Sequence Capture Lab Procedure
Contents
1. 1 Prepare PCR Master Mix on ice in a sa oi oo ala and mix by pipetting Component Amount 50 ul Nuclease free water 32 5 5x Herculase II Buffer 10 Nia in anew PCR dNTP mix 25mM each 0 5 Add Captured Library PCR primers mix 10 uM each and mix by pipetting Herculase II Fusion DNA Polymerase l Captured Library 5 2 Place the tubes in a thermocycler and run the following program Temperature Time 98 C TETT room 4218 Applied Biosystems 98 C 20 seconds The program is called See Appendix A 60 C 30 seconds FILIPE per MYBaits post 72 C See 1min capture But note Repeat step 2 through 4 for 14 times ZC 5 minutes 4 C 00 Extension time Step 4 will depend on the genomic library average fragment size Use 30 seconds for fragments shorter than 500 bp 45 seconds for fragments with size between 500 and 700 bp and 1 minute for fragment sizes ranging from 700 bp to 1 Kb 3 Purify the PCR product using QIAquick PCR purification kit following the manufacturer s instructions Use 30 ul of buffer EB or Molecular Biology grade water for the final elution step Perform the QlAquick PCR Purification as described for the previous PCR purfication Pool all 3 PCR products 4 Measure the DNA concentration with a spectrophotometer nanodrop i MYcroarray techsupport mycroarray com 734 998 0751 Make a master mix and Use the PCR machine in VII Appendix A Post capture PCR Amplification2. 998 0751 When to perform the capture during sequencing library preparation We strongly recommend performing the capture on fully prepared and validated sequencing library Your genomic DNA should have been fragmented size purified and the sequencing adaptor ligated This kit has been tested with 100 500 ng of genomic library Working with lower or larger amounts of starting material may require some optimization Denature DNA l mame Prepped gDNA sequencing library Sa Hybridize baits 2 __ pan to targets Biotinylated baits eee eee eee eee eee eee eee YETEN ea eee eee ee eee 7K Capture target 3 gt n on beads e Streptavidin beads a Recover k a Gt oe a eee E m captured targets oA Amplify _ 5 eo Sequence MYcroarray techsupport mycroarray com 734 998 0751 II Materials Reagents provided in MYbaits kits and storage conditions Box 1 Store at 4 C Stored in the fridge in room 4109 Product Amount Components Cap Color HYB 2 60 ul 500 mM EDTA Red HYB 4 750 ul 1 Sodium Dodecyl Sulfate Teal Binding Buffer 45 ml 1MNaCl 10 mM Tris HCl pH 7 5 1 mM EDTA Wash Buffer 1 30ml 1X SSC 0 1 SDS Wash Buffer 2 80 ml 0 1X SSC 0 1 SDS Neutralization Buffer 3 75 ml 1M Tris HCl pH 7 While the tube label may read Room Temperature we now recommend storage at 4 C Amounts shown here are based on a 50
3. Required equipment and supplies e1 BioRad C1000 Thermocycler or compatible thermocycler see Appendix C e Non sticl erie nuclease free tubes compatible with the thermocyele PCR strip e2 Magnetic particle stand Life Technologies 123 210 DynaMag 2 e Vortex mixer e3 Water bath set at 65 C e4 Lab rotator Thermo Scientific 400110Q 2 room 4109 drawer under desk 1 room 3310 opposite to fume hood 4 room 4109 on desk opposite to fume hood MYcroarray techsupport mycroarray com 734 998 0751 PCR strip Il Hybridization 4 Prepare Capture Baits Master Mix in a nuectease freetube and mix by pipetting Set aside until step 7 Note When the hybridization step is finnished you need to proceed with step IV at once i e you should PETRE Do not make a start the hybridization in the evening to have the procedure ready in the morning 36 h later a a kee a Component N E shored en This step involves denaturing and hybridizing the sequencing library to a pool of custom mixes will be Capture Probe baits 5 directly in a new complementary RNA baits MYbaits kit has been tested with 100 500 ng of input genomic transiered to RNase Block l PCR strip library Smaller or larger amounts may require optimization Before starting equilibrate HYB 4 tube at room temperature to fully dissolve SDS that may have precipitated during storage at 4 C 1 Set the following program on a thermocycler See Appe
4. When to perform the capture during sequencing library preparation MATERIALS Reagents provided in MYbaits kits and storage conditions Reagents to be provided by user Required equipment and supplies HYBRIDIZATION RECOVERY OF CAPTURED TARGETS ELUTION OF ENRICHED LIBRARY ENRICHED LIBRARY CLEANUP POST CAPTURE AMPLIFICATION APPENDIX A Post capture PCR Amplification Primers B Elution Buffer C Thermocyclers D MSDS Get the latest version at http www mycroarray com pdf MY baits manual pdf For research use ONLY Not intended for diagnostic use MYcroarray techsupport mycroarray com N e e gt A A U W 10 11 11 11 11 11 734 998 0751 I Introduction MyYbaits is a fully customizable liquid phase DNA capture system for targeted sequencing or any other applications requiring sequence enrichment Each kit is custom made to target your sequences of interest What does the bait library contain Each MYbaits kit contains a custom library of biotinylated single stranded RNA baits designed per your recommendation Each library can contain up to 100 000 different bait sequences We first synthesize a library of DNA oligonucleotides using our proprietary parallel DNA synthesis technology Then the DNA library is converted into biotinylated RNA baits by in vitro transcription Each sequence from the bait library is present in the pool at an average concentration of 50 pM Depending on the number of baits in you
5. S 4 MYcroarray techsupport mycroarray com 734 998 0751 Bring all mixes to the PCR machine room Transfer the tube containing the Library Master Mix to the thermocycler and start the program set in step 1 This will denature the DNA library for 5 minutes at 95 C press cancel and Once the thermocycler program reaches step 2 temperature 65 C transfer the tube containing the Hybridization Master Mix to the thermos ee raat the Library Master Mix in the thermocycler This will pre warm the Hybridization Master Mix for 3 minutes at 65 C press cancel and Once the thermocycler program reaches step 3 temperature 65 C transfer the tube containing the Capture Baits Master Mix to the thermo ycler T eave all other tubes in the thermocycler This will pre warm the Capture Baits Master Mix for 2 minutes at 65 C i e leave all samples in the PCR machine while performing this step While keeping tubes at 65 C transfer 7 ul of Library Master Mix and 13 ul of Hybridization Master Mix to Capture Baits Master Mix and mix via pipetting Briefly centrifuge your sample before next step Hybridize solution at 65 C for 36 hours Depending the application hybridization time may need some optimization between 24 and 48 hours When finnished proceed with step IV at once Sketch summarizing the hybridization procedure 65 C 36 hours ime MYcroarray techsupport mycroarray com 734 998 0751 IV Recovery of Captured Targ
6. com 734 998 0751 VI Enriched Library Cleanup This step consists of removing extra salts added during the release of the captured DNA molecules It also permits the captured DNA to be concentrated 1 Concentrate and desalt the solution using a QIAquick PCR Purification column following manufacturer s manual The bindine ee be adjusted if necessary Elute with 36 ul buffer EB or Molecular Biology grade water 20 Expected amounts of recovered material are very small and cannot be detected by spectrophotometry Note The sample can be stored at 20 C after this step if necessary Perform the QlAquick PCR Purification as described for the previous PCR purfication The only modification is that you elute in 20uL EB instead of 30uL MYcroarray techsupport mycroarray com 734 998 0751 VII Post Capture Amplification This step consists of amplifying the small amount of captured DNA recovered in the previous step in order to have enough material for sequencing It is important to limit the number of cycles to get just enough material while minimizing PCR amplification bias We recommend using the Herculase II Fusion DNA Polymerase which compare favorably to other DNA polymerases Dabney and Meyer BioTechniques 52 87 94 February 2012 0 5 To get a high concentration of your captured DNA run 3 PCR s on each sample You have 20yuL from the previous step you use 5uL in each PCR so in total you use 15yL of each sample
7. minutes easy cap tubes 19 Place the 96 welLPGR Plate on the magnetic stand at room temperature for 5 minutes 500 700 bp 650 800 bp 7 28 5 yb 20 Do not discard the supernatant in this step Transfer the Sample Volume of clear supernatant to a new well as in the table below for the desired final library size distribution Be careful not to disrupt the magnetic bead pellet or transfer any magnetic beads with the sample This is your size selected DNA The ja aaie ai now consists of 400 600 bp long fragments 300 aU bp 350 500bp 1 400600 bp 500 700p B50 800 bp Z L385 J 75ul Selection Range 75 uL 72 pl 21 Add Bead Volume of AMPure XP Beads to each well containing sample as in the table below for the desired final library size distribution Mix thoroughly until homogenized Incubate sample at room temperature for 5 minutes The beads now capture the 400 600 bp long fragments 300 400 bp 350 500 bp 400 600 bp 500 700 bp 650 800 bp wu Ng Z easy cap tubes 22 Place the 96 well PGR Plate on the magnetic stand at room temperature for 5 minutes 7 around 58 uL 23 Remove and discard the clear supernatant taking care not fo disturb beads Some liquid may remain in wells 100 24 With plate on stand add 280 uL of 80 ethanol to each magnetic bead pellet and incubate plate at room temperature for 30 seconds Carefully remove ethanol by pipette 25 Repeat step 24 for a total of 2 e
8. 514121 514122 514123 or NEXTflex 96 ChIP Seq Barcodes Cat ortex prior to 514124 d NEXTflex End Repair amp Adenylation Buffer Mix E ae ae NEXTflex End tical amp Adenylation Enzyme Mix ii Add milliQH2O to your dried DNA 25 80 ul of End Repaired DNA Grom STEP A2 sara ball ie samples 16yL 9 Thaw NEXTflex Ligase Enzyme Mix to room temperature and vortex for 5 10 seconds Do not iii Make a master mix of Buffer and spin down tube as this may cause components of the mix to separate and affect performance r Enzyme mix distribute in PCR strip Fragmented BNA in 32 pt feHess nuclease free wate CR Plate ea work on ice Adhesive PC penne iv Transfer DNA sample to PCR strip Agencourt AMPure XP Magnetic Beads and perform thermocycler program Microcentrifuge iceRoom 3315 User Supplied Dried DNA samples 6 The following table lists recommended adapter concentration dilutions for various input amounts face Usei Concentatan Desired Adapter Adapter Dilution Barcodes Used Concentration Required ChIP Seg 0 6 UM None None DNA Seq 25 uM A 1 83 DNA Seq 25 uM DNA Seq 25 uM 25 UM None Dilution likely not necessary DNA Seq 25 uM 25 UM Each sample will require 2 5 uL of adapter to be added Perform adapter dilutions if necessary depending on input amount and starting adapter concentration Master Mix 8 4 For each sample combine the following reagents on ice in a nuclease free 96 wel
9. Last updated 20 11 2014 SEQUENCE CAPTURE PROCEDURE AT BOTAN Keep in mind that this is just a general procedure and for individual samples you might have to do some adjustments e DNA extraction o The DNA extraction is usually carried out with a Dneasy Plant Kit from Qiagen Extractions are done in room 4230 Pre PCR rum DNA prep o Check the length of your DNA fragments on an agarose gel If you have worked with e g herbarium material the DNA 1s usually already fragmented but if you have fresh material you usually will have longer DNA fragments that must be sheared Electrophoresis is done in room 4235 Make a 1 agarose gel ex Agarose 0 7g 70mL 1 TAE Use 5 uL of a High Range Ladder in the first well in each row Mix 5 7 uL of DNA with 2 uL Loading Buffer and transfer to well Run the electrophoresis for 20 30 min Put gel 10 min in ethidium bromide bath Put gel 10 in in water bath Check gel in UV light machine o Use the Nanodrop instrument room 4237 to measure your DNA concentration For more accurate measurements you can check fragment length on TapeStation e sample here Thermo NANODROP 2000c m Sar mananam i Take 1 5 uL of your DNA sample in a PCR strip and take it to the nanodrop room this so that you do not take samples in and out of the extraction room Start computer User nanodrop Password nanodrop Lounge nanodrop 2000 Calibration Take 1 uL of the same sol
10. Place each singles column in a clean 1 5 ml eferecante was tubs 7 To elute DNA add 50 ul Buffer EB 10 mM Tris Cl pH 8 5 erwatertpH 0 8 al to the center of the QlAquick membrane and centrifuge the columntor ofthe GlAqick membrane let the ae stand 1 min aad then centrifuge 1 min 9 Use the nanodrop as previosly described and measure DNA concentration and 260 280 ratio DNA purity 10 If the DNA concentration is lt 13yL you will need to redo the amplification for this sample otherwise you do not have enough product for the sequence capture you still have 5uL left from the library so you can perform one more amplification For up to date licensing information and product specific disclaimers see the respective QIAGEN kit handbook or user manual Trademarks QIAGEN QlAquick QIAGEN Group 1069842 9 2011 2011 QIAGEN all rights reserved QIAGEN Last updated 20 11 2014 e After library construction Store your samples in room 4109 You can pool some of your DNA libraries before sequence capture How many samples to pool will depend on your total target size 1 e the total length of all selected regions Previous work has shown that pooling 8 samples works well for a total target size of about 150 200 Kbp You need to prepare so that you pool equimolar amounts of each amplified DNA library Use the nanodrop instrument to measure the DNA concentration as described above Calculate the
11. Primers MYbaits Box 1 50 reactions E E culate http www mycroarray com msds MY baits Box1 MSDS pdf PCR primers to use for the post capture amplification depend on sequencing platform to be used The sequence of the PCR primers should be obtained from your sequencer s representative ae MYbaits Box 2 50 reactions The annealing temperature during the PCR amplification should be set 5 C below the lowest Tm l http www mycroarray com msds MY baits Box2 MSDS pdf of the primers MYbaits Box 3 8 reactions B Elution Buffer http www mycroarray com msds MY baits Box3 MSDS pdf NaOH pellet is in the extraction lab room 4230 The recommended Elution Buffer is a solution of sodium hydroxide 100 mM Due to the limited shelf life of diluted NaOH solutions we do not include it in our kit We recommend preparing fresh Elution Buffer just prior to usage 1 Prepare a 10M NaOH stock solution in RNAse DNAse free water using NaOH pellets high purity or molecular biology grade This solution should be discarded after 1 week Example Take 10g NaOH pellet 25mL milliQH2O0 2 Prepare the Elution Buffer 100 mM NaOH from the stock solution using RNAse DNAse free water This solution should be discarded after 1 day Example Take 1mL of the stock solution step 1 99mL milliQH2O C Thermocyclers A thermocycler with a heated lid to prevent condensation on the tube cap MUST be used for capture hybridization We have successfully tested an
12. ample Add the same solution that you eluted your DNA with usually Elution Buffer or milliQ water to your sample to reach 130 uL Transfer the DNA sample to the special tubes Press with the pipet tip on the opening and carefully add the sample in the tube make sure there are no bubbles Cap 6x16mm 25 OTUBE AFA Fiber Pre Slit m o The Covaris sonication instrument uses ultrasound to shear the DNA Depending on intensity vibration and duration the DNA will be sheared in different length The program Filipe_400bp shear at 400 bp If you want other length you must adjust the program e After shearing DNA Store your samples in room 4109 transfer from the special covaris tubes to normal tubes Before starting the library construction you need to prepare so that you have the same amount of DNA for each sample Use the nanodrop instrument to measure your DNA concentration as described above For more accurate measurements you can check fragment length on TapeStation Calculate the amount of each sample to be transferred to a new tube for use in the library construction To get the right volume divide the DNA concentration ng uL with 500 E g if you have a DNA concentration of 20 ng uL you should transfer 500 20 25uL of your DNA sample to a new tube Last updated 20 11 2014 ee ae cnn ie n ae a a a a Dry your DNA samples with the Speed Vacuum i
13. brary This protocol starts on page 12 and is described below In this description we want to size select DNA fragments of 400 600bp In one day you can do step A2 C2 in fact you have to samples can be safely stored after step C2 Step D2 purification can also be done in one day NEXTflex Kits are in room 4109 Work Except from the kit you also need on the bench opposite to fume hood Agencourt AMPure XP Magnetic Beads in fridge NEXTflex DNA Barcodes in freezer Bi Gh NEXT flex Rapid DNA Seq Kit Manual 5144 02 OPTION TWO Option Two is designed for users who wish to size select their libraries from five selection ranges found in Step C2 Bead Size Selection The user can choose Note Step A2 C2 is if you do not wish to size select your libraries please follow Option 1 done in the same day STEP A2 End Repair amp Adenylation 1G NEXT flex Rapid DNA Seq Kit Manual 5144 02 STEP B2 Adapter Ligation Materials Bioo Scientific Supplied PURPLE CAP NEXTflex Ligase Enzyme Mix Thaw on ice WHITE CAP Nuciease free Water User Supplied Usually this Thermocycler i N Materials NEXTflex DNA Bar 6 12 24 148 Cat 514101 514102 514103 514104 or Bioo Scientific Supplied i Kits are in the freezer to the right NEXTflex 96 DNA Barcodes Cat 5141 06 or NEXTflex ChiP Seq Barcodes 6 12 24 Vort to _ CLEAR CAP Thaw Materials for step A2 B2 on ice Pe Ne 514120
14. d recommend the BioRad C1000 and S1000 thermocyclers with dual 48 blocks They show minimal evaporation over a period of 72 hours compared to competitors Before performing the first hybridization please validate that the combination of thermocycler and tubes you are using will not allow more than 15 evaporation over the planned duration of the hybridization D MSDS MY croarray 5692 Plymouth Road Ann Arbor MI 48105 USA Phone 734 998 0751 Fax 734 998 0750 techsupport mycroarray com To obtain an MSDS for a particular box click on the relevant link below MYcroarray techsupport mycroarray com 734 998 0751 Last updated 20 11 2014 e After sequence capture You can pool as many samples as the number of barcodes you have used in the library construction i e if you have used 48 unique barcodes you pool all 48 samples in one tube From the previous example it means that you pool the 6 samples from the sequence capture procedure You need to prepare so that you pool equimolar amounts of each sample Calculate the right amount by dividing the DNA concentration ng uL with 500 E g if you have a DNA concentration of 250 ng uL you should use 500 250 2uL of your sample Take the calculated amount and pool into the same tube Measure DNA concentration using the nanodrop instrument Store sample in freezer with parafilm on the lid until sending for sequencing Also make one dilution for use in the TapeStation 2uL
15. e and remove the lids caps ee Select your samples on the TapeStation Controller software Click START and specify a file to which the results will be saved It takes about 1 min for each sample to run o Checking results As the analysis is finished you can open your result file in the TapeStation Analysis software Here you can see the gel image sample information and chromatograms e NGS sequencing Samples have previously been sent to The Sahlgrenska Genomics Core Facility with a paired end 2 150 bp run on the Illumina MiSeq platform A large benefit of using this facility 1s the personal service You have direct contact with the people who work there and can be part of the whole procedure However pricing is higher than at 1 e the SciLifeLab and you have to pay for all labour hours whereas at SciLifeLab this is covered by public research funding Ifyou deliver sample to Sahlgrenska you can just talk to Ellen Hanson and deliver the sample directly to them on ice They want the following information e Mark the tube with Project name G14 XX name on the NGS pool date and concentration e Provide a file with adaptor sample information 1 e what sample name correspond to the specific adaptor e Send the result from the final TapeStation run
16. each sample Mix thoroughly until homogenized 30 incubate sample at room temperature for 5 minutes easy cap tubes Place the 96 well PGR Plate on the magnetic stand at room temperature for 5 minutes around 72 pL Remove and discard clear sional a care not to disturb beads Some liquid may remain in wells Keep samples on the stand while removing supernatant 100 With plate on stand add 280 uL 80 ethanol to each magnetic bead pellet and incubate plate at room temperature for 30 seconds Carefully remove ethanol by pipette Repeat step 5 for a total of 2 ethanol washes Ensure all ethanol has been removed Remove the plate from the magnetic stand and let dry at room temperature for 5 minutes or until bead pellet is visibly dry Put samples in fume hood turn on for faster drying 25 Resuspend dried beads with 50 pL Resuspension Buffer Mix thoroughly until homogenized Add Lower Cutoff volume of Sizing Solution to the resuspended bead pellet as in the table below for the desired final library size distribution For example if the desired selection range is 350 500 bp add 35 uL of Sizing Solution to the 50 uL resuspended bead pellet Mix thoroughly until homogenized The beads capture fragments from 400bp and above 300 400 bp 350 900 bp mice in Shig 700 bp 650 800 bp 10 Incubate sample at room temperature for 5 minutes easy cap tubes 11 Place the 96 well PCR Plate on the magnetic stand at room temperature f
17. eads using a magnetic particle stand and discard the supernatant i a j j 4 Pellet the beads and transfer supernatant to a tube containing 70 ul Neutralization 3 Add 200 ul Binding Buffer to beads to wash Vortex tube for 5 10 seconds place on Buffer magnetic particle stand for two minutes to pellet the beads and remove and discard supernatant Put your TAE in i 4 Repeat step 3 twice for a total of three washes h Eix ee opel 5 Resuspend the beads in 200 ul Binding Buffer Aee c2 on i e your product from the hybridization step GALL 6 Transfer the hybridization solution to the Binding Buffer Beads and incubate 30 minutes at room temperature on a rotator Pellet beads with magnetic particle stand for two minutes and remove supernatant 7 Add 500 ul Wash Buffer 1 to the beads and briefly vortex to resuspend Incubate 15 minutes at room temperature Pellet beads with magnetic particle stand for two minutes and remove supernatant 8 Add 500 ul 65 C Wash Buffer 2 to the beads and briefly vortex to mix Incubate for 10 minutes at 65 C Pellet beads with magnetic particle stand for two minutes and remove supernatant Note Continue to work in room 4109 i e move samples back and forth to the water bath in room 4236A 9 Repeat step 8 twice for a total of three 65 C washes After third wash make sure all additional buffer is removed MYcroarray techsupport mycroarray com 734 998 0751 MYcroarray techsupport mycroarray
18. er Mix z HL of Adapter Ligated DNA from STEP C2 8 uL Primer Mix 160 uL 20 uL in each well The following table lists recommended PCR cycles input DNA ng Ligated DNA uL Nuclease free HO uL PCR cycles Master Mix 16 a aaa 208 uL H20 ocr pi a nii aos 1000 5 34 320 yL gt 20 pL in each well 1 For each sample combine the following reagents on ice in the PCR plate Mix thoroughly S_uL Ligated DNA a l 3 uL Nuclease free H O ii Make a Master Mix of PCR master mix 6 ZuL NEXTflex PCR Master Mix and Primer mix and distribute in PCR strip 1 L__NEXTflex Primer Mix ni 3 uL TOTAL iii Add your ligated DNA and mix 5 2 Apply adhesive PCR plate seal and place in thermocycler for the following PCR cycles 2min 98 C 30sec 98 C PCR program 30sec 65 C Repeat 6 15 cycles as suggested in above table FILIPE pcr Illumina library eE 20 sec rat 15 cycles Amin 72 C 3 Proceed with PCR purification using the QlAquick PCR Purification Kit ba ie tat i i I6 BIOO LIFE SCIENCE PRODUCTS WWW BIOOSCIENTIFIC COM 17 Quick StartProtocol Room 4235 Kits in Kemikalieskap 2 QlAquick PCR Purification Kit The QlAquick PCR Purification Kit cat nos 28104 and 28106 can be stored at room temperature 15 25 C for up to 12 months For more information please refer to the QlAquick Spin Handbook which can be found at www qiagen com handbooks For technical assista
19. er for 15 minutes at 22 C Thermocycler program run now Filipe ligation Illumina s 8 Proceed to Step C2 Bead Size Selection EE mel ee eee BIOO LIFE SCIENCE PRODUCTS WWW BIOOSCIENTIFIC COM 12 BIOO LIFE SCIENCE PRODUCTS WWW BIOOSCIENTIFIC COM 13 Bih STEP C2 Bead Size Selection Materials Bioo Scientific Supplied User Supplied 50120 uL of Adapter Ligated DNA from STEP B2 NEXTflex Rapid DNA Seq Kit Manual 5144 02 i Make a fresh dilution of 80 ethanol Take 40mL of 99 5 ethanol and 10mL milliQH2O Extraction room WHITE CAP ii Transfer your DNA samples from the PCR NEXTflex Resuspension Buffer strip to easy cap tubes the magnetic stand CLEAR CAP BOTTLE can only have ependorf tubes iihi NEXTflex Sizing Solution iii Start with step 1 Agencourt AMPure XP Magnetic Beads room temperature 80 Ethanol freshly prepared room temperature In the drawer Magnetic Stand __ _ under bench Size Selection may not be optimal for inputs lt 5 ng The size ranges listed in tables below reflect the total library size including the insert and NEXTflex Barcode Adapters NEXTflex Barcode Adapters add 120bp to the insert length Ensure all reagents are at room temperature Vortex AMPure XP and Sizing Solution thoroughly prior to use Use a fresh dilution of 80 ethanol during wash steps 1 2 E Lower Cutotf 37 5 lL Add 68 uL of AMPure XP Beads to
20. ets V Elution of Enriched Library This step consists of recovering the captured targets from the hybridization solution Targeted This step consists of releasing the captured DNA target molecules from the RNA baits This is DNA sequences are hybridized to biotinylated RNA baits RNA baits either hybridized to a achieved by specifically degrading the RNA molecules by an alkaline treatment that will leave complementary DNA molecule or free are pulled out of the hybridization solution by the means DNA molecules unaffected All of the steps in this section are performed at room temperature of streptavidin coated magnetic beads Beads are then washed to remove any non specific carry au sper DNA mbolecales The Elution Buffer is not provided with this kit Fresh Elution Buffer should be prepared according to Appendix B Before starting equilibrate Wash Buffer 1 bottle at room temperature to fully dissolve A SDS that may have precipitated during storage at 4 C and preheat Wash Buffer 2 at 65 C 3 1 Add 50 ul freshly prepared Elution Buffer to beads from step 9 of Section IV in a water bath for at least 1 hour 0 5 Set water bath room 4236A at 65 C Add 1 5mL Wash Buffer 2 sample in a new tube 2 VorextorS 10 secoudsto mi wrap parafilm on the lid to prevent evaporation and preheat in water bath 1 Transfer 50 ul of MyOne Streptavidin C1 magnetic beads to a new 1 5 ml tube 3 Incubate 10 minutes at room temperature 2 Pellet b
21. l PCR Plate 60 yL Buffer Mix If the water in the kit is empty you find 12 HL Enzyme Mix __ 72uL gt 9 pLin each well 16 _uL Nuclease free Water ail l _ pL Fragmented DNA 1 ng 1 pg MilliQH2O in room 4236A 7 54 uL NEXTflex End Repair amp Adenylation Buffer Mix 1 5 4uL NEXTflex End Repair amp Adenylation Enzyme Mix Master Mix 16 2550 uL TOTAL 120 uL Buffer Mix 24 pL Enzyme Mix 144 uL gt QuLin each well The following reaction must be mixed thoroughly The NEXTflex Ligase Enzyme Mix is 5 Apply adhesive PCR plate seal and incubate on a thermocycler using the following program very viscous Thorough mixing of the reaction below is critical to obtaining optimal results Suggestion To mix pipette up and down 15 times visually inspect tubes to ensure proper 20min 22 C The thermocycler program homogenization 20 iy fe Ms is done on the Applied Biosystems machine in 6 Proceed to Step B2 Adapter Ligation OOM 4218 The program is called Filipe end repair RAPID Combine the following in the PCR plate and mix thoroughly by pipette 25 66 lL End Repaired DNA from Step A For optimal mixing Add barcode first 24 4 5 uL NEXTflex Ligase Enzyme Mix then enzyme 1 3 5 uL NEXTflex DNA Barcode or NEXTflex ChiP Seq Barcode 50 120 uL TOTAL Press Browse select H program view edit run reaction volume 25 Start 7 Apply adhesive PCR plate seal and incubate on a thermocycl
22. nce please call toll free O0800 22 44 6000 or find regional phone numbers at www qiagen com contact OBS Always make aliquots of reagents Buffer PB Buffer PE Ethanol added Notes before starting Buffer EB This protocol is for the purification of up to 10 Ug PCR products 100 bp to 10 kb in size Add ethanol 96 100 to Buffer PE before use see bottle label for volume All centrifugation steps are carried out at 17 900 x g 13 000 rpm in a conventional table top microcentrifuge at room temperature M Add 250 volume pH indicator to Buffer PB The ine color of Buffe ion of pH indicator Do not add pH indicator to buffer heuer HM Symbols centrifuge processing A vaevum preeessing Use the centrifuge processing For Material Safety Data Sheets see www giagen com safety S6S6e QIAGEN September 2011 3 To bind DNA apply the sila to the aves column and eeniriiuge for 30 60 s or through the column Discard flow Danh and place the QlAquick ee back in the same tube 4 To wash add 750 pl Buffer PE to the QIAquick column centrifuge for 30 60 s or A apply vacuum Discard flow through and place the QlAquick column back in the same tube Tap the collection tube and QlAquick column on a piece of paper to get rid of all fluid 5 Centrifuge the QlAquick column once more in the provided 2 ml collection tube for 1 min to remove residual wash buffer _ Safe ca 6
23. ndix C for recommended thermocyclers and validation procedure Room 3310 The program is called Filipe Note You need to book the PCR machine beforehand Step Temperature Time 1 9 2 2 65 C 3 Write your name on 3 the calender on the 3 65 C 2 notice board 4 65 C 36h PCR stri 2 Prepare Library Master Mix in a nuclease tres tube and mix by vortexing Set aside until step 5 Note It may be necessary to concentrate the genomic library by reducing the volume using a SpeedVac in order to have 100 500 ng of library DNA in 3 4 ul before preparing the Library Master Mix Make a master mix and distribute in a PCR strip Then add the calculated amount of each sequencing library Component ALKOT C M milliQH20 so that the total volume This is the master mix including your Block 1 2 5 equals 3 4uL See example sequencing library and Block 2 25 i blocking agents Block 3 o6 Example Calculated amount o Block 3 0 6 sequencing library 2 5yL Sequencing library 100 500 ng 3 4 3 4 2 5 0 9 i e add 0 9L milliQH20 PCR strip to your sample 3 Prepare Hybridization Master Mix in a and mix by vortexing Set aside until step 6 Use only half of the amounts per sample when you prepare the master mix it is more than enough for the next step E EEE EER Component Amount 36 8 ul Make a master mix and including the Hyb 1 27 10 aia in anew PCR hybridization buffers Hyb 2 08 0 4 l Hyb 3 A 4 Hyb 4
24. nstrument room 3204 Cyanolab e Open the lid of your samples and place them in the centrifuge Keep the lids towards the centre of the centrifuge Close the centrifuge lid e Turn on the centrifuge at Med drying rate and turn rotor on e Turn on the Refrigerated Vapor Trap e Set SAVANT Speedvac dryness controller on MANUAL ON e Turn on VP 100 e Close fume hood e When finished turn everything off in the opposite order Let your samples run until they are completely dry This will vary from about 30 min to a few hours depending on the volume of the sample When the samples are dry you will be left with the same DNA amount in each of your DNA samples and you are ready for library construction e Library construction We use the NEXTflex DNA Rapid Sequencing Kits for library construction Stored in the right freezer in room 4109 o Working with eight samples is a good option maximum 16 since the magnetic stand only takes 16 samples at a time Work on the bench opposite to the fume hood The procedure follows the NEXTflexTM Rapid DNA Seg Kit catalogue 5144 02 2013 V14 02 the manual comes with the kit check if it is the same version as last time Use only half of the reaction volumes You find the full manual in the drawer under the bench Read the full manual before you start good information about the reagents and so on Option two is used when you want to size select your li
25. of NGS sample and 4uL of milliQH20O e TapeStation Use the Agilent 2200 TapeStation instrument to validate your sample s fragment size NOTE DIK has been replaced by D1000 Online protocol here follow this protocol o Sample preparation room 3122 Pre pcr room All reagents are in the fridge Equilibrate all reagents 30 min before use Use the special TapeStation tubes you find them in the locker next to the fume hood Take 3uL of DIK Ladder in the first tube Mix 1 uL of your diluted NGS sample with 3uL DIK Sample Buffer Mix by vortexing and spin down You need to run an even number of samples so if necessary add a negative sample or a sample with only water o Prepare TapeStation room 3304 B Bring your samples to the TapeStation and also bring the ScreenTape from the fridge in room 3122 Turn on the computer user admin password 2200 Turn on the Agilent 2200 TapeStation Last updated 20 11 2014 Load the ScreenTape and the special loading tips they are in the shelf above the machine Even if you do not have a full run always load a full rack of tips The once that are not used you just place back again Make sure there are no bubbles in the separation channels by flicking the ScreenTape prior to loading Start the TapeStation software TapeStation Controller it is located on the Desktop on the computer o Sample Analysis Load a samples in the machin
26. or 5 minutes BIOO LIFE SCIENCE PRODUCTS WWW BIOOSCIENTIFIC COM an se While waiting you can prepare the _ new tubes with 23 uL of AMPure XP beads BIOO LIFE SCIENCE PRODUCTS 81 around 38 pL NEXTflex Rapid DNA Seq Kit Manual 5144 02 12 Remove and discard clear TETEE taking care not to disturb beads Some liquid may remain in wells The shortest fragments lt 400bp are discarded with the supernatant 00 13 With plate on stand add 280 uL of 80 ethanol to each magnetic bead pellet and incubate plate at room temperature for 30 seconds Carefully remove ethanol by pipette Repeat step 13 for a total of 2 ethanol washes Ensure all ethanol has been removed 15 Remove the plate from the magnetic stand and let dry at room temperature for 5 minutes or until bead pellet is visibly dry Put samples in fume hood turn on for faster drying 25 16 Resuspend dried beads with 50 uL Resuspension Buffer Mix thoroughly until homogenized Add Upper Cutoff volume of Sizing Solution to the resuspended bead pellet as in the table below for the desired final library size distribution For example if the desired selection range is 350 500 bp add 32 5 uL of Sizing Solution to the 50 uL resuspended bead pellet Mix thoroughly until homogenized The beads capture fragments gt 600 bp 300 400 bp 350 500 bp 7 00 600 600 bp _ Uppercut Tagg ea ATS 18 Incubate sample at room temperature for 5
27. r library a capture experiment will use from 0 25 to 0 5 fmole of each bait This represents 1 5 to 3 x 10 molecules per baits As a comparison 1 microgram of human genomic DNA library contains 3 x 10 copies of the genome How does it work Our approach is based on the work of Gnirke et al Solution Hybrid Selection with Ultra long Oligonucleotides for Massively Parallel Targeted Sequences 2009 Nature Biotechnology 27 2 182 189 The genomic DNA library is heat denatured and hybridized to the RNA baits in stringent conditions for 36 hours This gives enough time for a bait to hybridize to a complementary target sequence After hybridization the biotinylated baits hybridized to captured material are pulled out of the solution with streptavidin coated magnetic beads Any DNA molecule that may have bound non specifically to the magnetic beads are washed away and the captured genomic DNA is released by chemical degradation of the RNA baits Depending on the total length of the targeted sequences it may be necessary to perform a limited PCR amplification post capture to have enough material for sequencing For example when targeting 3 Mb of human sequence 1 1000 of human genome and starting from 5 micrograms of genomic library the theoretical amount of recoverable material is 5 nanograms But in practice the recovered amount will be lower due to inevitable loss of material at various steps MYcroarray techsupport mycroarray com 734
28. reactions kit size Formerly distributed with a PINK cap color Box 2 Store at 20 C in a non frost free freezer Stored in the freezer in room 4109 Product Amount Components Cap Color HYB 3 700 ul 50X Denhardt s Solution Yellow BLOCK 1 125 ul 1ug ul Human Cot 1 DNA Green BLOCK 2 125 ul 1 ug ul Salmon Sperm DNA Blue BLOCK 3 30 ul Proprietary Blocking Agent Gold RNase Block 70 ul SUPERase In 20 U ul Purple Amounts shown here are based on a 50 reactions kit size Formerly distributed with a BROWN cap color Stored in the freezer room 1207 First freezer to the right Product Amount Components Cap Color Capture Probe Library Biotinylated RNA Baits probes White The Capture Probe Library is sensitive to freeze thaw cycles If performing a small number of captures at a time it is recommended to aliquot the library to decrease its susceptibility to degradation variable MYcroarray techsupport mycroarray com 734 998 0751 Reagents to be provided by user Same primers as in the library construction PCR primers compatible with the sequencing platform to be used see Appendix A Elution Buffer see Appendix B Nuclease free Water Room 4236A Dynabeads MyOne Streptavidin C1 Invitrogen 650 01 Herculase II Fusion DNA Polymerase Stratagene 600677 QIAquick PCR purification Kit Qiagen 28704 Same procedure as described above Order seperately talk to Vivian
29. right amount by dividing the DNA concentration ng wL with 400 E g if you have a DNA concentration of 25 ng uL you should use 400 25 16uL of your DNA library Take the calculated amount and pool into the same tube for e g eight see above DNA libraries I e if you have worked with 48 unique barcodes you pool eight of these in the same tube and will end up with 46 8 6 tubes for sequence capture Dry your pooled DNA libraries with the Speed Vacuum instrument as described above Note from Lovisa I think that at least a subset of the samples should also be checked on TapeStation before Sequence Capture This to confirm DNA concentration and fragment length e Sequence capture Now it is time for the actual sequence capture procedure This procedure follows the MY Baits target enrichment system with some modifications as described below Read the whole document before starting First elute your dried DNA libraries with 8uL milliQH2O and once again measure the DNA concentration using the nanodrop instrument Divide the DNA concentration ng uL with 500 E g if you have a DNA concentration of 200 ng uL you should use 500 200 2 5uL of your pooled DNA libraries sequencing libraries in the sequence capture procedure ER MYcroarray MY baits Sequence Enrichment for Targeted Sequencing User Manual Version 1 3 8 06 14 2013 Table of Contents INTRODUCTION What does the bait library contain How does it work
30. thanol washes Ensure all ethanol has been removed easy cap tubes 26 Remove the plate from the magnetic stand and let dry at room temperature for 5 minutes or until bead pellet is visibly dry Put samples in fume hood turn on for faster drying WWW BIOOSCIENTIFIC COM 15 ore NEXTflex Rapid DNA Seq Kit Manual 5144 02 10 5 27 Resuspend dried beads with 24 uL Resuspension Buffer Mix thoroughly until homogenized 28 Incubate resuspended beads at room temperature for 5 minutes easy cap tubes 29 Place the 96 well PCR Plate on the magnetic stand at room temperature for 5 minutes 10 safe cap tube 30 Gently transfer 29 uL of clear sample to a 31 if you wish to pause your experiment the procedure may be safely stopped at this step with samples stored at 20 C To restart thaw frozen samples on ice before proceeding 32 Proceed to Step D2 PCR Amplification BIOO LIFE SCIENCE PRODUCTS WWW BIOOSCIENTIFIC COM Big NEXT flex Rapid DNA Seq Kit Manual 5144 02 STEP D2 PCR Amplification Materials Bioo Scientific Supplied GREEN CAP NEXTflex PCR Master Mix WHITE CAP Nuclease free Water GLEAR CAP BOTTLE NEXHiex Resuspension Buffer User Supplied NEXTflex Primer Mix found in NEXTflex DNA Barcode and ChIP Barcode kits Thermocycler Room 4218 96 Well PGR Plate PCR strip rt ASA i Thaw your ligated DNA PCR Master Mix and Primer Mix on ice Master Mix 8 104 uL H2O 48 uL PCR Mast
31. ution that you eluted your DNA with usually Elution Buffer or milliQ water and place on the instrument Press Blank and then Measure on the computer Check that the DNA concentration is ca 0 ng wL Add 1 uL of your DNA sample on the instrument Press Measure and note concentration ng uL and the 260 280 ratio the purity of your sample Optimal is 1 8 but above is fine lower is not that good Last updated 20 11 2014 Between each sample clean the instrument with soft tissue e Shearing DNA If your DNA fragments are too long use the Covaris 220 sonication instrument at the Sahlgrenska Genomics Core Facility to shear your DNA in appropriate length depending on your analysis you want fragments of different length o You need an introductory course at the core facility cost range from about 550 to 1100 SEK to be allowed to process the machine Contact Ellen Hanson for booking and details At the moment you can ask Filipe De Sousa or Jos Luis Blanco Pastor for help as they have the permission to use it o Your samples must be deposited in special tubes that are provided by the core facility Contact Ellen Hanson so that you can pick them up and prepare your samples before using the machine They are called microTUBE AFA Fiber Pre Slit Snap Cap 6 16mm The cost for one tube is 60SEK and this price includes the use of the Covaris machine Fach tube should have 130 uL of DNA s
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