Sciclone Ngs Workstation

USER’S GUIDE: AGILENT SURESELECTXT TARGET ENRICHMENT ON THE SCICLONE NGS WORKSTATION

Agilent® SureSelectXT 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 Agilent® SureSelectXT Target Enrichment on the Sciclone NGS Workstation

Table of Contents

Introduction 3 Maestro SureSelectXT 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 SureSelectXT 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 Agilent® SureSelectXT Target Enrichment on the Sciclone NGS Workstation

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

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The Maestro Workflow for the SureSelectXT DNA plate and stored at -20 ˚C for future use. A Post-PCR SPRI® Sample Preparation Consists of 5 Independent Cleanup step is integrated into the “SureSelectXT Library Maestro Applications Prep” application. This step requires fresh AMPure® XP beads and elution in water rather than EB. The full library Run Time preparation (End-Repair through Post-PCR SPRI® Cleanup) (IncludingRun Time Set-up (including can be completed for up to 96 samples in about 6 hours. Setup Thermocycler Maestro Application Time thermocycler The quality, size distribution, and concentration of library in Maestro Application Time steps)Steps) each sample must be checked prior to continuing to “SureSelectXT1 “SureSelectXT Initial Initial SPRI SPRI Cleanup” Cleanup” 1010 min min 1 hour1 hour the next steps. “SureSelectXT2 “SureSelectXT Library Library Prep” Prep” 1 hour1 hour 6 hours6 hours In the “SureSelectXT PreCapture Normalization” procedure, samples are diluted with water to a concentration of “SureSelectXT3 “SureSelectXT PreCapture PreCapture Normalization” Normalization” 10 10 min min upup toto 1.5 1.5 hours hours 18.9 ng/µL in 26.4 µL (total 500 ng DNA). This is undertaken 4 “SureSelectXT Hyb Setup” 20 min 15 min “SureSelectXT Hyb Setup” 20 min 15 min one sample at a time and requires up to 1.5 hours for a “SureSelectXT5 “SureSelectXT Target Target Selection” Selection” 3030 min min 7 hours7 hours 96 sample plate. The Maestro application simplifies sample tracking by importing concentration data directly from a Microsoft® Excel® spreadsheet and automatically calculating In the “SureSelectXT Initial SPRI Cleanup”, 130 µL sheared the proper sample and water volumes for each well. ® DNA samples are split into two aliquots for the AMPure XP The excess volume in the normalized sample is reduced bead cleanup to accommodate the necessary sample/bead appropriately by evaporation during the high temperature ® volumes in standard Hard-Shell PCR plates. After elution in denaturing step of hybridization setup. 15 µL each, the split samples are pooled back together for a total sample volume of 30 µL. The “SureSelectXT Initial SPRI For “SureSelectXT Hyb Setup”, hybridization buffer and Cleanup” application processes a maximum of 48 samples capture library are mixed by the user at room temperature, (6 columns); it is implemented twice if more than 48 samples then arrayed into a 96-well plate and heated to 65 ˚C on are being processed. the Sciclone. Samples are mixed with blocking solution, denatured on the deck at 95 ˚C, then transferred to the In the “SureSelectXT Library Prep” procedure, the volume 65 ˚C plate containing the capture library. The user is ® of the End Repair reaction is reduced from the Agilent - required to promptly seal and transfer the plate to a nearby recommended volume of 100 µL. Instead, the End Repair thermocycler for the overnight incubation. 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 In the “SureSelectXT Target Selection” application, wash SPRI® Cleanup (with 90 µL AMPure® XP beads) in a Bio- steps are carried out in 96-well PCR plates with 150 µL ® Rad 96-well PCR plate. A single aliquot of AMPure® XP wash buffer per well, rather than the Agilent recommended beads is used per sample for SPRI® Cleanups following the 200 and 500 µL per sample. Additional wash cycles are End-Repair, A-Tailing, and Ligation steps. The DNA in the used to compensate for reduced wash volumes. At the sample is driven on and off the beads via changes in PEG completion of the wash steps, samples (with streptavidin and NaCl concentrations. This strategy increases yield by beads) are resuspended in 50 µL 10 mM Tris pH 8.0. The XT XT limiting the number of times samples are transferred to SureSelect Elution Buffer and SureSelect Neutralization new wells/plates. For the library prep, use of Elution Buffer Buffer are not used. Half the resuspended beads/sample (Qiagen® EB), rather than water, is recommended for all (25 µL) is transferred to a plate containing PCR Master Mix ® pre-PCR elution steps to ensure proper buffering during for PCR enrichment followed by a post-PCR SPRI Cleanup. resuspension. When the post-ligation SPRI® Cleanup is The remaining 25 µL resuspended beads/sample (25 µL) is complete, the samples are split into two aliquots: 15 µL stored at -20 ˚C for future use. The “SureSelectXT Target ® is added directly to PCR master mix for enrichment and Selection” application, including the PCR and Post-PCR SPRI further processing and 15 µL is transferred to a clean PCR Cleanup steps, can process up to 96 libraries in 7 hours.

4 Agilent® SureSelectXT Target Enrichment on the Sciclone NGS Workstation

Flowcharts for Individual Steps of the Maestro Workflow for the SureSelectXT Protocol

Figure 2. “SureSelectXT Initial SPRI Cleanup” Application (Day 1).

Figure 3. “SureSelectXT Library Prep” Application (Day 2).

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Flowcharts for Individual Steps of the Maestro Workflow of the SureSelectXT 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 Agilent® SureSelectXT Target Enrichment on the Sciclone NGS Workstation

Required Materials and Reagents

Reagents

Reagent Vendor and Part No. Agilent® SureSelectXT Human All Exon Kit or Agilent® SureSelectXT Custom Target Enrichment System Kit Agilent® Herculase II Fusion DNA Polymerase* Agilent® 600677 (200 rxn) 600679 (400 rxn) Dynabeads MyOne Streptavidin T1 Invitrogen 656-01 (2 mL) 656-02 (10 mL) 656-03 (100 mL) Nuclease-Free Water Various Ampure® XP Beads Beckman Coulter A63880 (5 mL) A63881 (60 mL) A63882 (450 mL) 100% EtOH Sigma E7023 50% PEG 8000 Sigma 83271 5 M NaCl Sigma S5150 10 mM Tris pH 8.0 Various Elution Buffer (EB) Qiagen® 19086

General Laboratory Equipment and Supplies

Equipment Supplier Microfuge Various Vortexer Various 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 Various Microplate centrifuge Various Thermocycler** Bio-Rad Laboratories (MJ Research) DNA Engine PTC-200, or equivalent MicroAmp Clear Adhesive Plate Seals Applied Biosystems 4306311 LabChip GX or Agilent® Bioanalyzer with appropriate chips and reagents Various ** 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. Sciclone NGS Workstation Accessories

Accessory PerkinElmer Part No. Agencourt 96-ring Magnet (2) CLS 128316 Spacer Assembly for Agencourt 96-ring Magnet CLS 135277 (qty 4) and CLS 133514 (1) INHECO Deepwell plate adapter (Corning 1 mL) CLS 128806 INHECO Deepwell plate adapter (12-row Pyramid) CLS 128315 INHECO 384-well plate adapter CLS 128373 INHECO 96-well adapters (3) CLS 128372 INHECO 96-well adapter/shaker CLS 100852

7 Agilent® SureSelectXT Target Enrichment on the Sciclone NGS Workstation

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 127737 Bio-Rad HSP-9631 19 Boxed Tips Pipette Tip 150 µL, Art, Box, 10-96 Sterile Racks CLS 111426 PerkinElmer 58 (96 samples) Deepwell Plates Deepwell-96 POS, Square 2.0 mL Well, Polypro, CLS 133355 Seahorse Bioscience 201379-100 7 Seahorse Bioscience Reservoir- 12-Column Pyramid Bottom, 290 mL CLS 128477 Seahorse Bioscience 201250-100 6 Deepwell Seahorse Bioscience Lids 946 Lid – Universal, Robotic Friendly, Polystyrene CLS 112785 Seahorse Bioscience 200856-100 9 384-well Plates Microplate 384-well, Round Bottom, CLS 134399 Corning, Inc. 3672 1 Polypropylene (Pkg. 10) Deepwell Plates Microplate-96 well, 1 mL Well Volume, Polypropylene CLS 128805 Corning, Inc. 3959 1 (1 mL) Corning

Running the Maestro SureSelectXT Workflow Sample preparation Please read and familiarize yourself with all steps described Genomic DNA should be fragmented to an average size of in this section prior to beginning the run. A 4-day process 150-200 bp on a Covaris S2 or E210 instrument as described is suggested for running the full set of Maestro SureSelectXT in the Agilent® SureSelectXT Target Enrichment System for applications, but the process may be modified according Illumina Paired-Ended Sequencing Library with Multiplexing to the laboratory schedule. Approximate times for setting Protocol v1.2 May 2011. Samples should be presented for up and running each application are indicated in the the Maestro “SureSelectXT Initial SPRI Cleanup” run as up chart on Page 4. Be sure to plan properly for the 24 hour to 3 µg sheared genomic DNA in 130 µL TE in a Bio-Rad hybridization step to allow enough time to complete the Hard-Shell® 96-well PCR plate. subsequent Target Selection application during the following Note: AMPure® XP beads should be warmed at room working day. temperature for about 30 min before use. They may be taken out of 4 ˚C storage before beginning. “SureSelectXT Initial SPRI Cleanup” application If necessary, boot up the system by first starting the Sciclone (Day 1) NGS Workstation and the INHECO units, then starting the This application serves to concentrate the fragmented DNA PC controller. in order to reduce the volume of sample presented for the 1. Modify the “SS Initial SPRI®” worksheet in the library prep application. Because each sample is split into “SureSelectXT Workbook” to specify the number of two aliquots, the maximum sample number for each run samples to run of the “SureSelectXT Initial SPRI Cleanup” application is 48 samples. All subsequent applications in the workflow The workbook must be located in the filepath: C:\Program can process up to 96 samples. 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 Agilent® SureSelectXT Target Enrichment on the Sciclone NGS Workstation

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

9 Agilent® SureSelectXT Target Enrichment on the Sciclone NGS Workstation

6. Run the “SureSelectXT Initial SPRI Cleanup” Steps If properly sheared and size selected with the Initial SPRI® Confirm that the deck setup matches the final picture in application, the DNA samples will contain a majority of the setup window. Selecting “Finished” will prompt the fragments in the150-200 bp (±10%) range. Note that the ® application to begin the run. The Maestro “SureSelectXT Initial SPRI Step will select against fragments smaller than Initial SPRI” application will automatically proceed 100 bp, so it is likely that the profile of the smear will shift through the steps indicated in the flowchart on Page 5 as shown below. 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 e-mail 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.

7. Application complete When the Initial SPRI® is complete, the application will pause and show a message indicating that the sample plate should Figure 8. Representative data tracings showing LabChipGX analysis of 1:25 dilutions of a sheared total human DNA sample before (blue) and after (red) be removed from the deck. The samples may be stored the Initial SPRI Cleanup Step. at -20 ˚C, checked for quality/concentration, or passed immediately into Library Preparation steps. “SureSelectXT Library Prep” Application (Day 2) XT If processing > 48 samples through the SureSelect procedure, Please read and familiarize yourself with all steps described repeat steps 1-7 with the second plate of samples. in this section prior to beginning the run. The Library Preparation application includes a thermocycler step and Quality control of Initial SPRI® samples (optional) a post-PCR SPRI® Cleanup. It is recommended to run the entire application prior to storing samples. If time allows, For high throughput projects, a LabChip® GX Automated quantification of the library may be carried out on the same Electrophoresis System is recommended for assessing sample day as the library prep. 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 Sample preparation 1 sample per minute. Genomic DNA should be fragmented to an average size To set up the LabChip GX System, follow the instructions in of 150-200 bp on a Covaris® S2 or E210 instrument as the LabChip GX System user guides, making sure to allow described in the Agilent® SureSelectXT Target Enrichment 30 minutes for the chip and reagents to equilibrate to room System for Illumina® Paired-Ended Sequencing libraries with temperature prior to preparing the chip. multiplexing v1.2 May 2011. Samples should be purified DNA 1K assay: dilute sample 1:10 in a Bio-Rad 96-well and concentrated with the Maestro “SureSelectXT Initial PCR plate. SPRI Cleanup” application (or manual cleanup according to the Agilent® protocol) and presented in 30 µL EB Buffer or DNA High Sensitivity assay: dilute sample 1:25 in a 10 mM Tris pH 8.0 in a Bio-Rad 96-well PCR plate for the Bio-Rad 96-well PCR plate. Maestro “SureSelectXT Library Prep” run. Ensure the diluted sample is well mixed by pipetting up and Note: AMPure® XP beads should be warmed at room down at least 3 times. Centrifuge the plate at 1000 xg for temperature for about 30 min before use. They may 1 min to eliminate bubbles and pellet any beads prior to be taken out of 4 ˚C storage before beginning. Do not beginning the LabChip GX System run. thaw SureSelectXT reagents until steps 1 and 2 have been ® For lower throughput projects, the Agilent Bioanalyzer can completed. be used in place of the LabChip GX System. Use the Agilent® If necessary, boot up the system by first starting the Sciclone Bioanalyzer DNA 1000 kit for sheared DNA QC. Allow NGS Workstation and the INHECO units, then starting the 30 minutes for reagents to equilibrate to room temperature, PC controller. and prepare the chip according to the Agilent® protocol.

10 Agilent® SureSelectXT Target Enrichment on the Sciclone NGS Workstation

1. Modify the “SS XT Lib Prep” spreadsheet in the Verify that the INHECO units for positions A3 and A4 are “SureSelectXT Workbook” to specify the number of set to 4 ˚C and are cooling. Verify that the INHECO units for samples to run positions D2 is set to 22 ˚C and D4 is set to 20 ˚C. The workbook must be located in the filepath: Note: when the run is started, the instrument will complete C:\ProgramData\CaliperLS\Maestro\Workbooks and must all initialization steps for the hardware and the specific have the name “SureSelectXT Workbook”. If the file is application. The run will automatically pause and prompt moved or the name is changed, Maestro will not be able to the user to set up the deck and confirm proper setup prior find the information necessary to begin the run. to beginning the library preparation steps. Starting the The sample number must be set by indicating the number application prior to thawing and diluting reagents ensures of columns to run in the worksheet titled “SS XT Lib Prep”. that the cold blocks are pre-chilled and ready for on-deck The Maestro application only processes full columns of reagent storage. 8 samples each. 3. Thaw the SureSelectXT library preparation reagents and place on ice Using the “SS XT Lib Prep” spreadsheet as a guide, thaw the reagents from the SureSelectXT 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 XT Figure 9. “SS XT Lib Prep” worksheet. with all SureSelect reagents. Reagents should be aliquoted appropriately if planning to use a kit for more than 2 independent runs.

After modifying entries for the number of columns to 4. Prepare the PEG, Elution Buffer, AMPure® XP bead process, the spreadsheet will update the recipes for the plates, and 80% EtOH reservoir reagent mixes and the appropriate volumes for the reagent Make fresh 20% PEG/2.5 M NaCl solution according to plates. Save the modified spreadsheet with its original the recipe in the “SS XT Library Prep” spreadsheet. Using a name in its original file path. Print the “SS XT Lib multichannel pipettor, aliquot 190 µL 20% PEG/2.5 M NaCl Prep” spreadsheet to use as a guide while setting up solution per well into a Bio-Rad 96-well PCR plate for each reagents. column of samples to be run. Cover the plate with a lid and store at room temperature. 2. Start the Maestro “SureSelectXT Library Prep” run The Elution Buffer (EB Buffer from Qiagen®), should be Launch the Maestro software and open the “SureSelectXT placed in a Seahorse Deepwell 12-column reservoir. Starting Library Prep” Application. with column 1, add 3 mL buffer for each column of samples Ensure that the INHECO units have the correct adapters for to be run. For higher throughput runs, an open Seahorse the run: Deepwell reservoir with 50 mL Elution Buffer may be used Position A3 96-well PCR Plate Adapter instead. Position A4 384-well Plate Adapter Note: water is not recommended for elution during the Position D2 96-well PCR Plate Adapter library preparation steps, as the lack of buffering can Position D4 96-well PCR Plate Shaker Adapter interfere with proper performance. Water is only used as Start the run by selecting the play button. If running in Edit elution buffer in the Post-PCR SPRI® Cleanup just prior to mode, be sure to start the Main Method. 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 Agilent® SureSelectXT Target Enrichment on the Sciclone NGS Workstation

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

12 Agilent® SureSelectXT Target Enrichment on the Sciclone NGS Workstation

library amplification. The plate containing excess sample is Quality control and quantification of amplified sealed and stored at -20 ˚C. The “SureSelectXT Library Prep” libraries application will remain paused until the user indicates that It is necessary to quantify and normalize amplified DNA the plate containing the completed PCR reactions has been libraries to 500 ng per sample prior to hybridization. spun, unsealed, and returned to the deck of the Sciclone For a high throughput project, a LabChip GX System is NGS Workstation. recommended for quality control steps. Either the DNA 1K Agilent® recommends the following program for amplification Assay Kit or DNA High Sensitivity Assay Kit may be used. of libraries prepared from 3 µg input DNA. The number of To set up the LabChip GX System, follow the instructions in cycles may need to be modified according to the amount the LabChip GX user guides, making sure to allow of input DNA in the sample. Use a heated lid to prevent 30 minutes for the chip and reagents to equilibrate condensation. to room temperature. 98 ˚C for 2 minutes For DNA 1K assay, dilute the sample 1:10 in a Bio-Rad 4 cycles of: 96-well plate. 98 ˚C for 30 seconds 65 ˚C for 30 seconds For DNA High Sensitivity assay, dilute the sample 1:25 72 ˚C for 1 minute in a Bio-Rad 96-well plate. 72 ˚C for 10 minutes Mix the diluted sample by pipeting up and down at least Hold at 4 ˚C 3 times. Spin the plate at 1000 xg for 1 minute to eliminate While the samples are amplifying, prepare the reagents for any bubbles and pellet any beads. Load the plate into the Post-PCR SPRI® Cleanup. Ensure that AMPure® XP beads LabChip GX System and run the appropriate assay. are at room temperature and are well resuspended. Aliquot For lower numbers of samples, an Agilent® Bioanalyzer can 90 µL per well to a clean Bio-Rad 96-well PCR plate (see be used in place of the LabChip GX System. Use the Agilent® “SS XT Lib Prep” spreadsheet). Prepare a new Deepwell Bioanalyzer DNA 1000 kit for library quantitation. It is also Seahorse reservoir to replace the reservoir containing Elution possible to quantify the library using a fluorescence-based Buffer. Either an open reservoir containing 50 mL of water assay such as Pico Green or Qubit. Absorbance readings are or a 12-column reservoir containing 3 mL per column (one not recommended for library quantification, as they tend to column for each column of samples in the run) may be used. over-represent the concentration of DNA in the library. Quantification data, representing the concentration in ng/uL 11. Purify amplified libraries of each undiluted library sample, should be saved in an ® Because amplification is minimal at this stage, Agilent does Microsoft® Excel®-compatible format for import into the ® not find it necessary to recommend segregation of this SPRI “SureSelectXT Pre-Capture Normalization” worksheet. Cleanup to a specified Post-PCR area. Therefore, the Post- PCR SPRI® Cleanup step is integrated into the “SureSelectXT Library Prep” application. Expected results The application will prompt the user to place the new AMPure® The total amount of DNA will vary depending on amount of XP bead plate at location D4, the reservoir containing water at input DNA, how the input DNA was quantified, and quality position D3, and the plate containing the amplified library at of the input DNA. Generally, yields of 1-2 µg of library with position B4. Once the user has indicated that the samples are maximum concentration at 250-300 bp are expected. 500 ng back on the deck, the run will resume. is required to proceed to hybridization steps. 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 Figure 10. Representative data tracing from eluted in 30 µL water, the application will pause and show a LabChipGX analysis of a message indicating that the sample plate should be removed 1:25 dilution of adapter- from the deck. Click through the dialogs in Maestro to shut ligated library after 4 down the INHECO units and complete the run. cycles of amplification and post-PCR SPRI cleanup. The user may proceed directly to QC or store libraries at -20 ˚C.

13 Agilent® SureSelectXT Target Enrichment on the Sciclone NGS Workstation

1. Modify the “Normalization” spreadsheet in the “SureSelectXT Workbook” to specify the number of samples to run and the concentration of each sample. 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. Figure 11. Gel image from LabChipGX analysis of 1:25 dilutions of a. Specify the number of samples to be normalized 16 replicate libraries prepared from 1000 ng total human DNA. Libraries were amplified with 4 cycles of PCR. 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 26.4. 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 Figure 13. “Normalization” workbook. 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. 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 Sample preparation well location in the normalized sample plate. Columns Library samples prepared with Agilent® SureSelectXT with green headers provide additional information on the adapters and PCR amplification through 4-6 cycles with samples, and will be displayed by Maestro when validating the SureSelectXT Primer (forward) and SureSelectXT Indexing the workbook. They may be deleted if desired. Do not Pre-Capture Primer (reverse) must be used. After post-PCR delete any columns highlighted in yellow. 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.

14 Agilent® SureSelectXT Target Enrichment on the Sciclone NGS Workstation

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

15 Agilent® SureSelectXT Target Enrichment on the Sciclone NGS Workstation

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

16 Agilent® SureSelectXT Target Enrichment on the Sciclone NGS Workstation Agilent® SureSelectXT Target Enrichment on the Sciclone NGS Workstation

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

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. Aliquot 10 mM Tris pH 8.0 to Column 6 of the same 96-well Figure 15. “SS Target Selection” worksheet. Seahorse 2 mL Deepwell plate.

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

18 Agilent® SureSelectXT Target Enrichment on the Sciclone NGS Workstation Agilent® SureSelectXT Target Enrichment on the Sciclone NGS Workstation

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

19 Agilent® SureSelectXT Target Enrichment on the Sciclone NGS Workstation

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 Figure 17. Gel image from LabChipGX analysis of 1:10 dilutions of and estimate the concentration of fragments in the 16 captured libraries. Capture was performed with 500 ng adapter-ligated appropriate size range. library per sample (as determined per LabChipGX analysis) and captured libraries were amplified with 12 cycles of PCR. 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.

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 post- Figure 18. Total yield of captured amplified libraries as calculated from capture amplification cycles. Generally, libraries will have LabChipGX data shown in figure 17. 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 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 ®

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010554_01 Printed in USA Oct 2012