SureSelect Target Enrichment for Roche 454 GS FLX and GS Junior
Contents
1. 6 If precipitate forms warm the hybridization buffer at 65 C for 5 minutes 7 Ina PCR plate strip tubes or tubes prepare the SureSelect capture library mix for target enrichment a Keep tubes on ice until step 11 b For each sample add the amount of SureSelect capture library as listed in Table 17 based on the Mb target size of your design c Use nuclease free water to prepare a dilution of the SureSelect RNase Block purple cap as listed in Table 17 Prepare enough RNase Block dilution for all samples plus excess d Add the amount of diluted SureSelect RNase Block purple cap listed in Table 17 to each capture library and mix by pipetting Table 17 SureSelect Capture Library Capture Size Volume of SureSelect RNase Block Dilution Volume of RNase Library Parts RNase block Block Dilution to Add Parts water lt 3 0 Mb 2uL 1 2 5 ul gt 3 0 Mb 5 ul 1 9 2uL SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries 37 3 Hybridization CAUTION 8 Ina separate PCR plate prepare the prepped library for target enrichment a Add 9 uL of amplified library and blocker mix to the B row in the PCR plate Put each sample into a separate well b Seal the wells of row B with caps and put the PCR plate in the thermal cycler Do not heat the Hybridization Buffer or capture library yet only the prepped library with blockers c Start the thermal cycler program in Table 182. Reagent Kits 16 Reactions 96 Reactions 480 Reactions SureSelect TE Reagent kit RCH G9607A G9607B G9607C SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries 9 1 Before You Begin Table 3 SureSelect Capture Library select one Capture Libraries 16 Reactions 96 Reactions 480 Reactions SureSelect Human All Exon 50Mb 5190 4626 5190 4627 5190 4629 SureSelect Human All Exon V4 5190 4631 5190 4632 5190 4634 SureSelect Human All Exon V4 UTRs 5190 4636 5190 4637 5190 4639 SureSelect Mouse All Exon 5190 4641 5190 4642 5190 4644 SureSelect DNA Kinome 5190 4646 5190 4647 5190 4649 SureSelect X chromosome 5190 4651 5190 4652 5190 4653 SureSelect Custom 1 kb up to 499 Kb 5190 4806 5190 4807 5190 4809 reorder 5190 4811 5190 4812 5190 4814 SureSelect Custom 0 5 Mb up to 2 9Mb 5190 4816 5190 4817 5190 4819 reorder 5190 4821 5190 4822 5190 4824 SureSelect Custom 3 Mb up to5 9Mb 5190 4826 5190 4827 5190 4829 reorder 5190 4831 5190 4832 5190 4834 SureSelect Custom 6 Mb up to 11 9Mb 5190 4836 5190 4837 5190 4839 reorder 5190 4841 5190 4842 5190 4844 SureSelect Custom 12 Mb up to 24Mb 5190 4896 5190 4897 5190 4899 reorder 5190 4901 5190 4902 5190 4904 Table4 Required Reagents for Hybridization Description Vendor and part number Dynabeads MyOne Streptavidin T1 Life Technologies 2mL Cat 65601 10 mL Cat 65602 100 mL Cat 65603 Nuclease free
3. In a PCR tube strip tube or plate prepare the reaction mix in Table 13 on ice Mix well by gently pipetting up and down 2 For multiple libraries a Prepare the reaction mix in Table 13 on ice Mix well on a vortex mixer b Add 25 uL of the reaction mix to each well or tube c Add 25 uL of each DNA sample to each well or tube Mix by pipetting Change pipette tips between samples Table 13 Components for PCR mix Reagent Volume for 1 Volume for 12 reactions reaction includes excess Adaptor ligated library 25 uL Nuclease free water 12 5 pL 156 25 uL SureSelect RCH PCR Primer clear cap 1 0 uL 12 5 pL 5X Herculase II Rxn Buffer clear cap 10 uL 125 uL 100 mM dNTP Mix green cap 0 5 pL 6 25 uL Herculase II Fusion DNA Polymerase red cap 1 uL 12 5 pL Total 50 uL 312 5 pL 25 pL reaction Included in the SureSelect Target Enrichment Kit RCH Hyb Module Box 2 t Included with the Herculase II Fusion DNA Polymerase Agilent Do not use the buffer or dNTP mix from any other kit SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries 27 2 28 Sample Preparation 3 Run the program in Table 14 in a thermal cycler Table 14 Step Time Temperature Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 Step 7 98 C 98 C 60 C 72 C 72 C 4 C 2 minutes 20 seconds 45 seconds 1 minute and 30 seconds Repeat Step 2 through Step 4 for a total of 12 to 14 times 5 minut
4. Prepped Library Figure 5 Prepped library shown in red Table 18 PCR program Step Temperature Time Step 1 95 C 5 minutes Step 2 65 C Hold 9 Use a heated lid on the thermal cycler at 105 C to hold the temperature of the plate on the thermal cycler at 65 C The lid of the thermal cycler is hot and can cause burns Use caution when working near the lid 38 SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries Hybridization 3 10 Maintain the plate at 65 C while you load 40 uL of hybridization buffer per well into the A row of the PCR plate The number of wells filled in Row A is the number of libraries prepared The example in Figure 6 is for 12 captures Hybridization Buffer Prepped Library Figure 6 Hybridization buffer shown in blue Make sure that the plate is at 65 C for a minimum of 5 minutes before you go to step 11 11 Add the capture library mix from step 7 to the PCR plate a Add the capture library mix 7 uL to the C row in the PCR plate For multiple samples use a multi channel pipette to load the capture library mix into the C row in the PCR plate Keep the plate at 65 C during this time b Seal the wells with strip caps Use a capping tool to make sure the fit is tight c Incubate the samples at 65 C for 2 minutes 12 Maintain the plate at 65 C while you use a multi channel pipette to take 13 uL of Hybridization Buffer from the A row an
5. 1004 Hee 04 w qa 3 100 150 200 300 400 s 600 1000 200 10380 bp Figure 8 Analysis of Amplified Capture DNA using the High Sensitivity DNA Kit The electropherogram shows fragment sizes ranging from 400 bp to 2 kb and a peak height of approximately 700 bp 100 bp Step 4 Perform emPCR and sequencing with Roche 454 Titanium chemistry e Run samples on a GS FLX or GS Junior System Use the instrument that is suited to the size of the capture library and level of coverage needed See Table 21 Table 21 Capture Library Size Mb Instrument Run Type gt 3 GS FLX Full 1 5 up to 2 9 GS FLX 2 pad 0 5 up to 1 4 GS FLX 4 pad lt 0 4 GS Junior Full 50 SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries j SureSelect Target Enrichment System for Roche 454 GS FLX and GS Junior Sequencing Platforms Protocol ee 5 626 Reference e E e j SureSelect Reagent Kit Content 54 Other Reagent Kits Content 56 Alternative Capture Equipment Combinations 58 This chapter contains reference information ee Agilent Technologies 53 5 54 Reference SureSelect Reagent Kit Content Each SureSelect Reagent Kit contains one or more of each of these individual kits Table 22 SureSelect Reagent Kit Contents Product Storage 16 96 480 Condition Reactions Reactions Reactions SureSelect Target Enrichment Kit Room 5190 4393 5190 4394 5190 4395 Box 1 Temperature SureSelect Target
6. Enrichment Kit RCH 20 C 5190 4449 5190 4450 5190 4451 Hyb Module Box 2 The content of each of these kits are described in the next tables Table 23 SureSelect Target Enrichment Kit Box 1 Kit Component SureSelect Hyb 1 orange cap or bottle SureSelect Hyb 2 red cap SureSelect Hyb 4 black cap or bottle SureSelect Binding Buffer SureSelect Wash 1 SureSelect Wash 2 SureSelect Elution Buffer SureSelect Neutralization Buffer SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries Reference Table 24 SureSelect Target Enrichment Kit RCH Hyb Module Box 2 Kit Component SureSelect Hyb 3 yellow cap SureSelect RCH Block 1 green cap SureSelect Block 2 blue cap SureSelect RCH Block 3 brown cap SureSelect RNase Block purple cap SureSelect RCH PCR Primer clear cap SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries 55 5 56 Other Reagent Kits Content These reagents are from kits other than the SureSelect Reagent kit Make sure you use only the reagents listed here Table 25 Herculase II Fusion DNA Polymerase Agilent Component DMSO green cap 5X Herculase II Rxn Buffer clear cap 100 mM dNTP Mix green cap Herculase II Fusion DNA Polymerase red cap Table 26 D1K Reagents Agilent p n 5067 5362 Components D1K ladder D1K sample buffer Table 27 High Sensitivity D1K Reagents Agilent p
7. Herculase Il Master Mix Reagent Volume for 1 Volume for 12 reactions reaction includes excess Captured DNA 15 uL Nuclease free water 22 5 uL 281 25 pL 5X Herculase II Rxn Buffer clear cap 10 uL 125 uL 100 mM dNTP Mix green cap 0 5 pL 6 25 uL Herculase II Fusion DNA Polymerase red cap 1 pL 12 5 uL SureSelect RCH PCR Primer clear cap 1 uL 12 5 uL Total 50 pL 437 5 pL 35 pL reaction Included in the Herculase II Fusion DNA Polymerase Agilent Do not use the buffer or dNTP mix from any other kit t Included in the SureSelect Target Enrichment Kit RCH Hyb Module Box 2 3 Put the tubes in a thermal cycler and run the program in Table 20 Table 20 PCR program Step Temperature Time Step 1 98 C 2 minutes Step 2 98 C 20 seconds Step 3 60 C 45 seconds Step 4 72 C 1 minute and 30 seconds Step 5 Repeat Step 2 through Step 4 for a total of 12 to 14 times Step 6 72 C 5 minutes Step 7 4 C Hold SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries 47 4 Post Hybridization Amplification Stopping Point 48 Step 2 Purify the sample using Agencourt AMPure XP beads 1 Let the AMPure XP beads come to room temperature for at least 30 minutes Mix the reagent well so that the reagent appears homogeneous and consistent in color Do not freeze Add 90 uL of homogenous AMPure beads to a 1 5 mL LoBind tube and add amplified library 50 uL Mix well on a vor
8. assay As an alternative you can use the High Sensitivity D1K ScreenTape Agilent p n 5067 5363 and High Sensitivity D1K Reagents Agilent p n 5067 5364 For more information to do this step see the Agilent 2200 TapeStation User Manual Use a Bioanalyzer High Sensitivity DNA Assay to assess the quality and size range You may need to dilute your sample accordingly Refer to the Agilent High Sensitivity DNA Kit Guide at http www chem agilent com en US Search Library _layouts Agilent PublicationSummary aspx whid 59504 1 Check that the 2100 Bioanalyzer electrodes have been cleaned as instructed in the reagent kit guide 2 Open the Agilent 2100 Expert Software version B 02 07 or higher required to run the High Sensitivity Kit turn on the 2100 Bioanalyzer and check communication 3 Prepare the chip samples and ladder as instructed in the reagent kit guide 4 Load the prepared chip into the 2100 Bioanalyzer and start the run within five minutes after preparation 5 Within the instrument context choose the appropriate assay from the drop down list 6 Start the run Enter sample names and comments in the Data and Assay context 7 Verify the results Determine the concentration of the sample by integration under the peak SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries 49 4 Post Hybridization Amplification FU 4 os 14c 1 20 3504 TY 3004 j 250 l pe 2004 Ss 1504
9. library The hybridization reaction requires 500 ng of DNA with a maximum volume of 3 4 uL 1 Add 500 ng of the amplified library to a 1 5 mL LoBind tube 2 Mix the contents in Table 15 to make the correct amount of SureSelect Block mix for the number of samples used Table 15 SureSelect Block Mix Reagent Volume for 1 reaction Volume for 12 reactions includes excess SureSelect RCH Block 1 green cap 10 uL 125 uL SureSelect Block 2 blue cap 2 5 uL 31 25 uL SureSelect RCH Block 3 brown cap 1 8 uL 22 5 uL Total 14 3 pL 178 75 pL 3 Add 14 3 uL of Blocker Mix to the amplified library 4 Dry sample down to 9 uL at 45 C in a vacuum concentrator To process multiple libraries of varying concentration add water to equalize the total volumes of all samples so that sample drying time variance is minimized 5 Mix the components in Table 16 at room temperature to prepare the hybridization buffer 36 SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries Hybridization 3 Table 16 Hybridization Buffer Reagent Volume for 1 Volume for 6 Volume for 12 capture pL captures pL captures pL includes excess includes excess includes excess SureSelect Hyb 1 orange cap or 25 125 250 bottle SureSelect Hyb 2 red cap 1 5 10 SureSelect Hyb 3 yellow cap 10 50 100 SureSelect Hyb 4 black cap or 13 65 130 bottle Total 49 40 pL 245 40 490 40 pL sample needed ptL sample
10. n 5067 5364 Components High Sensitivity D1K ladder High Sensitivity D1K sample buffer SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries Reference 5 Table 28 GS FLX Titanium Rapid Library Preparation Kit Roche p n 05608228001 Components TE Buffer Sizing Solution RL 10 x Buffer RL dNTP Mix RL T4 Polymerase RL ATP RL PNK RL Tag DNA Polymerase SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries 57 5 58 Reference Alternative Capture Equipment Combinations Table 29 lists combinations of thermal cycler lid temperature plates or strips and sealing method strip caps or sealing tape other than those used in this protocol that have shown minimal evaporation Refer to this list for additional of equipment combination options for hybridization Note that minimal evaporation is needed to ensure good capture results Table 29 Tested options that show minimal evaporation PCR Machine Plate Strips Cover Comments Agilent Mx3005P Mx3000P Strip Tubes MX3000P Optical Strip Heated Lid QPCR 401428 Caps 401425 Agilent Mx3005P MicroAmp Optical MicroAmp Clear Heated Lid QPCR 96 well reaction plate Adhesive Film ABI compression pad N801 0560 4306311 4312639 Use two layers of film ABI GeneAmp 9700 MicroAmp Optical MicroAmp Caps Heated Lid 96 well Reaction Plate 8caps strip N801 0560 N801 0535 ABI Veriti 4375786 Micro
11. 202 3 Rights in Commercial Computer Software or Com puter Software Documentation Safety Notices CAUTION A CAUTION notice denotes a haz ard It calls attention to an operat ing procedure practice or the like that if not correctly performed or adhered to could result in damage to the product or loss of important data Do not proceed beyond a CAUTION notice until the indicated conditions are fully understood and met A WARNING notice denotes a hazard It calls attention to an operating procedure practice or the like that if not correctly per formed or adhered to could result in personal injury or death Do not proceed beyond a WARNING notice until the indicated condi tions are fully understood and met 2 SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries In this Guide This guide describes the recommended operational procedures to use the Agilent SureSelect Target Enrichment Kits with Roche 454 Rapid Libraries on the Roche 454 GS FLX and GS Junior Systems using Titanium chemistry This protocol is specifically developed and optimized to use biotinylated RNA oligomer libraries to enrich targeted regions of the genome from repetitive sequences and sequences unrelated to the research focus 1 Before You Begin This chapter contains information such as procedural notes safety information required reagents and equipment that you should read and understand before you start a
12. Amp Optical MicroAmp Clear Heated Lid 96 well Reaction Plate Adhesive Film ABI compression pad N801 0560 4306311 4312639 Use two layers of film Eppendorf Eppendorf 8 Tube PCR Attached lids Lid heating set to 75 C Mastercycler Tubes BioRad MJ Agilent strip tubes Agilent Optical cap Heated Lid Research PTC 200 410022 Mx4000 410024 Mx4000 BioRad MJ Agilent strip tubes Agilent Optical cap Heated Lid Research PTC 200 410022 Mx4000 401425 Mx3000 3005 BioRad MJ Agilent 96 well Plate Agilent Optical cap Heated Lid Research PTC 200 410088 Mx3000 3005 401425 Mx3000 3005 SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries www agilent com In This Book This guide contains information to run the SureSelect Target Enrichment System for Roche 454 GS FLX and GS Junior Sequencing Platforms protocol with the Roche 454 Rapid Libraries with Titanium Sequencing Chemistry Agilent Technologies Inc 2010 2012 Version 1 4 February 2012 G3360 90003 Revision A5 Ee Agilent Technologies
13. SureSelect Target Enrichment System for Roche 454 GS FLX and GS Junior Sequencing Platforms Protocol Version 1 4 February 2012 SureSelect platform manufactured with Agilent SurePrint Technology Research Use Only Not for use in Diagnostic Procedures Ee Agilent Technologies Notices Agilent Technologies Inc 2010 2012 No part of this manual may be reproduced in any form or by any means including elec tronic storage and retrieval or translation into a foreign language without prior agree ment and written consent from Agilent Technologies Inc as governed by United States and international copyright laws Manual Part Number G3360 90003 Edition Version 1 4 February 2012 Printed in USA Agilent Technologies Inc 5301 Stevens Creek Rd Santa Clara CA 95051 USA Acknowledgement Oligonucleotide sequences 2006 2008 and 2011 Illumina Inc All rights reserved Only for use with the Illumina sequencer systems and associated assays Technical Support For technical product support contact your local Agilent Support Services representa tive For US and Canada call 800 227 9770 option 3 4 4 For other countries find your support center telephone numbers at www agilent com chem contactus Or send an e mail to SureSelect Support agilent com Notice to Purchaser Research Use Only Not for use in diagnostic procedures Warranty The material contained in this document is provi
14. Titanium Sequencing Libraries Sample Preparation 2 Table 10 Covaris shear settings Setting Value Duty Cycle 5 Intensity 3 Cycles per Burst 200 Time 3 cycles of 30 seconds Set Mode Frequency sweeping Temperature 4 to7 C 7 Put the Covaris microTube back into the loading and unloading station 8 While keeping the snap cap on insert a pipette tip through the pre split septa then slowly remove the sheared DNA 9 Transfer the sheared DNA into a new 1 5 mL LoBind tube SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries 19 2 Sample Preparation Step 2 Purify the sample with the QIAGEN MinElute PCR Purification Kit 1 If you haven t already done so add the pH indicator to the Buffer PB 2 Add 650 uL of Buffer PB per sample and mix well by pipetting Check for the yellow color to make sure buffer PB pH is correct For more information on how to check buffer pH refer to the Qiagen MinElute Handbook If needed use 5 uL of the 3M Sodium Acetate included in the kit to adjust the pH to the proper range 4 Puta MinElute spin column in a 2 mL collection tube 5 Transfer the 780 uL sample to the MinElute column Spin the sample in a centrifuge for 60 seconds at 17 900g 13 000 rpm Discard the flow through Add 750 uL of buffer PE to the column Spin the sample in a centrifuge for 60 seconds at 17 900g 13 000 rpm Discard the flow through Put the MinElute column back in th
15. Water not DEPC treated Ambion Cat AM9930 10 SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries Required Equipment Table 5 Before You Begin 1 Required Equipment for Library Prep and Post Hybridization Amplification Description Vendor and part number 2100 Bioanalyzer Nuclease free 1 5 mL microfuge tubes sustainable at 95 C Thermal cycler Covaris S series Single Tube Sample Preparation System Model S2 Covaris microTUBE with AFA fiber and snap cap Nuclease free 0 2 mL PCR tubes thin walled Microcentrifuge Qubit Fluorometer P10 P20 P200 and P1000 pipettes Vacuum concentrator Ice bucket Powder free gloves Sterile nuclease free aerosol barrier pipette tips Timer Vortex mixer Heat block at 37 C Agilent p n G2938C Ambion p n AM12400 or equivalent Agilent SureCycler Life Technologies Veriti Thermal Cycler BioRad MJ Research DNA Engine PTC 200 or equivalent Covaris Covaris p n 520045 Eppendorf p n 951010006 or equivalent Eppendorf Microcentrifuge Model 5417C or equivalent Life Technologies p n 032857 Pipetman P10 P20 P200 P1000 or equivalent Savant SpeedVac or equivalent SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries 11 1 12 Before You Begin Table6 Required Equipment for Hybridization Description Vendor and part number Mx3000P Mx3005P 96 well tube plates Mx3000P Mx3005P
16. YB BUFFER BIOTINYLATED RNA LIBRARY BAITS O Hybridization 20000 90000 20000 STREPTAVIDIN COATED MAGNETIC BEADS Maan oe S JATA it Ras 20000 vie capture UNBOUND FRACTION Wash a DISCARDED and Digest RNA Amplify SAVY IW gt Sequencing Sequencing VT VS Figure 4 SureSelect Target Enrichment System Capture Process 34 SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries Hybridization 3 Refer to SureSelect Reagent Kit Content on page 54 for a complete content listing of each SureSelect Target Enrichment kit CAUTION You must avoid evaporation from the small volumes of the capture during the 24 hour or greater incubation If you want to use a different combination of thermal cycler lid temperature plates or strips and sealing method strip caps or sealing tape first test the conditions Incubate 27 uL of SureSelect Hybridization Buffer without DNA at 65 C for 24 hours or longer if applicable as a test Include buffer in each well that you might use including those in the center and those on the edges Check that you do not get extensive evaporation Evaporation should not exceed 3 to 4 pL For a partial list of tested options showing minimal evaporation refer to Alternative Capture Equipment Combinations on page 58 SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries 35 3 Hybridization Step 1 Hybridize the
17. d add it to the SureSelect capture library mix contained in row C of the PCR plate for each sample See Figure 7 SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries 39 3 Hybridization CAUTION Hybridization Buffer Prepped Library SureSelect Capture Library Figure 7 SureSelect Capture Library or Baits shown in Green 13 Maintain the plate at 65 C while you use a multi channel pipette to transfer the entire contents of each prepped library mix in row B to the hybridization solution in row C See Figure 7 Mix well by slowly pipetting up and down 8 to 10 times The hybridization mixture is now 27 to 29 uL depending on degree of evaporation during the preincubations 14 Seal the wells with strip caps or double adhesive film Make sure all wells are completely sealed Use new adhesive seals or strip caps The structural integrity of the seals and caps can be compromised during the previous incubation steps Wells must be adequately sealed to minimize evaporation or your results can be negatively impacted 40 If you use strip tubes test for evaporation before you do the first experiment to make sure the tube capping method is appropriate for the thermal cycler Check that no more than 3 to 4 uL is lost to evaporation 15 Incubate the hybridization mixture for 24 hours at 65 C with a heated lid at 105 C SureSelect Target Enrichment System for Roche 454 Ti
18. ded as is and is subject to being changed with out notice in future editions Fur ther to the maximum extent permitted by applicable law Agi lent disclaims all warranties either express or implied with regard to this manual and any information contained herein including but not limited to the implied warranties of merchant ability and fitness for a particular purpose Agilent shall not be lia ble for errors or for incidental or consequential damages in con nection with the furnishing use or performance of this document or of any information contained herein Should Agilent and the user have a separate written agreement with warranty terms covering the material in this doc ument that conflict with these terms the warranty terms in the separate agreement shall control Technology Licenses The hardware and or software described in this document are furnished under a license and may be used or copied only in accor dance with the terms of such license Restricted Rights Legend U S Government Restricted Rights Soft ware and technical data rights granted to the federal government include only those rights customarily provided to end user cus tomers Agilent provides this customary commercial license in Software and techni cal data pursuant to FAR 12 211 Technical Data and 12 212 Computer Software and for the Department of Defense DFARS 252 227 7015 Technical Data Commercial Items and DFARS 227 7
19. e 2 mL collection tube and spin in a centrifuge for 60 seconds at 17 900g 13 000 rpm Transfer the MinElute column to a new 1 5 mL collection tube to elute the cleaned sample Let sit for 2 minutes to completely evaporate residual ethanol All traces of ethanol must be removed 10 Add 17 uL of buffer EB 10 mM Tris Cl pH 8 5 directly onto the MinElute filter membrane 11 Wait 60 seconds then spin in a centrifuge for 60 seconds at 17 900g 13 000 rpm 12 Collect the eluate and transfer 17 uL to PCR tubes 20 SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries Sample Preparation 2 Step 3 Assess quality with the 2100 Bioanalyzer As an alternative you can use the D1K ScreenTape Agilent p n 5067 5361 and D1K Reagents Agilent p n 5067 5362 For more information to do this step see the Agilent 2200 TapeStation User Manual Use a Bioanalyzer High Sensitivity DNA chip and reagent kit See the Agilent High Sensitivity DNA Kit Guide at http www chem agilent com en US Search Library _layouts Agilent Public ationSummary aspx whid 59504 1 Check that the 2100 Bioanalyzer electrodes have been cleaned as instructed in the reagent kit guide 2 Open the Agilent 2100 Expert Software version B 02 07 or higher turn on the 2100 Bioanalyzer and check communication 3 Prepare the chip samples and ladder as instructed in the reagent kit guide 4 Load the prepared chip into the 2100 Bioanalyz
20. ely 3 to 5 minutes 10 Keep the tube in the magnetic stand Do not touch the beads while you carefully discard the cleared solution from the tubes 11 Add 25 uL of TE Buffer Mix on a vortex mixer for 5 seconds 12 Add 125 uL of Sizing Solution Mix on a vortex mixer for 5 seconds 13 Incubate at room temperature for 5 minutes 14 Put the tube in the magnetic stand Wait for the solution to clear approximately 3 to 5 minutes 15 Keep the tube in the magnetic stand Do not touch the beads while you carefully discard the cleared solution from the tubes 16 Repeat step 11 through step 15 once 17 Continue to keep the tube on the magnetic stand while you dispense 175 uL of 70 ethanol in each tube Use fresh 70 ethanol for optimal result SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries 29 2 Sample Preparation 18 Let the tube sit for 1 minute to let any disturbed beads settle and remove the ethanol 19 Repeat step 17 through step 18 once 20 Dry the samples on a 37 C heat block for 5 minutes until the residual ethanol is completely evaporated Do not dry the bead pellet to the point that the bead pellet appears cracked Elution efficiency is significantly decreased when the bead pellet is excessively dried 21 Add 28 uL of water Mix on a vortex mixer for 5 seconds and spin briefly 22 Put the tube in the magnetic stand and leave for 2 to 3 minutes until the solution is clear 23 Remo
21. er and start the run within five minutes after preparation 5 Within the instrument context choose the appropriate assay from the drop down list 6 Start the run Enter sample names and comments in the Data and Assay context 7 Verify the results Check that the electropherogram shows a distribution with a peak height of 800 bp 4100 bp as shown in Figure 2 SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries 21 2 22 Sample Preparation Step 3 Assess quality with the 2100 Bioanalyzer FU Post shearing purifica 35 100 150 200 300 400 500 600 1000 2000 10380 bp Figure 2 Analysis of sheared DNA using a Bioanalyzer High Sensitivity DNA assay SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries Sample Preparation 2 Step 4 Repair the ends To process multiple samples prepare master mixes with overage at each step without the DNA sample Master mixes for preparation of 12 samples including excess are shown in each table as an example Use the GS FLX Titanium Rapid Library Preparation Kit Roche p n 05608228001 Prepare the master mix on ice 1 For 1 library prepare on ice In a PCR tube strip tube or plate prepare the reaction mix in Table 11 Mix well by gently pipetting up and down 2 For multiple libraries prepare on ice a Prepare the reaction mix in Table 11 b Add 9 uL of the reaction mix to each well or tube c Add 16 uL o
22. es Hold Different library preparations can produce slightly different results based on varying DNA quality In most cases 12 cycles will produce an adequate yield for subsequent capture without introducing bias or non specific products If yield is too low or non specific high molecular weight products are observed adjust the number of cycles accordingly SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries Sample Preparation 2 Step 9 Purify the sample with the Agencourt AMPure XP beads The Sizing Solution and TE Buffer are part of the GS FLX Titanium Rapid Library Preparation Kit Roche p n 05608228001 1 Prepare a new batch of Agencourt AMPure XP beads and let it come to room temperature for at least 30 minutes 2 Mix the reagent well so that the reagent appears homogeneous and consistent in color Do not freeze 3 Add 125 uL of homogenous AMPure XP beads to a 200 uL PCR tube 4 Put the tube in the magnetic stand Wait for the solution to clear approximately 3 to 5 minutes 5 Keep the tube in the magnetic stand Do not touch the beads while you carefully discard the cleared solution from the tubes 6 Add 125 uL of Sizing Solution to the beads Mix on a vortex mixer for 5 seconds then spin briefly on a centrifuge 7 Add the amplified library to the AMPure beads 8 Incubate at room temperature for 5 minutes Put the tube in the magnetic stand Wait for the solution to clear approximat
23. et Enrichment System for Roche 454 Titanium Sequencing Libraries 13 1 Before You Begin Optional Equipment 14 SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries CAUTION SureSelect Target Enrichment System for Roche 454 GS FLX and GS Junior Sequencing Platforms Protocol ee 2 Sample Preparation Step 1 Shear DNA 18 Step 2 Purify the sample with the QIAGEN MinElute PCR Purification Kit 20 Step 3 Assess quality with the 2100 Bioanalyzer 21 Step 4 Repair the ends 23 Step 5 Prepare the Agencourt AMPure XP beads 25 Step 6 Ligate the adaptor 25 Step 7 Remove small fragments with AMPure XP beads 26 Step 8 Amplify adaptor ligated library 27 Step 9 Purify the sample with the Agencourt AMPure XP beads 29 Step 10 Assess quality and quantity with 2100 Bioanalyzer 31 This section contains instructions for prepped library production specific to the Roche 454 Rapid Library preparation It is intended for use with Roche 454 Titanium Chemistry The steps in this section differ from the Roche protocol in the use of the Covaris system for gDNA shearing and implementation of DNA library amplification after adaptor ligation Refer to the Roche Rapid Library Preparation Method Manual GS FLX Titanium Series for more information Use a non absorbance based quantitation system such as the Qubit system to quantify genomic DNA before library preparation Make sure genomic DNA samples are of high quali
24. f each DNA sample to each well or tube Mix by pipetting Change pipette tips between samples Table 11 Fragment End Repair Mix Reagent Volume for 1 Library Volume for 12 Libraries includes excess DNA sample 16 uL RL ATP 2 5 uL 31 25 uL RL 10 x Buffer 2 5 uL 31 25 uL RL dNTP Mix TpLl 12 5 pL RL T4 Polymerase Tul 12 5 pL RL PNK Tpl 12 5 pL RL Tag DNA Polymerase Tul 12 5 pL Total Volume 25 pL 112 5 pL 9 pL reaction These reagents are included in the GS FLX Titanium Rapid Library Preparation Kit Roche p n 05608228001 SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries 23 2 24 Sample Preparation 3 Run the End Repair program Table 12 on a thermal cycler with a heated lid Table 12 End Repair program Step Temperature Time Step 1 25 C 20 minutes Step 2 72 C 20 minutes Step 3 4 C Hold SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries Sample Preparation 2 Step 5 Prepare the Agencourt AMPure XP beads The Sizing Solution and TE Buffer are part of the Roche 454 Rapid Library Preparation Kit 1 Let the AMPure XP beads come to room temperature for at least 30 minutes 2 Mix the reagent well so that the reagent appears homogeneous and consistent in color Do not freeze 3 Add 125 uL of homogenous AMPure XP beads to a 200 uL PCR tube 4 Put the tube in the magnetic stand Wait for the solution to clear approximately 3 to 5
25. form emPCR and sequencing with Roche 454 Titanium chemistry 50 This chapter describes the steps to amplify purify assess quality of the library and dilute the sample appropriately for cluster amplification Agg Agilent Technologies 45 4 Post Hybridization Amplification CAUTION Step 1 Amplify the captured library Use reagents from Herculase II Fusion DNA Polymerase Agilent SureSelect Target Enrichment Kit RCH Hyb Module Box 2 Do not use amplification enzymes other than Herculase II Fusion DNA Polymerase Other enzymes have not been validated CAUTION To avoid cross contaminating libraries set up PCR reactions all components except the library DNA in a dedicated clean area or PCR hood with UV sterilization and positive air flow 46 Prepare 1 amplification reaction for each hybrid capture Include a negative no template control 1 For 1 library In a PCR tube strip tube or plate prepare the reaction mix in Table 19 on ice Mix well by gently pipetting up and down 2 For multiple libraries a Prepare the reaction mix in Table 19 on ice Mix well on a vortex mixer b Add 35 uL of the reaction mix to each well or tube c Use a pipette to add 15 uL of each DNA sample to each well or tube Mix by pipetting Change pipette tips between samples to avoid cross contamination SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries Post Hybridization Amplification 4 Table 19
26. in temperature c Briefly spin in a centrifuge d Separate the beads and buffer on a magnetic separator and remove the supernatant e Repeat step a through step d for a total of 3 washes Make sure all of the wash buffer has been removed 11 Mix the beads in 50 uL of SureSelect Elution Buffer on a vortex mixer for 5 seconds to resuspend the beads 12 Occasionally mix on a vortex mixer 13 Briefly spin in a centrifuge 14 Separate the beads and buffer on a magnetic separator 15 Use a pipette to transfer the supernatant to a new 1 5 mL microfuge tube The supernatant contains the captured DNA The beads can now be discarded 16 Add 50 uL of SureSelect Neutralization Buffer to the captured DNA 17 Briefly mix on a vortex mixer SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries 43 3 Hybridization Step 4 Purify the sample using Agencourt AMPure XP beads 1 Let the AMPure XP beads come to room temperature for at least 30 minutes 2 Mix the reagent well so that the reagent appears homogeneous and consistent in color Do not freeze 3 Add 180 uL of homogenous AMPure XP beads to a 1 5 mL LoBind tube and add 100 uL of captured DNA library Mix well on a vortex mixer and incubate for 5 minutes 4 Put the tube in the magnetic stand Wait for the solution to clear approximately 3 to 5 minutes 5 Keep the tube in the magnetic stand Do not touch the beads while you carefully discard the cleared
27. minutes 5 Keep the tube in the magnetic stand Do not touch the beads while you carefully discard the cleared solution from the tubes 6 Add 125 uL of Sizing Solution to the beads Mix on a vortex mixer for 5 seconds then spin briefly on a centrifuge 7 Keep the tube on ice until the ligation step is completed Step 6 Ligate the adaptor Add 1 uL of the RL to the reaction tube Add 1 uL of the RL Ligase to the reaction tube Mix on a vortex mixer for 5 seconds then spin briefly in a centrifuge Aa O N Incubate for 10 minutes at 25 C on a thermal cycler Do not use a heated lid SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries 25 2 26 Sample Preparation Stopping Point Step 7 Remove small fragments with AMPure XP beads 1 Add the sample to the AMPure beads that you prepared in Step 5 Prepare the Agencourt AMPure XP beads on page 25 2 Incubate at room temperature for 5 minutes 3 Put the tube in the magnetic stand Wait for the solution to clear approximately 3 to 5 minutes 4 Keep the tube in the magnetic stand Do not touch the beads while you carefully discard the cleared solution from the tubes Add 25 uL of TE Buffer Mix on a vortex mixer for 5 seconds Add 125 uL of Sizing Solution Mix on a vortex mixer for 5 seconds Incubate at room temperature for 5 minutes on ool Put the tube in the magnetic stand Wait for the solution to clear approximately 3 t
28. mised 18 1 Use the Qubit dsDNA BR Assay to determine the concentration of your gDNA sample Make sure the gDNA is of high quality non degraded Ag 0 A280 is 1 8 to 2 0 Follow the instructions for the instrument Set up the Covaris instrument a Check that the water in the Covaris tank is filled with fresh deionized water to fill line level 12 on the graduated fill line label b Check that the water covers the visible glass part of the tube c Set the chiller temperature to between 2 C to 5 C to ensure that the temperature reading in the water bath displays 5 C d Optional Supplement the circulated water chiller with ethylene glycol to 20 volume to prevent freezing e On the instrument control panel push the Degas button Degas the instrument for least 30 minutes before use Refer to the Covaris instrument user guide Dilute 500 ng of high quality gDNA with 1X Low TE Buffer in a 1 5 mL LoBind tube to a total volume of 130 uL Put a Covaris microTube into the loading and unloading station Keep the cap on the tube 5 Use a tapered pipette tip to slowly transfer the 130 uL DNA sample through the pre split septa Be careful not to introduce a bubble into the bottom of the tube Secure the microTube in the tube holder and shear the DNA with the recommended settings in Table 10 to get a 500 bp target peak size which results in an actual peak size of 800 bp SureSelect Target Enrichment System for Roche 454
29. n experiment 2 Sample Preparation This chapter describes the steps to prepare the DNA sample for target enrichment 3 Hybridization This chapter describes the steps to prepare and hybridize samples 4 Post Hybridization Amplification This chapter describes the steps to amplify purify and assess quality of the sample library 5 Reference This chapter contains information on alternative equipment that can be used with this protocol SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries 3 What s New in 1 4 e New product configuration and product numbers for SureSelect reagent kits and capture libraries up to 24 Mb e Support for the optional use of the Agilent 2200 TapeStation for DNA quantitation and qualification e Support for SureSelect Human All Exon v4 and All Exon v4 UTRs capture libraries What s New in 1 3 e Corrections to volume in Step 2 Purify the sample with the QIAGEN MinElute PCR Purification Kit e Corrections to Step 9 Purify the sample with the Agencourt AMPure XP beads What s New in 1 2 e Support for SureSelect Human Kinome Plus kits What s New in 1 1 e Support for SureSelect Mouse All Exon Kits 4 SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries Content 1 Before You Begin 7 Procedural Notes 8 Safety Notes 8 Required Reagents 9 Required Equipment 11 Optional Equipment 13 2 Sample Preparation 15 Ste
30. nts off of walls and lid 3 Store on ice or in a cold block until use e In general follow Biosafety Level 1 BL1 safety rules Safety Notes CAUTION Wear appropriate personal protective equipment PPE when working in the laboratory 8 SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries Required Reagents Before You Begin 1 Table 1 Required Reagents for Library Prep and Post Hybridization Amplification Description Vendor and part number DNA 7500 Kit High Sensitivity DNA Kit Herculase II Fusion DNA Polymerase includes dNTP mix and 5x Buffer 200 reactions 400 reactions Nuclease free Water not DEPC treated Agencourt AMPure XP Kit 5 mL 60 mL 450 mL 1X Low TE Buffer 10 mM Tris HCl pH 8 0 0 1 mM EDTA Qubit dsDNA BR Assay Kit 100 assays 2 1000 ng 500 assays 2 1000 ng 1000 assays 2 1000 ng Qubit assay tubes MinElute PCR Purification Kit 250 GS FLX Titanium Rapid Library Preparation Kit 100 Ethanol molecular biology grade Distilled water Agilent p n 5067 1506 Agilent p n 5067 4626 Agilent p n 600677 p n 600679 Ambion Cat AM9930 Beckman Coulter Genomics p n A63880 p n A63881 p n A63882 Life Technologies p n 4389764 Life Technologies p n 032850 Life Technologies p n 032853 Life Technologies p n 033130 Life Technologies p n 032856 Qiagen p n 28006 Roche p n 05608228001 Sigma Aldrich p n E7023 Table 2 SureSelect Reagent Kit
31. o 5 minutes 9 Keep the tube in the magnetic stand Do not touch the beads while you carefully discard the cleared solution from the tubes 10 Repeat step 5 through step 9 once 11 Continue to keep the tube on the magnetic stand while you dispense 175 uL of 70 ethanol in each tube Use fresh 70 ethanol for optimal result 12 Let the tube sit for 1 minute to let any disturbed beads settle and remove the ethanol 13 Repeat step 11 through step 12 once 14 Dry the samples on a 37 C heat block for 5 minutes until the residual ethanol is completely evaporated Do not dry the bead pellet to the point that the bead pellet appears cracked Elution efficiency is significantly decreased when the bead pellet is excessively dried 15 Add 28 uL of water Mix on a vortex mixer for 5 seconds and spin briefly 16 Put the tube in the magnetic stand and leave for 2 to 3 minutes until the solution is clear 17 Remove 25 uL of the supernatant to a fresh 1 5 mL LoBind tube You can discard the beads at this time If you do not continue to the next step store the samples at 20 C SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries CAUTION Sample Preparation 2 Step 8 Amplify adaptor ligated library To avoid cross contaminating libraries set up PCR reactions all components except the library DNA in a dedicated clean area or PCR hood with UV sterilization and positive air flow 1 For 1 library
32. optical strip caps MicroAmp Clear Adhesive Film BD Clay Adams Nutator Mixer Dynal DynaMag 2 magnetic stand P10 P20 P200 and P1000 pipettes Pipet Light Multichannel Pipette 12 channels Sterile nuclease free aerosol barrier pipette tips Thermal cycler Timer Vortex mixer Water bath or heat block set to 65 C Agilent p n 410088 or equivalent Agilent p n 401425 or equivalent Life Technologies p n 4306311 or equivalent BD Diagnostics p n 421105 or equivalent Life Technologies p n 123 21D or equivalent Pipetman P10 P20 P200 P1000 or equivalent Rainin p n L12 20 or equivalent Agilent SureCycler Life Technologies Veriti Thermal Cycler BioRad MJ Research DNA Engine PTC 200 or equivalent SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries Before You Begin 1 Optional Equipment Table 7 Optional Equipment for Hybridization Description Vendor and part number Tube strip capping tool Agilent p n 410099 Qubit Quantitation Starter Kit Life Technologies p n 032860 Table 8 Optional Equipment for Library Prep and Post Hybridization Amplification Description Vendor and part number 2100 Bioanalyzer System Agilent p n G2938C 2200 TapeStation System Agilent p n G2964AA or G2965AA D1K ScreenTape Agilent p n 5067 5361 D1K Reagents Agilent p n 5067 5362 High Sensitivity D1K ScreenTape Agilent p n 5067 5363 High Sensitivity D1K Reagents Agilent p n 5067 5364 SureSelect Targ
33. p 1 Shear DNA 18 Step 2 Purify the sample with the QIAGEN MinElute PCR Purification Kit 20 Step 3 Assess quality with the 2100 Bioanalyzer 21 Step 4 Repair the ends 23 Step 5 Prepare the Agencourt AMPure XP beads 25 Step 6 Ligate the adaptor 25 Step 7 Remove small fragments with AMPure XP beads 26 Step 8 Amplify adaptor ligated library 27 Step 9 Purify the sample with the Agencourt AMPure XP beads 29 Step 10 Assess quality and quantity with 2100 Bioanalyzer 31 3 Hybridization 33 Step 1 Hybridize the library 36 Step 2 Prepare magnetic beads 41 Step 3 Hybrid capture with SureSelect 42 Step 4 Purify the sample using Agencourt AMPure XP beads 44 SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries Contents 4 Post Hybridization Amplification 45 Step 1 Amplify the captured library 46 Step 2 Purify the sample using Agencourt AMPure XP beads 48 Step 3 Assess quality and quantity with the 2100 Bioanalyzer High Sensitivity DNA assay 49 Step 4 Perform emPCR and sequencing with Roche 454 Titanium chemistry 50 5 Reference 53 SureSelect Reagent Kit Content 54 Other Reagent Kits Content 56 Alternative Capture Equipment Combinations 58 6 SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries SureSelect Target Enrichment System for Roche 454 GS FLX and GS Junior Sequencing Platforms Protocol ee 1 Before You Begin Procedural Notes 8 Safety Notes 8 Required Reagent
34. s 9 Required Equipment 11 Optional Equipment 13 Make sure you have the most current protocol Go to the SureSelect Related Literature page on genomics agilent com and search for manual number G3360 90003 Make sure you read and understand the information in this chapter and have the necessary equipment and reagents listed before you start an experiment Agilent cannot guarantee the SureSelect Target Enrichment kits and cannot provide technical support for the use of non Agilent protocols to process samples for enrichment phe Agilent Technologies 1 Before You Begin Procedural Notes e Prolonged exposure of SureSelect Elution Buffer to air can decrease product performance by altering the pH of the solution Keep the Elution Buffer container tightly sealed when not in use To prevent contamination of reagents by nucleases always wear powder free laboratory gloves and use dedicated solutions and pipettors with nuclease free aerosol resistant tips e Maintain a clean work area e Do not mix stock solutions and reactions containing gDNA on a vortex mixer Instead gently tap the tube with your finger to mix the sample e Avoid repeated freeze thaw cycles of stock and diluted gDNA solutions e When preparing frozen reagent stock solutions for use 1 Thaw the aliquot as rapidly as possible without heating above room temperature 2 Mix briefly on a vortex mixer then spin in a centrifuge for 5 to 10 seconds to drive the conte
35. solution from the tubes 6 Continue to keep the tube in the magnetic stand while you dispense 0 5 mL of 70 ethanol in each tube Use fresh 70 ethanol for optimal result 7 Let the tube sit for 1 minute to allow any disturbed beads to settle and remove the ethanol 8 Repeat step 6 and step 7 step once Dry the samples on the 37 C heat block for 5 minutes or until the residual ethanol completely evaporates Do not dry the bead pellet to the point that the bead pellet appears cracked Elution efficiency is significantly decreased when the bead pellet is excessively dried 10 Add 33 uL nuclease free water mix well on a vortex mixer and incubate for 2 minutes at room temperature 11 Put the tube in the magnetic stand and leave for 2 to 3 minutes until the solution is clear 12 Remove the supernatant 30 uL to a fresh 1 5 mL LoBind tube You can discard the beads at this time Stopping Point If you do not continue to the next step store the samples at 20 C 44 SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries j SureSelect Target Enrichment System for Roche 454 GS FLX and GS Junior Sequencing Platforms Protocol ee 4 Post Hybridization Amplification e a e g Step 1 Amplify the captured library 46 Step 2 Purify the sample using Agencourt AMPure XP beads 48 Step 3 Assess quality and quantity with the 2100 Bioanalyzer High Sensitivity DNA assay 49 Step 4 Per
36. tanium Sequencing Libraries Hybridization 3 Step 2 Prepare magnetic beads Use these reagents from the SureSelect Target Enrichment Kit Box 1 SureSelect Binding Buffer SureSelect Wash 2 1 Prewarm SureSelect Wash 2 at 65 C in a circulating water bath for use in Step 3 Hybrid capture with SureSelect 2 Vigorously resuspend the Dynabeads MyOne Streptavidin T1 on a vortex mixer Magnetic beads settle during storage 3 For each hybridization add 50 uL of Dynabeads MyOne Streptavidin T1 toa 1 5 mL microfuge tube 4 Wash the beads a Add 200 uL of SureSelect Binding Buffer b Mix the beads on a vortex mixer for 5 seconds c Put the tubes into a magnetic device such as the Dynal magnetic separator Life Technologies d Remove and discard the supernatant e Repeat step a through step d for a total of 3 washes 5 Resuspend the beads in 200 uL of SureSelect Binding Buffer SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries 41 3 Hybridization Step 3 Hybrid capture with SureSelect CAUTION Use these reagents from the SureSelect Target Enrichment Kit Box 1 SureSelect Wash 1 SureSelect Wash 2 SureSelect Elution Buffer SureSelect Neutralization Buffer Keep the Elution Buffer container tightly sealed when not in use Prolonged exposure of SureSelect Elution Buffer to air can decrease product performance by altering the pH of the solution 42 Estimate and record the volume of h
37. tex mixer and incubate for 5 minutes Put the tube in the magnetic stand Wait for the solution to clear approximately 3 to 5 minutes Keep the tube in the magnetic stand Do not touch the beads while you carefully discard the cleared solution from the tubes Continue to keep the tube in the magnetic stand while you dispense 500 uL of 70 ethanol in each tube Use fresh 70 ethanol for optimal result Let the tube sit for 1 minute to allow any disturbed beads to settle and remove the ethanol Repeat step 6 and step 7 once Dry the samples on the 37 C heat block for 5 minutes or until the residual ethanol is completely evaporated Do not dry the bead pellet to the point that the bead pellet appears cracked Elution efficiency is significantly decreased when the bead pellet is excessively dried 10 Add 33 uL nuclease free water mix well on a vortex mixer and incubate for 2 minutes at room temperature 11 Put the tube in the magnetic stand and leave for 2 to 3 minutes until the solution is clear 12 Remove the supernatant 30 uL to a fresh 1 5 mL LoBind tube You can discard the beads at this time If you do not continue to the next step store the samples at 4 C for up toa week or at 20 C for longer periods SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries Post Hybridization Amplification 4 Step 3 Assess quality and quantity with the 2100 Bioanalyzer High Sensitivity DNA
38. ty with an OD 260 280 ratio ranging from 1 8 to 2 0 gig Agilent Technologies 15 2 Sample Preparation Genomic BNA Covaris shear 500 bp setting Sheared BNA End repair with 5 phosphorylated cron ocations SS ooo Ligate adaptors Bait Design in eArray Ada tor modified ends co bibrary y y AMPure XP bead purification Removal of small a AMENS y PCR and XP bead purification Pre m Libra ibra ridization 24 hours at 65 C ybri apture election Magnetic bead selection mplification PCR and purification Uall ssessment Bioanalyzer and Quantitation equencin Figure 1 Overall sequencing sample preparation workflow 16 SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries Sample Preparation 2 Table9 Overview and time requirements Step Time Roche 454 Sequencing Library Production 1 day Library Hybridization 24 hours optional up to 72 hours Bead preparation 30 minutes Capture Selection and Washing 2 hours DNA purification 30 minutes Post Hybridization Amplification 1 hour PCR purification 30 minutes Bioanalyzer QC 1 hour SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries 17 2 CAUTION Sample Preparation Step 1 Shear DNA For each DNA sample to be sequenced prepare 1 library Use a non absorbance based quantitation system or the library complexity and capture efficiency can be severely compro
39. ve 25 uL of the supernatant to a fresh 1 5 mL LoBind tube You can discard the beads at this time Make sure no beads remain in the sample Stopping Point If you do not continue to the next step store the samples at 20 C 30 SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries Sample Preparation 2 Step 10 Assess quality and quantity with 2100 Bioanalyzer As an alternative you can use the D1K ScreenTape Agilent p n 5067 5361 and D1K Reagents Agilent p n 5067 5362 For more information to do this step see the Agilent 2200 TapeStation User Manual Use the Bioanalyzer DNA 7500 chip and reagent kit to assess the quantity quality and size distribution of the PCR products 1 Check that the 2100 Bioanalyzer electrodes have been cleaned as instructed in the reagent kit guide 2 Open the Agilent 2100 Expert Software version B 02 07 or higher turn on the 2100 Bioanalyzer and check communication 3 Prepare the chip samples and ladder as instructed in the reagent kit guide 4 Load the prepared chip into the 2100 Bioanalyzer and start the run within five minutes after preparation 5 Within the instrument context choose the appropriate assay from the drop down list 6 Start the run Enter sample names and comments in the Data and Assay context 7 Verify the results Check that the electropherogram shows a distribution with a peak size between 600 to 800 bp Measure the concentration of the librar
40. y by integrating under the peak A minimum of 500 ng of library is required for hybridization SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries 31 32 2 Sample Preparation Step 10 Assess quality and quantity with 2100 Bioanalyzer FU Pre_Capture_Amoplification 1204 Varrena 50 300 500 700 1000 2000 10380 bp Figure 3 Analysis of amplified prepped library DNA using a Bioanalyzer DNA 7500 as say The electropherogram shows fragment sizes that range between 400 bp and 2 kb with a peak height of approximately 700 bp 100 bp SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries CAUTION SureSelect Target Enrichment System for Roche 454 GS FLX and GS Junior Sequencing Platforms Protocol ee 3 Hybridization Step 1 Hybridize the library 36 Step 2 Prepare magnetic beads 41 Step 3 Hybrid capture with SureSelect 42 Step 4 Purify the sample using Agencourt AMPure XP beads 44 This chapter describes the steps to combine the prepped library with the hybridization reagents blocking agents and the SureSelect capture library The ratio of SureSelect capture library to prepped library is critical for successful capture ee Agilent Technologies 33 3 Hybridization GENOMIC SAMPLE Set of chromosomes SureSelect Target Enrichment System Capture Process q Kit ERE 4 pace AAAS GENOMIC SAMPLE PREPPED SureSelect H
41. ybridization that remained after 24 hour incubation Keep the PCR plate or tubes at 65 C in the PCR machine while you add the hybridization mixture directly from the thermal cycler to the bead solution Invert the tube to mix 3 to 5 times Excessive evaporation such as when less than 20 uL remains after hybridization can indicate suboptimal capture performance See Table 29 on page 58 for tips to minimize evaporation Incubate the hybrid capture bead solution on a Nutator or equivalent for 30 minutes at room temperature Make sure the sample is properly mixing in the tube 4 Briefly spin in a centrifuge Separate the beads and buffer on a magnetic separator and remove the supernatant Resuspend the beads in 500 uL of SureSelect Wash 1 by mixing on a vortex mixer for 5 seconds Incubate the samples for 15 minutes at room temperature Occasionally mix on a vortex mixer Briefly spin in a centrifuge Separate the beads and buffer on a magnetic separator and remove the supernatant SureSelect Target Enrichment System for Roche 454 Titanium Sequencing Libraries Hybridization 3 10 Wash the beads a Resuspend the beads in 500 uL of 65 C prewarmed SureSelect Wash 2 and mix on a vortex mixer for 5 seconds to resuspend the beads b Incubate the samples for 10 minutes at 65 C in a recirculating water bath heat block or equivalent Occasionally mix on a vortex mixer Do not use a tissue incubator It cannot properly mainta
Download Pdf Manuals
Related Search