Mondrian SP Protocol
Contents
1. m T Mondrian SP Library Preparation Method 1 DNA Library Construction for Illumina SBS Sequencing Platforms I Introduction This application note describes the materials and methods for constructing DNA sequence libraries utilizing NEBNext DNA Sample Prep library construction reagents on the Mondrian SP digital microfluidics system The protocol requires 100 nanograms of fragmented DNA as input and produces indexed or non indexed libraries ready for quanti tation and normalization prior to cluster generation The system processes eight samples at a time on a sin gle Mondrian SP Cartridge Reagent master mixes and samples are prepared by the user and loaded into the cartridge After placing the cartridge into the Mondrian SP Workstation the protocol is selected on the touch screen user interface The system processes the samples through each enzyme step and purification steps with no further intervention by the user At the end of the approxi mately three and a half hour run the processed sample is extracted from the cartridge added to PCR master mix and placed into a thermal cycler for library enrichment Following library enrichment the library is quantitated and normalized for cluster generation The libraries created are suitable for sequencing on the Genome Analyzer IIx lle GAII HiScan SQ HiSeq and MiSeq systems ga NUGEN imagine more from lesse ll Materials Instrumentation and Consumables Mond2. Cat E7023 for purification e 10 mM Tris HCI 0 1 mM EDTA pH 8 0 e Water nuclease free e Nuclease free pipette tips e 1 5 mL and 0 5 mL RNase free microcentrifuge tubes e 0 2 mL individual thin wall PCR tubes e Disposable gloves e Kimwipes e Ice bucket e Cleaning solutions such as DNA OFF MP Biomedicals Cat QD0500 for preparing the work space Samples The DNA samples used in this study were genomic DNA from E coli and H sapiens gDNA samples were soni cally sheared using the Covaris 220 ultrasonicator system according to manufacturer s instructions to generate DNA fragments of approximately 150 to 200 bases lll Protocol A Overview The approximate time to complete a library run is summa rized below Time required for each step of a library preparation run Preparation of master mixes and cartridge set up 0 5 hours Running the protocol 3 25 hours PCR enrichment purification amp quantitation 2 25 hours Total time to pieces and anal amplified T mE library B Sample Concentration Prepare DNA samples as follows Sheared DNA 100 ng xx pL Sample concentration solution 25 uL AMPure XP magnetic beads 3 6 pL Water xx uL Total volume 50 pL Note adjust the DNA volume and water to ensure a final sample volume of 50 microliters Mix thoroughly and incubate at room temperature for at least 10 min
3. The additional required master mixes below can be prepared during this time DO NOT PLACE SAMPLES ON ICE C Preparation of Master Mixes 1 End Repair Mix prepare and keep on ice Total volume COMPONENT VOLUME End Repair Reaction Buffer 10X 1 5 uL End Repair Enzyme Mix 0 75 uL Reagent additive 1 5 uL Water 3 75 uL 7 5 pL 2 dA Tailing Mix prepare and keep on ice COMPONENT VOLUME dA Tailing Reaction Buffer 10X 15 pL Klenow Fragment 3 5 exo 0 9 uL Reagent additive 1 5 yL Water 3 6 uL Total volume 7 5 pL 3 Ligation Mix prepare and keep on ice COMPONENT VOLUME Quick Ligation Reaction Buffer 5X 4 5 uL Quick T4 DNA Ligase 2 25 uL Reagent Additive 0 75 uL Total volume 7 5 pL 4 a Indexed Adaptors one mix prepared for each library Prepare and keep at room temperature COMPONENT VOLUME 50 uM adaptor mix 1 5 uL Reagent Additive 1 0 uL Total volume 2 5 pL 4 b Non indexed Adaptors a single mix prepared for all eight libraries Prepare and keep at room temperature COMPONENT VOLUME 50 uM adaptor mix 12 uL Reagent Additive 8 uL Total volume 20 pL D Loading the Cartridge Refer to the Mondrian SP Universal Cartridge User Guide for instructions on how to prepare load reagent and mount the Universal Cartridge on the Mondrian SP Workstation Pipet 50 uL of each s
4. million reads per library are shown as an overlay illustrating very even coverage of the genome with no discernable regions of over or under representation FIGURE 3 Plot illustrating depth of coverage for a series of libraries generated from 100 ng E coli gDNA on the Mondrian SP Workstation using Library Preparation Method 1 500000 450000 ee ae EET IEE RENE REAR ESET ee ee 400000 bel i b eevee tee echo seee costes P 350000 ba 2 300000 teas ae p NN b 2 250000 e e E Se See be bc8 5 1000 00 Ma eR o 150000 4 100000 477 50000 4 04 1 0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 51 54 57 60 Depth of coverage V Conclusions The Library Preparation Method 1 protocol on the Mondrian SP Workstation is a robust hands free system for producing high quality libraries for sequencing on the Illumina SBS Sequencing Systems The first in a series of enabling molecular methods to be ported to the Mondrian SP digital microfluids platform this method allows for a substantial savings in hands on lab time while providing efficiencies of scale in reagent use and reproducibility by reducing reaction volumes and automating the most time consuming and effort intensive tasks Jonathan Benton Rahul Dhopeshwarkar Ph D Allen Eckhardt Ph D Bonnie Kwong Miriam Levy Ph D Brian Minie Garry Miyada Ph D Michael Pollack Ph D Arnaud Rival Ph D
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6. Jeremy Rouse Arjun Sudarsan Ph D Michael Weiand Uichong Yi Ph D NUGEN Technologies Inc San Carlos CA Advanced Liquid Logic Inc Research Triangle Park NC Broad Institute Cambridge MA Portions of this method utilize Broad proprietary technology The Mondrian SP Workstation and Cartridges were developed and manufactured for NuGEN by Advanced Liquid Logic NuGEN Technologies Inc Headquarters USA 201 Industrial Road Suite 310 San Carlos CA 94070 USA Toll Free Tel 888 654 6544 Toll Free Fax 888 296 6544 custserv nugeninc com techserv nugeninc com g NUGEN imagine more from less Europe P O Box 149 6680 AC Bemmel The Netherlands Tel 31 13 5780215 Fax 31 13 5780216 europe nugeninc com For our international distributors contact information visit our website www nugeninc com 2011 NuGEN Technologies Inc All rights reserved The Ovation and Applause families of products and methods are covered by U S Patent Nos 6 692 918 6 251 639 6 746 251 7 354 717 7 771 946 and Application Ser No 12 615958 issuance pending and other issued and pending patents in the U S and other countries NUGEN Ovation SPIA Ribo SPIA WT Ovation 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 M01
7. ample mix into S1 S8 ensure that the 10 minute incubation step as described above is performed prior to loading onto the cartridge f u ipet 50 uL of Bead Wash into E7 2 Pipet 50 uL of Elution Solution into E5 NOTE Well E6 is intentionally left empty ipet 50 uL of Bead Binding Solution into E4 pet 6 uL of End Repair mix into D7 pet 6 uL of dA Tailing mix into D pet 6 uL of Ligation mix into D5 P TOS ae a u ipet 1 5 uL of Adaptor 1 into A1 1 5 uL of Adaptor 2 into A2 and so forth until adaptors are loaded in all eight adaptor wells E Starting the Run 1 Refer to the Mondrian SP Workstation User Manual for instructions on how to turn on the workstation and select and run a protocol 2 Run the protocol Library Preparation Method 1 3 If desired fill in user and sample information for the run as described in the Mondrian SP Workstation User Manual 4 The run will be completed in 3 25 hours F Collecting the Library 1 When the run is completed the screen displays Run Successful Press the Done button and turn off the instrument by pressing the Power button below the touch screen Note If the screen indicates there were issues with the run continue to the Home screen and press the LOG button On the following screen select the current run and export the log csv file to a USB drive inserted on the right side of the Touch Screen 2 Open the lid if clos
8. d at 4 C H Library Purification and Quantification 1 Remove the Agencourt AMPure XP magnetic beads from the refrigerator and allow to warm to room tem perature before proceeding 2 Resuspend beads by inverting and tapping the bottle Ensure beads are fully resuspended before adding to sample 3 Prepare a 70 ethanol wash solution It is critical that this solution be prepared fresh on the same day of the experiment from a recently opened stock container Measure both the ethanol and the water components carefully prior to mixing Failure to do so can result in 10 11 12 13 14 15 16 a higher than anticipated aqueous content that may reduce yield Allow the PCR enriched libraries to come to room temperature Add 50 uL 1 volume of the AMPure XP bead suspen sion to each sample Mix thoroughly by pipetting up and down several times Incubate the samples at room temperature for 10 minutes Transfer tubes to a recommended magnet device and allow to stand 5 minutes or until the solution is com pletely cleared of beads Carefully remove 85 uL of the binding buffer from each sample and discard Leaving some of the volume behind minimizes bead loss at this step Note The beads should not disperse instead they will stay on the walls of the tubes Significant loss of beads at this stage will impact the yield of DNA from PCR amplification so ensure beads are not removed with the binding buffer
9. ed 3 Collect samples from the appropriate wells as described in the Mondrian SP Universal Cartridge User Guide 4 After all the prepared libraries have been extracted from the cartridge push the deck lever to disengage the cartridge and remove the cartridge from the instrument deck by gently pulling it toward the front of the instru ment Dispose the cartridge as appropriate and use a Kimwipe to clean up any spilled filler fluid on the deck 5 Add 20 uL of TE 10 mM Tris HCl 0 1 mM EDTA pH 8 0 to each tube containing the library droplet and filler fluid Mix and allow the aqueous lower phase to separate from the filler fluid G PCR Enrichment 1 Remove the PfuUltra II Hotstart PCR Master Mix and PCR primers and allow them to thaw 2 Prepare a PCR reaction Master Mix according to the fol lowing table Keep on ice COMPONENT VOLUME 2X PfuUltra Il Master Mix 225 uL PCR Primer 1 25 uM 18 uL PCR Primer 2 25 uM 18 uL Water 27 uL 3 Set up each library for enrichment according to the fol lowing table carefully recovering the 18 uL sample from the lower aqueous phase COMPONENT VOLUME PCR Master Mix 32 uL Sample 18 uL Total volume 50 pL 4 Mix each PCR reaction setup thoroughly 5 Process the samples in a thermocycler running the fol lowing program 95 C 1 min 10 cycles 95 C 30 sec 65 C 30 sec 72 C 1 min 72 C 10 min hol
10. or the wash With the tubes still on the magnet add 200 uL of freshly prepared 70 ethanol and allow to stand for 30 seconds Remove the 70 ethanol wash using a pipette Repeat the 70 ethanol wash one more time for a total of two washes Note With the final wash it is critical to remove as much of the ethanol as possible Use at least two pipet ting steps and allow excess ethanol to collect at the bottom of the tubes after removing most of the ethanol in the first pipetting step Air dry the beads on the magnet for 5 10 minutes Inspect each tube carefully to ensure that all the ethanol has evaporated It is critical that all residual ethanol be removed prior to continuing Remove tubes from magnet Add 22 uL TE 10 mM Tris HCl 0 1 mM EDTA pH 8 0 to the dried beads Mix thoroughly to ensure all the beads are resuspended and let stand on the bench top for 3 minutes Transfer tubes to the magnet and let stand for 3 minutes for the beads to clear the solution Carefully remove 20 uL of the eluate ensuring as few beads as possible are carried over transfer to a fresh set of PCR tubes and place on ice It is better to leave some of the solution behind rather than to carry over the beads 17 Remove 1 5 uL of the purified library and determine its concentration on a NanoDrop 8000 or equivalent IV Results The system produces sufficient material for library quan titation evaluation and cluster generation Fig
11. rian SP Workstation NUGEN Part No 8000 Mondrian SP Universal Cartridge NUGEN Part No 8010 The Universal Cartridge package includes Digital microfluidics cartridge Filler Fluid Sample Concentration Solution Bead Binding Solution Bead Wash Elution Solution Reagent Additive Cartridge Loading Guide Reagents NEBNext DNA Sample Prep Master Mix Set 1 NEB Cat E6040S PfuUltra Il Hotstart PCR Master Mix Agilent Cat 600850 AMPure XP magnetic bead DNA purification system Beckman Coulter Genomics Cat A63881 Appropriate adaptor mix and PCR enrichment primers must be obtained separately see http www current protocols com protocol hg1802 for reference Additional Equipment NanoDrop 8000 Spectrophotometer or equivalent for the quantification of DNA by UV absorbance Thermocycler for PCR amplification 0 5 10 uL pipette 2 20 uL pipette 20 200 uL pipette 200 1000 uL pipette Vortexer Microcentrifuge for individual 1 5 mL and 0 5 mL tubes and 0 2 mL PCR tubes Magnetic separation device options Agencourt SPRIPlate Ring Super Magnet Plate Beckman Coulter Genomics Cat A32782 Invitrogen DynaMag 96 Side Invitrogen Cat 123 31D Invitrogen DynaMag 96 Side Skirted Invitrogen Cat 120 27 Promega MagnaBot II Magnetic Separation Device Promega Cat V8351 Agencourt SPRIStand Beckman Coulter Genomics Cat A29182 Supplies and Labware e Ethanol Sigma Aldrich
12. ures 1A C show the average library yield obtained for samples across multiple cartridges Consistent and sufficient PCR yield was determined by Nanodrop measurement FIGURE 1 Enriched library yields plotted by sample type A N 12 cartridge number B N 8 and lane number C N 3 Library Yield By Sample A 3 0 Pe Ce A A o 2 0 E g 1 5 Pees 2 510 05 E 0 0 E coli Human Library Yield By Cartridge B 3 0 25 Micrograms ee Oo uw o in A01145 A01149 A01265 Library Yield By Lane Micrograms gt gt N N o oa o uw o w 0 0 Average is shown with standard deviation The libraries were further characterized by Bioanalzyer to determine size distribution and the presence or absence of adaptor dimer As seen in Figure 2 libraries from both human and E coli genomic DNA were produced of the expected size with little or no adaptor dimer FIGURE 2 _ Bioanalyzer traces of purified libraries from human A and E coli B genomic DNA A Cee Conroe Cero ES FI mappe gpaana OE OJ ZE ORe Shr Sequencing of barcoded E coli gDNA libraries show even coverage with no discernable bias in representation Figure 3 These results are similar to in house results with libraries processed manually The distribution of a theoretical randomly generated library is shown as the dotted line The distribution plots for six actual libraries 2 35
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