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HaloPlex HS Target Enrichment System for Illumina Sequencing

1. u c c01 c02 c03 c04 C05 C06 D D01 D02 D03 D04 D05 D06 u E E01 E02 E03 E04 E05 E06 u F F01 F02 F03 F04 F05 F06 _ G G01 G02 G03 G04 G05 G06 _ H H01 H02 H03 H04 H05 H06 Table 15 Plate map for HaloPlex HS Indexing Primers A01 through H12 provided in orange plate with 96 reaction kits 1 2 3 4 5 6 7 8 9 10 11 12 A A0 A02 A03 A04 A05 A06 A07 A08 A09 A10 A11 A12 B B01 B02 B03 B04 B05 B06 B07 B08 B09 B10 B11 B12 c c01 c02 c03 C04 C05 C06 C07 C08 c09 C10 C11 C12 D D01 D02 D03 D04 D05 D06 D07 D08 D09 D10 D11 D12 E E01 E02 E03 E04 E05 E06 E07 E08 E09 E10 E11 E12 F F01 F02 F03 F04 F05 F06 F07 F08 F09 F10 F11 F12 G G01 G02 G03 G04 G05 G06 G07 G08 G09 G10 G11 G12 H H01 H02 H03 H04 H05 H06 H07 H08 H09 H10 H11 H12 52 HaloPlex HS Target Enrichment System ILM Nucleotide Sequences of HaloPlex HS Indexes Reference The nucleotide sequence of the 8 nucleotide index portion of each HaloPlex HS Indexing Primer is provided in the table below Table 16 HaloPlex HS Indexes Sequence Sequence Sequence Sequence ATGCCTAA GAATCTGA C01 AACGTGAT D01 CACTTCGA E01 GCCAAGAC F01 GACTAGTA G01 ATTGGCTC H01 GATGAATC A02 AGCAGGAA B02 GAGCTGAA C02 AAACATCG D02 GAGTTAGC E02 CGAACTTA l F02 GATAGACA G02 AAGGACAC H02 GACAGTGC A03 ATCATTCC B03 GCCACATA c03 ACCACTGT D03 CTGG
2. 2200 TapeStation Platform and Consumables 2200 TapeStation High Sensitivity D1000 ScreenTape High Sensitivity D1000 Reagents 2100 Bioanalyzer Platform and Consumables 2100 Bioanalyzer Laptop Bundle 2100 Bioanalyzer Electrophoresis Set High Sensitivity DNA Kit Gel Electrophoresis Platform and Consumables XCell SureLock Mini cell Novex 6 Polyacrylamide TBE Pre cast Gels Novex TBE Running Buffer 5X Novex High density TBE Sample Buffer 5X GelRed Nucleic Acid Stain 3X in water DNA molecular weight markers UV transilluminator Vendor and part number Agilent p n G2964AA or G2965AA Agilent p n 5067 5584 Agilent p n 5067 5585 Agilent p n G2943CA Agilent p n G2947CA Agilent p n 5067 4626 Life Technologies p n E10001 Life Technologies p n EC62655B0X Life Technologies p n LC6675 Life Technologies p n LC6678 Biotium p n 41001 General laboratory supplier General laboratory supplier HaloPlex HS Target Enrichment System ILM HaloPlex HS Target Enrichment System Protocol ee 2 7 e Sample Preparation a Run Size Considerations 17 Run Time Considerations 17 Step 1 Digest genomic DNA with restriction enzymes 18 Step 2 Hybridize digested DNA to HaloPlex HS or ClearSeq HS probes 28 Step 3 Remove the hybridization buffer 31 Step 4 Ligate the circularized fragments 33 Step 5 Capture the target DNA 35 Step 6 PCR amplify the captured target library 38 Step 7 Purify the amplified target library 40 S
3. No 6 627 424 and EP Pat No 1 283 875 B1 owned by Bio Rad Labora tories Inc Purchase of this product con veys to the buyer the non transferable right to use the purchased amount of the product and components of the product in PCR but not real time PCR in the Research Field including all Applied Research Fields including but not limited to forensics ani mal testing and food testing HaloPlex HS Target Enrichment System ILM Safety Notices CAUTION A CAUTION notice denotes a hazard It calls attention to an operating 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 performed or adhered to could result in personal injury or death Do not proceed beyond a WARNING notice until the indicated conditions are fully understood and met HaloPlex HS Target Enrichment System ILM 3 In this Guide This guide describes an optimized protocol for using the HaloPlex HS target enrichment system to prepare sequencing library samples for Illumina paired end multiplexed sequencing platforms 1 Before You Begin This chapter contains information such as procedural notes safety information required reagents and e
4. 100 ul multichannel pipette set to 40 ul 5 Incubate the samples for 2 minutes at room temperature to elute the DNA samples from the beads 6 Spin briefly to collect the liquid then place the tubes in the magnetic plate Wait for the solution to clear approximately 5 minutes 7 For each sample transfer 47 5 ul of the cleared supernatant to a fresh tube Pipette slowly to minimize transfer of beads with the supernatant HaloPlex HS Target Enrichment System ILM 33 2 34 Sample Preparation 8 Add 2 5 ul of HS DNA Ligase to each sample tube Mix by gentle vortexing and then spin briefly to collect the liquid 9 Incubate the tubes in a thermal cycler at 55 C for 10 minutes using a heated lid Do not include a low temperature hold step in the thermal cycler program following the 10 minute incubation During the 10 minute incubation prepare the following components for later protocol steps e Dynabeads MyOne Streptavidin T1 magnetic beads prepare as described in step 1 and step 2 on page 35 e Wash 1 Mix prepare as described in step 3 on page 35 HaloPlex HS Target Enrichment System ILM Sample Preparation Step 5 Capture the target DNA In this step the circularized target DNA HaloPlex HS probe hybrids containing biotin are captured on streptavidin beads 1 Vigorously resuspend the Dynabeads MyOne Streptavidin T1 magnetic beads on a vortex mixer The magnetic beads settle during storage 2 For each sam
5. degenerate molecular barcode i5 requires a 10 nt index read For complete index sequence information see Table 16 on page 53 Before aligning reads to the reference genome trim the reads from Illumina adaptor sequences See page 48 for information on Agilent s SureCall data analysis software which may be used for this task If you are not using SureCall software for analysis you must manually trim the first base from Read 2 since this base originates from non target DNA HaloPlex HS Target Enrichment System ILM Sample Preparation 2 Setting up a custom Sample Sheet For each HaloPlex HS sequencing run generate a custom Sample Sheet with content based on the example shown in Figure 9 according to the guidelines below Enter Header data appropriate for your run and Sample data in columns 1 5 appropriate for each of your samples e In column 6 enter the specific HaloPlex HS Index sequence used for each individual sample See Table 16 on page 53 for nucleotide sequences of the HaloPlex HS indexes In column 8 enter text NNNNNNNNNN for all samples to represent the degenerate 10 nucleotide molecular barcode used to tag each fragment in the hybridization step Header Investigator Name NN Project Name Sequencing Project amp Experiment Name Experiment 1 Date 1812015 Workflow GenerateFASTQ Assay HaloPlex Chemistry Default Reads 151 151 Settings OnlyGenerateFASTQ 1 Data Sample_ID Sample_Nam
6. is available to simplify the sequencing data analysis workflow after HaloPlex HS target enrichment To learn more about this resource and download the SureCall software free of charge visit www agilent com genomics surecall HaloPlex HS Target Enrichment System ILM HaloPlex HS Target Enrichment System Protocol ee 3 Reference Kit Contents 50 Nucleotide Sequences of HaloPlex HS Indexes 53 phe Agilent Technologies 49 3 Reference Kit Contents The HaloPlex HS Target Enrichment System is supplied using the part numbers listed below Table 12 HaloPlex HS and ClearSeq HS Target Enrichment System Kit Part Numbers Design Type Reaction HaloPlex HS Target Enrichment Number System ILM Store at 20 C Custom 1 500 kb up to 20 000 probes ILMFST 48 Reactions 5190 7835 OR 5190 7837 96 Reactions 5190 7847 OR 5190 7849 Custom 0 5 2 5 Mb OR lt 0 5 Mb with gt 20 000 probes ILM 48 Reactions 5190 7839 OR 5190 7841 96 Reactions 5190 7851 OR 5190 7853 Custom 2 6 Mb 5 Mb ILM 48 Reactions 5190 7843 OR 5190 7845 96 Reactions 5190 7855 OR 5190 7857 ClearSeq Cancer HS ILM 16 Reactions G9933A 96 Reactions G9933B ClearSeq Cardiomyopathy HS ILM 16 Reactions G9943A 96 Reactions G9943B ClearSeq ICCG HS ILM 48 Reactions 5190 9182 96 Reactions 5190 9200 ClearSeq Connective Disorder HS ILM 48 Reactions 5190 9186 96 Reactions 5190 9220 ClearSeq Arrhythmia HS ILM 48 Reactions 5190 9180 96 Reactions 5190 9198 Cle
7. samples briefly then place the tubes in the magnetic plate and remove any residual ethanol with a 10 ul volume pipette 12 Air dry the samples by keeping tubes with open lids at room temperature until the residual ethanol completely evaporates approximately 5 minutes During this step prepare ligation reagents as instructed in step 1 and step 2 on page 33 HaloPlex HS Target Enrichment System ILM Sample Preparation 2 Step 4 Ligate the circularized fragments In this step DNA ligation reagents are added to the circularized hybridization products to close nicks in the probe target DNA hybrids 1 Prepare a 1 mM rATP solution by diluting the provided 10 mM rATP 1 10 with nuclease free water For 12 sample runs combine 1 ul of the provided 10 mM rATP and 9 ul of nuclease free water 2 Prepare a DNA ligation master mix by combining the reagents in the following table Mix the components thoroughly by gentle vortexing then spin the tube briefly Table 8 Preparation of DNA ligation master mix Reagent Volume for 1 Reaction Volume for 12 Reactions includes excess HS Ligation Solution 10 pl 130 ul 1 mM rATP prepared in step 1 0 6 pl 78 pl Nuclease free water 39 4 ul 512 2 pl Total Volume 50 pl 650 pl 3 Remove the DNA sample tubes from the magnetic plate see step 12 on page 32 then add 50 ul of the DNA ligation master mix to each sample 4 Resuspend the beads thoroughly by pipetting up and down 10 times using a
8. three predominant bands at approximately 125 225 and 450 bp These three bands correspond to the 800 bp PCR product derived restriction fragments and precise sizes will differ after digestion in each of the eight RE master mixes In addition to the three predominant bands at approximately 125 225 and 450 bp you may detect additional minor bands in the digested ECD sample lanes Successful digestion is indicated by the appearance of the three predominant bands The presence of additional minor bands with relative abundance similar to the additional bands visible in Figure 3 Figure 4 and Figure 5 does not impact enrichment results It is acceptable for band intensities in digestion reaction B to be slighltly reduced compared to the other digestion reactions 24 HaloPlex HS Target Enrichment System ILM Sample Preparation 2 Option 1 Validation by 2100 Bioanalyzer analysis Use a High Sensitivity DNA Kit p n 5067 4626 and the 2100 Bioanalyzer system with 2100 Expert Software version B 02 07 or higher required to run the High Sensitivity Kit See the reagent kit guide for general Bioanalyzer system setup instructions e Prepare an undigested DNA gel control by combining 1 ul of the Enrichment Control DNA stock solution and 1 ul of nuclease free water Prepare the chip samples and ladder as instructed in the reagent kit guide using 1 ul of each ECD sample and undigested DNA control dilution for the analysis e When loading
9. 0 Bioanalyzer with 2100 Expert Software version B 02 07 or higher required to run the High Sensitivity Kit See the reagent kit guide for general Bioanalyzer instrument and assay setup instructions 1 Prepare the chip samples and ladder as instructed in the reagent kit guide using 1 ul of enriched library sample for the analysis 2 Load the prepared chip into the 2100 Bioanalyzer and start the run within five minutes after preparation 3 Analyze the electropherogram for each sample using the analysis guidelines on page 45 See Figure 7 for a sample Bioanalyzer system electropherogram If the concentration determined by Bioanalyzer analysis is gt 10 ng ul repeat the analysis using a 1 10 dilution of the sample Dilute 1 yl of the sample in 9 ul of 10 mM Tris 1 mM EDTA and then mix well by vortexing at 2000 rpm on the IKA vortex supplied with the Bioanalyzer before analyzing the diluted sample 150 100 35 150 300 500 1000 10380 bp Figure 7 Validation of HaloPlex HS enrichment by 2100 Bioanalyzer analysis HaloPlex HS Target Enrichment System ILM 43 2 Sample Preparation CAUTION Option 2 Analysis using the 2200 TapeStation Use a High Sensitivity D1000 ScreenTape and reagent kit For more information to do this step see the Agilent 2200 TapeStation User Manual at www genomics agilent com 1 Prepare the TapeStation samples as instructed in the 2200 TapeStation User Manual Use 2 ul of each enriched library sample
10. 100 ul of PCR Master Mix to each sample tube Mix by pipetting up and down until the beads are in a homogeneous suspension 38 HaloPlex HS Target Enrichment System ILM Sample Preparation 2 Place the amplification reaction tubes in a thermal cycler and run the program in Table 10 using a heated lid The optimal amplification cycle number varies for each HaloPlex HS or ClearSeq HS probe design Consult the Certificate of Analysis provided with your kit for the PCR cycling recommendation for your probe Table 10 HaloPlex HS post capture DNA amplification PCR program Segment Number of Cycles Temperature Time 1 1 98 C 2 minutes 2 Obtain cycle number 98 C 30 seconds from Certificate of 2 Analysis 60 C 30 seconds 72 C 1 minute 3 1 72 C 10 minutes 4 1 8 C Hold During amplification remove the Agencourt AMPure XP magnetic beads from 4 C storage and allow the beads to come to room temperature for use on page 40 When the PCR program is complete briefly spin the tubes in a desktop centrifuge and then transfer the tubes to the magnetic plate Wait for the solution to clear then remove 40 ul of each PCR reaction sample to a fresh 0 2 ml tube for purification Store the remaining volume of each sample at 20 C for troubleshooting Stopping Point If you do not continue to the next step PCR products may be stored at 20 C for up to 72 hours or at 8 C overnight For best results however purify PCR products as soon as
11. CATA E03 ACCTCCAA F03 GCGAGTAA ACTATGCA CGGATTGC AACTCACC GCTAACGA C04 CAGATCTG D04 ATCCTGTA E04 CTGTAGCC F04 GCTCGGTA G04 ACACGACC H04 AGTCACTA A05 AACGCTTA B05 GGAGAACA C05 CATCAAGT D05 AAGGTACA E05 CGCTGATC F05 GGTGCGAA G05 CCTAATCC H05 CTGAGCCA A06 AGCCATGC B06 GTACGCAA C06 AGTACAAG D06 ACATTGGC E06 ATTGAGGA F06 GTCGTAGA AGAGTCAA CCGACAAC HaloPlex HS Target Enrichment System ILM ACGTATCA GTCTGTCA CTAAGGTC CGACACAC CCGTGAGA GTGTTCTA CAATGGAA AGCACCTC CAGCGTTA TAGGATGA AGTGGTCA ACAGCAGA CATACCAA TATCAGCA ATAGCGAC ACGCTCGA CTCAATGA TCCGTCTA AGGCTAAC CCATCCTC AGATGTAC TCTTCACA CCGAAGTA CGCATACA C10 D10 E10 F10 G10 H10 All B11 C11 D11 E11 F11 G11 H11 A12 B12 C12 D12 E12 F12 G12 AATGTTGC TGAAGAGA AGATCGCA AAGAGATC CAACCACA TGGAACAA CCTCTATC ACAGATTC CCAGTTCA TGGCTTCA CGACTGGA CAAGACTA CCTCCTGA TGGTGGTA AACAACCA AATCCGTC CAAGGAGC TTCACGCA CACCTTAC AAGACGGA ACACAGAA GAACAGGC AACCGAGA ACAAGCTA www agilent com In This Book This guide contains information to run the HaloPlex HS Target Enrichment System protocol for the Illumina sequencing platform Agilent Technologies Inc 2015 Version BO June 2015 G9931 90000 oe Agilent Technologies
12. HaloPlex HS Target Enrichment System For Illumina Sequencing Version BO June 2015 For Research Use Only Not for use in diagnostic procedures SEE Agilent Technologies Notices Agilent Technologies Inc 2015 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 G9931 90000 Edition Version BO June 2015 Printed in USA Agilent Technologies Inc 5301 Stevens Creek Blvd Santa Clara CA 95051 USA 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 For Research Use Only Not for use in diag nostic procedures Warranty The material contained in this document is provided 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 no
13. accurate results e Load the sample tube strip the High Sensitivity D1000 ScreenTape and loading tips into the 2200 TapeStation as instructed in the 2200 TapeStation User Manual Start the run See Figure 4 for sample TapeStation electrophoresis results MW bp 1500 1000 1 _ 200 _ me EEE 500 5 200 s a un 100 ee ee ee ee ee w Figure 4 Validation of restriction digestion by 2200 TapeStation analysis Lane 1 26 High Sensitivity Ladder Lane 2 Undigested Enrichment Control DNA Lanes 3 10 ECD digestion reactions A H HaloPlex HS Target Enrichment System ILM Sample Preparation 2 Option 3 Validation by gel electrophoresis Use a Novex 6 polyacrylamide TBE pre cast gel and 1X Novex TBE Running Buffer For more information to do this step consult the manufacturer s recommendations e Prepare an undigested DNA gel control by combining 0 8 ul of the Enrichment Control DNA stock solution and 3 2 ul of nuclease free water e Add 1 ul of Novex Hi Density TBE Sample Buffer 5X to each 4 ul ECD sample e Load 5 ul of each sample on the gel In one or more adjacent lanes load 200 ng of a 50 bp DNA ladder e Run the gel at 210 V for approximately 15 minutes e Stain the gel in 3X GelRed Nucleic Acid Stain for 10 minutes and visualize bands under UV radiation See Figure 5 for sample gel results Figure 5 Validation o
14. ar appropriate personal protective equipment PPE when working in the laboratory 10 HaloPlex HS Target Enrichment System ILM Before You Begin 1 Required Reagents Table 1 Required Reagents for HaloPlex HS Target Enrichment Description Vendor and part number HaloPlex HS Target Enrichment System Kit or Select the appropriate kit for your ClearSeq HS Target Enrichment System Kit probe design from Table 2 Nuclease free Water not DEPC treated Ambion Cat AM9930 Agencourt AMPure XP Kit Beckman Coulter Genomics 5 ml p n A63880 60 ml p n A63881 Dynabeads MyOne Streptavidin T1 Life Technologies 2mL p n 65601 10 mL p n 65602 100 mL p n 65603 10 M NaOH molecular biology grade Sigma p n 72068 10 mM Tris HCl pH 8 5 General laboratory supplier 100 Ethanol molecular biology grade Sigma Aldrich p n E7023 Quant iT dsDNA BR Assay Kit for use with the Qubit fluorometer 100 assays 2 1000 ng Life Technologies p n 032850 500 assays 2 1000 ng Life Technologies p n 032853 HaloPlex HS Target Enrichment System ILM 11 1 12 Before You Begin Before ordering a HaloPlex HS Target Enrichment System Reagent Kit use Agilent s SureDesign tool at www agilent com genomics suredesign to design a custom HaloPlex HS probe or to select a pre designed ClearSeq HS disease research probe Reagent kit ordering information is supplied as part of the SureDesign process and is summarized in Table 2 below Table 2 _HaloPlex HS Target Enrichmen
15. arSeq Noonan Syndrome HS ILM 48 Reactions 5190 9188 96 Reactions 5190 9222 ClearSeq Chromosome X HS ILM 48 Reactions 5190 9184 96 Reactions 5190 9218 See Table 13 for list of included reagents t Part number 5190 7835 5190 7847 5190 7839 5190 7851 5190 7843 or 5190 7855 is provided for the first order of a specific HaloPlex HS Custom Probe design Re order kits containing previously purchased Custom Probe designs include part num ber 5190 7837 5190 7849 5190 7841 5190 7853 5190 7845 or 5190 7857 50 HaloPlex HS Target Enrichment System ILM Reference 3 The contents of the HaloPlex HS Target Enrichment kits are detailed in the table below Table 13 HaloPlex HS Target Enrichment System Kit Contents Included Reagents RE Buffer BSA Solution Enzyme Strip 1 Enzyme Strip 2 Enrichment Control DNA Hybridization Solution HS Hybridization Stop Solution HS Ligation Solution HS DNA Ligase 10 mM rATP HS Wash 1 Solution HS Wash 2 Solution HS Capture Solution HS Elution Buffer Primer 1 Primer 2 Herculase Il Fusion DNA Polymerase Herculase II Reaction Buffer 100 mM dNTP Mix HaloPlex HS or ClearSeq HS Probe HaloPlex HS Indexing Primers 16 Reaction Kit tube with clear cap tube with clear cap 8 well strip with green label 8 well strip with red label tube with orange cap tube with clear cap tube with clear cap tube with black cap tube with green cap tube with clear cap bottle bottle t
16. ch corresponding tube A to H of the same Restriction Enzyme Master Mix Strip Reagent Volume for 1 Reaction Volume for 12 Reactions includes excess RE Enzymes from 0 35 ul 4 9 ul Red Enzyme Strip e Mix by gentle vortexing and then spin briefly f Keep the Restriction Enzyme Master Mix Strip on ice until it is used in step 4 4 Aliquot the Restriction Enzyme Master Mixes to the rows of a 96 well plate to be used as the restriction digest reaction plate a Align the Restriction Enzyme Master Mix Strip prepared in step 3 along the vertical side of a 96 well PCR plate as shown below b Using a multichannel pipette carefully distribute 3 5 ul of each RE master mix row wise into each well of the plate For runs with gt 12 samples continue distributing 3 5 ul from the same RE Master Mix strip row wise into each well of the additional plates Visually inspect pipette tips for equal volumes before dispensing to the plate s Restriction Digestion Reaction Plate C eo AAe eoeevoeeeevneeee KERN EN EEEEE ME ee A O O O Oe Oe OO C eeeeeeee ee e 2e2000000808080 v oe o u uuouo 12 3 45 6 7 8 9 10 11 12 RE Master Mix Strip TOATmTMVIOWS HaloPlex HS Target Enrichment System ILM 21 2 22 Sample Preparation Each row of the 96 well plate now contains 3 5 ul per well of the same restriction enzyme combination 5 Aliquot DNA samples into the 96 well Restric
17. ction Volume for 12 Reactions includes excess HS Wash 1 Solution 90 ul 1170 ul 1 M NaOH prepared in step a 10 pl 130 ul Total Volume 100 pl 1300 pl 4 Remove the ligation reactions from the thermal cycler and immediately add 40 ul of the HS Capture Solution streptavidin bead mixture prepared in step 2 to each 50 ul ligation reaction When adding beads to the ligation reactions visually inspect the bead preparation to ensure a homogeneous suspension with no aggregated bead mass at the bottom of the tube If aggregation is present thoroughly resuspend the beads by vortexing and pipetting up and down before use 5 Incubate samples for 15 minutes at room temperature with continuous shaking Make sure the samples are properly mixing in the wells during the 15 minute incubation During the 15 minute incubation prepare the PCR Master Mix as described on page 38 6 Briefly spin the tubes in a desktop centrifuge and then transfer the tubes to the magnetic plate 7 Wait for the solution to clear then remove and discard the supernatant using a pipette 36 HaloPlex HS Target Enrichment System ILM Sample Preparation 8 Wash the bead bound samples Remove the capture reaction tubes from the magnetic plate and add 100 ul of the Wash 1 Mix prepared in step 3 to each tube Resuspend the beads thoroughly by pipetting up and down 10 times using a 100 ul multichannel pipette set to 80 ul then briefly spin the tubes in a deskt
18. diluted with 2 ul of High Sensitivity D1000 sample buffer in separate wells of a tube strip for the analysis Make sure that you thoroughly mix the combined DNA sample and High Sensitivity D1000 sample buffer on a vortex mixer for 5 seconds for accurate results 44 2 Load the sample tube strip the High Sensitivity D1000 ScreenTape and loading tips into the 2200 TapeStation as instructed in the 2200 TapeStation User Manual Start the run 3 Analyze the electropherogram for each sample using the analysis guidelines on page 45 See Figure 8 for a sample TapeStation electropherogram Sample Intensity FU Figure 8 6000 5000 4000 3000 2000 1000 n A ie an N x Size bp 1 000 o sl s s alela g 23 slal 8 Validation of HaloPlex HS enrichment by 2200 TapeStation analysis HaloPlex HS Target Enrichment System ILM Sample Preparation 2 Analysis of Electropherogram Results e Check that the electropherogram shows a peak fragment size between approximately 225 to 525 bp e Determine the concentration of enriched target DNA in the sample by integration under the peak between 175 and 625 bp Peaks at lt 175 bp may be observed but should be excluded from quantitation e Some designs may generate a peak at about 140 bp This peak is associated with an adaptor dimer product which will cluster and generate sequence that does not map to the genome If the m
19. dization Stop Solution and 80 ul of the homogeneous AMPure XP bead suspension Mix well until the bead suspension mixture appears homogeneous Pipette the viscous HS Hybridization Stop Solution slowly to ensure that the full volume is aspirated and dispensed Verify that any residual volume of this solution or mixture containing the solution has been dispensed from the pipette tip 4 Add 100 ul of the homogeneous stop solution bead suspension prepared in step 3 to each 100 ul hybridized library sample Mix by pipetting up and down 10 times using a 200 ul pipette set to 150 ul 5 Incubate samples for 5 minutes at room temperature with continuous shaking at 1300 rpm Make sure the samples are properly mixing in the wells during the 5 minute incubation 6 Spin briefly to collect the liquid then place the tubes in the magnetic plate Wait for the solution to clear approximately 5 minutes 7 Keep the tubes in the magnetic plate Carefully remove and discard the cleared solution from each tube Do not touch the beads while removing the solution 8 Continue to keep the tubes in the magnetic plate while you add 200 ul of 70 ethanol into the tubes Use fresh 70 ethanol for optimal results HaloPlex HS Target Enrichment System ILM 31 2 32 Sample Preparation 9 Wait for 1 minute to allow any disturbed beads to settle then remove and discard the ethanol 10 Repeat step 8 and step 9 once for a total of two washes 11 Spin the
20. e Sample_Plate Sample_Well Sample_Project index I _Index_ID index 15_Index_ID Sample 1 Samplet Platel 1 01 HaloPlex AACGTGAT S00 NNNNNNNNNN molbe Sample 2 Sample2 Platel 1 01 HaloPlex AAACATCG 5002 NNNNNNNNNN molbe Figure 9 Sample sheet for HaloPlex HS library sample sequencing HaloPlex HS Target Enrichment System ILM 47 2 48 Sample Preparation Additional MiSeq platform sequencing run setup requirements Before the first use of the MiSeq instrument for HaloPlex HS library sequencing you must adjust the MiSeq Reporter settings to generate FASTQ files for index reads Once changed this setting is retained for future runs To change this setting open the file MiSeq Reporter exe config Under the lt appSettings gt tag add lt add key CreateFastqForlndexReads value 1 gt You must restart the instrument for this setting change to take effect Additional HiSeq platform sequencing run setup requirements Set up sequencing runs using the Cycles settings shown in Table 11 Cycle number settings can be specified on the Run Configuration screen of the instrument control software interface after choosing Custom from the index type selection buttons Table 11 HiSeq platform Run Configuration screen Cycle Number settings Run Segment Cycle Number Read 1 100 Index 1 i7 8 Index 2 i5 10 Read 2 100 Settings apply to v3 0 SBS chemistry Sequence analysis resources Agilent s SureCall data analysis software
21. e magnetic plate while you add 200 ul of 70 ethanol into the tubes Use fresh 70 ethanol for optimal results 10 Wait for 1 minute to allow any disturbed beads to settle then remove the ethanol using a 200 ul pipette set to 200 ul 11 Repeat step 9 and step 10 once for a total of two washes 12 Spin the samples briefly then place the tubes in the magnetic plate and remove any residual ethanol with a 10 ul volume pipette 13 Air dry the samples at room temperature with tube lids open until the residual ethanol completely evaporates HaloPlex HS Target Enrichment System ILM Sample Preparation 2 Make sure all ethanol has evaporated before continuing 14 Remove tubes from the magnetic plate and add 45 ul of HS Elution Buffer to each sample 15 Mix thoroughly by pipetting up and down 10 times using a 100 ul pipette set to 30 ul 16 Incubate the tubes at room temperature for 2 minutes to allow DNA elution 17 Put the tubes in the magnetic plate and leave for 2 minutes or until the solution is clear 18 Remove the cleared supernatant approximately 40 ul to a fresh tube You can discard the beads at this time Stopping Point If you do not continue to the next step samples may be stored at 20 C for long term storage up to one year Avoid subjecting the stored DNA samples to multiple freeze thaw cycles HaloPlex HS Target Enrichment System ILM 41 2 Sample Preparation Step 8 Validate enrichment and quantify e
22. e size distribution of DNA in each DNA preparation by gel electrophoresis Any smearing below 2 5 kb indicates sample degradation This protocol is compatible with FFPE derived DNA samples but enrichment performance and sequencing results may be impacted depending on the extent of DNA fragmentation in each FFPE sample 1 Use the Qubit dsDNA BR Assay or PicoGreen staining kit to determine the concentration of your gDNA samples Follow the manufacturers instructions for the kits and instruments Use a fluorometry based DNA quantitation method such as Qubit fluorometry or PicoGreen staining to accurately quantify the DNA starting material In the protocol below 50 ng genomic DNA is split among eight different restriction digests with additional excess DNA included to allow for pipetting losses Using lt 50 ng DNA in the enrichment protocol can result in low yield and can potentiate rare allele dropouts 2 Prepare the DNA samples for the run For 12 reaction runs prepare 11 gDNA samples and one Enrichment Control DNA sample a Dilute each gDNA sample to concentration of 1 8 ng ul in 10 mM Tris buffer pH 8 5 b In separate wells of a 0 2 ml tube strip dispense 32 ul of each SDNA sample prepared in step a Store the DNA sample strip on ice c Ina separate well of the DNA sample strip dispense 32 ul of the supplied Enrichment Control DNA ECD Store on ice Although specific instructions are provided for the typical 12 sample run si
23. es for four runs of 12 samples each for a total of 48 samples When processing samples using runs with fewer than 12 samples some reagents may be depleted before 48 samples are run A 16 reaction kit contains enough reagents to prepare master mix for one run of 16 samples When processing samples using runs with fewer than 16 samples some reagents may be depleted before 16 samples are run Run Time Considerations Before you begin refer to the Certificate of Analysis provided with your kit to determine the hybridization duration appropriate for your design After reviewing the duration of this and other steps in the protocol plan the start time for your experiment accordingly Designs containing lt 20 000 probes use a 2 hour hybridization time and DNA digestion through PCR steps see Figure 1 are typically run in the same day Designs containing gt 20 000 probes use a 16 hour hybridization time which is typically completed overnight with the DNA digestion step started in the afternoon HaloPlex HS Target Enrichment System ILM 17 2 Sample Preparation Step 1 Digest genomic DNA with restriction enzymes In this step SDNA samples are digested by 16 different restriction enzymes to create a library of gDNA restriction fragments Successful enrichment using the protocol in this guide requires high quality DNA samples Before you begin verify that the genomic DNA samples have an OD 260 280 ratio ranging from 1 8 to 2 0 Verify th
24. f restriction digestion by gel electrophoresis Lanes 1 8 ECD di gestion reactions A H Lane 9 Undigested Enrichment Control DNA Lane 10 25 bp DNA ladder Stopping Point If you do not continue to the next step samples may be stored at 20 C for long term storage There are no more long term stopping points until after the PCR amplification step on page 39 HaloPlex HS Target Enrichment System ILM 27 2 28 Sample Preparation Step 2 Hybridize digested DNA to HaloPlex HS or ClearSeq HS probes In this step the collection of gDNA restriction fragments is hybridized to the HaloPlex HS or ClearSeq HS probe library During the hybridization process molecular barcodes and Illumina sequencing motifs including index sequences are incorporated into the targeted fragments HaloPlex HS and ClearSeq HS probes are designed to hybridize selectively to fragments originating from target regions of the genome and to direct circularization of the targeted DNA fragments The duration of the hybridization reaction is determined by the probe density of your design Refer to the Certificate of Analysis provided with your kit to determine the hybridization conditions appropriate for your design 1 Prepare a Hybridization Master Mix by combining the reagents in Table 6 Mix well by gentle vortexing then spin the tube briefly Table 6 Hybridization Master Mix Reagent Volume for 1 Reaction Volume for 12 Reactions includes excess Hybridizati
25. get Enrichment System Protocol 1 Before You Begin Procedural Notes 10 Safety Notes 10 Required Reagents 11 Required Equipment 13 Optional Validation Reagents and Equipment 14 Make sure you read and understand the information in this chapter and have the necessary equipment and reagents listed before you start an experiment ee Agilent Technologies 1 Before You Begin Procedural Notes The HaloPlex HS protocol is optimized for digestion of 50 ng of genomic DNA split among 8 different restriction digestion reactions plus excess DNA for pipetting losses Using lower amounts of DNA in the enrichment protocol can adversely affect your results Use a fluorometry based DNA quantitation method such as PicoGreen stain or Qubit fluorometry to quantify the DNA starting material e Always keep pre amplification and post amplification DNA samples in separate work areas Perform the enrichment procedure in the pre amplification area Open and store the amplified enriched DNA samples only in the post amplification area Possible stopping points where DNA samples may be stored between steps are marked in the protocol Store the samples at 20 C but do not subject the samples to multiple freeze thaw cycles e Ensure that master mixes are thoroughly mixed by pipetting up and down or by gentle vortexing before distributing to the samples e In general follow Biosafety Level 1 BL1 safety rules Safety Notes CAUTION e We
26. he end of the Hybridization incubation remove reagents to be used in upcoming protocol steps from cold storage and allow the solutions to reach the appropriate temperature From 20 C storage remove the HS Hybridization Stop Solution HS Ligation Solution HS Capture Solution HS Wash 1 Solution HS Wash 2 Solution and HS Elution Buffer to room temperature Be sure to bring the HS Hybridization Stop Solution to room temperature before use The high viscosity of this solution impedes accurate pipetting at lower temperatures From 4 C storage remove the Agencourt AMPure XP magnetic beads and the Dynabeads MyOne Streptavidin T1 magnetic beads to room temperature From 20 C storage remove the HS Ligation Solution 10 mM rATP and HS DNA ligase to ice 30 HaloPlex HS Target Enrichment System ILM Sample Preparation 2 Step 3 Remove the hybridization buffer In this step the hybridization buffer is removed in preparation for the ligation step using AMPure XP beads The AMPure XP beads and HS Hybridization Stop Solution must be at room temperature for the purification steps Remove these reagents to room temperature at least 30 minutes before each use 1 Prepare 400 ul of 70 ethanol per sample plus excess for use in step 8 2 Mix the AMPure XP bead suspension well until the suspension appears homogeneous and consistent in color 3 For each sample to be purified prepare a bead mix by combining 20 ul of HS Hybri
27. lex HS Target Enrichment System ILM 19 2 Sample Preparation a Combine the amounts of RE Buffer and BSA Solution indicated in the table below in a 1 5 ml tube Mix by vortexing briefly Reagent Volume for 1 Reaction Volume for 12 Reactions includes excess RE Buffer 24 6 ul 344 ul BSA Solution 0 64 ul 9 ul Total Volume 25 2 pl 353 pl b To begin preparation of the Restriction Enzyme Master Mix Strip dispense the appropriate volume of the RE Buffer BSA mixture to each well of an 8 well strip tube Reagent Volume for 1 Reaction Volume for 12 Reactions includes excess RE Buffer BSA 2 8 pl 39 2 pl mixture It is important to use the restriction enzyme tube strip in the proper orientation when CAUTION preparing the RE Master Mixes as described below The red or green color marker on the tube strip and cap strip is positioned adjacent to well A of each enzyme strip c Using a multichannel pipette add the appropriate volume of each enzyme from the Green Enzyme Strip with green marker aligned with tube A to corresponding tubes A to H of the Restriction Enzyme Master Mix Strip Reagent Volume for 1 Reaction Volume for 12 Reactions includes excess RE Enzymes from 0 35 pl 4 9 ul Green Enzyme Strip 20 HaloPlex HS Target Enrichment System ILM Sample Preparation 2 d Using a multichannel pipette add the appropriate volume of each enzyme from the Red Enzyme Strip with red marker aligned with tube A to ea
28. nriched target DNA Prior to sample pooling and sequencing sample preparation validate enrichment and quantify the enriched target DNA in each library sample by microfluidic analysis using the 2100 Bioanalyzer see page 43 or the 2200 TapeStation see page 44 Expected Results Each amplicon in the prepared library contains one target insert surrounded by sequence motifs required for multiplexed sequencing using the Illumina platform Amplicons include 50 to 500 bp of target DNA insert and 140 bp of sequencing motifs as shown in Figure 6 Molecular Barcode 10 nt y Read 1 gt Index 1 gt Index 2 lt Read2 Sample Index 8 nt Figure 6 Content of HaloPlex HS enriched target amplicons Each amplicon contains one target insert blue surrounded by the Illumina paired end sequencing ele ments black the sample index red the molecular barcode green and the library bridge PCR primers yellow The amplicons should range from 175 to 625 bp in length with the majority of products sized 225 to 525 bp Amplicons in the 175 to 625 bp size range should be included for quantitation of the enriched target DNA in each sample Any spurious DNA products outside of this size range in any sample should be excluded from the target DNA quantitation results 42 HaloPlex HS Target Enrichment System ILM Sample Preparation 2 Option 1 Analysis using the 2100 Bioanalyzer Use a Bioanalyzer High Sensitivity DNA Assay kit and the 210
29. olar fraction of the 140 bp peak is greater than 10 do another round of AMPure purification after pooling samples First pool equimolar amounts of libraries to be multiplexed using concentrations determined for the 175 625 bp peak of each sample Using 40 ul of the pooled libraries purify the DNA using AMPure XP beads according to the protocol on page 40 HaloPlex HS Target Enrichment System ILM 45 2 Sample Preparation Step 9 Pool samples with different indexes for multiplexed sequencing Use the following guidelines to design your sample pooling and sequencing strategy 46 Use the Bioanalyzer or TapeStation measured concentration of 175 625 bp products in each sample to pool equimolar amounts of differentially indexed samples in order to optimize the use of sequencing capacity The final HaloPlex HS enrichment pool is ready for direct sequencing using standard Illumina paired end primers and chemistry on the Illumina HiSeq or MiSeq platform For both platforms set up a custom sample sheet as described on page 47 See page 48 for additional sequencing setup guidelines Use 100 100 bp or 150 150 bp paired end sequencing depending on the selection made during probe design Since the read length affects maximum achievable coverage check the design report to verify read length selected in probe design Set up sequencing runs using the Custom Index setting The sample level index i7 requires an 8 nt index read and the
30. on Solution 344 442 ul HaloPlex HS Probe 5 ul 65 ul Total Volume 39 pl 507 pl 2 Distribute 39 ul of the Hybridization Master Mix to each of 12 0 2 ml tubes 3 Add 5 ul of the appropriate HaloPlex HS Indexing Primer to each tube containing Hybridization Master Mix Be sure to add only one specific Indexing Primer to each hybridization tube using different indexes for each sample to be multiplexed Record the identity of the Indexing Primer added to each tube for later sequence analysis Components needed to incorporate a unique molecular barcode sequence into each target fragment prior to amplification are included in the HaloPlex HS Indexing Primer solutions and do not need to be added separately HaloPlex HS Target Enrichment System ILM Sample Preparation 4 Transfer digested DNA samples from the 96 well Restriction Digest Reaction Plate s directly into the hybridization reaction tubes prepared in step 3 Transfer all eight digestion reactions that correspond to one DNA sample into the appropriate hybridization reaction tube After addition of each individual digest reaction to the hybridization solution mix by pipetting before adding the next digest reaction to ensure complete inactivation of the enzymes CAUTION Do not pool the digestion samples before adding to the hybridization reaction mixture 2 as restriction enzymes are still active and may catalyze inappropriate cleavage events After pooling each 100 ul h
31. op centrifuge Incubate the samples at room temperature for 1 minute Transfer the tubes to the magnetic plate Wait for the solution to clear then remove and discard the supernatant using a pipette Add 150 ul of the provided HS Wash Solution 2 to each tube Resuspend the beads thoroughly by pipetting up and down 10 times then briefly spin the tubes in a desktop centrifuge Transfer the tubes to the magnetic plate Wait for the solution to clear then remove and discard the supernatant using a pipette HaloPlex HS Target Enrichment System ILM 37 2 Sample Preparation Step 6 PCR amplify the captured target library In this step the captured target libraries are amplified by PCR 1 Prepare the PCR master mix on ice by combining the reagents in the following table Table9 Preparation of PCR master mix Reagent Volume for 1 reaction Volume for 12 reactions includes excess Nuclease free water 53 2 ul 691 6 pl Herculase II Reaction Buffer 30 pl 390 ul dNTPs 100 mM 25 mM for each dNTP 0 8 pl 10 4 pl Primer 1 4 ul 52 ul Primer 2 8 ul 104 ul Herculase II Fusion DNA Polymerase 4ul 52 ul Total 100 pl 1300 pl 2 Mix the master mix components by gentle vortexing Store the mixture on ice until it is used in step 3 The PCR Master Mix is typically prepared during the 15 minute capture step on page 36 3 Remove the captured and washed DNA sample tubes from the magnetic plate see step g on page 37 then transfer
32. ple in the run prepare 40 ul 1 Volume of Dynabeads MyOne Streptavidin T1 magnetic beads in HS Capture Solution using the steps below a Transfer the appropriate volume of bead suspension to a 1 5 ml tube Reagent Volume for 1 Reaction Volume for 12 Reactions includes excess 2 Dynabeads MyOne Streptavidin T1 40 ul 520 ul bead suspension b Put the tube into a 1 5 ml tube compatible magnetic rack until the solution has cleared approximately 5 minutes c Carefully remove and discard the cleared supernatant using a pipette d Add an equivalent volume of HS Capture Solution to the beads and resuspend by pipetting up and down Reagent Volume for 1 Reaction Volume for 12 Reactions includes excess HS Capture Solution 40 ul 520 ul 3 Prepare Wash 1 Mix for use on page 37 using the following steps a Prepare 10 ul per sample plus excess of fresh 1 M NaOH for use in step b Prepare the 1 M NaOH solution from a 10 M NaOH stock solution CAUTION Using high quality NaOH is critical for optimal DNA sample quality Do not use stock NaOH solutions that were stored at concentrations below 10 M to prepare the 1 M NaOH solution Keep the 1 M NaOH solution container sealed when not in use especially when processing large numbers of samples per run HaloPlex HS Target Enrichment System ILM 35 2 Sample Preparation b Prepare Wash 1 Mix by combining the reagents in the following table Reagent Volume for 1 Rea
33. possible HaloPlex HS Target Enrichment System ILM 39 2 40 Sample Preparation Step 7 Purify the amplified target library In this step the amplified target DNA is purified using AMPure XP beads 1 Verify that the Agencourt AMPure XP beads have been kept at room temperature for at least 30 minutes Prepare 400 ul of 70 ethanol per sample plus excess for use in step 9 Mix the AMPure XP bead suspension well until the suspension appears homogeneous and consistent in color For each sample to be purified prepare a bead mix by combining 40 ul of nuclease free water and 100 ul of the homogeneous AMPure XP bead suspension Mix well until the bead mix suspension appears homogeneous Add 140 ul of the homogeneous bead suspension prepared in step 4 to each 40 ul amplified library sample Vortex thoroughly Using this bead to sample volume ratio is imperative to ensure optimal purification results Incubate samples for 5 minutes at room temperature with continuous shaking Make sure the samples are properly mixing in the wells during the 5 minute incubation Spin briefly to collect the liquid then place the tubes in the magnetic plate Wait for the solution to clear approximately 5 minutes Keep the tubes in the magnetic plate Carefully remove and discard the cleared solution from each tube using a 200 ul pipette set to 180 ul Do not touch the beads while removing the solution Continue to keep the tubes in th
34. pture uniquely barcoded targets Soe 06 Streptavidin r 4 Amplify enriched fragments by PCR p CR primers om Figure 1 Overall HaloPlex HS target enriched sequencing sample preparation workflow 16 HaloPlex HS Target Enrichment System ILM Sample Preparation 2 Run Size Considerations Kits contain enough reagents for 16 48 or 96 reactions total including control reactions using the provided Enrichment Control DNA ECD Each run that uses independently prepared reagent master mixes should include one ECD control enrichment reaction The following protocol includes volumes appropriate for 12 sample runs When planning a run size different from 12 samples you will need to adjust volumes of components accordingly Calculate the amount of each solution needed for the number of reactions in your run plus 2 reactions excess for the restriction digestion steps and 1 reaction excess for the remaining steps For example for a 16 sample run calculate amounts of each solution by multiplying the single reaction value by 18 for restriction digestion steps and by 17 for hybridization and later steps A 96 reaction kit contains enough reagents to prepare master mixes for eight runs of 12 samples each for a total of 96 samples When processing samples using runs with fewer than 12 samples some reagents may be depleted before 96 samples are run A 48 reaction kit contains enough reagents to prepare master mix
35. quipment that you should read and understand before you start an experiment 2 Sample Preparation This chapter describes the steps of the HaloPlex HS workflow to prepare target enriched sequencing libraries for the Illumina platform 3 Reference This chapter contains reference information including component kit contents and index sequences 4 HaloPlex HS Target Enrichment System ILM What s New in Version BO Updated product labeling statement HaloPlex HS Target Enrichment System ILM HaloPlex HS Target Enrichment System ILM Content Before You Begin Procedural Notes 10 Safety Notes 10 Required Reagents 11 Required Equipment 13 Optional Validation Reagents and Equipment 14 Sample Preparation Run Size Considerations 17 Run Time Considerations 17 Step 1 Digest genomic DNA with restriction enzymes 18 Step 2 Hybridize digested DNA to HaloPlex HS or ClearSeq HS probes 28 Step 3 Remove the hybridization buffer 31 Step 4 Ligate the circularized fragments 33 Step 5 Capture the target DNA 35 Step 6 PCR amplify the captured target library 38 Step 7 Purify the amplified target library 40 Step 8 Validate enrichment and quantify enriched target DNA 42 Step 9 Pool samples with different indexes for multiplexed sequencing 46 Reference Kit Contents 50 Nucleotide Sequences of HaloPlex HS Indexes 53 HaloPlex HS Target Enrichment System ILM HaloPlex HS Target Enrichment System ILM HaloPlex HS Tar
36. samples on the chip load the DNA ladder in the ladder sample well marked on the chip Load the eight ECD digest samples A to H in sample wells 1 to 8 and load the undigested ECD sample in sample well 9 Do not run the undigested ECD control in sample well 1 e Place the prepared chip into the 2100 Bioanalyzer instrument and start the run within five minutes after preparation See Figure 3 for sample Bioanalyzer electrophoresis results N w Fa a a N Oo 2 Figure 3 Validation of restriction digestion by 2100 Bioanalyzer system analysis Lane 1 50 bp DNA ladder Lanes 2 9 ECD digestion reactions A H Lane 10 Undi gested Enrichment Control DNA HaloPlex HS Target Enrichment System ILM 25 2 Sample Preparation Option 2 Validation by 2200 TapeStation analysis Use a High Sensitivity D1000 ScreenTape and reagent kit For more information to do this step see the Agilent 2200 TapeStation User Manual at www genomics agilent com Prepare an undigested DNA gel control by combining 1 ul of the Enrichment Control DNA solution and 1 ul of nuclease free water Prepare the TapeStation samples as instructed in the 2200 TapeStation User Manual Use 2 ul of each ECD sample diluted with 2 ul of High Sensitivity D1000 sample buffer in separate wells of a tube strip for the analysis CAUTION Make sure that you thoroughly mix the combined DNA and High Sensitivity D1000 sample buffer on a vortex mixer for 5 seconds for
37. t System Kits for Illumina Sequencing Part Number HaloPlex HS Probe Design 96 Reactions 48 Reactions 16 Reactions Custom Panel Tier 1 ILMFST G9931B G9931C Custom Panel Tier 2 ILM G9941B G9941C Custom Panel Tier 3 ILM G9951B G9951C ClearSeq Cancer HS ILM G9933B G9933A ClearSeq Cardiomyopathy HS ILM G9943B G9943A ClearSeq ICCG HS ILM 699548 G9954C ClearSeq Connective Disorder HS ILM G9954B G9954C ClearSeq Arrhythmia HS ILM G9954B G9954C ClearSeq Noonan Syndrome HS ILM G9954B G9954C ClearSeq Chromosome X HS ILM G9954B G9954C Tier 1 designs are 1 500 kb and up to 20 000 probes t Tier 2 designs are 0 5 2 5 Mb OR 1 500 kb with gt 20 000 probes t Tier 3 designs are 2 6 Mb 5 Mb Select the appropriate made to order probe option in SureDesign Kits contain enough reagents for 96 48 or 16 reactions total including one or more control reactions using Enrichment Control DNA ECD samples Each run of up to 96 samples should include one ECD control enrichment reaction HaloPlex HS Target Enrichment System ILM Required Equipment Table 3 Before You Begin 1 Required Equipment for HaloPlex HS Target Enrichment Description Thermal Cycler Thermal cycler compatible 96 well plates 8 well strip tubes and caps 12 well strip tubes and caps 96 well plate and strip tube compatible magnetic separator 1 5 ml tube compatible magnetic separator Bench
38. t 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 7202 3 Rights in Commercial Computer Software or Com puter Software Documentation Notice to Purchaser This product is provided under an agree ment between Bio Rad Laboratories and Agilent Technologies Inc and the manu facture use sale or import of this product is subjectto US Pat
39. t this time Restriction enzymes are still active and will catalyze inappropriate cleavage events if DNA samples are pooled before enzyme inactivation DNA samples are pooled during the hybridization step on page 29 upon which restriction enzymes are inactivated by the reaction conditions HaloPlex HS Target Enrichment System ILM 23 2 Sample Preparation 9 Validate the restriction digestion reaction by electrophoretic analysis of the Enrichment Control DNA ECD reactions Keep the Restriction Digest Reaction Plate on ice during validation a Briefly spin the digestion reaction plate to collect the digested DNA at the bottom of each well b Transfer 4 ul of each ECD digestion reaction from wells of the digestion reaction plate to fresh 0 2 ml PCR tubes c Incubate the removed 4 ul samples at 80 C for 5 minutes to inactivate the restriction enzymes d Analyze the prepared samples using microfluidic electrophoresis on the 2100 Bioanalyzer see page 25 or on the 2200 TapeStation see page 26 or by gel electrophoresis see page 27 The ECD sample contains genomic DNA mixed with an 800 bp PCR product that contains restriction sites for all the enzymes used in the digestion protocol When analyzing validation results the undigested control should have gDNA bands at gt 2 5 kbp and a PCR product band at 800 bp Each of the eight digested ECD samples should have a smear of gDNA restriction fragments between 100 and 2500 bp overlaid with
40. tep 8 Validate enrichment and quantify enriched target DNA 42 Step 9 Pool samples with different indexes for multiplexed sequencing 46 This section contains instructions for gDNA library target enrichment for sequence analysis using the Illumina platform For each sample to be sequenced an individual target enriched indexed library is prepared The target region can vary from 1 kb to 5 Mb Custom HaloPlex HS probes must be designed before purchasing the kit using Agilent s SureDesign tool at www agilent com genomics suredesign The HaloPlex HS Target Enrichment System amplifies thousands of targets in the same reaction incorporating standard Illumina paired end sequencing motifs in the process During hybridization each sample can be uniquely indexed allowing for pooling of up to 96 samples per sequencing lane The indexing primers incorporated during hybridization also include degenerate molecular barcode sequences allowing tracking of individual target amplicons during sequence analysis See Figure 1 for a summary of the overall HaloPlex HS target enrichment workflow SEE Agilent Technologies 15 2 Sample Preparation r Digest and denature sample DNA Target Region Qo Hybridize probe library to DNA targets Biotin sm Q Sequencing primer motif Index Bridge or emulsion PCR primer Target amp complementary probe sequence Molecular barcode oO Ligate and ca
41. tion Digest Reaction Plate s a Align the DNA sample strip prepared in step 2 containing 11 gDNA samples and the ECD sample along the horizontal side of the digestion reaction plate as shown below gDNA Samples 1 11 1 8 ng ul S1 2 S3 S4 S5 S6 S7 S8 S9 S10 S11 ECD 35ulperwell 4 4 4 FF 4 HH F ZzZoOnTmMOoouUu gt 123 45 6 7 8 9 10 11 12 b Carefully distribute 3 5 ul of DNA samples column wise into each well of the digestion reaction plate If using a multichannel pipette visually inspect pipette tips for equal volumes before dispensing Change tips after pipetting the DNA samples into each digestion reaction mix to prevent cross contamination of restriction enzymes c Seal the plate thoroughly with adhesive plastic film 6 Carefully vortex the plate to mix the digestion reactions 7 Briefly spin the plate in a plate centrifuge Wells of the prepared 96 well plate now contain complete 7 ul restriction digestion reactions In this format each column corresponds to one DNA sample digested in eight different restriction reactions HaloPlex HS Target Enrichment System ILM Sample Preparation 2 8 Place the Restriction Digest Reaction Plate in a thermal cycler and run the program in Table 5 using a heated lid Table5 Thermal cycler program for HaloPlex HS restriction digestion Step Temperature Time Step 1 37 C 30 minutes Step 2 4 C Hold Do not pool the eight restriction digests for a single DNA sample a
42. top microcentrifuge Benchtop plate centrifuge Multichannel pipettes 10 uL and 100 uL volume P10 P20 P200 and P1000 pipettes Adhesive seals for 96 well PCR plates Qubit 2 0 Fluorometer Qubit assay tubes Ice bucket Vortex mixer Vendor and part number Agilent SureCycler 8800 p n G8800A and 96 well plate module p n G8810A or equivalent thermal cycler and accessories Agilent p n 410088 for SureCycler 8800 or see manufacturer s recommendations Agilent p n 410092 strip tubes and Agilent p n 410096 strip tube caps Agilent p n 410082 strip tubes and Agilent p n 410086 strip tube caps DynaMag 96 Side magnet Life Technologies p n 12331D or equivalent DynaMag 2 magnet Life Technologies p n 12321D or equivalent VWR p n 93000 196 or equivalent Labnet International MPS1000 Mini Plate Spinner p n C1000 or equivalent Pipetman or equivalent Pipetman P10 P20 P200 P1000 or equivalent Agilent p n 410186 or equivalent Life Technologies p n 032866 Life Technologies p n 032856 General laboratory supplier IKA Vortex 4 digital vortex p n 4050100 or equivalent compatible with 0 2 ml tubes HaloPlex HS Target Enrichment System ILM Thermal cycler must have a maximum reaction volume specification of at least 100 uL and must be 13 1 14 Before You Begin Optional Validation Reagents and Equipment Table4 Reagents and Equipment for Optional Validation Methods Description
43. ube with clear cap tube with purple cap tube with yellow cap tube with blue cap tube with clear cap tube with black cap tube with clear cap tube with pink cap Indexing Primers A01 to H02 in white capped tubes 48 Reaction Kit tube with clear cap tube with clear cap 8 well strip with green label 8 well strip with red label tube with orange cap bottle tube with clear cap tube with black cap tube with green cap tube with clear cap bottle bottle bottle bottle tube with yellow cap tube with blue cap tube with clear cap bottle tube with clear cap tube with pink cap Indexing Primers A01 to H06 in orange 96 well plate 96 Reaction Kit bottle tube with clear cap 8 well strip with green label 8 well strip with red label tube with orange cap bottle bottle tube with black cap tube with green cap tube with clear cap bottle bottle bottle bottle tube with yellow cap tube with blue cap tube with clear cap bottle tube with clear cap tube with pink cap Indexing Primers A01to H12 in orange 96 well plate See Table 14 for a plate map t See Table 15 for a plate map HaloPlex HS Target Enrichment System ILM 51 3 Reference Table 14 Plate map for HaloPlex HS Indexing Primers A01 through H06 provided in orange plate with 48 reaction kits wells in columns 7 through 12 are empty 1 2 3 4 5 6 7 8 9 10 11 12 A AM A02 A03 A04 A05 A06 B B01 B02 B03 B04 B05 B06
44. ybridization reaction contains the following components e 39 ul Hybridization Master Mix 5 ul HaloPlex HS Indexing Primer e approximately 56 ul pooled digested DNA samples Due to partial evaporation of samples you may recover less than 7 ul of each restriction digest Minor reductions to the digested DNA pool volume will not impact hybridization performance you do not need to compensate for any sample evaporation volume losses in the final pool For the ECD sample add 2 5 ul of each digestion reaction and 36 ul of nuclease free water to the mixture of Hybridization master mix and Indexing Primer from step 3 for a total volume of 100 ul 5 Vortex the mixtures briefly and then spin tubes briefly HaloPlex HS Target Enrichment System ILM 29 2 Sample Preparation 6 Place the hybridization reaction tubes in a thermal cycler Run the appropriate program in Table 7 using the hybridization duration listed on the Certificate of Analysis Use a heated lid Do not include a low temperature hold step in the thermal cycler program Incubation at 58 C for more than the indicated time is not recommended Table 7 Thermal cycler program for HaloPlex HS probe hybridization Step Temperature Time Duration of Step Designs with lt 20 000 probes Designs with gt 20 000 probes see Certificate of Analysis see Certificate of Analysis Step 1 95 C 5 minutes 5 minutes Step 2 58 C 2 hours 16 hours 7 At least 30 minutes before t
45. ze runs may include up to 96 samples Include one ECD control sample per run of 2 to 96 samples See page 17 for additional run size considerations 18 HaloPlex HS Target Enrichment System ILM Sample Preparation 2 3 Prepare the Restriction Enzyme Master Mix strip The gDNA is digested in eight different restriction reactions each containing two restriction enzymes The 16 restriction enzymes are provided in two 8 well strip tubes that are distinguished by red and green color markers Enzymes are combined from corresponding wells of the red and green marked strip tubes along with restriction buffer and BSA to make eight different RE Master Mixes Figure 2 illustrates how to prepare the 8 well Restriction Enzyme Master Mix strip for a 12 sample run using the steps detailed on page 20 RE Buffer BSA y CO A 392p Hr Ir Hr HH perwell eco Fou RE Master Mix Strip I i Nil PAW VVVVVVVV Green Enzyme Strip Align green marker with tube A of RE Master Mix strip VY Y VYY y y 4 9 pl to ind Oa was corresponding well PE RE Master Mix Strip AA ll rin Red Enzyme Strip I I A I ji I I Align red marker with tube A of UUVUUUUUU RE Master Mix strip VAVA VA VAVA VA V 4 9 pl to LAA Ae Ae Zu Zu Zu zz correspondingwell a s co er c u ooga RE Master Mix Strip V y y y j U U Figure 2 Preparation of the Restriction Enzyme Master Mix Strip for 12 sample run HaloP

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