USER GUIDE. Ovation Rapid DR Multiplex System 1 96 PART NO. 0328

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1 USER GUIDE Ovation Rapid DR Multiplex System 1 96 PART NO. 0328

2 Patents, Licensing and Trademarks 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 ( 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 product 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 Phone or ; FAX or 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.

3 Table of Contents Contents I. Introduction... 1 A. Background... 1 B. Performance Specifications... 2 C. Library Quantification... 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... 4 III. Planning the Experiment... 6 A. Input DNA Requirements... 6 B. Using the Ovation Rapid DR Multiplex System 1 96 on Illumina NGS Platforms... 6 C. Working with the NGS DR Adaptor Plate D. Library Quantification... 7 E. Amplified Library Storage... 7 F. Data Analysis and Parsing Multiplex Libraries... 7 IV. Protocol... 8 A. Overview... 8 B. Programming the Thermal Cycler... 8 C. Constructing the Automation Script... 9 V. Quantitative and Qualitative Assessment of the Library A. Quantitative and Qualitative Assessment of the Library B. Gel Analysis of KAPA qpcr Product VI. Technical Support VII. Appendix A. Barcode Sequences for Ovation Rapid DR Multiplex System 1 96 Reactions.15 B. Using the Ovation Rapid DR Multiplex System 1 96 as a Manual Kit C. Frequently Asked Questions (FAQs) D. Update History... 17

4 I. Introduction A. Background The Ovation Rapid DR Multiplex System 1 96 provides a simple, fast and scalable solution for producing libraries used in next-generation sequencing. This system enables library preparation starting with as little as 100 ng of double-stranded DNA without PCR amplification. The library construction workflow is compatible with a wide range of sequencing applications including RNA-Seq, Digital Gene Expression (DGE), genomic DNA sequencing, amplicon sequencing and more. These libraries are suitable for sequencing on the Illumina Genome Analyzer IIx/IIe (GAII), HiScan SQ, MiSeq and HiSeq 2000/2500 Systems. While the Ovation Rapid DR Multiplex System 1 96 is primarily intended for use with a liquid handling robotics system, such as the Caliper Sciclone NGS workstation, it may also be used for manual library production. For manual processing, refer to Appendix B: Using the Ovation Rapid DR Multiplex System 1 96 as a Manual Kit. As shown in Figure 1, the streamlined workflow consists of four main steps: fragmentation 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 generally be completed in fewer than two hours and yields libraries ready for quantitation and cluster formation and either single read or paired-end sequencing. For large numbers of samples, DNA fragmentation is expected to require additional time, possibly making the entire library production process longer than two hours. The Ovation Rapid DR Multiplex System 1 96 has been designed for seamless integration with NuGEN s Ovation RNA-Seq System V2 and Ovation RNA-Seq FFPE System (Part Nos and 7150) to enable a complete end-to-end solution for transcriptome library construction starting with total RNA. The Ovation Rapid DR Multiplex System 1 96 (Part No. 0328) contains reagents for production of up to 96 barcoded libraries. Each kit provides 96 unique 8-base dedicated read (DR) barcoded adaptors to prepare libraries for multiplex sequencing using a dedicated read design strategy with a second sequencing primer. 1 Ovation Rapid DR Multiplex System 1 96

5 I. Introduction Figure 1. Ovation Rapid DR Multiplex System 1 96 workflow. Input DNA Fragment 5 3 End-repair 5 P 3 P Add adaptors and ligate Final repair Bead purification Cluster formation and sequencing AATCGGATCGGTAGGAT TCTCGATGCAAGTGATC GTAGCAAAATCCTGAGA B. Performance Specifications The Ovation Rapid DR Multiplex System 1 96 is 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 2000/2500 systems without gelbased size selection, using ng input of double-stranded DNA. The absence of PCR amplification in the workflow eliminates the risk of unequal amplification or PCR bias introduced as a result of differences in template composition. When preparing libraries using cdna, it is critical to use at least µg of ds-cdna prepared using the Ovation RNA-Seq System V2 (Part No. 7102) or Ovation RNA-Seq FFPE System (Part No. 7150). When preparing libraries using cdna, it is critical to use at least µg of ds-cdna prepared using the Ovation RNA-Seq System V2 (Part No. 7102) or Ovation RNA-Seq FFPE System (Part No. 7150). 2 Ovation Rapid DR Multiplex System 1 96

6 I. Introduction C. Library Quantification 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 quantification. See Section V. Quantitative and Qualitative Assessment of the Library for details. D. Quality Control Every lot of the Ovation Rapid DR Multiplex System 1 96 undergoes functional testing to meet specifications for library generation performance. E. Storage and Stability The Ovation Rapid DR Multiplex System 1 96 is shipped on dry ice and should be unpacked immediately upon receipt. 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 specifications for at least six months. F. Safety Data Sheet (SDS) If applicable, an SDS for this product is available on the NuGEN website at 3 Ovation Rapid DR Multiplex System 1 96

7 II. Components A. Reagents Provided Table 1. Ovation Rapid DR Multiplex System 1 96 Reagents (Part No ) COMPONENT 0328 PART NUMBER VIAL LABEL VIAL NUMBER MRV* End Repair Buffer Mix S01686 Blue ER1 ver µl End Repair Enzyme Mix S01687 Blue ER2 ver µl End Repair Enhancer S01688 Blue ER3 275 µl Ligation Buffer Mix S01689 Yellow L1 ver µl Ligation Enzyme Mix S01690 Yellow L3 ver µl Final Repair Buffer Mix S01695 Purple FR µl Final Repair Enzyme Mix S01696 Purple FR2 135 µl Nuclease-free Water S01001 Green D µl Ligation Adaptor Plate S01697 NGS DR Adaptor Plate µl/well *Minimum Recoverable Volume Note: SPRI beads (Agencourt RNAClean XP) are required for the final purification step in the Ovation Rapid DR Multiplex System 1 96 library process. This product must be obtained from Beckman Coulter. NuGEN does not supply SPRI beads in this kit due to the variances in purification method implementation on different liquid handling platforms. B. Additional Equipment, Reagents and Labware Required Materials Equipment Covaris S-series Sonication System 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 µl pipette, 2 20 µl pipette, µl pipette, µl pipette 4 Ovation Rapid DR Multiplex System 1 96

8 II. Components 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 quantification of fragmented DNA Reagents Agencourt RNAClean XP Kit (Beckman Coulter, Cat. #A63987) KAPA Library Quantification Kit specified for Illumina sequencing platforms and for the qpcr platform to be used Ethanol (Sigma-Aldrich, Cat. #E7023), for purification steps 1X TE buffer (low EDTA), ph 8.0 (Affymetrix USB products, Cat. #75793) 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. #12331D) for PCR strips, non-skirted or half-skirted PCR plates Invitrogen DynaMag-96 Side Skirted (Invitrogen, Cat. #12027) for skirted PCR plates Promega MagnaBot II Magnetic Separation Device (Promega, Cat. #V8351) for PCR plates AlumaSeal II foil seals for resealing the adaptor plate in the event that not all adaptor wells are used at one time (Excel Scientific, Cat. #AFS-25) Disposable gloves Kimwipes Ice bucket Cleaning solutions such as DNA OFF (MP Biomedicals, Cat. #QD0500) To Order Affymetrix, Inc., Beckman Coulter, Covaris, Excel Scientific, Invitrogen, KAPA Biosystems, MP Biomedicals, Promega, Sigma-Aldrich, Inc., 5 Ovation Rapid DR Multiplex System 1 96

9 III. Planning the Experiment A. Input DNA Requirements The Ovation Rapid DR Multiplex System 1 96 is designed to work with as little as ng of fragmented genomic dsdna or ds-cdna. When using ds-cdna generated by NuGEN s Ovation RNA-Seq System V2 (Part No. 7102) or Ovation RNA-Seq FFPE System (Part No. 7150), use at least 500 ng 1 µg of of ds-cdna. DNA samples must be free of contaminating proteins, RNA, organic solvents (including phenol and ethanol) and salts. Use of a commercially available system for DNA/cDNA isolation is recommended. The A260:A280 ratio for DNA samples should be in excess of 1.8. Use of DNA samples with lower ratios may result in low library yield. B. Using the Ovation Rapid DR Multiplex System 1 96 on Illumina NGS Platforms The Ovation Rapid DR Multiplex System 1 96 uses a dedicated read (second sequencing primer) strategy, as shown in Figure 2. Figure 2. Dedicated read (DR) barcoding strategy. Dedicated Read Barcode Design Illumina Standard Seq Primer Library Insert Illumina Index Seq Primer Flow cell surface Barcode The Ovation Rapid DR Multiplex System 1 96 uses 8-base barcodes and the same approach to multiplexing found in the standard Illumina method. The resulting libraries should be sequenced using the Illumina protocol for multiplex sequencing for 8-base barcodes (total of 9 cycles). The DR barcode sequences can be found by clicking the barcodes link on the Ovation Rapid DR Multiplex System 1 96 product page at and must be entered into the Illumina software prior to the analysis. 6 Ovation Rapid DR Multiplex System 1 96

10 III. Planning the Experiment C. Working with the NGS DR Adaptor Plate 1 96 The NGS DR Adaptor Plate 1 96 contains 96 ligation adaptor mixes, each with a unique eight-base barcode. Each well of the 96-well plate contains sufficient volume for preparation of a single library. The NGS DR Adaptor Plate 1 96 is sealed with a foil seal designed to provide for airtight storage. Each well is designed for a single use. Prior to thawing the adaptor plate, spin it for 5 to 10 minutes at room temperature at ~1000 X g in a centrifuge with a rotor appropriate for microwell plates. This will allow the plate to thaw while spinning and help to minimize cross-contamination between wells. To use only a portion of the plate, remove the seal covering only the portion of the plate to be used. The remaining wells of the plate should remain sealed for use at a later date. Used wells can be covered with a new foil seal (e.g., AlumaSeal II) to prevent any remaining adaptor-containing liquid from contaminating future reactions. D. Library Quantification Libraries created using Ovation Rapid DR Multiplex System 1 96 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 quantification. See Section V. Quantitative and Qualitative Assessment of the Library for details. E. Amplified Library Storage Amplified libraries may be stored at 20 C. F. Data Analysis and Parsing Multiplex Libraries Data analysis for next generation sequencing is an evolving field and the number of available analysis strategies and software tools is growing rapidly. The specific analysis workflow for a given experiment will depend on many variables, including the type of sequencing application (DNA-Seq, RNA-Seq, etc.) and the goals of the particular study. Follow the recommendations in the Illumina technical support documentation on parsing barcodes. The sequences of the Ovation Rapid DR Multiplex System 1 96 barcodes must be entered prior to parsing. These sequences can be found by clicking the barcodes link in the left hand menu of the Ovation Rapid DR Multiplex System 1 96 product page at Once the data have been parsed according to sample, additional sample-specific data analysis may be conducted according to the requirements of the experiment. 7 Ovation Rapid DR Multiplex System 1 96

11 IV. Protocol A. Overview The library preparation process used in the Ovation Rapid DR Multiplex System 1 96 is performed in three stages: 1. DNA end repair 0.75 hours 2. Adaptor ligation 0.5 hours 3. Final repair and purification 0.75 hours Total time to prepare amplified library ~2 hours Components in the Ovation Rapid DR Multiplex System 1 96 are color coded, with each color linked to a specific stage of the process. Each stage requires making a master mix then adding it to the reaction, followed by incubation. Master mixes are prepared by mixing components provided for each stage. B. Programming the Thermal Cycler Use a thermal cycler with a heat block designed for 0.2 ml tubes, equipped with a heated lid, and with a capacity of 100 µl reaction volume. Prepare the programs shown in Table 2, following the operating instructions provided by the manufacturer. For thermal cyclers with an adjustable heated lid, set the lid temperature to 100 C only when sample temperature reaches above 30 C. For thermal cyclers with a fixed temperature heated lid (e.g., ABI GeneAmp PCR 9600 and 9700 models), use the default settings (typically 100 to 105 C). Table 2. Thermal Cycler Programming END REPAIR Program 1 End Repair 25 C 30 min, 70 C 10 min, hold at 4 C LIGATION Program 2 Ligation 25 C 30 min, 70 C 10 min, hold at 4 C FINAL REPAIR Program 3 Final Repair 72 C 2 min, hold at 25 C 8 Ovation Rapid DR Multiplex System 1 96

12 IV. Protocol C. Constructing the Automation Script The Ovation Rapid DR Multiplex System 1 96 has been designed to enable construction of sequencing libraries in 48 or 96 sample batches. Reagent volumes have been scaled accordingly, taking into account the overages required for automation protocols. To assist in constructing automation scripts we have provided a minimum recoverable volume for each component (see Table 1). This volume can be used as a guideline to determine the feasibility of a particular automation schema. Take care in developing automation scripts that minimize reagent consumption above the nominal volume requirements (dead volumes) in order to ensure 96 full reactions. For automation, a minimum of 48 samples must be processed at once to ensure a full 96 reactions per kit. Producing Finished Libraries Using the Ovation Rapid DR Multiplex System Determine the number of samples to process and the starting address of the multiplex adaptors. (In the event that some wells in the adaptor plate have already been used, it is necessary to indicate the plate address of the next available unused adaptor.) 2. Set up the deck and prepare the master mixes (refer to Tables 3 5). 3. Place 10 µl of 100 ng 1 µg fragmented DNA in the PCR plate (reaction plate) wells. 4. Add 5 µl End Repair Master Mix (Table 3) to the reaction plate and mix (total volume = 15 µl). Table 3. End Repair Master Mix (volumes listed are for a single reaction) Mix by pipetting and spin down the master mix briefly. Place on ice. Use immediately. END REPAIR BUFFER MIX (BLUE: ER1 ver 3) END REPAIR ENZYME MIX (BLUE: ER2 ver 4) END REPAIR ENHANCER (BLUE: ER3) 3.5 µl 0.5 µl 1.0 µl 5. Remove the plate from the deck and seal. 6. Place the plate in a pre-warmed thermal cycler programmed to run Program 1 (End Repair; see Table 2): 25 C 30 min, 70 C 10 min, hold at 4 C 7. Remove the plate from the thermal cycler, spin down, unseal and return to the deck. 8. Unseal needed wells of the adaptor plate, transfer 15 µl of each sample into the appropriate well of the adaptor plate and mix. 9 Ovation Rapid DR Multiplex System 1 96

13 IV. Protocol 9. Transfer the entire volume from the adaptor plate wells back into the reaction plate wells (total volume = 18 µl). 10. Add 12 µl Ligation Master Mix (Table 4) to the reaction plate and mix (total volume = 30 µl). Table 4. Ligation Master Mix (volumes listed are for a single reaction) Mix by pipetting and spin down the master mix briefly. Place on ice. Use immediately. LIGATION BUFFER MIX (YELLOW: L1 ver 4) LIGATION ENZYME MIX (YELLOW: L3 ver 4) WATER (GREEN: D1) 6.0 µl 1.5 µl 4.5 µl 11. Remove the plate from the deck and seal. 12. Place the plate in a pre-warmed thermal cycler programmed to run Program 2 (Ligation; see Table 2): 25 C 30 min, 70 C 10 min, hold at 4 C 13. Remove the plate from the thermal cycler, spin down, unseal and return to the deck. 14. Add 20 µl Final Repair Master Mix (Table 5) to the reaction plate and mix (total volume = 50 µl) Table 5. Final Repair Master Mix (volumes listed are for a single reaction) Mix by pipetting and spin down the master mix briefly. Place on ice. Use immediately. FINAL REPAIR BUFFER MIX (PURPLE: FR1) FINAL REPAIR ENZYME MIX (PURPLE: FR2) 19.0 µl 1.0 µl 15. Remove the plate from the deck and seal. 16. Place the plate in a pre-warmed thermal cycler programmed to run Program 3 (Final Repair; see Table 2). 72 C 2 min, hold at 25 C 17. Remove the plate from the thermal cycler, spin down, unseal and return to the deck. 18. Add 50 µl Agencourt RNAClean XP beads to each sample and mix (total volume = 100 µl) 19. Transfer samples and beads to round bottom Costar plate to facilitate small volume elution required in steps below. 20. Incubate at room temperature for 10 minutes. 21. Transfer to the magnetic plate to clear for 5 minutes. 10 Ovation Rapid DR Multiplex System 1 96

14 IV. Protocol 22. Remove 80 µl of supernatant to waste. 23. Wash the beads 2 times with 150 μl 70% ethanol. 24. Air dry for 2 minutes. 25. Remove the Costar plate from the magnet plate. 26. Add 13 µl 1X TE buffer (low EDTA), ph 8.0, to the beads and mix to resuspend. 27. Transfer to the magnetic plate to clear for 5 minutes. 28. Carefully transfer 10 µl cleared supernatant to a fresh PCR plate. 29. Continue with Qualitative and Quantitative Assessment of the Library. For additional guidance or to contract the services of NuGEN Automation Scientists for developing scripts for your liquid handling system, contact NuGEN Technical Services or your NuGEN Account Executive. 11 Ovation Rapid DR Multiplex System 1 96

15 V. Quantitative and Qualitative Assessment of the Library Libraries created using Ovation Rapid DR Multiplex System 1 96 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 quantification. A. Quantitative and Qualitative Assessment of the Library Use the KAPA Library Quantification kit specific for the Illumina NGS platform and qpcr instrument being used. Follow the instructions described by KAPA BioSystems in the Technical Data Sheet for the KAPA Library Quantification kit being used. We recommend dilutions be performed with a volume of purified library not to exceed 1.0 μl. Problems may arise if too much sample is used when yields are low. Note: For the library qpcr reactions, we recommend loading at least duplicates of 1:100 and 1:1000 dilutions per library on the same plate as the KAPA Biosystems concentration standards (run in triplicate). For cbot cluster formation, use a final Molarity in the hybridization solution of 8 12 pm. B. Gel Analysis of KAPA qpcr Product Gel analysis of the library KAPA qpcr product is important for qualitative and quantitative analysis of the final libraries. Use the gel to determine the average size of the library, as this value is required for calculation of the concentration of the libraries (Step 6, detailed protocol; KAPA Library Quantification kit Technical Data Sheet). Also, use the gel image to determine if adaptor dimers are present. Adaptor dimers may reduce the efficiency of cluster formation. On the same 2% agarose gel, run the following lanes: 1. 5 µl of KAPA product from one well of the 1:100 dilution for the sample library 2. 5 µl of one KAPA concentration standard µl (10 ng/μl ) of 1 Kb Plus and 50 bp ladder in the same gel 12 Ovation Rapid DR Multiplex System 1 96

16 V. Quantitative and Qualitative Assessment of the Library Figure 3. 2% agarose gel analysis of libraries after KAPA Library Quantification kit processing. Lanes 1 and 10 are the 50 bp ladder; lanes 2 and 9 are the 1 Kb Plus ladder. For size reference, the position of migration of the 1 Kb, 0.5 Kb, and 0.1 Kb fragments in the 1 Kb ladder are noted with arrows. Duplicate KAPA products from libraries prepared with either 100 ng (Lanes 3 and 4) or 200 ng (Lanes 5 and 6) of fragmented genomic E. coli DNA are shown. Lanes 7 and 8 are duplicate qpcr reactions from a KAPA DNA standard. 13 Ovation Rapid DR Multiplex System 1 96

17 VI. Technical Support For help with any of our products, please contact NuGEN Technical Support at (direct) or , option 2 (toll-free, U.S. only). Send faxes to (toll-free) or techserv@nugen.com. In Europe contact NuGEN at +31(0) (Phone) or +31(0) (Fax) or europe@nugen.com. In all other locations, contact your NuGEN distributor for technical support. 14 Ovation Rapid DR Multiplex System 1 96

18 VII. Appendix A. Barcode Sequences for Ovation Rapid DR Multiplex System 1 96 Reactions Barcode sequences for the Ovation Rapid DR Multiplex System 1 96 can be found by clicking on the barcodes link in the left hand menu of the Ovation Rapid DR Multiplex System 1 96 product page at B. Using the Ovation Rapid DR Multiplex System 1 96 as a Manual Kit For manual use of this kit, follow the protocol given in this user guide with the following changes. 1. In order to maintain consistent incubation times, we recommend that batch reactions be limited to fewer than 32 reactions. 2. Final Repair (D. Final Repair, Step 16): If the library purification step cannot be performed within 60 minutes after the final repair step, change the hold step to 4 C. 3. Library Purification (E. Library Purification, Step 24): Air dry the beads on the magnet for 10 minutes to ensure that all the ethanol has evaporated. It is critical that all residual ethanol be removed prior to continuing. 4. Library Purification (E. Library Purification, Step 26): Add 11 µl room temperature 1X TE buffer (low EDTA) to the dried beads. Mix thoroughly to ensure all the beads are resuspended and let stand on the bench top for 5 minutes. 5. Library Purification (E. Library Purification, Step 28): Carefully transfer 9 µl of the supernatant to a fresh PCR tube, ensuring as few beads as possible are carried over. C. Frequently Asked Questions (FAQs) Q1. What kind of sequencing primers can I use with your library? The Ovation Rapid DR Multiplex System 1 96 is designed for use with the standard Illumina sequencing primers for both single end and paired-end sequencing applications. Q2. Can the Ovation Rapid DR Multiplex System 1 96 be used with pairedend sequencing? Yes, it can be used for both single end and paired-end sequencing. Special consideration should be given to the expected insert size in the paired-end assay. The expected distances between the 5 -most and 3 -most coordinates of paired-end reads will depend on the average fragment size of the insert pool. Q3. How much material should I load onto the cbot? Follow the Illumina recommendations for loading libraries onto the cbot. 15 Ovation Rapid DR Multiplex System 1 96

19 VII. Appendix Q4. Does the Ovation Rapid DR Multiplex System 1 96 work with the Illumina Cluster Station (predecessor of the cbot instrument)? Yes, it is also compatible with the Illumina Cluster Station. Q5. I don t have access to a Covaris instrument, can I use alternative fragmentation methods? We have evaluated only Covaris fragmented DNA during the development of the Ovation Rapid DR Multiplex System Other mechanical means of fragmentation, such as nebulization, may be suitable. Q6. How does your protocol improve the efficiency of ligation and avoid adaptor dimer formation? The Ovation Rapid DR Multiplex System 1 96 uses optimized chemistries to increase the efficiency of blunt-end adaptor ligation and minimize the amount of adaptor dimer in the library. Q7. Does NuGEN provide reagents for performing the fragmentation step of the protocol? We suggest using the Covaris instrument for DNA fragmentation, as recommended in the Materials section of this user guide. NuGEN does not provide the reagents used in the fragmentation steps. Q8. Which NuGEN amplification system kits can be used to produce dsdna for input to the Ovation Rapid DR Multiplex System 1 96? The Ovation RNA-Seq System V2 and Ovation RNA-Seq FFPE System (Part Nos and 7150) have been validated to work with the Ovation Rapid DR Multiplex System Q9. How can gel purification be eliminated from the workflow and still prevent adaptor dimer formation? The Ovation Rapid DR Multiplex System 1 96 workflow uses bead-based purification and efficient primer design, thus eliminating the need for gelbased purification. Q10. What kind of error correction is used to minimize the impact of sequencing errors in the barcodes? For experiments using the Ovation Rapid DR Multiplex System 1 96 with dedicated read barcodes, please follow the Illumina recommendations on parsing barcodes. The NuGEN dedicated read barcodes are eight-base unique barcode tags. The sequence of these NuGEN barcodes must be input into the Illumina sequencing analysis software prior to parsing. Q11. What is the expected yield of the library using the Ovation Rapid DR Multiplex System 1 96? The expected yield is >1000 pm (final library concentration), depending on the quality and quantity of the source cdna or genomic DNA. 16 Ovation Rapid DR Multiplex System 1 96

20 VII. Appendix D. Update History This document, the Ovation Rapid DR Multiplex System 1 96 user guide (M01300 v5), is an update to address the following topics: Description Section Page Provided clarification for input volumes III.A. 6 Changed Vial Cap to Vial Label II.A. 4 NuGEN Technologies, Inc. Headquarters USA 201 Industrial Road, Suite 310 San Carlos, CA USA Toll Free Tel: Toll Free Fax: custserv@nugen.com techserv@nugen.com Europe P.O. Box AC Leek The Netherlands Tel: Fax: europe@nugen.com For our international distributors contact information, visit our website 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 ( 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. M01300 v5