Home

"user manual"

1. Before setting up the pooling run you must determine the total amount of DNA to pool and the appropriate daughter plate type based on the starting concentrations of the DNA samples to be pooled Accurate normalization of pools requires a minimum pipetting volume of 2 uL for each sample Maximum DNA concentration values for a 1500 ng pool containing gt 2 uL of each sample are shown in Table 27 above When higher concentration DNA samples are included in the pooling run the DNA pool amount must be adjusted as described below 1 Check the DNA concentration of each sample in the set of source plates to be pooled to a single daughter plate to determine the appropriate amount of DNA per pool a If all samples contain DNA at concentrations below the maximum DNA concentration shown in Table 27 lt 94 ng uL for All Exon and SureSelect Automated Library Prep and Capture System 65 4 Hybridization Inherited Disease captures or at lt 47 ng uL for DNA Kinome and custom captures then prepare 1500 ng DNA pools If at least one of the samples is above the maximum DNA concentration shown in Table 27 gt 94 ng uL for All Exon and Inherited Disease captures or at gt 47 ng uL for DNA Kinome and custom captures then you need to calculate the appropriate DNA pool amount First identify the most concentrated DNA sample and calculate the amount of DNA contained in 2 uL of that sample This becomes the amount of each DNA sample used for p
2. ee 1 070 Before You Begin 2 Procedural Notes 10 Safety Notes 10 Required Reagents 11 Required Equipment 14 Optional Reagents 16 Optional Equipment 16 Make sure you read and understand the information in this chapter and have the necessary equipment and reagents listed before you start an experiment Agilent cannot guarantee the SureSelect Target Enrichment kits and cannot provide technical support for the use of non Agilent protocols or instruments to process samples for enrichment ee Agilent Technologies 1 Before You Begin Procedural Notes Certain protocol steps require the rapid transfer of sample plates between the Bravo deck and a thermal cycler Locate your thermal cycler in close proximity to the Agilent NGS Workstation to allow rapid and efficient plate transfer Prepare and load the Agilent NGS Workstation as detailed in each of the protocol steps before initiating each automated protocol run When loading plates in the workstation s Labware MiniHub always place plates in the orientation shown in Figure 6 on page 48 To prevent contamination of reagents by nucleases always wear powder free laboratory gloves and use dedicated solutions and pipettors with nuclease free aerosol resistant tips Maintain a clean work area Do not mix reactions containing gDNA on a vortex mixer Instead gently tap the tube with your finger to mix the sample Avoid repeated freeze thaw cycles of stock and diluted
3. Covaris Sample Preparation System E series or S series Covaris sample holders 96 microTUBE plate E series only microTUBE for individual sample processing DNA LoBind Tubes 1 5 mL PCR clean 250 pieces Centrifuge Qubit Fluorometer Qubit assay tubes Vendor and part number Contact Agilent Automation Solutions for ordering information Customerservice automation agilent com Agilent p n 19477 022 Eppendorf p n 951020401 or 951020619 Thermo Scientific p n 1064156 Thermo Scientific p n 260251 Axygen p n P 2ML SQ C E amp K Scientific p n EK 2440 SureCycler 8800 Thermal Cycler Agilent p n G8810A 96 well plate module Agilent p n G8810A and compression mats Agilent p n 410187 or equivalent Agilent p n 401334 Covaris Covaris p n 520078 Covaris p n 520045 Eppendorf p n 022431021 or equivalent Eppendorf Centrifuge model 5804 or equivalent Life Technologies p n 032857 Life Technologies p n 032856 SureSelect 2 Automated Library Prep and Capture System Before You Begin 1 Table 4 Required Equipment for SureSelect Automated Target Enrichment Description Vendor and part number P10 P20 P200 and P1000 pipettes Magnetic separator DNA Analysis Platform and Consumables 2100 Bioanalyzer Laptop Bundle 2100 Bioanalyzer Electrophoresis Set DNA 1000 Kit High Sensitivity DNA Kit OR 2200 TapeStation D1000 ScreenTape D1000 Reagents High Sensitivity D1000 ScreenTape D1000 Reagen
4. SureSelect 2 Reagent Volume for 1 Well Volume for 12 Columns of Wells Nuclease free water 4 0 uL 462 0 uL SureSelect RNase Block purple cap 0 5 pL 57 8 uL SureSelect or ClearSeq Capture Library 5 0 pL 577 5 uL Total Volume 9 5 pL 1097 3 pL 74 SureSelect 2 Automated Library Prep and Capture System Hybridization 4 b For runs that use different Capture Libraries in individual rows prepare a Capture Library Master Mix for each Capture Library as listed in Table 31 or Table 32 based on the Mb target size of your design The volumes listed in Table 31 and Table 32 are for a single row of sample wells If a given Capture Library will be hybridized in multiple rows multiply each of the values below by the number of rows assigned to that Capture Library Table 31 Preparation of Capture Library Master Mix for target sizes lt 3 0 Mb single row of wells Target size lt 3 0 Mb SureSelect 2 Volume for Volume for Volume for Volumefor Volumefor Volume for Volume for Reagent 1 Well 1 Column 2 Columns 3Columns 4 Columns 6 Columns 12 Columns Nuclease free 7 0 uL 14 0 uL 21 2 uL 28 4 uL 35 7 uL 53 7 uL 100 6 pL water SureSelectRNase 0 5 pL 1 0 pL 1 5 uL 2 0 pL 2 5 uL 3 8 uL 7 2 uL Block purple cap Capture Library 2 0 uL 4 0 uL 6 1 pL 8 1 uL 10 2 uL 15 3 uL 28 8 uL Total Volume 9 5 pL 19 0 pL 28 8 pL 38 6 pL 48 4 pL 72 9 pL 136 6 pL Table 32 Preparation of Capture Library Master Mix for target sizes gt 3 0 Mb single row of
5. Table 37 Thermal cycler program used for sample denaturation prior to hybridization Step Temperature Time Step 1 95 C 5 minutes Step 2 65 C Hold While the sample plate incubates on the thermal cycler the Agilent NGS Workstation combines aliquots of the Capture Library master mix and Hybridization Buffer SureSelect 2 Automated Library Prep and Capture System 81 4 Hybridization CAUTION You must complete step 20 to step 24 quickly and immediately after being prompted by the VWorks software It is important that sample temperature remains approximately 65 C during transfers between the Agilent NGS Workstation and thermal cycler 20 When the workstation has finished aliquoting the Capture Library master mixes and Hybridization Buffer you will be prompted by VWorks as shown below When the thermal cycler reaches the 65 C hold step click Continue Leave the sample plate in the thermal cycler until you are notified to move it Wait for plate in thermocycler When thermocycler has reached hold step at 65C dick Continue Leave DNA plate in thermocycler until you are prompted to transfer the plate I User data entry Pause and Diagnose Continue 82 SureSelect 2 Automated Library Prep and Capture System Hybridization 4 21 When prompted by VWorks as shown below quickly remove the sample plate from the thermal cycler unseal the plate carefully to avoid splashing and transfer the plate to posit
6. 1 2 Clear the Labware MiniHub and BenchCel of all plates and tip boxes Gently wipe down the Labware MiniHub Bravo decks and BenchCel with a Nucleoclean decontamination wipe Let the AMPure XP beads come to room temperature for at least 30 minutes Mix the bead suspension well so that the reagent appears homogeneous and consistent in color Do not freeze Prepare a Nunc DeepWell source plate containing AMPure XP beads For each well to be processed add 95 uL of homogeneous AMPure XP beads per well to the Nunc DeepWell plate Prepare a Thermo Scientific reservoir containing 15 mL of nuclease free water Prepare a separate Thermo Scientific reservoir containing 45 mL of freshly prepared 70 ethanol SureSelect Automated Library Prep and Capture System 113 5 114 Post Capture Sample Processing for Multiplexed Sequencing 8 Load the Labware MiniHub according to Table 58 using the plate orientations shown in Figure 6 Table 58 Initial MiniHub configuration for DNA cleanup using SPRI_XT_Illumina_v2 0 pro Post CaptureOnBeadPCR Cleanup Vertical Shelf Cassette 1 Cassette 2 Cassette 3 Cassette 4 Position Shelf 5 Top Empty Nunc Empty Empty Empty DeepWell plate Shelf 4 Empty Empty Empty Empty Shelf 3 Empty Empty Eppendorf Empty Empty twin tec Plate Shelf 2 Empty Nuclease free AMPure XP beads Empty water reservoir in Nunc DeepWell from step 6 plate from step 5 Shelf 1 Bottom Empty 70 ethanol Empty Empty t
7. 12 Select the number of columns of samples to be processed Runs must include 1 2 3 4 6 or 12 columns 13 Click Display Initial Workstation Setup Display Initial Workstation Setup 14 Verify that the NGS workstation has been set up as displayed in the Workstation Setup region of the form Workstation Setup MiniHub MiniHub Cassette 1 MiniHub Cassette 2 MiniHub Cassette 3 MiniHub Cassette 4 n Di e dandi PN aanl T ee te ee ee Oe E a DE ae TARY 15 When verification is complete click Run Selected Protocol Run Selected Protocol The purification protocol takes approximately 45 minutes When complete the amplified DNA samples are in the Eppendorf plate located on Bravo deck position 7 60 SureSelect 2 Automated Library Prep and Capture System Sample Preparation 3 Step 6 Assess Library DNA quantity and quality Option 1 Analysis using the Agilent 2100 Bioanalyzer and DNA 1000 Assay Use a Bioanalyzer DNA 1000 chip and reagent kit For more information to do this step see the Agilent DNA 1000 Kit Guide 1 Set up the 2100 Bioanalyzer as instructed in the reagent kit guide 2 Seal the sample plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 1 0 sec 3 Vortex the plate to mix samples in each well then centrifuge the plate for 30 seconds to drive the well contents off the walls and plate seal 4 Prepare the chip samples and ladder as instructed in the reagent kit g
8. Commercial Items and DFARS 227 7202 3 Rights in Commercial Computer Software or Com puter Software Documentation Notice to Purchaser This product is provided under an agree ment between Bio Rad Laboratories and Agilent Technologies Inc and the manu facture use sale or import of this product is subject to US Pat No 6 627 424 and EP Pat No 1 283 875 B1 owned by Bio Rad Labora tories Inc Purchase of this product con veys to the buyer the non transferable right to use the purchased amount of the product and components of the product in PCR but not real time PCR in the Research Field including all Applied Research Fields including but not limited to forensics ani mal testing and food testing SureSelect Automated Library Prep and Capture System Safety Notices CAUTION A CAUTION notice denotes a hazard It calls attention to an operating procedure practice or the like that if not correctly performed or adhered to could result in damage to the product or loss of important data Do not proceed beyond a CAUTION notice until the indicated conditions are fully understood and met A WARNING notice denotes a hazard It calls attention to an operating procedure practice or the like that if not correctly performed or adhered to could result in personal injury or death Do not proceed beyond a WARNING notice until the indicated conditions are fully understood and met SureSelect Automated Library P
9. 1 Number of Capture Libraries 2 Appropriate positioning rows vs columns of the Capture Libraries with respect to the DNA sample plate configuration In Hybridization_MMCol_v2 0 pro master mixes are organized in the source plate by column see Figure 11 and each row of the DNA sample plate may be hybridized to a different Capture library e In Hybridization_MMRow_v2 0 pro master mixes are organized in the source plate by row see Figure 12 and each column of the DNA sample plate may be hybridized to a different Capture library Table 28 Comparison of Hybridization protocol options Protocol Name Optimal Hybridization Run Size Number of Different Capture Instructions Start Libraries Allowed in Run Hybridization MMCol_v2 0 pro 12 column runs 8 page 73 Hybridization MMRow_v2 0 pro lt 6 column runs 12 page 86 72 SureSelect Automated Library Prep and Capture System Hybridization 4 Hybridization Option A Master Mixes in Columns Hybridization MMCol_v2 0 pro Prepare the workstation 1 Clear the Labware MiniHub and BenchCel of all plates and tip boxes 2 Gently wipe down the Labware MiniHub Bravo decks and BenchCel with a NucleoClean decontamination wipe Prepare one or more Capture Library Master Mixes 3 Prepare the appropriate volume of SureSelect or ClearSeq Capture Library Master Mix for each of the Capture Libraries that will be used for hybridization as indicated in Table 29 to Table 32 Mix the c
10. 22 5 uL 31 5 pL 40 5 uL 58 5 uL 117 0 pL Blocking Mix Column 1 Nuclease free A1 H1 3 75 pL 6 25 uL 8 75 uL 11 25 pL 16 25 pL 32 5 pL water Capture Library Column 2 18 4 uL 28 2 uL 38 0 uL 47 8 uL 72 3 uL 136 0 pL Master Mix A2 H2 SureSelect XT2 Column 3 55 5 uL 92 5 uL 129 5 uL 166 5 pL 240 5 pL 481 uL Hybridization A3 H3 Buffer 76 SureSelect 2 Automated Library Prep and Capture System Hybridization 4 VN ee be an 4 4 4 KA amp a X Figure 11 Configuration of the master mix source plate for Hybridization _M MCol_v2 0 pro Each well in column 2 may contain the same or different Cap ture Libraries 5 Seal the master mix source plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 1 0 sec 6 Vortex the plate for 5 seconds to ensure homogeneity of the Block Master Mix dilution 7 Centrifuge the plate for 30 seconds to drive the well contents off the walls and plate seal and to eliminate any bubbles Keep the master mix plate at room temperature SureSelect 2 Automated Library Prep and Capture System 71 4 78 Hybridization Load the Agilent NGS Workstation 8 Load the Labware MiniHub according to Table 34 using the plate orientations shown in Figure 6 Table 34 Initial MiniHub configuration for Hybridization MMCol_v2 0 pro Vertical Shelf Cassette 1 Cassette 2 Cassette 3 Cassette 4 Position Shelf 5 Top Empty Empty
11. B10 TGAAGAGA C01 AACGTGAT C04 CAGATCTG C07 CTAAGGTC C10 AGATCGCA D01 CACTTCGA D04 ATCCTGTA D07 CGACACAC D10 AAGAGATC E01 GCCAAGAC E04 CTGTAGCC E07 CCGTGAGA E10 CAACCACA F01 GACTAGTA F04 GCTCGGTA F07 GTGTTCTA F10 TGGAACAA G01 ATTGGCTC G04 ACACGACC G07 CAATGGAA G10 CCTCTATC H01 GATGAATC H04 AGTCACTA H07 AGCACCTC H10 ACAGATTC A02 AGCAGGAA A05 AACGCTTA A08 CAGCGTTA A11 CCAGTTCA B02 GAGCTGAA B05 GGAGAACA B08 TAGGATGA B11 TGGCTTCA C02 AAACATCG c05 CATCAAGT C08 AGTGGTCA C11 CGACTGGA D02 GAGTTAGC D05 AAGGTACA D08 ACAGCAGA D11 CAAGACTA E02 CGAACTTA E05 CGCTGATC E08 CATACCAA E11 CCTCCTGA F02 GATAGACA F05 GGTGCGAA F08 TATCAGCA F11 TGGTGGTA G02 AAGGACAC G05 CCTAATCC G08 ATAGCGAC G11 AACAACCA H02 GACAGTGC H05 CTGAGCCA H08 ACGCTCGA H11 AATCCGTC A03 ATCATTCC A06 AGCCATGC A09 CTCAATGA A12 CAAGGAGC B03 GCCACATA B06 GTACGCAA B09 TCCGTCTA B12 TTCACGCA C03 ACCACTGT C06 AGTACAAG c09 AGGCTAAC C12 CACCTTAC D03 CTGGCATA D06 ACATTGGC D09 CCATCCTC D12 AAGACGGA E03 ACCTCCAA E06 ATTGAGGA E09 AGATGTAC E12 ACACAGAA F03 GCGAGTAA F06 GTCGTAGA F09 TCTTCACA F12 GAACAGGC G03 ACTATGCA G06 AGAGTCAA G09 CCGAAGTA G12 AACCGAGA H03 CGGATTGC H06 CCGACAAC H09 CGCATACA H12 ACAAGCTA SureSelect 2 Automated Library Prep and Capture System 131 6 Reference Reference Informat
12. CAUTION 21 When prompted by VWorks as shown below quickly remove the PCR sample plate from Bravo deck position 4 leaving the red insert in place r Remove Plate from 4 Quickly remove plate from position 4 seal and place in thermocycler Click Continue after plate is in thermocycler for protocol to finish User data entry Pause and Diagnose Continue 22 Seal the sample plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 3 0 sec 23 Quickly transfer the plate back to the thermal cycler held at 65 C Place a compression mat over the PCR plate in the thermal cycler After transferring the plate click Continue on the VWorks screen 24 To finish the VWorks protocol click Continue in the Unused Tips and Empty Tip box dialogs and then click Yes in the Protocol Complete dialog The temperature of the plate in the thermal cycler should be held at 65 C using a heated lid at 105 C The lid of the thermal cycler is hot and can cause burns Use caution when working near the lid 96 25 Incubate the hybridization mixture in the thermal cycler for 24 hours at 65 C with a heated lid at 105 C Samples may be hybridized for up to 72 hours but you must verify that the extended hybridization does not cause extensive evaporation in the sample wells SureSelect 2 Automated Library Prep and Capture System Hybridization 4 Step 3 Capture the hybridized DNA In this ste
13. DNA Assay 118 SureSelect 2 Automated Library Prep and Capture System CAUTION Post Capture Sample Processing for Multiplexed Sequencing 5 Option 2 Analysis using the Agilent 2200 TapeStation and High Sensitivity D1000 ScreenTape Use a High Sensitivity D1000 ScreenTape p n 5067 5584 and reagent kit p n 5067 5585 to analyze the amplified captured DNA For more information to do this step see the Agilent 2200 TapeStation User Manual 1 Seal the DNA sample plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 1 0 sec Vortex the plate to mix samples in each well then centrifuge the plate for 30 seconds to drive the well contents off the walls and plate seal Prepare the TapeStation samples as instructed in the Agilent 2200 TapeStation User Manual Use 2 uL of each DNA sample diluted with 2 uL of High Sensitivity D1000 sample buffer for the analysis Make sure that you thoroughly mix the combined DNA and High Sensitivity D1000 sample buffer on a vortex mixer for 5 seconds for accurate quantitation Stopping Point Load the sample plate or tube strips from step 3 the High Sensitivity D1000 ScreenTape and loading tips into the 2200 TapeStation as instructed in the Agilent 2200 TapeStation User Manual Start the run Verify that the electropherogram shows an average DNA amplicon size of 250 to 300 bp A sample electropherogram is shown in Figure 15 Determine the concentration of each
14. Display Initial Workstation Setup Display Initial S Workstation Setup 15 Verify that the NGS workstation has been set up as displayed in the Workstation Setup region of the form Workstation Setup MiniHub Minit ub Cassette 1 MiniHub Cassette 2 MiniHub Cassette 3 MiniHub Cassette 4 Abs ae y a 4 A Bes _ s She ee ee ae ee ee eee _a P ae P T an tht ton anami 16 When verification is complete click Run Selected Protocol Run Selected Protocol SureSelect Automated Library Prep and Capture System 115 5 _ Post Capture Sample Processing for Multiplexed Sequencing The purification protocol takes approximately 45 minutes When complete the amplified DNA samples are in the Eppendorf plate located on Bravo deck position 7 116 SureSelect Automated Library Prep and Capture System Post Capture Sample Processing for Multiplexed Sequencing 5 Step 3 Assess quantity and quality of the amplified captured library pools Option 1 Analysis using the Agilent 2100 Bioanalyzer and High Sensitivity DNA Assay 1 Set up the 2100 Bioanalyzer as instructed in the High Sensitivity DNA Assay kit guide Version B 02 07 or higher of the Agilent 2100 Expert Software is required for High Sensitivity DNA Assay Kit runs 2 Seal the sample plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 1 0 sec 3 Vortex the plate to mix samples in each well then centrifuge the plate for 30 seconds
15. Empty Empty Shelf 4 Empty Empty Empty Empty Shelf 3 Empty Empty Empty Empty Shelf 2 Empty Empty Empty Empty tip box Shelf 1 Bottom Empty Empty Empty Empty 9 Load the BenchCel Microplate Handling Workstation according to Table 35 Table 35 Initial BenchCel configuration for Hybridization MMCol_v2 0 pro No of Columns Rack 1 Rack 2 Rack 3 Rack 4 Processed 1 2 Tip boxes Empty Empty Empty 2 2 Tip boxes Empty Empty Empty 3 2 Tip boxes Empty Empty Empty 4 2 Tip boxes Empty Empty Empty 6 3 Tip boxes Empty Empty Empty 12 4 Tip boxes Empty Empty Empty SureSelect 2 Automated Library Prep and Capture System Hybridization 4 10 Load the Bravo deck according to Table 36 Table 36 Initial Bravo deck configuration for Hybridization MMCol_v2 0 pro Location Content 4 Empty PCR plate seated on red insert PCR plate type must be specified on setup form under step 2 5 Empty Eppendorf twin tec plate 6 Hybridization Master Mix source plate unsealed seated on silver insert Master Mixes in Columns 1 3 8 Empty tip box 9 Indexed DNA pools in Eppendorf twin tec plate unsealed Run VWorks protocol Hybridization MMCol_v2 0 pro 11 On the SureSelect setup form under Select Protocol to Run select Hybridization_MMCol_v2 0 pro 12 Under Select PCR plate labware for Thermal Cycling select the specific type of PCR plate that was loaded on Bravo deck position 4 13 Select the number of columns of samples to be pr
16. Genomic DNA samples 1 2 n SureSelect 12 Shear DNA NGS Target Enrichment DNA fragments of 150 200 bp Workflow Prepare samples using SureSelect Library Prep Kit Indexed DNA libraries Gores ito i Design target Indexed DNA library amplicons sequences in SureDesign Pool 8 or 16 amplified indexed libraries Indexed library pools Hybridize using SureSelect reagents and protocol l Capture hybrids on magnetic beads l PCR amplify Pool captured samples Optional i 1 1 v Figure 2 Overall sequencing sample preparation workflow SureSelect Automated Library Prep and Capture System 29 2 Using the Agilent NGS Workstation for SureSelect Target Enrichment Table 8 Overview of VWorks protocols and runsets used during the workflow Workflow Step Substep VWorks Protocols Used for Agilent NGS Workstation Protocol Chapter automation Sample Preparation Hybridization Sample Processing for Multiplexed Sequencing Prepare indexing adaptor ligated DNA Amplify indexed DNA Purify indexed DNA amplicons using AMPure XP beads Prepare indexed DNA pools for hybridization Hybridize pooled indexed DNA to Capture Library Capture and wash DNA hybrids PCR amplify captured DNA Purify captured DNA amplicons using AMPure XP beads LibraryPrep_XT_Illumina_v2 0 rst Pre CapturePCR_XT_IIlumina_v2 0 pro SPRI_XT_Illumina_v2 0 pro Pre Capture PCR Cleanup PreCapture_Pooling_v1 0 pro initiated u
17. PCR master mixes in a dedicated clean area or PCR hood with UV sterilization and positive air flow 106 The post capture PCR master mix source plate must be a Nunc DeepWell plate with the PCR master mix for the run supplied in column 4 If the Hybridization protocol was run with master mixes configured by column Hybridization_MMCol_v2 0 pro reuse the Nunc DeepWell master mix source plate used for the Hybridization run The final configuration of the master mix source plate for this scenario is shown in Figure 13 If the Hybridization protocol was run with master mixes configured by row Hybridization_MMRow_v2 0 pro use a new Nunc DeepWell plate SureSelect Automated Library Prep and Capture System Post Capture Sample Processing for Multiplexed Sequencing 5 5 Prepare the Post capture PCR Master Mix by combining SureSelect Herculase II Master Mix and the XT2 Primer Mix in column 4 of the master mix source plate Add the volumes of both reagents shown in Table 52 to each well of column 4 of the master mix source plate Table 52 Preparation of the Master Mix Source Plate for Post CaptureOnBeadPCR_XT_Illumina_v2 0 pro SureSelect 2 Position on Volume of Reagents added per Well of Nunc Deep Well Source Plate Reagent Source Plate 1 Column 2 Column 3 Column 4 Column 6 Column 12 Column Runs Runs Runs Runs Runs Runs SureSelect Column 4 37 5 uL 62 5 uL 87 5 uL 112 5 uL 162 5 pL 325 uL Herculase II A4 H4 Master Mix XT2 P
18. amplified captured library pool by integration under the peak in the electropherogram If the yield is too low or non specific peaks are observed in the electropherogram repeat the PCR with more or fewer cycles The goal is to minimize cycles while you produce enough library for application to the flow cell If you do not continue to the next step seal the indexed DNA sample plate and store at 4 C overnight or at 20 C for prolonged storage SureSelect Automated Library Prep and Capture System 119 5 Post Capture Sample Processing for Multiplexed Sequencing Step 3 Assess quantity and quality of the amplified captured library pools Lower 253 400 w a 3 Sample Intensity FU N o o 100 MW bp 3 25 s s 2 1000 3 a a Figure 15 Analysis of amplified captured DNA using the 2200 TapeStation 120 SureSelect 2 Automated Library Prep and Capture System Post Capture Sample Processing for Multiplexed Sequencing 5 Step 4 Prepare samples for multiplexed sequencing The final SureSelect enriched samples contain pools of either 8 or 16 indexed libraries based on the Capture Library used and resulting pre capture pooling strategy When appropriate for your sequencing platform the 8 plex or 16 plex samples may be further multiplexed by post capture pooling Determine whether to do post capture pooling by calculating the number of indexes that can be combined p
19. and tip boxes 2 Turn on the chiller set to 0 C at position 9 of the Bravo deck Be sure that the chiller reservoir contains at least 300 mL of 25 ethanol 3 Pre set the temperature of Bravo deck position 6 to 4 C using the Inheco Multi TEC control touchscreen as described in Setting the Temperature of Bravo Deck Heat Blocks Bravo deck position 6 corresponds to CPAC 2 position 2 on the Multi TEC control touchscreen SureSelect Automated Library Prep and Capture System 51 3 Sample Preparation Prepare the pre capture PCR master mix and the master mix source plate 4 Prepare the Pre capture PCR Master Mix by combining SureSelect Herculase II Master Mix and the XT2 Primer Mix in column 4 of the master mix source plate Add the volumes of both reagents shown in Table 19 to each well of column 4 of the master mix source plate Use the same Nunc DeepWell master mix source plate that was used for the LibraryPrep_XT_Ilumina_v2 0 rst run The final configuration of the master mix source plate is shown in Figure 7 Table 19 Preparation of the Master Mix Source Plate for Pre CapturePCR_XT_Illumina_v2 0 pro SureSelect 2 Position on Volume of Reagents added per Well of Nunc Deep Well Source Plate Reagent Source Plate 1 Column 2 Column 3 Column 4 Column 6 Column 12 Column Runs Runs Runs Runs Runs Runs SureSelect 37 5 uL 62 5 pL 87 5 pL 112 5 pL 162 5 pL 325 uL Herculase II Column 4 Master Mix A4 H4 XT2 Primer Mix 1 5 uL 2 5 uL 3 5
20. contained in this document is provided as is and is subject to being changed with out notice in future editions Fur ther to the maximum extent permitted by applicable law Agi lent disclaims all warranties either express or implied with regard to this manual and any information contained herein including but not limited to the implied warranties of merchant ability and fitness for a particular purpose Agilent shall not be lia ble for errors or for incidental or consequential damages in con nection with the furnishing use or performance of this document or of any information contained herein Should Agilent and the user have a separate written agreement with warranty terms covering the material in this doc ument that conflict with these terms the warranty terms in the separate agreement shall control Technology Licenses The hardware and or software described in this document are furnished under a license and may be used or copied only in accor dance with the terms of such license Restricted Rights Legend U S Government Restricted Rights Soft ware and technical data rights granted to the federal government include only those rights customarily provided to end user cus tomers Agilent provides this customary commercial license in Software and techni cal data pursuant to FAR 12 211 Technical Data and 12 212 Computer Software and for the Department of Defense DFARS 252 227 7015 Technical Data
21. menu select the number of indexed DNA source plates to be provided for sample pooling If gt 8 plates will be used to create a single Hybridization sample plate run the pooling and normalization protocol in sets of 8 source plates e Under Concentration File use the browse button to specify the location of each csv file that provides sample position and concentration data for each plate Controls Load Sources To MiniHub Manually Sources Enter Sealed AEC A Show y Run XT2 Initialize all Full Screen Display SureSelect On Off f Gantt Chart Pooling and Elapsed Time 00 00 00 Normalization Pooling Options Destination Plate Number of Indexes to Pool 8 Plate ID Barcode Pooled DNA Quantity ng Destination1 1500ng Recommended Source Plates Number of Source Plates 2 Cancentratian File ID Barcode EEE Wor Worspace sopton Bet umire Pate 2 R SureSelect Automated Library Prep and Capture System 69 4 70 Hybridization Step 1 Pool indexed DNA samples for hybridization 5 When finished entering run parameters in the Form click Show Setup agp Show Setup 6 Load sample plates and labware as displayed in the Workstation Setup region of the form example shown below is for pooling run for two source plates e Load each indexed DNA source plates onto its assigned shelf on the MiniHub e Load the appropriate type of destination daughter plate on Bravo deck position 5 See step 2 on
22. on ice during preparation and aliquoting Each column of the indexed gDNA pool plate may be hybridized to a different Capture Library However Capture Libraries of different sizes require different post capture amplification cycles Plan experiments such that similar sized libraries are hybridized on the same plate For runs that use a single Capture Library for all columns of the plate prepare the master mix as described in Step a Table 38 or Table 39 below For runs that use different Capture Libraries for individual columns prepare each master mix as described in Step b Table 40 or Table 41 below 86 SureSelect Automated Library Prep and Capture System Hybridization 4 a For runs that use a single Capture Library for all columns prepare the Capture Library Master Mix as listed in Table 38 or Table 39 based on the Mb target size of your design Table 38 Preparation of Capture Library Master Mix for target sizes lt 3 0 Mb same Capture Library for all columns Target size lt 3 0 Mb SureSelect 2 Reagent Volume for Volumefor Volumefor Volumefor Volumefor Volume for 1 Well 1 Column 2 Columns 3Columns 4Columns 6 Columns Nuclease free water 7 0 pL 68 3 uL 143 3 uL 211 6 uL 279 8 uL 416 3 uL SureSelect RNase Block 0 5 pL 4 9 uL 10 2 uL 15 1 uL 20 0 uL 29 7 uL purple cap Capture Library 2 0 uL 19 5 pL 41 0 uL 60 5 pL 80 0 uL 119 0 pL Total Volume 9 5 pl 92 7 pL 194 5 pL 287 1 pL 379 8 pL 565 0 pL Table 39 Prepa
23. page 66 to determine plate type needed e Load an empty tip box on Bravo deck position 6 e Load the indicated number of tip boxes in the BenchCel stacker Workstation Setup Source Plate 2 User_source_2 Source Plate 1 User_source_1 Destination Plate f Empty Tip Box Hyb_Plate_Name orc Ff BenchCel 2 Tip Boxes SureSelect Automated Library Prep and Capture System Hybridization 4 7 When verification is complete click Run Protocol Run Protocol CAUTION When more than one indexed DNA source plate is used in the run a workstation operator must be present during the run to remove and replace plate seals during the run in response to NGS Workstation prompts Running the PreCapture_Pooling_v1 0 pro protocol takes approximately one hour per indexed DNA source plate Once complete the Hybridization sample plate containing indexed DNA pools is located at position 5 of the Bravo deck Adjust final concentration of pooled DNA 8 Remove the Hybridization sample plate from Bravo deck position 5 9 Use a vacuum concentrator held at lt 45 C to reduce the volume in each well to 1 2 uL 10 Add sufficient nuclease free water to each concentrated gDNA pool to bring the final DNA concentration to 214 3 ng uL For example for 1500 ng pools bring the final volume in each well to 7 uL for a final concentration of 214 3 ng uL 11 Seal the plate using the PlateLoc Thermal Microplate Sealer wi
24. runs t For kits that include SureSelect End Repair Master Mix add 125 uL of the pre combined master mix for 2 column runs For kits that include SureSelect End Repair Master Mix add 175 uL of the pre combined master mix for 3 column runs For kits that include SureSelect End Repair Master Mix add 225 pL of the pre combined master mix for 4 column runs tt For kits that include SureSelect End Repair Master Mix add 325 uL of the pre combined master mix for 6 column runs tt For kits that include SureSelect End Repair Master Mix add 650 uL of the pre combined master mix for 12 column runs May also be labeled as SureSelect End Repair Oligo Mix 44 SureSelect 2 Automated Library Prep and Capture System Sample Preparation 3 5 Seal the master mix source plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 1 0 sec 6 Vortex the plate for 5 seconds to ensure homogeneity of the mixtures 7 Centrifuge the plate for 30 seconds to drive the well contents off the walls and plate seal and to eliminate any bubbles Keep the master mix source plate on ice The final configuration of the master mix source plate is shown in Figure 5 The presence of bubbles in source plate solutions may cause inaccurate volume transfer by the Bravo liquid handling platform Ensure that the source plate is sealed and centrifuged prior to use in a run Figure 5 Configuration of the master mix source plate for Library
25. settings of 165 C and 1 0 sec 3 Vortex the plate to mix samples in each well then centrifuge the plate for 30 seconds to drive the well contents off the walls and plate seal 4 Prepare the chip samples and ladder as instructed in the reagent kit guide using 1 uL of each sample for the analysis 5 Load the prepared chip into the 2100 Bioanalyzer and start the run within five minutes after preparation 6 Verify that the electropherogram shows an average DNA fragment size of 150 to 200 bp A sample electropherogram is shown in Figure 3 Stopping Point If you do not continue to the next step seal the plate and store at 4 C overnight or at 20 C for prolonged storage fagon 1 Figure 3 Analysis of sheared DNA using a DNA 1000 Bioanalyzer assay SureSelect 2 Automated Library Prep and Capture System 41 3 Sample Preparation CAUTION Option 2 Analysis using the Agilent 2200 TapeStation and D1000 ScreenTape You can use Agilent s 2200 TapeStation for rapid analysis of multiple samples Use a D1000 ScreenTape p n 5067 5582 and associated reagent kit p n 5067 5583 to analyze the sheared DNA For more information to do this step see the Agilent 2200 TapeStation User Manual 1 Seal the sheared DNA sample plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 1 0 sec Vortex the plate to mix samples in each well then centrifuge the plate for 30 seconds to drive the well contents off the
26. tec source plate Shelf 2 Empty Nuclease free Empty Empty water reservoir from step 13 Shelf 1 Bottom Empty Empty Empty Empty tip box SureSelect Automated Library Prep and Capture System 99 4 100 Hybridization 15 Load the BenchCel Microplate Handling Workstation according to Table 50 Table 50 Initial BenchCel configuration for SureSelectCapture amp Wash_v2 0 rst No of Columns Rack 1 Rack 2 Rack 3 Rack 4 Processed 1 1 Tip box Empty Empty Empty 2 2 Tip boxes Empty Empty Empty 3 3 Tip boxes Empty Empty Empty 4 4 Tip boxes Empty Empty Empty 6 6 Tip boxes Empty Empty Empty 12 10 Tip boxes 2 Tip boxes Empty Empty 16 Load the Bravo deck according to Table 51 positions 5 and 6 should already be loaded Table 51 Initial Bravo deck configuration for SureSelectCapture amp Wash_v2 0 rst Location Content 1 Empty waste reservoir Axygen 96 Deep Well Plate square wells 4 Empty red aluminum insert PCR plate type used for Hybridization protocol must be specified on setup form under step 2 5 Streptavidin beads DeepWell source plate 6 Wash 2 DeepWell source plate seated on silver insert Run VWorks runset SureSelectCapture amp Wash_v2 0 rst 17 On the SureSelect setup form under Select Protocol to Run select SureSelectCapture amp Wash_v2 0 rst 18 Under Select PCR plate labware for Thermal Cycling select the specific type of PCR plate used to incubate the hybridization reactions in the therma
27. the VWorks screen 25 To finish the VWorks protocol click Continue in the Unused Tips and Empty Tip box dialogs and then click Yes in the Protocol Complete dialog The temperature of the plate in the thermal cycler should be held at 65 C using a heated lid at 105 C The lid of the thermal cycler is hot and can cause burns Use caution when working near the lid 84 26 Incubate the hybridization mixture in the thermal cycler for 24 hours at 65 C with a heated lid at 105 C SureSelect 2 Automated Library Prep and Capture System Hybridization Samples may be hybridized for up to 72 hours but you must verify that the extended hybridization does not cause extensive evaporation in the sample wells When hybridization is complete proceed to Step 3 Capture the hybridized DNA on page 97 SureSelect Automated Library Prep and Capture System 85 4 Hybridization Hybridization Option B Master Mixes in Rows Hybridization MMRow_v2 0 pro Prepare the workstation 1 Clear the Labware MiniHub and BenchCel of all plates and tip boxes 2 Gently wipe down the Labware MiniHub Bravo decks and BenchCel with a NucleoClean decontamination wipe Prepare one or more Capture Library Master Mixes 3 Prepare the appropriate volume of Capture Library Master Mix for each of the Capture Libraries that will be used for hybridization as indicated in Table 38 to Table 41 Mix the components by pipetting Keep the master mixes
28. the plate See page 28 for guidelines on selecting the appropriate hybridization run configuration Plan your experiment such that each indexed DNA library is placed in a pool in the row or column of the sample plate that corresponds to the appropriate Capture Library for hybridization e For post capture amplification see Figure 2 different Capture Libraries can require different amplification cycle numbers based on sizes of the captured targets It is most efficient to process similar sized Capture Libraries on the same plate See Table 57 on page 112 to determine which Capture Libraries may be amplified on the same plate 34 SureSelect Automated Library Prep and Capture System Using the Agilent NGS Workstation for SureSelect Target Enrichment 2 e After the SureSelect capture process DNA samples enriched using small Capture Libraries are typically pooled a second time before sequencing See page 121 for post capture secondary sample pooling guidelines When using such a secondary pooling strategy develop a pre capture indexed library pooling strategy that is compatible with post capture pooling and sequencing designs Considerations for Equipment Setup e Some workflow steps require the rapid transfer of sample plates between the Bravo deck and a thermal cycler Locate your thermal cycler in close proximity to the Agilent NGS Workstation to allow rapid and efficient plate transfer e Several workflow steps require that the sa
29. to amplify purify and assess quality and quantity of the sample libraries Samples are pooled by mass prior to sequencing 6 Reference This chapter contains reference information including component kit contents and index sequences SureSelect Automated Library Prep and Capture System What s New in Version B 1 Support for ClearSeq Capture Libraries including ClearSeq Comprehensive Cancer Libraries see Table 3 on page 13 Support for Human All Exon v6 Capture Libraries see Table 2 on page 12 Update to name of end repair reagent from SureSelect End Repair Oligo Mix to SureSelect End Repair Nucleotide Mix see Table 65 on page 129 Update to name of indexing adaptor reagent from SureSelect Pre Capture Index to SureSelect Pre Capture Indexed Adaptor see Table 65 on page 129 Updates to post capture pooling and sequencing setup guidelines including support for the NextSeq 500 platform see page 121 to page 122 What s New in Version B 0 Support for kits supplied with either of two indexing primer configurations Kits with revised index configuration typically received February 2015 or later include indexing primers A01 through H12 provided in a blue plate For kit content details see page 128 For nucleotide sequences of the 8 bp indexes in this revised configuration see Table 69 on page 131 Kits with original index configuration typically received before February 2015 include indexing primers 1
30. 0 SureSelect Human All Exon v5_ 5190 6218 5 x 5190 6218 SureSelect Human All Exon v5 UTRs 5190 6223 5 x 5190 6223 SureSelect Human All Exon v5 IncRNA 5190 6454 5 x 5190 6454 SureSelect Human All Exon v5 Plus 5190 6225 5 x 5190 6225 SureSelect Human All Exon v4 5190 4668 5190 4670 SureSelect Human All Exon v4 UTRs 5190 4673 5190 4675 SureSelect Mouse All Exon 5190 4683 5190 4685 SureSelect 2 Custom 1 kb up to 499 kb 5190 4848 5190 4850 reorder 5190 4853 5190 4855 SureSelect 2 Custom 0 5 Mb up to 2 9 Mb 5190 4858 5190 4860 reorder 5190 4863 5190 4865 SureSelect 2 Custom 3 Mb up to 5 9 Mb 5190 4868 5190 4870 reorder 5190 4873 5190 4875 SureSelect 2 Custom 6 Mb up to 11 9 Mb 5190 4878 5190 4880 reorder 5190 4883 5190 4885 SureSelect 2 Custom 12 Mb up to 24 Mb 5190 4888 5190 4890 reorder 5190 4893 5190 4895 Eight gDNA samples are enriched in one capture reaction after sample pooling Capture Libraries are provided for the number of capture reactions needed to enrich the indicated number of samples t Sixteen gDNA samples are enriched in one capture reaction after sample pooling Capture Libraries are provided for the number of capture reactions needed to enrich the indicated number of samples SureSelect 2 Automated Library Prep and Capture System Before You Begin 1 Table3 Compatible ClearSeq Automation Capture Libraries Capture Library 96 Reacti
31. 0 Load the Bravo deck according to Table 22 Table 22 Initial Bravo deck configuration for Pre CapturePCR_XT_IIlumina_v2 0 pro Location Content 6 Empty PCR plate seated on red insert PCR plate type must be specified on setup form under step 2 7 Indexing adaptor ligated DNA samples in Eppendorf twin tec plate 9 Master mix plate unsealed containing Pre Capture PCR Master Mix in Column 4 seated on silver insert Run VWorks protocol Pre CapturePCR_XT_Illumina_v2 0 pro 11 On the SureSelect setup form under Select Protocol to Run select Pre CapturePCR_XT_Tlumina_v2 0 pro 12 Under Select PCR plate labware for Thermal Cycling select the specific type of PCR plate that was loaded on Bravo deck position 6 13 Select the number of columns of samples to be processed Runs must include 1 2 3 4 6 or 12 columns SureSelect 2 Automated Library Prep and Capture System 55 3 Sample Preparation 14 Click Display Initial Workstation Setup O Display Initial Workstation Setup 15 Verify that the NGS workstation has been set up as displayed in the Workstation Setup region of the form Workstation Setup MiniHub MiniHub Cassette 1 MiniHub Cassette 2 MiniHub Cassette 3 MiniHub Cassette 4 Anda ON aten aA a a DES E aan bP e os Bo at ee en 16 When verification is complete click Run Selected Protocol Run Selected Protocol Running the Pre CapturePCR_XT_Illumina_v2 0 pro protocol takes approximately 15 min
32. 1 BX SureSelect XT2 Hybridization Buffer Row C 314 5 pL C1 CX SureSelect 2 Automated Library Prep and Capture System 89 4 Hybridization Figure 12 Configuration of the master mix source plate for Hybridization_M MRow_v2 0 pro Rows A C may contain 1 2 3 4 6 or 12 wells of reagents depending on run size example shown is for 6 column run size Each well in row B may contain the same or different Capture Libraries 5 Seal the master mix source plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 1 0 sec 6 Vortex the plate for 5 seconds to ensure homogeneity of the Block Master Mix dilution 7 Centrifuge the plate for 30 seconds to drive the well contents off the walls and plate seal and to eliminate any bubbles Keep the master mix plate at room temperature 90 SureSelect 2 Automated Library Prep and Capture System Hybridization 4 Load the Agilent NGS Workstation 8 Load the BenchCel Microplate Handling Workstation according to Table 43 Table 43 Initial BenchCel configuration for Hybridization MMRow_v2 0 pro No of Columns Rack 1 Rack 2 Rack 3 Rack 4 Processed 1 1 Tip box Empty Empty Empty 2 1 Tip box Empty Empty Empty 3 2 Tip boxes Empty Empty Empty 4 2 Tip boxes Empty Empty Empty 6 3 Tip boxes Empty Empty Empty 12 5 Tip boxes Empty Empty Empty 9 Load the Bravo deck according to Table 44 Table 44 Initial Bravo deck configuration for Hybridization MMRow_
33. 2 Pre Capture Automation ILM Module Box 2 Content Kit Component 96 Sample Kit 480 Sample Kit SureSelect XT2 Blocking Mix tube with blue cap tube with blue cap SureSelect XT2 Hybridization Buffer tube with yellow cap bottle SureSelect RNase Block tube with purple cap tube with purple cap SureSelect 2 Automated Library Prep and Capture System 129 6 Reference Table 68 Plate map for indexed adaptors containing indexes A01 through H12 blue plate in Library Prep kit p n 5500 0131 1 2 3 4 5 6 7 8 9 10 11 12 A A0 A02 A03 A04 A05 A06 A07 A08 A09 A10 A11 A12 B B01 B02 B03 B04 B05 B06 B07 B08 B09 B10 B11 B12 c c01 c02 c03 c04 c05 c06 c07 c08 c09 C10 C11 C12 D D01 D02 D03 D04 D05 D06 D07 D08 D09 D10 D11 D12 E E01 E02 E03 E04 E05 E06 E07 E08 E09 E10 E11 E12 F F01 F02 F03 F04 F05 F06 F07 F08 F09 F10 F11 F12 G G01 G02 G03 G04 G05 G06 G07 G08 G09 G10 G11 G12 H H01 H02 H03 H04 H05 H06 H07 H08 H09 H10 H11 H12 130 SureSelect Automated Library Prep and Capture System Reference 6 XT2 Nucleotide Sequences of SureSelect Indexes A01 to H12 Each index is 8 nt in length See page 121 for sequencing run setup information using 8 bp indexes Table 69 SureSelect Indexes for indexing primers provided in blue 96 well plate ndex Sequence ndex Sequence index Sequence Index Sequence A01 ATGCCTAA A04 AACTCACC A07 ACGTATCA A10 AATGTTGC B01 GAATCTGA B04 GCTAACGA B07 GTCTGTCA
34. 96 provided in clear capped tubes For kit content details see page 132 For nucleotide sequences of the 8 bp indexes in this original configuration see Table 75 on page 134 through Table 80 on page 139 Support for revised Library Prep kit configuration now including End Repair Enzyme Mix and End Repair Oligo Mix both replacing End Repair Master Mix For instructions for use of the revised kit components see page 44 See Table 65 on page 129 for updated kit contents SureSelect Automated Library Prep and Capture System Content Before You Begin 9 Procedural Notes 10 Safety Notes 10 Required Reagents 11 Required Equipment 14 Optional Reagents 16 Optional Equipment 16 Using the Agilent NGS Workstation for SureSelect Target Enrichment 17 About the Agilent NGS Workstation 18 About the Bravo Platform 18 VWorks Automation Control Software 22 Overview of the Workflow 28 Experimental Setup Considerations for Automated Runs 31 Considerations for Placement of gDNA Samples in 96 well Plates for Automated Processing 34 Considerations for Indexed DNA Sample Placement for Automated Hybridization and Post Hybridization Processing 34 Considerations for Equipment Setup 35 PCR Plate Type Considerations 36 Sample Preparation 37 Step 1 Shear DNA 38 Step 2 Assess sample quality and DNA fragment size 41 Step 3 Prepare indexed gDNA library samples 43 Step 4 Amplify the indexed libraries 51 Step 5 Purify amplified DNA using AMPu
35. All Form Selections to Defaults BenchCel Information BenchCel Stacker 1 BenchCel Stacker 2 BenchCel Stacker 3 BenchCel Stacker 4 Currently Running Protocol 1 Open the form using the XT2_ILM VWForm VWForm shortcut on your desktop 2 Use the drop down menus on the form to select the appropriate SureSelect workflow step PCR plate labware description and number of columns of samples for the run 3 Once all run parameters have been specified on the form click Display Initial Workstation Setup Display Initial Workstation Setup SureSelect Automated Library Prep and Capture System 23 gt I Onttw L i UUI if N 2 Using the Agilent NGS Workstation for SureSelect Target Enrichment 4 The Workstation Setup region of the form will then display the required placement of reaction components and labware in the NGS Workstation for the specified run parameters Workstation Setup MiniHub SureSelect li MiniHub Cassette 1 MiniHub Cassette 2 MiniHub Cassette 3 MiniHub Cassette 4 pre capture pooling Shelf 5 Empty Nunc Empty Nunc Index Adapters for Illumina sequencers DeepWell Plate DeepWell Plate Twin tec Shelf 4 Empty Eppendorf Empty Eppendorf Twin tec Plate Twin tec Plate Parameters Shelf3 Empty Eppendorf 1 Select Protocol to Run Twin tec Plate LibraryPrep_XT_Illumina_v2 0 rst gt Shelf 2 Empty Tip Box Nudease free E AmpureXP Beads _ T NAE Water Reservoir
36. Bravo deck Be sure that the chiller reservoir contains at least 300 mL of 25 ethanol 2 Pre set the temperature of Bravo deck position 6 to 4 C using the Inheco Multi TEC control touchscreen as described in Setting the Temperature of Bravo Deck Heat Blocks Bravo deck position 6 corresponds to CPAC 2 position 2 on the Multi TEC control touchscreen 3 Clear the Labware MiniHub and BenchCel of all plates and tip boxes SureSelect Automated Library Prep and Capture System 43 oo Sample Preparation Step 3 Prepare indexed gDNA library samples Prepare the Library Prep master mix source plate 4 Ina Nunc DeepWell plate prepare the master mix source plate by adding the volumes indicated in Table 15 of each reagent to all wells of the indicated column of the plate As indicated in the shaded portions of Table 15 Column 1 and Column 3 are prepared to contain mixtures of two reagents Keep the reagents and source plate on ice during the aliquoting steps Table 15 Preparation of the Master Mix Source Plate for LibraryPrep_XT_IIlumina_v2 0 rst Reagent Solution Position on Volume added per Well of Nunc Deep Well Source Plate Source Plate 1 Column 2 Column 3 Column 4 Column 6 Column 12 Column Runs Runs Runs Runs Runs Runs SureSelect Column 2 30 uL 50 uL 70 pL 90 pL 130 uL 260 uL dA Tailing Master A2 H2 Mix For kits that include SureSelect End Repair Master Mix add 75 uL of the pre combined master mix for 1 column
37. Cassette 1 Shelf 2 and clean tip box Cassette 1 Shelf 1 are retained from the LibraryPrep_XT_Illumina_v2 0 rst run and reused here If you are using a new box of tips on shelf 1 of cassette 1 for example when amplifying the second half of the indexed DNA sample first remove the tips from columns 1 to 3 of the tip box Any tips present in columns 1 to 3 of the clean tip box Cassette 1 Shelf 1 may be inappropriately loaded onto the Bravo platform pipette heads and may interfere with automated processing steps 54 Table 20 Initial MiniHub configuration for Pre CapturePCR_XT_Illumina_v2 0 pro Vertical Cassette 1 Cassette 2 Cassette 3 Cassette 4 Shelf Position Shelf 5 Empty Empty Empty Empty Top Shelf 4 Empty Empty Empty Empty Shelf 3 Empty Empty Empty Empty Shelf 2 Waste tip box Empty Empty Empty Shelf 1 Clean tip box Empty Empty Empty tip box Bottom Retained from the LibraryPrep_XT_Illumina_v2 0 rst run and reused here SureSelect 2 Automated Library Prep and Capture System Sample Preparation 3 9 Load the BenchCel Microplate Handling Workstation according to Table 21 Table 21 Initial BenchCel configuration for Pre CapturePCR_XT_IIlumina_v2 0 pro No of Columns Rack 1 Rack 2 Rack 3 Rack 4 Processed 1 1 Tip box Empty Empty Empty 2 1 Tip box Empty Empty Empty 3 1 Tip box Empty Empty Empty 4 1 Tip box Empty Empty Empty 6 1 Tip box Empty Empty Empty 12 1 Tip box Empty Empty Empty 1
38. GA 39 CCTCCTGA 40 CGAACTTA 41 CGACTGGA 42 CGCATACA 43 CTCAATGA 44 CTGAGCCA 45 CTGGCATA 46 GAATCTGA 41 GACTAGTA 48 GAGCTGAA 136 SureSelect Automated Library Prep and Capture System Table 78 SureSelect Pre capture Indexes 49 64 Index Number Sequence 49 GATAGACA 50 GCCACATA 51 GCGAGTAA 52 GCTAACGA 53 GCTCGGTA 54 GGAGAACA 55 GGTGCGAA 56 GTACGCAA 57 GTCGTAGA 58 GTCTGTCA 59 GTGTTCTA 60 TAGGATGA 61 TATCAGCA 62 TCCGTCTA 63 TCTTCACA 64 TGAAGAGA SureSelect Automated Library Prep and Capture System Reference 6 137 6 Reference Table 79 SureSelect Pre capture Indexes 65 80 Index Number Sequence 65 TGGAACAA 66 TGGCTTCA 67 TGGTGGTA 68 TTCACGCA 69 AACTCACC 70 AAGAGATC 71 AAGGACAC 72 AATCCGTC 73 AATGTTGC 74 ACACGACC 75 ACAGATTC 76 AGATGTAC 77 AGCACCTC 78 AGCCATGC 79 AGGCTAAC 80 ATAGCGAC 138 SureSelect 2 Automated Library Prep and Capture System Table 80 SureSelect 2 Pre capture Indexes 81 96 Index Number Sequence 81 ATCATTCC 82 ATTGGCTC 83 CAAGGAGC 84 CACCTTAC 85 CCATCCTC 86 CCGACAAC 87 CCTAATCC 88 CCTCTATC 89 CGACACAC 90 CGGATTGC 91 CTAAGGTC 92 GAACAGGC 93 GACAGTGC 94 GAGTTAGC 95 GATGAATC 96 GCCAAGAC SureSelect Automated Library Prep and Capture System Reference 6 139 www agilent com In This Book This guide contains information to run the SureSelect Automated Library Prep and Capture System protocol using the automation protocols
39. PCR plate from position 4 Leave Red Aluminum PCR plate insert at Position 4 for next protocol Pause and Diagnose The remainder of the SureSelectCapture amp Wash_v2 0 rst runset takes approximately 1 5 hours Once the runset is complete the captured bead bound DNA samples are located in the Eppendorf plate at position 9 of the Bravo deck When the runset is complete seal the plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 1 0 sec and store the plate on ice while setting up the next automation protocol Captured DNA is retained on the streptavidin beads during the post capture amplification steps SureSelect 2 Automated Library Prep and Capture System 103 4 Hybridization Step 3 Capture the hybridized DNA 104 SureSelect 2 Automated Library Prep and Capture System SureSelect 2 Automated Library Prep and Capture System Protocol 5 Post Capture Sample Processing for Multiplexed Sequencing Step 1 Amplify the captured libraries 106 Step 2 Purify the amplified captured libraries using AMPure XP beads 113 Step 3 Assess quantity and quality of the amplified captured library pools 117 Step 4 Prepare samples for multiplexed sequencing 121 Step 5 Optional Quantify captured library pools by QPCR 123 Step 6 Optional Pool captured libraries for sequencing 124 This chapter describes the steps to amplify purify and assess quality and quantity of the captured libraries Post c
40. Prep and Capture System 21 2 Using the Agilent NGS Workstation for SureSelect Target Enrichment VWorks Automation Control Software VWorks software included with your Agilent NGS Workstation allows you to control the robot and integrated devices using a PC The Agilent NGS Workstation is preloaded with VWorks software containing all of the necessary SureSelect system liquid handling protocols General instructions for starting up the VWorks software and the included protocols is provided below Each time a specific VWorks protocol is used in the SureSelect procedure any settings required for that protocol are included in the relevant section of this manual The instructions in this manual are compatible with VWorks software version 11 3 0 1195 If you have questions about VWorks version compatibility please contact service automation agilent com Logging in to the VWorks software 1 Double click the XT2_ILM VWForm shortcut on the Windows desktop to start the VWorks software 2 If User Authentication dialog is not visible click Log in on the VWorks window toolbar 3 In the User Authentication dialog type your VWorks user name and password and click OK If no user account is set up contact the administrator VWorks protocol and runset files VWorks software uses two file types for automation runs pro protocol files and rst runset files Runset files are used for automated procedures in which the workstation uses more
41. Prep_XT_Illumina_v2 0 rst SureSelect 2 Automated Library Prep and Capture System 45 3 Sample Preparation Prepare the Pre capture Indexed Adaptors source plate 8 Select the appropriate index for each sample Nucleotide sequence information for the index portion of each indexed adaptor is provided in the Reference chapter starting on page 127 Using an Eppendorf Twin tec plate prepare the indexed adaptors source plate by combining 5 uL of each SureSelect Pre capture Indexed Adaptor solution with 2 5 uL of nuclease free water Each pre capture index dilution is made in a separate well of the source plate corresponding to the well position of the sample to be indexed Prepare the purification reagents 9 Verify that the AMPure XP bead suspension is at room temperature 10 Mix the bead suspension well so that the reagent appears homogeneous and consistent in color Do not freeze 11 Prepare a separate Nunc DeepWell source plate for the beads by adding 250 uL of homogeneous AMPure XP beads per well for each well to be processed 12 Prepare a Thermo Scientific reservoir containing 20 mL of nuclease free water 13 Prepare a separate Thermo Scientific reservoir containing 100 mL of freshly prepared 70 ethanol 46 SureSelect Automated Library Prep and Capture System Sample Preparation 3 Load the Agilent NGS Workstation 14 Load the Labware MiniHub according to Table 16 using the plate orientations shown in Figure 6
42. SureSelect 2 Automated Library Prep and Capture System For Illumina Paired End Multiplexed Sequencing gilent NGS ion B Automated u Workstation Protocol Version B1 June 2 SureSelect platform manufacture SurePrint Technology For Research Use Only Not Procedures e in Diagnostic wie Agilent Technologies Notices Agilent Technologies Inc 2015 No part of this manual may be reproduced in any form or by any means including elec tronic storage and retrieval or translation into a foreign language without prior agree ment and written consent from Agilent Technologies Inc as governed by United States and international copyright laws Manual Part Number G9450 90000 Edition Version B1 June 2015 Printed in USA Agilent Technologies Inc 5301 Stevens Creek Blvd Santa Clara CA 95051 USA Acknowledgement Oligonucleotide sequences 2006 2008 and 2011 Illumina Inc All rights reserved Only for use with the Illumina sequencer systems and associated assays Technical Support For technical product support contact your local Agilent Support Services representa tive For US and Canada call 800 227 9770 option 3 4 4 For other countries find your support center telephone numbers at www agilent com chem contactus Or send an e mail to SureSelect Support agilent com Notice to Purchaser Research Use Only Not for use in diagnostic procedures Warranty The material
43. Table 16 Initial MiniHub configuration for LibraryPrep_XT_IIlumina_v2 0 rst Vertical Shelf Cassette 1 Cassette 2 Cassette 3 Cassette 4 Position Shelf 5 Top Empty Empty Nunc Empty Nunc Indexing DeepWell plate DeepWell plate Adaptors in Eppendorf twin tec plate Shelf 4 Empty Empty Eppendorf Empty Eppendorf Empty twin tec plate twin tec plate Shelf 3 Empty Empty Empty Eppendorf Empty twin tec plate Shelf 2 Empty tip box Nuclease free AMPure XP beads Empty water reservoir in Nunc DeepWell from step 12 plate from step 11 Shelf 1 Bottom New tip box 70 ethanol Empty Empty tip box reservoir from step 13 SureSelect Automated Library Prep and Capture System 47 3 48 Sample Preparation Figure 6 Labware MiniHub plate orientation For Thermo Scientific reservoirs place the notched corner facing the center of the hub 15 Load the BenchCel Microplate Handling Workstation according to Table 17 Table 17 Initial BenchCel configuration for LibraryPrep_XT_Illumina_v2 0 rst No of Columns Rack 1 Rack 2 Rack 3 Rack 4 Processed 1 2 Tip boxes Empty Empty Empty 2 3 Tip boxes Empty Empty Empty 3 4 Tip boxes Empty Empty Empty 4 5 Tip boxes Empty Empty Empty 6 7 Tip boxes Empty Empty Empty 12 11 Tip boxes 3 Tip boxes Empty Empty SureSelect Automated Library Prep and Capture System Sample Preparation 3 16 Load the Bravo deck according to Table 18 Table 18 Initial Bravo deck configuration for LibraryPrep_XT
44. _Illumina_v2 0 rst Location Content 1 Empty waste reservoir Axygen 96 Deep Well Plate square wells 6 Empty Eppendorf twin tec plate 7 Eppendorf twin tec plate containing sheared gDNA samples oriented with well A1 in the upper left 9 Library Prep Master Mix Source Plate unsealed seated on silver insert Run VWorks runset LibraryPrep_XT_Ilumina_v2 0 rst For this runset you are not required to select PCR Plate labware under step 2 on the setup form 17 On the SureSelect setup form under Select Protocol to Run select LibraryPrep_XT_Illumina_v2 0 rst 18 Select the number of columns of samples to be processed Runs must include 1 2 3 4 6 or 12 columns 19 Click Display Initial Workstation Setup Display Initial S Workstation Setup 20 Verify that the NGS workstation has been set up as displayed in the Workstation Setup region of the form Workstation Setup MiniHub MiniHub Cassette 1 MiniHub Cassette 2 MiniHub Cassette 3 MiniHub Cassette 4 EO N a a N S ae T eee and GR den Ann Am er ee ie am anam SureSelect Automated Library Prep and Capture System 49 3 Sample Preparation 21 When verification is complete click Run Selected Protocol Run Selected Protocol 22 When ready to begin the run click OK in the following window E VWorks G 1 This runset contains protocols that will start running as soon as N possible Before you click OK verify that the system is rea
45. abeads MyOne Streptavidin T1 2mL 10 mL 100 mL Quant iT dsDNA BR Assay Kit for use with the Qubit fluorometer 100 assays 2 1000 ng 500 assays 2 1000 ng 100 Ethanol molecular biology grade Select one library from Table 2 or Table 3 Agilent p n G9661B p n G9661C p n G9662B p n G9662C Ambion Cat AM9930 Life Technologies p n 4389764 Beckman Coulter Genomics p n A63881 p n A63882 Life Technologies Cat 65601 Cat 65602 Cat 65603 Life Technologies p n 032850 Life Technologies p n 032853 Sigma Aldrich p n E7023 SureSelect reagents and Capture Libraries must be used within one year of receipt Tt HiSeq and MiSeq Reagent Kits are also compatible with the NextSeq 500 platform SureSelect Automated Library Prep and Capture System 11 1 12 Before You Begin Table 2 SureSelect Automation Capture Libraries Capture Library 96 Reactions 480 Reactions SureSelect Human All Exon v6 5190 8874 5 x 5190 8874 SureSelect Human All Exon v6 UTRs 5190 9306 5 x 5190 9306 SureSelect Human All Exon v6 COSMIC 5190 9312 5 x 5190 9312 SureSelect Human All Exon v6 Plus 1 5190 8877 5 x 5190 8877 SureSelect Human All Exon v6 Plus 2 5190 8880 5 x 5190 8880 SureSelect Clinical Research Exome 5190 7347 5 x 5190 7347 SureSelect Focused Exome 5190 7799 5 x 5190 7799 SureSelect Focused Exome Plus 1 5190 7807 5 x 5190 7807 SureSelect Focused Exome Plus 2 5190 7810 5 x 5190 781
46. apture dilution and optional pooling instructions are provided to prepare the indexed samples for multiplexed sequencing ee Agilent Technologies 105 5 _ Post Capture Sample Processing for Multiplexed Sequencing CAUTION Step 1 Amplify the captured libraries In this step the Agilent NGS Workstation completes the liquid handling steps for PCR amplification of the SureSelect enriched DNA samples After the PCR plate is prepared by the Agilent NGS Workstation you transfer the plate to a thermal cycler for amplification Plan your experiments for amplification of libraries captured using Capture Libraries of similar sizes on the same plate See Table 57 for cycle number recommendations for different Capture Library size ranges Prepare the workstation 1 Clear the Labware MiniHub and BenchCel of plates and tip boxes 2 Gently wipe down the Labware MiniHub Bravo decks and BenchCel with a Nucleoclean decontamination wipe 3 Turn on the chiller set to 0 C at position 9 of the Bravo deck Be sure that the chiller reservoir contains at least 300 mL of 25 ethanol 4 Pre set the temperature of Bravo deck position 6 to 4 C using the Inheco Multi TEC control touchscreen as described in Setting the Temperature of Bravo Deck Heat Blocks Bravo deck position 6 corresponds to CPAC 2 position 2 on the Multi TEC control touchscreen Prepare the Post capture PCR master mix source plate To avoid cross contaminating libraries set up
47. ature is displayed press the rectangle to enter the temperature 4 Press the Temp button until the new temperature is displayed on the SET button and until the Temp button is darkened indicating that the selected heat block is heating or cooling to the new temperature setting The current temperature of the block is indicated in the center of the display CPAC 2 1 25 0 C KE Current temp 0200 rpm se 20 SureSelect 2 Automated Library Prep and Capture System Using the Agilent NGS Workstation for SureSelect Target Enrichment 2 Setting the Temperature of Bravo Deck Position 9 Using the ThermoCube Device Bravo deck position 9 is equipped with a ThermoCube thermoelectric temperature control system used to incubate components at a defined temperature during the run During protocols that require temperature control at position 9 you will be instructed to start and set the temperature of the ThermoCube device before starting the run ThermoCube temperature settings are modified using the control panel LCD display screen and four input buttons on the front panel of the device using the following steps 1 Turn on the ThermoCube and wait for the LCD screen to display TEMP 2 Press the UP or DOWN button to change SET TEMP 1 to the required set point 3 Press the START button The ThermoCube will then initates temperature control of Bravo deck position 9 at the displayed set point SureSelect Automated Library
48. circulated water chiller with ethylene glycol to 20 volume to prevent freezing Refer to the Covaris instrument user guide for more details 4 Put a Covaris microTube into the loading and unloading station Keep the cap on the tube You can use the 96 microTube plate see Table 4 on page 14 for the DNA shearing step when preparing multiple gDNA samples in the same experiment SureSelect Automated Library Prep and Capture System Sample Preparation 3 5 Use a tapered pipette tip to slowly transfer the 50 uL DNA sample through the pre split septa Be careful not to introduce a bubble into the bottom of the tube 6 Secure the microTube in the tube holder and shear the DNA with the settings in Table 13 or Table 14 depending on the Covaris instrument SonoLab software version used The target DNA fragment size is 150 to 200 bp Table 13 Shear settings for Covaris instruments using SonoLab software version 7 or newer Setting Value Duty Factor 10 Peak Incident Power PIP 175 Cycles per Burst 200 Treatment Time 360 seconds Bath Temperature 4 to 8 C Table 14 Shear settings for Covaris instruments using SonoLab software prior to version 7 Setting Value Duty Cycle 10 Intensity 5 Cycles per Burst 200 Time 6 cycles of 60 seconds each Set Mode Frequency sweeping Temperature 4 to7 C 7 Put the Covaris microTube back into the loading and unloading station 8 While keeping the snap cap on insert a pipette
49. ct 2 Automated Library Prep and Capture System 133 134 Reference Nucleotide Sequences of SureSelect Pre Capture Indexes Original Index Configuration The nucleotide sequence of each SureSelect Pre Capture Index provided with the original kit configuration is provided in the tables below Refer to the sequence information below only if your kit includes p n 5190 3936 and p n 5190 3937 with indexing primers provided in 96 individual clear capped tubes Each index is 8 nt in length Each index is 8 nt in length See page 121 for sequencing run setup information using 8 bp indexes Table 75 SureSelectX Pre capture Indexes 1 16 Index Number Sequence 1 AACGTGAT 2 AAACATCG 3 ATGCCTAA 4 AGTGGTCA 5 ACCACTGT 6 ACATTGGC 7 CAGATCTG 8 CATCAAGT 9 CGCTGATC 10 ACAAGCTA 11 CTGTAGCC 12 AGTACAAG 13 AACAACCA 14 AACCGAGA 15 AACGCTTA 16 AAGACGGA SureSelect 2 Automated Library Prep and Capture System Table 76 SureSelect 2 Pre capture Indexes 17 32 Index Number Sequence 17 AAGGTACA 18 ACACAGAA 19 ACAGCAGA 20 ACCTCCAA 21 ACGCTCGA 22 ACGTATCA 23 ACTATGCA 24 AGAGTCAA 25 AGATCGCA 26 AGCAGGAA 27 AGTCACTA 28 ATCCTGTA 29 ATTGAGGA 30 CAACCACA 31 CAAGACTA 32 CAATGGAA SureSelect 2 Automated Library Prep and Capture System Reference 6 135 6 Reference Table 77 SureSelect 2 Pre capture Indexes 33 48 Index Number Sequence 33 CACTTCGA 34 CAGCGTTA 35 CATACCAA 36 CCAGTTCA 37 CCGAAGTA 38 CCGTGA
50. dy for the runs to start If you are not ready to start a run immediately click Cancel cone Running the LibraryPrep_XT_Illumina_v2 0 rst runset takes approximately 3 hours Once complete the purified indexing adaptor ligated DNA samples are located in the Eppendorf twin tec plate at position 7 of the Bravo deck Stopping Point If you do not continue to the next step seal the plate and store at 4 C overnight or at 20 C for prolonged storage 50 SureSelect 2 Automated Library Prep and Capture System CAUTION Sample Preparation 3 Step 4 Amplify the indexed libraries In this step the Agilent NGS Workstation completes the liquid handling steps for amplification of the indexing adaptor ligated DNA samples Afterward you transfer the PCR plate to a thermal cycler for amplification In this protocol one half of the DNA sample is removed from the Eppendorf sample plate for amplification The remainder can be saved at 4 C for future use or amplification troubleshooting if needed Store the samples at 20 C for long term storage To avoid cross contaminating libraries set up PCR master mixes in a dedicated clean area or PCR hood with UV sterilization and positive air flow Prepare the workstation 1 Leave tip boxes on shelves 1 and 2 in cassette 1 of the Labware MiniHub from the previous LibraryPrep_XT_Illumina_v2 0 rst run Otherwise clear the remaining positions of the MiniHub and BenchCel of plates
51. e SureSelect Automated Library Prep and Capture System 97 4 Hybridization 5 Wash the magnetic beads a Inaconical vial combine the components listed in Table 47 The volumes below include the required overage Table 47 Components required for magnetic bead washing procedure Reagent Volume for Volumefor Volumefor Volumefor Volumefor Volumefor Volume for 1 Well 1 Column 2 Columns 3Columns 4 Columns 6 Columns 12 Columns Streptavidin bead 50 uL 425 uL 825 uL 1225 uL 1 65 mL 2 5 mL 5 0 mL suspension SureSelect XT2 0 2 mL 1 7 mL 3 3 mL 4 9 mL 6 6 mL 10 mL 20 mL Binding Buffer Total Volume 0 25 mL 2 125 mL 4 7125mL 6 125 mL 8 25 mL 12 5 mL 25 mL b Mix the beads on a vortex mixer for 5 seconds c Put the vial into a magnetic device such as the Dynal magnetic separator d Remove and discard the supernatant e Repeat step a through step d for a total of 3 washes Retain the beads after each wash and combine with a fresh aliquot of the indicated volume of SureSelect XT2 Binding Buffer 6 Resuspend the beads in SureSelect XT2 Binding Buffer according to Table 48 below Table 48 Preparation of magnetic beads for SureSelect Capture amp Wash_v2 0 rst Reagent Volume for Volumefor Volumefor Volumefor Volumefor Volumefor Volume for 1 Library 1 Column 2 Columns 3Columns 4 Columns 6 Columns 12 Columns SureSelect XT2 0 2 mL 1 7 mL 3 3 mL 4 9 mL 6 6 mL 10 mL 20 mL Binding Buffer 7 Prepare a Nunc DeepWell source
52. e Unseal the plate without tilting or jerking the plate to avoid sample splashing Make sure that the Agilent NGS Workstation is completely prepared with deck platforms at temperature and all components in place before you transfer the sample plate to the Bravo deck 24 When prompted by VWorks as shown below quickly remove the PCR plate containing the hybridization reactions held at 65 C from the thermal cycler Unseal the plate carefully to avoid splashing and quickly transfer the plate to position 4 of the Bravo deck seated in the red aluminum insert Click Continue to resume the runset Add Hyb Plate Complete the following steps as quickly as possible Retrieve Hybridization plate from thermocyder and place on carrier at Bravo position 4 and unseal Click Continue to resume protocol Use Caution Position 4 will be hot User data entry Pause and Diagnose Continue WARNING Bravo deck position 4 will be hot Use caution when handling components that contact heated deck positions 102 SureSelect 2 Automated Library Prep and Capture System Hybridization 4 25 When the hybridization samples have been transferred from the PCR plate to the capture plate wells you will be prompted by VWorks as shown below Remove the PCR plate from position 4 of the Bravo deck leaving the red insert in place When finished click Continue to resume the runset Update Bravo Deck Remove
53. e 1 Bravo platform deck SureSelect Automated Library Prep and Capture System Using the Agilent NGS Workstation for SureSelect Target Enrichment 2 Setting the Temperature of Bravo Deck Heat Blocks Bravo deck positions 4 and 6 are equipped with Inheco heat blocks used to incubate sample plates at defined temperatures during the run Runs that include high 85 C or low 4 C temperature incubation steps may be expedited by pre setting the temperature of the affected block before starting the run Bravo deck heat block temperatures may be changed using the Inheco Multi TEC Control device touchscreen as described in the steps below See Table 7 for designations of the heat block containing Bravo deck positions on the Multi TEC control device Table 7 Inheco Multi TEC Control touchscreen designations Bravo Deck Position Designation on Inheco Multi TEC Control Screen 4 CPAC 21 6 CPAC 22 1 Using the arrow buttons select the appropriate block CPAC 2 block 1 or CPAC 2 block 2 crac 2 lt gt 24 9 C 2 Shaker 0200 rpm set SureSelect Automated Library Prep and Capture System 19 2 Using the Agilent NGS Workstation for SureSelect Target Enrichment 2 To set the temperature of the selected block press the SET button CPAC 2 1 24 9 C Shaker 0200 rpm 3 Using the numeral pad enter the desired temperature The entered temperature appears in the top left rectangle Once the correct temper
54. e 41 Preparation of Capture Library Master Mix for target sizes gt 3 0 Mb single column of wells Target size gt 3 0 Mb SureSelect 2 Reagent Volume for 1 Well Nuclease free water 4 0 uL SureSelect RNase Block purple cap 0 5 pL SureSelect or ClearSeq Capture Library 5 0 uL Total Volume 9 5 pL Volume for 1 Column 39 0 pL 4 9 uL 48 8 uL 92 7 pL 88 SureSelect 2 Automated Library Prep and Capture System Hybridization 4 Prepare the master mix source plate 4 Ina Nunc DeepWell plate prepare the hybridization master mix source plate at room temperature Add the volumes indicated in Table 42 to the appropriate number of wells of the indicated row of the Nunc DeepWell plate Fill the number of wells that corresponds to the number of DNA sample columns in the run 1 2 3 4 6 or 12 As indicated in the shaded portion of Table 42 Blocking Mix and nuclease free water are combined in the wells of Row A When using multiple Capture Libraries in a run add each Capture Library Master Mix to the appropriate column s of the Nunc DeepWell plate The final configuration of the master mix source plate is shown in Figure 12 Table 42 Preparation of the Master Mix Source Plate for Hybridization MMRow_v2 0 pro Master Mix Solution Position on Source Volume of Master Plate Mix added per Well SureSelect XT2 Blocking Mix Row A 81 0 pL Nuclease free water A1 AX 22 5 uL Capture Library Master Mix Row B 92 7 uL B
55. e following plate configuration considerations for pooling gDNA samples for automated hybridization and capture runs e When using a single Capture Library for all wells on the plate fill the plate column wise in well order Al to H1 then A2 to H2 ending with Al2 to H12 SureSelect Automated Library Prep and Capture System Hybridization 4 e When using multiple Capture Libraries configure the plate such that all gDNA library pools to be hybridized to a particular Capture Library are positioned in appropriate rows or columns When using the Hybridization_MMCol_v2 0 pro protocol place samples to be enriched using the same library in the same row When using the Hybridization_MMRow_v2 0 pro protocol place samples to be enriched using the same library in the same column e Each 96 reaction library preparation run produces 6 or 12 gDNA pools For greatest efficiency of reagent use 3DNA pools from multiple library preparation runs may be placed on the same daughter plate for hybridization Prepare csv files for pooling and normalization Before starting the sample pooling automation protocol you must create comma separated value csv files containing instructions including the specific wells to be pooled and the concentration of each sample From this data the workstation calculates the volume of each sample required to prepare each concentration normalized pool for the Hybridization step See Figure 10 for required csv file cont
56. ed DNA and D1000 sample buffer on a vortex mixer for 5 seconds for accurate quantitation 4 Load the sample plate or tube strips from step 3 the D1000 ScreenTape and loading tips into the 2200 TapeStation as instructed in the Agilent 2200 TapeStation User Manual Start the run 5 Verify that the electropherogram shows an average DNA amplicon size of 250 to 300 bp A sample electropherogram is shown in Figure 9 6 Determine the DNA concentration ng uL by integrating under the peak Stopping Point If you do not continue to the next step seal the sheared DNA sample plate and store at 4 C overnight or at 20 C for prolonged storage MW Figure 9 Analysis of amplified DNA using the 2200 TapeStation 62 SureSelect Automated Library Prep and Capture System SureSelect 2 Automated Library Prep and Capture System Protocol ee 4 CAUTION 070 Hybridization Step 1 Pool indexed DNA samples for hybridization 64 Step 2 Hybridize the gDNA library and Capture Library 72 Step 3 Capture the hybridized DNA 97 This chapter describes the steps to pool indexed gDNA libraries and then hybridize the pooled gDNA libraries with a SureSelect or ClearSeq Capture Library Pools of 8 or 16 indexed samples are hybridized to the appropriate Capture Library and the targeted molecules are captured for sequencing See Table 27 for the recommended number of indexes per gDNA library pool for different types of Capture Librar
57. ed pond concentrations field enter the concentration in ng uL determined on page 61 for each indexed DNA sample In the Target WellID field enter the well position of the pool in which the indexed DNA sample should be included for the Hybridization plate See the guidelines on page 65 for Hybridization sample pool placement considerations Set up and run the PreCapture_Pooling_v1 0 pro automation protocol 1 Turn on the chiller set to 0 C at position 9 of the Bravo deck Be sure that the chiller reservoir contains at least 300 mL of 25 ethanol 2 Clear the Labware MiniHub and BenchCel of all plates and tip boxes 3 To set up the PreCapture_Pooling_v1 0 pro automation protocol open the VWorks Form XT2_Pooling VWForm using the shortcut on your desktop 68 SureSelect Automated Library Prep and Capture System Hybridization 4 Step 1 Pool indexed DNA samples for hybridization 4 In the Form enter the run information highlighted below e Under Controls specify whether the indexed DNA source plates will be loaded in the MiniHub and will be sealed at start of run recommended e From Number of Indexes to Pool menu select 8 or 16 see Table 27 for guidelines From Pooled DNA Quantity menu enter the required total amount of DNA in the pool typically 1500 ng See page 65 for guidelines e In Plate ID Barcode field enter the name or barcode of the daughter Hybridization sample plate From Number of Source Plates
58. eficial to consolidate the indexed library pools from multiple Library Prep runs to prepare full columns of samples for Hybridization runs and downstream workflow steps Table 9 Hybridization reaction numbers derived from each Library Prep run size Number of Columns Total Libraries Number of Hyb Reactions Processed in Library Prepared wi oe Prep Protocol Pools containing Pools containing 8 indexed libraries 16 indexed libraries 1 8 1 0 5 2 16 2 1 3 24 3 1 5 4 32 4 2 6 48 6 3 N co o N for SureSelect Automated Library Prep and Capture System 31 2 Using the Agilent NGS Workstation for SureSelect Target Enrichment Optimal reagent usage is obtained using Hybridization runs that include 3 6 or 12 columns Hybridization runs of this size result from processing indexed library samples from multiple 96 well plates in the same Hybridization run To determine the number of Library Prep reaction plates required for various Hybridization run sizes see Table 10 for All Exon captures and see Table 11 for all other captures Sample numbers required for optimal 3 6 and 12 column runs are highlighted in gray For greatest efficiency of reagent use plan experiments using at least 3 columns per run Each 2x96 reaction kit contains sufficient reagents for hybridization reactions configured as 1 run of 3 columns of samples per run Table 10 Sample number conversion using Exome libraries 8 sample pools Number of 96 Well T
59. elf 2 Empty Nuclease free AMPure XP beads Empty water reservoir in Nunc DeepWell from step 5 plate from step 4 Shelf 1 Bottom Empty 70 ethanol Empty Empty tip box reservoir from step 6 SureSelect 2 Automated Library Prep and Capture System Sample Preparation 3 8 Load the BenchCel Microplate Handling Workstation according to Table 25 Table 25 BenchCel configuration for SPRI_XT_Illumina_v2 0 pro Pre Capture PCR Cleanup No of Columns Rack 1 Rack 2 Rack 3 Rack 4 Processed 1 1 Tip box Empty Empty Empty 2 1 Tip box Empty Empty Empty 3 2 Tip boxes Empty Empty Empty 4 2 Tip boxes Empty Empty Empty 6 3 Tip boxes Empty Empty Empty 12 6 Tip boxes Empty Empty Empty 9 Load the Bravo deck according to Table 26 Table 26 Bravo deck configuration for SPRI_XT_Illumina_v2 0 pro Pre Capture PCR Cleanup Location Content 1 Empty waste reservoir Axygen 96 Deep Well Plate square wells 9 Amplified DNA libraries in unsealed PCR plate seated in red insert PCR plate type must be specified on setup form under step 2 Run VWorks protocol SPRI_XT_Illumina_v2 0 pro Pre Capture PCR Cleanup 10 On the SureSelect setup form under Select Protocol to Run select SPRI_XT_Ilumina_v2 0 pro Pre Capture PCR Cleanup 11 Under Select PCR plate labware for Thermal Cycling select the specific type of PCR plate that was loaded on Bravo deck position 9 SureSelect Automated Library Prep and Capture System 59 3 Sample Preparation
60. ent Pooling and normalization csv file templates are provided in the following directory C gt VWorks Workspace gt NGS Option B gt XT_IIlumina_2 0 gt Pooling and Normalization Templates Select the appropriate set of templates from the directory based on the intended pool composition 8 or 16 prepared samples and on the number of source plates to be consolidated in the run to prepare the single hybridization sample plate For example for 8 library pools use the template Pool8_01_SourcePlate csv for the first DNA source plate continuing with additional Pool8_0X_SourcePlate csv files for additional DNA source plates 1 Copy and rename the appropriate set of csv file templates for the run Make sure to retain the header text used in the template files without introducing spaces or other new characters If processing a partial plate of prepped gDNA samples delete the rows corresponding to the WellIDs of the empty wells on the plate SureSelect Automated Library Prep and Capture System 67 4 Hybridization A B G 1 WellID PreCap Amplified pond concentrations ng ul Target WelllD 2 A1 52 79 Al 3 B1 49 21 Al 4 C1 38 73 Al 5 D1 43 56 Al 6 E1 39 7 Al 7 F1 45 33 A1 8 Gl 53 38 A1 Sa H1 48 91 A1 10 A2 40 74 B1 11 B2 37 22 B1 ee eee ot a em i a Figure 10 Sample pooling and normalization csv file content 2 In each csv file edit the information for each DNA sample Well ID as follows e Inthe PreCap Amplifi
61. er lane according to the capacity of your platform together with the amount of sequencing required to achieve the needed coverage for your specific Capture Library for each indexed sample If doing post capture pooling use the guidelines provided in Step 6 Optional Pool captured libraries for sequencing on page 124 Prior to post capture pooling the DNA concentration of each sample may be accurately determined as described in Step 5 Optional Quantify captured library pools by QPCR on page 123 If samples will not be further combined in post capture pools proceed to cluster amplification using the Illumina Paired End Cluster Generation Kit Refer to the manufacturer s instructions for this step The optimal seeding concentration for SureSelect target enriched libraries is 6 to 8 pM on HiSeq or MiSeq instruments and 1 2 to 1 3 pM on the NextSeq platform Seeding concentration and cluster density may also need to be optimized based on the DNA fragment size range for the library and on the desired output and data quality Follow Ilumina s recommendation for a PhiX control in a low concentration spike in for improved sequencing quality control Sequencing run setup guidelines Sequencing runs must be set up to perform an 8 bp index read For complete 8 bp index sequence information see the Reference chapter starting on page 127 SureSelect Automated Library Prep and Capture System 121 5 Post Capture Sample Proces
62. fer to the Illumina protocol Preparing Samples for Multiplexed Paired End Sequencing p n1005361 or the appropriate Illumina protocol for more information oie Agilent Technologies 37 3 38 Sample Preparation Step 1 Shear DNA Before you begin you can use the SureSelect gDNA Extraction Kit to extract genomic DNA Refer to the gDNA Extraction Kit Protocol p n 5012 8701 Make sure genomic DNA samples are of high quality with an OD 260 280 ratio ranging from 1 8 to 2 0 For each DNA sample to be sequenced prepare 1 library 1 Use the Qubit dsDNA BR Assay to determine the concentration of your gDNA sample Follow the instructions for the instrument 2 Dilute 1 ug of high quality gDNA with 1X Low TE Buffer in a 1 5 mL LoBind tube to a total volume of 50 uL 3 Set up the Covaris E series or S series instrument a Check that the water in the Covaris tank is filled with fresh deionized water to the appropriate fill line level according to the manufacturer s recommendations for the specific instrument model and sample tube or plate in use b Check that the water covers the visible glass part of the tube c On the instrument control panel push the Degas button Degas the instrument for least 2 hours before use or according to the manufacturer s recommendations d Set the chiller temperature to between 2 C to 5 C to ensure that the temperature reading in the water bath displays 5 C e Optional Supplement the
63. g 10 nM DNA Component V f C i C f Volume to use pL Sample 1 20 pL 20 nM 10 nM 6 5 0 Sample 2 20 pL 15 nM 10 nM 6 6 7 Low TE 8 3 3 Ifyou store the library before sequencing add Tween 20 to 0 1 v v and store at 20 C short term Proceed to cluster amplification using the lumina Paired End Cluster Generation Kit refer to the manufacturer s instructions for this step The optimal seeding concentration for cluster amplification from SureSelect DNA libraries is approximately 6 to 8 pM The optimal seeding concentration may vary depending on the method used for library quantification and fragment size distribution See page 121 for sequencing run setup guidelines for SureSelect libraries SureSelect Automated Library Prep and Capture System 125 5 Post Capture Sample Processing for Multiplexed Sequencing Step 6 Optional Pool captured libraries for sequencing 126 SureSelect 2 Automated Library Prep and Capture System SureSelect 2 Automated Library Prep and Capture System Protocol ee 6 070 Reference z Reference Information for Kits with Revised Index Configuration indexing primers in blue plate 128 Reference Information for Kits with Original Index Configuration indexing primers in clear capped tubes 132 This chapter contains reference information including component kit contents and index sequences ot Agilent Technologies 127 6 Reference CAUTION This chapter contains two se
64. gDNA solutions Possible stopping points where gDNA samples may be stored overnight at 4 C are marked in the protocol When storing samples for gt 24 hours store the samples at 20 C but do not subject the samples to multiple freeze thaw cycles When preparing reagent stock solutions for use 1 Thaw the aliquot as rapidly as possible without heating above room temperature 2 Mix briefly on a vortex mixer then spin in a centrifuge for 5 to 10 seconds to drive the contents off of walls and lid Store vials used during an experiment on ice or in a cold block 4 If reagents will be used for multiple experiments aliquot to multiple vials to minimize freeze thaw cycles for each vial In general follow Biosafety Level 1 BL1 safety rules Safety Notes CAUTION Wear appropriate personal protective equipment PPE when working in the laboratory 10 SureSelect Automated Library Prep and Capture System Required Reagents Table 1 Before You Begin 1 Required Reagents for SureSelect Automated Target Enrichment Description Vendor and part number SureSelect or ClearSeq Capture Library SureSelect Automation Reagent Kit HiSeq platform HSQ 96 Samples HiSeq platform HSQ 480 Samples MiSeq platform MSQ 96 Samples MiSeq platform MSQ 480 Samples Nuclease free Water not DEPC treated 1X Low TE Buffer 10 mM Tris HCl pH 8 0 0 1 mM EDTA Agencourt AMPure XP Kit 60 mL 450 mL Dyn
65. ialization Ignore and Continue leaving device in current state Abort SureSelect 2 Automated Library Prep and Capture System Using the Agilent NGS Workstation for SureSelect Target Enrichment 2 Verifying the Simulation setting VWorks software may be run in simulation mode during which commands entered on screen are not completed by the NGS workstation If workstation devices do not respond when you start a run verify the simulation mode status in VWorks using the following steps 1 Verify that Simulation is off is displayed on the status indicator accessible by clicking View gt Control Toolbar 2 Ifthe indicator displays Simulation is on click the status indicator button to turn off the simulation mode g amp Log out compie D Start I pause a x Diagnostics B If you cannot see the toolbar above the VWorks form click Full Screen on off to exit full screen mode If the toolbar is still not visible right click on the form and then select Control Toolbar from the menu Finishing a protocol or runset The window below appears when each run is complete Click Yes to release the BenchCel racks to allow removal of components used in the current run in preparation for the next pro or rst run r Protocol complete 2 Release stacker racks used in protocols f e e SureSelect Automated Library Prep and Capture System 27 2 Using the Agilent NGS Workstation for Su
66. ies The size of your SureSelect or ClearSeq Capture Library determines the post capture amplification cycle number See Table 57 for cycle number recommendations for different Capture Library sizes Plan your experiments for capture using similar sized Capture Libraries on the same plate to facilitate post capture amplification The ratio of Capture Library to indexed gDNA library is critical for successful capture CAUTION You must avoid evaporation from the small volumes of the capture during the 24 hour or greater incubation If you want to use a duration of hybridization gt 24 hours first test the conditions Incubate 60 uL of SureSelect XT2 Hybridization Buffer without DNA at 65 C for 24 hours or longer if applicable as a test Include buffer in each well that you might use including those in the center and those on the edges Check that you do not get extensive evaporation Evaporation should not exceed 6 to 8 uL Be Agilent Technologies 63 4 64 Hybridization Step 1 Pool indexed DNA samples for hybridization In this step the workstation pools the prepped indexed gDNA samples before hybridization to the SureSelect or ClearSeq Capture Library This workflow step is set up using the VWorks Form XT2_Pooling VWForm shown below Controls Load Sources To MiniHub Manually Sources Enter Sealed Yes No Show Run ann XT2 Initialize all Full Screen Display SureSelect On Off Gantt Chart Pool
67. in Nunc DeepWell eRe Met Applicable Shelf1 New Tip Box 70 Ethanol g g Empty Tip Box 2 Select PCR Plate labware for Thermal Cycling Reservoir 96 ABI PCR half skirt in Red Alum Insert d Bravo Deck o 3 Select Number of Columns of Samples E EN A 4 Click button below to Display Initial Workstation Setup oo Eta lt Position 1 gt lt Position 2 gt lt Position 3 gt Display Initial Clear Workstation Waste Reservoir Workstation Setup Setup Display Axygen 96DW 5 Load labware according to Workstation Setup gt lt Pos 4 Peltier gt RT lt Pos 5 Shaker gt lt Pos 6 Peltier gt 4 C Controls Empty Eppendorf Twin tec Plat Once you have loaded labware according to Workstation RETER Setup on right click Run Selected Protocol to start run Run Selected lt Pos 7 Magnetic gt lt Position 8 gt lt Pos 9 Chiller gt 0 C Aree D Pause Eea Sheared DNA Plate Nunc Master Mix Twin tec Plate Col 1 3 on Full Screen Gantt Chart Elapsed Time 00 00 00 Silver Insert Reset All Form Selections to Defaults BenchCel Information BenchCel Stacker 1 Benchcel Stacker 2 BenchCel Stacker 3 BenchCel Stacker 4 Currently Running Protocol 2 Tip Boxes Empty Empty Empty 5 After verifying that the NGS Workstation has been set up correctly click Run Selected Protocol Run Selected Protocol 24 SureSelect 2 Automated Library Prep and Capture Sy
68. ing and Normalization Pooling Options Destination Plate Number of Indexes to Pool f3 X Plate ID Barcode podon Quit incl ETE re 1500ng Recommended Source Plates Number of Source Plates aoo o Concentration File ID Barcode Pate mae Pate 5 e Workstation Setup MiniHub Bravo Deck a E os Pate 7 es cesee el EEE Eee eee SureSelect 2 Automated Library Prep and Capture System Hybridization 4 Plan pooling run parameters The Hybridization reaction requires 1500 ng indexed gDNA made up of a pool containing equal amounts of 8 or 16 individual libraries See Table 27 for the recommended pool composition based on your SureSelect or ClearSeq Capture Library Where possible indexed DNA pools are prepared containing a total DNA amount of 1500 ng For some indexed DNA pools the initial library pool will contain gt 1500 ng DNA as detailed below with 1500 ng of the pooled DNA added to the Hybridization reaction at a later step Table 27 Pre capture pooling of indexed DNA libraries Capture Library Number of indexed Amount of each indexed gDNA libraries per pool gDNA library in pool SureSelect Custom Capture Libraries 16 93 75 ng ClearSeq Comprehensive Cancer 16 93 75 ng ClearSeq DNA Kinome 16 93 75 ng SureSelect Human or Mouse All Exon 8 187 5 ng SureSelect Clinical Research Exome 8 187 5 ng SureSelect Focused Exome 8 187 5 ng ClearSeq Inherited Disease 8 187 5 ng
69. ion 4 of the Bravo deck seated in the red insert Click Continue r Place DNA plate on Bravo Complete the following steps as quickly as possible Retrieve DNA plate from thermocycler and place on carrier at Bravo position 4 and unseal Click Continue to resume protocol Use Caution Position 4 will be hot User data entry Pause and Diagnose Continue WARNING Bravo deck position 4 will be hot Use caution when handling components that contact heated deck positions The Agilent NGS Workstation transfers the capture library hybridization buffer mixture to the wells of the PCR plate containing the mixture of indexed gDNA pools and blocking agents SureSelect Automated Library Prep and Capture System 83 4 Hybridization CAUTION 22 When prompted by VWorks as shown below quickly remove the PCR sample plate from Bravo deck position 4 leaving the red insert in place a Remove Plate from 4 Quickly remove plate from position 4 seal and place in thermocycler Click Continue after plate is in thermocycler for protocol to finish User data entry Pause and Diagnose Continue 23 Seal the sample plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 3 0 sec 24 Quickly transfer the plate back to the thermal cycler held at 65 C Place a compression mat over the PCR plate in the thermal cycler After transferring the plate click Continue on
70. ion for Kits with Original Index Configuration indexing primers in clear capped tubes Use the reference information in this section if your kit includes Pre Capture Indexes in tube format in p n 5190 3936 and p n 5190 3937 If your kit does not include these component kits see page 128 for kit content and indexing primer information Kit Contents SureSelect Automation Reagent Kits contain the following component kits Table 70 SureSelect Automation Reagent Kit Content Original Index Configuration Component Kits Storage Condition G9661B G9661C 96 Samples 480 Samples SureSelect XT2 Library Prep Kit ILM 20 C 5500 0103 5 x 5500 0103 SureSelect XT2 Pre Capture Indexes ILM 20 C 5190 3936 and 5 x 5190 3936 and 5190 3937 5 x 5190 3937 SureSelect XT2 Pre Capture Box 1 Room Temperature 5190 4076 5190 4077 SureSelect XT2 Pre Capture Automation ILM Module Box 2 20 C 5190 4462 5190 4463 See Table 71 through Table 74 for a list of reagents included in each component kit t Kits contain reagents to prepare indexed libraries from 96 gDNA samples and to enrich the samples in 6 or 12 hybridization and capture reactions as appropriate for the specific Capture Library size and sample pooling format Kits contain reagents to prepare indexed libraries from 480 gDNA samples and to enrich the samples in 30 or 60 hybridization and capture reactions as appropriate for the specific Capture Library size and sample
71. ip box reservoir from step 7 9 Load the BenchCel Microplate Handling Workstation according to Table 59 Table 59 Initial BenchCel configuration for DNA cleanup using SPRI_XT_Illumina_v2 0 pro Post CaptureOnBeadPCR Cleanup No of Columns Rack 1 Rack 2 Rack 3 Rack 4 Processed 1 1 Tip box Empty Empty Empty 2 1 Tip box Empty Empty Empty 3 2 Tip boxes Empty Empty Empty 4 2 Tip boxes Empty Empty Empty 6 3 Tip boxes Empty Empty Empty 12 6 Tip boxes Empty Empty Empty SureSelect 2 Automated Library Prep and Capture System Post Capture Sample Processing for Multiplexed Sequencing 5 10 Load the Bravo deck according to Table 60 Table 60 Initial Bravo deck configuration for DNA cleanup using SPRI_XT_Illumina_v2 0 pro Post CaptureOnBeadPCR Cleanup Location Content 1 Empty waste reservoir Axygen 96 Deep Well Plate square wells 9 Amplified captured library pools in unsealed PCR plate seated in red insert PCR plate type must be specified on setup form under step 2 Run VWorks protocol SPRI_XT_Illumina_v2 0 pro Post CaptureOnBeadPCR Cleanup 11 On the SureSelect setup form under Select Protocol to Run select SPRI_XT_Illumina_v2 0 pro Post CaptureOnBeadPCR Cleanup 12 Under Select PCR plate labware for Thermal Cycling select the specific type of PCR plate that was loaded on Bravo deck position 9 13 Select the number of columns of samples to be processed Runs must include 1 2 3 4 6 or 12 columns 14 Click
72. kirted plates MicroAmp Optical Life Technologies p n N8010560 plates 96 Agilent semi skirted PCR plate Agilent p n 401334 96 Eppendorf Twin tec half skirted PCR plates Eppendorf p n 951020303 96 Eppendorf Twin tec PCR plates full skirted Eppendorf p n 951020401 or 951020619 36 SureSelect 2 Automated Library Prep and Capture System SureSelect 2 Automated Library Prep and Capture System Protocol c D ee 3 7 e Sample Preparation J e Step 1 Shear DNA 38 Step 2 Assess sample quality and DNA fragment size 41 Step 3 Prepare indexed gDNA library samples 43 Step 4 Amplify the indexed libraries 51 Step 5 Purify amplified DNA using AMPure XP beads 58 Step 6 Assess Library DNA quantity and quality 61 This section contains instructions for indexed gDNA library preparation specific to the Illumina multiplexed paired end sequencing platform and to automated processing using the Agilent NGS Workstation For each sample to be sequenced an individual indexed library is prepared See the Reference chapter starting on page 127 for sequences of the index portion of the indexing adaptors ligated to gDNA libraries in this section The steps in this section differ from the Illumina protocol in the use of the Covaris system for gDNA shearing smaller target shear size elimination of size selection by gel purification implementation of AMPure XP beads SPRI beads for all purification steps and primers used for PCR Re
73. l cycler This plate will be transferred from the thermal cycler to the NGS workstation in step 24 below SureSelect 2 Automated Library Prep and Capture System Hybridization 19 Select the number of columns of samples to be processed Runs must include 1 2 3 4 6 or 12 columns 20 Click Display Initial Workstation Setup Display Initial Workstation Setup 21 Verify that the NGS workstation has been set up as displayed in the Workstation Setup region of the form Workstation Setup MiniHub MiniHub Cassette 1 MiniHub Cassette 2 MiniHub Cassette 3 MiniHub Cassette 4 Aand PS Men eae om Oe ee eee Oe e ON K a Lee 2 ae ae een 22 When verification is complete click Run Selected Protocol Run Selected Protocol 23 When ready to begin the run click OK in the following window If the temperature of Bravo deck position 4 was not pre set to 66 C the runset will pause while position 4 reaches temperature E VWorks G This runset contains protocols that will start running as soon as S possible Before you click OK verify that the system is ready for the runs to start If you are not ready to start a run immediately click Cancel cnet SureSelect 2 Automated Library Prep and Capture System 101 4 4 Hybridization CAUTION It is important to complete step 24 quickly and carefully Transfer the sample plate to the Bravo platform quickly to retain the 65 C sample temperatur
74. le denaturation prior to hybridization 92 SureSelect 2 Automated Library Prep and Capture System Hybridization 4 16 When prompted by VWorks as shown below remove the PCR plate from position 4 of the Bravo deck leaving the red insert in place Remove plate Remove plate from carrier seal and place in thermocycler User data entry Pause and Diagnose Continue 17 Seal the sample plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 3 0 sec 18 Transfer the sealed plate to a thermal cycler and run the following program shown in Table 37 After transferring the plate click Continue on the VWorks screen Table 45 Thermal cycler program used for sample denaturation prior to hybridization Step Temperature Time Step 1 95 C 5 minutes Step 2 65 C Hold While the sample plate incubates on the thermal cycler the Agilent NGS Workstation combines aliquots of the Capture Library master mix and Hybridization Buffer SureSelect 2 Automated Library Prep and Capture System 93 4 Hybridization CAUTION You must complete step 19 to step 23 quickly and immediately after being prompted by the VWorks software It is important that sample temperature remains approximately 65 C during transfers between the Agilent NGS Workstation and thermal cycler 19 When the workstation has finished aliquoting the Capture Library master mixes and Hybridization Buffer you will be prom
75. mple plate be sealed then centrifuged to collect any dispersed liquid before being transfered between instruments To maximize efficiency locate the PlateLoc thermal microplate sealer and the centrifuge in close proximity to the Agilent NGS Workstation and thermal cycler SureSelect 2 Automated Library Prep and Capture System 35 2 Using the Agilent NGS Workstation for SureSelect Target Enrichment PCR Plate Type Considerations Automation protocols include several liquid handling steps in which reagents are dispensed to PCR plates in preparation for transfer to a thermal cycler For these steps you must specify the PCR plate type to be used on the SureSelect_RNA_ILM VWForm to allow correct configuration of the liquid handling components for the PCR plate type Before you begin the automation protocol make sure that you are using a supported PCR plate type The PCR plate type to be used in the protocol is specified using the menu below Vendor and part number information is provided for the supported plate types in Table 12 2 Select PCR Plate labware for Thermal Cycling 96 ABI PCR half skirt in Red Alum Insert 3 Eas half skirt in Red Alum Insert 96 Agilent Semi skirted PCR in Red Alum Insert 96 Eppendorf Twin tec half skirt PCR in Red Alum Insert 4 96 Eppendorf Twin tec PCR in Red Alum Insert Table 12 Ordering information for supported PCR plates Description in VWorks menu Vendor and part number 96 ABI PCR half s
76. ning library template SureSelect 2 Automated Library Prep and Capture System 57 3 58 Sample Preparation Step 5 Purify amplified DNA using AMPure XP beads In this step the Agilent NGS Workstation transfers AMPure XP beads and amplified adaptor ligated DNA to a Nunc DeepWell plate and then collects and washes the bead bound DNA Prepare the workstation and reagents 1 2 Clear the Labware MiniHub and BenchCel of all plates and tip boxes Verify that the AMPure XP bead suspension is at room temperature If necessary allow the bead solution to come to room temperature for at least 30 minutes Mix the bead suspension well so that the reagent appears homogeneous and consistent in color Do not freeze Prepare a Nunc DeepWell source plate for the beads by adding 65 uL of homogeneous AMPure XP beads per well for each well to be processed Prepare a Thermo Scientific reservoir containing 15 mL of nuclease free water Prepare a separate Thermo Scientific reservoir containing 45 mL of freshly prepared 70 ethanol Load the Labware MiniHub according to Table 24 using the plate orientations shown in Figure 6 Table 24 MiniHub configuration for SPRI_XT_IIlumina_v2 0 pro Pre Capture PCR Cleanup Vertical Shelf Cassette 1 Cassette 2 Cassette 3 Cassette 4 Position Shelf 5 Top Empty Nunc Empty Empty Empty DeepWell plate Shelf 4 Empty Empty Empty Empty Shelf 3 Empty Empty Eppendorf Empty Empty twin tec plate Sh
77. ntification Kit p n G4880A for more details to do this step 1 Prepare a standard curve using the quantification standard included in the kit according to the instructions provided in the user guide 2 Dilute each captured library pool such that it falls within the range of the standard curve Typically this corresponds to approximately a 1 1000 to 1 10 000 dilution of the captured DNA 3 Prepare the QPCR master mix with Illumina adaptor specific PCR primers according to instructions provided in the kit 4 Add an aliquot of the master mix to PCR tubes and add template 5 Ona QPCR system such as the Mx3005p run the thermal profile outlined in the QPCR NGS Library Quantification kit user guide Use the SYBR Green instrument setting 6 Use the standard curve to determine the concentration of each unknown captured library pool in nM The concentration will be used to accurately pool samples for multiplexed sequencing In most cases the cycle numbers in Table 57 will produce an adequate yield for sequencing without introducing bias or non specific products If yield is too low or non specific products are observed adjust the number of cycles accordingly with the remaining captured DNA template SureSelect Automated Library Prep and Capture System 123 5 Post Capture Sample Processing for Multiplexed Sequencing Step 6 Optional Pool captured libraries for sequencing See page 121 for post capture pooling considerations ba
78. number of columns of samples to be processed Runs must include 1 2 3 4 6 or 12 columns 14 Click Display Initial Workstation Setup Display Initial Workstation Setup 15 Verify that the NGS workstation has been set up as displayed in the Workstation Setup region of the form Workstation Setup MiniHub Cassette 1 MiniHub Cassette 2 MiniHub Cassette 3 MiniHub Cassette 4 el A lion ee ee ee Ae ee ke eee Me T 110 SureSelect Automated Library Prep and Capture System Post Capture Sample Processing for Multiplexed Sequencing 5 16 When verification is complete click Run Selected Protocol a Run Selected Protocol Running the Post CaptureOnBeadPCR_XT_Illumina_v2 0 pro protocol takes approximately 15 minutes Once complete the PCR ready samples containing captured DNA and PCR master mix are located in the PCR plate at position 6 of the Bravo deck The Eppendorf plate containing the remaining captured DNA samples which may be stored for future use at 20 C is located at position 5 of the Bravo deck 17 When you see the following prompt remove the PCR plate from position 6 of the Bravo deck and seal the plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 3 0 seconds r Plate ready to seal Seal PCR plate and run thermocyder protocol User data entry Pause and Diagnose Continue 18 Centrifuge the plate for 30 seconds to drive the well contents off the
79. ocessed Runs must include 1 2 3 4 6 or 12 columns 14 Click Display Initial Workstation Setup Display Initial S Workstation Setup SureSelect 2 Automated Library Prep and Capture System 79 4 Hybridization 15 Verify that the NGS workstation has been set up as displayed in the Workstation Setup region of the form ee oe oY ore Workstation Setup MiniHub MiniHub Cassette 1 MiniHub Cassette 2 MiniHub Cassette 3 MiniHub Cassette 4 Pe Oe a Oe ee A ee ee eT ee andanan 16 When verification is complete click Run Selected Protocol A Run Selected Protocol The Agilent NGS Workstation transfers Blocking Mix and indexed gDNA pools to the PCR plate When this process is complete you will be prompted to transfer the plate to the thermal cycler for sample denaturation prior to hybridization SureSelect 2 Automated Library Prep and Capture System Hybridization 4 17 When prompted by VWorks as shown below remove the PCR plate from position 4 of the Bravo deck leaving the red insert in place Remove plate Remove plate from carrier seal and place in thermocycler User data entry Pause and Diagnose Continue 18 Seal the sample plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 3 0 sec 19 Transfer the sealed plate to a thermal cycler and run the following program shown in Table 37 After transferring the plate click Continue on the VWorks screen
80. omponents by pipetting Keep the master mixes on ice during preparation and aliquoting Each row of the indexed gDNA pool plate may be hybridized to a different Capture Library However Capture Libraries of different sizes require different post capture amplification cycles Plan experiments such that similar sized libraries are hybridized on the same plate For runs that use a single Capture Library for all rows of the plate prepare the master mix as described in Step a Table 29 or Table 30 below For runs that use different Capture Libraries for individual rows prepare each master mix as described in Step b Table 31 or Table 32 below SureSelect 2 Automated Library Prep and Capture System 73 4 Hybridization a For runs that use a single Capture Library for all rows prepare the Capture Library Master Mix as listed in Table 29 or Table 30 based on the Mb target size of your design Table 29 Preparation of Capture Library Master Mix for target sizes lt 3 0 Mb same Capture Library for all 8 rows of wells Target size lt 3 0 Mb SureSelect 2 Reagent Volume for 1 Well Volume for 12 Columns of Wells Nuclease free water 7 0 uL 808 5 uL SureSelect RNase Block purple cap 0 5 pL 57 8 uL SureSelect or ClearSeq Capture Library 2 0 pL 231 0 pL Total Volume 9 5 pL 1097 3 pL Table 30 Preparation of Capture Library Master Mix for target sizes gt 3 0 Mb same Capture Library for all 8 rows of wells Target size gt 3 0 Mb
81. ons 480 Reactions ClearSeq Comprehensive Cancer XTZ 5190 8019 5 x 5190 8019 ClearSeq Comprehensive Cancer Plus XT2 5190 8022 5 x 5190 8022 ClearSeq Inherited Disease XT2 5190 7526 5 x 5190 7526 ClearSeq Inherited Disease Plus xTa2t 5190 7529 5 x 5190 7529 ClearSeq DNA Kinome XT2 5190 4678 5190 4680 Sixteen gDNA samples are enriched in one capture reaction after sample pooling Capture Libraries are provided for the number of capture reactions needed to enrich the indicated number of samples t Eight gDNA samples are enriched in one capture reaction after sample pooling Capture Libraries are provided for the number of capture reactions needed to enrich the indicated number of samples SureSelect 2 Automated Library Prep and Capture System 13 1 14 Before You Begin Required Equipment Table 4 Required Equipment for SureSelect Automated Target Enrichment Description Agilent NGS Workstation Option B with VWorks software version 11 3 0 1195 Robotic Pipetting Tips Sterile Filtered 250 uL Eppendorf twin tec full skirted 96 well PCR plates Thermo Scientific Reservoirs Nunc DeepWell Plates sterile 1 3 mL well volume Axygen 96 Deep Well Plate 2 2 mL Square Well waste reservoirs Thermal cycler and accessories PCR plates compatible with selected Thermal Cycler e g Agilent semi skirted PCR plate for the SureCycler 8800 Thermal Cycler See page 36 for a list of supported PCR plates for automation protocols
82. ooling in the run For example if the highest DNA sample concentration is 100 ng uL then the final DNA pool will contain 200 ng of each indexed DNA Next determine the total amount of DNA per pool based on the Capture Library size Continuing with the same example an All Exon capture pool would contain 8 x 200 ng or 1600 ng DNA 2 Determine the appropriate daughter plate type based on DNA pool volumes First calculate the volume of each indexed DNA sample to be pooled using the concentration values for each sample and the amount of each DNA sample per pool from step 1 above Next calculate the expected total pool volume for each indexed DNA pool included on the daughter plate a Ifthe volume for all pools in the run is lt 180 uL then use an Eppendorf twin tec plate as the daughter destination plate for the pooling protocol This plate will be used directly as indexed DNA pool source plate in the Hybridization protocol If the volume for any pool in the run is gt 180 uL then use a Nunc DeepWell plate as the daughter destination plate for the pooling protocol After pool volumes are standardized see page 71 the indexed DNA pools must be transferred to an Eppendorf twin tec plate for the Hybridization protocol Plan daughter indexed DNA pool sample plate configuration The indexed gDNA samples should be pooled into the daughter plate using a pooled sample configuration appropriate for the subsequent Hybridization run Use th
83. otal Indexed Number of Columns of Samples Plates Processed Libraries Prepared Hybridization in Hybridization though Library Prep Reactions Protocol 1 96 12 15 2 192 24 3 3 288 36 4 5 4 384 48 6 5 480 60 7 5 6 576 72 gt 7 672 84 10 5 8 768 96 12 Not a valid run size Hybridization runs should include 1 2 3 4 6 or 12 complete columns of samples t When planning a run using 6 plates of gDNA samples to generate 9 columns of Hybridization samples split the Hybridization samples into one 6 column plate and one 3 column plate 32 SureSelect 2 Automated Library Prep and Capture System Using the Agilent NGS Workstation for SureSelect Target Enrichment 2 Table 11 Sample number conversion using 16 sample pools Number of 96 Well Total Indexed Number of Columns of Samples in Plates Processed Libraries Prepared Hybridization Hybridization Protocol though Library Prep Reactions 1 96 6 0 75 2 192 12 1 5 3 288 18 2 25 4 384 24 3 5 480 30 3 75 6 576 36 4 5 7 672 42 5 25 8 768 48 6 9 864 54 6 75 10 960 60 7 5 11 1056 66 8 25 12 1152 72 gt 13 1248 78 9 75 14 1344 84 10 5 15 1440 90 11 25 16 1536 96 12 Not a valid run size Hybridization runs should include 1 2 3 4 6 or 12 complete columns of sam ples t When planning a run using 12 plates of gDNA samples to generate 9 columns of Hybridization samples Split the Hybridization samples into one 6 column plate and one 3 column
84. p the indexed gDNA Capture Library hybrids are captured using streptavidin coated magnetic beads This step is run immediately after the 24 hour hybridization period This step is automated by the NGS workstation using the SureSelectCapture amp Wash_v2 0 rst runset with a total duration of approximately 2 hours A workstation operator must be present to complete two actions during the runset at the time points in the table below The times provided are approximate each action is completed in response to a VWorks prompt at the appropriate time in the runset Table 46 Operator action Approximate time after run start Transfer hybridization reaction plate lt 5 minutes from thermal cycler to NGS workstation Remove hybridization plate from 5 10 minutes position 4 after reactions transferred to capture plate Prepare the workstation 1 Clear the Labware MiniHub and BenchCel of all plates and tip boxes 2 Gently wipe down the Labware MiniHub Bravo decks and BenchCel with a NucleoClean decontamination wipe 3 Pre set the temperature of Bravo deck position 4 to 66 C using the Inheco Multi TEC control touchscreen as described in Setting the Temperature of Bravo Deck Heat Blocks On the Multi TEC control touchscreen Bravo deck position 4 corresponds to CPAC 2 position 1 Prepare the streptavidin coated beads 4 Vigorously resuspend the Dynal MyOne Streptavidin T1 magnetic beads on a vortex mixer Dynal beads settle during storag
85. plate SureSelect Automated Library Prep and Capture System 33 2 Using the Agilent NGS Workstation for SureSelect Target Enrichment Considerations for Placement of gDNA Samples in 96 well Plates for Automated Processing e The Agilent NGS Workstation processes samples column wise beginning at column 1 gDNA samples should be loaded into 96 well plates column wise in well order Al to H1 then A2 to H2 ending with A12 to H12 When processing partial runs with lt 12 sample columns do not leave empty columns between sample columns always load the plate using the left most column that is available e Samples are indexed during the LibraryPrep_XT_Illumina_v2 0 rst runset using indexing adaptors supplied in the corresponding well on a separate plate Assign the gDNA sample wells to be indexed with their respective indexing primers during experimental design Considerations for Indexed DNA Sample Placement for Automated Hybridization and Post Hybridization Processing Indexed DNA samples are pooled before the hybridization step see Figure 2 and captured DNA samples may be pooled again when preparing samples for sequencing It is important to develop a pooling strategy that is compatible with the specific Capture Library sizes and sequencing goals of the experiment using the following considerations e At the hybridization step see Figure 2 you can add a different SureSelect or ClearSeq Capture Library to different rows or columns of
86. plate for the washed bead suspension For each well to be processed add 200 uL of the homogeneous bead suspension to the Nunc DeepWell plate 8 Place the streptavidin bead source plate at position 5 of the Bravo deck 98 SureSelect Automated Library Prep and Capture System Hybridization 4 Prepare capture and wash solution source plates 9 Prepare an Eppendorf twin tec source plate labeled Wash 1 For each well to be processed add 160 uL of SureSelect XT2 Wash 1 10 Prepare a Nunc DeepWell source plate labeled Wash 2 For each well to be processed add 1150 uL of SureSelect XT2 Wash 2 11 Place the silver Nunc DeepWell plate insert on position 6 of the Bravo deck This insert is required to facilitate heat transfer to DeepWell source plate wells during the runset 12 Place the Wash 2 source plate on the silver insert at position 6 of the Bravo deck Make sure the plate is seated properly on the silver DeepWell insert 13 Prepare a Thermo Scientific reservoir containing 20 mL of nuclease free water Load the Agilent NGS Workstation 14 Load the Labware MiniHub according to Table 49 using the plate orientations shown in Figure 6 Table 49 Initial MiniHub configuration for SureSelect Capture amp Wash_v2 0 rst Vertical Shelf Cassette 1 Cassette 2 Cassette 3 Cassette 4 Position Shelf 5 Top Empty Empty Empty Empty Shelf 4 Empty Empty Empty Empty Shelf 3 Empty Eppendorf Empty Wash 1 Empty twin tec plate Eppendorf twin
87. pooling format See Table 75 on page 134 through Table 80 on page 139 for index sequence information SureSelect reagents and Capture Libraries must be used within one year of receipt 132 SureSelect 2 Automated Library Prep and Capture System Reference 6 The contents of each of the component kits listed in Table 70 are described in the tables below Table 71 SureSelect XT2 Library Prep Kit ILM Content Original Index Configuration Kit Component Format SureSelect End Repair Master Mix bottle SureSelect dA Tailing Master Mix bottle SureSelect Ligation Master Mix tube with purple cap SureSelect Herculase II Master Mix bottle XT2 Primer Mix tube with clear cap Table 72 SureSelect XT2 Pre Capture Indexes Content Original Index Configuration Kit Component Format Indexes 1 48 48 clear capped tubes supplied in component kit 5190 3936 Indexes 49 96 48 clear capped tubes supplied in component kit 5190 3937 Table 73 SureSelect XT2 Pre Capture Box 1 Content Kit Component Format SureSelect XT2 Binding Buffer bottle SureSelect XT2 Wash 1 bottle SureSelect XT2 Wash 2 bottle Table 74 SureSelect XT2 Pre Capture Automation ILM Module Box 2 Content Kit Component 96 Sample Kit 480 Sample Kit SureSelect XT2 Blocking Mix tube with blue cap tube with blue cap SureSelect XT2 Hybridization Buffer tube with yellow cap bottle SureSelect RNase Block tube with purple cap tube with purple cap SureSele
88. provided with the Agilent NGS Workstation Option B Agilent Technologies Inc 2015 Version B1 June 2015 G9450 90000 oe Agilent Technologies
89. pted by VWorks as shown below When the thermal cycler reaches the 65 C hold step click Continue Leave the sample plate in the thermal cycler until you are notified to move it Wait for plate in thermocycler When thermocycler has reached hold step at 65C click Continue Leave DNA plate in thermocycler until you are prompted to transfer the plate f User data entry Pause and Diagnose Continue 94 SureSelect 2 Automated Library Prep and Capture System Hybridization 4 20 When prompted by VWorks as shown below quickly remove the sample plate from the thermal cycler unseal the plate carefully to avoid splashing and transfer the plate to position 4 of the Bravo deck seated in the red insert Click Continue r Place DNA plate on Bravo Complete the following steps as quickly as possible Retrieve DNA plate from thermocycler and place on carrier at Bravo position 4 and unseal Click Continue to resume protocol Use Caution Position 4 will be hot User data entry Pause and Diagnose Continue WARNING Bravo deck position 4 will be hot Use caution when handling components that contact heated deck positions The Agilent NGS Workstation transfers the capture library hybridization buffer mixture to the wells of the PCR plate containing the mixture of indexed gDNA pools and blocking agents SureSelect Automated Library Prep and Capture System 95 4 Hybridization
90. pty Empty Empty Shelf 3 Empty Empty Empty Empty Shelf 2 Empty tip box Empty Empty Empty Shelf 1 New tip box Empty Empty Empty tip box Bottom 9 Load the BenchCel Microplate Handling Workstation according to Table 54 Table 54 Initial BenchCel configuration for Post CaptureOnBeadPCR_XT_Illumina_v2 0 pro No of Columns Rack 1 Rack 2 Rack 3 Rack 4 Processed 1 1 Tip box Empty Empty Empty 2 1 Tip box Empty Empty Empty 3 2 Tip boxes Empty Empty Empty 4 2 Tip boxes Empty Empty Empty 6 3 Tip boxes Empty Empty Empty 12 6 Tip boxes Empty Empty Empty SureSelect 2 Automated Library Prep and Capture System 109 5 Post Capture Sample Processing for Multiplexed Sequencing 10 Load the Bravo deck according to Table 55 Table 55 Initial Bravo deck configuration for Post CaptureOnBeadPCR_XT_Illumina_v2 0 pro Location Content 5 Eppendorf twin tec plate unsealed containing captured bead bound DNA samples 6 Empty PCR plate seated in red insert PCR plate type must be specified on setup form under step 2 9 Master mix plate unsealed containing PCR Master Mix in Column 4 seated in silver insert Run VWorks protocol Post CaptureOnBeadPCR_XT_Illumina_v2 0 pro 11 On the SureSelect setup form under Select Protocol to Run select Post CaptureOnBeadPCR_XT_Ilumina_v2 0 pro 12 Under Select PCR plate labware for Thermal Cycling select the specific type of PCR plate that was loaded on Bravo deck position 6 13 Select the
91. ration of Capture Library Master Mix for target sizes gt 3 0 Mb same Capture Library for all columns Target size gt 3 0 Mb SureSelect 2 Reagent Volume for Volumefor Volumefor Volumefor Volume for Volume for 1 Well 1 Column 2 Columns 3Columns 4Columns 6 Columns Nuclease free water 4 0 uL 39 0 uL 81 9 uL 120 9 uL 159 9 pL 237 9 uL SureSelect RNase Block 0 5 uL 4 9 uL 10 2 pL 15 1 pL 20 0 uL 29 7 uL purple cap Capture Library 5 0 uL 48 8 uL 102 4 pL 151 1 pL 199 9 pL 297 4 uL Total Volume 9 5 uL 92 7 pL 194 5 pL 287 1 pL 379 8 pL 565 0 pL SureSelect Automated Library Prep and Capture System 87 4 Hybridization b For runs that use different Capture Libraries in individual columns prepare a Capture Library Master Mix for each Capture Library as listed in Table 40 or Table 41 based on the Mb target size of your design The volumes listed in Table 40 and Table 41 are fora single column of sample wells If a given Capture Library will be hybridized in multiple columns multiply each of the values below by the number of columns assigned to that Capture Library Table 40 Preparation of Capture Library Master Mix for target sizes lt 3 0 Mb single column of wells Target size lt 3 0 Mb SureSelect 2 Reagent Volume for 1 Well Volume for 1 Column Nuclease free water 7 0 pL 68 3 uL SureSelect RNase Block purple cap 0 5 pL 4 9 uL SureSelect or ClearSeq Capture Library 2 0 pL 19 5 uL Total Volume 9 5 pL 92 7 pL Tabl
92. re XP beads 58 Step 6 Assess Library DNA quantity and quality 61 SureSelect Automated Library Prep and Capture System Contents 4 Hybridization 63 Step 1 Pool indexed DNA samples for hybridization 64 Step 2 Hybridize the gDNA library and Capture Library 72 Hybridization Option A Master Mixes in Columns Hybridization MMCol_v2 0 pro 73 Hybridization Option B Master Mixes in Rows Hybridization MMRow_v2 0 pro 86 Step 3 Capture the hybridized DNA 97 5 Post Capture Sample Processing for Multiplexed Sequencing 105 Step 1 Amplify the captured libraries 106 Step 2 Purify the amplified captured libraries using AMPure XP beads 113 Step 3 Assess quantity and quality of the amplified captured library pools 117 Step 4 Prepare samples for multiplexed sequencing 121 Step 5 Optional Quantify captured library pools by QPCR 123 Step 6 Optional Pool captured libraries for sequencing 124 6 Reference 127 Reference Information for Kits with Revised Index Configuration indexing primers in blue plate 128 Kit Contents 128 Nucleotide Sequences of SureSelect 2 Indexes A01 to H12 131 Reference Information for Kits with Original Index Configuration indexing primers in clear capped tubes 132 Kit Contents 132 Nucleotide Sequences of SureSelect Pre Capture Indexes Original Index Configuration 134 8 SureSelect 2 Automated Library Prep and Capture System SureSelect 2 Automated Library Prep and Capture System Protocol
93. reSelect Target Enrichment Overview of the Workflow Figure 2 summarizes the SureSelect pre capture indexing and target enrichment workflow For each sample to be sequenced an individual library indexing reaction is performed Indexed libraries are then pooled for hybridization and capture steps using a pooling strategy appropriate for the size of the Capture Library and the sequencing design Table 8 summarizes how the VWorks protocols are integrated into the SureSelect workflow See the Sample Preparation Hybridization and Post Capture Sample Processing for Multiplexed Sequencing chapters for complete instructions for use of the VWorks protocols for sample processing For greater flexibility two versions of the Hybridization automation protocol are available see Table 8 The Hybridization_MMCol_v2 0 pro protocol is used optimally when processing full plates of hybridization samples and may be set up using different Capture Libraries in each row allowing enrichment with up to 8 different libraries in a run The Hybridization_MMRow_v2 0 pro protocol is designed for optimal reagent usage when processing plates containing lt 6 columns of samples and may be set up using different Capture Libraries in each column allowing enrichment with up to 12 different libraries in a run 28 SureSelect Automated Library Prep and Capture System Using the Agilent NGS Workstation for SureSelect Target Enrichment 2 Overview of the Workflow
94. rep and Capture System 3 In this Guide This guide describes an optimized protocol for Illumina paired end multiplexed library preparation using the SureSelect Automated Library Prep and Capture System This protocol is specifically developed and optimized to capture the genomic regions of interest using Agilent s SureSelect system to enrich targeted regions of the genome from repetitive sequences and sequences unrelated to the research focus prior to sample sequencing Sample processing steps are automated using the NGS Workstation Before You Begin This chapter contains information such as procedural notes safety information required reagents and equipment that you should read and understand before you start an experiment Using the Agilent NGS Workstation for SureSelect Target Enrichment This chapter contains an orientation to the Agilent NGS Workstation an overview of the SureSelect target enrichment protocol and considerations for designing SureSelect experiments for automated processing using the Agilent NGS Workstation Sample Preparation This chapter describes the steps to prepare index tagged DNA samples for target enrichment Hybridization This chapter describes the steps to pool indexed libraries and then hybridize and capture the pooled DNA SureSelect Automated Library Prep and Capture System 5 Post Capture Sample Processing for Multiplexed Sequencing This chapter describes the steps
95. ridization and capture reactions as appropriate for the specific Capture Library size and sample pooling format Kits contain reagents to prepare indexed libraries from 480 gDNA samples and to enrich the samples in 30 or 60 hybridization and capture reactions as appropriate for the specific Capture Library size and sample pooling format SureSelect reagents and Capture Libraries must be used within one year of receipt 128 SureSelect 2 Automated Library Prep and Capture System Reference 6 The contents of each of the component kits listed in Table 64 are described in the tables below Table 65 SureSelect XT2 Library Prep Kit ILM Content Revised Configuration Kit Component Format SureSelect End Repair Enzyme Mix SureSelect End Repair Nucleotide Mix SureSelect dA Tailing Master Mix SureSelect Ligation Master Mix SureSelect Herculase I Master Mix XT2 Primer Mix SureSelect Pre Capture Indexed Adaptors bottle tube with green cap bottle tube with purple cap bottle tube with clear cap Indexes A01 through H12 provided in blue 96 well plate May also be labeled as SureSelect End Repair Oligo Mix Tt See Table 69 on page 131 for index sequences t See Table 68 on page 130 for a plate map Table 66 SureSelect XT2 Pre Capture Box 1 Content Kit Component SureSelect XT2 Binding Buffer SureSelect XT2 Wash 1 SureSelect XT2 Wash 2 Format bottle bottle bottle Table 67 SureSelect XT
96. rimer Mix 1 5 pL 2 5 uL 3 5 uL 4 5 uL 6 5 uL 13 0 uL 6 Seal the source plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 1 0 sec 7 Vortex the plate for 5 seconds then centrifuge the plate for 30 seconds to drive the well contents off the walls and plate seal and to eliminate any bubbles Keep the master mix source plate on ice SureSelect Automated Library Prep and Capture System 107 5 108 Post Capture Sample Processing for Multiplexed Sequencing Figure 13 CD lt lt lt gt lt gt lt gt SE 59396 OOOH Nal ds es AALA ce O A Configuration of the master mix source plate for Post Capture0nBeadPCR_XT_lllumina_v2 0 pro Columns 1 3 may have been used to dispense master mixes for the Hybridization MMCol_v2 0 pro protocol or may be empty Load the Agilent NGS Workstation 8 Load the Labware MiniHub according to Table 53 using the plate orientations shown in Figure 6 Load a new tip box in Cassete 1 Shelf 1 for the Post CaptureOnBeadPCR_XT_Illumina_v2 0 pro protocol Do not retain a partially filled tip box from previous runs SureSelect 2 Automated Library Prep and Capture System Post Capture Sample Processing for Multiplexed Sequencing 5 Table 53 Initial MiniHub configuration for Post CaptureOnBeadPCR_XT_IIlumina_v2 0 pro Vertical Cassette 1 Cassette 2 Cassette 3 Cassette 4 Shelf Position Shelf 5 Empty Empty Empty Empty Top Shelf 4 Empty Em
97. sed on your SureSelect or ClearSeq Capture Library size and sequencing design Pooling instructions are provided below 1 Combine the capture pools such that each index tagged sample is present in equimolar amounts in the final sequencing sample pool For each final pool use the formula below to determine the amount of each capture pool to use Volume of capture pool VP xC where x C i where V f is the final desired volume of the sequencing sample pool C f is the desired final concentration of all the DNA in the pool is the number of capture pool samples to be combined and C i is the initial concentration of each capture pool sample 2 Adjust the final volume of the pooled library to the desired final concentration If the final volume of the combined index tagged samples is less than the desired final volume V f add Low TE to bring the volume to the desired level If the final volume of the combined index tagged samples is greater than the final desired volume V f lyophilize and reconstitute to the desired volume Table 63 shows an example of the amount of 2 capture pool samples of different concentrations and Low TE needed for a final volume of 20 uL at 10 nM final DNA concentration 124 SureSelect Automated Library Prep and Capture System Post Capture Sample Processing for Multiplexed Sequencing 5 Table 63 Example of capture pool volume calculations for a 20 uL final sequencing sample pool containin
98. sing XT2_Pooling VWForm Hybridization MMCol_v2 0 pro OR Hybridization MMRow_v2 0 pro SureSelectCapture amp Wash_v2 0 rst Post CaptureOnBeadPCR_XT_Illumina_v2 0 pro SPRI_XT_IIlumina_v2 0 pro Post CaptureOnBeadPCR Cleanup 30 SureSelect 2 Automated Library Prep and Capture System Using the Agilent NGS Workstation for SureSelect Target Enrichment 2 Experimental Setup Considerations for Automated Runs SureSelect automated Library Prep runs may include 1 2 3 4 6 or 12 columns equivalent to 8 16 24 32 48 or 96 wells of gDNA samples Plan your experiments using complete columns of samples The number of columns or samples that may be processed using the supplied reagents see Table 1 will depend on the experimental design For greatest efficiency of reagent use plan experiments using at least 3 columns per run Each 96 reaction kit contains sufficient reagents for 96 Library Prep reactions configured as 4 runs of 3 columns of samples per run Prior to hybridization indexed library samples are pooled in sets of 8 samples or in sets of 16 samples based on the type of Capture Library to be used for hybridization see Table 27 on page 65 Thus one Library Prep run corresponds to 0 5 to 12 hybridization wells depending on the number of columns processed see Table 9 Hybridization runs are set up using 1 2 3 4 6 or 12 columns equivalent to 8 16 24 32 48 or 96 wells Accordingly it is typically ben
99. sing for Multiplexed Sequencing For the HiSeq 2500 and NextSeq 500 v1 platforms use the Cycles settings shown in Table 61 Cycle number settings can be specified on the Run Configuration screen of the instrument control software interface after choosing Custom from the index type selection buttons Table 61 Cycle Number settings for HiSeq and NextSeq platforms Run Segment Cycle Number Read 1 100 Index 1 i7 9 Index 2 i5 0 Read 2 100 For the MiSeq platform use the Illumina Experiment Manager IEM software to generate a Sample Sheet that includes the run parameters specified in Table 62 Table 62 Run parameters for MiSeq platform Sample Sheet Parameter Entry Workflow Cycles for Read 1 Cycles for Read 2 Index 1 i7 Sequence enter in Data Section for each sample GenerateFASTO 100 for v2 chemistry 75 for v3 chemistry 100 for v2 chemistry 75 for v3 chemistry Type the 8 nt index sequence for each individual sample see the index sequence tables in the Reference chapter starting on page 127 122 SureSelect 2 Automated Library Prep and Capture System Post Capture Sample Processing for Multiplexed Sequencing 5 Step 5 Optional Quantify captured library pools by QPCR For accurate determination of the DNA concentration in each captured library pool use the QPCR NGS Library Quantification Kit for Ilumina Refer to the protocol that is included with the QPCR NGS Library Qua
100. stem Using the Agilent NGS Workstation for SureSelect Target Enrichment Error messages encountered at start of run After starting the run you may see the error messages displayed below When encountered make the indicated selections and proceed with the run Encountering either or both of these error messages is not indicative of a problem with the NGS workstation or your run setup 1 If you encounter the G axis error message shown below select Ignore and Continue leaving device in current state Bravo 1 Error There appears to be a plate present in or in front of the gripper s plate presence sensor Choose Retry to check the plate presence sensor again Choose Ignore to continue to home theG axis Please note that any plate currently held by the Gripper will be dropped Choose Abort to cancel initialization SureSelect Automated Library Prep and Capture System 2 25 2 26 Using the Agilent NGS Workstation for SureSelect Target Enrichment VWorks Automation Control Software 2 If you encounter the W axis error message shown below select Retry Please verify that itis safe to home a aspirate dispense axis If there is fluid in the tips you kS may want to manually home the W axis in diagnostics over a waste position Choose Retry to continue homing the W axis Choose Ignore to leave the W axis unhomed Choose Abort to cancel init
101. th sealing settings of 165 C and 1 0 sec 12 Vortex the plate for 30 seconds to ensure complete reconstitution then centrifuge the plate for 1 minute to drive the well contents off the walls and plate seal 13 If indexed DNA pool samples are in a Nunc DeepWell plate carefully transfer the samples to an Eppendorf twin tec plate for use in the following Hybridization protocol SureSelect Automated Library Prep and Capture System 71 4 Hybridization Step 2 Hybridize the gDNA library and Capture Library In this step the Agilent NGS Workstation completes the liquid handling steps in preparation for hybridization of the indexed DNA pools to one or more SureSelect or ClearSeq Capture Libraries Afterward you transfer the sample plate to a thermal cycler held at 65 C to allow hybridization of the indexed DNA to the Capture Library Two versions of the Hybridization automation protocol are available See Table 28 for a summary of suggested usage and to locate the instructions for each protocol option in this manual For runs in which all samples will be hybridized to the same Capture Library hybridization protocol selection is based on run size where 12 column runs should use the Hybridization_MMCol_v2 0 pro protocol and 1 6 column runs should use the Hybridization_MMRow_v2 0 pro protocol For runs that include hybridization to multiple Capture Libraries hybridization protocol selection is based on the following considerations
102. than one automation protocol during the run 22 SureSelect Automated Library Prep and Capture System Using the Agilent NGS Workstation for SureSelect Target Enrichment 2 Using the XT2_ILM VWForm VWForm to setup and start a run Use the VWorks form XT2_ILM VWForm VWForm shown below to set up and start each SureSelect automation protocol or runset Workstation Setup MiniHub XT2 SureSelect MiniHub Cassette 1 MiniHub Cassette 2 MiniHub Cassette 3 MiniHub Cassette 4 pre capture pooling Shas for Illumina sequencers Shelf 4 Parameters Shas 1 Select Protocol to Run LibraryPrep_XxT _Illumina_v2 0 rst z Shelf 2 SPRI Case Shelf 1 2 Select PCR Plate labware for Thermal Cyding 96 ABI PCR half skirt in Red Alum Insert ET 3 Select Number of Columns of Samples DE LJ a 4 Click button below to Display Initial Workstation Setup Sie abe lt Position 1 gt lt Position 2 gt lt Position 3 gt Display Initial Clear Workstation workstation Setup Setup Display 5 Load labware according to Workstation Setup gt lt Pos 4 Peltier gt lt Pos 5 Shaker gt lt Pos 6 Peltier gt Controls Once you have loaded labware according to Workstation Setup on right click Run Selected Protocol to start run lt gt lt Position 8 gt lt I Chiller gt an Saeed D pm Eea lt Pos 7 Magnetic Position 8 Pos 9 Chiller Protocol Full Screen Gantt Chart Elapsed Time 00 00 00 Reset
103. tip through the pre split septa then slowly remove the sheared DNA SureSelect Automated Library Prep and Capture System 39 3 Sample Preparation 9 Transfer 50 uL of each sheared DNA sample to a separate well of a 96 well Eppendorf twin tec plate column wise for processing on the Agilent NGS Workstation in well order Al to H1 then A2 to H2 ending with A12 to H12 SureSelect Automated Library Prep and Capture System runs may include 1 2 3 4 6 or 12 columns of the plate See Using the Agilent NGS Workstation for SureSelect Target Enrichment for additional sample placement considerations 10 Seal the plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 1 0 sec 11 Centrifuge the plate for 30 seconds to drive the well contents off the walls and plate seal and to remove air bubbles Stopping Point If you do not continue to the next step store the sample plate at 4 C overnight or at 20 C for prolonged storage 40 SureSelect Automated Library Prep and Capture System Sample Preparation 3 Step 2 Assess sample quality and DNA fragment size Option 1 Analysis using the Agilent 2100 Bioanalyzer and DNA 1000 Assay Use a Bioanalyzer DNA 1000 chip and reagent kit For more information to do this step see the Agilent DNA 1000 Kit Guide 1 Set up the 2100 Bioanalyzer as instructed in the reagent kit guide 2 Seal the sample plate using the PlateLoc Thermal Microplate Sealer with sealing
104. to drive the well contents off the walls and plate seal 4 Prepare the chip samples and ladder as instructed in the reagent kit guide using 1 uL of a ten fold dilution of each sample for the analysis Dilute 1 uL of the sample in 9 pL of 10 mM Tris 1 mM EDTA prior to analysis Be sure to mix well by vortexing at 2000 rpm on the IKA vortex supplied with the Bioanalyzer before analyzing the diluted samples 5 Load the prepared chip into the 2100 Bioanalyzer and start the run within five minutes after preparation 6 Verify that the electropherogram shows an average DNA amplicon size of approximately 250 to 300 bp A sample electropherogram is shown in Figure 14 7 Determine the concentration of each amplified captured library pool by integration under the peak in the electropherogram If the yield is too low or non specific peaks are observed in the electropherogram repeat the PCR with more or fewer cycles The goal is to minimize cycles while you produce enough library for application to the flow cell Stopping Point If you do not continue to the next step seal the plate and store at 4 C overnight or at 20 C for prolonged storage SureSelect Automated Library Prep and Capture System 117 5 Post Capture Sample Processing for Multiplexed Sequencing Step 3 Assess quantity and quality of the amplified captured library pools Figure 14 Analysis of amplified captured DNA using the 2100 Bioanalyzer and the High Sensitivity
105. ts NucleoClean Decontamination Wipes Vacuum concentrator Ice bucket Powder free gloves Sterile nuclease free aerosol barrier pipette tips Vortex mixer Pipetman P10 P20 P200 P1000 or equivalent DynaMag 50 magnet Life Technologies p n 123 02D or equivalent Agilent p n G2943CA Agilent p n G2947CA Agilent p n 5067 1504 Agilent p n 5067 4626 Agilent p n G2964AA or G2965AA Agilent p n 5067 5582 Agilent p n 5067 5583 Agilent p n 5067 5584 Agilent p n 5067 5585 Millipore p n 3097 Savant SpeedVac model DNA120 with 96 well plate rotor model RD2MP or equivalent SureSelect Automated Library Prep and Capture System 15 1 16 Before You Begin Optional Reagents Table5 Reagents for Optional Quantitation Methods Description Vendor and part number QPCR NGS Library Quantification Kit Illumina Agilent p n G4880A Optional Equipment Table 6 Equipment for Optional Quantitation Methods Description Vendor and part number Mx3005P Real Time PCR System Agilent p n 401449 or equivalent Mx3000P Mx3005P 96 well tube plates Agilent p n 410088 or equivalent Mx3000P Mx3005P optical strip caps Agilent p n 401425 or equivalent SureSelect 2 Automated Library Prep and Capture System SureSelect 2 Automated Library Prep and Capture System Protocol ee 2 7 e Using the Agilent NGS Workstation for att SureSelect Target Enrichment About the Agilent NGS Workstation 18 Overvie
106. ts of index sequence and kit content information The first section covers kits with indexing primers supplied in a blue plate format in Library Prep Kit p n 5500 0131 typically received February 2015 or later The second section covers kits with indexing primers supplied in tube format in p n 5190 3936 and p n 5190 3937 typically received before February 2015 Verify that you are referencing the information appropriate for your kit version before you proceed Reference Information for Kits with Revised Index Configuration indexing primers in blue plate Use the reference information in this section if your kit includes Library Prep Kit p n 5500 0131 If your kit does not include this component kit see page 132 for kit content and indexing primer information Kit Contents SureSelect Automation Reagent Kits contain the following components Table 64 SureSelect Automation Reagent Kit Content Revised Index Configuration Component Kits Storage Condition G9661B G9661C 96 Samples 480 Samples SureSelect XT2 Library Prep Kit ILM 20 C 5500 0131 5 x 5500 0131 SureSelect XT2 Pre Capture Box 1 Room Temperature 5190 4076 5190 4077 SureSelect XT2 Pre Capture Automation ILM Module Box 2 20 C 5190 4462 5190 4463 See Table 65 through Table 67 for a list of reagents included in each component kit t Kits contain reagents to prepare indexed libraries from 96 gDNA samples and to enrich the samples in 6 or 12 hyb
107. uL 4 5 uL 6 5 uL 13 0 uL If you are using a new DeepWell plate for the pre capture PCR source plate for example when amplifying the second half of the indexing adaptor ligated DNA sample leave columns 1 to 3 empty and add the PCR Master Mix to column 4 of the new plate 52 SureSelect Automated Library Prep and Capture System Sample Preparation 3 LLIS V gt lt gt lt lt lt gt gt e608 S A Oe lt gt ee Figure 7 Configuration of the master mix source plate for Pre CapturePCR_XT_Illumina_v2 0 pro Columns 1 3 were used to dispense master mixes during the previous protocol 5 Seal the master mix source plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 1 0 sec 6 Vortex the plate for 5 seconds to ensure homogeneity of the PCR Master Mix 7 Centrifuge the plate for 30 seconds to drive the well contents off the walls and plate seal and to eliminate any bubbles Keep the master mix source plate on ice The presence of bubbles in source plate solutions may cause inaccurate volume transfer by the Bravo liquid handling platform Ensure that the source plate is sealed and centrifuged prior to use in a run SureSelect Automated Library Prep and Capture System 53 3 Sample Preparation CAUTION Load the Agilent NGS Workstation 8 Load the Labware MiniHub according to Table 20 using the plate orientations shown in Figure 6 The waste tip box
108. uide using 1 uL of each sample for the analysis 5 Load the prepared chip into the 2100 Bioanalyzer and start the run within five minutes after preparation 6 Verify that the electropherogram shows an average DNA amplicon size of 250 to 300 bp A sample electropherogram is shown in Figure 8 7 Determine the concentration of the library ng L by integrating under the peak Stopping Point If you do not continue to the next step seal the plate and store at 4 C overnight or at 20 C for prolonged storage Figure 8 Analysis of amplified prepped library DNA using a DNA 1000 assay SureSelect Automated Library Prep and Capture System 61 3 Sample Preparation Option 2 Analysis using the Agilent 2200 TapeStation and D1000 ScreenTape Use a D1000 ScreenTape p n 5067 5582 and associated reagent kit p n 5067 5583 to analyze the amplified prepped DNA For more information to do this step see the Agilent 2200 TapeStation User Manual 1 Seal the DNA library sample plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 1 0 sec 2 Vortex the plate to mix samples in each well then centrifuge the plate for 30 seconds to drive the well contents off the walls and plate seal 3 Prepare the TapeStation samples as instructed in the Agilent 2200 TapeStation User Manual Use 1 uL of each DNA sample diluted with 3 uL of D1000 sample buffer for the analysis CAUTION Make sure that you thoroughly mix the combin
109. utes Once complete the PCR ready samples containing indexed DNA and PCR master mix are located in the PCR plate at position 6 of the Bravo deck The Eppendorf plate containing the remaining prepped DNA samples which may be stored for future use at 4 C overnight or at 20 C for long term storage is located at position 7 of the Bravo deck 17 Remove the PCR plate from position 6 of the Bravo deck and seal the plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 3 0 seconds 18 Centrifuge the plate for 30 seconds to drive the well contents off the walls and plate seal and to eliminate air bubbles 19 Transfer the PCR plate to a thermal cycler and run the PCR amplification program shown in Table 23 The volume of each PCR amplification reaction is 50 uL 56 SureSelect 2 Automated Library Prep and Capture System Sample Preparation 3 Table 23 Pre Capture PCR cycling program Segment Number of Temperature Time Cycles 1 1 98 C 2 minutes 2 5 98 C 30 seconds 60 C 30 seconds 72 C 1 minute 3 1 72 C 10 minutes 4 1 4 C Hold Different library preparations can produce slightly different results based on varying DNA quality In most cases 5 cycles will produce an adequate yield for subsequent capture without introducing bias or non specific products If yield is too low or non specific high molecular weight products are observed adjust the number of cycles accordingly with the remai
110. v2 0 pro Location Content 1 Empty tip box 4 Empty PCR plate seated on red insert PCR plate type must be specified on setup form under step 2 5 Empty Eppendorf twin tec plate 6 Hybridization Master Mix source plate unsealed seated on silver insert Master Mixes in Rows A C 9 Indexed DNA pools in Eppendorf twin tec plate Run VWorks protocol Hybridization MMRow_v2 0 pro 10 On the SureSelect setup form under Select Protocol to Run select Hybridization_MMRow_v2 0 pro 11 Under Select PCR plate labware for Thermal Cycling select the specific type of PCR plate that was loaded on Bravo deck position 4 SureSelect Automated Library Prep and Capture System 91 4 Hybridization 12 Select the number of columns of samples to be processed Runs must include 1 2 3 4 6 or 12 columns 13 Click Display Initial Workstation Setup Display Initial Workstation Setup 14 Verify that the NGS workstation has been set up as displayed in the Workstation Setup region of the form Workstation Setup MiniHub MiniHub Cassette 1 MiniHub Cassette 2 MiniHub Cassette 3 MiniHub Cassette 4 ets ee Nhl ION linn NU ee Cece ee ee a CT Mae T 15 When verification is complete click Run Selected Protocol Run Selected Protocol The Agilent NGS Workstation transfers Blocking Mix and indexed gDNA pools to the PCR plate When this process is complete you will be prompted to transfer the plate to the thermal cycler for samp
111. w of the Workflow 28 Experimental Setup Considerations for Automated Runs 31 This chapter contains an orientation to the Agilent NGS Workstation an overview of the SureSelect target enrichment protocol and considerations for designing SureSelect experiments for automated processing using the Agilent NGS Workstation wit Agilent Technologies 17 2 Using the Agilent NGS Workstation for SureSelect Target Enrichment About the Agilent NGS Workstation CAUTION About the Bravo Platform The Bravo platform is a versatile liquid handler with a nine plate location platform deck suitable for handling 96 well 384 well and 1536 well plates The Bravo platform is controlled by the VWorks Automation Control software Fitted with a choice of seven interchangeable fixed tip or disposable tip pipette heads it accurately dispenses fluids from 0 1 uL to 250 uL Before you begin make sure that you have read and understand operating maintenance and safety instructions for using your Bravo platform Refer to the Bravo Platform User Guide G5409 90006 and the VWorks Software User Guide G5415 90063 18 Bravo Platform Deck The protocols in the following sections include instructions for placing plates and reagent reservoirs on specific Bravo deck locations Use Figure 1 to familiarize yourself with the location numbering convention on the Bravo platform deck Back B L Front Figur
112. walls and plate seal Prepare the TapeStation samples as instructed in the Agilent 2200 TapeStation User Manual Use 1 uL of each sheared DNA sample diluted with 3 uL of D1000 sample buffer for the analysis Make sure that you thoroughly mix the combined DNA and D1000 sample buffer on a vortex mixer for 5 seconds for accurate quantitation Stopping Point 42 Load the sample plate or tube strips from step 3 the D1000 ScreenTape and loading tips into the 2200 TapeStation as instructed in the Agilent 2200 TapeStation User Manual Start the run Verify that the electropherogram shows an average DNA fragment size of 150 to 200 bp A sample electropherogram is shown in Figure 4 If you do not continue to the next step seal the sheared DNA sample plate and store at 4 C overnight or at 20 C for prolonged storage 2000 4 Figure 4 Analysis of sheared DNA using the 2200 TapeStation SureSelect Automated Library Prep and Capture System Sample Preparation 3 Step 3 Prepare indexed gDNA library samples In this step the Agilent NGS Workstation completes the DNA end modification steps required for SureSelect pre capture indexing including end repair A tailing and indexing adaptor ligation Where required the Agilent NGS Workstation purifies the prepared DNA using AMPure XP beads as part of the Library Prep runset Prepare the workstation 1 Turn on the chiller set to 0 C at position 9 of the
113. walls and plate seal and to eliminate air bubbles SureSelect Automated Library Prep and Capture System 111 5 Post Capture Sample Processing for Multiplexed Sequencing 19 Transfer the PCR plate to a thermal cycler and run the PCR amplification program shown in Table 56 using the cycle number specified in Table 57 The volume of each PCR amplification reaction is 50 uL Table 56 Post Capture PCR cycling program Segment Number of Temperature Time Cycles 1 1 98 C 2 minutes 2 8 14 98 C 30 seconds see Table 57 60 C 30 seconds 72 C 1 minute 3 1 72 C 10 minutes 4 1 4 C Hold Table 57 Recommended cycle number based on Capture Library size Size of Capture Library Cycles lt 0 5 Mb 12 to 14 cycles 0 5 to 1 49 Mb 9 to 11 cycles gt 1 5 Mb including All Exon and Exome libraries 8 to 10 cycles Amplify the captured DNA using a minimal number of PCR cycles If yield is too low or non specific high molecular weight products are observed adjust the number of cycles accordingly with the remaining captured DNA template 112 SureSelect 2 Automated Library Prep and Capture System Post Capture Sample Processing for Multiplexed Sequencing 5 Step 2 Purify the amplified captured libraries using AMPure XP beads In this step the Agilent NGS Workstation transfers AMPure XP beads to the amplified captured DNA and then collects and washes the bead bound enriched DNA amplicons Prepare the workstation and reagents
114. wells Target size gt 3 0 Mb SureSelect 2 Volume for Volume for Volume for Volumefor Volumefor Volumefor Volume for Reagent 1 Well 1 Column 2 Columns 3Columns 4 Columns 6 Columns 12 Columns Nuclease free 4 0 uL 8 0 uL 12 1 uL 16 3 uL 20 4 uL 30 7 uL 57 5 uL water SureSelectRNase 0 5 uL 1 0 pL 1 5 uL 2 0 uL 2 5 uL 3 8 uL 7 2 yL Block purple cap Capture Library 5 0 uL 10 0 uL 15 2 uL 20 3 uL 25 5 uL 38 4 uL 71 9 uL Total Volume 9 5 pL 19 0 pL 28 8 pL 38 6 pL 48 4 pL 72 9 pL 136 6 pL SureSelect Automated Library Prep and Capture System 75 4 Hybridization Prepare the master mix source plate 4 Ina Nunc DeepWell plate prepare the hybridization master mix source plate at room temperature Add the volumes indicated in Table 33 to all wells of the indicated column of the Nunc DeepWell plate As indicated in the shaded portion of Table 33 Blocking Mix and nuclease free water are combined in the wells of Column 1 When using multiple Capture Libraries in a run add each Capture Library Master Mix to the appropriate row s of the Nunc DeepWell plate The final configuration of the master mix source plate is shown in Figure 11 Table 33 Preparation of the Master Mix Source Plate for Hybridization MMCol_v2 0 pro Master Mix Position on Volume of Master Mix added per Well of Nunc Deep Well Source Plate Solution Source Plate 1 Column 2 Column 3 Column 4 Column 6 Column 12 Column Runs Runs Runs Runs Runs Runs SureSelect XT2 13 5 uL

"user manual"

Contents

Download Pdf Manuals

Related Search

Related Contents

&quot;user manual&quot;

Contents

Download Pdf Manuals

Related Search

Related Contents

"user manual"