Illumina Sequencing Quantitation Using Droplet Digital
Illumina Sequencing Quantitation usingDroplet Digital PCR (ddPCR) ProtocolDocument No.:MCCRD-SOP0007Version:1.2Effective Date:12/01/2014Page No.:Page 1 of 11Table of Contents1.0PURPOSE/SCOPE . 12.0REFERENCE DOCUMENTATION. 23.0BACKGROUND . 24.0DEFINITIONS . 25.0EQUIPMENT . 36.0REAGENTS/MATERIALS . 37.0AUTOMATION METHODS . 48.0SAFETY . 49.0ASSAY GUIDELINES . 410.0PROCEDURE . 51.0PURPOSE/SCOPEThis Standard Operating Procedure (SOP) describes the steps necessary to use digitaldroplet PCR (ddPCR) to quantitate libraries and/or pools of libraries for sequencing ofPatient Derived Xenograft samples on the Illumina HiSeq 2500. This protocol beginswith sample dilution, PCR plate set-up and processing, and concludes with data analysis.This SOP is intended for processing up to 24 samples per instrument run. This SOP isfor research purposes only and no clinical samples will be processed using thisSOP. Any deviation from this SOP will be noted but will not be formallydocumented.
Illumina Sequencing Quantitation usingDroplet Digital PCR (ddPCR) Protocol2.0Document No.:MCCRD-SOP0007Version:1.2Effective Date:12/01/2014Page No.:Page 2 of 11REFERENCE DOCUMENTATIONDocumentNumber6407TitleDroplet Digital PCR Applications guide, BioRad /literature/Bulletin 6407.pdf10031906QX200 Droplet Reader and QuantaSoft Software Instruction iterature/10031906.pdf10023997PX1 PCR Plate Sealer Instruction terature/bulletin 10023997.pdf10021377C1000 Touch Thermal Cycler Instruction iterature/10021377.pdf6402ddPCR Library Quantification Kit for Illumina TruSeq terature/Bulletin 6402.pdf10043138Automated Droplet Generator Instruction iterature/10043138.pdf10031986ddPCR Library Quantification Kit for Illumina TruSeq Product terature/10031986.pdf3.0BACKGROUNDddPCR is used to reproducibly determine the concentrations of library samples andlibrary pools to achieve accurate cluster density for sequencing on the Illumina HiSeq2500. The ddPCR Library Quantification Kit for Illumina TruSeq is used on the BioRad QX200 AutoDG ddPCR systems and allows absolute quantification of both the P5and P7 adapter sequences. The multiplexed, probe-based method also provides libraryquality finitionDroplet Digital PCR
Illumina Sequencing Quantitation usingDroplet Digital PCR (ddPCR) Protocol5.0Document No.:MCCRD-SOP0007Version:1.2Effective Date:12/01/2014Page No.:Page 3 of 11EQUIPMENTDescription6.0Model #VendorSingle Channel Pipettes (p2, 20, 200, 1000)VariableRaininMulti-Channel Pipettes (LTS 2, 00-196VWRPX1 PCR Plate Sealer181-4000Bio-RadQX200 AutoDG ddPCR system includinglaptop and droplet reader186-4100Bio-RadPlate Centrifuge022628203EppendorfC1000 Touch thermal cycler1851197Bio-RadPCR WorkstationAC648LFUVCAirClean SystemsREAGENTS/MATERIALSDescriptionProduct No.VendorIon AmpliSeq Library Kit v2.0*4475345Life TechnologiesMPACT Custom Primer PanelIAD27536Life TechnologiesddPCR Library Quantification Kit forIllumina TruSeq1863040Bio-RadPCR Plate Heat Seal, foil, pieceable1814040Bio-RadDG32 Automated Droplet GeneratorCartridges1864108Bio-RadNuclease-free Molecular Biology GradeWater (not DEPC-Treated)AM9937Life TechnologiesMicroAmp Optical 96-well PCR PlatesN8010560Life TechnologiesMicroAmp 96-well Optical Adhesive Film 4311971Life Technologies1.5mL Lo-Bind TubesEppendorf022431021
Illumina Sequencing Quantitation usingDroplet Digital PCR (ddPCR) ProtocolDescription7.0Document No.:MCCRD-SOP0007Version:1.2Effective Date:12/01/2014Page No.:Page 4 of 11Product No.Vendor200-1000 µL Aerosol Barrier Pipette Tips RT-L1000FLRRainin20-200 µL Aerosol Barrier Pipette TipsRT-L-200FLRRainin0.2-20 µL Aerosol Barrier Pipette TipsRT-L10FLRRaininddPCR Droplet Reader Oil1863004Bio-RadAutomated Droplet Generation Oil forProbes1864110Bio-RadPipet Tip Waste Bins for the AutoDG System1864125Bio-RadPipet Tips for the AutoDG System1864121Bio-RadEppendorf 96-Well twin.tec PCRPlatesE951020346Fisher ScientificBuffer EB19086QiagenDisposable Pipetting Reservoirs89094-662VWR96 well 0.8mL Storage Plates(MIDIPlates)AB-0859Fisher ScientificAUTOMATION METHODSThe AutoDG droplet generator is pre-programmed. The user must load the instrumentwith the necessary consumables, select the columns to be processed, and the type ofprobes that are to be used.8.0SAFETY8.19.0Lab coats, safety glasses, gloves must be worn at all times when handlinghazardous or sensitive equipment, samples, reagents, and materials. These safetymeasures must also be followed when in close proximity to those who areworking with these items.ASSAY GUIDELINES9.1Once thawed, the library quantification kit can be stored at 4oC for 2 weeks.Repeated freeze thawing is not recommended.9.2Thaw reagents at room temperature and then store on ice during use. Afterthawing, vortex and centrifuge reagents prior to pipetting.
Illumina Sequencing Quantitation usingDroplet Digital PCR (ddPCR) Protocol10.0Document No.:MCCRD-SOP0007Version:1.2Effective Date:12/01/2014Page No.:Page 5 of 119.3For accurate quantitation, the ddPCR concentrations should be between 1005000 copies/ul.9.4Assaying 2 dilution points in duplicate ensures that a reliable concentrationmeasurement is obtained. In most cases 10-6 and 10-7 dilutions of libraries thatwere originally diluted to a target concentration of 5nM will yield data within therecommended range.PROCEDURE10.1Program the C1000 Thermal Cycler10.1.1 Program the C1000 thermal cycler with the cycler protocol outlinedbelow.TemperatureTime (mm:ss)# Cycles95oCHOLD195oC10:00194oC00:3040o61 C02:002oC/sec ramp rate9810:00112oCHOLD1*Use a heated lid set to 105oC and set the sample volume to 40 uL.Note: Use the above thermocycler protocol instead of the protocol outlined in the assaydocumentation. This protocol has been optimized to maximize the fluorescent intensitybetween positive and negative droplets.
Illumina Sequencing Quantitation usingDroplet Digital PCR (ddPCR) Protocol10.2Document No.:MCCRD-SOP0007Version:1.2Effective Date:12/01/2014Page No.:Page 6 of 11Preparation of ddPCR master mix10.2.1 In a clean room or PCR box, prepare the ddPCR master mix as outlinedin the table below.ReagentVolume/reaction, uL( 5% overage)Volume/96-well plate( 5% overage)Finalconc2x ddPCR Supermix forProbes (no dUTP)11.551108.81x20x ddPCR libraryquantification assay1.155110.881xNuclease-free water5.775554.4Total volume to add per well17.6 uL ( 5% overage)17.6 uL ( 5%overage)1xNote: The AutoDG requires complete columns of samples. If necessary, fill remainingwells in the column with NTC samples (17.6 uL master mix 4.4 uL nuclease-freewater).10.2.2 Add 17.6 uL master mix into each reaction well of a 96 well twin.tecplate. Seal the plate with adhesive film. Store the plate on ice in the darkuntil samples are added.10.3Create sample dilutions10.3.1 Using the concentration information from the quality control FragmentAnalyzer run, dilute the libraries or library pools with nuclease-freemolecular biology grade water. Dilute samples to a final targetconcentration of about 5nM. Total dilution volumes should be at least14uL. This ensures enough volume to use as a working stock for theremainder of the workflow.
Illumina Sequencing Quantitation usingDroplet Digital PCR (ddPCR) Protocol10.4Document No.:MCCRD-SOP0007Version:1.2Effective Date:12/01/2014Page No.:Page 7 of 11Perform serial dilutions of samples10.4.1 Create serial dilutions to be used for the ddPCR setup. Use EB buffer todilute samples in a 96 well MIDI plate. Thoroughly mix samplesbetween each step of the dilution series. Dilute each sample row-wise asoutlined in the table below.Dilution stepColumn 1Column 2Column 3Column 4Sample volume, uL2 uL working 2 uL fromstockcolumn 12 uL fromcolumn 210 uL fromcolumn 3EB buffer volume, uL198 uL198 uL198 uL90 uLDilution100x100x100x10xFinal dilution10-210-410-610-710.5Combine diluted samples and PCR master mix to create reaction plate (examplein image below)10.5.1 Add 4.4 uL diluted sample to each reaction well containing master mixaccording to the plate layout below. The total volume in each wellshould be 22 uL.Sample#1 106Sample#1 106Sample#1 107Sample#1 107Sample#9 106Sample#9 106Sample#9 107Sample#9 107Sample#17107Sample#1 7107Sample#17107Sample#1 7107Sample#2 106Sample#2 106Sample#2 107Sample#2 107Sample#10 106Sample#10 106Sample#10 107Sample#10 107Sample#18 107Sample#18 107Sample#18 107Sample#18 107Sample#3 106Sample#3 106Sample#3 107Sample#3 107Sample#11 106Sample#11 106Sample#11 107Sample#11 107Sample#19 107Sample#19 107Sample#19 107Sample#19 107Sample#4 106Sample#4 106Sample#4 107Sample#4 107Sample#12 106Sample#12 106Sample#12 107Sample#12 107Sample#20 107Sample#20 107Sample#20 107Sample#20 107Sample#5 106Sample#5 106Sample#5 107Sample#5 107Sample#13 106Sample#13 106Sample#13 107Sample#13 107Sample#21 107Sample#21 107Sample#21 107Sample#21 107Sample#6 106Sample#6 106Sample#6 107Sample#6 107Sample#14 106Sample#14 106Sample#14 107Sample#14 107Sample#22 107Sample#22 107Sample#22 107Sample#22 107Sample#7 106Sample#7 106Sample#7 107Sample#7 107Sample#15 106Sample#15 106Sample#15 107Sample#15 107Sample#23 107Sample#23 107Sample#23 107Sample#23 107Sample#8 106Sample#8 106Sample#8 107Sample#8 107Sample#16 106Sample#16 106Sample#16 107Sample#16 107Sample#24 107Sample#24 106Sample#24 107Sample#24 10710.5.2 Seal plate with an optical seal and vortex thoroughly. Quick spin theplate. Inspect the plate to ensure there are no bubbles in the wells.Note: Plate should be stored on ice away from light if it is not being immediately loadedonto the AutoDG for droplet generation.
Illumina Sequencing Quantitation usingDroplet Digital PCR (ddPCR) Protocol10.6Document No.:MCCRD-SOP0007Version:1.2Effective Date:12/01/2014Page No.:Page 8 of 11Generate droplets10.6.1 Load consumables on AutoDG instrument. For a run with 96 reactions:10.6.1.1 Place 3 unused droplet generation cartridges on the back row10.6.1.2 Remove lids and place 2 full boxes of droplet generation tips onthe middle row10.6.1.3 Place an empty waste bin on the middle row to the left10.6.1.4 Place the 96 well cold chill block to the front right. Place anunused twin-tec 96 well plate in the chill block. Droplets willbe deposited into this plate after they are formed.10.6.1.5 Ensure that there is enough droplet generation oil to completethe run10.6.2 Configure the AutoDG using the touchscreen on the instrument10.6.2.1 ‘Probes’ should be selected as oil type10.6.2.2 Configure the sample plate by selecting the columns containingsamples10.6.2.3 Place the reaction plate on the deck to the front left. Carefullyremove the adhesive film.10.6.2.4 Lower the lid of the AutoDG. Push start to generate droplets.Note: Droplet generation will take approximately 40 minutes for a full 96 well plate ofsamples. Final volume in each reaction well is approximately 40 uL.10.7Seal the reaction plate10.7.1 After droplet generation is complete, place a pierceable foil seal on the 96well plate containing the droplets.10.7.2 Remove the reaction plate from the AutoDG and seal the plate on theplate sealer.10.7.2.1 Seal at 180 oC for 5 seconds10.8Amplification10.8.1 Place the reaction plate on the thermal cycler and start the programoutlined in step 10.1.1.Note: The amplification protocol takes about 2.5 hours.Note: If droplets are not immediately read plate can be stored for up to 2 days at 4-12away from light.oC
Illumina Sequencing Quantitation usingDroplet Digital PCR (ddPCR) Protocol10.9Document No.:MCCRD-SOP0007Version:1.2Effective Date:12/01/2014Page No.:Page 9 of 11Configure the plate template10.9.1 Open the Quantasoft software and load the plate template file. If desired,update the sample names on the template to match the reaction platesetup.10.9.1.1 ABS should be selected as Experiment Type in the well editor10.9.1.2 ddPCR Supermix for Probes (no dUTP) should be selected asSupermix Type in the well editor10.9.1.3 Ch1 Unknown in Target 1 and Ch2 Unknown in Target 2should be selected in the well editor10.9.1.4 Load the reaction plate onto the droplet reader.10.9.1.5 In the software, click Run and select the FAM/HEX dye set.Note: A complete plate of 96 reactions will take approximately 2 hours to read.10.10 Data analysis10.10.1Click Analyze in the Quantasoft software. Select the dilutionscorresponding to a sample in the well selector under analyze. Examinethe automatic thresholding applied to the amplitude data. It may benecessary to manually set the threshold between the positive and negativedroplets using the amplitude and/or histogram plots. See below for anexample of thresholding. Repeat for each dilution of each sample.10.10.2Assess data quality
Illumina Sequencing Quantitation usingDroplet Digital PCR (ddPCR) ProtocolDocument No.:MCCRD-SOP0007Version:1.2Effective Date:12/01/2014Page No.:Page 10 of 1110.10.2.1 Concentration data for 106 dilutions should be about 10 timesconcentration data for 107 dilutions.10.10.2.2Replicate data should be similar.10.10.2.3Total number of droplets per reaction should be 12000-20000.10.10.2.4 Concentration data for Ch1 and Ch2 should be similar. If thesenumbers are not similar it may indicate an issue with libraryquality. Properly formed library molecules will be positive forboth the Ch1 and Ch2 targets.10.10.2.5 When looking at the 2D plot, ddPCR data can also be used toassess library quality.10.10.2.6 Inserts of varying sizes can be observed along a diagonal path.Larger fragments are nearer the negative droplets.10.10.2.7 Adaptor/adaptor ligations will appear as a population of dropletsin the upper right-hand corner. See below for an illustration of library quality information thatcan be determined from a 2D plot.10.10.3Save the post-analysis plate information in Setup, Plate, Save As.10.10.4Export both the merged and single well information and save as a csvfile.
Illumina Sequencing Quantitation usingDroplet Digital PCR (ddPCR) ProtocolDocument No.:MCCRD-SOP0007Version:1.2Effective Date:12/01/2014Page No.:Page 11 of 1110.11 Concentration calculations10.11.1Using the merged information that was exported and saved in step10.10.4, transfer sample name and concentration (in copies/uL) data to anExcel sheet to perform ddPCR calculations.10.11.2For each dilution for each sample, compare the Ch1 and Ch2 data andcopy and paste the lower concentration into the lowest conc, copies/uLcolumn. Once this concentration information is entered, the nMconcentration of the library will be automatically calculated. Calculation example: A library at the 106 dilution yielded 2000 copies/uL in theddPCR mixture. Multiply 2000 by 106 and by 5 to account for the dilution inthe reaction mixture (2000 x 106 x 5 1010 copies/uL oforiginal stock library). To obtain nM: 1010 copies/uL x 106 uL/L/6.023 x 1023copies/mole 1.66 x 10-8 M or 16.6 nM.10.11.3Use the ddPCR data from the least diluted reactions that yieldedconcentration information between 100 and 5000 copies per reaction.For most samples this means the 106 data will be used. Use theseconcentrations to further dilute the samples for pooling or clustering.
PX1 PCR Plate Sealer 181-4000 Bio-Rad QX200 AutoDG ddPCR system including laptop and droplet reader 186-4100 Bio-Rad Plate Centrifuge 022628203 Eppendorf C1000 Touch thermal cycler 1851197 Bio-Rad PCR Workstation AC648LFUVC AirClean Syst
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