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Ovation® Rapid Library Systems PDF

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USER GUIDE Ovation® Rapid Library Systems PART NOS. 0319, 0320 Patents, Licensing and Trademarks © 2011–2016 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 of 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, 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 product. 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 I. Introduction .........................................................................................................1 A. Background .......................................................................................................1 B. Performance Specifications ...............................................................................2 C. Library Quantitation ..........................................................................................3 D. Quality Control .................................................................................................3 E. Storage and Stability .........................................................................................3 F. Safety Data Sheet (SDS) ....................................................................................3 II. Components ........................................................................................................4 A. Reagents Provided ............................................................................................4 B. Additional Equipment, Reagents and Labware ................................................5 III. Planning the Experiment .....................................................................................7 A. Input DNA Requirements ..................................................................................7 B. Using Ovation Rapid Multiplex Systems on Illumina NGS Systems .................7 C. Library Quantitation ..........................................................................................8 D. Final Library Storage .........................................................................................8 E. Data Analysis and Parsing Multiplex Libraries ..................................................8 IV. Protocol ...............................................................................................................9 A. Overview ...........................................................................................................9 B. Protocol Notes ..................................................................................................9 C. Agencourt® Purification Beads ........................................................................10 D. Programming the Thermal Cycler ...................................................................11 E. DNA Fragmentation ........................................................................................12 V. Ovation Rapid DR Multiplex Systems 1–8 and 9–16 Protocol ...........................15 A. Setting Up a DR Multiplex Experiment ...........................................................15 B. End Repair .......................................................................................................15 C. Ligation ...........................................................................................................16 D. Final Repair .....................................................................................................17 E. Library Purification ..........................................................................................18 VI. Quantitative and Qualitative Assessment of the Library ....................................20 A. Quantitation Using KAPA Biosystems Products ..............................................20 B. Gel Analysis of KAPA qPCR Product ...............................................................20 VII. Technical Support ..............................................................................................22 VIII. Appendix ...........................................................................................................23 A. Preparation of Ovation Rapid Library Systems Libraries for Cluster Generation on Illumina Systems .....................................................................23 B. Barcode Sequences for Ovation Rapid Multiplex System Reactions ..............26 C. Frequently Asked Questions (FAQs) ...............................................................27 D. Update History ................................................................................................29 I. Introduction A. Background The Ovation® Rapid Library Systems provide a simple, fast and scalable solution for producing libraries used in next-generation sequencing. These systems enable library preparation starting with as little as 100 ng of double-stranded DNA without PCR amplification. The library construction workflow is suitable for a wide range of sequenc- ing applications including RNA-Seq, Digital Gene Expression (DGE), genomic DNA sequencing, amplicon sequencing, ChIP-Seq and more. As shown in Figure 1, the streamlined workflow consists of four main steps: fragmenta- tion of either genomic DNA or double-stranded cDNA, end repair to generate blunt ends, adaptor ligation, and final repair to produce the final library. The entire workflow requires only one bead purification step and no gel purification. Starting with as little as 100 ng of fragmented double-stranded DNA (dsDNA), the protocol can be completed in fewer than 2 hours and yields libraries ready for cluster formation and either single read or paired-end sequencing. In addition to genomic and other double-stranded DNA sources, the Ovation Rapid Library Systems have been designed for seamless integration with NuGEN’s Ovation products for cDNA generation, such as Ovation RNA-Seq System V2 (Part No. 7102), to enable a complete end-to-end solution for transcriptome library construction starting with total RNA. Importantly, for DNA sequencing applications, low abundance samples (100 ng or higher) can be input directly to the library construction workflow without the need for pre-amplification. The absence of PCR steps in the library construction work- flow makes this library construction method particularly well suited to either high- or low-GC genomes, which are often susceptible to PCR artifacts. The Ovation Rapid DR Multiplex Systems 1–8 (Part No. 0319) and 9–16 (Part No. 0320) each provide eight unique dedicated read adaptors to prepare libraries for multi- plex sequencing using a dedicated read design strategy with a second sequencing primer. Either kit may be used for up to 8-plex sequencing or the two kits may be used together to multiplex up to 16 samples. 1 Ovation Rapid Library Systems I. Introduction Figure 1. Ovation Rapid Library System Workflow. Input DNA Fragment 5´ 3´ End-repair 5´ P 3´ P Add adaptors and ligate Final repair Bead purification Cluster formation AATCGGATCGGTAGGAT … and sequencing TCTCGATGCAAGTGATC … GTAGCAAAATCCTGAGA … B. Performance Specifications The Ovation Rapid Library Systems are designed to produce DNA libraries suitable for either single read or paired-end sequencing on the Illumina Genome Analyzer IIx/ IIe (GAII), MiSeq, HiScan SQ or HiSeq NGS platforms without gel-based size selection, using 100–500 ng of double-stranded DNA. When preparing libraries using cDNA generated from the Ovation RNA-Seq System V2, it is critical to use at least 500 ng – 1 µg of ds-cDNA. The absence of PCR amplification in the workflow eliminates the risk of unequal amplification or PCR bias introduced as a result of differences in sequence composition. These systems generate libraries suitable for cluster generation in about 2 hours. 2 Ovation Rapid Library Systems I. Introduction C. Library Quantitation Libraries created using Ovation Rapid Library Systems must be quantified using qPCR. These libraries cannot be accurately quantified using other means. We recommend using KAPA Library Quantification kits from KAPA Biosystems for quantitation. See Section VII, Quantitative and Qualitative Assessment of the Library, for details. D. Quality Control Every lot of the Ovation Rapid Library Systems undergoes functional testing to meet specifications for library generation performance. E. Storage and Stability The Ovation Rapid Library Systems are shipped on dry ice and should be unpacked immediately upon receipt. Note: This product contains components with multiple storage temperature requirements. • Vials labeled Agencourt® Beads (clear cap) should be removed from the top of the shipping carton upon delivery and stored at 4°C. • All other components should be stored at –20°C on internal shelves of a freezer without a defrost cycle. The kit 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 specifi- cations for at least six months. F. Safety Data Sheet (SDS) If applicable, an SDS for this product is available on the NuGEN website at www.nugen.com/products/ngs/ovation-rapid-library-systems 3 Ovation Rapid Library Systems II. Components A. Reagents Provided Table 1. Ovation Rapid DR Multiplex System 1–8 and 9–16 Reagents (Part Nos. 0319-32 and 0320-32) COMPONENT PART NUMBER VIAL LABEL VIAL NUMBER End Repair Buffer Mix S01532 Blue ER1 ver 4 End Repair Enzyme Mix S01533 Blue ER2 ver 4 End Repair Enhancer S01503 Blue ER3 Ligation Buffer Mix S01534 Yellow L1 ver 4 0319-32 0319-32 S01591 L2DR-BC1 S01592 L2DR-BC2 S01593 L2DR-BC3 S01594 L2DR-BC4 S01595 L2DR-BC5 S01596 L2DR-BC6 S01597 L2DR-BC7 S01598 L2DR-BC8 Ligation Adaptor Mix Yellow 0320-32 0320-32 S01599 L2DR-BC9 S01600 L2DR-BC10 S01601 L2DR-BC11 S01602 L2DR-BC12 S01603 L2DR-BC13 S01604 L2DR-BC14 S01605 L2DR-BC15 S01606 L2DR-BC16 Ligation Enzyme Mix S01535 Yellow L3 ver 4 Final Repair Buffer Mix S01567 Purple FR1 Final Repair Enzyme Mix S01569 Purple FR2 Nuclease-free Water S01001 Green D1 Agencourt Beads S01307 Clear — 4 Ovation Rapid Library Systems II. Components B. Additional Equipment, Reagents and Labware Required Materials • Equipment - Covaris S-series Sonication System - Agilent 2100 Bioanalyzer (optional, for analysis of DNA fragmentation) - Materials and equipment for electrophoretic analysis of nucleic acids including 2% agarose gels, DNA ladders (1 kB and 50 bp recommended) - Real-time PCR System capable of SYBR Green detection - Microcentrifuge for individual 1.5 mL and 0.5 mL tubes - 0.5–10 µL pipette, 2–20 µL pipette, 20–200 µL pipette, 200–1000 µL pipette - Vortexer - Thermal cycler with 0.2 mL tube heat block, heated lid, and 100 µL reaction capacity - Appropriate spectrophotometer and cuvettes, or Nanodrop® UV-Vis Spectrophotometer for quantitation of fragmented DNA • Reagents - Ethanol (Sigma-Aldrich, Cat. #E7023), for purification steps - 1X TE buffer (low EDTA), pH 8.0 (Affymetrix USB products, Cat. #75793) for input DNA sample - KAPA Library Quantification kit specified for Illumina and the real-time PCR system to be used • Supplies and Labware - 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 or 0.2 mL thin-wall PCR plates - Magnetic separation device options: ° Agencourt® SPRIPlate Ring Super Magnet Plate (Beckman Coulter, Cat. #A32782) — for PCR strips, skirted, non-skirted or half-skirted PCR plates ° Invitrogen™ DynaMag™-96 Side (Invitrogen, Cat. #123-31D) — for PCR strips, non-skirted or half-skirted PCR plates ° Invitrogen DynaMag-96 Side Skirted (Invitrogen, Cat. #120-27) — for skirted PCR plates ° Promega MagnaBot® II Magnetic Separation Device (Promega, Cat. #V8351) — for PCR plates ° Agencourt SPRIStand (Beckman Coulter, Cat. #A29182) - Disposable gloves - Kimwipes - Ice bucket - Cleaning solutions such as DNA-OFF™ (MP Biomedicals, Cat. #QD0500) - OPTIONAL: PhiX Control (Illumina, Cat. #FC-110-3001) 5 Ovation Rapid Library Systems II. Components To Order • Affymetrix, Inc., www.affymetrix.com • Beckman Coulter, www.beckmancoulter.com • Covaris, www.covarisinc.com • Illumina, www.illumina.com • Invitrogen, www.invitrogen.com • KAPA Biosystems, www.kapabiosystems.com • MP Biomedicals, www.mpbio.com • Promega, www.promega.com • Sigma-Aldrich, Inc., www.sigmaaldrich.com 6 Ovation Rapid Library Systems

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Ribo-SPIA, Applause, Encore, Prelude, Mondrian and Imagine More From Less are trademarks or registered in Table 2, following the operating instructions provided by the manufacturer. For ther- .. NaOH (0.5N instead of 0.1N) in the library denaturation mix and adding distilled water to make up
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