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SureSelect Target Enrichment System for Illumina Paired

1. If you use strip tubes test for evaporation before you do the first experiment to make sure the tube capping method is appropriate for the thermal cycler Check that no more than 3 to 4 uL is lost to evaporation 14 Incubate the hybridization mixture for 16 or 24 hours at 65 C with a heated lid at 105 C SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing 67 4 68 Hybridization Step 2 Prepare magnetic beads Use these reagents from the SureSelect Target Enrichment Kit Box 1 SureSelect Binding Buffer SureSelect Wash 2 1 Prewarm SureSelect Wash 2 at 65 C in a circulating water bath or heat block for use in Step 3 Select hybrid capture with SureSelect 2 Vigorously resuspend the Dynabeads MyOne Streptavidin T1 on a vortex mixer Magnetic beads settle during storage 3 For each hybridization add 50 uL of Dynabeads MyOne Streptavidin T1 toa 1 5 mL LoBind Tube 4 Wash the beads a b c d e Add 200 uL of SureSelect Binding Buffer Mix the beads on a vortex mixer for 5 seconds Put the tubes into a magnetic device such as the Dynal magnetic separator Life Technologies Remove and discard the supernatant Repeat step a through step d for a total of 3 washes 5 Resuspend the beads in 200 uL of SureSelect Binding Buffer SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing Hybridization 4 Step 3 Select hybrid capture with SureSelect
2. Nuclease free Water 15 5 uL 193 75 uL 5x T4 DNA Ligase Buffer 10 uL 125 uL SureSelect Adaptor Oligo Mix brown cap 10 pL 125 uL T4 DNA Ligase red cap 1 5 uL 18 75 uL Total Volume 37 pL 462 5 pL 37 pL sample 2 Incubate for 15 minutes at 20 C on a thermal cycler Do not use a heated lid Stopping Point If you do not continue to the next step store the samples at 20 C SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing 29 Stopping Point Step 9 Purify the sample using Agencourt AMPure XP beads 1 Let the AMPure XP beads come to room temperature for at least 30 minutes Mix the reagent well so that the reagent appears homogeneous and consistent in color Do not freeze Add 90 uL of homogeneous AMPure XP beads to a 1 5 mL LoBind Tube and add the ligated library 50 uL Mix well on a vortex mixer and incubate for 5 minutes Put the tube in the magnetic stand Wait for the solution to clear approximately 3 to 5 minutes Keep the tube in the magnetic stand Do not touch the beads while you carefully discard the cleared solution from the tubes Continue to keep the tube in the magnetic stand while you dispense 500 uL of 70 Ethanol in each tube Use fresh 70 Ethanol for optimal result Let the tube sit for 1 minute to allow any disturbed beads to settle and remove the Ethanol Repeat step 6 and step 7 step once 9 Spin briefly in a centrifuge Return the tube to the m
3. SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing 11 1 12 Before You Begin Required Equipment Table 6 Required Equipment for Library Prep and Post Hybridization Amplification Description Vendor and part number 2100 Bioanalyzer or 2200 TapeStation System Mx3005P Real Time PCR System Thermal cycler Covaris Sample Preparation System S series or E series model Covaris microTUBE with AFA fiber and snap cap Microcentrifuge 1 5 mL LoBind Tube Qubit Fluorometer Dynal DynaMag 2 magnetic stand P10 P20 P200 and P1000 pipettes Vacuum concentrator Ice bucket Powder free gloves PCR tubes strips or plates Sterile nuclease free aerosol barrier pipette tips Timer Vortex mixer Heat block at 37 C Agilent p n G2938C Agilent p n G2964AA or G2965AA Agilent p n 401449 or equivalent Agilent SureCycler Life Technologies Veriti Thermal Cycler BioRad MJ Research DNA Engine PTC 200 or equivalent Covaris Covaris p n 520045 Eppendorf Microcentrifuge Model 5417C or equivalent Eppendorf p n 022431021 or equivalent Life Technologies p n 032857 Life Technologies p n 123 21D or equivalent Pipetman P10 P20 P200 P1000 or equivalent Savant SpeedVac or equivalent SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing Before You Begin 1 Table 7 Required Equipment for Hybridization Description Vendor and part number
4. SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing 69 4 Hybridization Do not use a tissue incubator It cannot properly maintain temperature c Briefly spin in a centrifuge d Separate the beads and buffer on a magnetic separator and remove the supernatant e Repeat step a through step d for a total of 3 washes Make sure all of the wash buffer has been removed 11 Mix the beads in 30 uL of nuclease free water on a vortex mixer for 5 seconds to resuspend the beads Captured DNA is retained on the streptavidin beads during the post capture amplification step 70 SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing SureSelect Target Enrichment System for Illumina Paired End Sequencing Protocol 5 Addition of Index Tags by Post Hybridization Amplification Step 1 Amplify the captured library to add index tags 72 Step 2 Purify the sample using Agencourt AMPure XP beads 76 Step 3 Assess quality 77 Step 4 Assess the quantity of each index tagged library by QPCR optional 80 Step 5 Pool samples for Multiplexed Sequencing 81 Step 6 Prepare sample for cluster amplification 82 This chapter describes the steps to add index tags by amplification purify assess quality and quantity of the libraries dilute the sample appropriately for cluster amplification and pool indexed samples for multiplexed sequencing ope Agilent Technologies P 5 Addition of Index Tags by
5. SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing 19 Table 11 Shear settings for Covaris instruments that use SonoLab 7 or newer Setting Value Duty Factor 10 Peak Incident Power PIP 175 Cycles per Burst 200 Treatment Time 360 seconds Bath Temperature 4 to 8 C Table 12 Shear settings for Covaris instruments that use SonoLab software previous to SonoLab 7 Setting Value Duty Cycle 10 Intensity 5 Cycles per Burst 200 Time 6 cycles of 60 seconds each Set Mode Frequency sweeping Temperature 4 to 7 C Put the Covaris microTube back into the loading and unloading station 8 While keeping the snap cap on insert a pipette tip through the pre split septa then slowly remove the sheared DNA 9 Transfer the sheared DNA into a new 1 5 mL LoBind Tube Sample Preparation 3 pg DNA Samples 2 Step 2 Purify the sample using Agencourt AMPure XP beads 1 Let the AMPure XP beads come to room temperature for at least 30 minutes 2 Mix the reagent well so that the reagent appears homogeneous and consistent in color Do not freeze 3 Add 180 uL of homogeneous AMPure XP beads to a 1 5 mL LoBind Tube and add the sheared DNA library 130 uL Mix well on a vortex mixer and incubate for 5 minutes 4 Put the tube in the magnetic stand Wait for the solution to clear approximately 3 to 5 minutes 5 Keep the tube in the magnetic stand Do not touch the beads while you carefully
6. Prepare enough RNase Block dilution for all samples plus excess d Add the amount of diluted SureSelect RNase Block purple cap listed in Table 27 to each capture library and mix by pipetting Table 27 SureSelect Capture Library Capture Size Volume of SureSelect RNase Block Dilution Volume of RNase Library Parts RNase block Block Dilution to Add Parts water lt 3 0 Mb 2 uL 1 9 10 5 uL gt 3 0 Mb 5 uL 1 3 25 2 uL SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing 63 4 Hybridization 6 Mix the contents in Table 28 to make the correct amount of SureSelect Block mix for the number of samples used Table 28 SureSelect Block Mix Reagent Volume for 1 reaction Volume for 12 reactions includes excess SureSelect Indexing Block 1 green cap 2 5 uL 31 25 uL SureSelect Block 2 blue cap 2 5 uL 31 25 uL SureSelect Indexing Block 3 brown cap 0 6 uL 7 5 pL Total 5 6 pL 70 pl 7 Ina separate PCR plate prepare the prepped library for target enrichment a Add 3 4 uL of 221 ng uL prepped library to the B row in the PCR plate Put each sample into a separate well b Add 5 6 uL of the SureSelect Block Mix to each well in row B c Mix by pipetting up and down d Seal the wells of row B with caps and put the PCR plate in the thermal cycler Do not heat the Hybridization Buffer or capture library yet only the prepped library with blockers e Start the thermal cycl
7. b Add 35 uL of the reaction mix to each well or tube c Add 15 uL of each DNA sample to each well or tube Mix by pipetting Change pipette tips between samples SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing 31 Table 16 Components for PCR mix Nuclease free Water 21 uL 262 5 pL SureSelect Primer brown cap 1 25 pL 15 625 pL SureSelect ILM Indexing Pre Capture PCR 1 25 uL 15 625 uL Reverse Primer clear cap 5x Herculase Il Rxn Buffer clear cap 10 uL 125 pL 100 mM dNTP Mix green cap 0 5 pL 6 25 pL Herculase II Fusion DNA Polymerase red cap 1L 12 5 pL Included in the SureSelect Library Prep Kit ILM t Included in the SureSelect Target Enrichment Kit ILM Indexing Hyb Module Box 2 Included in the Herculase II Fusion DNA Polymerase Agilent kit Do not use the buffer or dNTP mix from any other kit 2 Run the program in Table 17 in a thermal cycler Table 17 PCR program Step 1 98 C 2 minutes Step 2 98 C 30 seconds Step 3 65 C 30 seconds Step 4 72 C 1 minute Step 5 Repeat Step 2 through Step 4 for a total of 4 to 6 times Step 6 72 C 10 minutes Step 7 4 C Hold Sample Preparation 3 pg DNA Samples 2 Different library preparations can produce slightly different results based on varying DNA quality In most cases 5 cycles will produce an adequate yield for subsequent capture without introducing bias or non specific products
8. 12 Mb up to 24 Mb Human All Exon v5 Human All Exon v5 UTRs Human All Exon 50 Mb Human DNA Kinome Mouse All Exon 1 2 to 2 4 Gb 4 Gb 6 Gb 5 Gb 320 Mb 5 Gb For custom libraries Agilent recommends analyzing 100x amount of sequencing data compared to the Capture Library size for each sample Pool samples according to your expected sequencing out put 2 Put the tubes in a thermal cycler and run the program in Table 32 Store the unused bead bound captured DNA samples at 4 C overnight or at 20 C long term Table 32 PCR program Step Temperature Time Step 1 98 C 2 minutes Step 2 98 C 30 seconds Step 3 57 C 30 seconds Step 4 72 C 1 minute Step 5 Repeat Step 2 through Step 4 for a total of 12 to 16 times Step 6 72 C 10 minutes Step 7 4 c Hold 74 SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing Addition of Index Tags by Post Hybridization Amplification 5 As with the pre capture PCR amplification minimize the number of PCR cycles used to enrich the captured DNA The use of only half of the captured DNA for amplification lets you adjust the number of cycles by repeating the PCR if needed Table 33 Number of cycles Capture Size Cycles 1 kb up to 0 5 Mb 16 cycles 0 5 Mb up to 1 49 Mb 14 cycles gt 1 5 Mb 12 cycles All Exon 10 to 12 cycles As an alternative you can prepare one PCR master mix as outlined in Table 30 Sp
9. SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing 79 5 Addition of Index Tags by Post Hybridization Amplification Step 4 Assess the quantity of each index tagged library by QPCR optional Refer to the protocol that is included with the QPCR NGS Library Quantification Kit Illumina GA for more details to do this step 1 Use the QPCR NGS Library Quantification Kit lumina GA to determine the concentration of each index tagged captured library 2 Prepare a standard curve using the quantification standard included in the kit according to the instructions provided in the user guide 3 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 4 Prepare the QPCR master mix with Illumina adaptor specific PCR primers according to instructions provided in the kit 5 Add an aliquot of the master mix to PCR tubes and add template 6 Ona QPCR system such as the MX3005P run the thermal profile outlined in the QPCR NGS Library Quantification kit user guide Use the SYBR Green instrument setting 7 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 80 SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing Addi
10. Use the Qubit system to quantify genomic DNA before library preparation Sample Preparation 200 ng DNA Samples 3 Shear Repair ends Add Klenow and dATP Ligate diluted indexing specific adaptors y Bait Design in eArray AMPure XP bead purification SureSelect Capture Library PCR with SureSelect Primer and SureSelect Pre capture Reverse PCR primers y Library Hybridization 1 per sample y 16 or 24 hours at 65 C Hybrid Capture Selection y Magnetic bead selection Amplification and Index Tagging y PCR and purification Quality Assessment of each indexed sample y Bioanalyzer and Quantification by QPCR Pool samples 1 2 n Figure 6 Overall sequencing sample preparation workflow SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing 41 Table 18 Overview and time requirements Step Time Illumina Prepped library Production 1 day Library Hybridization 16 or 24 hours Bead preparation 30 minutes Capture Selection and Washing 2 hours DNA purification 30 minutes Post Hybridization Amplification 1 hour PCR purification 30 minutes Bioanalyzer QC and QPCR 2 to 3 hours Pool indexed samples by mass lt 1 hour Sample Preparation 200 ng DNA Samples 3 Step 1 Shear DNA For each DNA sample to be sequenced prepare 1 library 1 Use the Qubit dsDNA Assay to determine the concentration of your gDNA sample Make sure the gDNA is of high quality non degraded Aggo Aggo
11. solution is clear 13 Remove the supernatant 15 uL to a fresh 1 5 mL LoBind Tube You can discard the beads at this time 14 Proceed immediately to the next step Step 7 Ligate the paired end adaptor SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing 49 Stopping Point Step 7 Ligate the paired end adaptor Use the SureSelect Library Prep Kit ILM 1 Dilute SureSelect Adaptor Oligo Mix brown cap 1 10 in Nuclease free Water immediately before use Use the diluted Oligo mix in the next step 2 Prepare reaction mix and DNA sample prepare on ice a Prepare the reaction mix in Table 23 b Add 37 uL of the reaction mix to each well or tube c Add 13 uL of each DNA sample to each well or tube Mix by pipetting Change pipette tips between samples Table 23 Ligation master mix Reagent Volume for 1 Library Volume for 12 Libraries includes excess Nuclease free Water 15 5 uL 193 75 uL 5x T4 DNA Ligase Buffer 10 uL 125 uL Diluted SureSelect Adaptor Oligo Mix 10 pL 125 uL T4 DNA Ligase red cap 1 5 uL 18 75 uL Total Volume 37 pL 462 5 pL 37 pL sample 3 Incubate for 15 minutes at 20 C on a thermal cycler Do not use a heated lid If you do not continue to the next step store the samples at 20 C Sample Preparation 200 ng DNA Samples 3 Step 8 Purify the sample using Agencourt AMPure XP beads 1 Let the AMPure XP beads come to room temperature for at least 30 minut
12. Bioanalyzer and check communication 3 Prepare the chip samples and ladder as instructed in the reagent kit guide 4 Load the prepared chip into the 2100 Bioanalyzer and start the run within Fu five minutes after preparation Within the instrument context choose the appropriate assay from the drop down list Start the run Enter sample names and comments in the Data and Assay context Verify the results Check that the electropherogram shows a distribution with a peak size approximately 225 to 275 bp Measure the concentration of the library by integrating under the peak ia T T T T T T T 15 50 100 150 200 300 400 500 700 1500 bp Figure 4 Analysis of amplified library DNA using a DNA 1000 assay The electrophero gram shows a single peak in the size range of 225 to 275 bp SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing 35 CAUTION 2200 TapeStation and D1000 ScreenTape You can use the 2200 TapeStation to analyze the amplified libraries Use the D1000 ScreenTape and D1000 Reagents For more information to do this step see the Agilent 2200 TapeStation User Manual 1 Prepare the TapeStation samples as instructed in the Agilent 2200 TapeStation 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 D1000 sample buffer on a vortex mixer for 5 seconds
13. Mx3000P Mx3005P 96 well tube plates Agilent p n 410088 or equivalent Mx3000P Mx3005P optical strip caps Agilent p n 401425 or equivalent MicroAmp Clear Adhesive Film Life Technologies p n 4306311 BD Clay Adams Nutator Mixer BD Diagnostics p n 421105 or equivalent Dynal DynaMag 2 magnetic stand Life Technologies p n 123 21D or equivalent P10 P20 P200 and P1000 pipettes Pipetman P10 P20 P200 P1000 or equivalent Pipet Light Multichannel Pipette 12 channels Rainin p n L12 20 or equivalent PCR tubes strips or plates Sterile nuclease free aerosol barrier pipette tips Thermal cycler Agilent SureCycler Life Technologies Veriti Thermal Cycler BioRad MJ Research DNA Engine PTC 200 or equivalent Timer Vortex mixer Water bath or heat block set to 65 C Optional Equipment Table 8 Optional Equipment for Hybridization Description Vendor and part number Tube strip capping tool Agilent p n 410099 PlateLoc Thermal Microplate Sealer and Agilent p n G5402A and Peelable Aluminum Seal Agilent p n 24210 001 SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing 13 14 This page intentionally left blank SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing CAUTION This chapter contains instructions for prepped library production specific to SureSelect Target Enrichment System for Illumina Paired End Sequencing Protocol 2 Sample Preparation 3 pg DNA Sa
14. Notes 8 Safety Notes 8 Required Reagents 9 Optional Reagents 11 Required Equipment 12 Optional Equipment 13 Make sure you have the most current protocol Go to genomics agilent com and search for G7530 90000 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 differs from the Illumina Multiplexed Paired End sequencing manual and other SureSelect protocols at several steps Pay close attention to the primers used for each amplification step and the blocking agents used during hybridization Agilent cannot guarantee the use of the SureSelect Target Enrichment kits with non Agilent protocols nor provide technical support for non Agilent protocols to process samples for enrichment Eh Agilent Technologies 1 Before You Begin Procedural Notes e 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 Maintain a clean work area e Do not mix stock solutions and reactions containing gDNA on a vortex mixer Instead gently tap the tube with your finger to mix the sample e When preparing frozen reagent stock solutions for use 1 Thaw the aliquot as rapidly as possible without heating above room temperature 2 Mix briefly on a vortex mixer then spin in a centrifuge for 5 to 10 seconds to drive
15. Post Hybridization Amplification Step 1 Amplify the captured library to add index tags Use reagents from Herculase II Fusion DNA Polymerase Agilent SureSelect Target Enrichment Kit ILM Indexing Hyb Module Box 2 SureSelect Library Prep Kit ILM CAUTION Do not use amplification enzymes other than Herculase II Fusion DNA Polymerase Other enzymes have not been validated CAUTION To avoid cross contaminating libraries set up PCR reactions all components except the library DNA in a dedicated clean area or PCR hood with UV sterilization and positive air flow Prepare 1 amplification reaction for each hybrid capture Include a negative no template control To see the nucleotide sequence in each of the index included in SureSelect reagent kits see SureSelect Indexes for Ilumina on page 87 1 Prepare reaction mix and DNA samples a Prepare the reaction mix in Table 30 on ice Mix well on a vortex mixer b Add 35 uL of the reaction mix to each well or tube c Add 1 uL ofthe appropriate index PCR Primer Index 1 through Index 16 clear caps from the SureSelect Library Prep Kit ILM to each well and mix by pipetting 72 SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing Addition of Index Tags by Post Hybridization Amplification 5 Use a different index primer for each sample to be sequenced in the same lane Use Table 31 as a guide to determine the number of indexes to pool per sequenc
16. Use these reagents from the SureSelect Target Enrichment Kit Box 1 SureSelect Wash 1 SureSelect Wash 2 1 Estimate and record the volume of hybridization that remained after 16 or 24 hour incubation 2 Keep the PCR plate or tubes at 65 C in the PCR machine while you add the hybridization mixture directly from the thermal cycler to the bead solution Invert the tube to mix 3 to 5 times Excessive evaporation such as when less than 20 uL remains after hybridization can indicate suboptimal capture performance See Table 43 on page 88 for tips to minimize evaporation 3 Incubate the hybrid capture bead solution on a Nutator or equivalent for 30 minutes at room temperature Make sure the sample is properly mixing in the tube 4 Briefly spin in a centrifuge Separate the beads and buffer on a magnetic separator and remove the supernatant 6 Resuspend the beads in 500 uL of SureSelect Wash 1 by mixing on a vortex mixer for 5 seconds 7 Incubate the samples for 15 minutes at room temperature Occasionally mix on a vortex mixer 8 Briefly spin in a centrifuge Separate the beads and buffer on a magnetic separator and remove the supernatant 10 Wash the beads a Resuspend the beads in 500 uL of 65 C prewarmed SureSelect Wash 2 and mix on a vortex mixer for 5 seconds to resuspend the beads b Incubate the samples for 10 minutes at 65 C in a recirculating water bath heat block or equivalent Occasionally mix on a vortex mixer
17. and reagents must be used within one year of receipt Warranty The material contained in this docu ment is provided as is and is sub ject to being changed without notice in future editions Further to the max imum extent permitted by applicable law Agilent 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 merchantability and fitness for a par ticular purpose Agilent shall not be liable for errors or for incidental or consequential damages in connection with the furnishing use or perfor mance 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 document that conflict with these terms the warranty terms in the sep arate 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 accordance 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 Softwa
18. for Illumina paired end multiplexed library preparation 1 Before You Begin This chapter contains information such as procedural notes safety information required reagents and equipment that you should read and understand before you start an experiment 2 Sample Preparation 3 pg DNA Samples 3 Sample Preparation 200 ng DNA Samples These chapters describe the steps to prepare the DNA samples for target enrichment 4 Hybridization This chapter describes the steps to prepare and hybridize samples 5 Addition of Index Tags by Post Hybridization Amplification This chapter describes the steps to amplify purify and assess quality of the sample libraries Samples are pooled by mass prior to sequencing 6 Reference This chapter contains information on reagent kit content SureSelect index sequences and alternative equipment that can be used with this protocol SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing 3 What s new in 1 8 What s new in 1 7 What s new in 1 6 What s new in 1 5 What s new in 1 4 Support for SureSelect Focused Exome and Exome Plus 1 capture libraries Support for SureSelect Clinical Research Exome and SureSelect Inherited Disease capture libraries D1000 ScreenTape and Reagents replace D1K ScreenTape and Reagents High Sensitivity D1000 ScreenTape and Reagents replace High Sensitivity DIK ScreenTape and Reagents Support for V5 IncRNA SureSele
19. in the magnetic stand while you dispense 500 uL of 70 Ethanol in each tube Use fresh 70 Ethanol for optimal results 7 Let the tube sit for 1 minute to allow any disturbed beads to settle and remove the Ethanol 8 Repeat step 6 and step 7 once 9 Spin briefly in a centrifuge Return the tube to the magnetic stand for 30 seconds Remove residual Ethanol with a P20 pipette 10 Dry the samples on the 37 C heat block for 5 minutes or until the residual Ethanol is completely evaporated Do not dry the bead pellet to the point that the bead pellet appears cracked Elution efficiency is significantly decreased when the bead pellet is excessively dried 11 Add 30 uL of Nuclease free Water or 1X Low TE Buffer mix well on a vortex mixer and incubate for 2 minutes at room temperature 12 Put the tube in the magnetic stand and leave for 2 to 3 minutes until the solution is clear 13 Remove the supernatant 30 uL to a fresh 1 5 mL LoBind Tube You can discard the beads at this time Stopping Point If you do not continue to the next step store the samples at 4 C for up toa week or at 20 C for longer periods 76 SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing Addition of Index Tags by Post Hybridization Amplification 5 Step 3 Assess quality Use a Bioanalyzer High Sensitivity DNA Assay or the 2200 TapeStation to assess the quality and size range 2100 Bioanalyzer High Sensitivity DNA Assa
20. is 1 8 to 2 0 Follow the instructions for the instrument 2 Set up the Covaris instrument a Check that the water in the Covaris tank is filled with fresh deionized water to fill line level 12 on the graduated fill line label b Check that the water covers the visible glass part of the tube c Set the chiller temperature to between 2 C to 5 C to ensure that the temperature reading in the water bath displays 5 C d Optional Supplement the circulated water chiller with ethylene glycol to 20 volume to prevent freezing e On the instrument control panel push the Degas button Degas the instrument for least 30 minutes before use Refer to the Covaris instrument user guide 3 Dilute 200 ng of high quality gDNA with 1X Low TE Buffer in a 1 5 mL LoBind Tube for a total of 50 uL 4 Put a Covaris microTube into the loading and unloading station Keep the cap on the tube 5 Use a tapered pipette tip to slowly transfer the 50 uL of DNA sample through the pre split septa Be careful not to introduce a bubble into the bottom of the tube 6 Secure the microTube in the tube holder and shear the DNA with the settings in Table 19 or Table 20 The target peak for base pair size is 150 to 200 bp SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing 43 Table 19 Shear settings for Covaris instruments that use SonoLab 7 or newer Setting Value Duty Factor 10 Peak Incident Power PIP 175 Cycles p
21. n 600679 Ambion Cat AM9930 Beckman Coulter Genomics p n A63880 p n A63881 p n A63882 Life Technologies p n 12090 015 or equivalent Life Technologies p n 032851 Life Technologies p n 032850 Life Technologies p n 032853 Life Technologies p n 033130 Life Technologies p n 032856 Qiagen p n 19086 Sigma Aldrich p n E7023 SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing 9 1 Before You Begin Table 2 SureSelect Reagent Kit Reagent Kits 16 Reactions 96Reactions 480 Reactions SureSelect Reagent Kit HSQ G9611A G9611B G9611C SureSelect Reagent Kit MSQ G9612A G9612B G9612C SureSelect reagents must be used within one year of receipt Table 3 SureSelect Capture Library select one Capture Libraries 16 Reactions 96 Reactions 480 Reactions SureSelect Focused Exome 5190 7787 5190 7788 SureSelect Focused Exome Plus 1 5190 7790 5190 7791 SureSelect Clinical Research Exome 5190 7338 5190 7339 SureSelect Human All Exon V5 5190 6208 5190 6209 SureSelect Human All Exon V5 UTRs 5190 6213 5190 6214 SureSelect Human All Exon V5 IncRNA 5190 6446 5190 6447 SureSelect Human All Exon V5 Plus 5190 6211 5190 6212 SureSelect Human All Exon V4 5190 4631 5190 4632 5190 4634 SureSelect Human All Exon V4 UTRs 5190 4636 5190 4637 5190 4639 SureSelect Mouse All Exon 5190 4641 5190 4642 5190 4644 SureSelect Inherited Disease 5190 7518 5190 75
22. this time b Seal the wells with strip caps Use a capping tool to make sure the fit is tight c Incubate the samples at 65 C for 2 minutes 11 Maintain the plate at 65 C while you use a multi channel pipette to take 13 uL of Hybridization Buffer from the A row and add it to the SureSelect capture library mix contained in row C of the PCR plate for each sample See Figure 13 step 11 tep 1 Hybridization Buffer Prepped Library SureSelect Capture Library Figure 13 SureSelect Capture Library or Baits shown in Green 12 Maintain the plate at 65 C while you use a multi channel pipette to transfer the entire contents of each prepped library mix in row B to the hybridization solution in row C See Figure 13 Mix well by slowly pipetting up and down 8 to 10 times The hybridization mixture is now 27 to 29 uL depending on degree of evaporation during the preincubations 13 Seal the wells with strip caps or double adhesive film Make sure all wells are completely sealed Use new adhesive seals or strip caps The structural integrity of the seals and caps can be compromised during the previous incubation steps 66 SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing Hybridization 4 CAUTION Wells must be adequately sealed to minimize evaporation or your results can be negatively impacted You can use the PlateLoc Thermal Microplate Sealer to minimize evaporation
23. thoroughly mix the combined DNA and D1000 sample buffer on a vortex mixer for 5 seconds or more for accurate quantitation SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing 23 Stopping Point 2 Load the sample plate or tube strips from step 1 the D1000 ScreenTape and loading tips into the 2200 TapeStation as instructed in the Agilent 2200 TapeStation User Manual Start the run 3 Verify that the electropherogram shows an average DNA fragment size of about 185 bp A sample electropherogram is shown in Figure 3 If you do not continue to the next step seal the sheared DNA sample plate and store at 4 C overnight or at 20 C for prolonged storage Sample Intensity FU Figure 3 wn GH 300 o Analysis of sheared DNA using the 2200 TapeStation with a D1000 Screen Tape The electropherogram shows an average DNA fragment size between 150 to 200 bp Sample Preparation 3 pg DNA Samples 2 Step 4 Repair the ends To process multiple samples prepare master mixes with overage at each step without the DNA sample Master mixes for preparation of 12 samples including excess are shown in each table as an example Use the SureSelect Library Prep Kit ILM Prepare the master mix on ice 1 Prepare reaction mix and DNA sample prepare on ice a Prepare the reaction mix in Table 13 Mix well on a vortex mixer b Add 52 uL of the reaction mix to each well or tube c Add 48 uL of ea
24. using Agencourt AMPure XP beads 49 Step 7 Ligate the paired end adaptor 50 Step 8 Purify the sample using Agencourt AMPure XP beads 51 Step 9 Amplify adaptor ligated library 52 Step 10 Purify the sample with Agencourt AMPure XP beads 54 Step 11 Assess quality and quantity 55 This chapter contains instructions for prepped library production specific to 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 barcode sequence Depending on the target size of the SureSelect capture up to 16 samples can be pooled and sequenced in a single lane using Illumina multiplex index tags This protocol contains an option for 3 pg DNA samples standard input and an option for 200 ng DNA samples low input Make sure you follow the steps in the appropriate chapter hee Agilent Technologies The steps in this chapter differ from the Illumina protocol in the use of the Covaris system for gDNA shearing smaller target shear size elimination of size selection by gel purification and implementation of AMPure XP beads for all purification steps and primers used for PCR Refer to the Illumina protocol Preparing Samples for Multiplexed Paired End Sequencing p n 1005361 Rev C for more information Make sure genomic DNA samples are of high quality with an OD 260 280 ratio ranging from 1 8 to 2 0
25. while you dispense 500 uL of 70 Ethanol in each tube Use fresh 70 Ethanol for optimal result Let the tube sit for 1 minute to allow any disturbed beads to settle and remove the Ethanol Repeat step 6 and step 7 step once 9 Spin briefly in a centrifuge Return the tube to the magnetic stand for 30 seconds Remove residual Ethanol with a P20 pipette 10 Dry the samples on the 37 C heat block for 5 minutes or until the residual Ethanol completely evaporates Do not dry the bead pellet to the point that the bead pellet appears cracked Elution efficiency is significantly decreased when the bead pellet is excessively dried 11 Add 30 uL of Nuclease free Water mix well on a vortex mixer and incubate for 2 minutes at room temperature 12 Put the tube in the magnetic stand and leave for 2 to 3 minutes until the solution is clear 13 Remove the supernatant 30 uL to a fresh 1 5 mL LoBind Tube You can discard the beads at this time If you do not continue to the next step store the samples at 20 C Sample Preparation 3 pg DNA Samples 2 Step 12 Assess quality and quantity Quality assessment can be done with either the 2100 Bioanalyzer instrument or the 2200 TapeStation 2100 Bioanalyzer System and DNA 1000 Assay 1 Check that the 2100 Bioanalyzer electrodes have been cleaned as instructed in the reagent kit guide Open the Agilent 2100 Expert Software version B 02 02 or higher turn on the 2100
26. 0 Agilent strip tubes 410022 Mx4000 Agilent 96 well Plate 410088 Mx3000 3005 Adhesive Film 4306311 MicroAmp Caps 8caps strip N801 0535 MicroAmp Clear Adhesive Film 4306311 Attached lids Agilent Optical cap 410024 Mx4000 Agilent Optical cap 401425 Mx3000 3005 Agilent Optical cap 401425 Mx3000 3005 ABI compression pad 4312639 Use two layers of film Heated Lid Heated Lid ABI compression pad 4312639 Use two layers of film Lid heating set to 75 C Heated Lid Heated Lid Heated Lid SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing www agilent com In This Book This guide contains information to run the SureSelect Target Enrichment System for Illumina Paired End Sequencing protocol with the Illumina HiSeq and MiSeq Multiplexed Sequencing Platforms Agilent Technologies Inc 2014 Version 1 7 July 2014 G7530 90000 Revision A9 Ee Agilent Technologies
27. 19 SureSelect Inherited Disease Plus 5190 7521 5190 7522 SureSelect DNA Kinome 5190 4646 5190 4647 5190 4649 SureSelect X chromosome 5190 4651 5190 4652 5190 4653 SureSelect Custom 1 kb up to 499 Kb 5190 4806 5190 4807 5190 4809 reorder 5190 4811 5190 4812 5190 4814 SureSelect Custom 0 5 Mb up to 2 9 Mb 5190 4816 5190 4817 5190 4819 reorder 5190 4821 5190 4822 5190 4824 10 SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing Before You Begin 1 Table3 SureSelect Capture Library select one Continued Capture Libraries 16 Reactions 96 Reactions 480 Reactions SureSelect Custom 3 Mb up to 5 9 Mb 5190 4826 5190 4827 5190 4829 reorder 5190 4831 5190 4832 5190 4834 SureSelect Custom 6 Mb up to 11 9 Mb 5190 4836 5190 4837 5190 4839 reorder 5190 4841 5190 4842 5190 4844 SureSelect Custom 12 Mb up to 24 Mb 5190 4896 5190 4897 5190 4899 reorder 5190 4901 5190 4902 5190 4904 SureSelect capture libraries must be used within one year of receipt Table4 Required Reagents for Hybridization Description Vendor and part number Dynabeads MyOne Streptavidin T1 Life Technologies 2 mL Cat 65601 10 mL Cat 65602 100 mL Cat 65603 Nuclease free Water not DEPC treated Ambion Cat AM9930 Optional Reagents Table5 Optional Reagents Description Vendor and part number Ethylene glycol American Bioanalytical p n AB00455 Tween 20 Sigma Aldrich p n P9416 50ML
28. If yield is too low or non specific high molecular weight products are observed adjust the number of cycles accordingly with the remaining extra library template As an alternative you can prepare one PCR master mix as outlined in Table 16 Split the master mix into three small scale 10 uL PCR reactions and run for 4 5 or 6 cycles Clean these PCR reactions using the AMPure XP protocol outlined in Step 2 Purify the sample using Agencourt AMPure XP beads with these modifications Use 30 uL of AMPure XP beads and elute with 20 uL of Nuclease free Water Run these cleaned samples on a DNA1000 chip on the Bioanalyzer as described in Step 3 Assess quality optional SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing 33 Stopping Point Step 11 Purify the sample with Agencourt AMPure XP beads 1 Let the AMPure XP beads come to room temperature for at least 30 minutes Mix the reagent well so that the reagent appears homogeneous and consistent in color Do not freeze Add 90 uL of homogeneous AMPure XP beads to a 1 5 mL LoBind Tube and add the amplified library 50 uL Mix well on a vortex mixer and incubate for 5 minutes Put the tube in the magnetic stand Wait for the solution to clear approximately 3 to 5 minutes Keep the tube in the magnetic stand Do not touch the beads while you carefully discard the cleared solution from the tubes Continue to keep the tube in the magnetic stand
29. L LoBind Tube You can discard the beads at this time Stopping Point If you do not continue to the next step store the samples at 20 C SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing 51 Step 9 Amplify adaptor ligated library Use reagents from these kits SureSelect Library Prep Kit ILM SureSelect Target Enrichment Kit ILM Indexing Hyb Module Box 2 Herculase II Fusion DNA Polymerase Agilent CAUTION To avoid cross contaminating libraries set up PCR reactions all components except the library DNA in a dedicated clean area or PCR hood with UV sterilization and positive air flow 1 Prepare reaction mix and DNA sample a Prepare the reaction mix in Table 24 on ice Mix well on a vortex mixer b Add 20 uL of the reaction mix to each well or tube c Add 30 uL of each DNA sample to each well or tube Mix by pipetting Change pipette tips between samples Sample Preparation 200 ng DNA Samples 3 Step 9 Amplify adaptor ligated library Table 24 Components for PCR mix Reagent Volume for 1 Volume for 12 reactions reaction includes excess Nuclease free Water 6 uL 75 uL SureSelect Primer brown cap 1 25 pL 15 625 pL SureSelect ILM Indexing Pre Capture PCR 1 25 pL 15 625 uL Reverse Primer clear cap 5x Herculase II Rxn Buffer clear cap 10 pL 125 pL 100 mM dNTP Mix green cap 0 5 pL 6 25 pL Herculase II Fusion DNA Polymerase red cap TL 12 5 pL In
30. Let the tube sit for 1 minute to allow any disturbed beads to settle and remove the Ethanol Repeat step 6 and step 7 step once 9 Spin briefly in a centrifuge Return the tube to the magnetic stand for 30 seconds Remove residual Ethanol with a P20 pipette 10 Dry the samples on the 37 C heat block for 5 minutes or until the residual Ethanol completely evaporates Do not dry the bead pellet to the point that the bead pellet appears cracked Elution efficiency is significantly decreased when the bead pellet is excessively dried 11 Add 30 uL of Nuclease free Water mix well on a vortex mixer and incubate for 2 minutes at room temperature 12 Put the tube in the magnetic stand and leave for 2 to 3 minutes until the solution is clear 13 Remove the supernatant 30 uL to a fresh 1 5 mL LoBind Tube You can discard the beads at this time If you do not continue to the next step store the samples at 20 C Sample Preparation 200 ng DNA Samples 3 Step 11 Assess quality and quantity Quality assessment can be done with either the 2100 Bioanalyzer instrument or the 2200 TapeStation 2100 Bioanalyzer System and DNA 1000 Assay 1 Check that the 2100 Bioanalyzer electrodes have been cleaned as instructed in the reagent kit guide Open the Agilent 2100 Expert Software version B 02 02 or higher turn on the 2100 Bioanalyzer and check communication 3 Prepare the chip samples and ladder as instructed in t
31. SureSelect Target Enrichment System for Illumina Paired End Sequ Library Illumina His MiSeq Version 1 8 October 2 Before you begin view hands on SureSelect platform manufactured with Agilent videos of SureSelect procedures at SurePrint Technology http www agilent com genomics protocolvideos For Research Use Only Not for Use in Diagnostic Procedures RE Agilent Technologies Notices Agilent Technologies Inc 2014 No part of this manual may be reproduced in any form or by any means including electronic storage and retrieval or transla tion into a foreign language without prior agreement and written consent from Agi lent Technologies Inc as governed by United States and international copyright laws Manual Part Number 67530 90000 Edition Version 1 8 October 2014 Agilent Technologies Inc 5301 Stevens Creek Rd Santa Clara CA 95051 USA Acknowledgment Oligonucleotide sequences 2006 2008 and 2011 Illumina Inc All rights reserved Only for use with the Illumina sequencer systems and associated assays Technical Support For technical product support contact your local Agilent Support Services representa tive For US and Canada call 800 227 9770 option 3 4 4 For other countries find your support center telephone numbers at www agilent com chem contactus Or send an e mail to SureSelect Support agilent com Notice to Purchaser SureSelect capture libraries
32. agnetic stand for 30 seconds Remove residual Ethanol with a P20 pipette 10 Dry the samples on the 37 C heat block for 5 minutes until the residual Ethanol is completely evaporated Do not dry the bead pellet to the point that the bead pellet appears cracked Elution efficiency is significantly decreased when the bead pellet is excessively dried 11 Add 32 uL of Nuclease free Water mix well on a vortex mixer and incubate for 2 minutes at room temperature 12 Put the tube in the magnetic stand and leave for 2 to 3 minutes until the solution is clear 13 Remove the supernatant 32 uL to a fresh 1 5 mL LoBind Tube You can discard the beads at this time If you do not continue to the next step store the samples at 20 C Sample Preparation 3 pg DNA Samples 2 Step 10 Amplify adaptor ligated library Use reagents from these kits SureSelect Library Prep Kit ILM SureSelect Target Enrichment Kit ILM Indexing Hyb Module Box 2 Herculase II Fusion DNA Polymerase Agilent This protocol uses half of the adaptor ligated fragments for amplification The remainder can be saved at 20 C for future use if needed CAUTION To avoid cross contaminating libraries set up PCR reactions all components except the library DNA in a dedicated clean area or PCR hood with UV sterilization and positive air flow 1 Prepare reaction mix and DNA sample a Prepare the reaction mix in Table 16 on ice Mix well on a vortex mixer
33. ch DNA sample to each well or tube Mix by pipetting Change pipette tips between samples 2 Incubate in a thermal cycler for 30 minutes at 20 C Do not use a heated lid If the heated lid of your thermal cycler cannot be turned off incubate the sample with lid open Table 13 End Repair Mix Reagent Volume for 1 Library Volume for 12 Libraries includes excess Nuclease free Water 35 2 uL 440 uL 10x End Repair Buffer clear cap 10 uL 125 uL dNTP Mix green cap 1 6 uL 20 uL T4 DNA Polymerase purple cap 1 uL 12 5 uL Klenow DNA Polymerase yellow cap 2 uL 25 uL T4 Polynucleotide Kinase orange cap 2 2 uL 27 5 uL Total Volume 52 uL 650 uL 52 pL sample SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing 25 Stopping Point Step 5 Purify the sample using Agencourt AMPure XP beads 1 Let the AMPure XP beads come to room temperature for at least 30 minutes Mix the reagent well so that the reagent appears homogeneous and consistent in color Do not freeze Add 180 uL of homogeneous AMPure XP beads to a 1 5 mL LoBind Tube and add the end repaired DNA library 100 uL Mix well on a vortex mixer and incubate for 5 minutes Put the tube in the magnetic stand Wait for the solution to clear approximately 3 to 5 minutes Keep the tube in the magnetic stand Do not touch the beads while you carefully discard the cleared solution from the tubes Continue to keep the tube in the ma
34. cluded in the SureSelect Library Prep Kit ILM t Included in the SureSelect Target Enrichment Kit ILM Indexing Hyb Module Box 2 Included in the Herculase II Fusion DNA Polymerase Agilent kit Do not use the buffer or dNTP mix from any other kit 2 Run the program in Table 25 in a thermal cycler Table 25 PCR program Step Temperature Time Step 1 98 C 2 minutes Step 2 98 C 30 seconds Step 3 65 C 30 seconds Step 4 72 C 1 minute Step 5 Repeat Step 2 through Step 4 for a total of 10 times Step 6 72 C 10 minutes Step 7 4 C Hold SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing 53 Stopping Point Step 10 Purify the sample with Agencourt AMPure XP beads 1 Let the AMPure XP beads come to room temperature for at least 30 minutes Mix the reagent well so that the reagent appears homogeneous and consistent in color Do not freeze Add 90 uL of homogeneous AMPure XP beads to a 1 5 mL LoBind Tube and add the amplified library 50 uL Mix well on a vortex mixer and incubate for 5 minutes Put the tube in the magnetic stand Wait for the solution to clear approximately 3 to 5 minutes Keep the tube in the magnetic stand Do not touch the beads while you carefully discard the cleared solution from the tubes Continue to keep the tube in the magnetic stand while you dispense 500 uL of 70 Ethanol in each tube Use fresh 70 Ethanol for optimal result
35. ct capture libraries Support for 200 ng DNA Samples input option Details added for storage of on bead captured DNA samples Support for SureSelect Human All Exon V5 V5 Plus and V5 UTRs capture libraries More details provided for the Agilent 2200 TapeStation for DNA quantitation and qualification Option to hybridize for 16 hours On bead PCR procedure replaces in solution PCR procedure for post hybridization Support for Covaris E series Sample Preparation system and SonoLab 7 software 4 SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing Content 1 Before You Begin 7 Procedural Notes 8 Safety Notes 8 Required Reagents 9 Optional Reagents 11 Required Equipment 12 Optional Equipment 13 2 Sample Preparation 3 pg DNA Samples 15 Step 1 Shear DNA 19 Step 2 Purify the sample using Agencourt AMPure XP beads 21 Step 3 Assess quality optional 22 Step 4 Repair the ends 25 Step 5 Purify the sample using Agencourt AMPure XP beads 26 Step 6 Add A Bases to the 3 end of the DNA fragments 27 Step 7 Purify the sample using Agencourt AMPure XP beads 28 Step 8 Ligate the paired end adaptor 29 Step 9 Purify the sample using Agencourt AMPure XP beads 30 Step 10 Amplify adaptor ligated library 31 Step 11 Purify the sample with Agencourt AMPure XP beads 34 Step 12 Assess quality and quantity 35 3 Sample Preparation 200 ng DNA Samples 39 Step 1 Shear DNA 43 Step 2 Assess quality optio
36. discard the cleared solution from the tubes 6 Continue to keep the tube in the magnetic stand while you dispense 500 uL of 70 Ethanol in each tube Use fresh 70 Ethanol for optimal result 7 Let the tube sit for 1 minute to allow any disturbed beads to settle and remove the Ethanol 8 Repeat step 6 and step 7 step once 9 Spin briefly in a centrifuge Return the tube to the magnetic stand for 30 seconds Remove residual Ethanol with a P20 pipette 10 Dry the samples on the 37 C heat block for 5 minutes or until the residual Ethanol completely evaporates Do not dry the bead pellet to the point that the bead pellet appears cracked Elution efficiency is significantly decreased when the bead pellet is excessively dried 11 Add 50 uL of Nuclease free Water mix well on a vortex mixer and incubate for 2 minutes at room temperature 12 Put the tube in the magnetic stand and leave for 2 to 3 minutes until the solution is clear 13 Remove approximately 50 uL of the supernatant to a fresh 1 5 mL LoBind Tube You can discard the beads at this time Stopping Point If you do not continue to the next step store the samples at 20 C SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing 21 Step 3 Assess quality optional Quality assessment can be done with either the 2100 Bioanalyzer instrument or the 2200 TapeStation 2100 Bioanalyzer System with DNA 1000 Assay For the Standard Input option use
37. e tips between samples Table 14 Adding A Bases Reagent Volume for 1 Volume for 12 Libraries Library includes excess Nuclease free Water 11 uL 137 5 uL 10x Klenow Polymerase Buffer blue cap 5 pL 62 5 uL dATP green cap 1 uL 12 5 uL Exo Klenow red cap 3 ul 37 5 uL Total Volume 20 pL 250 uL 20 pL sample 2 Incubate in a thermal cycler for 30 minutes at 37 C If you use a heated lid make sure that the lid temperature does not exceed 50 C SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing 27 Step 7 Purify the sample using Agencourt AMPure XP beads 1 Let the AMPure XP beads come to room temperature for at least 30 minutes Mix the reagent well so that the reagent appears homogeneous and consistent in color Do not freeze Add 90 uL of homogeneous AMPure XP beads to a 1 5 mL LoBind Tube and add the A tailed DNA library 50 uL Mix well on a vortex mixer and incubate for 5 minutes Put the tube in the magnetic stand Wait for the solution to clear approximately 3 to 5 minutes Keep the tube in the magnetic stand Do not touch the beads while you carefully discard the cleared solution from the tubes Continue to keep the tube in the magnetic stand while you dispense 500 uL of 70 Ethanol in each tube Use fresh 70 Ethanol for optimal result Let the tube sit for 1 minute to allow any disturbed beads to settle and remove the Ethanol Repeat step 6 and ste
38. er Burst 200 Treatment Time 360 seconds Bath Temperature 4 to 8 C Table 20 Shear settings for Covaris instruments that use SonoLab software previous to SonoLab 7 Setting Value Duty Cycle 10 Intensity 5 Cycles per Burst 200 Time 6 cycles of 60 seconds each Set Mode Frequency sweeping Temperature 4 to 7 C Put the Covaris microTube back into the loading and unloading station 8 While keeping the snap cap on insert a pipette tip through the pre split septa then slowly remove the sheared DNA 9 Transfer the sheared DNA into a new 1 5 mL LoBind Tube Sample Preparation 200 ng DNASamples 3 Step 2 Assess quality optional Quality assessment can be done with the 2100 Bioanalyzer instrument For the 200 ng option use the High Sensitivity DNA assay 1 Check that the 2100 Bioanalyzer electrodes have been cleaned as instructed in the reagent kit guide 2 Open the Agilent 2100 Expert Software version B 02 02 or higher turn on the 2100 Bioanalyzer and check communication 3 Prepare the chip samples and ladder as instructed in the reagent kit guide 4 Load the prepared chip into the 2100 Bioanalyzer and start the run within five minutes after preparation 5 Within the instrument context choose the appropriate assay from the drop down list 6 Start the run Enter sample names and comments in the Data and Assay context 7 Verify the results Check that the electropherogram shows a distribution wit
39. er program in Table 29 Prepped Library Figure 11 Prepped library shown in red 64 SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing Hybridization 4 Table 29 PCR program Step Temperature Time Step 1 95 C 5 minutes Step 2 65 C Hold 8 Use a heated lid on the thermal cycler at 105 C to hold the temperature of the plate on the thermal cycler at 65 C CAUTION The lid of the thermal cycler is hot and can cause burns Use caution when working near the lid 9 Add the hybridization buffer to the PCR plate a Maintain the plate at 65 C while you load 40 uL of hybridization buffer per well into the A row of the PCR plate The number of wells filled in Row A is the number of libraries prepared The example in Figure 12 is for 12 captures Hybridization Buffer Prepped Library Figure 12 Hybridization buffer shown in blue b Seal the wells with strip caps Use a capping tool to make sure the fit is tight c Keep the plate at 65 C for a minimum of 5 minutes before you go to step 10 SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing 65 4 Hybridization 10 Add the capture library mix from step 5 to the PCR plate a Add the capture library mix 7 uL to the C row in the PCR plate For multiple samples use a multi channel pipette to load the capture library mix into the C row in the PCR plate Keep the plate at 65 C during
40. ers used for PCR Refer to the Illumina protocol Preparing Samples for Multiplexed Paired End Sequencing p n 1005361 Rev C for more information Make sure genomic DNA samples are of high quality with an OD 260 280 ratio ranging from 1 8 to 2 0 Use the Qubit system to quantify genomic DNA before library preparation Sample Preparation 3 pg DNA Samples 2 Shear Repair ends Add Klenow and dATP Ligate indexing specific adaptors v Bait Design in eArray AMPure XP bead purification SureSelect Capture Library PCR with SureSelect Primer and SureSelect Pre capture Reverse PCR primers ea Library Hybridization 1 per sample y 16 or 24 hours at 65 C Hybrid Capture Selection y Magnetic bead selection Amplification and Index Tagging 4 PCR and purification Quality Assessment of each indexed sample y Bioanalyzer and Quantification by QPCR Pool samples 1 2 n Figure 1 Overall sequencing sample preparation workflow SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing 17 Table 10 Overview and time requirements Step Time Illumina Prepped library Production 1 day Library Hybridization 16 or 24 hours Bead preparation 30 minutes Capture Selection and Washing 2 hours DNA purification 30 minutes Post Hybridization Amplification 1 hour PCR purification 30 minutes Bioanalyzer QC and QPCR 2 to 3 hours Pool indexed samples by mass lt 1 hour Sample Preparation 3 p
41. es 2 Mix the reagent well so that the reagent appears homogeneous and consistent in color Do not freeze 3 Add 90 uL of homogeneous AMPure XP beads to a 1 5 mL LoBind Tube and add the ligated library 50 uL Mix well on a vortex mixer and incubate for 5 minutes 4 Put the tube in the magnetic stand Wait for the solution to clear approximately 3 to 5 minutes 5 Keep the tube in the magnetic stand Do not touch the beads while you carefully discard the cleared solution from the tubes 6 Continue to keep the tube in the magnetic stand while you dispense 500 uL of 70 Ethanol in each tube Use fresh 70 Ethanol for optimal result 7 Let the tube sit for 1 minute to allow any disturbed beads to settle and remove the Ethanol 8 Repeat step 6 and step 7 step once 9 Spin briefly in a centrifuge Return the tube to the magnetic stand for 30 seconds Remove residual Ethanol with a P20 pipette 10 Dry the samples on the 37 C heat block for 5 minutes until the residual Ethanol is completely evaporated Do not dry the bead pellet to the point that the bead pellet appears cracked Elution efficiency is significantly decreased when the bead pellet is excessively dried 11 Add 32 uL of Nuclease free Water mix well on a vortex mixer and incubate for 2 minutes at room temperature 12 Put the tube in the magnetic stand and leave for 2 to 3 minutes until the solution is clear 13 Remove the supernatant 32 uL to a fresh 1 5 m
42. et is excessively dried 11 Add 32 uL Nuclease free Water mix well on a vortex mixer and incubate for 2 minutes at room temperature 12 Put the tube in the magnetic stand and leave for 2 to 3 minutes until the solution is clear Remove the supernatant 32 uL to a fresh 1 5 mL LoBind Tube You can discard the beads at this time Stopping Point If you do not continue to the next step store the samples at 20 C SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing 47 Step 5 Add A Bases to the 3 end of the DNA fragments Use the SureSelect Library Prep Kit ILM 1 Prepare reaction mix and DNA sample prepare on ice a Prepare the reaction mix in Table 22 Mix well on a vortex mixer b Add 20 uL of the reaction mix to each well or tube c Add 30 uL of each DNA sample to each well or tube Mix by pipetting Change pipette tips between samples Table 22 Adding A Bases Reagent Volume for 1 Volume for 12 Libraries Library includes excess Nuclease free Water 11 uL 137 5 uL 10x Klenow Polymerase Buffer blue cap 5 pL 62 5 uL dATP green cap 1 uL 12 5 uL Exo Klenow red cap 3 ul 37 5 uL Total Volume 20 pL 250 uL 20 pL sample 2 Incubate in a thermal cycler for 30 minutes at 37 C If you use a heated lid make sure that the lid temperature does not exceed 50 C Sample Preparation 200 ng DNASamples 3 Step 6 Purify the sample using Agencourt AMPure XP beads 1 L
43. et the AMPure XP beads come to room temperature for at least 30 minutes 2 Mix the reagent well so that the reagent appears homogeneous and consistent in color Do not freeze 3 Add 90 uL of homogeneous AMPure XP beads to a 1 5 mL LoBind Tube and add the A tailed DNA library 50 uL Mix well on a vortex mixer and incubate for 5 minutes 4 Put the tube in the magnetic stand Wait for the solution to clear approximately 3 to 5 minutes 5 Keep the tube in the magnetic stand Do not touch the beads while you carefully discard the cleared solution from the tubes 6 Continue to keep the tube in the magnetic stand while you dispense 500 uL of 70 Ethanol in each tube Use fresh 70 Ethanol for optimal result 7 Let the tube sit for 1 minute to allow any disturbed beads to settle and remove the Ethanol 8 Repeat step 6 and step 7 step once 9 Spin briefly in a centrifuge Return the tube to the magnetic stand for 30 seconds Remove residual Ethanol with a P20 pipette 10 Dry the samples on the 37 C heat block for 5 minutes or until the residual Ethanol completely evaporates Do not dry the bead pellet to the point that the bead pellet appears cracked Elution efficiency is significantly decreased when the bead pellet is excessively dried 11 Add 15 uL of Nuclease free Water mix well on a vortex mixer and incubate for 2 minutes at room temperature 12 Put the tube in the magnetic stand and leave for 2 to 3 minutes until the
44. g DNA Samples 2 Step 1 Shear DNA For each DNA sample to be sequenced prepare 1 library 1 Use the Qubit dsDNA Assay to determine the concentration of your gDNA sample Make sure the gDNA is of high quality non degraded Aggo Aggo is 1 8 to 2 0 Follow the instructions for the instrument 2 Set up the Covaris instrument a Check that the water in the Covaris tank is filled with fresh deionized water to fill line level 12 on the graduated fill line label b Check that the water covers the visible glass part of the tube c Set the chiller temperature to between 2 C to 5 C to ensure that the temperature reading in the water bath displays 5 C d Optional Supplement the circulated water chiller with ethylene glycol to 20 volume to prevent freezing e On the instrument control panel push the Degas button Degas the instrument for least 30 minutes before use Refer to the Covaris instrument user guide 3 Dilute 3 ug of high quality gDNA with 1X Low TE Buffer in a 1 5 mL LoBind Tube to a total volume of 130 uL 4 Put a Covaris microTube into the loading and unloading station Keep the cap on the tube 5 Use a tapered pipette tip to slowly transfer the 130 uL of DNA sample through the pre split septa Be careful not to introduce a bubble into the bottom of the tube 6 Secure the microTube in the tube holder and shear the DNA with the settings in Table 11 or Table 12 The target peak for base pair size is 150 to 200 bp
45. gnetic stand while you dispense 500 uL of 70 Ethanol in each tube Use fresh 70 Ethanol for optimal result Let the tube sit for 1 minute to allow any disturbed beads to settle and remove the Ethanol Repeat step 6 and step 7 step once 9 Spin briefly in a centrifuge Return the tube to the magnetic stand for 30 seconds Remove residual Ethanol with a P20 pipette 10 Dry the samples on the 37 C heat block for 5 minutes or until the residual Ethanol completely evaporates Do not dry the bead pellet to the point that the bead pellet appears cracked Elution efficiency is significantly decreased when the bead pellet is excessively dried 11 Add 32 uL of Nuclease free Water mix well on a vortex mixer and incubate for 2 minutes at room temperature 12 Put the tube in the magnetic stand and leave for 2 to 3 minutes until the solution is clear Remove the supernatant 32 uL to a fresh 1 5 mL LoBind Tube You can discard the beads at this time If you do not continue to the next step store the samples at 20 C Sample Preparation 3 pg DNA Samples 2 Step 6 Add A Bases to the 3 end of the DNA fragments Use the SureSelect Library Prep Kit ILM 1 Prepare reaction mix and DNA sample prepare on ice a Prepare the reaction mix in Table 14 Mix well on a vortex mixer b Add 20 uL of the reaction mix to each well or tube c Add 30 uL of each DNA sample to each well or tube Mix by pipetting Change pipett
46. h a peak height between 120 to 150 bp ee sa i Rnd Mai mT t t T T t T T T T T T 35 100 150 200 300 400 500 600 1000 2000 10380 bp Figure 7 Analysis of sheared DNA using a High Sensitivity DNA assay The electro pherogram shows a distribution with a peak size between 120 to 150 bp SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing 45 Step 3 Repair the ends To process multiple samples prepare master mixes with overage at each step without the DNA sample Master mixes for preparation of 12 samples including excess are shown in each table as an example Use the SureSelect Library Prep Kit ILM Prepare the master mix on ice 1 Prepare reaction mix and DNA sample prepare on ice a Prepare the reaction mix in Table 21 Mix well on a vortex mixer b Add 52 uL of the reaction mix to each well or tube c Add 48 uL of each DNA sample to each well or tube Mix by pipetting Change pipette tips between samples 2 Incubate in a thermal cycler for 30 minutes at 20 C Do not use a heated lid If the heated lid of your thermal cycler cannot be turned off incubate the sample with lid open Table 21 End Repair Mix Reagent Volume for 1 Library Volume for 12 Libraries includes excess Nuclease free Water 35 2 uL 440 uL 10x End Repair Buffer clear cap 10 uL 125 uL dNTP Mix green cap 1 6 uL 20 uL T4 DNA Polymerase purple cap 1 uL 12 5 uL Klenow DNA Polymerase yellow cap 2 uL 25
47. he edges Check that you do not get extensive evaporation Evaporation should not exceed 3 to 4 uL For a partial list of tested options showing minimal evaporation refer to Alternative Capture Equipment Combinations on page 88 SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing 61 4 62 Hybridization Step 1 Hybridize the library For each sample library prepared do one hybridization and capture Do not pool samples at this stage The hybridization reaction requires 750 ng of DNA with a maximum volume of 3 4 uL 1 Ifthe prepped library concentration is below 221 ng uL use a vacuum concentrator to concentrate the sample at lt 45 C a Add the entire 30 uL of prepped library to an Eppendorf tube Poke one or more holes in the lid with a narrow gauge needle You can also break off the cap cover with parafilm and poke a hole in the parafilm b Completely lyophilize Use a vacuum concentrator on low heat less than 45 C to dehydrate c Reconstitute with nuclease free water to bring the final concentration to 221 ng uL or greater if sample recovery is of concern Pipette up and down along the sides of the tube for optimal recovery d Mix well on a vortex mixer and spin in a microfuge for 1 minute Optional To test recovery after lyophilization reconstitute the sample to greater than 221 ng uL and check the concentration on a Bioanalyzer DNA 1000 chip See Step 12 Assess
48. he reagent kit guide 4 Load the prepared chip into the 2100 Bioanalyzer and start the run within FU 160 five minutes after preparation Within the instrument context choose the appropriate assay from the drop down list Start the run Enter sample names and comments in the Data and Assay context Verify the results Check that the electropherogram shows a distribution with a peak size approximately 225 to 275 bp Measure the concentration of the library by integrating under the peak T T T T T T T T 15 50 100 150 200 300 400 500 700 1500 bp Figure 8 Analysis of amplified prepped library DNA using a DNA 1000 assay The elec tropherogram shows a single peak in the size range of 225 to 275 bp SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing 55 CAUTION 2200 TapeStation and D1000 ScreenTape You can use the 2200 TapeStation to analyze the amplified libraries Use the D1000 ScreenTape and D1000 Reagents For more information to do this step see the Agilent 2200 TapeStation User Manual 1 Prepare the TapeStation samples as instructed in the Agilent 2200 TapeStation 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 D1000 sample buffer on a vortex mixer for 5 seconds or more for accurate quantitation Stopping Point 2 Load the sample plate or tube stri
49. imer clear cap SureSelect ILM Indexing Post Capture Forward PCR Primer orange cap Table 38 SureSelect Library Prep Kit ILM Kit Component 10x End Repair Buffer clear cap T4 Polynucleotide Kinase orange cap 10x Klenow Polymerase Buffer blue cap T4 DNA Ligase red cap Exo Klenow red cap T4 DNA Polymerase purple cap Klenow DNA Polymerase yellow cap dATP green cap dNTP Mix green cap SureSelect Adaptor Oligo Mix brown cap SureSelect Primer brown cap PCR Primer Index 1 through Index 16 clear caps 5x T4 DNA Ligase Buffer SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing 85 6 86 Reference Other Reagent Kits Content These reagents are from kits other than the SureSelect Reagent kit Make sure you use only the reagents listed here Table 39 Herculase II Fusion DNA Polymerase Agilent Component DMSO green cap 5x Herculase II Rxn Buffer clear cap 100 mM dNTP Mix green cap Herculase II Fusion DNA Polymerase red cap Table 40 D1000 Reagents Agilent p n 5067 5583 Components D1000 ladder D1000 sample buffer Table 41 High Sensitivity D1000 Reagents Agilent p n 5067 5585 Components High Sensitivity D1000 ladder High Sensitivity D1000 sample buffer SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing Reference 6 SureSelect Indexes for Illumina XT The nucleotide seque
50. ing lane d Pipette each DNA sample up and down to make sure that the bead solution is homogeneous e Use a pipette to add 14 uL of each DNA sample to each well or tube Mix by pipetting Change pipette tips between samples to avoid cross contamination If needed store the remaining bead at 20 C for future use Table 30 Herculase II Master Mix Reagent Volume for 1 Volume for 12 reactions reaction includes excess Nuclease free water 22 5 uL 281 25 uL 5x Herculase I Rxn Buffer clear cap i 10 uL 125 uL Herculase II Fusion DNA Polymerase red cap Tul 12 5 pL 100 mM dNTP Mix green cap 0 5 uL 6 25 uL SureSelect ILM Indexing Post Capture Forward Tul 12 5 uL PCR Primer orange cap Total 35 uL 437 5 pL 35 pL reaction Included in the Herculase II Fusion DNA Polymerase Agilent Do not use the buffer or dNTP mix from any other kit t Included in the SureSelect Target Enrichment Kit ILM Indexing Hyb Module Box 2 Table 31 Sequencing data requirement guidelines Capture size Optimal sequencing output per index 1 kb up to 0 5 Mb 0 1 to 50 Mb 0 5 Mb up to 2 9 Mb 50 to 290 Mb 3 Mb up to 5 9 Mb 300 to 590 Mb 6 Mb up to 11 9 Mb 600 to 1190 Mb SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing 73 5 Addition of Index Tags by Post Hybridization Amplification Table 31 Sequencing data requirement guidelines Capture size Optimal sequencing output per index
51. lit this master mix into three small scale 10 uL PCR reactions and cycle for 10 12 14 or 16 cycles Clean these PCR reactions using the AMPure XP protocol outlined in Step 2 Purify the sample using Agencourt AMPure XP beads on page 21 with these modifications use 30 uL of AMPure XP beads and elute with 20 uL of nuclease free water Run these cleaned samples on a DNA 1000 chip on the Bioanalyzer as described in Step 3 Assess quality optional on page 22 Use the optimal cycle number to repeat PCR at the 50 uL reaction scale See Table 33 for approximate number of cycles for a given library size Results may vary based on library content SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing 75 5 Addition of Index Tags by Post Hybridization Amplification Step 2 Purify the sample using Agencourt AMPure XP beads 1 Let the AMPure XP beads come to room temperature for at least 30 minutes 2 Mix the reagent well so that the reagent appears homogeneous and consistent in color Do not freeze 3 Add 90 uL of homogeneous AMPure beads to a 1 5 mL LoBind Tube and add amplified library 50 uL Mix well on a vortex mixer and incubate for 5 minutes 4 Put the tube in the magnetic stand Wait for the solution to clear approximately 3 to 5 minutes 5 Keep the tube in the magnetic stand Do not touch the beads while you carefully discard the cleared solution from the tubes 6 Continue to keep the tube
52. mples Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 Step 7 Step 8 Step 9 Shear DNA 19 Purify the sample using Agencourt AMPure XP beads 21 Assess quality optional 22 Repair the ends 25 Purify the sample using Agencourt AMPure XP beads 26 Add A Bases to the 3 end of the DNA fragments 27 Purify the sample using Agencourt AMPure XP beads 28 Ligate the paired end adaptor 29 Purify the sample using Agencourt AMPure XP beads 30 Step 10 Amplify adaptor ligated library 31 Step 11 Purify the sample with Agencourt AMPure XP beads 34 Step 12 Assess quality and quantity 35 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 barcode sequence Depending on the target size of the SureSelect capture up to 16 samples can be pooled and sequenced in a single lane using Illumina multiplex index tags This protocol contains an option for 3 pg DNA samples standard input and an option for 200 ng DNA samples low input Make sure you follow the steps in the appropriate chapter Eh Agilent Technologies The steps in this chapter differ from the Illumina protocol in the use of the Covaris system for gDNA shearing smaller target shear size elimination of size selection by gel purification and implementation of AMPure XP beads for all purification steps and prim
53. nal 45 Step 3 Repair the ends 46 Step 4 Purify the sample using Agencourt AMPure XP beads 47 Step 5 Add A Bases to the 3 end of the DNA fragments 48 Step 6 Purify the sample using Agencourt AMPure XP beads 49 Step 7 Ligate the paired end adaptor 50 SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing5 Contents Step 8 Purify the sample using Agencourt AMPure XP beads 51 Step 9 Amplify adaptor ligated library 52 Step 10 Purify the sample with Agencourt AMPure XP beads 54 Step 11 Assess quality and quantity 55 Hybridization 59 Step 1 Hybridize the library 62 Step 2 Prepare magnetic beads 68 Step 3 Select hybrid capture with SureSelect 69 5 Addition of Index Tags by Post Hybridization Amplification 71 Step 1 Amplify the captured library to add index tags 72 Step 2 Purify the sample using Agencourt AMPure XP beads 76 Step 3 Assess quality 77 Step 4 Assess the quantity of each index tagged library by QPCR optional 80 Step 5 Pool samples for Multiplexed Sequencing 81 Step 6 Prepare sample for cluster amplification 82 Reference 83 SureSelect Reagent Kit Content 84 Other Reagent Kits Content 86 SureSelect Indexes for Illumina 87 Alternative Capture Equipment Combinations 88 SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing SureSelect Target Enrichment System for Illumina Paired End Sequencing Protocol 1 Before You Begin Procedural
54. nce of each of the SureSelec index is listed in Table 42 Table 42 SureSelect Indexes 1 16 Index Number Sequence 1 ATCACG 2 CGATGT 3 TTAGGC 4 TGACCA 5 ACAGTG 6 GCCAAT 7 CAGATC 8 ACTTGA 9 GATCAG 10 TAGCTT 11 GGCTAC 12 CTTGTA 13 AAACAT 14 CAAAAG 15 GAAACC 16 AAAGCA SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing 87 6 88 Reference Alternative Capture Equipment Combinations Table 43 lists combinations of thermal cycler lid temperature plates or strips and sealing method strip caps or sealing tape other than those used in this protocol that have shown minimal evaporation Refer to this list for additional of equipment combination options for hybridization Note that minimal evaporation is needed to ensure good capture results Table 43 Tested options that show minimal evaporation PCR Machine Plate Strips Cover Comments Agilent Mx3005P Mx3000P Strip Tubes MX3000P Optical Strip Heated Lid QPCR 401428 Caps 401425 Agilent Mx3005P MicroAmp Optical MicroAmp Clear Heated Lid QPCR ABI GeneAmp 9700 ABI Veriti 4375786 Eppendorf Mastercycler BioRad MJ Research PTC 200 BioRad MJ Research PTC 200 BioRad MJ Research PTC 200 96 well reaction plate N801 0560 MicroAmp Optical 96 well Reaction Plate N801 0560 MicroAmp Optical 96 well Reaction Plate N801 0560 Eppendorf 8 Tube PCR Tubes Agilent strip tubes 410022 Mx400
55. ogies 83 6 84 Reference SureSelect Reagent Kit Content SureSelect capture libraries and reagents must be used within one year of receipt Each SureSelect Reagent Kit contains one or more of each of these kits Table 35 SureSelect Reagent Kit Contents Product Storage 16 96 480 Condition Reactions Reactions Reactions SureSelect Target Enrichment Kit Room 5190 4393 5190 4394 5190 4395 Box 1 Temperature SureSelect Target Enrichment Kit ILM 20 C 5190 4455 5190 4456 5190 4457 Indexing Hyb Module Box 2 SureSelect Library Prep Kit ILM 20 C 5500 0105 5500 0075 The content of each of these kits are described in the next tables Table 36 SureSelect Target Enrichment Kit Box 1 Kit Component SureSelect Hyb 1 orange cap or bottle SureSelect Hyb 2 red cap SureSelect Hyb 4 black cap or bottle SureSelect Binding Buffer SureSelect Wash 1 SureSelect Wash 2 SureSelect Elution Buffer SureSelect Neutralization Buffer These reagents are not used in this protocol SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing Reference 6 Table 37 SureSelect Target Enrichment Kit ILM Indexing Hyb Module Box 2 Kit Component SureSelect Hyb 3 yellow cap SureSelect Indexing Block 1 green cap SureSelect Block 2 blue cap SureSelect Indexing Block 3 brown cap SureSelect RNase Block purple cap SureSelect ILM Indexing Pre Capture PCR Reverse Pr
56. or more for accurate quantitation Stopping Point 2 Load the sample plate or tube strips from step 1 the D1000 ScreenTape and loading tips into the 2200 TapeStation as instructed in the Agilent 2200 TapeStation User Manual Start the run 3 Verify that the electropherogram shows a distribution with a peak size approximately 225 to 275 bp A sample electropherogram is shown in Figure 5 If you do not continue to the next step seal the sheared DNA sample plate and store at 4 C overnight or at 20 C for prolonged storage Sample Preparation 3 pg DNA Samples 2 Step 12 Assess quality and quantity Sample Intensity FU o 3l MW i 2 bp Figure5 Analysis of amplified library DNA using the 2200 TapeStation with a D1000 ScreenTape The electropherogram shows a single peak in the size range of 225 to 275 bp 300 500 700 ol n GH zi SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing 37 38 This page intentionally left blank SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing CAUTION SureSelect Target Enrichment System for Illumina Paired End Sequencing Protocol 3 Sample Preparation 200 ng DNA Samples Step 1 ShearDNA 43 Step 2 Assess quality optional 45 Step 3 Repair the ends 46 Step 4 Purify the sample using Agencourt AMPure XP beads 47 Step 5 Add A Bases to the 3 end of the DNA fragments 48 Step 6 Purify the sample
57. p 7 step once 9 Spin briefly in a centrifuge Return the tube to the magnetic stand for 30 seconds Remove residual Ethanol with a P20 pipette 10 Dry the samples on the 37 C heat block for 5 minutes or until the residual Ethanol completely evaporates Do not dry the bead pellet to the point that the bead pellet appears cracked Elution efficiency is significantly decreased when the bead pellet is excessively dried 11 Add 15 uL of Nuclease free Water mix well on a vortex mixer and incubate for 2 minutes at room temperature 12 Put the tube in the magnetic stand and leave for 2 to 3 minutes until the solution is clear 13 Remove the supernatant 15 uL to a fresh 1 5 mL LoBind Tube You can discard the beads at this time 14 Proceed immediately to the next step Step 8 Ligate the paired end adaptor Sample Preparation 3 pg DNA Samples 2 Step 8 Ligate the paired end adaptor This step uses a 10 1 molar ratio of adaptor to genomic DNA insert based on a starting quantity of 3 ug of DNA before shearing Use the SureSelect Library Prep Kit ILM 1 Prepare reaction mix and DNA sample prepare on ice a Prepare the reaction mix in Table 15 b Add 37 uL of the reaction mix to each well or tube c Add 13 uL of each DNA sample to each well or tube Mix by pipetting Change pipette tips between samples Table 15 Ligation master mix Reagent Volume for 1 Library Volume for 12 Libraries includes excess
58. prior to addition of the indexing tag by PCR The ratio of SureSelect capture library to prepped library is critical for successful capture eig Agilent Technologies e 4 Hybridization GENOMIC SAMPLE Set of chromosomes SureSelect 000000000000 Target Enrichment System 000000000000C Capture Process ie Kit Ane VV 00o 0000 B NN GENOMIC SAMPLE PREPPED SureSelect HYB BUFFER Seite LIBRARY O Hybridization 2000 9000F 20000 STREPTAVIDIN COATED MAGNETIC BEADS 9000F 0 00000 oo M ATA 00000 2A capture UNBOUND FRACTION Wash Beads DISCARDED Amplify DAVA Add Multiplex Tag IN Sequencing VW VW equimotar pooling of multiplexed samples Figure 10 SureSelect Target Enrichment System Capture Process 60 SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing Hybridization 4 Refer to SureSelect Reagent Kit Content on page 84 for a complete content listing of each SureSelect Target Enrichment kit CAUTION You must avoid evaporation from the small volumes of the capture during the 16 or 24 hour incubation If you want to use a different combination of thermal cycler lid temperature plates or strips and sealing method strip caps or sealing tape first test the conditions Incubate 27 uL of SureSelect Hybridization Buffer without DNA at 65 C for 24 hours as a test Include buffer in each well that you might use including those in the center and those on t
59. ps from step 1 the D1000 ScreenTape and loading tips into the 2200 TapeStation as instructed in the Agilent 2200 TapeStation User Manual Start the run 3 Verify that the electropherogram shows a distribution with a peak size approximately 225 to 275 bp A sample electropherogram is shown in Figure 9 If you do not continue to the next step seal the sheared DNA sample plate and store at 4 C overnight or at 20 C for prolonged storage Sample Preparation 200 ng DNA Samples 3 Step 11 Assess quality and quantity Sample Intensity FU o 3l MW 3l i 3 lbp Figure9 Analysis of amplified library DNA using the 2200 TapeStation with a D1000 ScreenTape The electropherogram shows a single peak in the size range of 225 to 275 bp o s ol o n GH m zi SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing 57 58 This page intentionally left blank SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing CAUTION SureSelect Target Enrichment System for Illumina Paired End Sequencing Protocol ee 4 Hybridization Step 1 Hybridize the library 62 Step 2 Prepare magnetic beads 68 Step 3 Select hybrid capture with SureSelect 69 This chapter describes the steps to combine the prepped library with the hybridization reagents blocking agents and the SureSelect capture library Each DNA library sample must be hybridized and captured individually
60. quality and quantity on page 35 After quantitation adjust the sample to 221 ng uL Alternatively concentrate a 750 ng aliquot at lt 45 C down to 3 4 uL If the sample dries up completely resuspend in 3 4 uL of water and mix ona vortex mixer If processing multiple samples adjust to equivalent volumes before concentrating Mix the components in Table 26 at room temperature to prepare the hybridization buffer SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing Hybridization 4 Table 26 Hybridization Buffer Reagent Volume for 1 Volume for 6 Volume for 12 capture pL captures pL captures pL includes excess includesexcess includes excess SureSelect Hyb 1 orange cap or 25 125 250 bottle SureSelect Hyb 2 red cap 1 5 10 SureSelect Hyb 3 yellow cap 10 50 100 SureSelect Hyb 4 black cap or 13 65 130 bottle Total 49 40 pL 245 40 490 40 needed pL sample pL sample 4 If precipitate forms warm the hybridization buffer at 65 C for 5 minutes 5 Ina PCR plate prepare the SureSelect capture library mix for target enrichment a Keep capture library mix tubes on ice during preparation and until they are used in step 10 b For each sample add the amount of SureSelect capture library as listed in Table 27 based on the Mb target size of your design c Use nuclease free water to prepare a dilution of the SureSelect RNase Block purple cap as listed in Table 27
61. re and for the Department of Defense DFARS 252 227 7015 Technical Data Commercial Items and DFARS 227 7202 3 Rights in Commercial Computer Software or Com puter Software Documentation Safety Notices CAUTION A CAUTION notice denotes a haz ard It calls attention to an operat ing procedure practice or the like that if not correctly performed or adhered to could result in damage to the product or loss of important data Do not proceed beyond a CAUTION notice until the indi cated conditions are fully under stood and met A WARNING notice denotes a hazard It calls attention to an operating procedure practice or the like that if not correctly per formed or adhered to could result in personal injury or death Do not proceed beyond a WARNING notice until the indicated condi tions are fully understood and met SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing In this Guide This guide describes the recommended operational procedures to capture the genomic regions of interest using the Agilent SureSelect Target Enrichment Kit for Illumina Multiplex Sequencing This protocol is specifically developed and optimized to use biotinylated RNA oligomer libraries to enrich targeted regions of the genome from repetitive sequences and sequences unrelated to the research focus specifically adjusted to provide high performance with SureSelect This guide uses an optimized protocol
62. the DNA 1000 Assay 1 Check that the 2100 Bioanalyzer electrodes have been cleaned as instructed in the reagent kit guide Open the Agilent 2100 Expert Software version B 02 02 or higher turn on the 2100 Bioanalyzer and check communication 3 Prepare the chip samples and ladder as instructed in the reagent kit guide 4 Load the prepared chip into the 2100 Bioanalyzer and start the run within five minutes after preparation Within the instrument context choose the appropriate assay from the drop down list Start the run Enter sample names and comments in the Data and Assay context Verify the results Check that the electropherogram shows a distribution with a peak height between 150 to 200 bp CAUTION Sample Preparation 3 pg DNA Samples 2 FU 100 80 60 40 20 15 100 200 300 400 700 1500 bp Figure2 Analysis of sheared DNA using a DNA 1000 Bioanalyzer assay The electro pherogram shows a distribution with a peak size between 150 to 200 bp 2200 TapeStation and D1000 ScreenTape You can use the 2200 TapeStation for rapid analysis of multiple samples Use the D1000 ScreenTape and D1000 Reagents For more information to do this step see the Agilent 2200 TapeStation User Manual 1 Prepare the TapeStation samples as instructed in the Agilent 2200 TapeStation User Manual Use 1 uL of each sheared DNA sample diluted with 3 pL of D1000 sample buffer for the analysis Make sure that you
63. the High Sensitivity D1000 ScreenTape and High Sensitivity D1000 Reagents For more information to do this step see the Agilent 2200 TapeStation User Manual 1 Prepare the TapeStation samples as instructed in the Agilent 2200 TapeStation User Manual Use 2 uL of each amplified library 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 or more for accurate quantitation 2 Load the sample plate or tube strips from step 1 the High Sensitivity D1000 ScreenTape and loading tips into the 2200 TapeStation as instructed in the Agilent 2200 TapeStation User Manual Start the run 3 Verify that the electropherogram shows an average DNA fragment size of about 300 bp A sample electropherogram is shown in Figure 15 Stopping Point If you do not continue to the next step seal the sample plate and store at 4 C overnight or at 20 C for prolonged storage 78 SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing Addition of Index Tags by Post Hybridization Amplification 5 Step 3 Assess quality Sample Intensity FU MW lbp 8 g x J oad adla Figure 15 Analysis of amplified library DNA using the 2200 TapeStation with a High Sensitivity D1000 ScreenTape The electropherogram shows a peak size range of 250 to 350 bp
64. the contents off of walls and lid 3 Store on ice or in a cold block until use e In general follow Biosafety Level 1 BL1 safety rules Safety Notes CAUTION Wear appropriate personal protective equipment PPE when working in the laboratory 8 SureSelectX Target Enrichment System Kit for Ilumina Multiplexed Sequencing Required Reagents Table 1 Before You Begin 1 Required Reagents for Library Prep and Post Hybridization Amplification Description Vendor and part number For use with 2100 Bioanalyzer DNA 1000 Kit High Sensitivity DNA Kit QPCR NGS Library Quantification Kit Illumina GA For use with 2200 TapeStation System D1000 ScreenTape D1000 Reagents High Sensitivity D1000 ScreenTape High Sensitivity D1000 Reagents Herculase II Fusion DNA Polymerase includes dNTP mix and 5x Buffer 200 reactions processes 100 XT libraries 400 reactions Nuclease free Water not DEPC treated Agencourt AMPure XP Kit 5 mL 60 mL 450 mL 1X Low TE Buffer 10 mM Tris HCI pH 8 0 0 1 mM EDTA Qubit dsDNA HS Assay Kit or Qubit dsDNA BR Assay Kit 100 assays 2 1000 ng 500 assays 2 1000 ng 1000 assays 2 1000 ng Qubit assay tubes Buffer EB 10mM Tris Cl ph 8 5 Ethanol 100 for molecular biology Agilent p n 5067 1504 Agilent p n 5067 4626 Agilent p n G4880A or equivalent Agilent p n 5067 5582 Agilent p n 5067 5583 Agilent p n 5067 5584 Agilent p n 5067 5585 Agilent p n 600677 p
65. tion of Index Tags by Post Hybridization Amplification 5 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 index sample to use Vif x C f ETON where Volume of Index V f is the final desired volume of the pool C f is the desired final concentration of all the DNA in the pool for example 10 nM for the standard Illumina protocol is the number of index and C is the initial concentration of each index sample Table 34 shows an example of the amount of 4 index tagged of different concentrations and 1X Low TE Buffer needed for a final volume of 20 uL at 10 nM Table 34 Example of index volume calculation for a total volume of 20 uL 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 1X Low TE Buffer 7 6 2 Adjust the final volume of the pooled library to the desired final concentration If the final volume of the combined index tagged samples is less than the desired final volume V f add 1X Low TE Buffer 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 S
66. uL T4 Polynucleotide Kinase orange cap 2 2 uL 27 5 uL Total Volume 52 uL 650 uL 52 pL sample Sample Preparation 200 ng DNA Samples 3 Step 4 Purify the sample using Agencourt AMPure XP beads 1 Let the AMPure XP beads come to room temperature for at least 30 minutes 2 Mix the reagent well so that the reagent appears homogeneous and consistent in color Do not freeze 3 Add 180 uL of homogeneous AMPure XP beads to a 1 5 mL LoBind Tube and add the end repaired DNA library 100 uL Mix well on a vortex mixer and incubate for 5 minutes 4 Put the tube in the magnetic stand Wait for the solution to clear approximately 3 to 5 minutes 5 Keep the tube in the magnetic stand Do not touch the beads while you carefully discard the cleared solution from the tubes 6 Continue to keep the tube in the magnetic stand while you dispense 500 uL of 70 Ethanol in each tube Use fresh 70 Ethanol for optimal result 7 Let the tube sit for 1 minute to allow any disturbed beads to settle and remove the Ethanol 8 Repeat step 6 and step 7 step once 9 Spin briefly in a centrifuge Return the tube to the magnetic stand for 30 seconds Remove residual Ethanol with a P20 pipette 10 Dry the samples on the 37 C heat block for 5 minutes or until the residual Ethanol completely evaporates Do not dry the bead pellet to the point that the bead pellet appears cracked Elution efficiency is significantly decreased when the bead pell
67. ureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing 81 5 Addition of Index Tags by Post Hybridization Amplification 3 If you store the library before sequencing add Tween 20 to 0 1 v v and store at 20 C short term 4 Proceed to template denaturation and flow cell preparation Refer to the appropriate Illumina protocol Exact library pool dilution and processing can vary based on the flow cell capacity and analysis pipeline versions being used Refer to the appropriate Illumina user guide for instructions This protocol has been validated with 100 base paired end reads However read length can be adjusted to achieve the desired research goals Step 6 Prepare sample for cluster amplification e Use the appropriate Illumina Paired End Cluster Generation Kit to do cluster amplification Refer to the instructions that are included with the Illumina Paired End Cluster Generation Kit The optimal seeding concentration for SureSelec libraries is 6 to pM depending on the desired output and data quality XT 82 SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing SureSelect Target Enrichment System for Illumina Paired End Sequencing Protocol 6 Reference SureSelect Reagent Kit Content 84 Other Reagent Kits Content 86 SureSelect Indexes for Illumina 87 Alternative Capture Equipment Combinations 88 This chapter contains reference information 5 Agilent Technol
68. y You may need to dilute your sample accordingly Refer to the Agilent High Sensitivity DNA Kit Guide at http www chem agilent com en US Search Library _layouts Agilent PublicationSummary aspx whid 59504 1 Check that the 2100 Bioanalyzer electrodes have been cleaned as instructed in the reagent kit guide 2 Open the Agilent 2100 Expert Software version B 02 07 or higher required to run the High Sensitivity Kit turn on the 2100 Bioanalyzer and check communication 3 Prepare the chip samples and ladder as instructed in the reagent kit guide 4 Load the prepared chip into the 2100 Bioanalyzer and start the run within five minutes after preparation 5 Within the instrument context choose the appropriate assay from the drop down list 6 Start the run Enter sample names and comments in the Data and Assay context 7 Verify the results Determine the concentration of the sample by integration under the peak SureSelect Target Enrichment System Kit for Illumina Multiplexed Sequencing 71 5 Addition of Index Tags by Post Hybridization Amplification Bl ees es 5 B ee a e 35 100 150 200 300 400 500 600 i 1000 2000 i 10380 bp Figure 14 Analysis of Amplified Capture DNA using the High Sensitivity DNA Kit The electropherogram shows a peak in the size range of approximately 250 to 350 bp 2200 TapeStation and High Sensitivity D1000 ScreenTape Use the 2200 TapeStation to analyze the barcoded DNA Use

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