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1. each lot of GeneRead DNA Library L Core Kit GeneRead DNA L Amp Kit GeneRead Adapter L Set 1 plex and GeneRead Adapter L Set 12 plex are tested against predetermined specifications to ensure consistent product quality 6 QIAGEN GeneRead Library Prep L Handbook 03 2013 Introduction Next generation sequencing NGS is a driving force for numerous applications including cancer research stem cell research metagenomics population genetics and medical research While NGS technology is continuously improving library preparation is one of the biggest bottlenecks in the NGS workflow and includes several time consuming steps that can result in considerable sample loss and the potential to introduce handling errors QIAGEN GeneRead Library Prep Kits use a streamlined optimized one tube protocol that does not require sample cleanup between each step saving time and preventing handling errors as well as loss of valuable samples The efficient procedure includes an optional high fidelity amplification step to ensure high yields of DNA library that are reproducibly generated with minimal sequence bias and low error rates 80 min Adapter ligation Cleanup and de Le werd End repair amplification and nick repair size selection optional peo 4 One tube Three steps Total time 45 min Hands on time 3 min Principle and procedure QIAGEN GeneRead Library Prep L Kits use a fast one tube procedure w2. QIAGEN GeneRead Library Prep L Handbook 03 2013 3 Kit Contents GeneRead DNA Library L Core Kit 12 Catalog no 180462 Number of reactions 12 End Repair Buffer 10x 50 ul Ligation Buffer 2x 600 ul End Repair Enzyme Mix 24 ul Ligation and Nick Repair Mix 48 ul dNTP Mix 10 mM 55 pl RNase free Water 1 9 ml Quick Start Protocol GeneRead DNA L Amp Kit 100 Catalog no 180485 Number of reactions 100 Primer Mix 10 UM 150 ul HiFi PCR Master Mix 2x 2x 1 25 ml RNase free Water 1 9 ml Quick Start Protocol GeneRead DNA Adapter L Set 1 plex 12 Catalog no 180922 Number of reactions 12 Adapter 50 uM 24 ul Quick Star Protocol QIAGEN GeneRead Library Prep L Handbook 03 2013 GeneRead DNA Adapter L Set 12 plex 72 Catalog no 180994 Number of reactions 72 Adapter BcGen 25 UM 144 ul Adapter Bc1 25 uM 12 ul Adapter Bc2 25 UM 12 ul Adapter Bc3 25 uM 12 ul Adapter Bc4 25 uM 12 ul Adapter Bc5 25 uM 12 ul Adapter Bec 25 uM 12 ul Adapter Bc7 25 uM 12 ul Adapter Bc8 25 uM 12 ul Adapter Bc9 25 uM 12 ul Adapter Bc10 25 uM 12 pl Adapter Bc11 25 UM 12 ul Adapter Bc12 25 uM 12 ul Quick Start Protocol For adapter sequences refer to Appendix A page 19 QIAGEN GeneRead Library Prep L Handbook 03 2013 5 Shipping and Storage The GeneRead DNA Library L Core Kit and GeneRead DNA L Amp Kit are shipped on dry ice and should be stored immediately
3. e g GelPilot 50 bp Ladder cat no 239025 or between100 bp and 1500 bp for larger fragment sizes e g GelPilot 100 bp Plus Ladder cat no 239045 IMPORTANT When handling multiple libraries in parallel avoid cross contamination during gel excision by using a new scalpel for each sample 9 Isolate the DNA from the gel using the MinElute Gel Extraction Kit cat no 28604 Note Dissolve the gel at room temperature as this will result in higher library yields Note Following isolation purified DNA can be stored at 20 C 10a If sequencing the library directly i e without further amplification proceed directly to step 10 10b If amplifying the library prior to sequencing proceed to step 1 of the protocol Optional Amplification of Library DNA page 15 a Clean up the amplified DNA using the MinElute PCR Purification Kit not provided cat no 28004 b Assess the quality of the library using an Agilent Bioanalyzer or a comparable method Check for the correct size distribution see Figure 1 page 16 of library fragments and for the absence of free library adapters Note The median fragment size can be used for subsequent qPCR based quantification methods step 12 11 Quantify the library using the GeneRead Library Quantification Kit not provided cat no 180612 or a comparable method Note Store the DNA at 20 C until ready to sequence 14 QIAGEN GeneRead Library Prep L Handbook 03 2013 Protoc
4. 1 Prepare a reaction mix for end repair according to Table 1 dispensing the reagents into a PCR tube of the well of a PCR plate Note The reaction mix should be prepared on ice QIAGEN GeneRead Library Prep L Handbook 03 2013 11 Table 1 Reaction mix for end repair Component Volume reaction pl DNA 100 ng 1 ug sheared DNA Variable RNase free water Variable End Repair Buffer 10x 2 5 End Repair Enzyme Mix 2 Total reaction volume 25 Contains dNTPs 2 Mix thoroughly 3 Program a thermocycler to incubate for 20 min at 25 C followed by 10 min at 70 C Adapter ligation 4 Prepare a reaction mix for adapter ligation according to Table 2 adding the components to the PCR tube containing the end repaired DNA step 3 E Note When using barcode adapters open one adapter tube at a time and change gloves between pipetting the different barcode adapters to avoid cross contamination SS IMPORTANT Only one of the 12 adapters Adapter Bc1 Bc12 should be used per ligation reaction in combination with the universal adapter BcGen QIAGEN GeneRead Library Prep L Handbook 03 2013 Table 2 Reaction setup for adapter ligation Singleplex adapter Multiplex adapter mix Adapter mix singleplex Variable 0 5 OM Universal Adapter Component mix Volume reaction Volume reaction pl End repaired DNA 25 25 from step 3 Ligation Buffer 2x 40 40 Variable 0 5 OM Bc
5. EN GeneRead Library Prep I Kits Use of this product signifies the agreement of any purchaser or user of the product to the following terms 1 The product may be used solely in accordance with the protocols provided with the product and this handbook and for use with components contained in the kit only QIAGEN grants no license under any of its intellectual property to use or incorporate the enclosed components of this kit with any components not included within this kit except as described in the protocols provided with the product this handbook and additional protocols available at www giagen com Some of these additional protocols have been provided by QIAGEN users for QIAGEN users These protocols have not been thoroughly tested or optimized by QIAGEN QIAGEN neither guarantees them nor warrants that they do not infringe the rights of third parties Other than expressly stated licenses QIAGEN makes no warranty that this kit and or its use s do not infringe the rights of third parties This kit and its components are licensed for one time use and may not be reused refurbished or resold QIAGEN specifically disclaims any other licenses expressed or implied other than those expressly stated ir ode 00 a The purchaser and user of the kit agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above QIAGEN may enforce the prohibitions of this Limited License Agreement in any Court and sh
6. Gen Barcode Adapter 1 12 Variable 0 5 OM Ligation and Nick 4 4 Repair Mix dNTP Mix 10 mM RNase free water Variable Variable Total volume 80 80 Use 0 5 uM final concentration of GeneRead Adapter Set 1 plex or GeneRead Adapter L Set 12 plex Alternatively add the correct amount of adapter according to supplier s directions 5 Mix thoroughly 6 Program a thermocycler to incubate for 10 min at 25 C followed by 5 min at 72 C IMPORTANT Do not use a thermocycler with a heated lid 7 Purify adapter ligated library fragments For libraries with a median fragment size below 200 bp use the MinElute PCR Purification Kit not supplied cat no 28004 For libraries with a median size of gt 200 bp QIAGEN s GeneRead Size Selection Kit not supplied cat no 180514 can be used Fine size selection 8 For DNA that was sheared to a median size of 150 bp select adapter ligated DNA in the 210 250 bp range for 100 bp read lengths or select adapter ligated DNA in the range of 280 320 bp for 200 bp read lengths Yields may vary depending on the size selection method used Size selection can be performed using a QIAGEN GeneRead Library Prep L Handbook 03 2013 13 standard 2 agarose gel or alternative gel based separation methods Note Ensure that the library is sufficiently well separated to allow selection of an exact fragment size Use a DNA ladder with size markers between 50 bp and 500 bp
7. March 2013 QIAGEN GeneRead Library Prep L Handbook For preparation of DNA libraries for next generation sequencing NGS applications that use lon Torrent instruments from Life Technologies QIAGEN Sample amp Assay Technologies QIAGEN Sample and Assay Technologies QIAGEN is the leading provider of innovative sample and assay technologies enabling the isolation and detection of contents of any biological sample Our advanced high quality products and services ensure success from sample to result QIAGEN sets standards in Purification of DNA RNA and proteins Nucleic acid and protein assays microRNA research and RNAi Automation of sample and assay technologies Our mission is to enable you to achieve outstanding success and breakthroughs For more information visit www qiagen com Contents Kit Contents 4 Shipping and Storage 6 Intended Use 6 Safety Information 6 Quality Control 6 Introduction 7 Principle and procedure 7 Description of protocols 8 Equipment and Reagents to Be Supplied by User 9 Important Notes 10 DNA preparation and quality control 10 High quality DNA is essential for obtaining good sequencing results 10 Protocols E End Repair Adapter Ligation and Cleanup and Size Selection of DNA 11 E Optional Amplification of Library DNA 15 Troubleshooting Guide 17 References 18 Appendix A Barcode Sequences for the GeneRead Adapter L Set 12 Plex 19 Ordering Information 20
8. all recover all its investigative and Court costs including attorney fees in any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the kit and or its components For updated license terms see www giagen com 2013 QIAGEN all rights reserved SEH www qiagen com Australia techservice au qiagen com Austria techservice at qiagen com Belgium techservice bnI giagen com Brazil suportetecnico brasil qiagen com Canada techservice ca Qqiagen com China techservice cn Qqiagen com Denmark techservice nordic Qqiagen com Finland techservice nordic qiagen com France techservice fr qiagen com Germany techservice de qiagen com Hong Kong techservice hk qiagen com India techservice india qiagen com Ireland techservice uk giagen com Italy techservice it qiagen com Japan techservice jp qiagen com Korea South techservice kr qiagen com Luxembourg techservice bnI giagen com Mexico techservice mx qiagen com The Netherlands techservice bnli qiagen com Norway techservice nordic qiagen com Singapore techservice sg qiagen com Sweden techservice nordic qiagen com UK techservice uk qiagen com E e e e o USA techservice us qiagen com QIAGEN Switzerland techservice ch qiagen com Sample amp Assay Technologies
9. an optional high fidelity amplification step that can be used to ensure high amounts of DNA library from minimum amounts of starting material SSS SS EH 8 QIAGEN GeneRead Library Prep L Handbook 03 2013 Equipment and Reagents to Be Supplied by User When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate safety data sheets SDSs available from the product supplier M Enzymatic or physical method e g sonication to shear DNA PCR tubes or plates Pipette tips and pipettes Microcentrifuge Thermocycler MinElute PCR Purification Kit cat no 28004 Agilent Bioanalyzer or a comparable method to assess the quality of DNA library Optional GeneRead Library Quantification Kit cat no 180612 Optional GelPilot 50 bp Ladder cat no 239025 or GelPilot 100 bp Plus Ladder cat no 239045 e QIAGEN GeneRead Library Prep L Handbook 03 2013 9 Important Notes DNA preparation and quality control High quality DNA is essential for obtaining good sequencing results The most important prerequisite for any DNA sequence analysis experiment is consistent high quality DNA from every experimental sample Therefore sample handling and DNA isolation procedures are critical to the success of the experiment Residual traces of proteins salts or other contaminants will degrade the DNA or decrease the efficiency of if not block comple
10. for next generation sequencing applications GeneRead Size For 50 reactions Spin columns and 180514 Selection Kit 50 buffers GeneRead Library Laboratory verified forward and reverse 180612 Quant Kit primers for 500 x 25 ul reactions 500 ul DNA Standard 100 ul Dilution Buffer 30 ml 1 35 ml x 5 GeneRead qPCR SYBR Green Mastermix QlAamp Kits for genomic DNA purification QlAamp DNA Mini Kit For 50 DNA preps 50 QlAamp Mini 51304 Spin Columns QIAGEN Proteinase K Reagents Buffers Collection Tubes 2 ml QlAamp DNA FFPE For 50 DNA preps 50 QlAamp 56404 Tissue Kit 20 MinElute Columns Proteinase K Buffers Collection Tubes 2 ml QIAGEN GeneRead Library Prep L Handbook 03 2013 For up to date licensing information and product specific disclaimers see the respective QIAGEN kit handbook or user manual QIAGEN kit handbooks and user manuals are available at www giagen com or can be requested from QIAGEN Technical Services or your local distributor E l l EE QIAGEN GeneRead Library Prep L Handbook 03 2013 21 Notes 22 QIAGEN GeneRead Library Prep L Handbook 03 2013 Trademarks QIAGEN QlAamp QIAGEN GeneRead GelPilot GeneRead MinElute QIAGEN Group Agilent Bioanalyzer Agilent Technologies Inc Covaris Covaris Inc Life Technologies lon Torrent lon Proton Life Technologies Corporation SYBR Molecular Probes Inc Limited License Agreement for QIAG
11. ion amplification If the final library yield is not sufficient for the expected number of sequencing runs a library amplification step can be performed following fragment size selection c Insufficient amount of RNA from the sample material is co purified with starting DNA genomic DNA This contaminating RNA will affect the accuracy of DNA quantification To remove RNA during the sample preparation protocol it is recommended to perform RNase A treatment of the DNA Unexpected signal peaks in Agilent Bioanalyzer traces a Library fragments are Make sure to excise the recommended size of unexpected size after ranges for subsequent emulsion PCR and gel size selection sequencing Fragments that are too large may negatively influence the efficiency of the downstream procedures QIAGEN GeneRead Library Prep L Handbook 03 2013 17 References QIAGEN maintains a large up to date online database of scientific publications utilizing QIAGEN products Comprehensive search options allow you to find the articles you need either by a simple keyword search or by specifying the application research area title etc For a complete list of references visit the QIAGEN Reference Database online at www giagen com RefDB search asp or contact QIAGEN Technical Services or your local distributor SESEEEoo a 18 QIAGEN GeneRead Library Prep L Handbook 03 2013 Appendix A Barcode Sequences for the GeneRead Adapter L Set 12 Plex The ba
12. ith fewer cleanup steps than library preparation workflows from other suppliers and an optional high fidelity library amplification step Samples consisting of longer DNA fragments are first sheared into a random library of fragments that are a median fragment size of 400 bp when using the lon Torrent PGM instrument or 200 bp when using the lon Proton instrument in length Following fragmentation the ends of the DNA fragments are repaired and adaptors which are necessary for amplification and sequencing are ligated to both ends of the DNA fragments Barcode adapters which contain a unique identifying sequence are also available with the GeneRead Library Prep L Kit and enable multiplex sequencing reactions to be performed The fragments are then size selected and purified To ensure maximum yields from minimum amounts of starting material an optional high fidelity amplification step can also be performed that provides highly accurate amplification of library DNA with low error rates and minimum bias QIAGEN GeneRead Library Prep L Handbook 03 2013 7 Description of protocols This handbook contains 2 protocols for generation of DNA libraries that are for use on instruments from Life Technologies The first protocol page 11 describes end repair adapter ligation and cleanup and size selection of DNA to generate libraries that are ready to quantify and use in next generation sequencing The second protocol page 15 describes
13. ol Optional Amplification of Library DNA This protocol is for optional high fidelity amplification of the DNA library Important points before starting SS The Primer Mix for library enrichment see Table 3 is provided as a ready to use premix with a final concentration of 10 UM Things to do before starting E Prepare library DNA using the protocol End Repair Adapter Ligation and Cleanup and Size Selection of DNA page 11 Procedure 1 Prepare a reaction mix according to Table 3 Table 3 Reaction mix for library enrichment Component Volume reaction pl HiFi PCR Master Mix 2x 25 Primer Mix 10 uM each 1 5 Library DNA from step 10b page 14 Variable RNase free water Variable Total reaction volume 50 2 Program a thermocycler according to Table 4 QIAGEN GeneRead Library Prep L Handbook 03 2013 15 Table 4 Cycling conditions Time Temperature Number of cycles 2 min 98 C 20s 98 C 30s 60 C 8 10 30s 72 C 1 min 72 C 1 We recommend using 8 10 amplification cycles Additional cycles may be required to ensure robust performance However too many cycles should be avoided to prevent over amplification 3 Clean up the amplified DNA using the MinElute PCR Purification Kit not provided cat no 28004 4 Assess the quality of the library using an Agilent Bioanalyzer or a comparable method Check for the correct size distribution Figure 1 of library fragmen
14. rcode sequences used in the GeneRead Adapter L set 12 plex are listed in Table 5 Barcodes 1 12 correspond to the respective lon Torrent adapter barcodes Table 5 Barcode adapter name Barcode adapter name Barcode sequence Adapter Bcl lon Torrent CTAAGGTAAC Adapter Bc2 lon Torrent TAAGGAGAAC Adapter Bc3 lon Torrent AAGAGGATTC Adapter Bc4 lon Torrent TACCAAGATC Adapter Bc5 lon Torrent CAGAAGGAAC Adapter Bc lon Torrent CTGCAAGTTC Adapter Bc7 lon Torrent TTCGTGATTC Adapter Bc8 lon Torrent TTCCGATAAC Adapter Bc9 lon Torrent TGAGCGGAAC Adapter Bc10 lon Torrent CTGACCGAAC Adapter Bc11 lon Torrent TCCTCGAATC Adapter Bc12 lon Torrent TAGGTGGTTC QIAGEN GeneRead Library Prep L Handbook 03 2013 19 Ordering Information Product Contents Cat no GeneRead DNA Library For 12 reactions Buffers and reagents 180462 L Core Kit 12 for end repair ligation and nick repair for use with lon Torrent Instruments from Life Technologies GeneRead DNA L Amp For 100 reactions Buffers and reagents 180485 Kit 100 for library amplification for use lon Torrent Instruments from Life Technologies GeneRead Adapter For 12 reactions Adapters for ligation 180922 Set 1 plex 12 to DNA library for use lon Torrent Instruments from Life Technologies GeneRead Adapter L For 72 reactions 12 barcoded 180994 Set 12 plex 72 adapters for ligation to DNA library for use with Illumina instruments Related products QIAGEN GeneRead Kits
15. tely the enzyme activities necessary for optimal library preparation Recommended genomic DNA preparation method The QlAamp DNA Mini Kit cat no 51304 and QlAamp DNA FFPE Tissue Kit cat no 56404 are highly recommended for the preparation of genomic DNA samples from fresh tissues and FFPE tissue samples Ensure that samples have been treated for the removal of RNA as RNA contamination will cause inaccuracies in DNA concentration measurements Do not omit the recommended RNase treatment step to remove RNA Recommendations for DNA fragmentation To ensure complete fragmentation of the DNA that is needed for library preparation only use the recommended parameters given in the manufacturer s instructions Using too much DNA in Covaris instrument may for example lead to incomplete shearing of the DNA Check the fragmented DNA for the correct size distribution using an agarose gel or Agilent Bioanalyzer Recommended library quantification method QIAGEN s GeneRead Library Quant Kit cat no 180612 which contains laboratory verified forward and revers primers together with a DNA standard is highly recommended for quantification of the prepared library 10 QIAGEN GeneRead Library Prep L Handbook 03 2013 Protocol End Repair Adapter Ligation and Cleanup and Size Selection of DNA This protocol describes the end repair A addition adapter ligation and cleanup and size selection of DNA and generates libraries that are ready
16. to quantify and use in next generation sequencing on instruments from Illumina Important points before starting E The median fragment size of DNA should be compatible with the read length of the sequencing platform you are using e g when using the Life Technologies lon Torrent POM instrument use a median fragment length of 400 bp When using the lon Proton instrument use a median fragment length of 200 bp Specific median fragment length sizes of DNA can be prepared using a Covaris instrument according to the manufacturer s instructions M GeneRead Adapter L Set 1 plex as well as the GeneRead Adapter L Set 12 plex are dissolved in duplex buffer 30 mM Hepes pH 7 5 100 mM Potassium Acetate The adapters are pre annealed and are provided ready to use E GeneRead Adapter L Set 1 plex contains both adapter duplices mixed together in one tube at a concentration of 50 uM for each adapter duplex GeneRead Adapter L Set 12 plex contains the universal adapter BcGen pre annealed and ready to use as well as the barcode adapters 1 12 in 13 separate tubes at a concentration of 25 UM E The library adapters are fully compatible with Life Technologies instruments such as the lon Torrent PGM or the lon Proton and do not require nick translation during the enrichment step Things to do before starting M Shear 100 ng 1 ug DNA using either an enzymatic method or a physical method e g sonication Procedure End repair of DNA
17. ts and for the absence of free library adapters Note The median fragment size can be used for subsequent qPCR based quantification methods step 5 5 Quantify the library using the GeneRead Library Quantification Kit cat no 180612 not provided or a comparable method Note Store the library DNA at 20 C until ready to sequence Figure 1 Agilent trace data showing the correct size distribution of library fragments and the absence of adapters or adapter dimers 16 QIAGEN GeneRead Library Prep L Handbook 03 2013 Troubleshooting Guide This troubleshooting guide may be helpful in solving any problems that may arise For more information see also the Frequently Asked Questions page at our Technical Support Center www giagen com FAQ FAQList aspx The scientists in QIAGEN Technical Services are always happy to answer any questions you may have about either the information and protocols in this handbook or sample and assay technologies for contact information see back cover or visit www giagen com Comments and suggestions Low library yields a Suboptimal reaction Make sure to use high quality DNA to ensure conditions due to low optimal activity of the library enzymes DNA quality b Insufficient amount of Typically 800 1000 ng of genomic DNA starting DNA for direct generates enough lon Torrent compatible library sequencing without to use the library directly for sequencing without library amplificat
18. upon receipt at 20 C in a constant temperature freezer The GeneRead Adapter L Set 1 plex and the GeneRead Adapter L Set 12 plex are shipped at ambient temperature and should be stored immediately upon receipt at 20 C in a constant temperature freezer If stored under these conditions the kits are stable until the date indicated on the QC label inside the kit lid Intended Use GeneRead DNA Library Prep Kits are intended for molecular biology applications These products are not intended for the diagnosis prevention or treatment of a disease All due care and attention should be exercised in the handling of the products We recommend all users of QIAGEN products to adhere to the NIH guidelines that have been developed for recombinant DNA experiments or to other applicable guidelines Safety Information When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information please consult the appropriate safety data sheets SDSs These are available online in convenient and compact PDF format at www giagen com safety where you can find view and print the SDS for each QIAGEN kit and kit component 24 hour emergency information Emergency medical information in English French and German can be obtained 24 hours a day from Poison Information Center Mainz Germany Tel 49 6131 19240 Quality Control In accordance with QIAGEN s ISO certified Quality Management System
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