User Guide
Contents
1. or email europe nugen com In all other locations contact your NUGEN distributor for technical support 12 Ovation Target Capture Module VI Appendix 13 Ovation Target Capture Module A Using the Ovation Target Capture Module with Agilent SureSelect Target Enrichment The following instructions describe how to use NUGEN NGS libraries and the Ovation Target Capture Module Part No 0332 with Agilent SureSelect Target Enrichment Agilent User Guide G7530 90000 We recommend reviewing section A of this user guide for a list of compatible NuGEN NGS library kits 1 When preparing libraries prior to target capture follow the Library Preparation pro tocol for the appropriate NUGEN NGS library kit rather than following the protocol in Chapter 2 Sample Preparation of the Agilent SureSelect User Guide When performing target enrichment of the NuGEN libraries follow Chapter 3 Hybridization of the SureSelect User Guide with the following modifications e If you intend to perform a multiplex capture experiment you may pool samples at this stage However you must ensure that the total input of pooled material for hybridization step is 500 ng in 3 4 uL e When making the SureSelect Block Mix Step 1 6 Table 20 page 42 replace the SureSelect Indexing Block 3 brown cap with an equal volume 0 6 uL per reaction of Library Adaptor Blocking Mix yellow cap from NuGEN s Ovation Target Capture Module kit e Co2. Netherlands Tel 31 13 5780215 Fax 31 13 5780216 europe nugen com For our international distributors contact information visit our website www nugen com 2012 2013 NuGEN Technologies Inc All rights reserved The Encore Ovation and Applause families of products and methods of their use are covered by several issued U S and International patents and pending applications www nugen com NuGEN Ovation SPIA Ribo SPIA Applause Encore Prelude Mondrian and Imagine More From Less are trademarks or registered trademarks of NUGEN Technologies Inc Other marks appearing in these materials are marks of their respective owners For research use only M01291 v6
3. genomic regions of interest The resulting enriched products are utilized for sequencing resulting in cost reduction simplified data analysis and increased sample throughput as compared to whole genome sequenc ing The Ovation Target Capture Module has been optimized for the SureSelect Target Enrichment System from Agilent Technologies Key benefits of the Ovation Target Capture Module include e Low DNA input In conjunction with the Ovation Ultralow Library Systems perform target capture starting with as little as 1 ng DNA Library pooling before capture Combine barcoded libraries prior to target capture reaction for significant cost reduction and increased throughput e Multiplexing options Several barcoding options are available with NuGEN s library solutions to optimize workflow needs The Ovation Target Capture Module contains sufficient blocking reagent mix to per form 16 capture reactions using individual or pooled libraries These reagents may be used with any of NUGEN s library solutions including multiplex systems utilizing either inline IL or dedicated read DR barcode designs The objective of this protocol is to enable target enrichment of libraries generated using NuGEN kits that have adaptors specific for sequencing on Illumina platforms The reagents provided in the module are compatible with the following NuGEN library kits for Illumina sequencing e Ovation Ultralow Library Systems Part Nos 0303 0304
4. no right to modify reverse engineer distribute offer to sell or sell NUGEN s product is conveyed or implied by buyer s purchase of this NUGEN prod uct The buyer agrees to use NuGEN products accompanying the product insert in accordance with the intended use and the written instructions provided Table of Contents Contents Ik NEFOGUCHION s ccssssssciscesctsssessssvsssascassssssssecasessessssesssosnesasovsaasossuscnsasesssvscasscssseasssssess 1 As BACK COUN oases tear Say E A E sands A E E E 1 B Target Capture Process istics csscastidicntesvncascnnsiicecdstidacchdenadicesnnishesiedebimneduetaes RS 1 C Quality Controla ai ene a a ena Ea 3 D Storage and Stabilityscccccccasicsacssteseccuctsnncssevidees conse cnuvsvaneestesouawsounecdaesneeeseeedy 3 E Material Safety Data Sheet MSDS 0 cecceeceeeceeeeeeeeeeeeseceeeceeeeeeaeeeseeneeeeeeees 3 Ie COMPONCNES cs 0 scssesccssrctessscessatesascucssavenvecuesdsvesassecsesvesassusasdsesvasstactstsvasececcessvasessess 4 A Reagents Provided ceecceesceseseeencesseecencersaeessceceaeeceeesseeeaeeseeesaeseneeseneeeeeee 4 B Additional Equipment Reagents and Labware cccceceeeeeeeseeseeeeseeeees 4 lll Planning the Experiment s c 0ccccccccesesseccccecsecsecccteesecseccossesessccostessssoncseceseassneses 6 A Planning the Target Capture Experiment cccscscsesssesecssesssneseeseneeeannas 6 B Using Nuclease free Techniques ccccceeecceeeceeteceeteseeseeescee
5. 0305 0329 0330 0331 e Encore 384 Library System Part Nos 0315A 0315B 0315C 0315D 0315E e Ovation SP Ultralow Library System and Ovation SP Ultralow DR Multiplex Systems Part Nos 8030 8033 8034 B Target Capture Process The Ovation Target Capture Module protocol starts with 500 ng of sequencing library prepared with one of the NuGEN library preparation systems listed This sample may be from a single non multiplexed sample or may represent a pool of barcoded librar ies intended to be multiplex sequenced on a single lane of the flow cell The Library Adaptor Blocking Mix is used in the appropriate target capture workflow This blocking mix consists of a cocktail of reagents designed to block the specific adaptor sequences used in our sequencing libraries so these sequences do not impede efficient sequence capture Introduction Figure 1 Workflow diagram Input DNA 47 4 calla Se Q Add adaptors and ligate CUTE TTT TTT TTT TT Fill in and PCR Optional pooling of barcoded libraries ap a gt lt gt Add Library Adaptor e Blocking Mix from the od Ovation Target Capture Module Continue with standard target capture workflow and sequencing 2 Ovation Target Capture Module 3 Introduction Ovation Target Capture Module C Quality Control Each Ovation Target Capture Module lot is tested to meet minimum performance specifications D Storage and Stability The Ov
6. Manuals such as in the table Alternative Capture Equipment Combinations Additionally if PCR plates are used for the hybridization we recommend that you partially fill surrounding non reaction wells with nuclease free water This will help to minimize evaporation of your samples IV Protocol 10 Ovation Target Capture Module D Post capture Amplification The following amplification protocol has been used successfully with the combination of NuGEN library kits this Ovation Target Capture Module and the Agilent SureSelect Target Enrichment System You may also use the amplification recommendations from Agilent SureSelect but be sure to use the amplification primer mix that is supplied with this kit not the primers supplied with third party capture kits Use the Amplification Primer Mix supplied in the Ovation Target Capture Module to perform the post capture PCR amplification step It is important to use our Amplification Primer Mix as other post amplification PCR primers may not work with NuGEN sequenc ing libraries In addition many systems use the post amplification step to add multiplex ing barcodes When using the NuGEN sequencing libraries the multiplex barcodes have already been added during the library preparation step during adaptor ligation Table 3 Thermal Cycler Programming AMPLIFICATION Program 1 72 C 2 min 18 cycles 94 C 30 sec Amplification 60 C 30 sec 72 C 1 min hold a
7. Ovation Target Capture Module PART NO 0332 t NUGEN imagine more from lesse Patents Licensing and Trademarks 2012 2013 NuGEN Technologies Inc All rights reserved The Encore Ovation and Applause families of products and methods of their use are covered by several issued U S and International patents and pending applications www nugen com NUGEN Ovation SPIA Ribo SPIA Applause Encore Prelude Mondrian and Imagine More From Less are trademarks or registered trademarks of NUGEN Technologies Inc Other marks appearing in these materials are marks of their respective owners The purchase of this product conveys to the buyer the limited non exclusive non transferable right without the right to modify reverse engineer resell repackage or further sublicense under these patent applications and any patents issuing from these patent applications to use this prod uct and methods accompanying this user guide for research and development purposes solely in accordance with the intended use described and the written instructions provided in this user guide No license to make or sell products by use of this product is granted to the buyer whether expressly by implication by estoppels or otherwise In particular the purchase of this product does not include or carry any right or license to use develop or otherwise exploit this product commercially and no rights are conveyed to the buyer to use the product or components o
8. ation Target Capture Module is shipped on dry ice and should be unpacked immediately upon receipt All components should be stored at 20 C on internal shelves of a freezer without a defrost cycle This product has been tested to perform to specifications after as many as six freeze thaw cycles Kits handled and stored according to the above guidelines will perform to specifications for at least six months from date of receipt in your lab E Material Safety Data Sheet MSDS An MSDS for this product is available on the NuGEN website at http www nugen com nugen index cfm support user guides ll Components 4 Ovation Target Capture Module A Reagents Provided Library Adaptor Blocking Mix a proprietary mixture of adaptor blocking reagents designed to prevent cross hybridization of target and non target library molecules that would otherwise significantly impair the efficacy of the enrichment procedure PCR primers for post capture amplification these amplification primers are compat ible with all NuGEN generated Illumina libraries Table 1 Reagents provided Mix 20 uM each 0332 COMPONENT PART NUMBER VIAL CAP VIAL NUMBER Library Adaptor Blocking Mix 300 uM aula Yellow 7 Amplification Primer 501664 Red P2 ver 5 B Additional Equipment Reagents and Labware Required Materials e Equipment Tube rotator Microcentrifuge for individual 1 5 mL and 0 5 mL tubes Microcentrifug
9. e for 0 2 mL individual and 8 X 0 2 mL strip PCR tubes 0 5 10 uL pipette 2 20 uL pipette 20 200 uL pipette 200 1000 uL pipette Vortexer Thermal cycler with 0 2 mL tube heat block heated lid and 100 uL reaction capacity Appropriate spectrophotometer and cuvettes or Nanodrop UV Vis Spectrophotometer e Reagents Third party Target Enrichment Kit and protocol for example Agilent SureSelect Dynal MyOne Streptavidin T1 beads Invitrogen Agencourt AMPure XP beads Nuclease free water 5 Components Ovation Target Capture Module Reagents for Post Capture Amplification these reagents can be used for post capture amplification or you may choose to use the reagents recommended in the third party capture protocol you are following with the NuGEN provided PCR primers Taq DNA Polymerase New England Biolabs Cat M0273 10x Standard Taq Buffer included with New England Biolabs Cat M0273 dNTP mixture 10mM each New England Biolabs Cat N0447 or equivalent MgCl DMSO Supplies and Labware O Nuclease free pipette tips 1 5 mL and 0 5 mL RNase free microcentrifuge tubes 0 2 mL individual thin wall PCR tubes or 8 X 0 2 mL strip PCR tubes Appropriate spectrophotometer cuvettes Disposable gloves Kimwipes Ice bucket ptional Materials Agilent 2100 bioanalyzer or other equipment for electrophoretic analysis of RNA Real time PCR system To Order Agilent www agilent c
10. f the product for purposes including commercial services or clinical diagnostics For information on purchasing a license to the NuGEN patents for uses other than in conjunction with this product or to use this product for purposes other than research please contact NUGEN Technologies Inc 201 Industrial Road Suite 310 San Carlos CA 94070 Phone 888 654 6544 or 650 590 3600 FAX 888 296 6544 or 650 590 3630 Warranty NuGEN warrants that this product meets the performance standards described in the Company s product and technical literature for a period of six months from the date of purchase Provided that the product is handled and stored according to published instructions and that the product is not altered or misused If the product fails to meet these performance standards NuGEN will replace the product free of charge or issue a credit for the purchase price NUGEN s liability under this warranty shall not exceed the purchase price of the product NUGEN shall assume no liability for direct indirect consequential or incidental damages arising from the use results of use or inability to use its products NUGEN reserves the right to change alter or modify any product to enhance its performance and design NuGEN s products are developed designed and sold FOR RESEARCH USE ONLY This product Is not to be used for diagnostic or therapeutic purposes nor is it to be administered to humans or animals Except as expressly set forth herein
11. generated from NuGEN library systems The following table contains a list of third party target enrichment protocols that are compatible with the Ovation Target Capture Module The table contains information on how the Library Adaptor Blocking Mix should be used instead of or in addition to the blocking mix included with the third party target enrichment kit IV Protocol 9 Ovation Target Capture Module Table 2 Third party enrichment protocols that are compatible with the Ovation Target Capture Module THIRD PARTY USE 0 6 pL CAPTURE LIBRARY REAGENT THIRD PARTY KIT USER MANUAL ADAPTOR PROVIDER BLOCKING MIX SureSelect Target taal SureSelect Indexing Enrichment System Block 3 brown Agilent for Illumina Paired G7530 90000 cap 0 6 uL For End Sequencing libra details see Y Appendix A Agilent SureSelect instead of Automated Library SureSelect Indexing Agilent Prep and Capture 67330 20000 Block 3 brown System cap 0 6 uL XT2 vureo ect in addition Target Enrichment to aes Agilent System for Illumina G9630 90000 i Blocking Mix tube Multiplexed with blue cap 9 uL Sequencing Continue with the sequence capture platform protocol until reaching the PCR step C Minimizing Evaporation During Incubation It is critical to take steps to minimize evaporation during the hybridization process Recommended equipment and consumables can be found in Agilent SureSelect User
12. module is intended for use only with NUGEN sequencing libraries designed for the Illumina sequencing platforms and it is not appropriate for use with sequencing libraries produced using other manufacturers reagent systems B Using Nuclease free Techniques Nuclease contamination from equipment and work environment will lead to experimental failure Follow these guidelines to minimize contamination e Wear disposable gloves and change them frequently e Avoid touching surfaces or materials that could introduce DNases e Use only the reagents provided and recommended e Prior to initiating protocol clean and decontaminate work areas and instruments including pipettes with commercially available decontamination reagents e Use only new DNase free pipette tips and microcentrifuge tubes C Sequencing Library Storage The sequencing libraries to be used as input for the target capture protocol may be stored at 20 C for up to six months prior to labeling Ill Planning the Experiment D Post capture Library Storage The post capture sequencing library mixture should be handled and stored in accor dance with the guidelines published by the target capture platform manufacturer 7 Ovation Target Capture Module IV Protocol 8 Ovation Target Capture Module A Preparing the Sequencing Libraries Sequencing libraries may be prepared using any of the following NuGEN library prepa ration methods e Ovation Ultralow Librar
13. mplete the steps in Chapter 3 including the final bead purification During post capture library amplification use the NuGEN provided Amplification Primer Mix P2 ver 5 red cap from the Ovation Target Capture Module instead of the amplification primers provided by Agilent You may use either a the amplification reagents and conditions recommended in the Ovation Target Capture Module User Guide or b the amplification conditions in Step 1 Chapter 4 Addition of Index Tags by Post Hybridization Amplification of the SureSelect User Guide using 2 5 uL of the NuGEN Amplification Primer Mix for a 50 uL reaction Note For the first few times performing this protocol and whenever changes are made such as changing the input amount or hybridization time we recommend monitoring PCR amplification both pre and post capture in order to avoid unnec essary amplification cycles and minimize PCR bias This can be accomplished by measuring DNA concentration using an intercalating dye such as Invitrogen Qubit or Picogreen or by running an aliquot on an agarose gel between cycles After post amplification purification analysis and sequencing follow the protocol in Chapter 4 of the SureSelect User Guide from step 2 onwards VI Appendix g NuGEN imagine more from lesse B Frequently Asked Questions FAQs Q1 Can use the PCR primers provided with the target capture system No The Amplification Primer Mix provided with the Ovation Ta
14. om Beckman Coulter Genomics www beckmangenomics com In N vitrogen www invitrogen com ew England Biolabs www neb com Ill Planning the Experiment 6 Ovation Target Capture Module A Planning the Target Capture Experiment The Ovation Target Capture Module allows the use of sequencing libraries prepared using a NUGEN sequencing library reagent system in commercially available target capture systems such as the Agilent SureSelect sequence capture kits A list of the NuGEN library systems that are compatible with this module is given on pg 1 A list of third party protocols that can be used for the target enrichment can be found in Table 2 The Ovation Target Capture Module contains a Blocking Reagent Mixture designed to block the adaptor sequences used in NuGEN library reagent systems enabling high efficiency capture of targeted genomic sequences in the library The presence of multiplex barcodes either DR or IL types or both will not interfere with efficient block ing therefore it is possible to generate multiplexed sequencing libraries prior to the capture process pool them and run a single target capture for each flow cell lane The provided Amplification Primer Mix can then be used to amplify the captured material prior to loading on the flow cell By enabling multiplexed libraries to be pooled prior to target capture this module will provide for simplified workflows and significant cost savings Please note that this
15. rget Capture Module is specifically designed for use with the workflow described in this user guide Many systems use the post capture PCR step to add multiplex ing barcodes This is not required with the NUGEN Ovation Target Capture Module workflow Q2 Is the Ovation Target Capture Module compatible with sequencing librar ies prepared by other methods No The Ovation Target Capture Module is only compatible with the NuGEN sequencing library preparation kits listed in this user guide Q3 Can use the Ovation Target Capture Module with Ovation Rapid Library Systems The yield of the Ovation Rapid Library Systems is not sufficient for the Target Capture methods discussed in this user guide If the end user has a Target Capture method for which Ovation Rapid Library Systems produce sufficient amounts of library the Ovation Target Capture Module would be suitable for blocking and subsequent PCR enhancement C Update History This document the Ovation Target Capture Module user guide M01291 v6 includes the following updates Description Section Page s Changed product name from Encore Target Capture Module to Ovation Target Capture Module Throughout Thiroughigut NuGEN Technologies Inc Headquarters USA 201 Industrial Road Suite 310 San Carlos CA 94070 USA Toll Free Tel 888 654 6544 Toll Free Fax 888 296 6544 custserv nugen com techserv nugen com Europe P O Box 109 9350 AC Leek The
16. seseeseeeeteseetanees 6 C Sequencing Library Storage sssr tics ete aa ee eea E KEAS 6 Ds Post capture Library Storage isc sssessussecstiniesigssiusteadeccpenscadessaastascsshsestgedsndyecvents 7 IV Protocol viccscsiscssssssssssescsscssssssvecsssessscssesaccasescesssscscassscsassocasssosussssasesssobeasscnseassadeess 8 A Preparing the Sequencing Libraries c cece ee eeeeeeeeeeeeeseeseeseseeseesenseeeanas 8 Be Tar Qet Capture s csicesateveuschiss covess ea eeri sth Mand pied EEEREN EE EAEE EEE ENE EE EEn 8 C Minimizing Evaporation During IMe bati i sissrsarssnersrrinsenisinssrsieatcusesiserie 9 D Post capture Amplification i 2 cs2 css ssacssacestuegeeacetaneguassestaeceesacesteeetaen ane sacs 10 V Technical SUPPOFt 0 eee E E E EE EEEE 12 VIS AP PONIXs3 iscssscessccd acescssesdsasstacsssuccsesasscagesuadessontsdsndaesssuaces E AOTEA SEE EEEN 13 A Using the Ovation Target Capture Module with Agilent SureSelect Target EMM IMIS Mites 25025 Sess e O e EE E E E EA EE 13 B Frequently Asked Questions FAQS n erinanoserosssireninna tieseeneteeeeee 14 Ge Update itean E A OTA 14 1 Introduction Ovation Target Capture Module A Background The Ovation Target Capture Module provides a library adaptor specific blocking reagent mix and a PCR Primer mix to enable target capture workflows with NuGEN library solutions for the Illumina NGS platforms Target or Sequence Capture meth ods allow researchers to capture
17. t 10 C Table 4 Amplification Mixture REAGENT VOLUME Purified selected library 30 uL 19 Standard Taq Reaction Buffer 8 uL included with New England Biolabs Cat M0273 25 mM MgCl 8 uL 5 DMSO 4 uL Taq DNA polymerase 1 pL New England Biolabs Cat M0273 10 mM dNTP mix l 4 uL each New England Biolabs Cat N0447 Amplification Primer Mix red P2 ver 5 4 uL Nuclease free Water 21 uL Total volume 80 uL IV Protocol 11 Ovation Target Capture Module Prepare a PCR enrichment mix by combining the reagents in a PCR tube according to the volumes shown in Table 4 Place the tubes in a pre warmed thermal cycler programmed to run Program 1 Library Amplification see Table 3 Purify amplified library sample using Agencourt AMPure XP beads following the Library Purification protocol from the appropriate NUGEN library system user guide Validate the library as described in the Illumina user guides for DNA library con struction Refer to the appropriate NUGEN library system user guide for informa tion regarding analysis of multiplexed libraries V Technical Support For help with any of our products please contact NUGEN Technical Support at 650 590 3674 direct or 888 654 6544 option 2 toll free U S only You may also send faxes to 888 296 6544 toll free or email techserv nugen com In Europe contact NUGEN at 31 0 135780215 Phone or 31 0 135780216 Fax
18. y Systems Part Nos 0303 0304 0305 0329 0330 0331 e Ovation SP Ultralow Library System and Ovation SP Ultralow DR Multiplex Systems Part Nos 8030 8033 8034 Libraries may be prepared using either the Mondrian SP or SP digital microfluidics system or manual protocols Sequencing libraries should be fragmented to the appropriate size then prepared in accordance with the protocols given in their respective user guides If multi plex sequencing will be used the multiplexing barcodes will be added during this step Quantify the library yield using a NanoDrop and check size distribution on a Bioanalyzer as described in the appropriate user guide If multiplexing determine the molar concentration of each library using an assay such as the KAPA Biosystems Library Quant kit or other suitable method for accurately determining sequencing library molarity Use these data to create an equimolar mixture of libraries to be multiplexed in each lane Ensure the mass of the mixture is sufficient for input into the hybridization step of the appropriate sequence capture protocol B Target Capture The sequencing libraries prepared in A above are used as input into the library hybrid ization step of the appropriate sequence capture protocol using the Library Adaptor Blocking Mix from the Ovation Target Capture Module It is critical to use the Library Adaptor Blocking Mix as only the NuGEN blocking reagents are designed to work with libraries
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