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SureSelect Automated Target Enrichment for Illumina Multiplexed

1. 8 Empty tip box 9 70 ethanol in Matrix reservoir 14 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 23 for more information on using this segment of the form during the run SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A 103 5 Indexing 15 When setup and verification is complete click Run Selected Protocol Run Selected Protocol Running the 10 Cleanup_Post CapturePCR_QXT_ILM_v1 0 pro protocol takes approximately 45 minutes An operator must be present during the run to complete tip box replacement and to transfer the water reservoir to the Bravo deck when directed by the VWorks prompt shown below Replace reservoir Remove the Ethanol Reservoir from Position 9 Place the Water reservoir at Position 9 When finished dick Continue below Once the protocol is complete the purified DNA samples are located in the Eppendorf plate at position 3 of the Bravo deck 104 SureSelect Target Enrichment for Illumina Sequencing NGS Bravo Option A Indexing 5 Step 3 Assess indexed DNA quality Option 1 Analysis using the 2100 Bioanalyzer and High Sensitivity DNA Assay 1 Set up the 2100 Bioanalyzer as instructed in the High Sensitivity DNA Kit Guide at www genomics agilent com Version B 02 07 or higher of the Agilent 2100 Expert Software is r
2. Volume for Volume for Volume for Volumefor Volumefor Volumefor Volume for Reagent 1 Library 1 Column 2 Columns 3 Columns 4 Columns 6 Columns 12 Columns Nuclease free water 2 5 pl 53 1 ul 74 4 ul 95 6 ul 116 9 pl 159 4 ul 297 5 pl SureSelect OXT Fast 6 0 pl 127 5 pl 178 5 pl 229 5 ul 280 5 pl 382 5 pl 714 0 pl Hybridization Buffer yellow cap Total Volume 8 5 pl 180 6 pl 252 9 pl 325 1 pl 397 4 pl 541 9 pl 1011 5 pl Prepare the master mix source plate 8 Ina Nunc DeepWell plate prepare the master mix source plate containing the master mixes prepared in step 5 to step 7 at room temperature Add the volumes indicated in Table 36 of each master mix to each well of the indicated column of the Nunc DeepWell plate When using multiple Capture Libraries in a run add each Capture Library Master Mix to the appropriate row s of the Nunc DeepWell plate The final configuration of the master mix source plate is shown in Figure 8 Table 36 Preparation of the Master Mix Source Plate for 06 Hybridization _OXT_v1 0 pro Master Mix Position on Volume of Master Mix added per Well of Nunc Deep Well Source Plate Solution Source Plate 1 Column 2 Column 3 Column 4 Column 6 Column 12 Column Runs Runs Runs Runs Runs Runs Block Master Mix Column 1 11 0 pl 19 0 pl 27 0 ul 34 9 ul 50 9 pl 102 7 pl A1 H1 Capture Library Column 2 14 0 pl 21 4 ul 28 9 ul 36 3 ul 58 6 ul 114 4 pl Master Mix A2 H2 Hybridization Column 3 19 9 pl 29 0 ul 38 0 ul 47 0 ul 65 1 pl
3. NGS Bravo Option A 3 In this Guide This guide describes an optimized protocol for Illumina paired end multiplexed library preparation using the SureSelect Automated Target Enrichment system This protocol is specifically developed and optimized to enrich targeted regions of the genome from repetitive sequences and sequences unrelated to the research focus prior to sample sequencing Sample processing steps are automated using Agilent s NGS Bravo Option A Before You Begin This chapter contains information that you should read and understand before you start an experiment Using the Agilent NGS Bravo Option A for SureSelect Target Enrichment This chapter contains an orientation to the Agilent NGS Bravo an overview of the SureSelect target enrichment protocol and considerations for designing SureSelect experiments for automated processing using the Agilent NGS Bravo Option A Sample Preparation This chapter describes the steps to prepare gsDNA sequencing libraries for target enrichment Hybridization This chapter describes the steps to hybridize and capture the prepared DNA library using a SureSelect or ClearSeq Capture Library Indexing This chapter describes the steps for post capture amplification and guidelines for sequencing sample preparation SureSelect Target Enrichment for Illumina Sequencing NGS Bravo Option A 6 Reference This chapter contains reference information including comp
4. V f add Low TE to bring the volume to the desired level If the final volume of the combined index tagged samples is greater than the final desired volume V f lyophilize and reconstitute to the desired volume 3 If you store the library before sequencing add Tween 20 to 0 1 v v and store at 20 C short term SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A Indexing 5 Step 6 Prepare sequencing samples The optimal seeding concentration for SureSelect XT target enriched libraries is 8 to 12 pM on HiSeq or MiSeq instruments and 1 2 to 1 4 pM on the NextSeq platform Seeding concentration and cluster density may also need to be optimized based on the DNA fragment size range for the library and on the desired output and data quality Follow Illumina s recommendation for a PhiX control in a low concentration spike in for improved sequencing quality control Proceed to cluster amplification using the appropriate Illumina Paired End Cluster Generation Kit See Table 57 for kit configurations compatible with the recommended read length plus reads for the SureSelect 8 bp dual indexes To do this step refer to the manufacturer s instructions using the modifications described in Using the SureSelect Read Primers with Illumina s Paired End Cluster Generation Kits on page 112 Table 57 Illumina Kit Configuration Selection Guidelines Platform Run Type Read Length SBS Kit Configura
5. 0 2 ml 1 7 ml 3 3 ml 4 9 ml 6 6 ml 10 ml 20 ml Binding Buffer 84 SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A Hybridization 8 Prepare a Nunc DeepWell source plate for the washed streptavidin bead suspension For each well to be processed add 200 ul of the homogeneous bead suspension to the Nunc DeepWell plate 9 Place the streptavidin bead source plate at position 5 of the Bravo deck 10 Prepare a Nunc DeepWell source plate labeled Wash 2 For each well to be processed add 1150 ul of SureSelect Wash Buffer 2 Place the Wash 2 source plate at position 6 of the Bravo deck Setup VWorks protocol 07 SureSelectOXT_Capture_v1 0 pro 11 On the SureSelect setup form under Select Protocol select 07 SureSelectQXT_Capture_v1 0 pro 12 Under Select PCR Plate Labware select the specific type of PCR plate used for hybridization This plate will be transferred from the thermal cycler to Bravo deck position 4 when prompted by VWorks 13 Select the number of columns of samples to be processed Runs must include 1 2 3 4 6 or 12 columns 14 Click Display Initial Bravo Deck Setup Display Initial Bravo Deck Setup SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A 4 85 4 Hybridization 15 Load the Bravo deck according to the Bravo Deck Setup region of the form and as shown in Table 42 Table 42 Initial Bravo deck configuration for 07 SureSelectOXT_Capture_v1
6. 2015 Version B0 November 2015 p n G9681 90020 oe Agilent Technologies
7. 5 Pool samples for Multiplexed Sequencing 110 Step 6 Prepare sequencing samples 111 Step 7 Set up the sequencing run and trim adaptors from the reads 115 Reference 119 Kit Contents 120 Nucleotide Sequences of SureSelect Dual Indexes 123 Guidelines for Multiplexing with Dual Indexed Samples 125 SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A SureSelect Automated Target Enrichment for Illumina Multiplexed Sequencing Protocol ee 1 070 Before You Begin s Procedural Notes 10 Safety Notes 11 Required Reagents 12 Optional Reagents 14 Required Equipment 15 Make sure you read and understand the information in this chapter and have the necessary equipment and reagents listed before you start an experiment This protocol describes automated sample processing using the Agilent NGS Bravo Option A For automated sample processing using the Agilent NGS Workstation Option B see publication G9681 90010 For non automated sample processing procedures see publication G9681 90000 Agilent cannot guarantee the SureSelect Target Enrichment kits and cannot provide technical support for the use of non Agilent protocols or instruments to process samples for enrichment ee Agilent Technologies 1 10 Before You Begin Procedural Notes The SureSelect X system requires high quality DNA samples for optimal performance Use best practices for verifying DNA sample quality before ini
8. Agilent p n 5067 5583 Agilent p n 5067 5584 Agilent p n 5067 5585 Agilent p n G2964AA or G2965AA Agilent p n 5067 5582 Agilent p n 5067 5583 Agilent p n 5067 5584 Agilent p n 5067 5585 Eppendorf Centrifuge model 5804 or equivalent Labnet International MPS1000 Mini Plate Spinner p n C1000 requires adapter p n C1000 ADAPT for use with strip tubes or equivalent Pipetman or equivalent SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A SureSelect Automated Target Enrichment for Illumina Multiplexed Sequencing Protocol 2 ee 2 7 e Using the Agilent NGS Bravo Option A for SureSelect Target Enrichment About the Agilent NGS Bravo OptionA 18 Overview of the SureSelect Target Enrichment Procedure 29 Experimental Setup Considerations for Automated Runs 32 This chapter contains an orientation to the Agilent NGS Bravo Option A an overview of the SureSelect X target enrichment protocol and considerations for designing SureSelect T experiments for automated processing using the Agilent NGS Bravo Option A wit Agilent Technologies 17 2 Using the Agilent NGS Bravo Option A for SureSelect X Target Enrichment About the Agilent NGS Bravo Option A About the Bravo Platform CAUTION 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 contr
9. Bravo Deck Setup region of the form and as shown in Table 26 Table 26 Initial Bravo deck configuration for 05 Aliquot_Libraries_v1 0 pro Location Content 1 empty 2 empty 3 empty 4 empty 5 Empty Eppendorf plate 6 Empty tip box 7 empty 8 New tip box 9 Prepped library DNA in Eppendorf plate CAUTION This protocol does not use the Current Tip State indicator function Be sure to place a completely full box of tips at position 8 and a completely empty tip box at position 6 7 When verification is complete click Run Selected Protocol Run Selected Protocol SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A 67 4 Hybridization 8 When prompted by the dialog below browse to the csv file created for the source plate of the current run and then click OK to start the run a amp Select Hit Pick Input File a Please select the hit pick input file for the hit pick replication task at task 5 of subprocess Aliquot Libraries C vWorks Workspace NGS Option A QXT_ILM_v1 0 Aliquot Library Input f E Ce lea The library aliquoting protocol takes approximately 1 hour for 96 samples When complete the DNA sample plate is on Bravo deck position 5 9 Remove the sample plate from the Bravo deck and use a vacuum concentrator to dry the samples at lt 45 C 10 Reconstitute each dried sample with 12 ul of nuclease free water Pipette up an
10. P5 Index 19 P5 i19 P5 Index 20 P5 i20 Sequence TAGATCGC CTCTCTAT TATCCTCT AGAGTAGA GTAAGGAG ACTGCATA AAGGAGTA CTAAGCCT Table 71 SureSelect P5 Indexes 13 to 20 for NextSeq platform Index Number Sequence P5 Index 13 P5 i13 GCGATCTA P5 Index 14 P5 i14 ATAGAGAG P5 Index 15 P5 i15 AGAGGATA P5 Index 16 P5 i16 TCTACTCT P5 Index 17 P5 i17 CTCCTTAC P5 Index 18 P5 i18 TATGCAGT P5 Index 19 P5 i19 TACTCCTT P5 Index 20 P5 i20 AGGCTTAG When doing NextSeq runs through BaseSpace use the reverse complement se quences provided in Table 70 SureSelect T Target Enrichment for Illumina Sequencing NGS Bravo Option A Reference 6 Guidelines for Multiplexing with Dual Indexed Samples Agilent recommends following the dual index sample pooling guidelines and shown in Table 72 These are designed to maintain color balance at each cycle of the index reads on both ends They also provide flexibility of demultiplexing as single or dual indexed samples in low plexity experiments One base mismatches should also be allowed during demultiplexing in order to maximize sequencing output per sample Table 72 Dual index sample pooling guidelines for 96 Reaction Kits Plexity of Sample Recommended SureSelect P7 Recommended SureSelect P5 Indexes Pool Indexes 1 plex Any P7 index i1 to i11 Any P5 index i13 to i20 2 plex P7 i1 and P7 i2 OR P5 i13 and P5 i14 OR P7 i2 and P7 i4 P5 i15 and P5 i16 OR P5 i17 and
11. P5 i18 3 plex P7 i1 P7 i2 and P7 i4 OR P5 i13 and P5 i14 OR P7 i3 P7 i4 and P7 i6 OR P5 i15 and P5 i16 OR P7 i5 P7 i7 and P7 i8 P5 i17 and P5 i18 as needed 4 plex P7 i1 P7 i2 P7 i3 and P7 i4 OR P5 i13 and P5 i14 OR P7 i3 P7 i4 P7 i5 and P7 i6 OR P5 i15 and P5 i16 OR P7 i5 P7 i6 P7 i7 and P7 i8 P5 i17 and P5 i18 as needed 5 plex P7 i1 P7 i2 P7 i3 P7 i4 and P7 i5 OR P5 i13 and P5 i14 OR P7 i3 P7 i4 P7 i5 P7 i6 and p7 i7 OR P5 i15 and P5 i16 OR P7 i5 P7 i6 P7 i7 P7 i8 and p7 i9 P5 i17 and P5 i18 as needed 6 to 11 plex Any combination of P7 indexes i1 to i11 P5 i13 and P5 i14 OR using each index only once P5 i15 and P5 i16 OR P5 i17 and P5 i18 as needed 12 to 88 plex Any combination of P7 indexes i1 to i11 P5 i13 and P5 i14 and any third P5 index OR as needed P5 i15 and P5 i16 and any third P5 index OR P5 i17 and P5 i18 and any third P5 index as needed 89 to 96 plex All twelve P7 indexes i1 to i12 Any P5 indexes i13 to i20 as needed differs from all others used in the sample pool The indicated indexes may be substituted with another index within range of P7 i1 to P7 i11 as long as the substitute index SureSelect T Target Enrichment for Illumina Sequencing NGS Bravo Option A 125 www agilent com In This Book This guide contains information to run the SureSelect Automated Library Prep and Target Enrichment protocol using Agilent s NGS Bravo Option A Agilent Technologies Inc
12. Primer 1 brown cap 1741 2 pl HP10 1 75 ml Index 8 8 pl SureSelect QXT Index Read Primer clear cap 1741 2 ul HP12 1 75 ml Read 2 8 8 pl SureSelect QXT Read Primer 2 black cap 1741 2 ul HP11 1 75 ml Aliquot the mixture as directed for HP10 in Illumina s cluster generation protocol Table 60 MiSeq platform custom sequencing primer preparation Sequencing Volume of SureSelect Primer Volume of Illumina TruSeq Total Final Read Primer Volume Cartridge Position Read 1 3 pl SureSelect QXT Read Primer 1 brown cap 597 pl HP10 well 12 0 6 ml well 18 Index 3 pl SureSelect QXT Index Read Primer clear cap 597 pl HP12 well 13 0 6 ml well 19 Read 2 3 pl SureSelect QXT Read Primer 2 black cap 597 pl HP11 well 14 0 6 ml well 20 SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A 113 5 Indexing Table 61 NextSeq 500 550 High Output v2 Kit custom sequencing primer preparation Sequencing Volume of SureSelect Primer Volume of Illumina Primer Total Final Read Volume Cartridge Position Read 1 3 9 ul SureSelect QXT Read Primer 1 brown cap 1296 1 ul BP10 from well 20 1 3 ml well 7 Read 2 4 2 ul SureSelect QXT Read Primer 2 black cap 1395 8 ul BP11 from well 21 1 4 ml well 8 Index 6 pl SureSelect QXT Index Read Primer clear cap 1988 pl BP14 from well 22 2 ml well 9 Index 2 6 pl SureSelect QXT Index 2 Read Primer NSO purple cap Table 62 NextSeq 500 550 Mid Output v2 Kit cust
13. 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 23 for more information on using this segment of the form during the run SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A Sample Preparation 3 15 When setup and verification is complete click Run Selected Protocol Run Selected Protocol Running the 04b Cleanup_Pre CapturePCR_QXT_ILM_v1 0 pro protocol takes approximately 45 minutes An operator must be present during the run to complete tip box replacement and to transfer the water reservoir to the Bravo deck when directed by the VWorks prompt shown below Replace reservoir Remove the Ethanol Reservoir from Position 9 Place the Water reservoir at Position 9 When finished dick Continue below Once the protocol is complete the purified DNA samples are located in the Eppendorf plate at position 3 of the Bravo deck SureSelect Target Enrichment for Illumina Sequencing NGS Bravo Option A 57 3 58 Sample Preparation Stopping Point Step 6 Assess Library DNA quantity and quality Measure the concentration of each library using one of the methods detailed below Option 1 Analysis using the Agilent 2100 Bioanalyzer and DNA 1000 Assay Use a Bioanalyzer DNA 1000 chip and reagent kit to analyze the amplified libraries For more information to
14. blue cap SureSelect QXT Primer Mix tube with clear cap SureSelect RNase Block tube with purple cap 122 SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A Reference 6 Nucleotide Sequences of SureSelect Dual Indexes The nucleotide sequence of each SureSelect XT tables below index is provided in the Note that some index number assignments of the SureSelect P5 and P7 indexes differ from the index number assignments used by Illumina for indexes of similar or identical sequence Each index is 8 bases in length Refer to llumina s sequencing run setup instructions for sequencing libraries using 8 base indexes Table 69 SureSelect T P7 Indexes 1 to 12 Index Number Sequence P7 Index 1 P7 i1 TAAGGCGA P7 Index 2 P7 i2 CGTACTAG P7 Index 3 P7 i3 AGGCAGAA P7 Index 4 P7 i4 TCCTGAGC P7 Index 5 P7 i5 GTAGAGGA P7 Index 6 P7 i6 TAGGCATG P7 Index 7 P7 i7 CTCTCTAC P7 Index 8 P7 i8 CAGAGAGG P7 Index 9 P7 i9 GCTACGCT P7 Index 10 P7 i10 CGAGGCTG P7 Index 11 P7 i11 AAGAGGCA P7 Index 12 P7 i12 GGACTCCT SureSelect T Target Enrichment for Illumina Sequencing NGS Bravo Option A 123 6 124 Reference Table 70 SureSelect P5 Indexes 13 to 20 for HiSeq platform MiSeq platform or NextSeq platform runs through BaseSpace Index Number P5 Index 13 P5 i13 P5 Index 14 P5 i14 P5 Index 15 P5 i15 P5 Index 16 P5 i16 P5 Index 17 P5 i17 P5 Index 18 P5 i18
15. complete mixing then centrifuge the plate for 30 seconds to drive the well contents off the walls and plate seal and to eliminate any bubbles 98 SureSelect Target Enrichment for Illumina Sequencing NGS Bravo Option A Indexing 5 Setup and run VWorks protocol 09 Post CapturePCR_QXT_ILM_v1 0 pro 12 On the SureSelect setup form under Select Protocol to Run select 09 Post CapturePCR_QXT_ILM_v1 0 pro 13 Under Select PCR plate Labware select the specific type of PCR plate used at position 6 of the Bravo deck 14 Select the number of columns of samples to be processed Runs must include 1 2 3 4 6 or 12 columns 15 Click Display Initial Bravo Deck Setup Display Initial Bravo Deck Setup 16 Load the Bravo deck according to the Bravo Deck Setup region of the form and as shown in Table 52 Table 52 Initial Bravo deck configuration for 01 Tn_OXT_ILM_v1 0 pro Location Content 1 empty 2 New tip box 3 empty 4 empty 5 Captured DNA bead suspensions in Eppendorf twin tec plate 6 Diluted P7 indexing primers in PCR plate seated in red insert PCR plate type must be specified on setup form under step 2 7 empty 8 Empty tip box 9 Master mix plate containing P5 indexing primers and PCR Master Mix in Column 4 unsealed seated in silver insert 17 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 p
16. d Adjust each gDNA sample with nuclease free water to a final concentration of 10 ng ul in a 1 5 ml LoBind tube 5 Transfer 5 ul of the 10 ng ul DNA samples into the wells of a 96 well Eppendorf plate column wise for processing on the NGS Bravo in well order A1 to H1 then A2 to H2 ending with A12 to H12 SureSelect Automated Library Prep and Capture System runs may include 1 2 3 4 6 or 12 columns of the plate See Experimental Setup Considerations for Automated Runs on page 32 for additional sample placement considerations 6 Seal the plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 1 0 sec 7 Centrifuge the plate for 30 seconds to drive the well contents off the walls and plate seal and to remove air bubbles Store the sample plate on ice until it is used on page 43 SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A 39 3 Sample Preparation Step 2 Fragment and adaptor tag the genomic DNA samples In this step automation protocol 01 Tn_QXT_ILM_v1 0 pro is used to enzymatically fragment the gDNA and to add adaptors to ends of the fragments in a single reaction This step uses the SureSelect X Reagent Kit components listed in Table 9 in addition to reagents prepared for use on page 38 to page 39 Table9 Reagents for DNA fragmentation and adaptor tagging Kit Component Storage Location Where Used SureSelect QXT Buffer SureSelect QXT Library Prep Kit Bo
17. do this step see the Agilent DNA 1000 kit Guide at www genomics agilent com 1 Set up the 2100 Bioanalyzer as instructed in the reagent kit guide 2 Seal the sample plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 1 0 sec 3 Vortex the plate to mix samples in each well then centrifuge the plate for 30 seconds to drive the well contents off the walls and plate seal 4 Prepare the chip samples and ladder as instructed in the reagent kit guide using 1 ul of each sample for the analysis 5 Load the prepared chip into the 2100 Bioanalyzer and start the run within five minutes after preparation 6 Verify that the electropherogram shows the peak of DNA fragment size positioned between 245 to 325 bp Sample electropherograms are shown in Figure 5 Variability of fragmentation profiles may be observed A peak DNA fragment size significantly less than 245 bp may indicate too little gDNA in the fragmentation reaction and may be associated with increased duplicates in the sequencing data In contrast a peak DNA fragment size significantly greater than 325 bp may indicate too much gDNA in the fragmentation reaction and may be associated with decreased percent on target performance in sequencing results 7 Measure the concentration of each library by integrating under the entire peak For accurate quantification make sure that the concentration falls within the linear range of the assay If you do not con
18. done on the same day when stored in a sealed container 3 Obtain the bottle of SureSelect QXT Stop Solution from SureSelect QXT Hyb Module Box 1 stored at room temperature Verify that the SureSelect QXT Stop Solution contains 25 ethanol by referring to the container label and the instructions below Before the first use of a fresh container add 1 5 ml of ethanol to the provided bottle containing 4 5 ml of stop solution for a final ethanol concentration of 25 Seal the bottle then vortex well to mix After adding the ethanol be sure to mark the label for reference by later users Keep the prepared 1X SureSelect QXT Stop Solution at room temperature tightly sealed until it is used on page 41 38 SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A Sample Preparation 3 4 Quantify and dilute gDNA samples using two serial fluorometric assays a Use the Qubit dsDNA BR Assay or Qubit dsDNA HS Assay to determine the initial concentration of each gDNA sample Follow the manufacturer s instructions for the specific assay kit and the Qubit instrument This step is critical for successful preparation of input DNA at the required concentration to ensure optimal fragmentation b Dilute each gDNA sample with nuclease free water to a final concentration of 100 ng ul in a 1 5 ml LoBind tube c Carefully measure the DNA concentration of each of the 100 ng ul dilutions using a second Qubit dsDNA BR or HS Assay
19. is retained on the streptavidin beads during the post capture amplification step SureSelect Target Enrichment for Illumina Sequencing NGS Bravo Option A 91 4 Hybridization Step 4 Wash the captured DNA 92 SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A SureSelect Automated Target Enrichment for Illumina Multiplexed Sequencing Protocol 5 Indexing Step 1 Amplify the captured DNA libraries to add index tags 94 Step 2 Purify the amplified indexed libraries using Agencourt AMPure XP beads 102 Step 3 Assess indexed DNA quality 105 Step 4 Quantify each index tagged library by QPCR optional 109 Step 5 Pool samples for Multiplexed Sequencing 110 Step 6 Prepare sequencing samples 111 Step 7 Set up the sequencing run and trim adaptors from the reads 115 This chapter describes the steps to add index tags by amplification purify assess quality and quantity of the libraries and pool indexed samples for multiplexed sequencing ait Agilent Technologies 93 5 Indexing Step 1 Amplify the captured DNA libraries to add index tags In this step the Agilent NGS Bravo completes the liquid handling steps for PCR based addition of dual indexing tags to the captured DNA samples using automation protocol 09 Post CapturePCR_QXT_ILM_v1 0 pro After the PCR plate is prepared by the NGS Bravo you transfer the plate to a thermal cycler for amplification This step uses the components listed in
20. primer solution using the platform specific instructions below For the HiSeq platform combine the primers as shown in Table 58 or Table 59 on page 113 For the MiSeq platform combine the primers as shown in Table 60 on page 113 For the NextSeq platform combine the primers as shown in Table 61 or Table 62 on page 114 It is important to combine the primers precisely in the indicated ratios Be sure to use measured volumes of each solution do not use volumes reported on vial labels when preparing the mixtures Vortex each mixture vigorously to ensure homogeneity for proper detection of the indexes using the custom read primers SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A Indexing 5 Table 58 HiSeq2000 and HiSeq 2500 High Output custom sequencing primer preparation Sequencing Volume of SureSelect Primer Volume of Illumina TruSeq Primer Total Volume Read Read 1 5 pl SureSelect QXT Read Primer 1 brown cap 995 pl HP6 or HP10 1m Index 15 ul SureSelect OXT Index Read Primer clear cap 2985 pl HP8 or HP12 3 ml Read 2 15 ul SureSelect OXT Read Primer 2 black cap 2985 pl HP7 or HP11 3 ml Aliquot the mixture as directed for HP6 or HP10 in Illumina s cluster generation protocol Table 59 HiSeq 2500 Rapid Mode custom sequencing primer preparation Sequencing Volume of SureSelect Primer Volume of Illumina TruSeq Primer Total Volume Read Read 1 8 8 pl SureSelect QXT Read
21. specified on setup form under step 2 Empty Eppendorf twin tec plate Master Mixes in Columns 1 3 of Nunc DeepWell seated in silver Nunc DeepWell insert empty Empty tip box Prepped library aliquots in Eppendorf twin tec plate 16 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 23 for more information on using this segment of the form during the run 17 When setup and verification is complete click Run Selected Protocol Run Selected Protocol SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A 71 4 Hybridization Running the 06 Hybridization_QXT_v1 0 pro protocol takes approximately 30 minutes An operator must be present during the run to complete tip box replacement and other labware transfer steps as directed by the VWorks prompts detailed below The NGS Bravo combines the prepped gDNA in the wells of the sample plate with the aliquotted SureSelect Block Master Mix When this process is complete you will be prompted to transfer the plate to the thermal cycler for sample denaturation and blocking prior to hybridization 18 When prompted by VWorks as shown below remove the PCR plate from position 4 of the Bravo deck leaving the red insert in place After removing the sample plate click Continue m Remove plate Remove plate from insert at posit
22. step see Figure 2 you can add a different SureSelect or ClearSeq Capture Library to each row of the plate Plan your experiment such that each prepared DNA library plate position corresponds to the appropriate Capture Library e For post capture amplification see Figure 2 different SureSelect or ClearSeq Capture Libraries can require different amplification cycle numbers based on sizes of the captured targets It is most efficient to process similar sized Capture Libraries on the same plate See Table 53 on page 101 to determine which Capture Libraries may be amplified on the same plate e Post capture dual index assignments for the DNA samples can affect sample placement decisions at the beginning of the workflow For example all samples on the same row of the DNA sample plate must be assigned to the same P5 indexing primer during sample indexing after hybridization to the Capture Library see Figure 2 It is important to review and understand the guidelines for assignment of dual indexing primers on page 96 while planning sample placement for the run to ensure that the indexing design is compatible with the initial DNA sample placement Considerations for Equipment Setup e Some workflow steps require the rapid transfer of sample plates between the Bravo deck and a thermal cycler Locate your thermal cycler in close proximity to the Agilent NGS Bravo to allow rapid and efficient plate transfer SureSelect X Target Enrichment for
23. temperature of Bravo deck position 4 was not pre set to 66 C the protocol will pause while position 4 reaches temperature CAUTION It is important to complete step 17 quickly and carefully Transfer the sample plate to the Bravo platform quickly to retain the 65 C sample temperature Unseal the plate without tilting or jerking the plate to avoid sample splashing Make sure that the NGS Bravo is completely prepared with deck platforms at temperature and all components in place before you transfer the sample plate to the Bravo deck 17 When prompted by VWorks as shown below quickly remove the PCR plate containing the hybridization reactions held at 65 C from the thermal cycler Unseal the plate carefully to avoid splashing and quickly transfer the plate to position 4 of the Bravo deck seated in the red insert Click Continue to resume the protocol Add Hyb Plate Complete the following steps as quickly as possible Retrieve Hybridization plate from thermocyder and place on RED INSERT at Bravo Position 4 and unseal Click Continue to resume protocol Use Caution Position 4 will be hot User data entry WARNING Bravo deck position 4 will be hot Use caution when handling components that contact heated deck positions SureSelect Target Enrichment for Illumina Sequencing NGS Bravo Option A 87 4 Hybridization 18 When the capture incubation period is complete you will be prompted by VWo
24. the Capture Library and Hybridization Buffer Master Mixes you will be prompted by VWorks as shown below When the thermal cycler reaches the 65 C hold step click Continue Leave the sample plate in the thermal cycler until you are notified to move it F Wait for plate in thermocycler When thermocyder has reached hold step at 65C dick Continue Leave DNA plate in thermocyder until you are prompted to transfer the plate SureSelect Target Enrichment for Illumina Sequencing NGS Bravo Option A Hybridization 4 22 When prompted by VWorks as shown below quickly remove the sample plate from the thermal cycler unseal the plate carefully to avoid splashing and transfer the plate to position 4 of the Bravo deck seated in the red insert Click Continue Place DNA plate on Bravo Complete the following steps as quickly as possible Retrieve DNA plate from thermocyder and place on carrier at Bravo position 4 and unseal Click Continue to resume protocol Use Caution Position 4 will be hot User data entry WARNING Bravo deck position 4 will be hot Use caution when handling components that contact heated deck positions The NGS Bravo transfers the Capture Library Hybridization Buffer mixture to the wells of the PCR plate that contain the mixture of prepped gDNA samples and blocking agents SureSelect Target Enrichment for Illumina Sequencing NGS Bravo Option A 81 4 Hybridizat
25. 0 pro Location Content 1 empty 2 New tip box 3 empty 4 Empty red insert 5 Prepared Dynabeads streptavidin bead DeepWell source plate Wash 2 DeepWell source plate seated on silver Nunc DeepWell insert 7 empty 8 Empty tip box 9 empty 16 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 23 for more information on using this segment of the form during the run Run VWorks protocol 07 SureSelectO0XT_Capture_v1 0 pro Start the 07 SureSelectQXT_Capture_v1 0 pro protocol upon completion of the hybridization incubation that was started on page 82 when the thermal cycler program reaches the 65 C Hold step in Segment 5 After verifying that the hybridization step is complete and that all NGS Bravo setup steps for capture are complete click Run Selected Protocol Leave the hybridization plate in the thermal cycler until you are prompted to transfer the plate to the NGS Bravo wa Run Selected Protocol The total duration of the 07 SureSelectQXT_Capture_v1 0 pro protocol is approximately 35 minutes An operator must be present to transfer the hybridization plate from the thermal cycler when prompted by VWorks as shown in step 17 below lt 5 minutes after starting the protocol SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A Hybridization 4 If the
26. 0 seconds Capture Libraries 1 to 3 Mb 12 Cycles 58 C 30 seconds Capture Libraries lt 1 Mb 14 Cycles 72 C minute 3 1 72 C 5 minutes 4 1 4 C Hold SureSelect T Target Enrichment for Illumina Sequencing NGS Bravo Option A 101 5 102 Indexing Step 2 Purify the amplified indexed libraries using Agencourt AMPure XP beads In this step the Agilent NGS Bravo transfers AMPure XP beads to the indexed DNA sample plate and then collects and washes the bead bound DNA This step uses protocol 10 Cleanup_Post CapturePCR_QXT_ILM_v1 0 pro Prepare the NGS Bravo and reagents 1 Clear the Bravo deck of all plates and tip boxes then wipe it down with a DNA Away decontamination wipe Pre set the temperature of Bravo deck positions 4 and 6 as indicated in Table 54 See page 19 for more information on how to do this step Table 54 Bravo Deck Temperature Presets Bravo Deck Position Temperature Preset Preset Method 4 6 45 C Inheco Multi TEC control touchscreen CPAC 2 1 4 C Inheco Multi TEC control touchscreen CPAC 2 2 Verify that the AMPure XP bead suspension is at room temperature If necessary allow the bead solution to come to room temperature for at least 30 minutes Do not freeze the beads at any time Mix the AMPure XP bead suspension well so that the reagent appears homogeneous and consistent in color Prepare a Nunc DeepWell source plate for the beads by adding 60 ul of homogeneous AMPure XP beads per
27. 123 8 pl Buffer Master Mix A3 H3 SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A 75 4 76 4 6 oe 4 oe Figure 8 Configuration of the master mix source plate for 06 Hybridization_OXT_v1 0 pro 9 Seal the master mix source plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 1 0 sec 10 Centrifuge the plate for 30 seconds to drive the well contents off the walls and plate seal and to eliminate any bubbles Keep the master mix plate at room temperature Setup and run VWorks protocol 06 Hybridization _QXT_v1 0 pro 11 On the SureSelect setup form under Select Protocol select 06 Hybridization_QXT_v1 0 pro 12 Under Select PCR Plate Labware select the plate type to be used for the hybridization step to be loaded at Bravo deck position 4 SureSelect Target Enrichment for Illumina Sequencing NGS Bravo Option A Hybridization 4 13 Select the number of columns of samples to be processed Runs must include 1 2 3 4 6 or 12 columns 14 Click Display Initial Bravo Deck Setup Display Initial Bravo Deck Setup 15 Load the Bravo deck according to the Bravo Deck Setup region of the form and as shown in Table 37 Table 37 Initial Bravo deck configuration for 06 Hybridization OXT_v1 0 pro Location 1 gt wo N Content empty New tip box empty Empty PCR plate seated in red insert PCR plate type must be
28. 15 ml of nuclease free water 7 Prepare a separate Thermo Scientific Matrix reservoir containing 45 ml of freshly prepared 70 ethanol 8 Centrifuge the amplified DNA sample plate for 30 seconds to drive the well contents off the walls and plate seal Setup and run VWorks 04b Cleanup_Pre CapturePCR_OXT_ILM_v1 0 pro 9 On the SureSelect setup form under Select Protocol select 04b Cleanup_Pre CapturePCR_QXT_ILM_v1 0 pro SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A 55 3 56 Sample Preparation 10 Under Select PCR plate Labware select the specific type of PCR plate used for pre capture amplification 11 Select the number of columns of samples to be processed Runs must include 1 2 3 4 6 or 12 columns 12 Click Display Initial Bravo Deck Setup Display Initial Bravo Deck Setup 13 Load the Bravo deck according to the Bravo Deck Setup region of the form and as shown in Table 15 Table 23 Initial Bravo deck configuration for 04b Cleanup_Pre CapturePCR_OXT_ILM_v1 0 pro Location Content 1 Empty waste reservoir Axygen 96 Deep Well Plate square wells 2 New tip box 3 empty 4 empty 5 AMPure XP beads in Nunc DeepWell plate 50 pl beads per processing well Amplified DNA libraries in unsealed PCR plate seated in red insert PCR plate type must be specified on setup form under step 2 7 empty 8 Empty tip box 9 70 ethanol in Matrix reservoir 14
29. 2 Columns Nuclease free 4 5 ul 76 5 ul 114 8 pl 153 0 pl 191 3 pl 306 0 pl 592 9 ul water RNase Block 0 5 pl 8 5 pl 12 8 pl 17 0 pl 21 3 pl 34 0 ul 65 9 pl purple cap Capture Library 2 0 pl 34 0 ul 51 0 ul 68 0 ul 85 0 ul 136 0 pl 263 5 pl Total Volume 7 0 pl 119 0 pl 178 6 pl 238 0 pl 297 6 pl 476 0 pl 922 3 pl Table 32 Preparation of Capture Library Master Mix for Capture Libraries gt 3 Mb 8 rows of wells Target size gt 3 0 Mb Reagent Volume for Volumefor Volumefor Volumefor Volumefor Volumefor Volume for 1 Library 1 Column 2 Columns 3Columns 4Columns 6 Columns 12 Columns Nuclease free 1 5 pl 25 5 ul 38 3 ul 51 0 pl 63 8 pl 102 0 pl 197 6 pl water RNase Block 0 5 pl 8 5 pl 12 8 pl 17 0 pl 21 3 pl 34 0 ul 65 9 ul purple cap Capture Library 5 0 pl 85 0 ul 127 5 pl 170 0 pl 212 5 pl 340 0 ul 658 8 pl Total Volume 7 0 pl 119 0 pl 178 6 pl 238 0 pl 297 6 pl 476 0 pl 922 3 pl Includes ClearSeq DNA Kinome XT Library 3 2 Mb SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A 73 4 Hybridization b For runs that use different Capture Libraries in individual rows prepare a Master Mix for each Capture Library as listed in Table 33 or Table 34 according to the library size The volumes listed in Table 33 and Table 34 are for a single row of sample wells If a given Capture Library will be hybridized in multiple rows multiply each of the values below by the number of rows assigned to tha
30. 51 Axygen p n P 2ML SQ C E amp K Scientific p n EK 2440 Eppendorf p n 022431021 or equivalent DNA Away Surface Decontaminant Wipes Thermo Scientific p n 7008 or equivalent Life Technologies p n 032857 Life Technologies p n 032856 DynaMag 50 magnet Life Technologies p n 123 02D or equivalent SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A 15 1 16 Before You Begin Table 4 Required Equipment for SureSelect T Target Enrichment Description Vendor and part number Vacuum concentrator DNA Analysis Platform and Consumables Agilent 2100 Bioanalyzer Laptop Bundle Agilent 2100 Bioanalyzer Electrophoresis Set Agilent DNA 1000 Kit Agilent High Sensitivity DNA Kit OR Agilent 4200 TapeStation Agilent D1000 ScreenTape Agilent D1000 Reagents Agilent High Sensitivity D1000 ScreenTape Agilent D1000 Reagents OR Agilent 2200 TapeStation Agilent D1000 ScreenTape Agilent D1000 Reagents Agilent High Sensitivity D1000 ScreenTape Agilent D1000 Reagents Centrifuge Plate or strip tube centrifuge Pipettes multichannel pipette and P10 P20 P200 and P1000 pipettes Vortex mixer Ice bucket Powder free gloves Sterile nuclease free aerosol barrier pipette tips Savant SpeedVac model DNA120 with 96 well plate rotor model RD2MP or equivalent Agilent p n G2943CA Agilent p n G2947CA Agilent p n 5067 1504 Agilent p n 5067 4626 Agilent p n G2991AA Agilent p n 5067 5582
31. AC 2 2 3 Place an empty square well plate waste reservoir at position 1 of the Bravo deck 4 Prepare an Eppendorf source plate labeled Wash 1 For each well to be processed add 160 ul of SureSelect Wash Buffer 1 Place the Wash 1 source plate at position 3 of the Bravo deck 5 Prepare a Thermo Scientific reservoir containing 15 ml of nuclease free water and place the reservoir at position 9 of the Bravo deck SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A 89 4 Hybridization Setup and Run VWorks protocol 08 SureSelectO0XT_Wash_v1 0 pro 6 On the SureSelect setup form under Select Protocol select 08 SureSelectQXT_Wash_v1 0 pro 7 Select the number of columns of samples to be processed Runs must include 1 2 3 4 6 or 12 columns 8 Click Display Initial Bravo Deck Setup Display Initial Bravo Deck Setup 9 Verify that the Bravo deck has been set up according to the Bravo Deck Setup region of the form and as shown in Table 45 Table 45 Initial Bravo deck configuration for 08 SureSelectOXT_Wash_v1 0 pro Location Content 1 Empty waste reservoir Axygen 96 Deep Well Plate square wells 2 New tip box 3 Wash 1 Eppendorf source plate 4 Empty red insert 5 DNA RNA hybrids captured on streptavidin beads in DeepWell plate 6 Wash 2 DeepWell source plate seated on silver Nunc DeepWell insert pre heated during Capture protocol 7 empty 8 Empty tip box 9 Nuclease free w
32. Deck Position 9 Using the ThermoCube Device Bravo deck position 9 is equipped with a ThermoCube thermoelectric temperature control system used to incubate components at a defined temperature during the run During protocols that require temperature control at position 9 you will be instructed to start and set the temperature of the ThermoCube device before starting the run ThermoCube temperature settings are modified using the control panel LCD display screen and four input buttons on the front panel of the device using the following steps 1 Turn on the ThermoCube and wait for the LCD screen to display TEMP 2 Press the UP or DOWN button to change SET TEMP 1 to the required set point 3 Press the START button The ThermoCube then initiates temperature control of Bravo deck position 9 at the displayed set point SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A 21 2 Using the Agilent NGS Bravo Option A for SureSelect X Target Enrichment VWorks Automation Control Software VWorks software included with your Agilent NGS Bravo Option A allows you to control the integrated devices using a PC The Agilent NGS Bravo Option A is preloaded with VWorks software containing all of the necessary SureSelect system liquid handling protocols General instructions for starting up the VWorks software and the included protocols is provided below Each time a specific VWorks protocol is used in the SureSelect proce
33. Fest Tine ooo rs Advanced Settings Enable audio alerts p Ignore all incubation times TESTING ONLY SureSelect Target Enrichment for Illumina Sequencing NGS Bravo Option A Using the Agilent NGS Bravo Option A for SureSelect 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 VvWvwvw vw wv i i i iv Select columns of used tips Box 8 DOOOOOOODOOOD 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 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 t
34. Illumina Sequencing NGS Bravo Option A 33 2 Using the Agilent NGS Bravo Option A for SureSelect X Target Enrichment CAUTION e Several workflow steps require that the sample plate be sealed using the PlateLoc thermal microplate sealer and then centrifuged to collect any dispersed liquid To maximize efficiency locate the centrifuge in close proximity to the Agilent NGS Bravo PCR Plate Type Considerations Automation protocols include several liquid handling steps in which reagents are dispensed to PCR plates in preparation for transfer to a thermal cycler For these steps you must specify the PCR plate type to be used on the QXT_ILM_OptA_v1 0 VWForm to allow correct configuration of the liquid handling components for the PCR plate type Before you begin the automation protocol make sure that you are using a supported PCR plate type The PCR plate type to be used in the protocol is specified using the menu below Vendor and part number information is provided for the supported plate types in Table 8 2 Select PCR Plate labware for Thermal Cycling 96 ABI PCR half skirt in Red Alum Insert 3 96 ABI PCR half skirt in Red Alum Insert 96 Agilent Semi skirted PCR in Red Alum Insert 96 Eppendorf Twin tec half skirt PCR in Red Alum Insert 4 96 Eppendorf Twin tec PCR in Red Alum Insert The plates listed in Table 8 are compatible with the Agilent NGS Bravo and associated VWorks automation protocols designed to support use of var
35. In this step the Agilent NGS Bravo completes the liquid handling steps for amplification of the adaptor ligated DNA samples using automation protocol 03b Pre CapturePCR_QXT_ILM_v1 0 pro Afterward you transfer the PCR plate to a thermal cycler for amplification This step uses the SureSelect X Reagent Kit components listed in Table 16 Table 16 Reagents for precapture amplification Kit Component Storage Location Where Used Herculase II Fusion DNA Polymerase SureSelect QXT Library Prep Kit Box 2 20 C page 49 Herculase II 5x Reaction Buffer SureSelect QXT Library Prep Kit Box 2 20 C page 49 100 mM dNTP Mix 25 mM each dNTP SureSelect QXT Library Prep Kit Box 2 20 C page 49 SureSelect QXT Primer Mix SureSelect QXT Hyb Module Box 2 20 C page 49 DMSO Transferred to Room Temperature storage on page 38 page 49 CAUTION To avoid cross contaminating libraries set up PCR master mixes in a dedicated clean area or PCR hood with UV sterilization and positive air flow Prepare the NGS Bravo 1 Clear the Bravo deck of all plates and tip boxes then wipe it down with a DNA Away decontamination wipe 2 Pre set the temperature of Bravo deck positions 6 and 9 as indicated in Table 17 See page 19 to page 21 for more information on how to do this step Table 17 Bravo Deck Temperature Presets Bravo Deck Position Temperature Preset Preset Method 6 4 C Inheco Multi TEC control touchscreen CPAC 2 2 9 0 C Th
36. Position 8 gt lt Pos 9 Chiller gt 96 Agilent Semi skirted PCR in Red Alum Insert Si 3 Select Number of Columns of Samples to Process 3 v 4 Display Initial Bravo Deck Setup Reference 5 Load labware according to Bravo Deck Setup Final DN A lees Nei Protocol Temperature 6 Update current tip state omit for Protocol 05 Location Duration Presets Current Tip State Information Select columns of unused tips VMMM MMM ee Select columns of used tips DOOOCeCoOoOooo Reset Clear Controls Run Se ented fi Pause Full Screen Status Advanced Settings M Enable audio alerts f Ignore all incubation times TESTING ol 1 Open the form using the QXT_ILM_OptA_v1 0 VWForm shortcut on your desktop 2 Use the drop down menus on the form to select the appropriate SureSelect workflow step and other Parameters for the run 3 Once all run parameters have been specified on the form click Display Initial Bravo Deck Setup Display Initial Bravo Deck Setup SureSelect Target Enrichment for Illumina Sequencing NGS Bravo Option A 23 2 24 Using the Agilent NGS Bravo Option A for SureSelect Target Enrichment 4 The form will then display the NGS Bravo deck configuration needed for the specified run parameters Load the Bravo Deck with labware and reagents as specified in the Bravo Deck Setup region of the form Review the temperature preset and in run labware transfer information shown in the Inf
37. Station as instructed in the instrument user manual Start the run 3 Verify that the electropherogram shows the peak of DNA fragment size positioned between 245 to 325 bp Sample electropherograms are shown in Figure 6 Variability of fragmentation profiles may be observed A peak DNA fragment size significantly less than 245 bp may indicate too little gDNA in the fragmentation reaction and may be associated with increased duplicates in the sequencing data In contrast a peak DNA fragment size significantly greater than 325 bp may indicate too much gDNA in the fragmentation reaction and may be associated with decreased percent on target performance in sequencing results 4 Measure the concentration of each library by integrating under the entire peak If you do not continue to the next step seal the library DNA sample plate and store at 4 C overnight or at 20 C for prolonged storage SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A Sample Preparation 3 25 20 lt gt g Ss w 15 E g 2 S a 10 a E G N 5 o ele Size n o s s 8 8 8 s aj bp N a N M n Aja A S we 25 a 09 a lt gt a gt 15 2 E amp v 10 a E 5 Nn 5 o ele Size n o s Ss 8 8 8 j sya bp N n a N mM n a a Figure 6 Representative sample electropherograms showing pre capture analysis of am
38. SureSelect Automated Target Enrichment for Illumina Multiplexed Sequencing Featuring Transposase Based Library Prep Technolog Automated usi gilent NGS Bravo Option A Proto Version BO Nove SureSelect platform manufactufgm with Agilent SurePrint Technology For Research Use Only Not for use in diagnostic procedures Apg Agilent Technologies Notices Agilent Technologies Inc 2015 No part of this manual may be reproduced in any form or by any means including elec tronic storage and retrieval or translation into a foreign language without prior agree ment and written consent from Agilent Technologies Inc as governed by United States and international copyright laws Manual Part Number 69681 90020 Edition Version B0 November 2015 Printed in USA Agilent Technologies Inc 5301 Stevens Creek Blvd Santa Clara CA 95051 USA Acknowledgement Oligonucleotide sequences 2006 2008 and 2011 Illumina Inc All rights reserved Only for use with the Illumina sequencer systems and associated assays Technical Support For technical product support contact your local Agilent Support Services representa tive For US and Canada call 800 227 9770 option 3 4 4 For other countries find your support center telephone numbers at www agilent com chem contactus Or send an e mail to SureSelect Support agilent com Warranty The material contained in this document is provided a
39. Table 46 Thaw then vortex to mix the reagents listed below and keep on ice Table 46 Reagents for post capture indexing by PCR amplification Kit Component Storage Location Where Used Herculase II Fusion DNA Polymerase SureSelect QXT Library Prep Kit Box 2 20 C page 95 Herculase II 5x Reaction Buffer SureSelect QXT Library Prep Kit Box 2 20 C page 95 100 mM dNTP Mix 25 mM each dNTP SureSelect QXT Library Prep Kit Box 2 20 C page 95 SureSelect QXT P7 and P5 dual indexing primers SureSelect QXT Library Prep Kit Box 2 20 C page 96 94 Prepare the NGS Bravo 1 Clear the Bravo deck of all plates and tip boxes then wipe it down with a DNA Away decontamination wipe 2 Pre set the temperature of Bravo deck positions 6 and 9 as indicated in Table 47 See page 19 to page 21 for more information on how to do this step Table 47 Bravo Deck Temperature Presets Bravo Deck Position Temperature Preset Preset Method 6 4 C Inheco Multi TEC control touchscreen CPAC 2 2 9 0 C ThermoCube control panel 3 Place a red PCR plate insert at Bravo deck position 6 and a silver deep well plate insert at Bravo deck position 9 SureSelect T Target Enrichment for Illumina Sequencing NGS Bravo Option A Indexing 5 Prepare the PCR master mix CAUTION To avoid cross contaminating libraries set up PCR master mixes in a dedicated clean area or PCR hood with UV sterilization and positive air flow 4 Prepare the
40. analyze SureSelect generated data you first need to define an analysis workflow This analysis workflow identifies the libraries as SureSelect libraries and enables automated adaptor trimming The trimmed FASTQ files can then be used for alignment to generate BAMs for downstream analysis To create the analysis workflow refer to Figure 13 on page 116 Upon starting SureCall click the Analysis Workflow tab Choose the appropriate analysis type single sample paired or trio analysis and then click the Import Unaligned Files button Within the Select Unaligned Sample Files window specify your read 1 and read 2 files using the Add buttons Using the menus near the bottom of the screen select Default SureSelect QXT Method from the Analysis Method menu choose the appropriate design description from the Design menu and select Ilumina from the Platform menu Once done refer to the SureCall guide for next steps on alignment and variant calling If using another pipeline for alignment and downstream analysis refer to the platform specific guidelines starting on page 116 SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A 115 5 Indexing SureCall Figure 13 116 impart your One 7 Semple Configure Sepporting a Review Settings Files DB Home tamper aad apply one of te default anahi metheds as defined in Condiqure Setting sr chose a diferent anadyih method Select the deug and requesc
41. appropriate volume of PCR master mix according to Table 48 Mix well using a vortex mixer and keep on ice Table 48 Preparation of PCR Master Mix for 09 Post CapturePCR_OXT_ILM_v1 0 pro SureSelect Volume for Volume for Volume for Volume for Volume for Volume for Volume for Reagent 1 Library 1 Column 2 Columns 3 Columns 4 Columns 6 Columns 12 Columns Nuclease free water 9 5 ul 121 1 pl 201 9 pl 282 6 pl 363 4 pl 524 9 ul 1049 8 pl Herculase II 5x 10 0 pL 127 5 pl 212 5 pl 297 5 ul 382 5 pl 552 5 ul 1105 0 pl Reaction Buffer 100 mM dNTP Mix 0 5 pL 6 4 pl 10 6 pl 14 9 ul 19 1 pl 27 6 ul 55 3 pl Herculase Il Fusion 1 0 pl 12 8 pl 21 3 pl 29 8 ul 38 3 pl 55 3 pl 110 5 pl DNA Polymerase Total Volume 21 0 pl 267 8 pl 446 3 pl 624 8 pl 803 3 pl 1160 3 pl 2320 6 pl 5 Using the same Nunc DeepWell master mix source plate that was used for the 06 Hybridization_QXT_v1 0 pro protocol add the volume of PCR master mix indicated in Table 49 to all wells of column 4 of the plate Keep the source plate on ice until it is used on page 97 Table 49 Preparation of the Master Mix Source Plate for 09 Post CapturePCR_OXT_ILM_v1 0 pro Master Mix Position on Volume of Master Mix added per Well of Nunc Deep Well Source Plate Solution Source Plate 1 Column 2 Column 3 Column 4 Column 6 Column 12 Column Runs Runs Runs Runs Runs Runs PCR Master Mix Column 4 30 8 ul 53 2 ul 75 5 pl 97 8 ul 142 4 pl 287 4 ul A4 H4 If you are using a new DeepWell
42. ated in Table 19 to all wells of column 2 of the master mix source plate The final configuration of the master mix source plate is shown in Figure 4 Table 19 Preparation of the Master Mix Source Plate for 03b Pre CapturePCR_OXT_ILM_v1 0 pro Master Mix Position on Volume of Master Mix added per Well of Nunc Deep Well Source Plate Solution Source Plate ee S 1 Column 2 Column 3 Column 4 Column 6 Column 12 Column Runs Runs Runs Runs Runs Runs Pre CapturePCR Column 2 41 9 ul 72 1 ul 102 4 pl 132 7 pl 193 3 pl 390 1 ul Master Mix A2 H2 If you are using a new DeepWell plate for the pre capture PCR source plate leave column 1 empty and add the PCR Master Mix to column 2 of the new plate 50 SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A Sample Preparation 3 LXXND C060 ddd ded lt gt lt eee8 Case sea 6 6s ee 200 gt ee JS e oe e EE Figure 4 Configuration of the master mix source plate for 03b Pre CapturePCR_OXT_ILM_v1 0 pro Column 1 was used to dispense master mix during the previous protocol 6 Seal the master mix source plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 1 0 sec 7 Centrifuge the plate for 30 seconds to drive the well contents off the walls and plate seal and to eliminate any bubbles The presence of bubbles in source plate solutions may cause inaccurate volume transfer by the Bravo l
43. ater reservoir 10 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 23 for more information on using this segment of the form during the run 11 When setup and verification is complete click Run Selected Protocol Run Selected Protocol SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A Hybridization 4 Running the 08 SureSelectQXT_Wash_v1 0 pro protocol takes approximately 60 minutes An operator must be present during the run to complete tip box replacement as directed by VWorks prompts Once complete you will be prompted as shown below 12 When the wash protocol is complete the captured bead bound DNA samples are located in the Eppendorf plate at position 5 of the Bravo deck and you will be prompted by VWorks as shown below Click Continue on the VWorks screen to finish the protocol Remove the DNA sample plate from position 5 and seal the wells using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 1 0 sec Store the plate on ice until it is used on page 101 Proceed immediately to the 09 Post CapturePCR_QXT_ILM_v1 0 pro protocol starting on page 94 Proceed to Amplification The captured DNA RNA hybrids are ready at Position 5 in an Eppendorf plate Proceed immediately to 09 Post CapturePCR_OXT_ILM_v1 0 pro Captured DNA
44. creen of the instrument control software interface after choosing Custom from the index type selection buttons Table 63 Run Configuration screen Cycle Number settings Run Segment Cycle Number Read 1 100 Index 1 i7 8 Index 2 i5 8 Read 2 100 After the sequencing run is complete generate demultiplexed FASTQ data following Illumina s instructions and then trim adaptor sequences from the reads using Agilent s Read Trimmer tool This tool takes in data in FASTQ format and removes the adaptor sequence from the ends of the sequencing reads generating trimmed FASTQ data as output To download the Read Trimmer tool free of charge and for additional information on this resource visit www agilent com genomics SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A 117 5 Indexing Step 7 Set up the sequencing run and trim adaptors from the reads 118 SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A SureSelect Automated Target Enrichment for Illumina Multiplexed Sequencing Protocol ee 6 070 Reference J e Kit Contents 120 Nucleotide Sequences of SureSelect T Dual Indexes 123 Guidelines for Multiplexing with Dual Indexed Samples 125 This chapter contains reference information including component kit contents and reference information for use during the downstream sample sequencing steps os Agilent Technologies 119 6 Reference Kit Conten
45. d down along the sides of each well for optimal recovery 11 Seal the plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 1 0 sec 12 Vortex the plate for 30 seconds to ensure complete reconstitution then centrifuge the plate for 1 minute to drive the well contents off the walls and plate seal SureSelect Target Enrichment for Illumina Sequencing NGS Bravo Option A Hybridization 4 Step 2 Hybridize the gDNA library and SureSelect or ClearSeq Capture Library In this step automation protocol 06 Hybridization_QXT_v1 0 pro is used to complete the liquid handling steps to set up the hybridization reactions Afterward you transfer the sample plate to a thermal cycler held at 65 C to allow hybridization of the DNA samples to the Capture Library This step uses the SureSelect XT Reagent Kit components listed in Table 27 Thaw each component under the conditions indicated in the table Vortex each reagent to mix then spin briefly to collect the liquid Table 27 Reagents for Hybridization and Capture Kit Component Storage Location Thawing Conditions Where Used SureSelect QXT Fast SureSelect QXT Hyb Module Warm to Room page 75 Hybridization Buffer Box 2 20 C Temperature RT then keep at RT SureSelect QXT Fast SureSelect QXT Hyb Module Thaw on ice page 72 Blocker Mix Box 2 20 C SureSelect RNase Block SureSelect QXT Hyb Module Thaw on ice page 73 or Box 2 20 C page 74 Capt
46. ds on streptavidin coated magnetic beads PCR amplify using Dual Indexing primers i Pool libraries for multiplex sequencing I Optional Figure 2 Overall sequencing sample preparation workflow 30 SureSelect Target Enrichment for Illumina Sequencing NGS Bravo Option A Using the Agilent NGS Bravo Option A for SureSelect Target Enrichment 2 Table 6 Overview of VWorks protocols used for SureSelectOX Target Enrichment using NGS Bravo Option A Workflow Step Substep Protocol Chapter VWorks Protocols Used for Agilent NGS Bravo Option A automation Prepare fragmented and adaptor tagged DNA Purify DNA using AMPure XP beads Sample Preparation Amplify adaptor tagged DNA Purify DNA using AMPure XP beads Aliquot prepped libraries for hybridization Hybridize prepped DNA to Capture Library Hybridization Capture DNA hybrids Wash captured DNA hybrids Indexing Add index tags by PCR Purify DNA using AMPure XP beads 01 Tn_OXT_ILM_v1 0 pro 02 Cleanup_Tn_v1 0 pro 03b Pre CapturePCR_OXT_ILM_v1 0 pro 04b Cleanup_Pre CapturePCR_QXT_ILM_v1 0 pro 05 Aliquot_Libraries_v1 0 pro 06 Hybridization_OXT_v1 0 pro 07 SureSelectOXT_Capture_v1 0 pro 08 SureSelectOXT_Wash_v1 0 pro 09 Post CapturePCR_OXT_ILM_v1 0 pro 10 Cleanup_Post CapturePCR_OXT_ILM_v1 0 pro SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A 31 2 Using the Agilent NGS Bravo Option A for SureSelect X Target Enrich
47. dure any settings required for that protocol are included in the relevant section of this manual The instructions in this manual are compatible with VWorks software version 11 3 0 1195 including SureSelect T automation protocols version 1 0 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 QXT_ILM_OptA_v1 0 VWForm shortcut on the Windows desktop to start the VWorks software 2 If User Authentication dialog is not visible click Log in on the VWorks window toolbar 3 In the User Authentication dialog type your VWorks user name and password and click OK If no user account is set up contact the administrator 22 SureSelect T Target Enrichment for Illumina Sequencing NGS Bravo Option A Using the Agilent NGS Bravo Option A for SureSelect X Target Enrichment 2 Using the OXT_ILM_OptA_v1 0 VWForm to setup and start a run Use the VWorks form QXT_ILM_OptA_v1 0 VWForm shown below to set up and start each SureSelect automation protocol Bravo Deck Setup z P SureSelect Li A ood Transposase Library Prep lt Position 1 gt lt Position 2 gt lt Position 3 gt for Illumina sequencers Parameters lt Pos 4 Peltier gt lt PosS Shaker gt lt Pos 6 Peltier gt 1 Select Protocol none pes 2 Select PCR Plate Labware Protocols 3 4 6 7 9 and 10 lt Pos 7 Magnet gt lt
48. ect Human All Exon v5 Plus 5190 6224 5 x 5190 6224 SureSelect Human All Exon v4 5190 4633 5190 4635 SureSelect Human All Exon v4 UTRs 5190 4638 5190 4640 SureSelect Mouse All Exon 5190 4643 5190 4645 SureSelect Custom 1 kb up to 499 kb 5190 4808 5190 4810 reorder 5190 4813 5190 4815 SureSelect Custom 0 5 Mb up to 2 9 Mb 5190 4818 5190 4820 reorder 5190 4823 5190 4825 SureSelect Custom 3 Mb up to 5 9 Mb 5190 4828 5190 4830 reorder 5190 4833 5190 4835 SureSelect Custom 6 Mb up to 11 9 Mb 5190 4838 5190 4840 reorder 5190 4843 5190 4845 SureSelect Custom 12 Mb up to 24 Mb 5190 4898 5190 4900 reorder 5190 4903 5190 4905 SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A 13 1 Before You Begin Table3 Compatible ClearSeq Automation Capture Libraries Capture Library 96 Reactions 480 Reactions ClearSeq Comprehensive Cancer XT 5190 8013 5 x 5190 8013 ClearSeq Comprehensive Cancer Plus XT 5190 8016 5 x 5190 8016 ClearSeq Inherited Disease XT 5190 7520 5 x 5190 7520 ClearSeq Inherited Disease Plus XT 5190 7523 5 x 5190 7523 ClearSeq DNA Kinome XT 5190 4648 5190 4650 Optional Reagents Description Vendor and part number Agilent QPCR NGS Library Quantification Kit Illumina GA Agilent p n G4880A 14 SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A Required Equipment Table 4 Before You Begin 1 Required Equipment fo
49. ecular biology grade Qubit dsDNA HS Assay Kit or Qubit dsDNA BR Assay Kit 100 assays 500 assays Nuclease free Water not DEPC treated Select one library from Table 2 or Table 3 Agilent p n G9681B p n G9683B Beckman Coulter Genomics p n A63880 p n A63881 p n A63882 Life Technologies p n 65601 p n 65602 p n 65603 Life Technologies p n 12090 015 or equivalent Sigma Aldrich p n E7023 Life Technologies p n 032851 Life Technologies p n 032850 p n 032853 Ambion Cat AM9930 SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A Before You Begin 1 Table2 SureSelect Automation Capture Libraries Capture Library 96 Reactions 480 Reactions SureSelect Human All Exon v6 5190 8865 5 x 5190 8865 SureSelect Human All Exon v6 UTRs 5190 8883 5 x 5190 8883 SureSelect Human All Exon v6 COSMIC 5190 9309 5 x 5190 9309 SureSelect Human All Exon v6 Plus 1 5190 8868 5 x 5190 8868 SureSelect Human All Exon v6 Plus 2 5190 8871 5 x 5190 8871 SureSelect Clinical Research Exome 5190 7344 5 x 5190 7344 SureSelect Focused Exome 5190 7789 5 x 5190 7789 SureSelect Focused Exome Plus 1 5190 7792 5 x 5190 7792 SureSelect Focused Exome Plus 2 5190 7796 5 x 5190 7796 SureSelect Human All Exon v5 5190 6210 5 x 5190 6210 SureSelect Human All Exon v5 UTRs 5190 6215 5 x 5190 6215 SureSelect Human All Exon v5 IncRNA 5190 6448 5 x 5190 6448 SureSel
50. ee Table 5 for designations of the heat block containing Bravo deck positions on the Multi TEC control device Table5 Inheco Multi TEC Control touchscreen designations Bravo Deck Position Designation on Inheco Multi TEC Control Screen 4 CPAC 21 6 CPAC 22 1 Using the arrow buttons select the appropriate block CPAC 2 block 1 or CPAC 2 block 2 crac 2 lt gt 24 9 C Shaker 0200 rpm SureSelect T Target Enrichment for Illumina Sequencing NGS Bravo Option A 19 2 Using the Agilent NGS Bravo Option A for SureSelect X Target Enrichment 2 To set the temperature of the selected block press the SET button CPAC 2 1 24 9 C 3 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 center of the display CPAC 2 1 25 0 C KE Current temp 0200 rpm se 20 SureSelect Target Enrichment for Illumina Sequencing NGS Bravo Option A Using the Agilent NGS Bravo Option A for SureSelect Target Enrichment 2 Setting the Temperature of Bravo
51. equired for High Sensitivity DNA Assay Kit runs 2 Seal the sample plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 1 0 sec 3 Vortex the plate to mix samples in each well then centrifuge the plate for 30 seconds to drive the well contents off the walls and plate seal 4 Prepare the chip samples and ladder as instructed in the reagent kit guide using 1 ul of each sample for the analysis For some samples Bioanalyzer results are improved by diluting 1 ul of the sample in 9 ul of 10 mM Tris 1 mM EDTA prior to analysis Be sure to mix well by vortexing at 2000 rpm on the IKA vortex supplied with the Bioanalyzer before analyzing the diluted samples 5 Load the prepared chip into the 2100 Bioanalyzer and start the run within five minutes after preparation 6 Verify that the electropherogram shows the peak of DNA fragment size positioned between 325 and 450 bp A sample electropherogram is shown in Figure 11 Stopping Point If you do not continue to the next step seal the plate and store at 4 C overnight or at 20 C for prolonged storage SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A 105 5 Indexing Step 3 Assess indexed DNA quality 35 100 150 200 300 40 500 600 700 1000 2000 10380 bo Figure 11 Analysis of indexed DNA using the High Sensitivity DNA Assay 106 SureSelect T Target Enrichment for Illumina Sequencing NGS Bravo Option A Indexing 5 Op
52. ermoCube control panel 48 SureSelect T Target Enrichment for Illumina Sequencing NGS Bravo Option A Sample Preparation 3 3 Place a red PCR plate insert at Bravo deck position 6 and a silver deep well plate insert at Bravo deck position 9 Prepare the pre capture PCR master mix and master mix source plate 4 Prepare the appropriate volume of pre capture PCR Master Mix according to Table 18 Mix well using a vortex mixer and keep on ice Table 18 Preparation of Pre Capture PCR Master Mix SureSelect Volumefor Volumefor Volumefor Volumefor Volumefor Volumefor Volume for Reagent 1Library 1 Column 2 Columns 3 Columns 4Columns 6 Columns 12 Columns Nuclease free 13 5 pl 172 1 pl 286 9 ul 401 6 ul 516 4 pl 745 9 ul 1491 8 ul water Herculase II 5X 10 0 pL 127 5 pl 212 5 pl 297 5 pl 382 5 ul 552 5 pl 1105 ul Reaction Buffer DMSO 2 5 uL 31 9 pl 53 1 ul 74 4 ul 95 6 ul 138 1 pl 276 3 pl dNTP mix 0 5 pL 6 4 pl 10 6 pl 14 9 ul 19 1 pl 27 6 ul 55 3 pl SureSelect QXT 1 0 pL 12 8 pl 21 3 ul 29 8 ul 38 3 pl 55 3 pl 110 5 pl Primer Mix Herculase II 1 0 pL 12 8 ul 21 3 pl 29 8 pl 38 3 pl 55 3 pl 110 5 pl Fusion DNA Polymerase Total Volume 28 5 pl 363 4 pl 605 6 pl 847 9 pl 1090 1 pl 1574 6 pl 3149 3 pl SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A 49 3 Sample Preparation 5 Using the same Nunc DeepWell master mix source plate that was used for the 01 Tn_QXT_ILM_v1 0 pro run add the volume of PCR Master Mix indic
53. et enriched DNA library plate See step 7 and step 8 below for P7 primer source plate setup details e For sample multiplexing Agilent recommends maximizing index diversity on both P7 and P5 primers as required for color balance For example when 8 plexing use eight different P7 index primers with two P5 index primers See Table 72 on page 125 for additional details 96 SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A oa Indexing 7 Dilute each P7 indexing primer P7 il through P7 i12 to be used in the run according to Table 50 The volumes below include the required excess Table 50 Preparation of P7 indexing primer dilutions Reagent Volume to Index 1 Sample Volume to Index 8 Samples Nuclease free water 4 0 ul 34 ul SureSelect QXT P7 dual indexing 1 0 pl 8 5 ul primer P7 i1 to P7 i12 Total Volume 5 0 pl 42 5 pl 8 Ina fresh PCR plate aliquot 5 ul of the appropriate P7 indexing primer dilution from Table 50 to the intended sample indexing well position s Keep the plate on ice 9 Obtain the Nunc DeepWell master mix source plate containing the PCR Master Mix in column 4 prepared in step 5 above Add each P5 indexing primer P5 i13 through P5 i20 to be used in the run to the master mix in the appropriate well of column 4 Add the volume listed in Table 51 to each well of column 4 according to the number of sample columns in the run Each well of column 4 can contain the same or di
54. fferent P5 indexing primers The final configuration of the master mix source plate is shown in Figure 10 on page 98 Keep the source plate on ice Table 51 Addition of P5 indexing primers to the post capture PCR master mix source plate Solution added to Position on Volume of Primer added per Well of Nunc Deep Well Source Plate Source Plate Source Plate 1 Column 2 Column 3 Column 4 Column 6 Column 12 Column Runs Runs Runs Runs Runs Runs SureSelect QXT Column 4 1 5 pl 2 5 pl 3 6 pl 47 ul 6 8 pl 13 7 pl P5 dual indexing A4 H4 primer s Each well of column 4 may contain the same or different P5 indexing primer Typical 12 column runs include all eight of the provided SureSelect QXT P5 dual indexing primers P5 i13 through P5 i20 resulting in a different P5 primer assignment to each row of the PCR indexing plate SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A 97 5 Indexing lt gt lt Oe OO ane ae 000 lt a gt 000 SSS ISOS Bee Coes Oo TRS LAA Q x ST S Xe S amp CS x BN xX amp amp FS YLT LT QF Fr SS Of c Ww Q S amp SF LT SL S N A S S g S V Q e S N S ge Figure 10 Configuration of the master mix source plate for 09 Post CapturePCR_OXT_ILM_v1 0 pro 10 Seal the master mix source plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 1 0 sec 11 Vortex the plate to ensure
55. ights in Commercial Computer Software or Com puter Software Documentation Notice to Purchaser This product is provided under an agree ment between Bio Rad Laboratories and Agilent Technologies Inc and the manu facture use sale or import of this product is subject to US Pat No 6 627 424 and EP Pat No 1 283 875 B1 owned by Bio Rad Labora tories Inc Purchase of this product con veys to the buyer the non transferable right to use the purchased amount of the product and components of the product in PCR but not real time PCR in the Research Field including all Applied Research Fields including but not limited to forensics ani mal testing and food testing 2 SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A Safety Notices CAUTION A CAUTION notice denotes a hazard It calls attention to an operating procedure practice or the like that if not correctly performed or adhered to could result in damage to the product or loss of important data Do not proceed beyond a CAUTION notice until the indicated conditions are fully understood and met A WARNING notice denotes a hazard It calls attention to an operating procedure practice or the like that if not correctly performed or adhered to could result in personal injury or death Do not proceed beyond a WARNING notice until the indicated conditions are fully understood and met SureSelect T Target Enrichment for Illumina Sequencing
56. ing platform that was uiad F the ceden fle hin nat been prevouily iapeeted ga ts Supporting Fies to domiad the Mile from Seretiouign Oich Nest to proceed lo ste import unaligned Files amport Atigned Files Cotapse Al File Plame E SC VAVA NOGI S ACAGTG Al Masta es Pies ol Type Fal File fag fasta Cash EA SCS Va WONNI A ACAGTG At Anata ge E E T O Chear AA Lists OK Gnod Ota tet Analysis workflow setup in SureCall MiSeq platform sequencing run setup and adaptor trimming guidelines Use the Illumina Experiment Manager IEM software to generate a custom primer Sample Sheet Set up the run to include adapter trimming using the IEM Sample Sheet Wizard When prompted by the wizard select the Use Adapter Trimming option and specify CTGTCTCTTGATCACA as the adapter sequence This enables the MiSeq Reporter software to identify the adaptor sequence and trim the adaptor from reads HiSeq or NextSeq 500 platform sequencing run setup and adaptor trimming guidelines Set up sequencing runs using the Custom setting Since custom primers are spiked into the standard sequencing primer tubes no additional specialized settings are required to accommodate the use of custom primers in the run SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A Indexing 5 Use the Cycles settings shown in Table 63 Cycle number settings can be specified on the Run Configuration s
57. ion CAUTION 23 When prompted by VWorks as shown below quickly remove the PCR sample plate from Bravo deck position 4 leaving the red insert in place r Remove Plate from 4 Quickly remove plate from position 4 seal and place in thermocyder Click Continue after plate is in thermocyder for protocol to finish 24 Seal the sample plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 3 0 sec 25 Quickly transfer the plate back to the thermal cycler held at 65 C Press the Play button to initiate the hybridization segment of the pre programmed thermal cycling program segment 4 from Table 28 on page 70 During this step the prepared DNA samples are hybridized to the SureSelect or ClearSeq Capture Library The thermal cycler is held at 65 C using a heated lid at 105 C The lid of the thermal cycler is hot and can cause burns Use caution when working near the lid 82 26 After initiating hybridization on the thermal cycler click Continue on the VWorks screen to finish the NGS Bravo protocol During the thermal cycler incubation for hybridization approximately 1 5 hour duration complete the reagent and NGS Bravo setup steps for the capture automation protocol as described on page 83 to page 85 SureSelect T Target Enrichment for Illumina Sequencing NGS Bravo Option A Hybridization 4 Step 3 Capture the hybridized DNA This step uses automation protocol 07 SureSelec
58. ion Prepare the Dynabeads streptavidin beads and Wash Buffer 2 source plates 5 Vigorously resuspend the Dynabeads MyOne Streptavidin T1 magnetic beads on a vortex mixer The beads settle during storage 6 Wash the magnetic beads a Inaconical vial combine the components listed in Table 40 The volumes below include the required overage Table 40 Magnetic bead washing mixture Reagent Volume for Volumefor Volumefor Volumefor Volumefor Volumefor Volume for 1 Library 1 Column 2 Columns 3Columns 4 Columns 6 Columns 12 Columns Dynabeads 50 pl 425 ul 825 ul 1225 ul 1 65 ml 2 5 ml 5 0 ml MyOne Streptavidin T1 bead suspension SureSelect 0 2 ml 1 7 ml 3 3 ml 4 9 ml 6 6 ml 10 ml 20 ml Binding Buffer Total Volume 0 25 ml 2 125 ml 4 125 ml 6 125 ml 8 25 ml 12 5 ml 25 ml b Mix the beads on a vortex mixer for 5 seconds c Put the vial into a magnetic device such as the Dynal magnetic separator d Remove and discard the supernatant e Repeat step a through step d for a total of 3 washes Retain the beads after each wash and combine with a fresh aliquot of the indicated volume of SureSelect Binding Buffer 7 Resuspend the beads in SureSelect Binding buffer according to Table 41 below Table 41 Preparation of magnetic beads for 07 SureSelectOXT_Capture_v1 0 pro Reagent Volume for Volumefor Volumefor Volumefor Volumefor Volumefor Volume for 1 Library 1 Column 2 Columns 3Columns 4 Columns 6 Columns 12 Columns SureSelect
59. ion 4 seal and place in thermocyder to begin denaturation step 19 Seal the sample plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 3 0 sec 78 SureSelect T Target Enrichment for Illumina Sequencing NGS Bravo Option A Hybridization 4 20 Transfer the sealed plate to a thermal cycler and initiate the preprogrammed thermal cycling program described in Table 28 on page 70 The denaturation and blocking segments of the preprogrammed thermal cycler program are shown in Figure 9 below for reference Table 28 Thermal cycler program for Hybridization Segment Purpose Number of Cycles Temperature Time Number 1 Denaturation 1 95 C 5 minutes 2 Blocking 1 65 C 10 minutes 3 Hold for NGS 1 65 C Hold workstation steps Figure 9 Preprogrammed thermal cycler segments used for sample denaturation and blocking prior to hybridization While the sample plate incubates on the thermal cycler the NGS Bravo combines aliquots of the Capture Library Master Mix and Hybridization Buffer Master Mix SureSelect Target Enrichment for Illumina Sequencing NGS Bravo Option A 79 4 Hybridization CAUTION You must complete step 21 to step 25 quickly and immediately after being prompted by the VWorks software It is important that sample temperature remains approximately 65 C during transfers between the NGS Bravo and thermal cycler 21 When the NGS Bravo has finished aliquoting
60. ious thermal cyclers Accordingly some plates listed in Table 8 are not compatible with the recommended SureCycler 8800 Thermal Cycler When using the SureCycler 8800 Thermal Cycler in the SureSelect automation workflow use 96 Agilent semi skirted PCR plates When using a different thermal cycler in the workflow be sure to select a PCR plate that is compatible with your thermal cycler and that is listed in Table 8 34 SureSelect Target Enrichment for Illumina Sequencing NGS Bravo Option A Using the Agilent NGS Bravo Option A for SureSelect Target Enrichment 2 Table 8 Ordering information for supported PCR plates Description in VWorks menu Vendor and part number 96 ABI PCR half skirted plates MicroAmp Optical Life Technologies p n N8010560 plates 96 Agilent semi skirted PCR plate Agilent p n 401334 96 Eppendorf Twin tec half skirted PCR plates Eppendorf p n 951020303 96 Eppendorf Twin tec PCR plates full skirted Eppendorf p n 951020401 or 951020619 SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A 35 2 Using the Agilent NGS Bravo Option A for SureSelect X Target Enrichment PCR Plate Type Considerations 36 SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A SureSelect Automated Target Enrichment for Illumina Multiplexed Sequencing Protocol c D ee 3 7 e Sample Preparation J e Step 1 Prepare the genomic DNA sample
61. ip boxes for NGS Bravo Option A automation protocols as long as positions of available tips are accurately indicated during run setup 6 After verifying that the NGS Bravo has been set up correctly click Run Selected Protocol Run Selected Protocol SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A 25 2 Using the Agilent NGS Bravo Option A for SureSelect Target Enrichment Error messages encountered at start of run After starting the run you may see the error messages displayed below When encountered make the indicated selections and proceed with the run Encountering either or both of these error messages is not indicative of a problem with the NGS Bravo 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 frontof the 4 gripper s plate presence sensor gf Choose Retry to check the plate presence sensor Choose Ignore to continue to home theG axis Please note that any plate currently held by the Gripper will be dropped Choose Abort to cancel initialization 26 SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A Using the Agilent NGS Bravo Option A for SureSelect Target Enrichment 2 VWorks Automation Control Software 2 If you encounter the W axis error message sho
62. iquid handling platform Ensure that the source plate is sealed and centrifuged prior to use in a run SureSelect Target Enrichment for Illumina Sequencing NGS Bravo Option A 51 3 52 Sample Preparation Setup and run VWorks protocol 03b Pre CapturePCR_OQXT_ILM_v1 0 pro 8 On the SureSelect setup form under Select Protocol to Run select 03b Pre CapturePCR_QXT_ILM_v1 0 pro 9 Under Select PCR plate Labware select the specific type of PCR plate to be used for thermal cycling placed at position 6 of the Bravo deck 10 Select the number of columns of samples to be processed Runs must include 1 2 3 4 6 or 12 columns 11 Click Display Initial Bravo Deck Setup Display Initial Bravo Deck Setup 12 Load the Bravo deck according to the Bravo Deck Setup region of the form and as shown in Table 20 Table 20 Initial Bravo deck configuration for 01 Tn_OXT_ILM_v1 0 pro Location Content 1 Empty waste reservoir Axygen 96 Deep Well Plate square wells 2 New tip box 3 empty 4 empty 5 empty 6 Empty PCR plate seated in red insert PCR plate type must be specified on setup form under step 2 7 Purified adaptor tagged DNA samples in Nunc DeepWell plate 8 Empty tip box 9 Master mix plate containing PCR Master Mix in Column 2 unsealed seated in silver insert 13 Verify that the Current Tip State indicator on the form matches the configuration of unused and used tips in the tip boxes at Brav
63. ize variation The SureSelect QXT Buffer and Enzyme Mix solutions are highly viscous Thorough mixing of the reagents is critical for optimal performance 6 Prepare the appropriate volume of Library Prep Master Mix according to Table 11 Mix well by vortexing for 20 seconds and then keep on ice Table 11 Preparation of Library Prep Master Mix SureSelect T Reagent Volume for Volume for Volume for Volumefor Volume for Volumefor Volume for 1 Library 1Column 2 Columns 3Columns 4Columns 6 Columns 12 Columns SureSelect QXT Buffer 17 0 pl 216 8 ul 361 3 pl 505 8 ul 650 3 pl 939 3 ul 1878 5 pl SureSelect OXT Enzyme 2 0 uL 25 5 ul 42 5 ul 59 5 ul 76 5 ul 110 5 pl 221 0 ul Mix ILM Total Volume 19 pl 242 3 pl 403 8 pl 565 3 pl 726 8 pl 1049 8 pl 2099 5 pl SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A 41 3 Sample Preparation 7 Prepare the Library Prep master mix source plate using a Nunc DeepWell plate containing the mixture from step 6 Add the volume indicated in Table 12 to all wells of column 1 of the Nunc DeepWell plate Keep the master mix on ice during the aliquoting steps The final configuration of the master mix source plate is shown in Figure 3 Table 12 Preparation of the Master Mix Source Plate for 01 Tn_QXT_ILM_v1 0 pro Master Mix Solution Position on Source Plate Volume of Master Mix added per Well of Nunc Deep Well Source Plate 1 Column 2 Column 3 Column 4 Column 6 Column 12 Co
64. l_plate_template csv file may be copied and used as a template for creating the csv files for each 05 Aliquot_Libraries_v1 0 pro run If you are using the sample file as a template for runs with fewer than 12 columns be sure to retain rows for all 96 wells and populate the Volume column with 0 for unused wells SureSelect Target Enrichment for Illumina Sequencing NGS Bravo Option A 65 4 Hybridization 3 Load the csv file onto the PC containing the VWorks software into a suitable folder such as C gt VWorks Workspace gt NGS Option A gt QXT_ILM_v1 0 gt Aliquot Library Input Files Setup and Run VWorks protocol 05 Aliquot_Libraries_v1 0 pro 1 Clear the Bravo deck of all plates and tip boxes then wipe it down with a DNA Away decontamination wipe 2 Pre set the temperature of Bravo deck position 9 as indicated in Table 25 See page 21 for more information on how to do this step Table 25 Bravo Deck Temperature Presets Bravo Deck Position Temperature Preset Preset Method 9 0 C ThermoCube control panel 3 On the SureSelect setup form under Select Protocol select 05 Aliquot_Libraries_v1 0 pro 4 Select the number of columns of samples to be processed Runs must include 1 2 3 4 6 or 12 columns 5 Click Display Initial Bravo Deck Setup Display Initial Bravo Deck Setup 66 SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A Hybridization 4 6 Load the Bravo deck according to the
65. lumn Runs Runs Runs Runs Runs Runs Library Prep Master Mix Column 1 A1 H1 27 9 ul 48 1 ul 68 3 ul 88 5 ul 128 8 pl 260 1 pl 42 Figure en Oren Me oseo amp Configuration of the master mix source plate for 01 Tn_QXT_ILM_v1 0 pro SureSelect T Target Enrichment for Illumina Sequencing NGS Bravo Option A Sample Preparation 3 8 Seal the master mix source plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 1 0 sec 9 Centrifuge the plate for 30 seconds to drive the well contents off the walls and plate seal and to eliminate any bubbles Keep the master mix source plate on ice The presence of bubbles in source plate solutions may cause inaccurate volume transfer by the Bravo liquid handling platform Ensure that the source plate is sealed and centrifuged prior to use in a run Setup and run VWorks protocol 01 Tn_QXT_ILM_v1 0 pro 10 On the SureSelect setup form under Select Protocol to Run select 01 Tn_QXT_ILM_v1 0 pro 11 Select the number of columns of samples to be processed Runs must include 1 2 3 4 6 or 12 columns 12 Click Display Initial Bravo Deck Setup Display Initial Bravo Deck Setup 13 Load the Bravo deck according to the Bravo Deck Setup region of the form and as shown in Table 13 Table 13 Initial Bravo deck configuration for 01 Tn_OXT_ILM_v1 0 pro Location Content 1 empty 2 New tip box 3 Stop Solution in Ep
66. ment Experimental Setup Considerations for Automated Runs Agilent SureSelect Automated Library Prep and Capture System runs may include 1 2 3 4 6 or 12 columns equivalent to 8 16 24 32 48 or 96 wells of gDNA samples to be enriched for sequencing on the Illumina platform Plan your experiments using complete columns of samples Table 7 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 will depend on the experimental design For greatest efficiency of reagent use plan experiments using at least 3 columns per run Each 96 reaction kit contains sufficient reagents for 96 reactions configured as 4 runs of 3 columns of samples per run 32 SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A Using the Agilent NGS Bravo Option A for SureSelect Target Enrichment 2 Considerations for Placement of gDNA Samples in 96 well Plates for Automated Processing e The NGS Bravo processes samples column wise beginning at column 1 gDNA samples should be loaded into 96 well plates column wise in well order A1 to H1 then A2 to H2 ending with A12 to H12 When processing partial runs with lt 12 sample columns do not leave empty columns between sample columns always load the plate using the left most column that is available e At the hybridization
67. nds 72 C 1 minute 4 1 72 C 5 minutes 5 1 4 C Hold SureSelect T Target Enrichment for Illumina Sequencing NGS Bravo Option A Sample Preparation 3 Step 5 Purify amplified DNA using AMPure XP beads In this step the Agilent NGS Bravo transfers AMPure XP beads and amplified adaptor tagged DNA to a Nunc DeepWell plate and then collects and washes the bead bound DNA This step uses protocol 04b Cleanup_Pre CapturePCR_QXT_ILM_v1 0 pro Prepare the NGS Bravo and reagents 1 Clear the Bravo deck of all plates and tip boxes then wipe it down with a DNA Away decontamination wipe 2 Pre set the temperature of Bravo deck positions 4 and 6 as indicated in Table 14 See page 19 for more information on how to do this step Table 22 Bravo Deck Temperature Presets Bravo Deck Position Temperature Preset Preset Method 4 45 C Inheco Multi TEC control touchscreen CPAC 2 1 6 4 C Inheco Multi TEC control touchscreen CPAC 2 2 3 Verify that the AMPure XP bead suspension is at room temperature If necessary allow the bead solution to come to room temperature for at least 30 minutes Do not freeze the beads at any time 4 Mix the AMPure XP bead suspension well so that the reagent appears homogeneous and consistent in color 5 Prepare a Nunc DeepWell source plate for the beads by adding 50 ul of homogeneous AMPure XP beads per well for each well to be processed 6 Prepare a Thermo Scientific Matrix reservoir containing
68. o Deck positions 2 and 8 respectively See page 23 for more information on using this segment of the form during the run SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A Sample Preparation 3 14 When setup and verification is complete click Run Selected Protocol a Run Selected Protocol 15 Running the 03b Pre CapturePCR_QXT_ILM_v1 0 pro protocol takes approximately 10 minutes Once complete the PCR ready samples containing prepped DNA and PCR master mix are located in the PCR plate at position 6 of the Bravo deck 16 When you see the following prompt remove the PCR plate from position 6 of the Bravo deck and seal the plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 3 0 seconds a Remove Plates The PCR plate at Position 6 is ready to be sealed and placed in the thermocycler User data entry Pause and Diagnose Continue i e e 17 Centrifuge the plate for 30 seconds to drive the well contents off the walls and plate seal and to eliminate air bubbles 18 Transfer the PCR plate to a thermal cycler and run the PCR amplification program shown in Table 21 SureSelect T Target Enrichment for Illumina Sequencing NGS Bravo Option A 53 3 54 Sample Preparation Table 21 Pre Capture PCR cycling program Segment Number of Temperature Time Cycles 1 1 68 C 2 minutes 2 1 98 C 2 minutes 3 8 98 C 30 seconds 57 C 30 seco
69. olled by the VWorks Automation Control software Fitted with a choice of seven interchangeable fixed tip or disposable tip pipette heads it accurately dispenses fluids from 0 1 ul to 250 ul Before you begin make sure that you have read and understand operating maintenance and safety instructions for using your Bravo platform Refer to the Bravo Platform User Guide G5409 90006 and the VWorks Software User Guide G5415 90063 18 Bravo Platform Deck The protocols in the following sections include instructions for placing plates and reagent reservoirs on specific Bravo deck locations Use Figure 1 to familiarize yourself with the location numbering convention on the Bravo platform deck Back B L j Front Figure 1 Bravo platform deck SureSelect T Target Enrichment for Illumina Sequencing NGS Bravo Option A Using the Agilent NGS Bravo Option A for SureSelect Target Enrichment 2 Setting the Temperature of Bravo Deck Heat Blocks Bravo deck positions 4 and 6 are equipped with Inheco heat blocks used to incubate sample plates at defined temperatures during the run Runs that include high 85 C or low 4 C temperature incubation steps may be expedited by pre setting the temperature of the affected block before starting the run Bravo deck heat block temperatures may be changed using the Inheco Multi TEC Control device touchscreen as described in the steps below S
70. om sequencing primer preparation Sequencing Volume of SureSelect Primer Volume of Illumina Primer Total Final Read Volume Cartridge Position Read 1 2 7 ul SureSelect QXT Read Primer 1 brown cap 897 3 ul BP10 from well 20 0 9 ml well 7 Read 2 3 3 ul SureSelect QXT Read Primer 2 black cap 1096 7 ul BP11 from well 21 1 1 ml well 8 Index 4 8 ul SureSelect QXT Index Read Primer clear 1590 4 ul BP14 from well 22 1 6 ml well 9 Index 2 cap 4 8 ul SureSelect OXT Index 2 Read Primer NSO purple cap 114 SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A Indexing 5 Step 7 Set up the sequencing run and trim adaptors from the reads Refer to Illumina protocols to set up custom sequencing primer runs using the additional guidelines outlined below For SureSelect dual index sequence information see tables starting on page 123 Before aligning reads to the reference genome SureSelect T adaptor sequences must be trimmed from the reads You can use SureCall Agilent s NGS data analysis software to perform adaptor trimming alignment of reads and variant calling of sequencing data generated from either the HiSeq or the MiSeq platform To download SureCall free of charge and for additional information including tutorials on this software visit the SureCall page at www genomics agilent com SureCall is compatible with FASTQ files generated by both the HiSeq and MiSeq platforms To use SureCall to
71. onent kit contents and index sequences SureSelect T Target Enrichment for Illumina Sequencing NGS Bravo Option A What s New in Version BO Updates to custom sequencing primer mixtures for the NextSeq 500 v2 platform seeTable 61 and Table 62 on page 114 Update to P5 Index details for NextSeq 500 platform runs using BaseSpace see Table 70 on page 124 Update to Qubit dsDNA Assay Kit nomenclature Table 1 on page 12 and step 4 on page 39 Updates to SureCycler 8800 PCR plate compatibility considerations see Caution on page 34 Support for Agilent 4200 TapeStation see Table 4 on page 16 Correction to ordering information for Axygen 96 Deep Well plates see Table 4 on page 15 Revised ordering information for nucleic acid surface decontamination wipes Table 4 on page 15 What s New in Version A1 Support for ClearSeq Capture Libraries including ClearSeq Comprehensive Cancer Libraries see Table 3 on page 14 Support for Human All Exon v6 Capture Libraries see Table 2 on page 13 Update to SBS Kit Configuration details for HiSeq 2500 Rapid Run sequencing see Table 57 on page 111 Support for sequencing using NextSeq 500 v2 see Table 61 on page 111 Update to Qubit dsDNA Assay Kit ordering information see Table 1 on page 12 6 SureSelect Target Enrichment for Illumina Sequencing NGS Bravo Option A Content SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A Before Yo
72. ons 2 Maintain clean work areas Clean pre PCR surfaces that pose the highest risk of contamination daily using a 10 bleach solution 3 Wear powder free gloves Use good laboratory hygiene including changing gloves after contact with any potentially contaminated surfaces Possible stopping points where samples may be stored at 20 C are marked in the protocol Do not subject the samples to multiple freeze thaw cycles SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A Before You Begin 1 To prevent contamination of reagents by nucleases always wear powder free laboratory gloves and use dedicated solutions and pipettors with nuclease free aerosol resistant tips e In general follow Biosafety Level 1 BL1 safety rules Safety Notes CAUTION e Wear appropriate personal protective equipment PPE when working in the laboratory SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A 11 Before You Begin Required Reagents Table 1 Required Reagents for SureSelect 2X Target Enrichment Description Vendor and part number SureSelect or ClearSeq Capture Library SureSelect Reagent Kit Illumina HiSeq or MiSeq platform ILM 96 Samples Illumina NextSeq platform NSQ 96 Samples Agencourt AMPure XP Kit 5 ml 60 ml 450 ml Dynabeads MyOne Streptavidin T1 2ml 10 ml 100 ml 1X Low TE Buffer 10 mM Tris HCI pH 8 0 0 1 mM EDTA 100 Ethanol mol
73. ontamination wipe 2 Pre set the temperature of Bravo deck positions 4 and 6 as indicated in Table 14 See page 19 for more information on how to do this step Table 14 Bravo Deck Temperature Presets Bravo Deck Position Temperature Preset Preset Method 4 45 C Inheco Multi TEC control touchscreen CPAC 2 1 6 20 C Inheco Multi TEC control touchscreen CPAC 2 2 3 Mix the AMPure XP bead suspension well so that the reagent appears homogeneous and consistent in color 4 Prepare a Nunc DeepWell source plate for the beads by adding 52 ul of homogeneous AMPure XP beads per well for each well to be processed 5 Prepare a Thermo Scientific Matrix reservoir containing 15 ml of nuclease free water 6 Prepare a separate Thermo Scientific Matrix reservoir containing 45 ml of freshly prepared 70 ethanol Setup and Run VWorks protocol 02 Cleanup_Tn_v1 0 pro 7 On the SureSelect setup form under Select Protocol select 02 Cleanup_Tn_v1 0 pro 8 Select the number of columns of samples to be processed Runs must include 1 2 3 4 6 or 12 columns SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A 45 3 Sample Preparation 9 Click Display Initial Bravo Deck Setup Display Initial Bravo Deck Setup 10 Load the Bravo deck according to the Bravo Deck Setup region of the form and as shown in Table 15 Table 15 Initial Bravo deck configuration for 02 Cleanup_Tn_v1 0 pro Location Content 1 Emp
74. or prolonged storage SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A 107 5 108 Indexing Step 3 Assess indexed DNA quality Figure 12 SureSelect Target Enrichment for Illumina Sequencing NGS Bravo Option A 700 600 500 400 Sample Intensity FU 200 100 Size bp 25 50 100 1 000 200 300 1 500 o 5S 7 500 700 Analysis of indexed DNA using the 2200 TapeStation Indexing Step 4 Quantify each index tagged library by QPCR optional Refer to the protocol that is included with the Agilent QPCR NGS Library Quantification Kit p n G4880A for more details to do this step 1 Use the Agilent QPCR NGS Library Quantification Kit for Illumina to determine the concentration of each index tagged captured library Prepare a standard curve using the quantification standard included in the kit according to the instructions provided in the user guide Dilute each index tagged captured library such that it falls within the range of the standard curve Typically this corresponds to approximately a 1 1000 to 1 10 000 dilution of the captured DNA Prepare the QPCR master mix with Illumina adaptor specific PCR primers according to instructions provided in the kit Add an aliquot of the master mix to PCR tubes and add template On a QPCR system such as the Mx3005p run the thermal profile outlined in the QPCR NGS Libra
75. ormation section of the form Set the temperature of Bravo Deck positions as needed Bravo Deck Setup SureSelect 7 Lm l Transposase Library Pre a P ry P m lt Position 1 gt lt Position 2 gt lt Position 3 gt i New tip box Stop Solution in 96 for Illumina sequencers Sekera Parameters m lt Pos 4 Peltier gt j lt Pos5 Shaker gt j lt Pos 6 Peltier gt 1 Select Protocol RED INSERT Nunc DW Master Mix Plate Col 1 o1 Tn_QxT_ILM_v1 0 pro z 52 C 4 C 2 Select PCR Plate Labware Protocols 3 4 6 7 9 and 10 m m lt Pos 7 Magnet gt lt Position 8 gt lt Pos 9 Chiller gt o6 ABI PCR half skirt in Red Alum Insert 7 50ng of gDNA in 96 Empty tip box Empty 96 Eppendorf Eppendorf Twin tec Twin tec on RED INSERT 3 Select Number of Columns of Samples to Process 4 TC 4 Display Initial Bravo Deck Setup Reference 5 Load labware according to Bravo Deck Setup i Final DNA lamare Neal Protocol Temperature 6 Update current tip state omit for Protocol 05 Location Duration Presets Current Tip State Information Select columns of unused tips Box 2 VVMMM VM Seca i Need ee Bix 8 Position 4 can be pre heated to 52 C Position 6 can be pre chilled to 4 C and miuisiuiui nininini niuia Position 9 can be pre chilled to 0 C Reset Clear Controls gt Run Selected f Pause Full Screen Status Protocol C VWorks Workspace NGS Option A QXT_ILM_v1 0 Protocol Files 01 Tn_QXT_ILM_v1 0 pro
76. ositions 2 and 8 respectively See page 23 for more information on using this segment of the form during the run SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A 99 5 Indexing 18 When setup and verification is complete click Run Selected Protocol Run Selected Protocol 19 Running the 09 Post CapturePCR_QXT_ILM_v1 0 pro protocol takes approximately 15 minutes Once complete the PCR ready samples containing captured DNA PCR master mix and indexing primers are located in the PCR plate at position 6 of the Bravo deck 20 When you see the following prompt remove the PCR plate from position 6 of the Bravo deck and seal the plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 3 0 seconds r Remove Plates The PCR plate at Bravo Position 6 is ready to be sealed and placed in the thermocycler Follow the amplification recommendations in the user quide based on your capture size 21 Centrifuge the plate for 30 seconds to drive the well contents off the walls and plate seal and to eliminate air bubbles 100 SureSelect Target Enrichment for Illumina Sequencing NGS Bravo Option A Indexing 5 22 Transfer the PCR plate to a thermal cycler and run the PCR amplification program shown in Table 53 Table 53 Post Capture PCR cycling program Segment Number of Cycles Temperature Time 1 1 98 C 2 minutes 2 Capture Libraries gt 3 Mb 10 Cycles 98 C 3
77. pendorf plate 4 Empty red insert 5 empty 6 Library Prep Master Mix source plate unsealed 7 gDNA samples 5 pl of 10 ng pl DNA per well in Eppendorf plate unsealed 8 Empty tip box 9 Empty Eppendorf plate on red insert SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A 43 3 Sample Preparation 14 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 23 for more information on using this segment of the form during the run 15 When setup and verification is complete click Run Selected Protocol Run Selected Protocol Running the 01 Tn_QXT_ILM_v1 0 pro protocol takes approximately 20 minutes Once complete the adaptor tagged DNA samples are located in the Eppendorf plate at position 6 of the Bravo deck 44 SureSelect T Target Enrichment for Illumina Sequencing NGS Bravo Option A Sample Preparation 3 Step 3 Purify adaptor tagged DNA using AMPure XP beads This step uses automation protocol 02 Cleanup_Tn_v1 0 pro In this step the NGS Bravo combines the adaptor tagged samples with AMPure XP beads and then collects and washes the bead bound DNA The purified eluted DNA is transferred to an Eppendorf twin tec plate for further processing Prepare the NGS Bravo and reagents 1 Clear the Bravo deck of all plates and tip boxes then wipe it down with a DNA Away dec
78. plate for the post capture PCR source plate leave columns 1 to 3 empty and add the PCR Master Mix to column 4 of the new plate SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A 95 5 Indexing Assign and aliquot indexing primers 6 Determine the appropriate index assignments for each sample See the Reference section for sequences of the index portion of the P7 and P5 indexing primers used to amplify the DNA libraries in this step See Table 69 and Table 70 for sequencing on HiSeq and MiSeq platforms or see Table 69 and Table 71 for sequencing on the NextSeq platform Use the following guidelines for dual index assignments e Use a different indexing primer combination for each sample to be sequenced in the same lane e All samples on the same row of the target enriched DNA library plate must be assigned to the same P5 indexing primer P5 i13 through P5 i20 This design results from the automation protocol configuration in which the P5 indexing primer is dispensed from a single source plate column to all columns of the indexing PCR plate Each row of samples may be assigned to the same or different P5 primers depending on run size and multiplexing requirements See step 9 below for details of P5 primer addition to the master mix source plate e The automation protocol configuration allows for any of the provided P7 indexing primers P7 il through P7 i12 to be assigned to any sample position of the targ
79. plified library DNA using the 2200 TapeStation with a D1000 ScreenTape SureSelect Target Enrichment for Illumina Sequencing NGS Bravo Option A 61 3 Sample Preparation Step 6 Assess Library DNA quantity and quality 62 SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A SureSelect Automated Target Enrichment for Illumina Multiplexed Sequencing Protocol ee 4 CAUTION 070 Hybridization 2 Step 1 Aliquot prepped DNA samples for hybridization 64 Step 2 Hybridize the gDNA library and SureSelect or ClearSeq Capture Library 69 Step 3 Capture the hybridized DNA 83 This chapter describes the steps to combine the prepped library with the blocking agents and the SureSelect or ClearSeq Capture Library Each DNA library sample must be hybridized and captured individually prior to addition of the indexing tag by PCR The ratio of Capture Library to prepped library is critical for successful capture Agg Agilent Technologies 63 4 64 Step 1 Aliquot prepped DNA samples for hybridization For each sample library prepared do one hybridization and capture Do not pool samples at this stage The amount of prepared gDNA library used in the hybridization reaction varies according to the size of the Capture Library used for hybridization as outlined in Table 24 below Use the maximum possible amount of each prepped DNA within the range listed in Table 24 Table 24 Amount of adapto
80. ption A Using the Agilent NGS Bravo Option A for SureSelect Target Enrichment 2 Overview of the SureSelect Target Enrichment Procedure Figure 2 summarizes the SureSelect XT target enrichment workflow for samples to be sequenced using the Illumina paired read sequencing platform For each sample to be sequenced individual library preparations hybridizations and captures are performed The samples are then tagged by PCR with an index sequence Depending on the target size of the SureSelect capture up to 96 samples can be pooled and sequenced in a single lane using the dual index tags that are provided with SureSelect T Library Prep kits Table 6 summarizes how the VWorks protocols are integrated into the SureSelect workflow See Sample Preparation Hybridization and Indexing chapters for complete instructions for use of the VWorks protocols for sample processing SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A 29 2 Using the Agilent NGS Bravo Option A for SureSelect Target Enrichment Overview of the SureSelect Target Enrichment Procedure SureSelect X NGS Target Enrichment Workflow gDNA samples 1 2 n Fragment DNA and adaptor tag DNA ends Genomic locations of interest in single enzymatic step Design target sequences Adaptor tagged DNA library in SureDesign PCR amplify Prepared DNA library amplicons Hybridize using SureSelect or ClearSeq Capture Library Capture hybri
81. r SureSelect T Target Enrichment Description Agilent NGS Bravo Option A with VWorks software version 11 3 0 1195 Robotic Pipetting Tips Sterile Filtered 250 uL PlateLoc Thermal Microplate Sealer with Small Hotplate Clear Peelable Seal plate seals for use with the PlateLoc Thermal Plate Sealer Thermal cycler and accessories PCR plates compatible with selected Thermal Cycler e g Agilent semi skirted PCR plate for the SureCycler 8800 Thermal Cycler When selecting plates for another thermal cycler see Table 8 on page 35 for the list of PCR plates supported in automation protocols Eppendorf twin tec full skirted 96 well PCR plates Thermo Scientific Reservoirs Nunc DeepWell Plates sterile 1 3 ml well volume Axygen 96 Deep Well Plate 2 mL Square Well waste reservoirs working volume 2 2 mL DNA LoBind Tubes 1 5 ml PCR clean 250 pieces Nucleic acid surface decontamination wipes Qubit Fluorometer Qubit Assay Tubes Magnetic separator Vendor and part number Contact Agilent Automation Solutions for ordering information Customerservice automation agilent com Agilent p n 19477 022 Agilent p n G5402 226 Agilent p n 16985 001 SureCycler 8800 Thermal Cycler Agilent p n G8810A 96 well plate module Agilent p n G8810A and compression mats Agilent p n 410187 or equivalent Agilent p n 401334 Eppendorf p n 951020401 or 951020619 Thermo Scientific p n 1064156 Thermo Scientific p n 2602
82. r tagged DNA libraries used for hybridization Capture Library Size Amount of prepared gDNA library used in hybridization Libraries gt 3 0 Mb except ClearSeq 750 to 1500 ng DNA DNA Kinome see below Libraries lt 3 0 Mb and ClearSeqDNA 500 to 750 ng DNA Kinome 3 2 Mb Using the DNA concentration for each sample determined on page 58 to page 60 calculate the volume of each sample to be used for hybridization using the appropriate formula below Volume ul 750 ng concentration ng ul OR Volume ul 1500 ng concentration ng ul If the concentration of any sample is not sufficient to allow use of the recommended amount of DNA 750 ng for ClearSeq DNA Kinome and libraries lt 3 0 Mb or 1500 ng for libraries gt 3 0 Mb then use the full remaining volume of DNA sample approximately 12 ul for the hybridization step The automation protocol 05 Aliquot_Libraries_v1 0 pro is used to prepare a new sample plate containing the appropriate amount of each DNA sample for hybridization Before running the automation protocol you must create a table containing instructions for the NGS Bravo indicating the volume of each sample to aliquot as described in the steps below SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A Hybridization 4 1 Create a csv comma separated value file with the headers shown in Figure 7 The header text must not contain spaces The table may be created using a spreadshee
83. rks as shown below Retain the hybrid capture bead suspension plate at position 5 and proceed immediately to automation protocol 08 SureSelectQXT_Wash_v1 0 pro Proceed to Cleanup The captured DNA RNA hybrids are ready at Position 5 Proceed immediately to 08 SureSelectQXT_Wash_v1 0 pro 88 SureSelect Target Enrichment for Illumina Sequencing NGS Bravo Option A Hybridization 4 Step 4 Wash the captured DNA This step uses automation protocol 08 SureSelectQXT_Wash_v1 0 pro to automate washing of the captured DNA RNA hybrids This step uses the SureSelect X Reagent Kit component in Table 38 in addition to components retained from the previous automation protocol Table 43 Reagents for capture wash protocol Kit Component Storage Location Where Used SureSelect Wash Buffer 1 SureSelect QXT Hyb Module Box1 RT page 89 Prepare the NGS Bravo 1 Retain all plates inserts and tip boxes used in the previous automation protocol on the Bravo deck including the hybrid capture bead suspension plate at position 5 and the pre warmed Wash Buffer 2 source plate at position 6 2 Pre set the temperature of Bravo deck positions 4 and 6 as indicated in Table 29 See page 19 for more information on how to do this step Table 44 Bravo Deck Temperature Presets Bravo Deck Position Temperature Preset Preset Method 4 69 C Inheco Multi TEC control touchscreen CPAC 2 1 6 74 C Inheco Multi TEC control touchscreen CP
84. ry Quantification kit user guide Use the SYBR Green instrument setting Use the standard curve to determine the concentration of each unknown index tagged library in nM The concentration will be used to accurately pool samples for multiplexed sequencing SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A 5 109 5 110 Step 5 Pool samples for Multiplexed Sequencing 1 Combine the libraries such that each index tagged sample is present in equimolar amounts in the pool For each library use the formula below to determine the amount of indexed sample to use Volume of Index Vx CP x CC where V f is the final desired volume of the pool C f is the desired final concentration of all the DNA in the pool is the number of indexes and C i is the initial concentration of each indexed sample Table 56 shows an example of the amount of 4 index tagged samples of different concentrations and Low TE needed for a final volume of 20 ul at 10 nM Table 56 Example of index volume calculation for a total volume of 20 pl Component V f C i C f Volume to use pl Sample 1 20 ul 20 nM 10 nM 4 2 5 Sample 2 20 ul 10 nM 10 nM 4 5 Sample 3 20 ul 17 nM 10 nM 4 2 9 Sample 4 20 ul 25 nM 10 nM 4 2 Low TE 76 2 Adjust the final volume of the pooled library to the desired final concentration If the final volume of the combined index tagged samples is less than the desired final volume
85. s and Library Prep reagents 38 Step 2 Fragment and adaptor tag the genomic DNA samples 40 Step 3 Purify adaptor tagged DNA using AMPure XP beads 45 Step 4 Amplify adaptor ligated libraries 48 Step 5 Purify amplified DNA using AMPure XP beads 55 Step 6 Assess Library DNA quantity and quality 58 This section contains instructions for gDNA library preparation specific to the Illumina paired read sequencing platform and to automated processing using the Agilent NGS Bravo Option A oie Agilent Technologies 37 3 Sample Preparation Step 1 Prepare the genomic DNA samples and Library Prep reagents It is important to have all materials prepared in advance of use in the SureSelect2 automated Library Prep protocol In this step the gDNA is carefully quantified and dispensed into the sample plate Additional reagents that require modification or temperature equilibration before use are also prepared in this step 1 Remove the DMSO vial from the SureSelect QXT Library Prep Kit Box 2 in 20 C storage Leave the DMSO vial at room temperature in preparation for use on page 50 2 Prepare reagents for the purification protocols on page 45 and page 55 a Transfer the AMPure XP beads to room temperature The beads should be held at room temperature for at least 30 minutes before use Do not freeze the beads at any time b Prepare 150 ml of fresh 70 ethanol for use in the purification steps The 70 ethanol may be used for multiple steps
86. s is and is subject to being changed with out notice in future editions Fur ther to the maximum extent permitted by applicable law Agi lent disclaims all warranties either express or implied with regard to this manual and any information contained herein including but not limited to the implied warranties of merchant ability and fitness for a particular purpose Agilent shall not be lia ble for errors or for incidental or consequential damages in con nection with the furnishing use or performance of this document or of any information contained herein Should Agilent and the user have a separate written agreement with warranty terms covering the material in this doc ument that conflict with these terms the warranty terms in the separate agreement shall control Technology Licenses The hardware and or software described in this document are furnished under a license and may be used or copied only in accor dance with the terms of such license Restricted Rights Legend U S Government Restricted Rights Soft ware and technical data rights granted to the federal government include only those rights customarily provided to end user cus tomers Agilent provides this customary commercial license in Software and techni cal data pursuant to FAR 12 211 Technical Data and 12 212 Computer Software and for the Department of Defense DFARS 252 227 7015 Technical Data Commercial Items and DFARS 227 7202 3 R
87. s with yellow caps one tube per primer P5 i13 through P5 i20 provided in 8 tubes with blue caps one tube per primer Table 66 SureSelect QXT Library Prep NSO Box 2 Content Kit Component SureSelect QXT Buffer SureSelect QXT Enzyme Mix ILM Herculase II Fusion DNA Polymerase Herculase II 5x Reaction Buffer 100 mM dNTP Mix 25 mM each dNTP DMSO SureSelect QXT Read Primer 1 SureSelect QXT Read Primer 2 SureSelect QXT Index Read Primer SureSelect QXT Index 2 Read Primer NSO SureSelect QXT P7 dual indexing primers SureSelect QXT P5 dual indexing primers NextSeq NSQ 96 Reactions bottle tube with orange cap tube with red cap tube with clear cap tube with green cap tube with green cap tube with amber cap tube with black cap tube with clear cap tube with purple cap P7 i1 through P7 i12 provided in 12 tubes with yellow caps one tube per primer P5 i13 through P5 i20 provided in 8 tubes with blue caps one tube per primer SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A 121 6 Reference Table 67 SureSelect QXT Hyb Module Box 1 Content Kit Component 96 Reactions SureSelect QXT Stop Solution bottle SureSelect Binding Buffer bottle SureSelect Wash Buffer 1 bottle SureSelect Wash Buffer 2 bottle Table 68 SureSelect QXT Hyb Module Box 2 Content Kit Component 96 Reactions SureSelect QXT Fast Hybridization Buffer bottle SureSelect QXT Fast Blocker Mix tube with
88. t Capture Table 33 Preparation of Capture Library Master Mix for Capture Libraries lt 3 Mb single row of wells Target size lt 3 0 Mb Reagent Volume for Volume for Volumefor Volumefor Volumefor Volume for 1 Library 2 Columns 3Columns 4Columns 6 Columns 12 Columns Nuclease free 4 5 ul 13 8 pl 18 6 pl 23 3 pl 37 7 ul 73 5 ul water RNase Block 0 5 pl 1 5 pl 2 1 pl 2 6 pl 4 2 ul 8 2 pl purple cap Capture Library 2 0 pl 6 1 pl 8 3 pl 10 4 pl 16 8 pl 32 7 ul Total Volume 7 0 pl 21 4 pl 28 9 pl 36 3 pl 58 6 pl 114 4 pl Table 34 Preparation of Capture Library Master Mix for Capture Libraries gt 3 Mb single row of wells Target size gt 3 0 Mb Reagent Volume for Volume for Volume for Volumefor Volumefor Volume for 1 Library 2Columns 3Columns 4Columns 6 Columns 12 Columns Nuclease free 1 5 pl 4 6 ul 6 2 pl 7 8 pl 12 6 pl 24 5 ul water RNase Block 0 5 pl 1 5 pl 2 1 pl 2 6 pl 4 2 ul 8 2 pl purple cap Capture Library 5 0 pl 15 3 pl 20 6 pl 25 9 ul 41 9 ul 81 7 ul Total Volume 7 0 pl 21 4 pl 28 9 pl 36 3 pl 58 6 pl 114 4 pl Includes ClearSeq DNA Kinome XT Library 3 2 Mb 74 SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A Hybridization 4 Prepare the Hybridization Buffer master mix 7 Prepare the appropriate volume of Hybridization Buffer Master Mix at room temperature as indicated in Table 35 Table 35 Preparation of Hybridization Buffer Master Mix SureSelect
89. t application such as Microsoft Excel software and then saved in csv format The file must include rows for all 96 wells of the plate 2 Enter the information requested in the header for each DNA sample e In the SourceBC field enter the sample plate description or barcode The SourceBC field contents must be identical for all rows e In the SourceWell and DestinationWell fields enter each well position for the plate SourceWell and DestinationWell field contents must be identical for a given sample In the Volume field enter the volume in ul of each DNA sample to be used in the hybridization step see page 64 for guidelines For all empty wells on the plate enter the value 0 as shown in Figure 7 do not delete rows for empty wells A B C D 1 SourceBC SourceWell DestinationWell Volume 2 SamplePlatexYZ Al Al 5 35 3 _ SamplePlatexYZ 81 B1 4 28 4 SamplePlatexYZ C1 C1 476 5 SamplePlatexYZ 01 D1 5 19 6 SamplePlatexYZ E1 E1 5 49 7 _ SamplePlatexYZ F1 F1 4 86 8 SamplePlatexYZ G1 Gi 5 05 9 SamplePlatexYZ H1 H1 4 37 10 SamplePlatexYZ A2 A2 0 11 SamplePlatexYZ 82 B2 0 12 SamplePlatexYZ C2 c2 0 SB Kamiah Pln Ann Aaa an am EL e lt an MAA Figure 7 Sample spreadsheet for 1 column run You can find a sample spreadsheet in the directory C gt VWorks Workspace gt NGS Option A gt QXT_ILM_v1 0 gt Aliquot Library Input Files gt Aliquot_Libraries_full_plate_template csv The Aliquot_Libraries_ful
90. tQXT_Capture_v1 0 pro to automate capture of the gDNA Capture Library hybrids using streptavidin coated magnetic beads Setup tasks for the Capture protocol step 1 below through step 16 on page 86 should be completed during the thermal cycler incubation for hybridization approximately 1 5 hour duration started on page 82 This step uses the SureSelect2X Reagent Kit components in Table 38 in addition to streptavidin coated magnetic beads obtained from another supplier see Table 1 on page 12 Table 38 Reagents for hybrid capture Kit Component Storage Location Where Used SureSelect Binding Buffer SureSelect QXT Hyb Module Box1 RT page 84 SureSelect Wash Buffer 2 SureSelect QXT Hyb Module Box1 RT page 85 Prepare the NGS Bravo 1 Clear the Bravo deck of all plates and tip boxes then wipe it down with a DNA Away decontamination wipe 2 Pre set the temperature of Bravo deck position 4 as indicated in Table 39 See page 19 for more information on how to do this step Table 39 Bravo Deck Temperature Presets Bravo Deck Position Temperature Preset Preset Method 4 66 C Inheco Multi TEC control touchscreen CPAC 2 1 3 Place a red PCR plate insert at Bravo deck position 4 4 Place the silver Nunc DeepWell plate insert at Bravo deck position 6 This insert is required to facilitate heat transfer to DeepWell source plate wells SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A 83 4 Hybridizat
91. the Bravo deck of all plates and tip boxes then wipe it down with a DNA Away decontamination wipe 2 Pre set the temperature of Bravo deck positions 4 and 6 as indicated in Table 29 See page 19 for more information on how to do this step Table 29 Bravo Deck Temperature Presets Bravo Deck Position Temperature Preset Preset Method 4 23 C Inheco Multi TEC control touchscreen CPAC 2 1 6 23 C Inheco Multi TEC control touchscreen CPAC 2 2 3 Place a red PCR plate insert at Bravo deck position 4 4 Place the silver Nunc DeepWell plate insert on position 6 of the Bravo deck This insert is required to facilitate heat transfer to DeepWell source plate wells during the Hybridization protocol When loading a source plate on the silver insert make sure the plate is seated properly to ensure proper heat transfer SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A 71 4 Hybridization Prepare the Block Master Mix 5 Prepare the appropriate volume of Block Master Mix on ice as indicated in Table 30 Table 30 Preparation of Block Master Mix SureSelect T Volume for Volume for Volume for Volume for Volumefor Volumefor Volume for Reagent 1 Library 1 Column 2 Columns 3 Columns 4Columns 6 Columns 12 Columns Nuclease free 2 5 pl 31 9 ul 53 1 pl 74 4 ul 95 6 pl 138 1 pl 276 3 pl water SureSelect QXT 5 0 pl 63 8 ul 106 3 pl 148 8 pl 191 3 pl 276 3 pl 552 5 pl Fast Blocker Mix blue cap Total Vol
92. tiating the workflow For best practice store diluted DNA solutions at 4 C to avoid repeated freeze thaw cycles which may compromise DNA quality Performance of the SureSelect library preparation protocol is very sensitive to variations in amounts of DNA sample and other reaction components It is important to quantify and dilute DNA samples as described on page 39 Carefully measure volumes for all reaction components and combine components as described in this instruction manual Use best practices for liquid handling including regular pipette calibration to ensure precise volume measurement Use care in handling the SureSelect QXT Enzyme Mix After removing the vial from storage at 20 C keep on ice or in a cold block while in use Return the vial to storage at 20 C promptly after use Certain protocol steps require the rapid transfer of sample plates between the Bravo deck and a thermal cycler Locate your thermal cycler in close proximity to the Agilent NGS Bravo to allow rapid and efficient plate transfer Use best practices to prevent PCR product contamination of samples throughout the workflow 1 Assign separate pre PCR and post PCR pipettors supplies and reagents In particular never use materials designated to post PCR segments for the pre PCR segments of the workflow For the pre PCR workflow steps always use dedicated pre PCR pipettors with nuclease free aerosol resistant tips to pipette dedicated pre PCR soluti
93. tinue to the next step seal the plate and store at 4 C overnight or at 20 C for prolonged storage SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A Iu Figure 5 SureSelect T Target Enrichment for Illumina Sequencing NGS Bravo Option A Sample Preparation Step 6 Assess Library DNA quantity and quality M Z Mi I Ste SS 1s so 100 150 200 300 400 500 700 850 1500 bp 15 3 10 15 XO 300 w w 700 1500 foe Representative sample electropherograms showing pre capture analysis of amplified library DNA using the Agilent 2100 Bioanalyzer and a DNA 1000 As say 59 3 3 Sample Preparation CAUTION Option 2 Analysis using an Agilent TapeStation and D1000 ScreenTape Use a D1000 ScreenTape p n 5067 5582 and associated reagent kit p n 5067 5583 to analyze the amplified libraries using the Agilent 4200 TapeStation or 2200 TapeStation For more information to do this step see the appropriate TapeStation user manual at www genomics agilent com 1 Prepare the TapeStation samples as instructed in the instrument user manual Use 1 ul of each amplified library DNA sample diluted with 3 ul of D1000 sample buffer for the analysis Make sure that you thoroughly mix the combined DNA and sample buffer on a vortex mixer for 5 seconds for accurate quantitation Stopping Point 2 Load the sample plate or tube strips from step 1 the D1000 ScreenTape and loading tips into the Tape
94. tion 2 Analysis using an Agilent TapeStation and High Sensitivity D1000 ScreenTape Use a High Sensitivity D1000 ScreenTape p n 5067 5584 and reagent kit p n 5067 5585 to analyze the indexed DNA using the Agilent 4200 TapeStation or 2200 TapeStation For more information to do this step see the appropriate TapeStation user manual at www genomics agilent com 1 Seal the DNA sample plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 1 0 sec 2 Vortex the plate to mix samples in each well then centrifuge the plate for 30 seconds to drive the well contents off the walls and plate seal 3 Prepare the TapeStation samples as instructed in the instrument user manual Use 2 ul of each indexed DNA sample diluted with 2 ul of High Sensitivity D1000 sample buffer for the analysis CAUTION Make sure that you thoroughly mix the combined DNA and High Sensitivity D1000 sample buffer on a vortex mixer for 5 seconds for accurate quantitation 4 Load the sample plate or tube strips from step 3 the High Sensitivity D1000 ScreenTape and loading tips into the TapeStation as instructed in the instrument user manual Start the run 5 Verify that the electropherogram shows the peak of DNA fragment size positioned between 325 and 450 bp A sample electropherogram is shown in Figure 12 Stopping Point If you do not continue to the next step seal the indexed DNA sample plate and store at 4 C overnight or at 20 C f
95. tion Chemistry HiSeq 2500 Rapid Run 2 x 100 bp 200 Cycle Kit vl or v2 HiSeq 2500 High Output 2 x 100 bp 4x 50 Cycle Kit v3 HiSeq 2500 High Output 2 x 100 bp 250 Cycle Kit v4 HiSeq 2000 All Runs 2 x 100 bp 4 x 50 Cycle Kit v3 MiSeq All Runs 2 x 100 bp 300 Cycle Kit v2 MiSeq All Runs 2 x 76 bp 150 Cycle Kit v3 NextSeq 500 All Runs 2 x 100 bp 300 Cycle Kit v2 If your application requires a different read length verify that you have sufficient sequencing re agents to complete Reads 1 and 2 in addition to the dual 8 bp index reads t A single 200 cycle kit does not include enough reagents to complete Reads 1 and 2 in addition to the dual 8 bp index reads in this format If preferred the additional reads may be supported by using one 200 cycle kit plus one 50 cycle kit SureSelect T Target Enrichment for Illumina Sequencing NGS Bravo Option A 111 5 112 Indexing Using the SureSelect Read Primers with Illumina s Paired End Cluster Generation Kits To sequence the SureSelect libraries on Illumina s sequencing platforms you need to use the following custom sequencing primers provided in SureSelect QXT Library Prep Kit Box 2 e SureSelect QXT Read Primer 1 e SureSelect QXT Read Primer 2 e SureSelect QXT Index Read Primer e SureSelect QXT Index 2 Read Primer NSQ NextSeq platform only These SureSelect custom sequencing primers are provided at 100 uM and must be diluted 1 200 in the corresponding Illumina
96. ts SureSelect X Reagent Kits contain the following component kits Table 64 SureSelect T Reagent Kit Contents Component Kits Storage Condition HiSeq or NextSeq MiSeq ILM NSQ 96 Samples 96 Samples SureSelect QXT Library Prep Kit ILM Box 2 20 C 5500 0121 5500 0127 SureSelect QXT Target Enrichment Kit ILM Room Temperature 5190 7335 5190 7335 Hyb Module Box 1 SureSelect QXT Target Enrichment Kit ILM 20 C 5190 7334 5190 7334 Hyb Module Box 2 SureSelect QXT Library Prep Kit ILM Box 1 is not required for the workflow described in this man ual The contents of each of the component kits listed in Table 64 are described in Table 66 to Table 68 below 120 SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A Reference 6 Table 65 SureSelect QXT Library Prep ILM Box 2 Content Kit Component HiSeq or MiSeq ILM 96 Reactions SureSelect QXT Buffer SureSelect QXT Enzyme Mix ILM Herculase II Fusion DNA Polymerase Herculase II 5x Reaction Buffer 100 mM dNTP Mix 25 mM each dNTP DMSO SureSelect QXT Read Primer 1 SureSelect QXT Read Primer 2 SureSelect QXT Index Read Primer SureSelect QXT P7 dual indexing primers SureSelect QXT P5 dual indexing primers bottle tube with orange cap tube with red cap tube with clear cap tube with green cap tube with green cap tube with amber cap tube with black cap tube with clear cap P7 i1 through P7 i12 provided in 12 tube
97. ty waste reservoir Axygen 96 Deep Well Plate square wells 2 New tip box 3 empty 4 empty 5 AMPure XP beads in Nunc DeepWell plate 52 ul beads per processing well 6 Adaptor tagged DNA samples in Eppendorf twin tec plate 7 empty 8 Empty tip box 9 70 ethanol in Matrix reservoir 11 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 23 for more information on using this segment of the form during the run 12 When setup and verification is complete click Run Selected Protocol Run Selected Protocol 46 SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A Sample Preparation 3 Running the 02 Cleanup_Tn_v1 0 pro protocol takes approximately 45 minutes An operator must be present during the run to complete tip box replacement and to transfer the water reservoir to the Bravo deck when directed by the VWorks prompt shown below Replace reservoir Remove the Ethanol Reservoir from Position 9 Place the Water reservoir at Position 9 When finished dick Continue below Once the protocol is complete the purified DNA samples are located in the Nunc DeepWell plate at position 7 of the Bravo deck SureSelect Target Enrichment for Illumina Sequencing NGS Bravo Option A 47 3 Sample Preparation Step 4 Amplify adaptor ligated libraries
98. u Begin 9 Procedural Notes 10 Safety Notes 11 Required Reagents 12 Optional Reagents 14 Required Equipment 15 Using the Agilent NGS Bravo Option A for SureSelect Target Enrichment 17 About the Agilent NGS Bravo OptionA 18 About the Bravo Platform 18 VWorks Automation Control Software 22 Overview of the SureSelect T Target Enrichment Procedure 29 Experimental Setup Considerations for Automated Runs 32 Considerations for Placement of gDNA Samples in 96 well Plates for Automated Processing 33 Considerations for Equipment Setup 33 PCR Plate Type Considerations 34 Sample Preparation 37 Step 1 Prepare the genomic DNA samples and Library Prep reagents 38 Step 2 Fragment and adaptor tag the genomic DNA samples 40 Step 3 Purify adaptor tagged DNA using AMPure XP beads 45 Step 4 Amplify adaptor ligated libraries 48 Step 5 Purify amplified DNA using AMPure XP beads 55 Step 6 Assess Library DNA quantity and quality 58 Contents 4 Hybridization 63 Step 1 Aliquot prepped DNA samples for hybridization 64 Step 2 Hybridize the gDNA library and SureSelect or ClearSeq Capture Library 69 Step 3 Capture the hybridized DNA 83 Step 4 Wash the captured DNA 89 Indexing 93 Step 1 Amplify the captured DNA libraries to add index tags 94 Step 2 Purify the amplified indexed libraries using Agencourt AMPure XP beads 102 Step 3 Assess indexed DNA quality 105 Step 4 Quantify each index tagged library by QPCR optional 109 Step
99. ume 7 5 pl 95 6 pl 159 4 pl 223 1 pl 286 9 pl 414 4 ul 828 8 pl Prepare one or more Capture Library Master Mixes 6 Prepare the appropriate volume of Capture Library Master Mix for each library that will be used for hybridization as indicated in Table 31 to Table 34 Mix thoroughly by vortexing at high speed then spin down briefly Keep the Capture Library Master Mix es on ice Each row of the prepped gDNA sample plate may be hybridized to a different Capture Library However libraries of different sizes require different post capture amplification cycles Plan experiments such that similar sized libraries are hybridized on the same plate For runs that use a single Capture Library for all rows of the plate prepare the master mix as described in Step a Table 31 or Table 32 on page 73 For runs that use different Capture Libraries for individual rows prepare each master mix as described in Step b Table 33 or Table 34 on page 74 72 SureSelect X Target Enrichment for Illumina Sequencing NGS Bravo Option A Hybridization 4 a For runs that use a single Capture Library for all rows prepare a Master Mix as described in Table 31 or Table 32 according to the size of the Capture Library Table 31 Preparation of Capture Library Master Mix for Capture Libraries lt 3 Mb 8 rows of well Target size lt 3 0 Mb Reagent Volume for Volumefor Volumefor Volumefor Volumefor Volumefor Volume for 1 Library 1 Column 2 Columns 3Columns 4Columns 6 Columns 1
100. ure Library 80 C Thaw on ice page 73 or page 74 SureSelect T Target Enrichment for Illumina Sequencing NGS Bravo Option A 69 4 Hybridization Program the thermal cycler 1 Pre program the thermal cycler for the Hybridization workflow by entering the thermal cycling program shown in Table 28 below It is critical to pre program the thermal cycler before starting the automation protocol for Hybridization in order to maintain the required sample and reagent temperatures during the workflow Table 28 Thermal cycler program for Hybridization Segment Purpose Number of Cycles Temperature Time Number 1 Denaturation 1 95 C 5 minutes 2 Blocking 1 65 C 10 minutes 3 Hold for NGS Bravo 1 65 C Hold steps 4 Hybridization 60 65 C 1 minute 37 C 3 seconds 5 Hold until start of 1 65 C Hold Capture When setting up the thermal cycling program use a reaction volume setting of 35 ul final volume of hybridization reactions during cycling in Segment 4 Samples are transferred to the NGS Bravo during this Hold step when prompted by the VWorks software Samples are held at 65 C until they are processed in the Capture amp Wash automation protocol that begins on page 83 CAUTION The lid of the thermal cycler is hot and can cause burns Use caution when working near the lid 70 SureSelect T Target Enrichment for Illumina Sequencing NGS Bravo Option A Hybridization 4 Prepare the NGS Bravo 1 Clear
101. well for each well to be processed Prepare a Thermo Scientific Matrix reservoir containing 20 ml of nuclease free water Prepare a separate Thermo Scientific Matrix reservoir containing 45 ml of freshly prepared 70 ethanol Centrifuge the amplified DNA sample plate for 30 seconds to drive the well contents off the walls and plate seal SureSelect T Target Enrichment for Illumina Sequencing NGS Bravo Option A Indexing 5 Setup and run VWorks 10 Cleanup_Post CapturePCR_QXT_ILM_v1 0 pro 9 On the SureSelect setup form under Select Protocol select 10 Cleanup_Post CapturePCR_QXT_ILM_v1 0 pro 10 Under Select PCR plate Labware select the specific type of PCR plate used for post capture amplification 11 Select the number of columns of samples to be processed Runs must include 1 2 3 4 6 or 12 columns 12 Click Display Initial Bravo Deck Setup Display Initial Bravo Deck Setup 13 Load the Bravo deck according to the Bravo Deck Setup region of the form and as shown in Table 55 Table 55 Initial Bravo deck setup for 10 Cleanup_Post CapturePCR_OXT_ILM_v1 0 pro Location Content 1 Empty waste reservoir Axygen 96 Deep Well Plate square wells 2 New tip box 3 Empty Eppendorf plate 4 empty 5 AMPure XP beads in Nunc DeepWell plate 60 ul beads per processing well 6 Amplified DNA libraries in unsealed PCR plate seated in red insert PCR plate type must be specified on setup form under step 2 7 empty
102. wn below select Retry Please verify that it is safe to home a aspirate dispense axis If there is fluid in the tips you kS may want to manually home the W axis in diagnostics over a waste position Choose Retry to continue homing the W axis Choose Ignore to leave the W axis unhomed Choose Abort to cancel initialization Ignore and Continue leaving device in current state Abort SureSelect Target Enrichment for Illumina Sequencing NGS Bravo Option A 27 2 Using the Agilent NGS Bravo Option A for SureSelect X 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 Bravo If NGS Bravo 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 S Log out Compile gt Start i Pause a 2 Ifthe indicator displays Simulation is on click the status indicator button to turn off the simulation mode af Diagnostics 3 If you cannot see the toolbar above the VWorks form click the Full Screen button 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 28 SureSelect T Target Enrichment for Illumina Sequencing NGS Bravo O
103. x 2 20 C page 40 SureSelect QXT Enzyme Mix ILM SureSelect QXT Library Prep Kit Box 2 20 C page 40 Prepare the NGS Bravo 1 Clear the Bravo deck of all plates and tip boxes then wipe it down with a DNA Away decontamination wipe 2 Pre set the temperature of Bravo deck positions 4 6 and 9 as indicated in Table 10 See page 19 to page 21 for more information on how to do this step Table 10 Bravo Deck Temperature Presets Bravo Deck Position Temperature Preset Preset Method 4 52 C Inheco Multi TEC control touchscreen CPAC 2 1 6 4 C Inheco Multi TEC control touchscreen CPAC 2 2 9 0 C ThermoCube control panel 3 Place red PCR plate inserts at Bravo deck positions 4 and 9 40 SureSelect T Target Enrichment for Illumina Sequencing NGS Bravo Option A Sample Preparation 3 Prepare the Library Prep Master Mix and Stop Solution source plates 4 Prepare the Stop Solution source plate using an Eppendorf twin tec full skirted PCR plate Add 35 ul of 1X SureSelect QXT Stop Solution per well for each well to be processed Place the source plate on Bravo deck position 3 5 Before use vortex the SureSelect QXT Buffer and SureSelect QXT Enzyme Mix ILM tubes vigorously at high speed These components are in liquid form when removed from 20 C storage and should be returned to 20 C storage promptly after use Minor variations in volumes of the solutions combined in step 6 below may result in CAUTION ae DNA fragment s

SureSelect Automated Target Enrichment for Illumina Multiplexed

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