Agilent Technologies HaloPlex Target Enrichment System for Illumina Sequencing guide
Contents
1. gt 3 Capture and ligate targets Biotinylated probe DNA fragment hybrids are captured on streptavidin beads Open circles are ligated by DNA ligase 4 PCR amplify targetted fragments Indexes are incorporated during PCR of the circular DNA resulting in a sequencing ready target enriched sample Figure 1 Overall HaloPlex target enriched sequencing sample preparation workflow 12 HaloPlex Target Enrichment System Sample Preparation 2 Step 1 Digest genomic DNA with restriction enzymes In this step the gDNA sample is aliquoted to eight digestion reactions each containing two restriction enzymes After digestion the eight reactions are pooled resulting in a single DNA sample containing a 16 enzyme restriction fragment library that includes both target and non target gDNA regions Successful enrichment requires high quality and carefully quantified DNA samples Make sure genomic DNA samples are of high quality with an OD 260 280 ratio ranging from 1 8 to 2 0 Verify the size distribution of DNA in each DNA preparation by gel electrophoresis Any smearing below 2 5 kb indicates sample degradation In the protocol below 800 ng genomic DNA is split among eight different restriction digests with an additional 100 ng excess DNA included to allow for pipetting losses Using 900 ng DNA in the enrichment protocol can result in low yield and can potentiate rare allele dropouts Use a fluorometry based2. OOOO OO OOOO OO OOOO OOOOOOO OOOOOOO 5 pl per well N 7 N C 2 U Aw C lt 2 F8 IS IS 0 UU CY VJ OO IOOC DO VW gt looo 6 7 89 1011 12 b Using a multichannel pipette carefully distribute 5 uL of DNA samples column wise into each well of the digestion reaction plate Visually inspect pipette tips for equal volumes before dispensing c Seal the plate thoroughly with adhesive plastic film The prepared 96 well plate now contains complete 10 uL restriction digestion reactions In this format each column of the plate corresponds to one DNA sample digested in eight different restriction reactions 7 Briefly spin the sealed plate in a plate centrifuge 8 Carefully vortex the plate to mix the digestion reactions 9 Briefly spin the plate in a plate centrifuge 10 Place the plate in a thermal cycler and run the program in Table 3 using a heated lid Table3 Thermal cycler program for HaloPlex restriction digestion Step Temperature Time Purpose Step 1 37 4 hours DNA digestion Step 2 80 20 minutes Enzyme inactivation Step 3 4 C Hold Hold HaloPlex Target Enrichment System 17 2 18 Sample Preparation 11 Validate the restriction digestion reaction by gel analysis of the Enrichment Control DNA reaction The Enrichment Control DNA sample contains genomic DNA mixed with an 800 bp PCR product that co
3. 11 HaloPlex Target Enrichment System1 500 kb ILM Box 1 Contents Included Reagents 16 Reaction Kit 96 Reaction Kit 480 Reaction Kit Hybridization Solution tube with clear cap bottle bottle Ligation Solution tube with clear cap bottle bottle Wash Solution tube with clear cap bottle bottle Capture Solution tube with clear cap bottle bottle DNA Ligase RE Buffer Haloase A1 Haloase A2 Haloase A Buffer Haloase B Haloase B Buffer Enrichment Control DNA Primer Cassette Primer 1 0 Index Primers Enzyme Strip 1 Enzyme Strip 2 HaloPlex Probe tube with red cap tube with clear cap tube with green cap tube with blue cap tube with clear cap tube with black cap tube with clear cap tube with orange cap tube with purple cap tube with yellow cap 16 tubes containing Index Primers A1 H2 8 well strip tube with green label 8 well strip tube with red label tube with pink cap tube with red cap bottle tube with green cap tube with blue cap bottle tube with black cap bottle tube with orange cap tube with purple cap tube with yellow cap 96 well plate with Index Primers A1 H12 8 well strip tube with green label 8 well strip tube with red label tube with pink cap tube with red cap bottle tube with green cap tube with blue cap bottle tube with black cap bottle tube with orange cap tube with purple cap tube with yellow cap 5 x 96 well plates with Index Primers A1 H12 8 well strip tube wi
4. Enrichment Control DNA to a fresh 0 2 mL PCR tube 2 Transfer 5 uL of a selection of enriched libraries prepared from your samples to separate fresh 0 2 mL PCR tubes 3 Add 2 uL of Novex Hi Density TBE Sample Buffer 5X to each sample 4 Prepare the XCell SureLock Mini Cell with a 12 well Novex 6 6 polyacrylamide pre cast gel Fill with Novex TBE Running Buffer 5 Load 7 uL of each sample on the gel In one or more adjacent lanes load 200 ng of a DNA ladder 6 Run the gel at 210 V for 15 minutes 7 Stain the gel in 3X GelRed Nucleic Acid Stain for 10 minutes and visualize bands under UV radiation Successful enrichment is indicated by the presence of a smear of amplicons from approximately 225 to 525 bp in each enrichment library lane See Figure 3 for a sample lane image Figure 5 Validation of HaloPlex enrichment process Lane 1 25 bp DNA ladder Lane 2 Enrichment Control DNA enriched library sample HaloPlex Target Enrichment System 31 2 Sample Preparation Figure 6 32 Step 9 Optional Validate and quantify target libraries using the 2100 Bioanalyzer Use a Bioanalyzer High Sensitivity DNA Assay kit and the Agilent 2100 Bioanalyzer with Agilent 2100 Expert Software version B 02 07 or higher required to run the High Sensitivity Kit See the reagent kit guide for general Bioanalyzer instrument and assay setup instructions 1 Prepare the chip samples and ladder as instructed in the reagent kit
5. Ligate the captured circularized fragments 24 Step 5 Remove non target DNA by Haloase A and B digestion 25 Step 6 Amplify and index captured target libraries 27 Step 7 Purify the amplified target libraries 29 Step 8 Optional Validate enrichment by gel analysis 31 Step 9 Optional Validate and quantify target libraries using the 2100 Bioanalyzer 32 Step 10 Pool samples with different indexes for multiplexed sequencing 33 This section contains instructions for gDNA library target enrichment for sequence analysis using the Illumina platform For each sample to be sequenced an individual target enriched indexed library is prepared The target region can vary from 1 to 500 kb The custom HaloPlex probe supplied with each kit must be designed before purchasing the kit using the online HaloPlex Design Wizard at www agilent com genomics ngs The HaloPlex Target Enrichment System amplifies thousands of targets in the same reaction incorporating standard Illumina paired end sequencing motifs in the process During the amplification each sample can be uniquely indexed allowing for pooling of up to 96 samples per sequencing lane See Figure 1 for a summary of the overall HaloPlex target enrichment workflow RE Agilent Technologies 2 Sample Preparation 1 Digest and denature DNA D mm Oe 2 Hybridize oligonucleotide probe library Hybridization results in DNA fragment circularization and incorporation of sequencing motifs
6. pipette add 6 5 uL of each enzyme from tubes A to H of Enzyme Strip 2 with red marker on tube A to each corresponding tube A to H of the master mix strip tube d Mix by gentle vortexing and then spin briefly to collect the liquid e Keep the Restriction Enzyme Master Mix Strip on ice until it is used in step 5 5 Aliquot the Restriction Enzyme Master Mixes to a 96 well restriction digest reaction plate a Align the Restriction Enzyme Master Mix Strip prepared in step 4 along the vertical side of a 96 well PCR plate as shown below Restriction Digestion Reaction Plate QOOOOOOQO00O OO m oo o gt RE Master Mix Strip Tonmnmoouo m 9 w DOOOOOO b Using a multichannel pipette carefully distribute 5 uL of each RE master mix row wise into each well of the digestion reaction plate Visually inspect pipette tips for equal volumes before dispensing to the plate Each row of the 96 well plate now contains 5 uL per well of the same restriction enzyme combination HaloPlex Target Enrichment System Sample Preparation 6 Aliquot gDNA samples into the 96 well restriction digest reaction plate a Align the 12 DNA samples 11 gDNA samples and the ECD sample prepared in step 2 and step 3 along the horizontal side of the digestion reaction plate as shown below gDNA Samples 1 11 20 ng uL ECD Vv vvvVVVVVY Zn Zu Zu ZZ Zu v
7. 2 minutes or until the solution is clear 18 Remove the cleared supernatant approximately 40 uL to a fresh tube You can discard the beads at this time Stopping Point If you do not continue to the next step samples may be stored at 20 C for long term storage up to one year Avoid subjecting the stored DNA samples to multiple freeze thaw cycles The HaloPlex target enriched sequencing library samples are now ready for validation and pooling for multiplex sequencing See page 31 to page 33 for recommended validation procedures and for pooling guidelines Each amplicon in the prepared library contains one target insert surrounded by sequence motifs required for multiplexed sequencing using the Illumina platform The amplicons should range from 225 to 525 bp in length including 100 to 400 bp of target DNA insert and 125 bp of sequencing motifs Figure 4 Content of HaloPlex enriched target amplicons Each amplicon contains one target insert blue surrounded by the Illumina paired end sequencing ele ments black the sample index red and the library bridge PCR primers yel low HaloPlex Target Enrichment System Sample Preparation 2 Step 8 Optional Validate enrichment by gel analysis Prior to sample pooling and sequencing sample preparation verify successful enrichment by gel analysis of the Enrichment Control DNA sample and a selection of your library samples 1 Transfer 5 uL of the captured library prepared from the
8. ATAGAA D7 TATGCG E7 CTCCGA F7 GCGAGG G7 GACCGC H7 GCGGTT A8 TCCGCT B8 CGATTC C8 CTGGTT D8 GTACCG E8 TTAAGA F8 TTCTCG G8 ATAAGC H8 TTAGTT HaloPlex Target Enrichment System 41 3 Reference Table 16 HaloPlex Indexes A9 H10 Index Number Sequence A9 GGTCCT B9 ACCGTC C9 GGAGTT D9 CAGACG E9 GGTTCA F9 GCTGCG G9 CATTAC H9 AGAATT A10 TCGTAT B10 TAAGTC C10 TACCTT D10 CCTGAG E10 ACGCCA F10 CTGGCG G10 TGATAC H10 ATCAGT 42 HaloPlex Target Enrichment System Reference 3 Table 17 HaloPlex Indexes A11 H12 Index Number Sequence A11 GTCCAT B11 TTCATC C11 TCTACT D11 TATTGC E11 CGACCA F11 CGAACG G11 CTAGAC H11 GGCGCT A12 GATTGG B12 AGCAGC C12 ATAACT D12 GAGAGC E12 TCGGAA F12 ATTCAG G12 TAGAAC H12 ACTTAT HaloPlex Target Enrichment System 43 www agilent com In This Book This guide contains information to run the HaloPlex Target Enrichment System protocol Agilent Technologies Inc 2012 Version A February 2012 G9900 90000 RE Agilent Technologies
9. DNA quantitation method such Qubit fluorometry or PicoGreen staining to accurately quantify the DNA starting material 1 Use the Qubit dsDNA BR Assay or PicoGreen staining kit to determine the concentration of your gDNA sample Follow the manufacturers instructions for the kits and instruments 2 Transfer 45 uL of the supplied Enrichment Control DNA to a 0 2 mL PCR tube Store on ice HaloPlex Target Enrichment System 13 2 14 Sample Preparation 3 In separate 0 2 mL PCR tubes dilute 900 ng of each gDNA sample in 45 uL nuclease free water for a final DNA concentration of 20 ng uL Store sample tubes on ice Prepare the appropriate number of gDNA samples for a single run 96 reaction kits and 480 reaction kits are configured for sample processing in sets of 12 samples 11 gDNA samples 1 control DNA sample per run 16 reaction kits may be used for sample processing in sets of up to 16 samples 15 gDNA samples 1 control DNA sample per run All instructions in the following section apply to 12 sample runs When processing gt 12 samples adjust the volumes of components added to master mixes accordingly and set up an additional restriction digest reaction plate for samples 13 to 16 Volumes specified in the following protocol are for 12 samples plus 1 reaction volume excess 13 reaction equivalents total Calculate the amount of each solution needed for the number of reactions in your run plus one reaction excess F
10. HaloPlex Target Enrichment System For Illumina Sequencing Protocol Version A February 2012 Research Use Only Not for use in Diagnostic Procedures Agilent Technologies Notices Agilent Technologies Inc 2012 No part of this manual may be reproduced in any form or by any means including elec tronic storage and retrieval or translation into a foreign language without prior agree ment and written consent from Agilent Technologies Inc as governed by United States and international copyright laws Manual Part Number G9900 90000 Edition Version A February 2012 Printed in USA Agilent Technologies Inc 5301 Stevens Creek Blvd Santa Clara CA 95051 USA Technical Support For technical product support contact your local Agilent Support Services representa tive For US and Canada call 800 227 9770 option 3 4 4 For other countries find your support center telephone numbers at www agilent com chem contactus Or send an e mail to SureSelect Support agilent com Notice to Purchaser Research Use Only Not for use in diagnostic procedures Warranty The material contained in this document is provided as is and is subject to being changed with out notice in future editions Fur ther to the maximum extent permitted by applicable law Agi lent disclaims all warranties either express or implied with regard to this manual and any information contained herein including bu
11. System 21 2 Sample Preparation Step 3 Capture the target DNA In this step the circularized target DNA HaloPlex probe hybrids containing biotin are captured on streptavidin beads 1 Remove reagents to be used in upcoming protocol steps from cold storage and allow the solutions to reach room temperature From 20 C storage remove the Capture Solution Wash Solution Ligation Solution Haloase A Buffer and Haloase B buffer From 4 C storage remove the Magnetic Beads 2 Vigorously resuspend the provided Magnetic Beads on a vortex mixer The magnetic beads settle during storage 3 Prepare 20 uL 1 Volume of Magnetic Beads per hybridization sample plus excess for the capture reaction a For 12 sample runs transfer 250 uL of the bead suspension to a 1 5 mL tube For runs that include greater or fewer than 12 samples adjust the volumes of the bead suspension and Capture Solution accordingly b Putthe tube into a 1 5 mL tube compatible magnetic rack for 5 minutes c Wait for the solution to clear then carefully remove and discard the supernatant using a pipette d Add 2 Volumes 500 uL for 12 sample runs of Capture Solution to the beads and resuspend by pipetting up and down 4 Add 40 uL of the prepared bead suspension to each 160 uL hybridization reaction When adding beads to the hybridization reactions visually inspect the bead preparation to ensure a homogenous suspension with no aggregated bead mass at
12. column wise into a 0 2 mL PCR tube as shown in the figure below Due to partial evaporation of samples you may recover less than 10 uL of each restriction digest Minor reductions to the digested DNA pool volume will not impact hybridization performance you do not need to compensate for any sample evaporation volume losses in the final pool 13 Pool the remaining 6 uL from each Enrichment Control DNA digest into a separate tube as shown in the figure below Add 32 uL of nuclease free water to compensate for the volume removed for gel analysis UU y l 811 IAF VP M Ay Y V B KICK IK IK I IK IKK IK I Fi CIO OOO OOOO OOD G OFO O O OO O OOG OO 1 2 34 5 6 7 8 9 10 1 12 u BU Ul uly M MI M u VJ VJ V WM Vi Vi Stopping Point If you do not continue to the next step samples may be stored at 20 C for long term storage There are no more stopping points until after the PCR amplification step on page 28 HaloPlex Target Enrichment System 19 2 20 Sample Preparation Step 2 Hybridize digested DNA to HaloPlex probes In this step the collection of gDNA restriction fragments is hybridized to the HaloPlex probe captur
13. ctions 480 reactions Phusion Hot Start High Fidelity DNA Polymerase dNTPs 25 mM for each nucleotide Nuclease free Water not DEPC treated Agencourt AMPure XP Kit 5 mL 60 mL 450 mL 10 mM Tris HCl pH 8 0 or 10 mM Tris acetate pH 8 0 10096 Ethanol molecular biology grade High Sensitivity DNA Kit Novex 6 Polyacrylamide TBE Pre cast Gels Novex TBE Running Buffer 5X Novex High density TBE Sample Buffer 5 GelRed Nucleic Acid Stain 3X in water DNA molecular weight markers Quant iT dsDNA BR Assay Kit for use with the Qubit fluorometer 100 assays 2 1000 ng 500 assays 2 1000 ng Vendor and part number Agilent p n G9900A p n G9900B p n G9900C Finnzymes p n F 549L Agilent p n 200415 Ambion Cat AM9930 Beckman Coulter Genomics p n A63880 p n A63881 p n A63882 General laboratory supplier Sigma Aldrich p n E7023 Agilent p n 5067 4626 Life Technologies p n EC62655BOX Life Technologies p n LC6675 Life Technologies p n LC6678 Biotium p n 41001 General laboratory supplier Life Technologies p n 032850 Life Technologies p n 032853 HaloPlex Target Enrichment System 1 10 Before You Begin Required Equipment Table2 Required Equipment for HaloPlex Target Enrichment Description Thermal Cycler Thermal cycler compatible 96 well plates 8 well PCR strip tubes with caps 2100 Bioanalyzer Laptop Bundle 2100 Bioanalyzer Electrophoresis Set XCell SureLock Mini cell for gel elect
14. d DNA to HaloPlex probes 20 Step 3 Capture the target DNA 22 Step 4 Ligate the captured circularized fragments 24 Step 5 Remove non target DNA by Haloase A and B digestion 25 Step 6 Amplify and index captured target libraries 27 Step 7 Purify the amplified targetlibraries 29 Step 8 Optional Validate enrichment by gel analysis 31 Step 9 Optional Validate and quantify target libraries using the 2100 Bioanalyzer 32 Step 10 Pool samples with different indexes for multiplexed sequencing 33 3 Reference Kit Contents 36 Nucleotide Sequences of HaloPlex Indexes 38 HaloPlex Target Enrichment System HaloPlex Target Enrichment System HaloPlex Target Enrichment System Protocol 1 et Before You Begin Procedural Notes 8 Safety Notes 8 Required Reagents 9 Required Equipment 10 Make sure you read and understand the information in this chapter and have the necessary equipment and reagents listed before you start an experiment Agilent cannot guarantee the HaloPlex Target Enrichment Kits and cannot provide technical support for the use of non Agilent protocols or reagents to process samples for enrichment X Agilent Technologies 7 1 Before You Begin Procedural Notes The HaloPlex target enrichment protocol is configured for processing samples in sets of 12 samples per run A 96 reaction kit contains enough reagents to prepare master mixes for eight runs of 12 samples each When processing samples us
15. e to the product or loss of important data Do not proceed beyond a CAUTION notice until the indicated conditions are fully understood and met A WARNING notice denotes a hazard It calls attention to an operating procedure practice or the like that if not correctly per formed or adhered to could result in personal injury or death Do not proceed beyond a WARNING notice until the indicated condi tions are fully understood and met HaloPlex Target Enrichment System In this Guide HaloPlex Target Enrichment System This guide describes an optimized protocol for using the Agilent HaloPlex target enrichment system to prepare sequencing library samples for Illumina paired end multiplexed sequencing platforms 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 Sample Preparation This chapter describes the steps of the HaloPlex workflow to prepare target enriched sequencing libraries for the Illumina platform Reference This chapter contains reference information including component kit contents and index sequences HaloPlex Target Enrichment System Content 1 Before You Begin Procedural Notes 8 Safety Notes 8 Required Reagents 9 Required Equipment 10 2 Sample Preparation Step 1 Digest genomic DNA with restriction enzymes 13 Step 2 Hybridize digeste
16. e library HaloPlex probes are designed to hybridize selectively to fragments originating from target regions of the genome and to direct circularization of the targeted DNA fragments During the hybridization process Illumina sequencing motifs are incorporated into the targeted fragments 1 Prepare a Hybridization master mix by combining the reagents in the following table Mix well by gentle vortexing then spin the tube briefly Table4 Hybridization Master Mix Reagent Volume for 1 Reaction Volume for 12 Reactions includes excess Hybridization Solution 65 uL 845 uL Primer Cassette 1 HL 13 HL HaloPlex Probes 14 uL 182 uL Total Volume 80 pL 1040 pL 2 Add 80 uL of the Hybridization Master Mix to each digested DNA sample 3 Vortex the reactions briefly and then spin tubes briefly 4 Place the tubes in a thermal cycler and run the program in Table 5 using a heated lid HaloPlex Target Enrichment System Sample Preparation 2 Table5 Thermal cycler program for HaloPlex probe hybridization Step Temperature Time Step 1 95 C 10 minutes Step 2 75 C 30 minutes Step 3 68 C 30 minutes Step 4 62 C 30 minutes Step 5 55 C 30 minutes Step 6 46 C 10 hours Step 7 8 C Hold Thermal cyclers that use calculated temperature methods cannot be set to 160 uL reaction vol umes In that case enter the maximum possible volume t Samples can be held at 8 C or 4 C for up to 72 hours prior to capture HaloPlex Target Enrichment
17. erative to ensure optimal purification results 5 Mix the reactions thoroughly by pipetting up and down 15 times using a 100 uL pipette set to 80 uL 6 Incubate samples for 5 minutes at room temperature 7 Putthe tube into a magnetic rack Wait for the solution to clear approximately 2 minutes 8 Keep the tube in the magnetic rack Carefully remove and discard the cleared solution from each well using a 100 uL pipette set to 100 uL Do not touch the beads while removing the solution 9 Continue to keep the tube in the magnetic rack while you add 100 uL of 70 ethanol into the tube Use fresh 70 ethanol for optimal results 10 Wait for 1 minute to allow any disturbed beads to settle then remove the ethanol using a 100 uL pipette set to 100 uL 11 Repeat step 9 and step 10 step once for a total of two washes 12 Remove any residual ethanol with a 20 uL volume pipette 13 Air dry the tubes with open lids at room temperature until the residual ethanol completely evaporates Make sure all ethanol has evaporated before continuing 14 Remove tubes from the magnetic rack and add 40 uL of 10 mM Tris acetate or Tris HCl buffer pH 8 0 to each sample 15 Mix thoroughly by pipetting up and down 15 times using a 100 uL pipette set to 30 uL HaloPlex Target Enrichment System 29 30 Sample Preparation 16 Incubate for 2 minutes at room temperature to allow elution of DNA 17 Put the tube in the magnetic rack and leave for
18. ette set to 50 uL If necessary carefully remove any residual liquid with a 20 uL volume pipette 7 Add 21 5 uL of Haloase Buffer to the beads in each DNA capture reaction tube HaloPlex Target Enrichment System 25 2 26 Sample Preparation 8 Resuspend the beads thoroughly by pipetting up and down 10 times using a 100 uL multichannel pipette set to 15 uL 9 Incubate the tubes in a thermal cycler at 80 for 20 minutes using heated lid to inactivate any residual Haloase A activity 10 Remove the tubes from the thermal cycler and wait for the reactions to cool to room temperature 11 Add 3 5 uL of Haloase B to the 21 5 uL bead suspension in each reaction tube 12 Resuspend the beads thoroughly by pipetting up and down 10 times using a 100 uL multichannel pipette set to 15 uL 13 Incubate the tubes in a thermal cycler at 37 C for 30 minutes using a heated lid The thermal cycler may be programmed to include a 4 C hold step following the 30 minute incubation During the 30 minute incubation prepare the PCR master mix used in the following step HaloPlex Target Enrichment System Sample Preparation 2 Step 6 Amplify and index captured target libraries In this step the captured DNA is amplified and index tagged in PCR reactions containing Illumina Primer 1 0 along with the appropriate Index Primer For sample indexing primer assignments see Nucleotide Sequences of HaloPlex Indexes on page 38 for n
19. guide using 1 uL of captured library samples for the analysis 2 Load the prepared chip into the 2100 Bioanalyzer and start the run within five minutes after preparation 3 Check that the electropherogram shows a fragment size distribution between approximately 225 to 525 bp Sample electropherograms for libraries prepared using three different probe designs are shown in Figure 6 4 Determine the concentration of each library pool by integration under the peak in the electropherogram Design 1 Fu Design 2 Fu Design 3 200 150 50 100 50 0 0 150 300 500 1000 10380 bp 35 150 300 500 1000 10380 bp 35 150 400 10380 bp Analysis of amplified enrichment products from three custom probe designs using the Agilent Bioan alyzer High Sensitivity DNA Assay The electropherograms show a DNA size distribution of approxi mately 225 to 525 bp Some iterated designs have non default size distributions such as the sample enriched using Design 2 which contains amplicons from 175 to 525 bp HaloPlex Target Enrichment System Sample Preparation 2 Step 10 Pool samples with different indexes for multiplexed sequencing Use the following guidelines to design your sample pooling strategy Determine the DNA concentration in each enriched sample The expected output concentration is at least 10 nM Pool equimolar amounts of each sample to optimize the use of sequencing capacity The average amplicon length in any design is approximate
20. ing runs with fewer than 12 samples some reagents may be depleted before 96 samples are run The HaloPlex protocol is optimized for digestion of 800 ng of genomic DNA split among 8 different restriction digestion reactions plus 100 ng excess DNA for a total of 900 ng genomic DNA Using lower amounts of DNA in the enrichment protocol can adversely affect your results Use a fluorometry based DNA quantitation method such as PicoGreen stain or Qubit fluorometry to quantify the DNA starting material Always keep pre amplification and post amplification DNA samples in separate work areas Perform the enrichment procedure in the pre amplification area Open and store the amplified enriched DNA samples only in the post amplification area Possible stopping points where DNA samples may be stored overnight are marked in the protocol Store the samples at 20 C but do not subject the samples to multiple freeze thaw cycles Ensure that master mixes are thoroughly mixed by pipetting up and down or by gentle vortexing before distributing to the samples n general follow Biosafety Level 1 BL1 safety rules Safety Notes CAUTION Wear appropriate personal protective equipment PPE when working in the laboratory 8 HaloPlex Target Enrichment System Before You Begin 1 Required Reagents Table 1 Required Reagents for HaloPlex Target Enrichment Description HaloPlex Target Enrichment System Kit 16 reactions 96 rea
21. ly 335 bp The final HaloPlex enrichment pool is ready for direct sequencing using standard Illumina paired end primers and chemistry on the Illumina HiSeq MiSeq or GAIIx platform depending on the platform selected during probe design HaloPlex Target Enrichment System 33 2 Sample Preparation Step 10 Pool samples with different indexes for multiplexed sequencing 34 HaloPlex Target Enrichment System HaloPlex Target Enrichment System Protocol 3 et Reference Kit Contents 36 Nucleotide Sequences of HaloPlex Indexes 38 This chapter contains reference information including component kit contents and index sequences ES Agilent Technologies 35 3 Reference Kit Contents The HaloPlex Target Enrichment System includes the following component kits Table 10 HaloPlex Target Enrichment System Kit Contents Component Kits Storage Condition 16 Reactions 96 Reactions 480 Reactions HaloPlex Target Enrichment System 20 5190 5017 5190 5021 5 x 5190 5311 1 500 kb ILM Box 1 HaloPlex Target Enrichment System 4 C 5190 5015 5190 5019 5 x 5190 5019 1 500 kb ILM Box 2 See Table 11 for list of included reagents t For long term storage gt 30 days store Enzyme Strip 1 and Enzyme Strip 2 at 70 C t Contains Magnetic Beads 36 HaloPlex Target Enrichment System Reference The contents of the HaloPlex Target Enrichement System Box 1 are detailed in the table below Table
22. ntains restriction sites for all the enzymes used in the digestion protocol Using a multichannel pipette carefully transfer 4 uL of each Enrichment Control DNA digestion reaction from wells A12 to H12 of the reaction plate to fresh 0 2 mL PCR tubes Prepare an undigested DNA gel control by combining 2 uL of the Enrichment Control DNA stock solution and 2 uL of nuclease free water in a separate tube Add 1 uL of Novex Hi Density TBE Sample Buffer 5X to each sample Prepare the XCell SureLock Mini Cell with a 12 well Novex 6 polyacrylamide TBE pre cast gel Fill with 1X Novex TBE Running Buffer Load 5 uL of each sample on the gel In one or more adjacent lanes load 200 ng of a 50 bp DNA ladder Run the gel at 210 V for 15 minutes Stain the gel in 3X GelRed Nucleic Acid Stain for 10 minutes and visualize bands under UV radiation The undigested control should have gDNA bands gt 2 5 kbp and a PCR product band at 800 bp Digested samples should have a smear of gDNA restriction fragments between 100 and 2500 bp overlaid with three distinct bands at 125 225 and 450 bp See Figure 3 for a sample gel Figure 3 M2 Validation of restriction digestion Lane 1 50 bp DNA ladder Lane 2 Undigest ed Enrichment Control DNA Lanes 3 10 ECD digestion reactions 1 8 HaloPlex Target Enrichment System Sample Preparation 2 12 Pool the eight 10 restriction digest reactions for each gDNA sample
23. or example preparation of the Restriction Enzyme Master Mix strip on page 16 requires 52 uL of RE buffer for 13 reaction equivalents or 4 uL per reaction For 16 reaction runs use 17 reaction equivalents or 68 uL of RE Buffer HaloPlex Target Enrichment System Sample Preparation 2 4 Prepare the Restriction Enzyme Master Mix strip In this step eight separate restriction enzyme master mixes are prepared by combining two enzymes from Enzyme Strips 1 and 2 and restriction buffer in each well of an 8 well strip tube Figure Z illustrates how to prepare the Restriction Enzyme Master Mix strip using the steps detailed on page 16 RE Buffer INE amp WV VV V ME Uu N WV RE Master Mix Strip Enzyme Strip 1 i al A B c D E F G H UU 6 5 ul to corre sponding well A B Ww RE Master Mix Strip Enzyme Strip 2 4 A C DE GH I 6 5 ul to corre sponding well D E F 6 RE Master Mix Strip Figure 2 Preparation of the Restriction Enzyme Master Mix Strip HaloPlex Target Enrichment System 15 2 16 Sample Preparation a Add 52 uL of RE Buffer to each tube of an 8 well strip tube b Using a multichannel pipette add 6 5 uL of each enzyme from tubes A to H of Enzyme Strip 1 with green marker on tube A to each corresponding tube A to H of the master mix strip tube c Using a multichannel
24. pernatant using a pipette set to 120 uL If necessary carefully remove any residual liquid with a 20 uL volume pipette HaloPlex Target Enrichment System 23 2 Sample Preparation Step 4 Ligate the captured circularized fragments In this step DNA ligase is added to the capture reaction to catalyze the formation of closed circular DNA from the circularized target DNA HaloPlex probe hybrids Non target DNA in the capture reaction liquid phase remains in linear fragment form 1 Prepare a DNA ligation master mix by combining the reagents in the following table Mix the components thoroughly by pipetting up and down Table 6 Preparation of DNA ligation master mix Reagent Volume for 1 Reaction Volume for 12 Reactions includes excess Ligation Solution 47 5 uL 617 5 uL DNA Ligase 2 5 uL 32 5 uL Total Volume 50 pL 650 pL 2 Add 50 uL of the DNA ligation master mix to the beads in each DNA capture reaction tube 3 Resuspend the beads thoroughly by pipetting up and down 10 times using a 100 uL multichannel pipette set to 40 uL 4 Incubate the tubes in a thermal cycler at 55 for 10 minutes using heated lid The thermal cycler may be programmed to include a 4 C hold step following the 10 minute incubation 5 Briefly spin the tubes in a desktop centrifuge and then transfer the tubes to the magnetic rack 6 Wait for the solution to clear about 30 seconds then carefully remove and discard the supernatant using a
25. pipette set to 70 uL If necessary carefully remove any residual liquid with 20 uL volume pipette 24 HaloPlex Target Enrichment System Sample Preparation 2 Step 5 Remove non target DNA by Haloase A and B digestion In this step linear non target DNA fragments are digested by the exonuclease activity of the provided Haloase A and B enzymes During the Haloase B treatment circularized target DNA is released from the streptavidin beads into the liquid phase 1 Prepare the Haloase A master mix by combining the reagents in the following table Mix the components thoroughly by pipetting up and down Table7 Preparation of Haloase A master mix Reagent Volume for 1 Reaction Volume for 12 Reactions includes excess Haloase A Buffer 22 5 HL 315 uL Haloase A1 0 5 uL 7 uL Haloase A2 2 0 uL 28 uL Total Volume 25 pL 350 pL 2 Add 25 uL of the Haloase A master mix to the beads in each DNA capture reaction tube 3 Resuspend the beads thoroughly by pipetting up and down 10 times using a 100 uL multichannel pipette set to 15 uL 4 Incubate the tubes in a thermal cycler at 37 C for 30 minutes using a heated lid The thermal cycler may be programmed to include a 4 C hold step following the 30 minute incubation 5 Briefly spin the tubes in a desktop centrifuge and then transfer the tubes to the magnetic rack 6 Wait for the solution to clear about 30 seconds then carefully remove and discard the supernatant using a 100 uL pip
26. ppropriate Index Primer Mix by pipetting up and down or by gentle vortexing 6 Runthe program in Table 9 in a thermal cycler using a heated lid The optimal amplification cycle number varies for each custom HaloPlex Probe design Consult the Certificate of Analysis provided with HaloPlex Target Enrichment System Box 1 for the PCR cycling recommendation for your custom probe Table 9 Segment Number of Cycles Temperature Time 1 1 98 30 seconds 2 Obtain cycle number 98 C 10 seconds from Certificate of Analysis 65 30 seconds 72 30 seconds 3 1 72 5 minutes 4 1 8 C Hold Stopping Point If you do not continue to the next step PCR products may be stored at 20 C for up to 72 hours For best results however purify PCR products as soon as possible 28 HaloPlex Target Enrichment System Sample Preparation Step 7 Purify the amplified target libraries In this step the amplified target DNA is purified using AMPure XP beads 1 Let the AMPure XP beads come to room temperature for at least 30 minutes 2 Transfer 40 uL of each PCR reaction sample to a fresh tube Store the remaining volume of each sample at 20 C for troubleshooting 3 Mix the bead suspension well until the suspension appears homogeneous and consistent in color 4 Add 60 uL 1 5 Volumes of homogenous AMPure XP beads to each 40 uL amplified library sample Pipette up and down to mix Using a bead to sample volume ratio of 1 5 1 is imp
27. rophoresis Benchtop microcentrifuge Benchtop plate centrifuge Qubit 2 0 Fluorometer Qubit assay tubes 96 well plate compatible magnetic separator 1 5 mL tube compatible magnetic separator Multichannel pipettes 10 uL and 100 uL volume P10 P20 P200 and P1000 pipettes Adhesive seals for 96 well PCR plates Ice bucket Vortex mixer Vendor and part number Agilent SureCycler 8800 p n G8800A 96 well plate module p n G8810A compression mats p n 410087 or equivalent thermal cycler and accessories Agilent p n 401333 for SureCycler 8800 or see manufacturer s recommendations Nippon Genetics p n FG 088WF or equivalent Agilent p n G2943CA Agilent p n G2947CA Life Technologies p n E10001 VWR p n 93000 196 or equivalent Labnet International MPS1000 Mini Plate Spinner p n C1000 or equivalent Life Technologies p n 032866 Life Technologies p n 032856 Agencourt SPRIPlate Super Magnet Plate p n A32782 DynaMag 2 magnet Life Technologies p n 12321D or equivalent Pipetman or equivalent Pipetman P10 P20 P200 P1000 or equivalent Agilent p n 410186 or equivalent General laboratory supplier General laboratory supplier HaloPlex Target Enrichment System HaloPlex Target Enrichment System Protocol 2 200 Sample Preparation e Step 1 Digest genomic DNA with restriction enzymes 13 Step 2 Hybridize digested DNA to HaloPlex probes 20 Step 3 Capture the target DNA 22 Step 4
28. t not limited to the implied warranties of merchant ability and fitness for a particular purpose Agilent shall not be lia ble for errors or for incidental or consequential damages in con nection with the furnishing use or performance of this document or of any information contained herein Should Agilent and the user have a separate written agreement with warranty terms covering the material in this doc ument that conflict with these terms the warranty terms in the separate agreement shall control Technology Licenses The hardware and or software described in this document are furnished under a license and may be used or copied only in accor dance with the terms of such license Restricted Rights Legend U S Government Restricted Rights Soft ware and technical data rights granted to the federal government include only those rights customarily provided to end user cus tomers Agilent provides this customary commercial license in Software and techni cal data pursuant to FAR 12 211 Technical Data and 12 212 Computer Software and for the Department of Defense DFARS 252 227 7015 Technical Data Commercial Items and DFARS 227 7202 3 Rights in Commercial Computer Software or Com puter Software Documentation Safety Notices CAUTION A CAUTION notice denotes a haz ard It calls attention to an operat ing procedure practice or the like that if not correctly performed or adhered to could result in damag
29. th green label 8 well strip tube with red label tube with pink cap HaloPlex Target Enrichment System 3 37 3 38 Reference Nucleotide Sequences of HaloPlex Indexes The nucleotide sequence of the index portion of each HaloPlex Index Primer is provided in the tables below HaloPlex 16 reaction kits include tubes containing the 16 primers listed in Table 12 The 96 and 480 reaction kits include plates containing the 96 indexes listed in column wise order in Table 12 to Table 17 Table 12 HaloPlex Indexes A1 H2 Index Number Sequence Al CTCGGT B1 TTACGG C1 GCGTCC D1 GAGTAT E1 ATATAC F1 GGCCTT G1 CCGTTC H1 ATGGTA A2 AATCGT B2 CCTTCG C2 AGGTAC D2 AGCTAT E2 GCCGAC F2 AGACGT G2 TACTTC H2 GTACGA HaloPlex Target Enrichment System Reference 3 Table 13 HaloPlex Indexes A3 H4 Index Number Sequence A3 GCGCGT B3 GGAGCG C3 ACGTTA D3 CAAGAT E3 CTTAAC F3 CATAGT G3 GAGGTC H3 AAGAGA A4 CGAAGT B4 ACGCAG C4 AACCTA D4 TCGTTG E4 GTTCTA F4 GATGAT G4 ATCCTC H4 GGCAGA HaloPlex Target Enrichment System 39 3 Reference Table 14 HaloPlex Indexes A5 H6 Index Number Sequence Ab TATTCT B5 TGCCAG C5 TGGATA D5 ACTCTG E5 CAGCTA F5 CCTATG G5 TCAATC H5 GGAGAA A6 AGATCT B6 GAGAAG C6 TTATCA D6 GATATG E6 ACCGGA F6 AACTGG G6 CTTCGC H6 GCGCAA 40 HaloPlex Target Enrichment System Reference 3 Table 15 HaloPlex Indexes A7 H8 Index Number Sequence 7 CAGGCT B7 ATCAAG C7
30. the bottom of the tube If aggregation is present thoroughly resuspend the beads by vortexing and pipetting up and down before use 5 After adding the magnetic beads mix the capture reactions thoroughly by pipetting up and down 10 times using a 100 uL multichannel pipette set to 80 uL 6 Incubate the capture reactions at room temperature for 15 minutes 22 HaloPlex Target Enrichment System Sample Preparation 7 Briefly spin the tubes in a desktop centrifuge and then transfer the tubes to the Agencourt SPRIPlate Super Magnet magnetic rack 2 Use the Agencourt SPRIPlate Super Magnet magnetic rack for the remainder of magnetic bead collection steps for samples in tube strips or PCR tubes 8 Wait for the solution to clear about 30 seconds then remove and discard the supernatant using a pipette set to 200 uL 9 Wash the bead bound samples Remove the capture reaction tubes from the magnetic rack and add 100 uL of Wash Solution to each tube Resuspend the beads thoroughly by pipetting up and down 10 times using a 100 uL multichannel pipette set to 80 uL Incubate the tubes in a thermal cycler at 46 for 10 minutes using a heated lid The thermal cycler may be programmed to include a 4 C hold step following the 10 minute incubation Briefly spin the tubes in a desktop centrifuge and then transfer the tubes to the magnetic rack Wait for the solution to clear about 30 seconds then carefully remove and discard the su
31. ucleotide sequences of the 96 indexes used in the HaloPlex Target Enrichment System For low plexity pooling assign indexes row wise from the plate starting with Index A1 Steps 1 3 below should be completed during the 30 minute Haloase B reaction period described on page 26 When the Haloase B incubation is complete transfer the supernatant directly into the prepared PCR reaction tubes 1 Prepare the PCR master mix by combining the reagents in the following table Table8 Preparation of PCR master mix Reagent Stock Concentration Volume for 1 Volume for 12 reactions reaction includes excess Phusion HF Buffer 5x 6 uL 84 uL dNTPs 25 mM 0 4 HL 5 6 uL Primer 1 0 25 uM 1 uL 14 uL Phusion HF DNA 2 U pL 0 5 uL 7 uL Polymerase Nuclease free water 12 1 uL 169 4 uL Total 20 pL 280 pL 2 Mix the master mix components by gentle vortexing then distribute 20 uL aliquots to fresh 0 2 mL reaction tubes 3 Add 10 uL of the appropriate Index Primer to each tube Be sure to record the specific Index Primer added to each tube for later sequence analysis HaloPlex Target Enrichment System 21 2 Sample Preparation 4 When the 30 minute Haloase B reaction period is complete briefly spin the Haloase B reaction tubes in a desktop centrifuge and then transfer the tubes to the magnetic rack 5 Wait for the solution to clear about 30 seconds then carefully transfer 20 uL of the supernatant to the 30 uL PCR reaction tube that contains the a
Download Pdf Manuals
Related Search