User Guide
Contents
1. Table 3 Sample Solution Preparation COMPONENT VOLUME 100 to 400 ng sheared ds DNA in water or 21 4 pL low EDTA TE Agencourt RNAClean XP beads 3 6 uL Sample Concentration Solution 25 uL Total volume 50 uL Ensure Agencourt RNAClean XP beads are at room temperature and completely resus pended prior to use Mix each sample solution well and incubate at room tempera ture approximately 23 C for 10 minutes The above recipe is meant for one sample Prepare sample solution for each sample to be processed 2 Encore SP Rapid Library Systems Reagent Master Mix Preparation Prepare End Repair Master Mix 1 Thaw End Repair Buffer Mix ER1 ver 6 at room temperature vortex to mix well and spin down briefly Keep End Repair Enzyme Mix ER2 ver 4 on ice 2 Prepare master mix in a 0 5 mL microcentrifuge tube or 0 2 mL PCR tube accord ing to the volumes shown in Table 4 Label the tube D IV Protocol Mix by pipetting and spin down the master mix briefly Place on ice Use immediately Mix by pipetting and spin down the master mix briefly Place on ice Use immediately Mix by pipetting and spin down the master mix briefly Place on ice Use immediately 16 Encore SP Rapid Library Systems Table 4 End Repair Master Mix COMPONENT VIAL CAP VOLUME End Repair Buffer Mix ER1 ver 6 Blue 7 0 uL End Repair Enzyme Mix ER2 ver 4 Blue 3 0 uL Total volume 10 0 pL Pr2. Thermo Scientific NanoDrop Spectrophotometer to quantify the Encore SP Rapid Library Systems libraries You must quantitate these libraries using qPCR We recommend the KAPA Biosystem Library Quantification kits Failure to quantitate the Encore SP Rapid Library Systems libraries by qPCR will result in inaccurate concentration readings which in turn will lead to low cluster density and poor quality sequencing results Can the Encore SP Rapid Library Systems kits be used on the Mondrian SP Workstation Yes The Encore SP Rapid Library Systems kits can be used with both the Mondrian SP and SP Workstation When using the Mondrian SP Workstation select the correct Encore SP Rapid protocol VII Appendix D Update History This document the Encore SP Rapid Library Systems user guide M01295 v5 is an update to address the following topics Description Section Page s Removed references to integration with Ovation RNA Seq System V2 and Ovation RNA Seq FFPE System Part Nos LA VIC 3 29 7102 and 7150 Updated Table 3 IV E 15 Updated Mondrian SP Cartridge Loading Instructions IV E 18 19 Changed loading volume for D ports IV E 19 Updated Library Collection from Mondrian SP Cartridge IVE 20 21 section NuGEN Technologies Inc Headquarters USA Europe 201 Industrial Road Suite 310 P O Box 109 For our international distributors contact San Carlos CA 94070 USA 9350 AC Leek information visit our web
3. 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 l NEFOGUCHON ss scsssssscsssessssssssssvecsescsssssscssacasessessesssssssvesasovsaasossusensasssssvecasscssseasssssess 1 As Background yenne S E sands a E E EE 1 B Performance Specifications ces sxcssicssacessncveuscsaccescuccssecshsessncsacneytcbevavuctadlechovachiens 3 C brary QuantifiCationic sasini neiaa eE a a anina 3 D Quality Control eieiei sae inini 3 E StOrage amd Stabilityiss cascssscssenccesasentesiecsstedssdsessae depend danedacdeaieastengsousevsashenesanen td 4 F Material Safety Data Sheet MSDS cecceeceeeceeeeeeeeeeeeseeeeeceeeneeeseeeseeneeeeeneees 4 ll COMPOMGNES 3 5e22ss55855cevadsesescccsassesgieccsevsnssieccsabensssoeszauseasescep cuseaaee
4. 5 S01677 DR Multiplex Ligation Adaptors 1of2 Yellow L2V9DR BC9 S01678 9 16 L2V9DR BC10 S01679 L2V9DR BC11 S01680 L2V9DR BC12 S01681 L2V9DR BC13 S01682 L2V9DR BC14 S01683 L2V9DR BC15 S01684 L2V9DR BC16 S01467 Ligation Enzyme Mix 1 of 2 Yellow L3 ver 4 01703 Final Repair Buffer Mix 1 of 2 Purple FR1 ver 2 01704 Final Repair Enzyme Mix 1 of 2 Purple FR2 ver 2 S01707 Library Dilution Buffer Mix 1of2 N A LD1 7 Components Encore SP Rapid Library Systems Encore SP Rapid DR Multiplex System 9 16 Components and Reagents Part No 8042 32 continued 8042 8042 PART E a MIAR E T NUMBER CAP S01001 Nuclease free Water 1of2 Green D1 P01190 Mondrian SP Cartridges X4 2of2 N A N A S01561 Cartridge Filler Fluid X4 2of2 N A N A 01556 Sample Concentration Solution 2 of 2 N A N A se Saes earel NA a 01588 Bead Binding Solution 2 of 2 N A N A 01589 Bead Wash Solution 2 of 2 N A N A 01590 Elution Buffer 2 of 2 N A N A P0119 ueo Ao 2of2 N A N A B Additional Equipment Reagents and Labware Required Materials e Equipment Mondrian SP Workstation NuGEN Part No 8000 or Mondrian SP Workstation NuGEN Part No 8100 Nanodrop UV Vis Spectrophotometer or appropriate spectrophotometer and cuvettes for quantification of fragmented DNA Covaris S series Sonication System to fragment input DNA Agilent 2100 Bioanalyzer optional for analysis of DN
5. VI Technical SUPPOrtscsivnjssessesessscassonetessansiessnnascenssned snpacssensscovanasesedaasscsensagensncaasuates 24 VIN AP PONiGIX eisene eae sovnescnvaaadsntacassnsetesscasecsucasesabaassenbasavansess 25 A Preparation of Encore SP Rapid Library Systems Libraries for Cluster Generation on Illumina SysteMs ccccseesessesseeeeeeseerseseeeeesenseeeens 25 By Barcode SEQUENCES acc eniesssa te ciciea ats tacit tere eevee OT O O 28 C Frequently Asked Questions FAQS urease 29 D Update Histo tiy scciscists iste aaa E E E iu EI AER 31 1 Introduction Encore SP Rapid Library Systems A Background The Encore SP Rapid Library Systems composed of Encore SP Rapid DR Multiplex Systems 1 8 and 9 16 Part Nos 8041 and 8042 are complete reagent cartridge and protocol packages for the simple automation of DNA library preparation protocols for next generation sequencing using the Mondrian SP or SP Workstation These systems enable library preparation from 100 400 ng of sheared double stranded DNA dsDNA and do not require any post Mondrian PCR enrichment or purification prior to library quantitation and sequencing The resulting libraries are suitable for a range of sequencing applications including RNA Seq Digital Gene Expression DGE genomic DNA sequencing and amplicon sequencing As shown in Figure 1 the streamlined workflow consists of five steps 1 Fragmentation of either genomic DNA or double stranded cDNA to produ
6. applications Can the Encore SP Rapid Library Systems be used with paired end sequencing Yes they 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 How much material should I load for cluster generation Please follow manufacturer s recommendations and see Appendix A Preparation of Encore SP Rapid Library Systems Libraries for Cluster Generation on the Illumina System Do the Encore SP Rapid Library Systems work with the Illumina Cluster Station predecessor of the cBot instrument Yes the Encore SP Rapid Library Systems are also compatible with the Illumina Cluster Station don t have access to a Covaris instrument Can I use alternative frag mentation methods We have evaluated only Covaris fragmented DNA during the development of the Encore SP Rapid Library Systems Other mechanical means of frag mentation such as nebulization may be suitable as well How does your protocol improve the efficiency of ligation and avoid adaptor dimer formation The Encore SP Rapid Library Systems use optimized chemistries to increase the efficiency of blunt end adaptor ligation and minimize the amount of adaptor dimer in the library Does NuGEN provide reagents for performing the fragmentation step of the protocol We suggest using the Covaris instrument as indicated in the materials section of this user guide NUGEN does n
7. the Workstation moving carefully to avoid spilling the fluid or disturbing the reagent 1 Locate the Cartridge Loading Guide that is provided with each Mondrian SP Cartridge and place the loading guide on the cartridge Note it is only possible to place the loading guide on the cartridge in a single orientation 2 Optional Carefully move the Filler Fluid containing cartridge and its Cartridge Loading Guide to the deck of the Mondrian SP or SP Workstation and insert the cartridge into the deck 3 Remove the Elution Buffer reagent tube shipped and stored at room temperature from the Encore SP Rapid Library System kit 4 Using a 50 or 100 uL single channel pipette set to 50 pL load 50 uL of Elution Buffer into port E5 of the Mondrian SP Cartridge When adding sample or reagent lower the pipette tip to the bottom of the port Do not press the tip into the bottom of the cartridge if the tip contacts the bottom of the cartridge withdraw the pipette tip slightly upwards Slowly depress the plunger to the first stopping point to dispense the reagent completely from the pipette tip do not depress the plunger completely as this will introduce bubbles into the cartridge Once all of the reagent is dispensed from the pipette tip pull the pipette tip back out of the port and dispose of the tip Note Do NOT add any samples or other reagents to the cartridge at this time Ensure that only Elution Buffer has been loaded 5 If the ca
8. 9 16 use the same approach to multiplexing used in the standard Illumina method These libraries should be sequenced using the Illumina protocol for multiplex sequencing The DR barcode sequences are found in Appendix B and must be entered into the Illumina software prior to the analysis 9 Encore SP Rapid Library Systems Ill Planning the Experiment C Library Quantification Libraries created using Encore 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 Purified Libraries for details D Final Library Storage Libraries may be stored at 20 C 10 Encore SP Rapid Library Systems IV Protocol A Overview The library preparation process used in the Encore SP Rapid Library Systems is per formed on the Mondrian SP or SP Workstation The process includes sample con centration DNA end repair purification adaptor ligation and final end repair It takes approximately 4 hours to complete The end product is a purified library that is ready for quantification prior to cluster generation on Illumina systems Library quantification via qPCR takes approximately 3 hours including qPCR KAPA product gel and quantifi cation calculations so the total time to prepare purified and quantified libraries ready for seq
9. A 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 uL pipette 2 20 uL pipette 20 200 uL pipette Vortexer 8 Components Encore SP Rapid Library Systems e Reagents 1X TE buffer low EDTA pH 8 0 Affymetrix Cat 75793 for input DNA samples KAPA Library Quantification Kit specified for Illumina and the real time PCR system to be used Quant iT PicoGreen dsDNA Assay Kit for quantifying fragmented DNA e 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 Disposable gloves Kimwipes Ice bucket Cleaning solutions such as DNA OFF MP Biomedicals Cat QD0500 To Order e Affymetrix www affymetrix com e Agilent www agilent com e Covaris www covarisinc com e Invitrogen www invitrogen com e KAPA Biosystems www kapabiosystems com e MP Biomedicals www mpbio com e Nanodrop www nanodrop com Ill Planning the Experiment A Input DNA Requirements The Encore SP Rapid Library Systems are designed to work with 100 to 400 ng of frag mented genomic DNA or ds cDNA DNA samples must be free of contaminating proteins RNA organic solv
10. C3 01672 L2V9DR BC4 01673 L2V9DR BC5 01674 L2V9DR BC6 S01675 L2V9DR BC7 S01676 L2V9DR BC8 S01467 Ligation Enzyme Mix 1 of 2 Yellow L3 ver 4 01703 Final Repair Buffer Mix 1 of 2 Purple FR1 ver 2 01704 Final Repair Enzyme Mix 1 of 2 Purple FR2 ver 2 S01707 Library Dilution Buffer Mix 1 of 2 N A LD1 01001 Nuclease free Water 1 of 2 Green D1 PO1190 Mondrian SP Cartridges X4 2 of 2 N A N A S01561 Cartridge Filler Fluid X4 2 of 2 N A N A 01556 Sample Concentration Solution 2 of 2 N A N A 501698 Agencourt RNAClean XP Shipped N A N A Purification Beads separately S01588 Bead Binding Solution 2of2 N A N A 6 Components Encore SP Rapid Library Systems Encore SP Rapid DR Multiplex System 1 8 Components and Reagents Part No 8041 32 continued ee 8041 8041 ya 8041 NUMBER DESCRIPTION BOX CAP VIAL NUMBER 01589 Bead Wash Solution 2 of 2 N A N A 01590 Elution Buffer 2 of 2 N A N A 01199 Encore SP Rapid Cartridge 2 of 2 N A N A Loading Guide X4 Table 2 Encore SP Rapid DR Multiplex System 9 16 Components and Reagents Part Number 8042 32 ees 8042 8042 ne 8042 NUMBER DESCRIPTION BOX CAP VIAL NUMBER 01705 End Repair Buffer Mix 1 of 2 Blue ER1 ver 6 01706 End Repair Enzyme Mix 1 of 2 Blue ER2 ver 4 S01626 End Repair Enhancer 1 of 2 Blue ER3 501625 End Repair Enhancer Buffer 1of2 Blue ER4 Mix S01662 Ligation Buffer Mix 1of2 Yellow L1 ver
11. E Storage and Stability The Encore SP Rapid Library Systems reagents are shipped in two boxes Box 1 is shipped on dry ice and should be stored at 20 C on an internal shelf of a freezer without a defrost cycle upon receipt Box 2 is shipped and should be stored at room tempera ture The Agencourt RNAClean XP Beads clear cap are shipped separately and should be removed from the top of the Box 2 shipping carton upon delivery and stored at 4 C The kit has been tested to perform to specifications after as many as four freeze thaw cycles Kits handled and stored according to the above guidelines will perform to specifi cations for at least six months F 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 5 Components Encore SP Rapid Library Systems A Reagents Provided Table 1 Encore SP Rapid DR Multiplex System 1 8 Components and Reagents Part No 8041 32 se 8041 8041 Re 8041 NUMBER DESCRIPTION BOX CAP VIAL NUMBER S01705 End Repair Buffer Mix 1of2 Blue ER1 VER 6 S01706 End Repair Enzyme Mix 1of2 Blue ER2 ver 4 01626 End Repair Enhancer 1 of 2 Blue ER3 501625 End Repair Enhancer Buffer 1of2 Blue ER4 Mix S01662 Ligation Buffer Mix 1 of 2 Yellow L1 ver 5 S01669 DR Multiplex Ligation Adaptors 1of2 Yellow L2V9DR BC1 S01670 1 8 L2V9DR BC2 S01671 L2V9DR B
12. Encore SP Rapid Library Systems PART NOS 8041 AND 8042 E imagine more GEI less 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 NuUGEN 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 Specific information on patents trademarks and licenses related to the Mondrian SP Universal Cartridge the Mondrian SP Cartridge the Mondrian SP Workstation and the Mondrian SP Workstation may be found in the Mondrian SP Universal Cartridge User Guide M01265 the Mondrian SP Cartridge User Guide M01344 the Mondrian SP Workstation User Manual Part No M01264 and the Mondrian SP Workstation User Manual M01322 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 u
13. GAII MiSeq HiScan SO or HiSeq NGS platforms without gel based size selection using 100 to 400 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 The Encore SP Rapid Library Systems generate quantified libraries ready for cluster generation on an Illumina System in about 7 hours C Library Quantification Libraries created using Encore 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 Purified Libraries for details D Quality Control Every lot of the Encore SP Rapid Library Systems undergoes functional testing to con firm that the products meet the specifications for library generation performance Introduction This product contains components with multiple 4 storage temperatures Encore SP Rapid Library Systems We recommend the use of control samples when beginning experiments and or using a new source of samples For RNA based experiments such as RNA Seq we recom mend the use of the MicroArray Quality Control MAQC reference samples A and B For DNA based experiments such as WGS we recommend the use of a control DNA sample from the HapMap project
14. NUGEN kits should not be used with the Encore SP Rapid Library Systems C DNA Fragmentation We recommend using a Covaris S series instrument to fragment DNA or ds cDNA to the desired size prior to starting the Encore SP Rapid Library Systems protocol 1 Dilute appropriate amount of intact DNA into 120 uL of 1X TE buffer low EDTA pH 8 0 2 Transfer 120 uL to Covaris snap cap microtube 11 Encore SP Rapid Library Systems IV Protocol 12 Encore SP Rapid Library Systems 3 Fragment to desired insert size following Covaris recommended settings 4 Quantify fragmented samples by either UV absorbance or Quant iT PicoGreen dsDNA assay preferred D Cartridge Quality Control Check The Mondrian SP Cartridge QC protocol is a QC check that we recommend be per formed prior to adding samples and reagents to the Mondrian SP Cartridge This QC check confirms the functionality of the Mondrian SP Cartridges prior to use The Mondrian SP Cartridge QC protocol requires inserting the Filler Fluid containing cartridge into the deck of the Mondrian SP or SP Workstation Some customers may find it easier to place the fluid containing cartridge into the deck of the workstation and then pipet the Elution Buffer into the cartridge Alternatively it may be easier to pipet the Elution Buffer into the fluid containing cartridge while the cartridge rests on the bench top and then insert the reagent and fluid filled cartridge into the deck of
15. RY PROTOCOL Volume of Library Denaturation Mix already f 10 pL in tube Pre chilled HT1 490 uL Final volume of library denaturation mix 500 pL for lt 2 nM libraries The final concentrations of NaOH and library in the library denaturation mix are as indicated in Table 13 Table 13 Final NaOH and library concentrations in the Library Denaturation Mix for lt 2 nM libraries REAGENT lt 2 nM LIBRARY PROTOCOL NaOH 1 0 mM Encore SP Rapid Library Systems library 20 pM 27 Encore SP Rapid Library Systems VII Appendix 28 Encore SP Rapid Library Systems B Barcode Sequences Table 14 Barcode sequences for dedicated read DR adaptors LIGATION ADAPTOR MIX BARCODE SEQUENCE L2V9DR BC1 AAGGGA L2V9DR BC2 CCTTCA L2V9DR BC3 GGACCC L2V9DR BC4 TTCAGC L2V9DR BC5 AAGACG L2V9DR BC6 CCTCGG L2V9DR BC7 GGATGT L2V9DR BC8 TTCGCT L2V9DR BC9 ACACGA L2V9DR BC10 CACACA L2V9DR BC11 GTGTTA L2V9DR BC12 TGTGAA L2V9DR BC13 ACAAAC L2V9DR BC14 CACCTC L2V9DR BC15 GTGGCC L2V9DR BC16 TGTTGC Vil 29 Appendix Encore SP Rapid Library Systems C Frequently Asked Questions FAQs Q1 Q2 Q3 Q4 Q5 Q6 Q7 What kind of sequencing primers can use with your library The Encore SP Rapid Library Systems are designed for use with the standard Illumina sequencing primers for both single end and paired end sequencing
16. ce the assay template 2 Addition of the template and reagents to the Mondrian SP Universal Cartridge see Figure 1A 3 Hands free automation of the following assay steps on the Mondrian SP or SP Workstation see Figure 1B e Sample concentration e End repair e Sample purification e Adaptor ligation with optional sample multiplexing e Sample purification e Final repair 4 Library quantification with KAPA Library Quantification Kit 5 Cluster formation and sequencing see Figure 1C Introduction Figure 1 The five steps of the Encore SP Rapid Library System workflow A Steps 1 2 Covaris fragmentation of the dsDNA template and loading the Mondrian SP Cartridge with template and reagents B Step 3 assay steps automated on the Mondrian SP Cartridge C Steps 4 5 library quantification cluster formation and sequencing Mondrian SP Cartridge Step 2 Filler E4 ES E7 Make master mix ge 0080 load in reagent ports 4 9990 Step 1 Sample Collection 2100 400 ng fragmented 000 000P DNA generated on a Covaris instrument 2 2 A i Encore SP Rapid Library Systems Single use only Encore SP Rapid Library System reagents Step 3 Mondrian SP or SP Workstation Sample concentration performs the following steps in 4 hours SOULE TIA e End repair steps 1 and 2 Add adaptors and ligate optional multiplexing Sample purification Final repair Steps 4 5 Step 4 Pe
17. cepsaseoiensesedsseuees 5 A Reagents Provided iiss ceicciiessanssadescnacteccessmcessdsasendesseestdee silage senebeaeestaceagdsenesteved 5 B Additional Equipment Reagents and Labware ou ceceeeeeeeeeseseeeeeneenes 7 lll Planning the Experiment ccciscscsssccscsessescecocssssesestoctsastesctactiessesscaceisssssocectinsnasoceces 9 A Input DNA REGUIFEMONES tc sscdicctedesasctsvedsedesscabdesdarsosasssessaactesa couse sneavereasesense 9 B Using Encore SP Rapid Library Systems on Illumina NGS Systems 0004 9 Ca brary QuantiticatiO i sinine an ienen Oa EER NR 10 D Final Library Storage sc eccciescsscssstieesuscssncssceuenevceudnesncnschuessuadonaesnandaoddesvevecsestenes 10 IV lt Protocol P TTT TO sees suue st iedy sbecesseedy seucees 11 As QVEIVIOW einantis cous tact son cde sucdndauchek paai aia NANEN EEE 11 B Protocol Notes agieren eee Ae a EEE ESE AA ERIEN SEA 11 C DNA Fragmentation eireiensannas ennen aea EER 11 D Cartridge Quality Control Check sssrinin arcsiiinea 12 E Protocol for the Encore SP Rapid Library Systems ON MOmanian SP Cart AGes essscccsisveseesossdesievonancatencesazans shaun tanewaasivensteataebeaseveetes 15 V Quantitative and Qualitative Assessment of the Purified Libraries 22 A POVEM EW Speen ings tan tes ducsa OEE TEE E N ETERN 22 B Quantification Using KAPA Biosystems Products seeereeerrererersrsn 22 G Gel Analysis of KAPA gPCR Product asisnuntonuinsernennnani a causeslts 22
18. chnologies Inc Support Mondrian The results are Repeat the Mondrian SP Cartridge QC protocol one SP cartridge inconclusive more time status is and must be undetermined Please consult Mondrian SP Universal Cartridge User Guide or appropriate NuGEN SP Library Systems User Guide for further instructions repeated one more time prior to making a determination on the quality of the cartridge e Do not add any additional Filler Fluid or Elution Buffer to the cartridge e Press OK on the Run Complete screen to return to the main menu e Proceed to re run the Mondrian SP Cartridge QC protocol starting from Step 10 in the protocol above If the message after the second Mondrian SP Cartridge QC protocol is e Mondrian SP cartridge passed then proceed as outlined in Next Step for passing cartridges above e Mondrian SP Cartridge failed or Mondrian SP cartridge status is undetermined do not use the cartridge and contact NUGEN Technical Support Encore SP Rapid Library Systems IV Protocol 15 Encore SP Rapid Library Systems E Protocol for the Encore SP Rapid Library Systems on Mondrian SP Cartridges 1 Sample Solution Preparation Prepare sample solution for loading onto the cartridge this is done on a per sample basis and not as a master mix according to the volumes shown in Table 3 You must prepare and process no fewer than eight samples on each cartridge
19. ding to the volumes shown in Table 7 Label the tube D4 Table 7 Final Repair Master Mix COMPONENT VIAL CAP VOLUME Final Repair Buffer Mix FR1 ver 2 Purple 8 0 uL Final Repair Enzyme Mix FR2 ver 2 Purple 2 0 uL Total volume 10 0 pL Prepare Adaptors 1 Thaw ligation adaptors at room temperature vortex well to mix and spin down briefly Use the desired barcode adaptors L2V9DR BC1 16 yellow vial cap 2 The adaptors will be loaded onto the cartridge as indicated below Step 8 of the Mondrian SP Cartridge Loading Instructions 3 Mondrian SP Cartridge Loading Instructions IV Protocol Figure 4 Mondrian SP Cartridge loading guide for the Encore SP Rapid Library System protocol Fille va 23 OC O on OO0O000 COO e ORCKOROHCHOES OCOO0S00 G Encore SP Rapid Library Systems Single use only ge NUGEN C 0096099 Loading Reagents and Samples in the Mondrian SP Cartridge P01199 v1 Using Figure 4 as a guide follow the instructions below to load reagents into their appropriate cartridge ports e Usea 10 uL pipette to add all reagent master mixes and the adaptors e Use a 100 or 200 uL pipette for adding the samples Bead Binding Solution Elution Buffer and Bead Wash Solution e Load the ports in a steady manner to avoid overflow of the Filler Fluid e When adding sample or reagent lower the pipette tip to the bottom of the port Do no
20. e and insert the tip all the way to the bottom of the sample collection port perpendicular to the cartridge to make a seal between the cartridge and the pipet tip Maintaining the seal formed between the pipette tip and the bottom of the cartridge release the plunger Immediately lift the pipette slightly off the bottom of the cartridge to release the seal rapidly drawing Filler Fluid and the sample droplet into the pipette tip Examine the pipette tip to ensure that the appropriately sized droplet is sus pended in the Filler Fluid IV Protocol 21 Encore SP Rapid Library Systems 10 11 Dispense the collected fluid including the small sample droplet directly into the 11 uL of Library Dilution Buffer in the collection tube Pipette up and down a few times in the aqueous phase to ensure that all the sample transfers into the buffer Discard the tip and repeat steps 2 5 to ensure complete collection of the entire sample This is important because occasionally a portion of the sample droplet is left behind in the sample collection port Continue to the next sample collection port and repeat this process until all eight libraries have been collected and placed in separate tubes Remove the cartridge from the workstation and dispose of as appropriate in labo ratory waste Cap the library droplet containing PCR tubes and vortex briefly to mix Spin down briefly to bring the aqueous phase to the bottom of the tube The Filler Flu
21. ease contact NUGEN Technical Support at 650 590 3674 direct or 888 654 6544 option 2 toll free US 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 or email europe nugen com In all other locations contact your NUGEN distributor for technical support 24 Encore SP Rapid Library Systems Vil Appendix 25 Encore SP Rapid Library Systems A Preparation of Encore SP Rapid Library Systems Libraries for Cluster Generation on Illumina Systems Guidance for preparation of Encore SP Rapid Library Systems libraries for cluster gen eration is provided below based upon the final library concentration Using reduced volume modified recipes may enable you to save an aliquot of the library for later studies Follow the standard protocol for creation of library denaturation mix if you would like to use the entire aliquot of Encore SP Rapid Library Systems library in the sequencing reaction We recommend following the half volume protocol for the library denaturation mix if you would prefer to retain half the Encore SP Rapid Library Systems library aliquot for later use If your final library concentration is between 1 11 and 1 95 nM we recommend following the instructions below under the header For Library Concentrations Lower than 2 nM For Library Concentrations of 2 nM or Greater 1 Dilute libraries to a concentrati
22. ents including phenol and ethanol and salts We recommend using a commercially available system for DNA cDNA isolation To obtain optimal library yields we recommend that the Encore SP Rapid Library System input be determined based on both the fragment size of the sample and the method of quantification For 200 bp fragments the recommended input is 200 ng if measured using either NanoDrop or UV absorbance or 100 ng if measured using PicoGreen quantification For 500 bp fragments the recommended input is 350 ng if measured using either NanoDrop or UV absorbance or 250 ng if measured using PicoGreen quantification Note We recommend PicoGreen quantification of the input DNA as it provides greater library yield consistency If using NanoDrop or UV absorbance the A260 A280 ratio for DNA samples should be in excess of 1 8 Using DNA samples with lower ratios may result in low library yield B Using Encore SP Rapid Library Systems on Illumina NGS Systems The Encore SP Rapid Library Systems use a Dedicated Read DR design with a second sequencing primer for multiplex sequencing Figure 2 depicts the DR multiplex barcode strategy Figure 2 Dedicated read multiplexing strategy used by the Encore SP Rapid Library Systems Dedicated Read Barcode Design Illumina Standard Seq Primer Library Insert Illumina Index Seq Primer Jeees m Flow cell surface IIIIIIIT The Encore SP Rapid DR Multiplex Systems 1 8 and
23. epare End Repair Enhancer Master Mix 1 Thaw End Repair Enhancer Buffer Mix ER4 at room temperature vortex to mix well and spin down briefly Keep End Repair Enhancer ER3 on ice 2 Prepare master mix in a 0 5 mL microcentrifuge tube or 0 2 mL PCR tube accord ing to the volumes shown in Table 5 Label the tube D7 Table 5 End Repair Enhancer Master Mix COMPONENT VIAL CAP VOLUME End Repair Enhancer Buffer Mix ER4 Blue 7 0 uL End Repair Enhancer ER3 Blue 3 0 uL Total volume 10 0 pL Prepare Ligation Master Mix 1 Thaw Ligation Buffer Mix L1 ver 5 at room temperature vortex to mix well and spin down briefly Keep Ligation Enzyme Mix L3 ver 4 on ice 2 Prepare master mix in a 0 5 mL microcentrifuge tube or 0 2 mL PCR tube according to the volumes shown in Table 6 Label the tube D5 Table 6 Ligation Master Mix COMPONENT VIAL CAP VOLUME Ligation Buffer Mix L1 ver 5 Yellow 7 0 uL Ligation Enzyme Mix L3 ver 4 Yellow 3 0 uL Total volume 10 0 pL IV Protocol Mix by pipetting and spin down the master mix 17 briefly Place on ice Use immediately Encore SP Rapid Library Systems Prepare Final Repair Master Mix 1 Thaw Final Repair Buffer Mix FR1 ver 2 at room temperature vortex to mix well and spin down briefly Keep Final Repair Enzyme Mix FR2 ver 2 on ice 2 Prepare master mix in a 0 5 mL microcentrifuge tube or 0 2 mL PCR tube accor
24. id oil should remain as a separate layer on top of the aqueous phase Use a pipette to remove most of the Filler Fluid floating on top of the aqueous phase This will aid the aqueous phase collection Use a fresh tip to transfer the aqueous phase 11 uL from beneath the remaining Filler Fluid and into a fresh tube 12 The aqueous phase contains the purified library Store the library at 20 C or pro ceed immediately to library quantitation V Quantitative and Qualitative Assessment of the Purified Libraries 22 Encore SP Rapid Library Systems A Overview This section details how to prepare Encore SP Rapid libraries for quantification and qualification prior to cluster generation and sequencing B Quantification Using KAPA Biosystems Products Use the KAPA Library Quantification kit specific for the Illumina NGS platform and your available qPCR instrument Follow the instructions described by KAPA BioSystems in the Technical Data Sheet for the KAPA Library Quantification kit you are using We recommend dilutions be performed with a volume of purified library not to exceed 1 0 uL 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 a 1 1 000 and a 1 10 000 dilution on the same plate as the KAPA Biosystems concentra tion standards run in triplicate See Appendix A for recommendations on the final pMolarity of the hybridizatio
25. n solution C Gel Analysis of KAPA qPCR Product Gel analysis of the library KAPA qPCR product is important for qualitative and quan titative 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 Also use the gel image to determine if adaptor dimers are present Adaptor dimers may reduce the number of useful sequence reads On the same 2 agarose gel run the following lanes 1 5 uL of KAPA product from one well of the 1 1 000 dilution and one well of the 1 10 000 dilution for the sample library 2 5 uLof one KAPA concentration standard 3 10 pL 10 ng uL of 1 Kb Plus and or 50 bp ladder See Figure 5 for an example of KAPA qPCR products generated with the Encore SP Rapid Library System run on a 2 agarose gel V Quantitative and Qualitative Assessment of the Purified Libraries 23 Encore SP Rapid Library Systems Figure 5 2 agarose gel analysis of Encore SP Rapid Library System libraries after KAPA qPCR analysis Lane 1 is the 1 Kb Plus ladder For size reference the position of migration of the 1 Kb and 500 bp fragments in the 1 Kb ladder are noted with arrows Lanes 2 through 5 are KAPA products from libraries made from increasing amounts 250 to 400 ng of fragmented human male DNA average size of frag ment was 500 bp 1000 bp 500 bp VI Technical Support For help with any of our products pl
26. o D7 Port D7 is high D7 lighted with a blue rim O Load 50 uL Bead Binding Solution in E4 Port E4 is not highlighted in E4 any way Load 50 uL Elution Buffer into E5 Port E5 is highlighted with a grey rim E5 IV Protocol 20 Encore SP Rapid Library Systems 10 Load 50 uL Bead Wash Solution into E7 Port E7 is highlighted with a E7 black rim Load 50 uL of sample mix into S1 S8 ensure that the 10 minute incubation step described above is performed prior to loading onto the cartridge Sample input ports are highlighted with red rims OOOSS000 If the cartridge is not already inserted into the Mondrian SP Workstation deck insert the cartridge into the deck and pull the locking lever forward to engage the cartridge with the control electronics and close the lid P01199 v1 Mondrian SP Workstation Initialization Instructions iE If not already ON locate the workstation ON OFF switch at the back of the work station and turn ON Press the On button on the front of the workstation Select Run on the touch screen menu select Encore SP Rapid from the list of available protocols and follow the instructions on the screen to begin the run Library Collection from the Mondrian SP Cartridge Place 8 low binding 0 2 mL or 0 5 mL microcentrifuge tubes in a rack Add 11 uL of Library Dilution Buffer LD1 to each tube Use a 100 or 200 uL pipette set to 20 pL Depress the plunger on the pipett
27. omplete screen and one of the following messages 13 Encore SP Rapid Library Systems IV Protocol MESSAGE MEANING NEXT STEP Mondrian No errors Press OK on the Run Complete screen to return SP cartridge detected to the main menu Proceed to section C Loading passed Droplet transport Samples and Reagents below in the Mondrian Continue was normal SP Universal Cartridge user guide or follow the to intended instructions for 3rd party reagents in the appropri protocol ate applications note The user may remove the cartridge from the Workstation to load samples and remaining reagents on the bench top taking care that the cartridge remains level at all times Note It is not necessary to add additional Elution Buffer or Filler Fluid to the cartridge prior to loading reagents and samples Mondrian A problem was Press OK on the Run Complete screen to return SP cartridge detected with to the main menu Remove the cartridge from the failed Remove cartridge from instrument deck and set aside prior to contact droplet trans port within the cartridge deck and set it aside do not discard and contact NuGEN Technical Support for further instructions Users who proceed with SP protocols and or who load additional SP reagents and samples onto failed cartridges are doing so at their own risk and will not ing NuGEN be compensated for loss of reagents samples or Technical cartridges by NuGEN Te
28. on 2 nM using Library Dilution Buffer Mix LD1 2 Mix the NaOH and the 2 nM Encore SP Rapid Library Systems library together in a microcentrifuge tube according to the volumes shown in Table 8 Table 8 Library Denaturation Mix for 2 nM libraries ILLUMINA STANDARD HALF VOLUME REAGENT PROTOCOL PROTOCOL 0 1N NaOH 10 uL 5 uL 2 nM Encore SP Rapid Library Systems Library vee me Initial volume of Library Denaturation Mix for 2 nM 20 pL 10 pL libraries 3 Incubate the tube at room temperature for 5 minutes 4 Add pre chilled HT1 reagent from the Illumina Sequencing Reagent kit to the microcentrifuge tube as indicated in Table 9 Vil Appendix 26 Encore SP Rapid Library Systems Table 9 Final Library Denaturation Mix for 2 nM libraries REAGENT ILLUMINA STANDARD HALF VOLUME denaturation mix PROTOCOL PROTOCOL Volume of Library Denaturation Mix already 20 uL 10 uL in tube Pre chilled HT1 980 uL 490 uL Final volume of library 1000 pL 500 pL The final concentrations of NaOH and library in the library denaturation mix are the same regardless of which volume protocol is used as indicated in Table 10 Table 10 Final NaOH and DNA concentrations in the Library Denaturation Mix for 2 nM libraries ILLUMINA STANDARD HALF VOLUME REAGENT PROTOCOL PROTOCOL NaOH 1 0 mM 1 0 mM Encore SP Rapid Library 20 pM 20 pM Systems library For Library Concentra
29. ot provide the reagents used in the fragmentation steps Vil 30 Appendix Encore SP Rapid Library Systems Q8 Q9 Q10 Q11 Are the libraries from the Encore SP Rapid Library Systems compatible with downstream target capture methods like Agilent s SureSelect or Nimblegen s SeqCap EZ No the libraries from the Encore SP Rapid Library Systems do not yield enough library material to be compatible with downstream target cap ture methods such as Agilent s SureSelect and Nimblegen s SeqCap EZ kits These protocols usually require a minimum of 500 ng library input Customers who wish to perform target selection on their samples should use the Ovation SP Ultralow Library Systems Part Nos 8033 and 8034 together with the Encore Target Capture Module Part No 0332 to prepare samples for target selection with the Agilent and Nimblegen products How can gel purification be eliminated from the workflow and still pre vent adaptor dimer formation The Encore SP Rapid Library Systems workflow uses bead based purifica tion and efficient primer design thus eliminating the need for gel based purification Can I use an Agilent 2100 Bioanalyzer or a Thermo Scientific NanoDrop Spectrophotometer to determine the final concentration of an Encore SP Rapid Library Systems library and use this measurement to calculate how much library need for cluster generation No We do not recommend using the Agilent 2100 Bioanalyzer or the
30. rformed off Library quantification PREA Mondrian SP or eal SP Workstation ae j AATCGGATCGGTAGGAT user anew TCTCGATGCAAGTGATE and sequencing GTAGCAAAATCCTGAGA 2 Encore SP Rapid Library Systems 3 Introduction Encore SP Rapid Library Systems The entire workflow requires no manual bead purification or gel purification steps Starting with 100 400 ng of fragmented double stranded DNA dsDNA the protocol can be completed in approximately 4 hours and yields libraries ready for quantification prior to cluster formation and either single read or paired end sequencing Importantly for DNA sequencing applications samples can be input directly to the library construction workflow without the need for pre amplification The absence of PCR steps in the library construction workflow makes this library construction method particularly well suited to either high or low GC genomes which are often susceptible to PCR artifacts The Encore SP Rapid DR Multiplex Systems 1 8 and 9 16 each provide eight unique dedicated read barcoded adaptors to prepare libraries for multiplex sequencing using a dedicated read design strategy with a second sequencing primer Together these two multiplex kits enable up to 16 plex sequencing B Performance Specifications The Encore SP Rapid Library Systems are designed to produce DNA libraries suitable for either single read or paired end sequencing on the Illumina Genome Analyzer IIx lle
31. rtridge is not already inserted into the Mondrian SP or SP Workstation deck carefully transport the cartridge to the Mondrian SP or SP Workstation and insert the cartridge into the deck 6 If not already ON locate the workstation ON OFF switch at the back of the workstation and turn ON IV Protocol 7 Press the On button Figure 3 on the front of the workstation to turn the instrument on Figure 3 The Mondrian SP or SP Workstation On button located on the front of the workstation 8 Pull the cartridge lever of the Mondrian SP or SP Workstation forward to the locked position and close the lid of the workstation 9 Select Run on the touch screen menu choose the Mondrian SP Cartridge QC protocol from the list of protocols and then select Next to proceed to the Protocol Information screen 10 Select Next to proceed to the Run Information screen 11 Optional Enter run details as required in the Run Information screen 12 Select Next to proceed to the Run Confirmation screen and select Start Run The Mondrian SP Cartridge QC protocol will take about nine minutes to complete During this test Elution Buffer droplets will be dispensed from the E5 port and trans ported around the cartridge prior to being discarded The purpose of this test is to confirm the successful transport of droplets across all lanes of the cartridge At the end of the protocol the instrument will display the Run C
32. se 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
33. site Toll Free Tel 888 654 6544 The Netherlands U Toll Free Fax 888 296 6544 Tel 31 13 5780215 giidi eT custserv nugen com Fax 31 13 5780216 imagine more from less techserv nugen com europe 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 M01295 v5
34. t press the tip into the bottom of the cartridge If the tip contacts 18 Encore SP Rapid Library Systems IV Protocol 19 Encore SP Rapid Library Systems the bottom of the cartridge withdraw the pipette tip slightly upwards Slowly depress the plunger to the first stopping point to dispense the reagent com pletely from the pipette tip As you raise the pipette tip from the port you should complete a gentle blow out by fully depressing the pipette plunger just prior to exiting the Filler Fluid to ensure all the reagent is dispensed Using a desktop lamp to illuminate your work area may facilitate sample and reagent loading We recommend loading the reagents and samples with the cartridge on the deck of the Mondrian SP or SP Workstation Load 1 5 uL of each adaptor mix into ports A1 A8 using a 10 uL pipette Use L2 ver 10 for all samples Adaptor ports A1 through A8 are high lighted with yellow rims C0000 00C 4 Note If the adaptor droplet appears to be floating above the surface of the bottom plate of the cartridge use a clean pipette tip to gently push the droplet down to the surface Load 8 uL Final Repair Master Mix into D4 Port D4 is highlighted with a D4 white rim circled with a dark line Load 8 uL Ligation Master Mix into D5 Port D5 is highlighted with an D5 orange rim O Load 8 uL End Repair Master Mix into D Port D is highlighted with a D6 green rim O Load 8 uL End Repair Enhancer Master Mix int
35. tions Lower than 2 nM If the final concentration of the Encore SP Rapid Library Systems library is lower than 2 nM between 1 11 and 1 95 nM we recommend using a higher concentration of NaOH 0 5N instead of 0 1N in the library denaturation mix and adding distilled water to make up the difference in volume 5 Calculate the volume of library required using the following formula Library volume uL 10 library concentration nM For example if the final Encore SP Rapid Library System library concentration is 1 5 nM the required volume is 10 1 5 nM 6 67 uL 6 Aliquot the calculated volume of Encore SP Rapid Library System library into a microcentrifuge tube and add 0 5N NaOH and distilled water as required to gen erate 10 uL of Library Denaturation Mix as shown in Table 11 Vil Appendix Table 11 Library Denaturation Mix for lt 2 nM libraries REAGENT lt 2 nM LIBRARY PROTOCOL 0 5N NaOH 1 0 uL 1 11 to 1 95 nM Encore SP Rapid Library Systane library Volume uL 10 library concentration nM dH O To 10 uL final volume Initial volume of Library Denaturation Mix for lt 2 nM libraries TOL 7 Incubate the tube at room temperature for 5 minutes 8 Add pre chilled HT1 reagent from the Illumina Sequencing Reagent kit to the microcentrifuge tube as indicated in Table 12 Table 12 Final Library Denaturation Mix for lt 2 nM libraries REAGENT lt 2 nM LIBRA
36. uencing is approximately 7 hours B Protocol Notes e The system is designed and intended for processing eight samples at a time Do not attempt to prepare smaller volume master mixes or process fewer than eight samples using the Encore SP Rapid Library Systems e We recommend the routine use of a positive control DNA Especially the first time you set up a reaction the use of a positive control DNA will allow you to establish a performance baseline e Use the water provided with the kit green cap vial D1 or an alternate source of nuclease free water We do not recommend the use of DEPC treated water with this protocol e Thaw components used in each step and immediately place them on ice e Always keep thawed reagents and reaction tubes on ice unless otherwise instructed e After thawing and mixing buffer mixes if any precipitate is observed re dissolve it completely prior to use You may gently warm the buffer mix for 2 minutes at room temperature followed by brief vortexing Do not warm any enzyme or primer mixes e When placing small amounts of reagents into the reaction mix pipet up and down several times to ensure complete transfer e When instructed to pipet mix gently aspirate and dispense a volume that is at least half of the total volume of the reaction mix e Always allow the thermal cycler to reach the initial incubation temperature prior to placing the tubes or plates in the block e Components and reagents from other
Download Pdf Manuals
Related Search