setup matches the final picture in the setup window. Selecting Finished will prompt the application to begin the library preparation steps. While the application is running, the green light at the top of the instrument will blink. If there is a problem with the run, the light will change to yellow and an alarm will sound to indicate that user intervention is necessary. The application may be set up so that an is sent if an error occurs or when additional tip boxes are needed on the deck. The Maestro SureSelectXT Library Prep application will automatically proceed through End Repair, A-tailing, Ligation, and PCR setup steps as indicated in the flowchart on Page 5 and Appendix B. 9. Prepare the PCR Master Mix During the Post-Ligation SPRI Cleanup (Step 4), the PCR Master Mix should be prepared according to the recipe in the SS XT Lib Prep spreadsheet. Thaw the reagents, prepare the master mix, and store it on ice or at 4 C. At the start of Step 5 PCR Setup, the application will prompt the user to add the PCR Master Mix to the reagent plate at A3. The Sciclone NGS Workstation will then distribute the Master Mix from the plate at A3 to the appropriate wells of a clean plate for the PCR reactions. At the completion of the post-ligation SPRI Cleanup, the sample is eluted in 30 µl total volume. 15 µl is transferred to a clean plate for storage and 15 µl is transferred to a plate containing PCR mix. The stored sample may be used in the event that later steps in the SureSelect XT workflow need to be repeated. 10. Amplify Adapter-Ligated Library Note: the PCR reaction will be setup in a Bio-Rad Hard-Shell skirted 96-well PCR plate. Please ensure that your thermocycler is compatible with this plate type. If it is not, the application will need to be modified to transfer samples to a thermocycler-compatible plate for the amplification step and then to transfer amplified libraries back to a Bio-Rad Hard-Shell skirted 96-well PCR plate for post-pcr purification. When the PCR setup is complete, the application will pause and prompt the user to remove the plate containing the PCR reactions, seal, spin, and transfer it to a thermocycler for 12

13 library amplification. The plate containing excess sample is sealed and stored at -20 C. The SureSelectXT Library Prep application will remain paused until the user indicates that the plate containing the completed PCR reactions has been spun, unsealed, and returned to the deck of the Sciclone NGS Workstation. Agilent recommends the following program for amplification of libraries prepared from 3 µg input DNA. The number of cycles may need to be modified according to the amount of input DNA in the sample. Use a heated lid to prevent condensation. 98 C for 2 minutes 4 cycles of: 98 C for 30 seconds 65 C for 30 seconds 72 C for 1 minute 72 C for 10 minutes Hold at 4 C While the samples are amplifying, prepare the reagents for Post-PCR SPRI Cleanup. Ensure that AMPure XP beads are at room temperature and are well resuspended. Aliquot 90 µl per well to a clean Bio-Rad 96-well PCR plate (see SS XT Lib Prep spreadsheet). Prepare a new Deepwell Seahorse reservoir to replace the reservoir containing Elution Buffer. Either an open reservoir containing 50 ml of water or a 12-column reservoir containing 3 ml per column (one column for each column of samples in the run) may be used. 11. Purify amplified libraries Because amplification is minimal at this stage, Agilent does not find it necessary to recommend segregation of this SPRI Cleanup to a specified Post-PCR area. Therefore, the Post- PCR SPRI Cleanup step is integrated into the SureSelectXT Library Prep application. The application will prompt the user to place the new AMPure XP bead plate at location D4, the reservoir containing water at position D3, and the plate containing the amplified library at position B4. Once the user has indicated that the samples are back on the deck, the run will resume. Note: it is critical that Nuclease-Free water is used in place of Elution Buffer during this cleanup step. Samples will be evaporated prior to hybridization, and it is important not to concentrate any buffer/salts during the evaporation. 12. Application complete When the Post-PCR SPRI is complete and the library has been eluted in 30 µl water, the application will pause and show a message indicating that the sample plate should be removed from the deck. Click through the dialogs in Maestro to shut down the INHECO units and complete the run. The user may proceed directly to QC or store libraries at -20 C. Quality control and quantification of amplified libraries It is necessary to quantify and normalize amplified DNA libraries to 500 ng per sample prior to hybridization. For a high throughput project, a LabChip GX System is recommended for quality control steps. Either the DNA 1K Assay Kit or DNA High Sensitivity Assay Kit may be used. To set up the LabChip GX System, follow the instructions in the LabChip GX user guides, making sure to allow 30 minutes for the chip and reagents to equilibrate to room temperature. For DNA 1K assay, dilute the sample 1:10 in a Bio-Rad 96-well plate. For DNA High Sensitivity assay, dilute the sample 1:25 in a Bio-Rad 96-well plate. Mix the diluted sample by pipeting up and down at least 3 times. Spin the plate at 1000 xg for 1 minute to eliminate any bubbles and pellet any beads. Load the plate into the LabChip GX System and run the appropriate assay. For lower numbers of samples, an Agilent Bioanalyzer can be used in place of the LabChip GX System. Use the Agilent Bioanalyzer DNA 1000 kit for library quantitation. It is also possible to quantify the library using a fluorescence-based assay such as Pico Green or Qubit. Absorbance readings are not recommended for library quantification, as they tend to over-represent the concentration of DNA in the library. Quantification data, representing the concentration in ng/ul of each undiluted library sample, should be saved in an Microsoft Excel -compatible format for import into the SureSelectXT Pre-Capture Normalization worksheet. Expected results The total amount of DNA will vary depending on amount of input DNA, how the input DNA was quantified, and quality of the input DNA. Generally, yields of 1-2 µg of library with maximum concentration at bp are expected. 500 ng is required to proceed to hybridization steps. Figure 10. Representative data tracing from LabChipGX analysis of a 1:25 dilution of adapterligated library after 4 cycles of amplification and post-pcr SPRI cleanup. 13

14 1. Modify the Normalization spreadsheet in the SureSelectXT Workbook to specify the number of samples to run and the concentration of each sample. Figure 11. Gel image from LabChipGX analysis of 1:25 dilutions of 16 replicate libraries prepared from 1000 ng total human DNA. Libraries were amplified with 4 cycles of PCR. The workbook must be located in the filepath: C:\ ProgramData\CaliperLS\Maestro\Workbooks and must have the name SureSelectXT Workbook. If the file is moved or the name is changed, Maestro will not be able to find the information necessary to begin the run. a. Specify the number of samples to be normalized by setting the number of columns to run in cell D2 (the application only processes full columns of 8 samples each). b. Ensure the value for Total Volume (µl) entered in cell D6 is c. Fill in the Sample, Source Well and Conc. (ng/µl) columns with information specific for the samples to be run. d. Save the modified spreadsheet with its original name in its original file path. Figure 12. Total yield of amplified libraries as calculated from LabChipGX data shown in figure 10. SureSelectXT Pre-Capture Normalization Application (Day 3) Please read and familiarize yourself with all steps described in this section prior to beginning the run. Libraries are normalized one at a time (by transfer to a clean plate and dilution with water) to a concentration of 18.9 ng/µl in 26.4 µl for a total of 500 ng DNA per library. Evaporation during the denaturing step of hybridization setup will further concentrate the sample prior to the hybridization incubation. Normalization will take up to 90 minutes for 96 samples. Sample preparation Library samples prepared with Agilent SureSelect XT adapters and PCR amplification through 4-6 cycles with the SureSelect XT Primer (forward) and SureSelect XT Indexing Pre-Capture Primer (reverse) must be used. After post-pcr purification, the concentration of each amplified library must be determined. Analyzing libraries using a LabChip GX System or Agilent 2100 Bioanalyzer is recommended in order to visualize the size distribution of the library fragments and quantify the DNA in the appropriate size range. If necessary, boot up the system by first starting the Sciclone NGS Workstation and the INHECO units, then starting the PC controller. Figure 13. Normalization workbook. The Volume for 500 ng value will be calculated by Microsoft Excel based on the value for Conc. (ng/µl). The Source well location will also be the destination well location in the normalized sample plate. Columns with green headers provide additional information on the samples, and will be displayed by Maestro when validating the workbook. They may be deleted if desired. Do not delete any columns highlighted in yellow. 14

15 2. Start the Maestro SureSelectXT Pre-Capture Normalization run Launch the Maestro software and open the SureSelectXT Pre-Capture Normalization Application. Ensure that the INHECO units have the correct adapters for the run: Position A3 Position A4 Position D2 Position D4 96-well PCR Plate Adapter 96-well PCR Plate Adapter 96-well PCR Plate Adapter 96-well PCR Plate Shaker Adapter Start the run by selecting the play button. If running in Edit mode, be sure to start the Main Method. Verify that the INHECO units for positions A3 and A4 are set to 4 C and are cooling. Verify that the INHECO units for positions D2 and D4 are set to 22 C. 3. Set Up the Sciclone NGS Workstation Deck Confirm that the software has correctly read the normalization worksheet, is set to run the correct number of samples, and is displaying data specific for the samples to be normalized. Step through the pictures, placing the indicated consumables/tip boxes/prepared plates in the indicated locations. Take care to note whether or not a lid is needed at each location. Note: the buffer reservoir for sample dilution must contain water, as no additional salts should be added to the library samples prior to the hybridization setup. Note: when the SureSelectXT Pre-Capture Normalization application is started, the variables used for tip tracking are reset. The run must be started with a new, full tip box at C3, as Maestro will not retain tip tracking information from the library prep run. An empty tip box must be placed at position C5, as shown in the setup pictures. 4. Run the SureSelectXT Pre-Capture Normalization application The head of the Sciclone NGS Workstation will transfer a single column of tips to the empty tip box at position C5, then load tips one at a time on mandrel A1. The tip will first aspirate the appropriate volume of water, then the appropriate volume of sample. Both water and sample will be dispensed to the clean plate at position A4, and the tip will be ejected. The application will continue through the total number of samples, taking about 1 minute to process each sample. 5. Application complete When prompted to do so, seal the original library plate (position A3) for long-term storage at -20 C. The normalized plate (position A4) may be sealed and stored at -20 C or may be used immediately for hybridization setup. Click through the dialogs in Maestro to shut down the INHECO units and complete the run. SureSelectXT Hyb Setup Application (Day 3) Please read and familiarize yourself with all steps described in this section prior to beginning the run. The minimum incubation time for SureSelect XT Hybridization is 24 hours. The subsequent washing, PCR amplification and cleanup steps require 6 hours to complete. If planning to run the target capture steps the day following hybridization setup, it is best to complete the SureSelectXT Hyb Setup Application no later than 11:00 am to allow sufficient time during normal working hours to complete the target capture. The SureSelectXT Hyb Setup Application involves adding block mix to the libraries and denaturing at > 95 C on the deck. Water is expected to evaporate from the samples during this incubation. The denatured and concentrated library/block mix is then added to a plate held at > 65 C containing capture probes in hybridization buffer. The user must immediately seal the plate and transfer it to a thermocycler for extended incubation at 65 C. Actual run time for the application is about 15 minutes. The user is advised to attend the entire run to ensure that no delays occur during the hybridization setup steps and subsequent transfer to thermocycler. Note: it is important that the thermocycler be located in immediate proximity to the Sciclone. The hybridization reactions must not be allowed to cool below 65 C while transferring from the Sciclone deck to the thermocycler. Prior to running SureSelectXT Hyb Setup Application for the first time, it is advised to check thermocycler incubation conditions with appropriate volumes of water to ensure that evaporation is not excessive. Please consult the Agilent SureSelect XT protocol for information on sealing plates and testing your specific incubation conditions for the 24 hr (or longer) hybridization. 15

16 Sample preparation Samples must be presented for hybridization setup in a Bio-Rad 96-well Hard-Shell PCR plate as 500 ng of sample in a total volume of 26.4 µl of water. If necessary, boot up the system by first starting the Sciclone NGS Workstation and the INHECO units, then starting the PC controller. 1. Modify the SS Hyb Setup spreadsheet in the SureSelectXT Workbook to specify the number of samples to run. The workbook must be located in the filepath: C:\Program Files\CaliperLS\Maestro\Workbooks and must have the name SureSelectXT Workbook. If the file is moved or the name is changed, Maestro will not be able to find the information necessary to begin the run. The sample number must be set by indicating the number of columns to run in the worksheet titled SS Hyb Setup. The Maestro application only processes full columns of 8 samples each. Start the run by selecting the play button. If running in Edit mode, be sure to start the Main Method. Verify that the INHECO unit for position A3 is set to 105 C, and D4 is set to 75 C. Verify that the INHECO units for positions D2 and A4 are set to 22 C. A timer will appear to prevent the user from continuing with the run before the INHECO units at A3 and D4 have equilibrated for a full 30 minutes. 3. Thaw the SureSelect XT hybridization reagents and place on ice The following reagents need to be thawed at this time: Indexing Block #1, Indexing Block #2, Indexing Block #3, Hyb#3, RNase Block, SureSelect Capture Library. Additional reagents: Hyb #1, Hyb #2, Hyb #4 are stored at room temperature. Note: the SureSelect XT Capture library is composed of RNA baits. To prevent degradation, be sure to handle the baits and all hybridization reagents under RNAse-free conditions. Upon first use of the capture library, aliquot the library appropriately to avoid multiple freeze-thaw cycles for subsequent runs. 4. Prepare the thermocycler The thermocycler must be located in immediate proximity to the Sciclone NGS Workstation. Turn on the thermocycler and set it to hold temperature indefinitely at 65 C with a heated lid set to > 75 C. 5. Prepare the hybridization reagents Figure 14. SS Hyb Setup worksheet. After modifying entries for the number of columns to process, the spreadsheet will update the recipes for the reagent mixes and the appropriate volumes for the reagent plates. Save the modified spreadsheet with its original name in its original file path. Print the SS Hyb Setup spreadsheet to use as a guide while setting up reagents. 2. Start the Maestro SureSelectXT Hyb Setup run Launch the Maestro software and open the SureSelectXT Hyb Setup Application. Ensure that the INHECO units have the correct adapters for the run: Position A3 Position A4 Position D2 Position D4 96-well PCR Plate Adapter 96-well PCR Plate Adapter 96-well PCR Plate Adapter 96-well PCR Plate Shaker Adapter Using the SS Hyb Setup spreadsheet as a guide, prepare the appropriate volumes of Block Mix and Hybridization Buffer in RNase-Free microtubes. Mix by inversion and spin briefly. If a precipitate forms in the hybridization buffer mix, warm the buffer to 65 C for 5 minutes then allow it to cool to room temperature. Store the reagent mixes at room temperature. In a RNase-Free 1.5 ml tube, dilute an aliquot of RNase block 1:3 with Nuclease-free water to reach the volume required for the Capture Probe Library mix. Mix by inversion or gentle vortexing and spin briefly. Keep the diluted RNase block at room temperature. In a separate RNase-Free 1.5 ml tube, mix the indicated volumes of diluted RNase block and capture library. Allow this tube with the Baits/RNase dilution to equilibrate to room temperature. Return the stock of capture library to -80 C storage. 16

17 Once the hybridization buffer and Baits/RNase mixture are at room temperature, add the indicated volume of hybridization buffer to the tube containing the Baits/RNase mixture. Mix gently by inversion and spin briefly. Keep at room temperature. Aliquot the block mix and the capture library in hyb buffer to the 96-well PCR plate as specified on the worksheet. Note: the worksheet only accounts for dead volume in the PCR plate, but, to conserve capture library, no additional volume is added for pipetting error. To avoid shortage of reagents, transfer 0.3 µl less than the indicated aliquot amount. 6. Set Up the Sciclone NGS Workstation Deck Confirm that the software has correctly read the workbook and is set to run the correct number of columns. Step through the pictures, placing the indicated consumables/ prepared plates in the indicated locations. Place new tip boxes in the indicated locations. Note: when the SureSelectXT Hyb Setup application is started, the variables used for tip tracking are reset. The run must be started with new, full tip boxes in the indicated positions, as Maestro will not retain tip tracking information from the previous run. 7. Run the SureSelectXT Hyb Setup application Note: the user is advised to attend the entire run to ensure that no delays occur during the hybridization setup steps and subsequent transfer to thermocycler. Block mix (5.6 µl) will be aliquoted from the plate at C4 into the normalized library plate at B4. After the blockers are aliquoted, the library plate at B4 will be moved to location A3 for denaturation. It is critical that the samples are denatured for exactly 5 minutes. To ensure that the incubation is completed correctly, it is recommend to manually run a timer for 5 minutes starting when the sample plate is moved to position A3. During the incubation, 20 µl of Capture Library/Hybridization Buffer mix will be arrayed to the appropriate number of columns in a clean PCR plate at D4. When the 5 minute incubation is complete, the sample plate at A3 will be moved back to B4 and the denatured libraries will be transferred from B4 to the Capture Library/Hybridization buffer mix at D4 (75 C). After mixing denatured library with capture library at D4, the head of the Sciclone NGS Workstation will move out of the way and the user will be prompted to seal the plate while on the deck at D4. For successful hybridization, the plate must be double-sealed IMMEDIATELY after mixing and BEFORE removing the plate from the 65 C INHECO block at D4. When completely sealed, quickly transfer the plate to the pre-heated thermocycler. Note the time. Samples should hybridize for at least 24 hours, but can incubate up to 72 hours if necessary. 8. Application complete Click through the dialogs in Maestro to shut down the INHECO units and complete the run. SureSelectXT Target Selection Application (Day 4) Please read and familiarize yourself with all steps described in this section prior to beginning the run. This application will take 6-7 hours and should be started 1 hour before the end of the hybridization incubation. The first step in the Target Selection application involves preparing the MyOne Streptavidin T1 beads for target capture. When the bead washing step is complete, the application will prompt the user to transfer the hybridized samples directly from the thermocycler to the deck of the Sciclone NGS Workstation. The samples will be immediately transferred onto the streptavidin beads. Sample preparation Samples must be hybridized for 24 hours in a 96-well PCR plate incubating at 65 C in a thermocycler. The samples will be transferred from the thermocycler to the deck of the Sciclone NGS Workstation when prompted by the application at the start of Step 2. If necessary, boot up the system by first starting the Sciclone NGS Workstation and the INHECO units, then starting the PC controller. 1. Modify the SS Target Selection spreadsheet in the SureSelectXT Workbook to specify the number of samples to run The workbook must be located in the filepath: C:\Program Data\CaliperLS\Maestro\Workbooks and must have the name SureSelectXT Workbook. If the file is moved or the name is changed, Maestro will not be able to find the information necessary to begin the run. 17

18 4. Prepare the streptavidin beads and resuspension buffer plate Thoroughly resuspend the MyOne Streptavidin T1 beads by rotating the bottle. Using the SS Target Selection spreadsheet as a guide, aliquot the specified volumes of streptavidin beads into Column 1 of a 96-well Seahorse 2 ml Deepwell plate. Figure 15. SS Target Selection worksheet. The sample number must be set by indicating the number of columns to run in the spreadsheet titled SS Target Selection. The Maestro application only processes full columns of 8 samples each. After modifying entries for the number of columns to process, the spreadsheet will update the recipes for the reagent mixes and the appropriate volumes for the reagent plates. Save the modified spreadsheet with its original name in its original file path. Print the SS Hyb Setup spreadsheet to use as a guide while setting up reagents. 2. Start the Maestro SureSelectXT Target Selection run Launch the Maestro software and open the SureSelectXT Target Selection Application. Start the run by selecting the play button. If running in Edit mode, be sure to start the Main Method. Ensure that the INHECO units have the correct adapters for the run: Position A3 Position A4 Position D2 Position D4 96-Deepwell Corning 3959 Adapter 12-Row Pyramid Reservoir Adapter 96-well PCR Plate Adapter 96-well PCR Plate Shaker Adapter Verify that the INHECO units for positions D2 and A3 are set to 65 C. Verify that the INHECO units for position D4 is set to 22 C and A4 is set to 4 C. 3. Prepare the Binding Buffer, Wash Buffer I, and Wash Buffer II plates Using the SS Target Selection spreadsheet as a guide, aliquot 775 µl of Binding Buffer and 350 µl of Wash Buffer I into the indicated columns of separate 96-well Seahorse 2 ml Deepwell plates. Aliquot 1000 µl of Wash Buffer II to the indicated columns of a 1 ml Costar 96 Deepwell plate. Cover the plates with lids, inspect for bubbles, spin if necessary, and store at room temperature. Aliquot 10 mm Tris ph 8.0 to Column 6 of the same 96-well Seahorse 2 ml Deepwell plate. Do not prepare the PCR Master Mix, PCR Master Mix plate, or Indexing Primers plate at this time. The PCR Master Mix and Indexing Primer dilutions should be made and aliquoted during Step 4 (Wash Buffer II) of the application. The application will prompt the user to place the PCR Master Mix plate and the Indexing Primers plate on the deck at the start of Step 5 (PCR Setup) Do not aliquot the AMPure XP beads to this plate at this time. Provided that the Post-PCR SPRI Cleanup will be performed on the same instrument, the application will prompt for the addition of AMPure XP beads to Column 4 of the Seahorse 2 ml Deepwell plate when the PCR setup has been completed. 5. Set Up The Sciclone NGS Workstation Deck Confirm that the software has correctly read the workbook and is set to run the correct number of columns. Step through the pictures, placing the indicated consumables/ prepared plates in the indicated locations. Take care to note whether or not a lid is needed at each location. Place new tip boxes in the indicated locations. Note: when the SureSelectXT Target Selection application is started, the variables used for tip tracking are reset. The run must be started with new, full tip boxes in the indicated positions, as Maestro will not retain tip tracking information from the previous run. 6. Run the Maestro SureSelectXT Target Selection application Confirm that the deck setup matches the final picture in the setup window. Selecting Finished will prompt the application to begin the target selection steps. While the application is running, the green light at the top of the instrument will blink. If there is a problem with the run, the light will change to yellow and an alarm will sound to indicate that user intervention is necessary. The application may be set up so that an is sent if an error occurs or when additional tip boxes are needed on the deck. 18

19 The Maestro SureSelectXT Target Selection application will automatically proceed through the streptavidin bead preparation. The user will be prompted to place the hybridized samples on the deck when the instrument has finished preparing the beads. Once the user has indicated that the samples have been added to the deck, the application will continue through the DNA Capture, Wash Buffer I and Wash Buffer II steps. At the completion of washing, the streptavidin beads will be resuspended in 10 mm Tris ph 8.0, and the user will be prompted to place the PCR Master Mix and Indexing Primers plates on the deck for PCR Setup. 7. PCR setup During Step 4 (Wash Buffer II), prepare the PCR Master Mix on ice according to the recipe on the SS Target Selection spreadsheet. Aliquot the indicated volume of PCR Master Mix to the indicated wells of the 96-well PCR Master Mix plate. Keep the plate on ice, pipette carefully into the bottom of the wells, and avoid trapping air or creating bubbles. Cover the plate with a lid and store on ice or at 4 C. Note: if more than 6 columns of samples are run, PCR Master Mix is added to column 7 of the plate, in addition to column 1. Dilute appropriate volumes of the desired set of SureSelect XT Indexing Primers 1:5 with nuclease-free water. Aliquot 5 µl per well of the appropriate diluted indexing primer to each well of the Indexing Primers Plate. One well should be filled for each sample to be run. Pipette carefully into the bottom of the wells, and avoid trapping air or creating bubbles. Cover the plate with a lid and store on ice or at 4 C. Note: the SureSelectXT Target Selection application is written for pre-arrayed indexing primers. The Indexing spreadsheet in the SureSelectXT Workbook may be used to record which indexes are to be used with each sample, but it is not used directly by the Maestro application. The user must create the Indexing Primers Plate with the appropriate indexing primer in each well. Inspect the PCR Master Mix Plate and the Indexing Primers Plate to ensure that air has not been trapped in the wells. If necessary, spin the plates briefly to bring reagents to the bottom of the wells. Place the plates in the specified locations on the deck of the Sciclone NGS Workstation when prompted to do so. Continue with the PCR Setup step. The Sciclone NGS Workstation will array the PCR Master Mix across the PCR Master Mix plate, add the Indexing Primers, and transfer 25 µl resuspended streptavidin beads/sample to the plate for PCR. The 25 µl resuspended streptavidin beads/sample remaining in the sample plate should be saved and stored at -20 C for future use. 8. PCR enrichment of the captured library When PCR Setup is complete, the application will prompt the user to seal the PCR reaction plate and run the appropriate program on the thermocycler. Agilent recommends the following program for enrichment of libraries prepared from 3 µg input DNA. The number of cycles should be adjusted according to the amount of input DNA in the sample and the capture library size (consult the Agilent SureSelect XT protocol for guidance). Use a heated lid to prevent condensation. 98 C for 2 minutes 10 to 16 cycles of: 98 C for 30 seconds 65 C for 30 seconds 72 C for 1 minute 72 C for 10 minutes Hold at 4 C 9. Post-PCR SPRI Cleanup If desired, the application may be modified by a PerkinElmer field application scientist to stop after PCR Setup. This allows Post-PCR SPRI Cleanup to be carried out on a separate instrument to avoid potential cross-contamination of pre-pcr samples with post-pcr samples. This section assumes that the Post-PCR SPRI Cleanup step will be performed on the Sciclone NGS Workstation as Step 6 of the SureSelectXT Target Selection application. Note: AMPure XP beads should be warmed at room temperature for about 30 min before use. They should be taken out of 4 C storage during the PCR Setup step and resuspended thoroughly prior to use. Make 50 ml fresh 80% EtOH solution by diluting 40 ml 100% Ethanol with 10 ml nuclease-free molecular biology grade water. Pour into a Seahorse Deepwell reservoir, cover with a lid and store at room temperature. Follow the prompts in the Maestro dialog boxes to reset the deck for the SPRI Cleanup step. Discard the Deepwell plates at A5, B4, and B5. Place a clean Deepwell plate for liquid waste at A5. Place the lidded Seahorse Deepwell reservoir with 80% EtOH at B5. Aliquot the appropriate volume of SPRI beads (refer to the SS Target Selection spreadsheet) to each well in column 4 of the Seahorse Deepwell plate at A4. When the PCR program is complete, unseal the sample plate, place it on the magnet at the indicated location on the deck, and continue with the run. The Sciclone NGS Workstation will broadcast the AMPure XP beads to a clean plate, and transfer the samples from the streptavidin beads to the AMPure XP beads. After the SPRI Cleanup, the samples will be resuspended in 30 µl 10 mm Tris ph

20 10. Application complete Seal the library plate and store at -20 C for up to 7 days, or proceed directly into library validation prior to storing. Click through the dialogs in Maestro to shut down the INHECO units and complete the run. Quality control and quantification of amplified libraries The LabChip GX System may be used to check the size distribution of fragments in the enriched captured library and estimate the concentration of fragments in the appropriate size range. Make a 1:20 dilution of library into molecular biology grade water in a Bio-Rad 96-well PCR plate. Mix well by pipetting up and down, and spin the plate to remove any bubbles. Run the samples on the LabChip GX System using a High Sensitivity DNA chip and kit according to the standard LabChip protocol. Additional/alternative validation and quantification, including qpcr quantification, should be done according to the user s standard practices. Figure 17. Gel image from LabChipGX analysis of 1:10 dilutions of 16 captured libraries. Capture was performed with 500 ng adapter-ligated library per sample (as determined per LabChipGX analysis) and captured libraries were amplified with 12 cycles of PCR. Expected Results The total amount of DNA in the final library will vary depending on the quality and quantity of the input DNA, the size of the capture library, and the number of postcapture amplification cycles. Generally, libraries will have concentrations of 3-4 pg/µl and will contain approximately 100 ng total DNA in 30 µl (as measured by smear analysis with the LabChip GX). CV values across replicate samples should be less than 25%. Figure 18. Total yield of captured amplified libraries as calculated from LabChipGX data shown in figure 17. Figure 16. Representative data tracing from LabChipGX analysis of a 1:10 dilution of captured library after 12 cycles of amplification and post-pcr SPRI cleanup. 20