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HaloPlex Target Enrichment System

1. empty Empty 384 well Eppendorf twin tec plate seated on 384 well insert empty Empty tip box RE Master Mix source plate full skirted 96 well Eppendorf twin tec plate seated on red insert 7 Verify that the NGS workstation has been set up as displayed in the Bravo Deck Setup and Information regions of the form HaloPlex Target Enrichment System Automation Protocol Sample Preparation 3 8 Verify that the Current Tip State indicator on the form matches the configuration of unused and used tips in the tip boxes at Bravo Deck positions 2 and 8 respectively See page 19 for more information on using this segment of the form during the run 9 When verification is complete click Start to start the run Controls B Start 19 Pause Screen If workstation devices do not respond when vou start the run but activitv is recorded in the Log verifv that VWorks is not running in Simulation mode See page 22 for more information 10 When prompted by VWorks as shown below replace the tip box at position 2 with a new tip box and replace the used tip box at position 8 with an empty tip box After both tip boxes are in place click Reset under Current Tip State on the form Verifv that the tip state was updated and then click Continue on the prompt shown below Depending on the run size you may be prompted to change tip boxes multiple times during the run Check Tips CHANGE TIP BOXES Press RESET on form and CONTINU
2. 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 Commercial Items and DFARS 227 7202 3 Rights in Commercial Computer Software or Com puter Software Documentation Safety Notices CAUTION A CAUTION notice denotes a haz ard It calls attention to an operat ing procedure practice or the like that if not correctly performed or adhered to could r
3. the new plate HaloPlex Target Enrichment System Automation Protocol Sample Preparation 3 Load the Agilent NGS Workstation and Run the Capture_v1 1 pro VWorks Protocol 3 4 On the VWorks HaloPlex form under Step select 03 Capture v1 1 pro Select the number of columns of samples to be processed Runs must include 1 2 3 4 6 or 12 columns Click Update layout and information Load the Bravo deck according to Table 26 Table 26 Initial Bravo deck configuration for Capture v1 1 pro Location Content 1 2 3 Empty Axygen 96 Deep Well Plate square wells for waste New tip box Wash Solution source plate full skirted 96 well Eppendorf twin tec plate Hybridized sample plate seated on red insert HaloPlex magnetic streptavidin bead source plate Nunc DeepWell plate Empty half skirted 96 well Eppendorf twin tec plate seated on red insert empty Empty tip box Master Mix source plate Nunc DeepWell plate seated on silver insert 5 Verify that the NGS workstation has been set up as displayed in the Bravo Deck Setup and Information regions of the form Verify that the Current Tip State indicator on the form matches the configuration of unused and used tips in the tip boxes at Bravo Deck HaloPlex Target Enrichment System Automation Protocol 53 3 54 Sample Preparation positions 2 and 8 respectively See page 19 for more information on using this segment of the form during the run 7 When
4. water Prepare a Thermo Scientific reservoir containing 15 mL of the final sample elution buffer nuclease free 10 mM Tris acetate or Tris HCl buffer pH 8 0 Prepare a separate Thermo Scientific reservoir containing 45 mL of freshly prepared 70 ethanol Load the Agilent NGS Workstation and Run the Purification_v1 1 pro VWorks Protocol 1 On the VWorks HaloPlex form under Step select 04 Purification v1 1 pro Select the number of columns of samples to be processed Runs must include 1 2 3 4 6 or 12 columns Click Update layout and information HaloPlex Target Enrichment System Automation Protocol 63 3 Sample Preparation 4 Load the Bravo deck according to Table 31 Table 31 Initial Bravo deck configuration for Purification_v1 1 pro Location Content 1 Empty Axygen 96 Deep Well Plate square wells for waste New tip box 3 Empty full skirted 96 well Eppendorf twin tec plate 4 empty 5 AMPure XP beads in Nunc DeepWell source plate 6 Amplified DNA samples in half skirted 96 well Eppendorf twin tec plate seated on red insert 7 empty 8 Empty tip box 9 Nuclease free water in Thermo Scientific reservoir 5 Verify that the NGS workstation has been set up as displayed in the Bravo Deck Setup region of the form 6 Verify that the Current Tip State indicator on the form matches the configuration of unused and used tips in the tip boxes at Bravo Deck positions 2 and 8 respectively See p
5. 18 0 pL 23 3 pL 33 9 uL 68 4 uL Column 7 A7 H7 7 3 uL 12 7 uL 18 0 uL 23 3 uL 33 9 uL 68 4 uL Column 8 A8 H8 7 3 uL 12 7 uL 18 0 uL 23 3 uL 33 9 uL 68 4 uL RE Master Mixes V V V 4 RE Master Mix Source Plate VANAN 7 C C OO SAW gt ii OOOOOOOO I 12 3456 7 891011 12 Figure 3 Preparation of the RE Master Mix source plate for automation protocol Digestion pro HaloPlex Target Enrichment System Automation Protocol 33 3 34 Sample Preparation Load the NGS Workstation and Run the Digestion pro VWorks Protocol 1 Open the HaloPlex setup form using the HaloPlex VWForm shortcut on your desktop Log in to the VWorks software On the setup form under Step select 01 Digestion pro Parameters 1 Step Digestion pro T 2 Number of columns of samples 12 Update layout and information 3 4 Update current tip state 4 Select the number of columns of samples to be processed Runs must include 1 2 3 4 6 or 12 columns 5 Click Update layout and information 6 Load the Bravo deck according to Table 12 Table 12 Initial Bravo deck configuration for Digestion pro Location 1 2 3 Content empty New tip box For 12 column runs only Empty 384 well Eppendorf twin tec plate no 384 well insert required For 1 to 6 column runs empty gDNA samples in full skirted 96 well Eppendorf twin tec plate seated on red insert
6. 27 35 43 51 59 67 75 83 91 D Index Index Index Index Index Index Index Index Index Index Index Index 4 12 20 28 36 44 52 60 68 76 84 92 E Index Index Index Index Index Index Index Index Index Index Index Index 5 13 21 29 37 45 53 61 69 77 85 93 F Index Index Index Index Index Index Index Index Index Index Index Index 6 14 22 30 38 46 54 62 70 78 86 94 G Index Index Index Index Index Index Index Index Index Index Index Index 7 15 23 31 39 47 55 63 71 79 87 95 H Index Index Index Index Index Index Index Index Index Index Index Index 8 16 24 32 40 48 56 64 72 80 88 96 84 HaloPlex Target Enrichment System Automation Protocol Nucleotide Sequences of HaloPlex Indexes Reference The nucleotide sequence of the 8 nucleotide index portion of each HaloPlex Indexing Primer Cassette is provided in the tables below HaloPlex 96 reaction kits include plates containing the 96 indexes listed in Table 36 to Table 41 Table 36 HaloPlex 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 HaloPlex Target Enrichment System Automation Protocol 5 85 5 86 Reference Table 37 HaloPlex 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 AGC
7. 71 Custom Primer for Read 2 Project Name Test Proyect E t 71 Use Adapter Tamming Investigator Name Test Descroton Test Date 62012 Ge Read Pared End 5 Reed Cycles Read 1 Cycles Read 2 field 72 HaloPlex Target Enrichment System Automation Protocol Sample Preparation 3 Using the Sample Plate Wizard set up a New Plate entering the required information for each sample to be sequenced In the Index 1 17 column of the TrueSeq LT Assay Plate table assign each sample to any of the Illumina 17 indexes The index will be corrected to a HaloPlex index at a later stage sample Plate Wizard Plate Samples ruSeq LT Assay Plate Table Plate Plate Graphic indicates invalid samples SamplelD Sample Name Sample Project Description A01 1 Sample A001 ProjectX Tumor 02 2 Sample2 A002 ProjectX Normal SJ TU n 4 Finish the sample plate setup tasks and save the sample plate file 5 Using the Sample Sheet Wizard select the samples to include in the run and save the Sample Sheet file HaloPlex Target Enrichment System Automation Protocol 3 73 3 Sample Preparation Editing the Sample Sheet to Include HaloPlex indexes 1 Open the Sample Sheet file in a text editor For each sample select the text for the 6 nucleotide index highlighted below and replace with the appropriate 8 nucleotide HaloPlex index sequence
8. H may be prepared in a 8 x 0 2 mL well strip tube using a multichannel pipette to transfer volumes from Enzyme Strips 1 and 2 to the RE master mix strip For 6 or 12 column runs prepare the master mixes in 1 5 mL tubes 4 Mix by gentle vortexing and then spin briefly Keep on ice 32 HaloPlex Target Enrichment System Automation Protocol Sample Preparation 3 Prepare the RE master mix source plate 1 Aliquot the Restriction Enzyme Master Mixes to a full skirted 96 well Eppendorf twin tec plate as shown in Figure 3 Add the volumes indicated in Table 11 of each master mix to each well of the indicated column of the twin tec plate Keep the master mixes on ice during the aliquoting steps Table 11 Preparation of the RE Master Mix Source Plate for Digestion pro Master Mix Solution Position on Source Volume of Master Mix added per Well of Source Plate Plate 1 Column 2 Column 3 Column 4 Column 6 Column 12 Column Runs Runs Runs Runs Runs Runs RE Master Mix A RE Master Mix B RE Master Mix C RE Master Mix D RE Master Mix E RE Master Mix F RE Master Mix G RE Master Mix H Column 1 A1 H1 7 3 uL 12 7 uL 18 0 uL 23 3 uL 33 9 uL 68 4 uL Column 2 A2 H2 7 3 uL 12 7 pL 18 0 pL 23 3 pL 33 9 uL 68 4 uL Column 3 A3 H3 7 3 uL 12 7 uL 18 0 uL 23 3 uL 33 9 uL 68 4 uL Column 4 A4 H4 73 12 7 uL 18 0 uL 23 3 uL 33 9 uL 68 4 uL Column 5 5 5 7 3 uL 12 7 uL 18 0 uL 23 3 uL 33 9 uL 68 4 uL Column 6 A6 H6 7 3 uL 12 7 pL
9. Volume for Volume for Volume for 1 Library 1 Column 2Columns 3Columns 4Columns 6Columns 12 Columns Nuclease free water 16 1 uL 205 3 uL 342 1 uL 479 uL 615 8 pL 889 5 uL 1779 uL 5X Herculase Il 10 uL 127 5 pL 212 5 uL 297 5 uL 382 5 uL 552 5 uL 1105 uL Reaction Buffer dNTPs 100 mM 0 4 uL 5 1 uL 8 5 uL 11 9 uL 15 3 uL 22 1 uL 44 2 uL Primer 1 1 uL 12 75 pL 21 3 uL 29 8 uL 38 3 uL 55 3 uL 110 5 uL Primer 2 1 uL 12 75 uL 21 3 uL 29 8 uL 38 3 uL 55 3 uL 110 5 uL 2 M Acetic acid 0 5 uL 6 4 uL 10 6 uL 14 9 uL 19 1 uL 27 6 uL 55 3 uL Herculase II Fusion 1 uL 12 75 uL 21 3 uL 29 8 uL 38 3 uL 55 3 uL 110 5 uL DNA Polymerase Total Volume 30 uL 382 5 uL 637 6 uL 892 7 uL 1147 6 pL 1657 6 uL 3315 pL Enzyme with dNTPs Agilent p n 600677 or 600679 HaloPlex Target Enrichment System Automation Protocol Be sure to use dNTPs at 100 mM concentration 25 mM for each nucleotide like those provided with the Herculase Fusion 51 3 Sample Preparation 2 Prepare the appropriate amount of Ligation Master Mix according to the table below Mix well by gentle vortexing then spin the tube briefly Store the master mix on ice until it is used on page 56 The Ligation Master Mix is added to the Master Mix Source Plate just before it is used in the Capture v1 1 pro protocol Do not add this master mix to the source plate before starting the run Table 24 Preparation of Ligation Master Mix for Capture_v1 1 pro Reagent Ligation S
10. direct sequencing using standard Illumina paired end primers and chemistry on the Illumina HiSeq MiSeq or GAIIx platform See additional guidelines for the MiSeq platform below and HiSeq platform page 75 Use 100 100 bp or 150 150 bp paired end sequencing depending on the selection made during probe design Since the read length affects maximum achievable coverage check the design report to verify read length selected in probe design Sequencing runs must be set up to perform an 8 nt index read For complete index sequence information see tables starting on page 85 Before aligning reads to the reference genome trim the reads from Illumina adaptor sequences MiSeq platform sequencing run setup guidelines Setting up a custom Sample Sheet 1 In the IEM software create a Sample Sheet for the MiSeq platform using the following Workflow selections Under Category select Other Under Application select FASTQ Only HaloPlex Target Enrichment System Automation Protocol 71 3 Sample Preparation Step 6 Pool samples with different indexes for multiplexed sequencing 2 On the Workflow Parameters screen enter the run information making sure to specify the key parameters highlighted below Illumina Experiment Manager sample Sheet Wizard Workflow Parameters FASTQ Only Fun Settings FASTQ Orly Wordlow Spectic Settings Reagent Caride Barcode 52000000300 EY Custom Primer for Read 1 Seve Po Fiat index Reads 0 2
11. material Always keep pre amplification and post amplification DNA samples in separate work areas Perform the enrichment procedure in the pre amplification area Open and store the amplified enriched DNA samples only in the post amplification area Possible stopping points where DNA samples may be stored between steps are marked in the protocol Store the samples at 20 C but do not subject the samples to multiple freeze thaw cycles Ensure that master mixes are thoroughly mixed by pipetting up and down or by gentle vortexing before distributing to the samples In general follow Biosafety Level 1 BL1 safety rules Safety Notes CAUTION Wear appropriate personal protective equipment PPE when working in the laboratory HaloPlex Target Enrichment System Automation Protocol Required Reagents Before You Begin 1 Table 1 Required Reagents for HaloPlex Target Enrichment Description HaloPlex Target Enrichment System Kit Herculase II Fusion Enzyme with dNTPs 100 mM 25 mM for each nucleotide 200 reactions Nuclease free Water not DEPC treated Agencourt AMPure XP Kit 5 mL 60 mL 450 mL 10 M NaOH molecular biology grade 2 M acetic acid 10 mM Tris HCl pH 8 0 or 10 mM Tris acetate pH 8 0 10096 Ethanol molecular biology grade Quant iT dsDNA BR Assay Kit for use with the Qubit fluorometer 100 assays 2 1000 ng 500 assays 2 1000 ng Vendor and part number Select the appropriate kit f
12. set the temperature of Bravo deck positions 4 and 6 to 4 C using the Inheco Multi TEC control touchscreen as described in Setting the Temperature of Bravo Deck Heat Blocks To expedite thermal cycler warm up for the restriction digest incubation on page 36 you can enter and initiate the digestion program on the thermal cycler now and then pause the program until you are instructed to transfer the reaction plate Be sure to pause the thermal cycler before the initiation of the 37 C incubation segment Release the pause immediately after transferring the plate to the thermal cycler in step 14 on page 36 Be sure that the 384 well block is in the thermal cycler before initiating the program for warm up HaloPlex Target Enrichment System Automation Protocol Sample Preparation 3 Prepare the DNA Sample Source Plate In the protocol below 200 ng genomic DNA is split among eight different restriction digests with an additional 25 ng excess DNA included to allow for pipetting losses Using lt 225 ng DNA in the enrichment protocol can result in low yield and can potentiate rare allele dropouts Use a fluorometry based DNA quantitation method such as Qubit fluorometry or PicoGreen staining to accurately quantify the DNA starting material 1 Use the Qubit dsDNA BR Assay or PicoGreen staining kit to determine the concentration of your gDNA samples Follow the manufacturers instructions for the kits and instruments 2 Prepare the DNA sample pla
13. v3 0 SBS chemistry Sequence analysis resources Agilent s SureCall data analysis software is available to simplify the sequencing data analysis workflow after HaloPlex target enrichment To learn more about this resource and download the SureCall software free of charge visit www agilent com genomics surecall HaloPlex Target Enrichment System Automation Protocol 75 3 Sample Preparation Step 6 Pool samples with different indexes for multiplexed sequencing 76 HaloPlex Target Enrichment System Automation Protocol HaloPlex Target Enrichment System Protocol 1 200 Appendix Provisional Adaptor Dimer 8 Removal Protocol Purify the enriched library pool using AMPure XP beads 78 This section contains a protocol for purification of the target enriched library pool to remove adaptor dimer molecules of approximately 125 bp size Only do this protocol if electrophoretic analysis of the target enriched library samples shows a peak at approximately 125 bp which represents a molar fraction of gt 10 of DNA in the sample see page 68 to page 70 E Agilent Technologies TI 4 78 Appendix Provisional Adaptor Dimer Removal Protocol Purify the enriched library pool using AMPure XP beads In this step a 40 pool of target enriched DNA libraries is purified using AMPure XP beads using manual sample processing This protocol requires a 0 2 mL tube compatible magnetic separation device such as the Agenco
14. 0 5538 or 5190 5540 HaloPlex Target Enrichment System Automation Protocol Reference 5 The contents of the HaloPlex Target Enrichement System Box 1 are detailed in the table below Table 34 HaloPlex Target Enrichment System for Illumina Box 1 Contents Included Reagents Formal Hybridization Solution bottle Ligation Solution bottle Wash Solution bottle Capture Solution bottle SSC Buffer bottle RE Buffer bottle BSA Solution tube with clear cap DNA Ligase tube with red cap Enrichment Control DNA tube with orange cap Primer 1 tube with yellow cap Primer 2 tube with blue cap HaloPlex Indexing Primer Cassettes 96 well plate with Indexing Primer Cassettes 1 96 Enzyme Strip 1 8 well strip tube with green label Enzyme Strip 2 8 well strip tube with red label HaloPlex Probe tube with pink cap See Table 35 for a plate map HaloPlex Target Enrichment System Automation Protocol 83 5 Reference Placement of each HaloPlex Indexing Primer Cassette in the index cassette plate provided with p n G9901B is shown in Table 35 Table 35 HaloPlex Indexing Primer Cassette plate map 1 2 3 4 5 6 7 8 9 10 11 12 A Index Index Index Index Index Index Index Index Index Index Index Index 1 9 17 25 33 41 49 57 65 73 81 89 Index Index Index Index Index Index Index Index Index Index Index Index 2 10 18 26 34 42 50 58 66 74 82 90 Index Index Index Index Index Index Index Index Index Index Index Index 3 11 19
15. 12321D or equivalent DynaMag 15 magnet p n 12301D or equivalent VWR p n 93000 196 or equivalent Labnet International MPS1000 Mini Plate Spinner p n C1000 or equivalent Pipetman P10 P20 P200 P1000 or equivalent Millipore p n 3097 Life Technologies p n 032866 Life Technologies p n 032856 General laboratory supplier Protocols are also compatible with Agilent NGS Workstation Option after purchase of accessories Table 5 t Thermal cycler must have a maximum reaction volume specification of at least 100 uL and must be compatible with 0 2 mL tubes t Compatible with Agilent SureCycler 8800 Select the appropriate device based on run size See page 53 to determine magnetic bead volume to be used for your run size HaloPlex Target Enrichment System Automation Protocol 11 1 12 Before You Begin Optional Validation Reagents and Equipment Table4 Reagents and Equipment for Optional Validation Methods Description Vendor and part number 2200 TapeStation Platform and Consumables 2200 TapeStation Agilent p n G2964AA or G2965AA Agilent p n 5067 5363 Agilent p n 5067 5364 High Sensitivity D1K ScreenTape High Sensitivity D1K Reagents 2100 Bioanalyzer Platform and Consumables 2100 Bioanalyzer Laptop Bundle Agilent p n G2943CA Agilent p n G2947CA Agilent p n 5067 4626 2100 Bioanalyzer Electrophoresis Set High Sensitivity DNA Kit Gel Electrophoresis Platform and Consumables XCell SureLock M
16. 2 5 uL 5525 uL Solution Total Volume 70 uL 892 5 uL 1487 5 2082 5 uL 2677 5 uL 3867 5 uL 7735 pL 2 Ina Nunc DeepWell plate prepare the Hybridization Master Mix source plate Add the volumes indicated in Table 16 of the Hybridization Master Mix to all wells of the indicated column of the Nunc DeepWell plate Table 16 Preparation of the Master Mix Source Plate for Hybridization pro Master Mix Position on Volume of Master Mix added per Well of Nunc DeepWell Source Plate Solution Source Plate 1 Column 2 Column 3 Column 4 Column 6 Column 12 Column Runs Runs Runs Runs Runs Runs Hybridization Column 1 102 8 uL 177 2 uL 251 6 uL 325 9 uL 474 7 uL 958 1 uL Master Mix A1 H1 HaloPlex Target Enrichment System Automation Protocol 43 3 44 Sample Preparation Prepare the Hybridization Reaction Plate with Indexing Primer Casettes 1 Ina half skirted 96 well Eppendorf twin tec plate aliquot 10 uL of the appropriate Indexing Primer Cassette to each intended sample indexing well position Keep the plate on ice Be sure to add only one specific Indexing Primer Cassette to each well using different indexes for each sample to be multiplexed Record the identity of the Indexing Primer Cassette assigned to each well for later sequence analysis 2 Ifthe run includes an ECD control sample that was analyzed as described on page 37 add 32 uL of nuclease free water to well Al of the hybridization reaction plate Well Al should al
17. 3 Thermal cycler program for HaloPlex restriction digestion Step Temperature Time Step 1 37 C 30 minutes Step 2 8 C Hold Preparation of each restriction digest reaction plate takes approximately 30 45 minutes HaloPlex Target Enrichment System Automation Protocol Sample Preparation 3 For 12 column runs the two 384 well plates are prepared sequentially for a total run time of approximately 90 minutes Run the thermal cycler digestion program for each plate as soon as prompted During the 30 minute incubation of plate 1 in the thermal cycler the workstation begins preparation of the digestion reactions in plate 2 Once the thermal cycler program is complete for plate 1 store the digested DNA in plate 1 on ice until the Digestion pro protocol and thermal cycler program for plate 2 is finished 15 Validate the restriction digestion reaction by electrophoretic analysis of the Enrichment Control DNA ECD reactions a Transfer 4 uL of each ECD digestion reaction from the wells of the 384 well reaction plate indicated in Table 14 to fresh 0 2 mL PCR tubes Note that for 12 column runs four of the eight ECD digests are found on the first 384 well plate and the remaining four digests are on the second 384 well plate Table 14 Position of ECD digestion reactions for obtaining validation samples Restriction Enzyme Master Position of ECD Digestion Reaction in 384 Well Plates Mix to be Validated 1 6 Column Runs 12 Column Ru
18. AGGAA 27 AGTCACTA 28 ATCCTGTA 29 ATTGAGGA 30 CAACCACA 31 CAAGACTA 32 CAATGGAA HaloPlex Target Enrichment System Automation Protocol Table 38 HaloPlex Indexes 33 48 Index Number Sequence 33 CACTTCGA 34 CAGCGTTA 35 CATACCAA 36 CCAGTTCA 37 CCGAAGTA 38 CCGTGAGA 39 CCTCCTGA 40 CGAACTTA 41 CGACTGGA 42 CGCATACA 43 CTCAATGA 44 CTGAGCCA 45 CTGGCATA 46 GAATCTGA 47 GACTAGTA 48 GAGCTGAA HaloPlex Target Enrichment System Automation Protocol 5 87 5 Reference Table 39 HaloPlex 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 HaloPlex Target Enrichment System Automation Protocol Reference 5 Table 40 HaloPlex 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 HaloPlex Target Enrichment System Automation Protocol 89 5 Reference Table 41 HaloPlex 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 HaloPlex Target Enrich
19. E to reset to Full boxes or press PAUSE to manually edit User data entry Pause and Diagnose Continue N The NGS Workstation combines each gDNA sample with each RE Master Mix in wells of a 384 well reaction plate For 1 to 6 column runs a single 384 well restriction digest plate is prepared for 12 column runs two 384 well restriction digest plates are prepared HaloPlex Target Enrichment System Automation Protocol 35 3 36 Sample Preparation 11 When the workstation has finished preparing each 384 well restriction digest plate for the run you will be prompted by VWorks as shown below The final Bravo deck position of the prepared restriction digest plate varies for different run sizes Transfer plate to thermal cycler Get plate from position 6 seal at 165C for 3 06 Place in thermal cvder and run the digestion program outlined in User Guide After transferring the plate dick Continue below User data entry and Doe 12 Remove the 384 well plate from the Bravo deck position indicated in the prompt 13 Seal the sample plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 3 0 sec Spin the plate briefly to release any bubbles trapped in the liquid 14 Transfer the sealed plate to a thermal cycler and run the digestion program shown in Table 13 using a heated lid After transferring the plate click Continue on the prompt Table 1
20. Enrichment Control DNA Lanes 3 10 ECD digestion reactions HaloPlex Target Enrichment System Automation Protocol Sample Preparation 3 Option 3 Validation by gel electrophoresis Use a Novex 6 polyacrylamide TBE pre cast gel and 1X Novex TBE Running Buffer For more information to do this step consult the manufacturer s recommendations Prepare an undigested DNA gel control by combining 2 uL of the Enrichment Control DNA stock solution and 2 uL of nuclease free water Add 1 uL of Novex Hi Density TBE Sample Buffer 5X to each 4 uL ECD sample Load 5 uL of each sample on the gel In one or more adjacent lanes load 200 ng of a 50 bp DNA ladder Run the gel at 210 V for approximately 15 minutes Stain the gel in 3X GelRed Nucleic Acid Stain for 10 minutes and visualize bands under UV radiation See Figure 6 for sample gel results Figure 6 Validation of restriction digestion by gel electrophoresis Lanes 1 8 ECD di gestion reactions A H Lane 9 Undigested Enrichment Control DNA Lane 10 25 bp DNA ladder Stopping Point If you do not continue to the next step samples may be stored at 20 C for long term storage There are no more long term stopping points until after the PCR amplification step on page 62 HaloPlex Target Enrichment System Automation Protocol 41 3 42 Sample Preparation Step 2 Hybridize digested DNA to HaloPlex probes for target enrichment and sample indexing In t
21. Final DNA Location Labware Needs Controls Information insert Dec Protocol Duration Temperature Presets Advanced Settings Itis okay toturn on the MTC ta pre heat position4 and pre chill position 6 F Enable audio alerts B Start 1 Pause Screen Turn on Thermocubeto chill position 9 Ignore all incubation times testing only C VWorks Workspace NGS Option A HaloPlex Protocol Files 03 Capture v1 1 pro HaloPlex Target Enrichment System Automation Protocol Using the Agilent NGS Workstation for HaloPlex Target Enrichment 2 5 Verify that the Current Tip State indicator on the form shown below matches the configuration of unused tips in the tip box at Bravo Deck position 2 For a fresh tip box containing 12 columns of tips all positions of the Current Tip State unused tip indicator top portion Box 2 should be selected as shown below Clicking Reset selects all columns for position 2 Current Tip State Select columns of unused tips Box 2 iv iv iviv iv iv iv iv Select columns of used tips Box 8 rrrrrrrrrrr EF Reset Clear Also verify that the used tip indicator bottom portion Box 8 matches the configuration of used tips in the tip box at Bravo Deck position 8 For an empty tip box all positions of the Current Tip State used tip indicator bottom portion Box 8 should be cleared as shown above Clicking Reset clears all columns for position 8 It is important that the Current Tip State
22. HaloPlex Target Enrichment System Automation Protocol For Illumina Sequencing Protocol Version D 4 May 2013 Research Use Only Not for use in Diagnostic Procedures OE Agilent Technologies Notices Agilent Technologies Inc 2013 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 G9900 90020 Edition Version D 4 May 2013 Printed in USA Agilent Technologies Inc 5301 Stevens Creek Blvd Santa Clara CA 95051 USA 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 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
23. Start Pause a 2 If the indicator displays Simulation is on click the status indicator button to turn off the simulation mode If you cannot see the toolbar above the HaloPlex VWorks form click Screen in the Controls section of the form 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 22 HaloPlex Target Enrichment System Automation Protocol Using the Agilent NGS Workstation for HaloPlex Target Enrichment 2 Overview of the HaloPlex Target Enrichment Procedure Figure 2 summarizes the HaloPlex target enrichment workflow For each sample to be sequenced individual HaloPlex enriched indexed libraries are prepared Depending on the specific sequencing platform used up to 96 samples can be pooled and sequenced in a single lane Table 7 summarizes how the VWorks automation protocols are integrated into the HaloPlex workflow See the Sample Preparation chapter for complete instructions for use of the VWorks protocols for sample processing Table 7 Overview of VWorks protocols used during the workflow Workflow Step VWorks Protocol used for Automation Digest genomic DNA Digestion pro Hybridize to HaloPlex probe and index Hybridization pro samples Capture and amplify enriched DNA Capture v1 1 pro Purify amplified libraries Purification v1 1 pro HaloPlex Target Enrichment System Automation Protocol 23 2 Using the Agilent NGS W
24. age 19 for more information on using this segment of the form during the run 7 When verification is complete click Start to start the run Controls B start Pause Screen 64 HaloPlex Target Enrichment Svstem Automation Protocol Sample Preparation 3 8 When prompted by VWorks as shown below remove the water reservoir from position 9 of the Bravo deck and replace it with the 70 ethanol reservoir When finished click Continue on the VWorks prompt Replace reservoir Remove the water reservoir from position Place the ethanol reservoir at position 9 When finished dick Continue below Pause and Diagnose L 9 When prompted by VWorks as shown below remove the 70 ethanol reservoir from position 9 of the Bravo deck and replace it with the final sample elution buffer reservoir When finished click Continue on the VWorks prompt Replace reservoir Remove the ethanol reservoir from position 9 Place the elution buffer reservoir at position 9 When finished dick Continue below User data entry mr mineros MN HaloPlex Target Enrichment System Automation Protocol 65 3 Sample Preparation The NGS Workstation completes the liquid handling steps for elution of the captured target DNA 10 When the workstation has finished preparing the final eluted sample plate you will be prompted by VWorks as shown below Click Continue on the VWorks prompt to finish the protocol Samp
25. anol for optimal results HaloPlex Target Enrichment System Automation Protocol Appendix Provisional Adaptor Dimer Removal Protocol 4 11 Wait for 30 seconds to allow any disturbed beads to settle then remove the ethanol using a 200 uL pipette set to 200 uL 12 Repeat step 10 and step 11 once for a total of two washes 13 Remove any residual ethanol with a 20 uL volume pipette 14 Air dry the tubes with open lids at room temperature until the residual ethanol completely evaporates Make sure all ethanol has evaporated before continuing 15 Remove tubes from the magnetic device and add 40 uL of 10 mM Tris acetate or Tris HCl buffer pH 8 0 to each sample Use room temperature Tris acetate or Tris HCl buffer for elution at this step 16 Mix thoroughly by pipetting up and down 15 times using a 100 uL pipette set to 30 uL 17 Incubate for 2 minutes at room temperature to allow elution of DNA 18 Put the tube in the magnetic device and leave for 2 minutes or until the solution is clear 19 Remove the cleared supernatant approximately 40 uL to a fresh tube You can discard the beads at this time Stopping Point If you do not continue to the next step samples may be stored at 20 C for long term storage up to one year Avoid subjecting the stored DNA samples to multiple freeze thaw cycles HaloPlex Target Enrichment System Automation Protocol 79 4 Appendix Provisional Adaptor Dimer Removal Protocol Purify the enriched libr
26. ary pool using AMPure XP beads 80 HaloPlex Target Enrichment System Automation Protocol HaloPlex Target Enrichment System Protocol 5 et Reference e Kit Contents 82 Nucleotide Sequences of HaloPlex Indexes 85 Qualitative analysis of enrichment by gel electrophoresis 91 This chapter contains reference information including component kit contents index sequences and optional gel validation instructions RE Agilent Technologies 81 5 82 Reference Kit Contents The HaloPlex Target Enrichment System includes the following component kits Table 33 HaloPlex Target Enrichment System Kit Contents Design Type HaloPlex Target Enrichment HaloPlex Magnetic System ILM Box 1 Beads Box 2 Store at 20 C Store at 4 C Custom 1 500 kb up to 20 000 probes 5190 5385 OR 5190 5436 5190 5386 ILMFST 96 Reactions Custom 0 5 2 5 Mb OR 0 5 Mb with 5190 5534 OR 5190 5538 5190 5386 220 000 probes ILM 96 Reactions Custom 2 6 Mb 5 Mb ILM 96 Reactions 5190 5536 OR 5190 5540 5190 5386 Cancer Research Panel ILM 96 5190 6236 5190 5386 Reactions Cardiomyopathy Research Panel ILM 96 5190 6529 5190 5386 Reactions See Table 34 for list of included reagents t Part number 5190 5385 5190 5534 or 5190 5536 is provided for the first order of a specific HaloPlex Probe design Re order kits containing previously purchased HaloPlex Probe designs include Box 1 part number 5190 5436 519
27. ation See Figure 4 for sample Bioanalyzer electrophoresis results Figure 4 Validation of restriction digestion by 2100 Bioanalyzer system analysis Lane 1 50 bp DNA ladder Lanes 2 9 ECD digestion reactions A H Lane 10 Undi gested Enrichment Control DNA HaloPlex Target Enrichment System Automation Protocol 39 3 3 Sample Preparation Option 2 Validation by 2200 TapeStation analysis Use a High Sensitivity DIK ScreenTape p n 5067 5363 and reagent kit p n 5067 5364 For more information to do this step see the 2200 TapeStation User Manual Prepare an undigested DNA gel control by combining 1 uL of the Enrichment Control DNA stock solution and 1 uL of nuclease free water Prepare the TapeStation samples as instructed in the 2200 TapeStation User Mamual Use 2 uL of each ECD sample diluted with 2 uL of High Sensitivity D1K sample buffer in separate wells of a tube strip for the analysis Load the sample tube strip the High Sensitivity D1K ScreenTape and loading tips into the 2200 TapeStation as instructed in the 2200 TapeStation User Manual Start the run See Figure 5 for sample TapeStation electrophoresis results MW bp 1500 1000 1 200 500 k k 300 __ 100 ww Figure 5 Validation of restriction digestion by 2200 TapeStation analysis Lane 1 40 TapeStation D1K High Sensitivity Ladder Lane 2 Undigested
28. bel the color marked tube with A then continue labeling the remaining tubes with B through H in order Keep the strips on ice CAUTION It is important to use the restriction enzyme tube strips in the proper orientation when preparing the RE Master Mixes as described below The red or green color marker on the tube strip and cap strip are used to mark well A of each enzyme strip 3 In eight individual tubes prepare the eight Restriction Enzyme Master Mixes A B C D E F G and H according to the table below To prepare Master Mix A combine RE Buffer BSA from step 1 with the indicated volumes of enzyme solution from well A of the Green Enzyme Strip and from well A of the Red Enzyme Strip Prepare Master Mixes B H by repeating this process using enzyme solutions from the corresponding wells B H of each provided Enzyme Strip Table 10 Preparation of RE Master Mixes for Digestion pro protocol Reagent Volume for Volumefor Volumefor Volumefor Volumefor Volume for Volume for 1 Library 1 Column 2Columns 3Columns 4Columns 6Columns 12 Columns RE Buffer BSA 4 0 51 0 uL 85 0 uL 119 0 uL 153 0 uL 221 0 uL 442 0 uL Green Enzyme Strip 0 5 6 4 uL 10 6 uL 14 9 uL 19 1 uL 21 6 uL 55 25 uL enzyme A H Red Enzyme Strip 0 5 6 4 uL 10 6 uL 14 9 uL 19 1 uL 27 6 uL 55 25 uL enzyme A H Total Volume foreach 51 63 8 pL 106 2 pL 148 8 pL 191 2 pL 276 2 pL 552 5 uL Master Mix A B C D E or H For 1 4 column runs RE master mixes A
29. bridize for 16 hours Make sure that the thermal cycler has a maximum reaction volume specification of at least 100 uL The 160 uL HaloPlex hybridization reaction conditions have been optimized with the SureCycler thermal cycler with volume specification of 10 100 uL for PCR reactions The performance of other thermal cyclers for this application should be verified before use HaloPlex Target Enrichment System Automation Protocol 47 3 Sample Preparation Step 3 Capture and amplify the target DNA In this step the circularized target DNA HaloPlex probe hybrids containing biotin are captured on streptavidin beads After capture DNA ligase is added to seal nicks then target DNA is eluted and PCR amplified Assemble reagents for the run 1 Remove reagents to be used in upcoming protocol steps from cold storage and allow the solutions to reach room temperature From 20 C storage remove the Capture Solution Wash Solution Ligation Solution and SSC Buffer From 4 C storage remove the HaloPlex Magnetic Beads 2 Prepare 30 uL per sample plus excess of fresh 50 mM NaOH for use in the DNA elution step on page 50 Prepare the 50 mM NaOH solution from a 10M NaOH stock solution CAUTION Using high quality NaOH is critical for optimal DNA elution and recovery Do not use stock NaOH solutions that were stored at concentrations below 10 M to prepare the 50 mM NaOH solution e Keep the 50 mM NaOH solution container sea
30. clude low temperature 4 C or high temperature 54 C 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 6 for designations of the heat block containing Bravo deck positions on the Multi TEC control device Table 6 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 1 lt gt 24 9 C Shaker 0200 HaloPlex Target Enrichment System Automation Protocol 15 2 Using the Agilent NGS Workstation for HaloPlex 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 temperature 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 ce
31. d User Guide Replace the Nunc plate at position 5 witha fresh Nunc plate Place a fresh 96 Eppendorf Twin tec half skirted plate at position 6 When finished dick Continue below User data entry wa a Get the sample plate from position 6 and seal the plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 3 0 sec Transfer the sealed plate to a thermal cycler and run the ligation program shown in Table 29 using a heated lid Table 29 Thermal cycler program for Capture_v1 1 pro ligation step Step Temperature Time 3 Step Step 1 55 C 10 minutes 2 4 C Hold Remove and discard the Nunc DeepWell plate from position 5 of the Bravo deck Place a fresh Nunc DeepWell plate at position 5 for use in the ligation purification steps of the protocol Place a fresh half skirted 96 well Eppendorf twin tec plate at position 6 for use in the following PCR protocol steps After completing all steps click Continue on the VWorks prompt to continue the automation protocol Do not wait for the conclusion of the thermal cycler ligation program to continue the protocol HaloPlex Target Enrichment System Automation Protocol 59 3 Sample Preparation 14 Once the ligation program in Table 29 is finished and you are prompted by VWorks transfer the plate from the thermal cycler to Bravo deck position 4 Carefully unseal the plate then click Continue on the VWork
32. d in User Guide Replace the plate at position 3 with the SSC Buffer source plate Replace the Nunc plate at position 5 with a fresh Nunc plate When finished dick Continue below User data entry massa Get the sample plate from position 4 and seal the plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 3 0 sec Transfer the sealed plate to a thermal cycler and run the wash program shown in Table 27 using a heated lid Do not include a low temperature hold step in the thermal cycler program following the 10 minute incubation Table 27 Thermal cycler program for Capture_v1 1 pro wash step Step Temperature Time Step 1 46 C 10 minutes b Remove and discard the Wash Solution plate from position 3 of the Bravo deck Place the SSC Buffer source plate at position 3 for use in the ligation segment of the protocol c Remove and discard the bead source plate from position 5 of the Bravo deck Place a fresh Nunc DeepWell plate at position 5 d After completing all steps click Continue on the VWorks prompt to continue the automation protocol Do not wait for the conclusion of the thermal cycler wash program to continue the protocol HaloPlex Target Enrichment System Automation Protocol 55 3 Sample Preparation 10 During the 10 minute incubation of the sample plate on the thermal cycler you will be prompted to add the Ligation Master Mix to the Master Mix source plat
33. duct purification remove the Agencourt AMPure XP Beads from 4 C storage for use on page 63 Let the beads come to room temperature for the remainder of the amplification program Stopping Point If you do not continue to the next step PCR products may be stored at 20 C for up to 72 hours or at 8 C overnight For best results however purify PCR products as soon as possible 62 HaloPlex Target Enrichment System Automation Protocol Sample Preparation 3 Step 4 Purify the amplified target DNA In this step the NGS Workstation does the liquid handling steps to purify the amplified target DNA sample using AMPure XP beads Prepare the workstation and reagents 1 Gently wipe down the Bravo deck with a NucleoClean decontamination wipe Let the AMPure XP beads come to room temperature for at least 30 minutes Do not freeze the AMPure XP beads at any time Mix the room temperature bead suspension well so that the reagent appears homogeneous and consistent in color Prepare a Nunc DeepWell source plate containing AMPure XP beads For each well to be processed add 100 uL of homogenous AMPure beads per well to the Nunc DeepWell plate 5 Place a red insert on Bravo deck position 6 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 Prepare a Thermo Scientific reservoir containing 15 mL of nuclease free
34. e Agilent NGS Workstation for HaloPlex Target Enrichment Using the HaloPlex VWForm to setup and start a run Use the VWorks form HaloPlex VWForm shown below to set up and start each HaloPlex automation protocol Open the form using the HaloPlex VWForm shortcut on your desktop Use the drop down menus on the form to select the appropriate HaloPlex workflow step and number of columns of samples for the run Once all run parameters have been specified on the form click Update layout and information The displayed protocol will not run unless the Update layout and information button has been clicked 4 i Agilent Technologies Q 18 The Bravo Deck Setup region of the form will then display the required placement of reaction components and labware on the workstation Bravo deck for the specified run parameters Agilent HaloPlex Automation 3 4 wi Parameters Bravo Deck Setup Current Tip State Select columns of unused tips Box 2 1 Step os Capture v1 1 pro Empty deepwell Newtip box Wash Solution in eed WMV WR RRR RR 2 Number of columns of samples 2 ERE i j SGB OO 3 Pi aie Hybridization plate HaloPlexMagnetic Eppendorf Twin tec 4 Update current tip state Status Elapsed Time 00 02 28 onred insert Beads Nuncplate half skirted plate onred insert Hose Gear 54 sec Reference T Empty tip box Master Mixes in SSS Nunc plate on silver
35. e as shown below Add Ligation Master Mix Add the appropriate amount of ligation master mix for the number of columns processed to column 2 of the Nunc master mix plate at position 9 When finished click Continue below User data entry pause and Dao Add the volume of Ligation Master Mix indicated in Table 28 to all wells of column 2 of the Nunc DeepWell Master Mix source plate on Bravo deck position 9 Table 28 Addition of Ligation Master Mix to the Master Mix Source Plate for Capture_v1 1 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 Ligation Master Column 2 73 4 uL 126 6 pL 179 7 pL 232 8 uL 339 1 uL 684 4 uL Mix A2 H2 The Master Mix source plate at postition 9 should already contain the PCR Master Mix in Column 3 and the depleted Hybridization Master Mix from the Hybridization pro protocol in Column 1 Be sure to add the Ligation Master Mix to Column 2 of the source plate at this step 56 HaloPlex Target Enrichment System Automation Protocol Sample Preparation 3 11 Once the wash program in Table 27 has finished and you are prompted by VWorks transfer the plate from the thermal cycler to Bravo deck position 4 Carefully unseal the plate then click Continue on the VWorks prompt to resume the Capture_v1 1 pro protocol Get plate from th
36. e washed HaloPlex streptavidin bead suspension Add 40 uL of the homogenous bead suspension to all wells of the Nunc DeepWell plate that correspond to sample containing wells on the hybridization plate 4 Place the streptavidin bead source plate at position 5 of the Bravo deck Prepare wash and elution solution source plates Prepare a separate source plate for each of the solutions listed in Table 22 Use full skirted 96 well Eppendorf twin tec plates to prepare all three source plates For all sample containing wells of the hybridization plate add the specified volume of solution to all corresponding wells of the solution source plate Table 22 Preparation of solution source plates for Capture v1 1 pro protocol Solution Volume to dispense per well of source plate Wash Solution 110 uL SSC Buffer 110 uL 50 mM NaOH 30 uL Seal the 50 mM NaOH source plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 1 0 sec Leave sealed until you are prompted to add the plate to the Bravo deck in step 15 on page 60 50 HaloPlex Target Enrichment System Automation Protocol Prepare the Master Mixes for Capture_v1 1 pro protocol Sample Preparation 3 1 Prepare the appropriate amount of PCR Master Mix according to the table below Mix well by gentle vortexing then spin the tube briefly Table 23 Preparation of PCR Master Mix for Capture_v1 1 pro Reagent Volume for Volume for Volume for Volume for
37. eck position 4 to 4 C using the Inheco Multi TEC control touchscreen as described in Setting the Temperature of Bravo Deck Heat Blocks For 12 column runs only place a second 384 well adapter insert on Bravo deck position 6 and pre set the temperature of Bravo deck position 6 to 4 C HaloPlex Target Enrichment System Automation Protocol Sample Preparation 3 To expedite thermal cycler warm up for the hybridization reaction on page 47 you can enter and initiate the hybridization program on the thermal cycler now and then pause the program until you are instructed to transfer the reaction plate Be sure to pause the thermal cycler before the initiation of the 95 C incubation segment Release the pause immediately after transferring the plate to the thermal cycler in step 11 on page 47 Be sure that the 96 well block is in the thermal cycler before initiating the program for warm up Prepare the Master Mix Source Plate for Hybridization pro 1 Prepare the appropriate amount of Hybridization Master Mix according to the table below Mix well by gentle vortexing then spin the tube briefly Table 15 Preparation of Hybridization Master Mix for Hybridization pro Reagent Volume for Volumefor Volumefor Volumefor Volumefor Volumefor Volume for 1 Library 1 Column 2 Columns 3Columns 4 Columns 6 Columns 12 Columns HaloPlex Probe 20 uL 255 uL 425 uL 595 uL 765 uL 1105 uL 2210 uL Hybridization 50 uL 637 5 uL 1062 5 uL 1487 5 uL 1912 5 uL 276
38. ed sample 250 lal Vi J f MAN a d m Un MEY A 10044 H HA y 1 Nf Fal 1 504 Ww VA LR JA N VN 2994 N la ll i Jl Pv EVEN AN 35 100 150 200 300 400 500 600 700 1000 2000 10380 bp Figure 8 Validation of HaloPlex enrichment by 2100 Bioanalyzer system analysis HaloPlex Target Enrichment System Automation Protocol Sample Preparation 3 Option 2 Analysis using the 2200 TapeStation Use a High Sensitivity D1K ScreenTape p n 5067 5363 and reagent kit p n 5067 5364 to analyze the enriched library samples For more information to do this step see the 2200 TapeStation User Manual 1 Prepare the TapeStation samples as instructed in the 2200 TapeStation User Manual Use 2 uL of each enriched library sample diluted with 2 uL of High Sensitivity DIK sample buffer in separate wells of a tube strip for the analysis 2 Load the sample tube strip the High Sensitivity D1K ScreenTape and loading tips into the 2200 TapeStation as instructed in the 2200 TapeStation User Manual Start the run 3 Analyze the electropherogram for each sample according to the analysis guidelines on page 70 See Figure 9 for a sample TapeStation electropherogram 5 Sample Intensity FU um MW Figure 9 Validation of HaloPlex enrichment by 2200 TapeStation analys
39. ermal cycler Once the wash program has finished retrieve plate from thermal cycler and place at position 4 Unseal the plate then click Continue below to resume the protocol User data entry pause nd Doe The NGS Workstation completes the liquid handling steps for ligation of the captured target DNA To expedite thermal cycler warm up for the subsequent ligation program on page 59 you can enter and initiate the wash program on the thermal cycler now and then pause the program until you are instructed to transfer the reaction plate Be sure to pause the thermal cycler before the initiation of the 55 C incubation segment Release the pause immediately after transferring the plate to the thermal cycler in step 13 HaloPlex Target Enrichment System Automation Protocol 57 3 Sample Preparation Step 3 Capture and amplify the target DNA 12 When prompted by VWorks as shown below remove and discard the plate from position 4 Click Continue on the prompt to resume the protocol Remove plate Remove and discard plate from position 4 When finished dick Continue below 58 HaloPlex Target Enrichment System Automation Protocol Sample Preparation 13 When the workstation has finished preparing the ligation plate you will be prompted by VWorks as shown below Transfer plate to thermal cycler Get plate from position 6 seal at 165C for 3 06 Place thermal cyder and run the ligation program outline
40. es Successful digestion is indicated by the appearance of the three predominant bands The presence of additional minor bands with relative abundance similar to the additional bands visible in Figure 4 Figure 5 and Figure 6 does not impact enrichment results It is acceptable for band intensities in digestion reaction B to be slighltly reduced compared to the other digestion reactions 38 HaloPlex Target Enrichment System Automation Protocol Sample Preparation Option 1 Validation by 2100 Bioanalyzer analysis Use a High Sensitivity DNA Kit p n 5067 4626 and the 2100 Bioanalyzer system with 2100 Expert Software version B 02 07 or higher required to run the High Sensitivity Kit See the reagent kit guide for general Bioanalyzer system setup instructions Prepare an undigested DNA gel control by combining 0 5 uL of the Enrichment Control DNA stock solution and 3 5 uL of nuclease free water Prepare the chip samples and ladder as instructed in the reagent kit guide using 1 uL of each ECD sample and undigested DNA control for the analysis When loading samples on the chip load the DNA ladder in the ladder sample well marked on the chip Load the eight ECD digest samples A to H in sample wells 1 to 8 and load the undigested ECD sample in sample well 9 Do not run the undigested ECD control in sample well 1 Place the prepared chip into the 2100 Bioanalyzer instrument and start the run within five minutes after prepar
41. esence sensor again Ignore to continue to home the Please note that any plate currently held by the Gripper will be dropped Choose Abort to cancel initialization 20 HaloPlex Target Enrichment System Automation Protocol Using the Agilent NGS Workstation for HaloPlex Target Enrichment VWorks Automation Control Software 2 If you encounter the W axis error message shown below select Retry Please verify that it is safe to home aspirate dispense axis If there is fluid in the tips you tj may want to manually home the W axis in diagnostics over a waste position Choose Retrv to continue homing the W axis Choose Ignore to leave the W axis unhomed Choose Abort to cancel initialization Ignore and Continue leaving device in current state Abort HaloPlex Target Enrichment System Automation Protocol 2 21 2 Using the Agilent NGS Workstation for HaloPlex Target Enrichment 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 Simulation is off f gt 2 Diagnostics a e Log out g Compile
42. esult 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 per formed or adhered to could result in personal injury or death Do not proceed beyond a WARNING notice until the indicated condi tions are fully understood and met HaloPlex Target Enrichment System Automation Protocol In this Guide This guide describes an optimized automation protocol for using the HaloPlex target enrichment system to prepare sequencing library samples for Illumina paired end multiplexed sequencing platforms Sample processing steps are automated using the Agilent 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 HaloPlex Target Enrichment This chapter contains an orientation to the Agilent NGS Workstation an overview of the HaloPlex target enrichment protocol and considerations for designing HaloPlex experiments for automated processing using the Agilent NGS Workstation Sample Preparation This chapter describes the steps of the automated HaloPlex workflow to prepare target enriched sequencing libraries fo
43. et Enrichment System Protocol 1 Before You Begin Procedural Notes 8 Safety Notes 8 Required Reagents 9 Required Equipment for HaloPlex Target Enrichment Automated Protocols 11 Optional Validation Reagents and Equipment 12 Required Accessories for Adaptation of NGS Workstation Option B for HaloPlex Automation 12 Make sure you read and understand the information in this chapter and have the necessary equipment and reagents listed before you start an experiment RE Agilent Technologies 1 Before You Begin Procedural Notes The protocols in this manual are for use with Agilent s G9901B G9911B G9921B and G9903B HaloPlex Target Enrichment System kits The protocol is not compatible with earlier versions of HaloPlex reagent kits including Agilent part numbers G9900A G9900B and G9900C The 96 reaction kit contains enough reagents to prepare master mixes for four runs of 3 columns of samples 24 samples per run When processing samples using runs with fewer than 24 samples some reagents may be depleted before 96 samples are run The HaloPlex protocol is optimized for digestion of 200 ng of genomic DNA split among 8 different restriction digestion reactions plus 25 ng excess DNA for a total of 225 ng genomic DNA Using lower amounts of DNA in the enrichment protocol can adversely affect your results Use a fluorometry based DNA quantitation method such as PicoGreen stain or Qubit fluorometry to quantify the DNA starting
44. fik Ede Sewc View Encoding Language Settings Macro Min Plugins Window 7 ote Re in H XD otv mader IEMFileVersion t ia F Investigator Mane Test Project Nana Test Proj Experiment Test Experiment Date 9 28 2012 Workflow Ganarate PASTI Application FASTQ Only 9 Assay Truteq LT Description Test Chanistry Default Reads 151 151 Settings Data Sanple ID Sample Nams Sanrle Plate Sanple Well 17 Index ID indax Sample Project Description 21 1 Sarcle 01 001 le Tumor 2 Sarpla 02 0 Normal 3 Sarpla A03 A003 Tumor 4 Sarpla A04 A006 Normal 5 SarzleS testplate AUS A005 Tumor 24 Sample tastplatu A06 A006 ProjectX Normal 2 Save the edited Sample Sheet in an appropriate file location for use in the MiSeq platform run 74 HaloPlex Target Enrichment System Automation Protocol Sample Preparation 3 HiSeq platform sequencing run setup guidelines Set up sequencing runs to perform an 8 nt index read using the Cycles settings shown in Table 32 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 32 HiSeq platform Run Configuration screen Cycle Number settings Run Segment Cycle Number Read 1 100 Index 1 i7 9 Index 2 15 0 Read 2 100 Settings apply to
45. his step the collection of gDNA restriction fragments is hybridized to the HaloPlex probe capture library HaloPlex probes are designed to hybridize selectively to fragments originating from target regions of the genome and to direct circularization of the targeted DNA fragments During the hybridization process lumina sequencing motifs including index sequences are incorporated into the targeted fragments For sample indexing primer assignments see Nucleotide Sequences of HaloPlex Indexes on page 85 for nucleotide sequences of the 96 indexes used in the HaloPlex Target Enrichment System The duration of the hybridization reaction is determined by the probe density of your design Refer to the Certificate of Analysis provided with Box 1 of your kit to determine the hybridization conditions appropriate for your design If the Certificate of Analysis provided with your custom probe does not include the hybridization time please see the Note on page 47 for more information Prepare the workstation 1 Gently wipe down the Bravo deck with a NucleoClean decontamination wipe 2 Place a red instert on Bravo deck position 1 3 Place a silver Nunc plate insert on Bravo deck position 9 4 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 2526 ethanol 5 For all run sizes place a 384 well adapter insert on Bravo deck position 4 Pre set the temperature of Bravo d
46. hould range from 175 to 625 bp in length with the majority of products sized 225 to 525 bp Amplicons in the 175 to 625 bp size range should be included for quantitation of the enriched target DNA in each sample Any spurious DNA products outside of this size range in any sample should be excluded from the target DNA quantitation results HaloPlex Target Enrichment System Automation Protocol 67 3 Sample Preparation Option 1 Analysis using the 2100 Bioanalyzer System Use a Bioanalyzer High Sensitivity DNA Assay kit and the 2100 Bioanalyzer instrument with 2100 Expert Software version B 02 07 or higher required to run the High Sensitivity Kit See the reagent kit guide for general Bioanalyzer instrument and assay setup instructions 1 Prepare the chip samples and ladder as instructed in the reagent kit guide using 1 uL of enriched library sample for the analysis Load the prepared chip into the 2100 Bioanalyzer instrument and start the run within five minutes after preparation Analyze the electropherogram for each sample according to the analysis guidelines on page 70 See Figure 8 for a sample Bioanalyzer electropherogram If the concentration determined by Bioanalyzer analysis is gt 10 ng uL repeat the analysis using a 1 10 dilution of the sample Dilute 1 uL of the sample in 9 uL of 10 mM Tris 1 mM EDTA and then mix well by vortexing at 2000 rpm on the IKA vortex supplied with the Bioanalyzer before analyzing the dilut
47. ies 2 Using the Agilent NGS Workstation for HaloPlex 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 90004 and the VWorks Software User Guide G5415 90002 14 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 L Front Figure 1 Bravo platform deck HaloPlex Target Enrichment System Automation Protocol Using the Agilent NGS Workstation for HaloPlex 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 in
48. indicator matches the configuration of tips present at Bravo Deck positions 2 and 8 when initiating the run Tips that are inappropriately loaded onto the Bravo platform pipette head or tips missing from the pipette head will interfere with automated processing steps You can use partial tip boxes for HaloPlex automation protocols as long as positions of available tips are accurately indicated during run setup 6 After verifying that the NGS Workstation has been set up correctly click Start in the Controls section of the form to begin the run Do not use the Start button on the VWorks Control Toolbar runs must be initiated using the start button on the HaloPlex VW Form shown below Controls start 1 Pause Screen HaloPlex Target Enrichment System Automation Protocol 19 2 Using the Agilent NGS Workstation for HaloPlex 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 pr
49. ini cell Life Technologies p n E10001 Novex 6 Polyacrylamide TBE Pre cast Gels Novex TBE Running Buffer 5X Novex High density TBE Sample Buffer 5X GelRed Nucleic Acid Stain 3X in water DNA molecular weight markers UV transilluminator Life Technologies p n 62655 0 Life Technologies p n LC6675 Life Technologies p n LC6678 Biotium p n 41001 General laboratory supplier General laboratory supplier Table 5 HaloPlex Automation Protocols Description Required Accessories for Adaptation of NGS Workstation Option B for HaloPlex Automation Accessories Required for Adaptation of Agilent NGS Workstation Option B for Vendor and part number Custom hardware 384 well plate inserts quantity of two required 96 well PCR plate insert red quantity of one required Agilent p n G5420A Agilent p n G5498B 13 HaloPlex Target Enrichment System Automation Protocol HaloPlex Target Enrichment System Protocol 2 200 Using the Agilent NGS Workstation for e HaloPlex Target Enrichment About the Agilent NGS Workstation 14 Overview of the HaloPlex Target Enrichment Procedure 23 Experimental Setup Considerations for Automated Runs 25 This chapter contains an orientation to the Agilent NGS Workstation an overview of the HaloPlex target enrichment protocol and considerations for designing HaloPlex experiments for automated processing using the Agilent NGS Workstation E Agilent Technolog
50. is HaloPlex Target Enrichment System Automation Protocol 69 3 Sample Preparation Analysis of Electropherogram Results Check that the electropherogram shows a peak fragment size between approximately 225 to 525 bp Determine the concentration of enriched target DNA in the sample by integration under the peak between 175 and 625 bp Peaks at 150 bp may be observed but should be excluded from quantitation Some designs may generate a peak at about 125 bp This peak is associated with an adaptor dimer product which will cluster and generate sequence that does not map to the genome If the molar fraction of the 125 bp peak is greater than 10 do another round of AMPure purification after pooling samples First pool equimolar amounts of libraries to be multiplexed using concentrations determined for the 175 625 peak of each sample Using 40 uL of the pooled libraries purify the DNA using AMPure XP beads according to the protocol starting on page 77 70 HaloPlex Target Enrichment System Automation Protocol Sample Preparation 3 Step 6 Pool samples with different indexes for multiplexed sequencing Use the following guidelines to design your sample pooling strategy Use the Bioanalyzer or TapeStation measured concentration of 175 625 bp products in each sample to pool equimolar amounts of differentially indexed samples in order to optimize the use of sequencing capacity The final HaloPlex enrichment pool is ready for
51. led when not in use especially when processing large numbers of samples per run Table 19 Amount of 50mM NaOH required per run size Volume for Volume for Volume for Volume for Volume for Volume for Volume for 1 Library 1 Column 2 Columns 3 Columns 4 Columns 6 Columns 12 Columns 30 uL 270 uL 510 uL 750 uL 990 uL 1470 uL 2940 pL 3 Obtain or prepare 0 5 uL per sample plus excess of 2 M acetic acid for use in the PCR master mix on page 51 CAUTION It is critical to use high quality acetic acid at 2 M concentration in this step to ensure neutralization of the NaOH used for elution See Table 1 on page 9 for 2 M acetic acid supplier information or prepare 2 M acetic acid from high quality glacial acetic acid 48 HaloPlex Target Enrichment System Automation Protocol Sample Preparation 3 Prepare the workstation 1 Gently wipe down the Bravo deck with a NucleoClean decontamination wipe 2 Place a red insert on Bravo deck position 4 3 Pre set the temperature of Bravo deck position 4 to 54 C using the Inheco Multi TEC control touchscreen as described in Setting the Temperature of Bravo Deck Heat Blocks 4 Place a second red insert on Bravo deck position 6 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 6 Place the silver Nunc plate insert on Bravo deck position 9 7 Turn on the chiller set
52. les are ready The eluted samples are ready Get the sample plate from position 3 Click Continue below to finish the protocol User data entry Stopping Point If you do not continue to the next step samples may be stored at 20 C for long term storage up to one year Avoid subjecting the stored DNA samples to multiple freeze thaw cycles HaloPlex Target Enrichment System Automation Protocol Sample Preparation 3 Step 5 Validate enrichment and quantify enriched target DNA Prior to sample pooling and sequencing sample preparation validate enrichment and quantify the enriched target DNA in each library sample by microfluidics analysis using the 2100 Bioanalyzer see page 68 or the 2200 TapeStation see page 69 Enriched library samples may also be qualitatively analyzed using gel electrophoresis Sample gel electrophoresis results are provided in the Reference section on page 91 Expected Results Each amplicon in the prepared library contains one target insert surrounded by sequence motifs required for multiplexed sequencing using the Illumina platform Amplicons include 50 to 500 bp of target DNA insert and 125 bp of sequencing motifs as shown in Figure 7 Figure 7 Content of HaloPlex enriched target amplicons Each amplicon contains one target insert blue surrounded by the Illumina paired end sequencing ele ments black the sample index red and the library bridge PCR primers yel low The amplicons s
53. m Automation Protocol Required Equipment Table 3 Before You Begin 1 Required Equipment for HaloPlex Target Enrichment Automated Protocols Description Agilent NGS Workstation Option A with VWorks software version 11 0 1 1032 or later Robotic Pipetting Tips Sterile Filtered 250 uL Thermal Cycler Eppendorf twin tec full skirted 96 well PCR plates Eppendorf twin tec full skirted 384 well PCR plates Eppendorf twin tec half 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 Magnetic separator j 1 5 mL tube compatible separator or Conical vial compatible separator Benchtop microcentrifuge Benchtop plate centrifuge P10 P20 P200 and P1000 pipettes NucleoClean Decontamination Wipes Qubit 2 0 Fluorometer Qubit assay tubes Vortex mixer Vendor and part number Contact Agilent Automation Solutions for ordering information Customerservice automation agilent com Agilent p n 19477 022 Agilent SureCycler 8800 p n G8800A 96 well plate module p n G8810A 384 well plate module p n G8820A or equivalent thermal cycler and accessories Eppendorf p n 951020401 or 951020619 Eppendorf p n 951020702 Eppendorf p n 951020303 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 Life Technologies DynaMag 2 magnet p n
54. ment System Automation Protocol Reference Qualitative analysis of enrichment by gel electrophoresis Enrichment products may be qualitatively analyzed by gel electrophoresis Analyze 5 uL of each enriched library sample enriched ECD sample or experimental enriched libraries by elecrophoresis on a Novex 6 polyacrylamide TBE pre cast gel See page 41 for additional gel analysis protocol recommendations Successful enrichment is indicated by the presence of a smear of amplicons from approximately 225 to 525 bp in each enrichment library lane For some probe designs low molecular weight lt 150 bp bands may also be visible but should not be included in enriched sample quantitation See Figure 10 for a sample gel analysis image 225 bp 125 bp 25 bp 1 2 3 4 5 6 7 Figure 10 Validation of HaloPlex enrichment process by gel electrophoresis Lane 1 25 bp DNA ladder Lanes 2 7 enriched library samples HaloPlex Target Enrichment System Automation Protocol 91 5 www agilent com In This Book This guide contains information to run the HaloPlex Target Enrichment System automation protocol Agilent Technologies Inc 2013 Version D 4 May 2013 G9900 90020 OE Agilent Technologies
55. mns per run Each 96 reaction kit contains sufficient reagents for 96 reactions configured as 4 runs of 3 columns of samples per run HaloPlex Target Enrichment System Automation Protocol 25 2 26 Using the Agilent NGS Workstation for HaloPlex 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 1 to then A2 to H2 ending with 12 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 For sample indexing during hybridization to the HaloPlex probe see Figure 2 you will need to prepare a separate plate containing the HaloPlex Indexing Primer Cassettes Assign the wells to be indexed with their respective indexing primers during experimental design See Nucleotide Sequences of HaloPlex Indexes on page 85 for nucleotide sequences of the 96 indexes used in the HaloPlex Target Enrichment System Considerations for Equipment Setup 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 Some workflow steps re
56. ns two 384 well plates produced RE Master Mix A A1 A1 plate 1 RE Master Mix B A2 A2 plate 1 RE Master Mix C B1 B1 plate 1 RE Master Mix D B2 B2 plate 1 RE Master Mix E A13 A1 plate 2 RE Master Mix F A14 A2 plate 2 RE Master Mix G B13 B1 plate 2 RE Master Mix H B14 B2 plate 2 b Incubate the removed 4 uL samples at 80 C for 5 minutes to inactivate the restriction enzymes c Analyze the prepared samples using microfluidic electrophoresis on the 2100 Bioanalyzer see page 39 or on the 2200 TapeStation see page 40 or using gel electrophoresis see page 41 HaloPlex Target Enrichment System Automation Protocol 37 3 Sample Preparation The ECD sample contains genomic DNA mixed with an 800 bp PCR product that contains restriction sites for all the enzymes used in the digestion protocol When analyzing validation results the undigested control should have gDNA bands at gt 2 5 kbp and a PCR product band at 800 bp Each of the eight digested ECD samples should have a smear of gDNA restriction fragments between 100 and 2500 bp overlaid with three predominant bands at approximately 125 225 and 450 bp These three bands correspond to the 800 bp PCR product derived restriction fragments and precise sizes will differ after digestion in each of the eight RE master mixes In addition to the three predominant bands at approximately 125 225 and 450 bp you may detect additional minor bands in the digested ECD sample lan
57. nter of the display CPAC 2 1 25 0 C 2 Current temp 0200 rpm ser 16 HaloPlex Target Enrichment Svstem Automation Protocol Using the Agilent NGS Workstation for HaloPlex Target Enrichment 2 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 HaloPlex 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 HaloPlex 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 0 1 1032 or later If you have questions about VWorks version compatibility please contact service automation agilent com Logging in to the VWorks software 1 Double click the VWorks icon or the HaloPlex VWForm shortcut on the Windows desktop to start the VWorks software 2 If User Authentication dialog is not visible click Log in 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 HaloPlex Target Enrichment System Automation Protocol 17 2 Using th
58. olution DNA Ligase Total Volume Volume for Volume for Volumefor Volumefor Volumefor X Volumefor Volume for 1 Library 1 Column 2 Columns 3Columns 4Columns 6 Columns 12 Columns 47 5 uL 605 6pL 1009 pL 1413 pL 1817 pL 2624 uL 5249 uL 2 5 uL 31 9 uL 53 1 uL 744 uL 95 6 uL 138 1 uL 276 3 uL 50 uL 6375 uL 1066211 14874pL 19126pL 276211 55253 Prepare the Master Mix Source Plate for Capture v1 1 pro Using the same Nunc DeepWell plate that was used for the Hybridization pro run prepare the Master Mix source plate for Capture v1 1 pro Add the volume indicated in Table 25 of PCR Master Mix to all wells of column 3 of the Nunc DeepWell plate Table25 Preparation of the Master Mix Source Plate for Capture v1 1 pro Master Mix Solution PCR Master Mix Position on Volume of Master Mix 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 Column 3 44 1 uL 75 9 uL 107 8 uL 139 7 uL 203 4 uL 410 6 uL A3 H3 Column 2 of the Master Mix source plate must remain empty at this step You will be prompted to add Ligation Master Mix to Column 2 at the appropriate time during the Capture v1 1 pro protocol Column 1 was used during the Hybridization pro protocol If you are using a new DeepWell plate for the Capture v1 1 pro Master Mix source plate be sure to leave columns 1 and 2 empty at this time adding the PCR Master Mix to column 3 of
59. onsiderations 28 HaloPlex Target Enrichment System Automation Protocol HaloPlex Target Enrichment System Protocol eo D 3 200 Sample Preparation e Step 1 Digest genomic DNA with restriction enzymes 30 Step 2 Hybridize digested DNA to HaloPlex probes for target enrichment and sample indexing 42 Step 3 Capture and amplify the target DNA 48 Step 4 Purify the amplified target DNA 63 Step 5 Validate enrichment and quantify enriched target DNA 67 Step 6 Pool samples with different indexes for multiplexed sequencing 71 This section contains instructions for gDNA library target enrichment for sequence analysis using the Illumina platform For each sample to be sequenced an individual target enriched indexed library is prepared The target region can vary from 1 kb to 5 Mb Custom HaloPlex probes must be designed before purchasing the kit using Agilent s SureDesign tool at www agilent com genomics suredesign The HaloPlex Target Enrichment System amplifies thousands of targets in the same reaction incorporating standard Illumina paired end sequencing motifs in the process During hybridization each sample can be uniquely indexed allowing for pooling of up to 96 samples per sequencing lane E Agilent Technologies 28 3 30 Sample Preparation Step 1 Digest genomic DNA with restriction enzymes In this step gDNA samples are digested by 16 different restriction enzymes to create a library of gDNA
60. or your probe design from Table 2 Agilent p n 600677 Ambion Cat AM9930 Beckman Coulter Genomics p n A63880 p n A63881 p n A63882 Sigma p n 72068 Sigma p n A8976 General laboratory supplier Sigma Aldrich p n E7023 Life Technologies p n 032850 Life Technologies p n 032853 HaloPlex Target Enrichment System Automation Protocol 1 10 Before You Begin To select a HaloPlex Target Enrichment System Reagent Kit use Agilent s SureDesign tool at www agilent com genomics suredesign to design a custom panel or to select a pre designed panel Reagent kit ordering information is supplied as part of the SureDesign process and is summarized in Table 2 below Table 2 Target Enrichment System Kits for Illumina Sequencing HaloPlex Probe Design Part Number Custom Panel Tier 1 ILM 96 reactions G9901B Custom Panel Tier 2 ILM 96 reactions G9911B Custom Panel Tier 3 ILM 96 reactions G9921B Cancer Research Panel ILM 96 reactions G9903B Cardiomyopathy Research Panel ILM 96 reactions G9908B Tier 1 designs are 1 500 kb and up to 20 000 probes t Tier 2 designs 0 5 2 5 Mb OR 1 500 kb with gt 20 000 probes t Tier 3 designs are 2 6 Mb 5 Mb Kits contain enough reagents for 96 reactions total including one or more control reactions using Enrichment Control DNA ECD samples Each run of up to 96 samples should include one ECD control enrichment reaction HaloPlex Target Enrichment Syste
61. orkstation for HaloPlex Target Enrichment Overview of the HaloPlex Target Enrichment Procedure 1 Digest genomic DNA E 2 Hybridize the HaloPlex probe library in presence of the Indexing Primer Cassette Hybridization results in gDNA fragment circularization and incorporation of indexes and Illumina sequencing motifs 3 Capture target DNA probe hybrids Biotinylation of probe DNA allows capture using streptavidin coated magnetic beads 4 PCR amplify targeted fragments to produce a sequencing ready target enriched sample Figure 2 Overall HaloPlex target enriched sequencing sample preparation workflow 24 HaloPlex Target Enrichment System Automation Protocol Using the Agilent NGS Workstation for HaloPlex Target Enrichment 2 Experimental Setup Considerations for Automated Runs HaloPlex Automated Target Enrichment System runs may include 1 2 3 4 6 or 12 columns equivalent to 8 16 24 32 48 96 wells of SDNA samples to be enriched for sequencing on the Illumina platform Plan your experiments using complete columns of samples Table 8 Columns to Samples Equivalency Number of Columns Processed Total Number of Samples Processed 1 8 2 16 3 24 4 32 6 48 12 96 The number of columns or samples that may be processed using the supplied reagents see page 82 will depend on the experimental design For greatest efficiency of reagent use plan experiments using at least 3 colu
62. ot include a low temperature hold step in the thermal cycler program Incubation at 54 C for more than the indicated time is not recommended Table 18 Thermal cycler program for HaloPlex probe hybridization Step Temperature Step 1 95 C Step 2 54 C Time Duration of Step Cancer Research Panel Custom Designs with 20 000 Custom Designs with gt 20 000 or Cardiomyopathy probes probes Research Panel see Certificate of Analysis see Certificate of Analysis 10 minutes 10 minutes 10 minutes 3 hours 3 hours 16 hours Thermal cyclers that use calculated temperature methods cannot be set to 160 uL reaction volumes In that case enter the maximum possible volume t Typical 1 500 kb desi gns contain lt 20 000 probes Please refer to the Certificate of Analysis included with your probe to determine the appropriate hybridization time t Typical 501 kb 5 Mb designs and some 1 500 kb designs contain gt 20 000 probes Please refer to the Certificate of Analysis included with your probe to determine the appropriate hybridization time CAUTION If the Certificate of Analysis provided with your custom probe does not include hybridization time information use the following guidelines For 1 500 kb designs hybridize for 3 hours unless you were notified by Agilent to use hybridization conditions for larger designs 16 hour hybridization For 501 kb 2 5 Mb designs hybridize for 16 hours For 2 5 5 Mb designs hy
63. quire that the sample plate be sealed using the PlateLoc thermal microplate sealer on the Agilent NGS Workstation and then centrifuged to collect any dispersed liquid To maximize efficiency locate the centrifuge in close proximity to the Agilent NGS Workstation Run Time Considerations Before you begin refer to the Certificate of Analysis provided with Box 1 of your kit to determine the hybridization duration appropriate for your design After reviewing the duration of this and other steps in the protocol plan the start time for your experiment accordingly HaloPlex Target Enrichment System Automation Protocol Using the Agilent NGS Workstation for HaloPlex Target Enrichment Designs containing lt 20 000 probes including the Cancer Research and Cardiomyopathy Research Panels use a 3 hour hybridization time For these designs DNA digestion through PCR protocols see Figure 2 are typically run on the same day with the DNA digestion protocol initiated early in the day Designs containing gt 20 000 probes use a 16 hour hybridization time which is typically completed overnight Calculate the appropriate start time for the DNA digestion protocol based on your run size and the run time estimates provided in the HaloPlex form in the VWorks software HaloPlex VWForm to allow overnight hybridization HaloPlex Target Enrichment System Automation Protocol 27 2 Using the Agilent NGS Workstation for HaloPlex Target Enrichment Run Time C
64. r the Illumina platform Appendix Provisional Adaptor Dimer Removal Protocol This chapter describes a protocol used to remove adaptor dimer 125 bp molecules that may be observed for some designs Reference This chapter contains reference information including component kit contents and index sequences HaloPlex Target Enrichment System Automation Protocol What s New in Version D 4 Support for HaloPlex Cardiomvopathv Research Panel designs Table 2 on page 10 Table 18 on page 47 and Table 33 on page 82 What s New in Version D 3 Support for HaloPlex Cancer Research Panel designs Table 2 on page 10 Table 18 on page 47 and Table 33 on page 82 Updated Custom Kit ordering information page 10 Updated supplier information for NaOH and acetic acid Table 1 on page 9 and updated preparation instructions for NaOH page 48 New Run Time Considerations section page 26 Support for FFPE derived DNA samples see Note on page 30 Updated instructions for preparation of RE Master Mixes from the provided Enzvme Strips page 32 Instructions for obtaining Agilent s SureCall analvsis software page 75 Support for NGS Workstation error messages that mav be encountered during run setup page 20 What s New in Version D 2 Updated instructions for determination of hvbridization time by referral to Box 1 Certificate of Analysis page 47 Updated instructions for use of Enzvme Strips 1 and 2 page 30 and page 32 Upda
65. restriction fragments The gDNA is digested in eight different restriction reactions each containing two restriction enzymes The 16 restriction enzymes are provided in two 8 well strip tubes that are distinguished by red and green color markers Enzymes are combined from corresponding wells of the red and green marked strip tubes to make eight different RE Master Mixes which are then combined with each DNA sample in the run Successful enrichment using the protocol in this guide requires high quality DNA samples Before you begin verify that the genomic DNA samples have an OD 260 280 ratio ranging from 1 8 to 2 0 Verify the size distribution of DNA in each DNA preparation by gel electrophoresis Any smearing below 2 5 kb indicates sample degradation For HaloPlex target enrichment of FFPE derived DNA samples see Agilent publication no G9900 90050 available at http www genomics agilent com This publication provides a PCR based protocol for assessment of DNA integrity and provides HaloPlex protocol modifications for improved performance from lower quality DNA samples Prepare the workstation 1 Gently wipe down the Bravo deck with a NucleoClean decontamination wipe 2 Place red aluminum inserts on Bravo deck positions 4 and 9 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 Place a 384 well adapter insert on Bravo deck position 6 Pre
66. s prompt to resume the Capture 1 1 protocol Get plate from thermal cycler Once the ligation program has finished retrieve plate from thermal cycler and place at postion 4 Unseal the plate then dick Continue below to resume the protocol User data entry sem 15 When prompted by VWorks as shown below remove and discard the SSC Buffer plate from position 3 of the Bravo deck Place the 50 mM NaOH source plate at position 3 for use in the elution steps of the protocol After carefully unsealing the source plate click Continue on the prompt to resume the protocol Replace plate Remove and discard the SSC Buffer plate from position 3 Replace with the 50mM NaOH source plate Carefully unseal the NaOH plate then dick Continue below User data entry er The NGS Workstation completes the liquid handling steps for elution of the captured target DNA followed by preparation of PCR reactions for amplification 60 HaloPlex Target Enrichment System Automation Protocol Sample Preparation 3 To expedite thermal cycler warm up for the subsequent PCR program on page 62 you can enter and initiate the PCR program on the thermal cycler now and then pause the program until you are instructed to transfer the reaction plate Be sure to pause the thermal cycler before the initiation of the 98 C denaturation segment Release the pause immediately after transferring the plate to
67. so contain 10 uL of indexing primer cassette from step 1 above 32 uL of water added back here compensates for the combined volume removed from the eight ECD digest wells during validation Load the Agilent NGS Workstation and Run the Hybridization pro VWorks Protocol 1 the VWorks HaloPlex form under Step select 02 Hybridization pro 2 Select the number of columns of samples to be processed Runs must include 1 2 3 4 6 or 12 columns 3 Click Update layout and information HaloPlex Target Enrichment System Automation Protocol Sample Preparation 3 4 Load the Bravo deck according to Table 17 Table 17 Initial Bravo deck configuration for Hybridization pro Location Content 1 Indexing Primer Cassette source plate half skirted 96 well Eppendorf twin tec plate seated on red insert 2 New tip box 3 empty 4 Digested DNA in 384 well plate seated on 384 well insert 5 Empty full skirted 96 well Eppendorf twin tec plate 6 For 12 column runs only Digested DNA in 384 well plate digest plate 2 seated on 384 well insert For 1 to 6 column runs empty 7 empty 8 Empty tip box 9 Hybridization Master Mix source plate Nunc DeepWell plate seated on silver insert 5 Verify that the NGS workstation has been set up as displayed in the Bravo Deck Setup and Information regions of the form 6 Verify that the Current Tip State indicator on the form matches the configuration of unused and
68. te for the run containing up to 95 gDNA samples and the Enrichment Control DNA sample using a full skirted 96 well Eppendorf twin tec plate HaloPlex Automated Target Enrichment System runs may include 1 2 3 4 6 or 12 columns of the plate Use full columns of DNA samples for each run a In well Al of a 96 well twin tec plate dispense 45 uL of the supplied Enrichment Control DNA ECD Store on ice b Inseparate wells of the same 96 well twin tec plate dilute 225 ng of each gDNA sample in 45 uL nuclease free water for a final DNA concentration of 5 ng uL Continue to store on ice For automated processing fill plate wells column wise in well order 1 to HI then 2 to H2 ending with A12 to H12 Prepare the RE Master Mix Source Plate 1 Prepare the appropriate amount of RE Buffer BSA mixture according to the table below Table9 Preparation of RE Buffer BSA mixture for Digestion pro protocol Reagent Volume for Volume for Volumefor Volumefor Volumefor Volumefor Volume for 1 Library 1 Column 2 Columns 3Columns 4 Columns 6 Columns 12 Columns RE Buffer 34 uL 408 uL 680 uL 952 uL 1224 uL 1768 uL 3536 uL BSA Solution 0 85 pL 10 2 pL 17 uL 23 8 uL 30 6 uL 44 2 uL 88 4 uL Total Volume 34 85 pL 418 2 pL 697 uL 975 8 uL 1254 6 pL 1812 2 pL 3624 4 uL HaloPlex Target Enrichment System Automation Protocol 31 3 Sample Preparation 2 Obtain the two provided green and red marked Enzyme Strips from Box 1 For each strip la
69. ted provisional purification protocol step 9 on page 78 Updated URL for design of HaloPlex probes www agilent com genomics suredesign see page 29 HaloPlex Target Enrichment Svstem Automation Protocol Content 1 Before You Begin Procedural Notes 8 Safety Notes 8 Required Reagents 9 Required Equipment 11 Optional Validation Reagents and Equipment 12 Required Accessories for Adaptation of NGS Workstation Option B for HaloPlex Automation 12 2 Using the Agilent NGS Workstation for HaloPlex Target Enrichment About the Agilent NGS Workstation 14 Overview of the HaloPlex Target Enrichment Procedure 23 Experimental Setup Considerations for Automated Runs 25 3 Sample Preparation Step 1 Digest genomic DNA with restriction enzymes 30 Step 2 Hybridize digested DNA to HaloPlex probes for target enrichment and sample indexing 42 Step 3 Capture and amplify the target DNA 48 Step 4 Purify the amplified target DNA 63 Step 5 Validate enrichment and quantify enriched target DNA 67 Step Pool samples with different indexes for multiplexed sequencing 71 4 Appendix Provisional Adaptor Dimer Removal Protocol Purify the enriched library pool using AMPure XP beads 78 5 Reference Kit Contents 82 Nucleotide Sequences of HaloPlex Indexes 85 Qualitative analysis of enrichment by gel electrophoresis 91 HaloPlex Target Enrichment System Automation Protocol 5 HaloPlex Target Enrichment System Automation Protocol HaloPlex Targ
70. the thermal cycler in step 16 16 When the workstation has finished preparing the PCR amplification reactions you will be prompted by VWorks as shown below Transfer plate to thermal cycler Get plate from position 6 seal at 165C for 3 06 Place thermal cyder and run the PCR Program outlined in User Guide After transferring the plate cick Continue below User data entry l a Get the sample plate from position 6 and seal the plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 3 0 sec b Transfer the sealed plate to a thermal cycler and run the PCR program in Table 30 using a heated lid HaloPlex Target Enrichment System Automation Protocol 61 3 Sample Preparation The optimal amplification cycle number varies for each HaloPlex Probe design Consult the Certificate of Analysis provided with HaloPlex Target Enrichment System Box 1 for the PCR cycling recommendation for your probe Table 30 HaloPlex post capture DNA amplification PCR program Segment Number of Cycles Temperature Time 1 1 98 C 2 minutes 2 Obtain cycle number 98 30 seconds from Certificate of 2 Analysis 60 C 30 seconds 72 C 1 minute 3 1 72 C 10 minutes 4 1 8 C Hold c After initiating the PCR program in the thermal cycler click Continue on the VWorks prompt to finish the automation protocol d If you are continuing to the next step of PCR pro
71. to 0 C at position 9 of the Bravo deck Be sure that the chiller reservoir contains at least 300 mL of 25 ethanol Prepare the HaloPlex Magnetic Beads Source Plate 1 Vigorously resuspend the HaloPlex Magnetic Beads on a vortex mixer The beads settle during storage 2 Wash the magnetic beads a Transfer 40 uL per sample of the HaloPlex Magnetic Beads suspension to a 1 5 mL tube or conical vial using volumes provided in Table 20 Table 20 Volume of HaloPlex Magnetic Bead suspension for capture Reagent Volume for Volume for Volume for Volumefor Volumefor Volumefor Volume for 1 Library 1 Column 2 Columns 3Columns 4 Columns 6 Columns 12 Columns HaloPlex 0 04 mL 0 36 mL 0 68 mL 1 0 mL 1 32 mL 1 96 mL 3 92 mL Magnetic Beads HaloPlex Target Enrichment System Automation Protocol 49 3 Sample Preparation b Put the vial into a compatible magnetic device for 5 minutes c After verifying that the solution has cleared carefully remove and discard the supernatant using a pipette d Add an equivalent volume of Capture Solution see Table 21 to the beads and resuspend by pipetting up and down Table 21 Volume of Capture Solution used for bead resuspension Reagent Volume for Volume for Volumefor Volumefor Volumefor Volumefor Volume for 1 Library 1 Column 2Columns 3Columns 4 Columns 6 Columns 12 Columns Capture Solution 0 04 mL 0 36 mL 0 68 mL 1 0 mL 1 32mL 1 96 mL 3 92 mL 3 Prepare a Nunc DeepWell source plate for th
72. urt SPRIPlate Super Magnet Plate Agencourt p n A32782 or equivalent Let the AMPure XP beads come to room temperature for at least 30 minutes Prepare 400 uL of 70 ethanol per sample plus excess for use in step 10 Transfer 40 uL of each target enriched library pool to a fresh 0 2 mL tube Mix the AMPure XP bead suspension well until the suspension appears homogeneous and consistent in color For each sample to be purified prepare a bead mix by combining 40 uL of nuclease free water and 100 uL of the homogenous AMPure XP bead suspension Mix well until the bead mix suspension appears homogeneous Add 140 uL of the homogenous bead suspension prepared in step 5 to each 40 uL DNA sample Vortex thoroughly Using this bead to sample volume ratio is imperative to ensure optimal purification results Incubate samples for 5 minutes at room temperature with continuous shaking Make sure the samples are properly mixing in the wells during the 5 minute incubation Spin briefly to collect the liquid then place the tubes in the magnetic separation device Wait for the solution to clear approximately 5 minutes Keep the tubes in the magnetic device Carefully remove and discard the cleared solution from each tube using a 200 uL pipette set to 180 uL Do not touch the beads while removing the solution 10 Continue to keep the tubes in the magnetic device while you add 200 uL of 70 ethanol into the tubes Use fresh 70 eth
73. used tips in the tip boxes at Bravo Deck positions 2 and 8 respectively See page 19 for more information on using this segment of the form during the run 7 When verification is complete click Start to start the run Controls Start ID Pause Screen The NGS Workstation combines all eight digestion reactions for each gDNA sample with Hybridization Master Mix and the appropriate Indexing Primer Cassette in wells of a 96 well plate HaloPlex Target Enrichment System Automation Protocol 45 3 46 Sample Preparation 8 When the workstation has finished preparing the hybridization plate for the run you will be prompted by VWorks as shown below Transfer plate to thermal cycler Get plate from position 1 seal at 165C for 3 06 Place in thermal cyder and run the hybridization program outlined in User Guide After transferring the plate Continue below User data entry uester Gm p 9 Sealthe sample plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 3 0 sec 10 Spin the plate briefly HaloPlex Target Enrichment System Automation Protocol Sample Preparation 3 11 Transfer the sealed plate to a thermal cycler and run the appropriate hybridization program in Table 18 using the hybridization duration listed on the Certificate of Analysis After transferring the plate click Continue on the VWorks prompt to finish the protocol Use a heated lid Do n
74. verification is complete click Start to start the run Controls 93 start 11 Pause Screen The NGS Workstation completes the liquid handling steps for capture of the target DNA HaloPlex probe hybrids on the streptavidin beads To expedite thermal cycler warm up for the subsequent wash program on page 55 you can enter and initiate the wash program on the thermal cycler now and then pause the program until you are instructed to transfer the reaction plate Be sure to pause the thermal cycler before the initiation of the 46 C incubation segment Release the pause immediately after transferring the plate to the thermal cycler in step 9 8 When prompted by VWorks as shown below remove and discard the hybridization plate from position 4 of the Bravo deck Place a fresh half skirted 96 well Eppendorf twin tec plate at position 4 for use in the wash segment of the protocol Replace plate Remove and discard the hybridization plate from position 4 Replace with a fresh 96 Eppendorf twin tec half skirted plate When finished dick Continue below User data entry HaloPlex Target Enrichment System Automation Protocol Sample Preparation 3 9 When the workstation has finished preparing the capture wash plate you will be prompted by VWorks as shown below Transfer plate to thermal cycler Get plate from position 4 seal at 165C for 3 06 Place in thermal cycler and run the wash program outline

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