2SureSelect Methyl-Seq Target Enrichment System - PDF document

2SureSelect Methyl-Seq Target Enrichment System notice until the indicated met. formed or adhered to, could result in personal injury or death. Do not proceed beyond a WARNING and 2011 Illumina, Inc. All rights reserved. Call (800) 227-9770 (option 3,4,4) Or send an e-mail to: For Europe, Middle East, Africa, and IndiaCall 00800 345 600 (toll free) or +49 69 8679 7730Or send an e-mail to: For all other regionsAgilent’s world-wide sales and support cen-ter telephone numbers can be obtained at under Contact Us.Or send an e-mail to: SureSelect Methyl-Seq Target Enrichment System3system to prepare bisulfite-sequencing samples for the Illumina paired-end multiplexed sequencing platform. 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 an experiment. Sample Preparation (3 µg DNA Samples) target enrichment from 3-µg gDNA samples. 3 Sample Preparation (1 µg DNA Samples) target enrichment from 1-µg gDNA samples. 4 Hybridization Human Methyl-Seq 5 Bisulfite Conversion This chapter describes the steps for bisulfite treatment of unmethylated DNA segments. 6 Indexing and Sample Pooling for Multiplexed Sequencing This chapter describes the steps to index the captured DNA libraries that were modified by bisulfite conversion and to 7 Reference This chapter contains reference information, including component kit contents and index sequences. 4SureSelect Methyl-Seq Target Enrichment SystemSupport for use of freshly-prepared 0.1 M NaOH, instead library samples from Streptavidin T1 magnetic beads. See on 10, instructions for preparation and use of 0.1 M 53 and note on 34 on page 78.Information on use of non-supported Capture Libraries 10 and 49Support for Agilent 4200 TapeStation (see on 12 and revised instructions on 21 27 37 44 68Updates to Agilent 2100 Bioanalyzer system ordering on 12 20 26 36 43 66Updates to product guarantee and support statement (see on Updates to description of dA-tailing step (see 22 38). This is a description-only update with no Updates to ordering information for materials purchased from Thermo Fisher Scientific (see on page 10 on page 11)Updates to chapter to remove information in clear-capped tubes). To obtain sequence information or Updates to Technical Support contact information (see SureSelect Methyl-Seq Target Enrichment System51Before You Begin 2Sample Preparation (3 µg DNA Samples) 3Sample Preparation (1 µg DNA Samples) 6SureSelect Methyl-Seq Target Enrichment System4Hybridization 5Bisulfite Conversion 6Indexing and Sample Pooling for Multiplexed Sequencing 7Reference 7 SureSelectXT Methyl-Seq Target Enrichment System for Illumina Multiplexed Sequencing ProtocolAgilent Technologies 1Before You BeginOverview of the Workflow Safety Notes Required Reagents Required Equipment Make sure you read and understand the information in this chapter and have the necessary equipment and reagents listed before you start an Agilent guarantees performance and provides technical support for the SureSelect reagents required for this workflow only when used as directed in this Protocol. NOTE 1Before You Begin8SureSelect Methyl-Seq Target Enrichment System Methyl-Seq target enrichment workflow is summarized in Figure 1Overall target-enriched sequencing sample preparation workflow. Before You Begin1 SureSelect Methyl-Seq Target Enrichment System9with nuclease-free aerosol-resistant tips. CAUTION 1Before You Begin10SureSelect Methyl-Seq Target Enrichment System Table 1Required Reagents for SureSelectXT Methyl-Seq Target Enrichment *Use of other SureSelect Capture Libraries is not supported by this protocol and requires optimiza-10 M NaOH, molecular biology gradeSigma, p/n 72068Nuclease-free Water (not DEPC-treated) Thermo Fisher Scientific p/n AM99301X Low TE Buffer (10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA)Thermo Fisher Scientific 12090015, or equivalent 5 mL 60 mL 450 mL p/n A63880 p/n A63881 p/n A63882 2 mL 10 mL 50 mL p/n 65601 p/n 65602 p/n 65604D 100 assays 500 assays p/n Q32850 p/n Q32853100% Ethanol, molecular biology gradeSigma-Aldrich p/n E7023 Before You Begin1 SureSelect Methyl-Seq Target Enrichment System11 Table 2Required Equipment for SureSelectXT Methyl-Seq Target Enrichment SureCycler 8800 Thermal CyclerAgilent p/n G8800ATube cap strips, domedAgilent p/n 410096DNA LoBind Tubes, 1.5-mL PCR clean, 250 piecesEppendorf p/n 022431021 or equivalentCentrifugeEppendorf Centrifuge model 5804 or Qubit FluorometerThermo Fisher Scientific p/n Q33226 or Qubit assay tubesThermo Fisher Scientific p/n Q32856Vacuum concentratorSavant SpeedVac, model DNA120, with Nutator plate mixerBD Diagnostics p/n 421105 or equivalentMultichannel pipettePipetman or equivalentP10, P20, P200 and P1000 pipettesPipetman P10, P20, P200, P1000 or 1Before You Begin12SureSelect Methyl-Seq Target Enrichment SystemMagnetic separatorThermo Fisher Scientific p/n 12331D or *Select a magnetic separator configured to collect magnetic particles on one side of each well. Do †DNA samples may also be analyzed using the Agilent 2200 TapeStation, p/n G2964AA or G2965AA. 13 XT Methyl-Seq Target Enrichment System for Illumina Multiplexed Sequencing Protocol Sample Preparation (3 µg DNA Samples) enrichment for methyl-C sequence analysis using the Illumina platform. For each sample to be sequenced, an individual methylated adapter-ligated This section contains instructions for the preparation of gDNA libraries from 3 29 2Sample Preparation (3 µg DNA Samples) 5012-8701).quality gDNA with 1X Low TE Buffer in a 1.5-mL series or S-series instrument. NOTEMake sure genomic DNA samples are of high quality with an OD 260/280 ratio ranging NOTE Sample Preparation (3 µg DNA Samples)2 SureSelect Methyl-Seq Target Enrichment System15through the pre-split septa. Table 3Shear settings for Covaris instruments using SonoLab software version 7 or pre-split septa, then slowly remove the sheared DNA. Setting Duty Factor10%Peak Incident Power (PIP)175Cycles per Burst200Treatment Time360 secondsBath Temperature4° to 8° C Setting Duty Cycle 10%Cycles per Burst200Time6 cycles of 60 seconds eachSet ModeFrequency sweepingTemperature4° to 7° C 2Sample Preparation (3 µg DNA Samples) 16SureSelect Methyl-Seq Target Enrichment System Assess sample quality and quantity using the 2100 Bioanalyzer system and DNA 1000 Assay, as described on page 20 , or using the 4200 TapeStation, as described on page 21 Verify that the electropherogram shows a DNA fragment size peak Sample Preparation (3 µg DNA Samples)2 SureSelect Methyl-Seq Target Enrichment System17Use the SureSelect Methyl-Seq Library Prep Kit for this step. Table 5Preparation of End Repair master mix 2Add 52 µL of the master mix to each sample well containing 48 µL of sheared DNA. Mix by vortexing for 5 seconds then spin the samples 3Incubate the samples in the thermal cycler and run the program in 6. Do not use a heated lid. Table 6End Repair Thermal Cycler Program Reagent Volume for 1 reaction Nuclease-free water35.2 µL580.8 µL10× End Repair Buffer (clear cap)10 µL165 µLdNTP Mix (green cap)1.6 µL26.4 µLT4 DNA Polymerase (purple cap)1 µL16.5 µLKlenow DNA Polymerase (yellow cap)2 µL33 µLT4 Polynucleotide Kinase (orange cap)2.2 µL36.3 µL Total 52 µL 858 µL Step Temperature Step 120°C 30 minutesStep 24°C Hold 2Sample Preparation (3 µg DNA Samples) NOTE Sample Preparation (3 µg DNA Samples)2 SureSelect Methyl-Seq Target Enrichment System19 Seal the wells with strip caps, then mix well on a vortex mixer and briefly spin the samples to collect the liquid. Incubate for 2 minutes at room temperature. 16Put the plate or tube strip in the magnetic stand and leave for 2 to 3 minutes, until the solution is clear. 17Remove the cleared supernatant (approximately 42 µL) to a fresh well. You can discard the beads at this time. Stopping Point If you do not continue to the next step, seal the wells and store at –20°C. 2Sample Preparation (3 µg DNA Samples)end-repaired DNA samples. For more information to do this step, see the Sample Preparation (3 µg DNA Samples)2 SureSelect Methyl-Seq Target Enrichment System21end-repaired DNA samples using the 4200 TapeStation. For more Prepare the TapeStation samples as instructed in the instrument user manual. Use 1 µL of each DNA sample diluted with 3 µL of D1000 sample buffer for the analysis. 2Load the sample plate or tube strips from step 1 , the D1000 ScreenTape, and loading tips into the 4200 TapeStation as instructed in the instrument user manual. Start the run. Verify that the electropherogram shows a DNA fragment size peak Figure 3 Figure 3Analysis of end-repaired DNA using a D1000 ScreenTape. mixer for 5 seconds for accurate quantitation. 2Sample Preparation (3 µg DNA Samples)Use the SureSelect Methyl-Seq Library Prep Kit for this step. Table 7Preparation of dA-Tailing master mix Dispense 9 µL of the dA-Tailing master mix into each sample well containing end-repaired, purified DNA (approximately 41 µL). Table 8dA-Tailing Thermal Cycler Program Reagent Volume for 1 reaction 10× Klenow Polymerase Buffer (blue cap)5 µL82.5 µLdATP (green cap)1 µL16.5 µLExo(–) Klenow (red cap)3 µL49.5 µL Total 9 µL 148.5 µL Step Temperature Step 137°C 30 minutesStep 24°C Hold Sample Preparation (3 µg DNA Samples)2 SureSelect Methyl-Seq Target Enrichment System23 1Let the AMPure XP beads come to room temperature for at least 30 minutes. Do not freeze the beads at any time. 2Mix the bead suspension well so that the reagent appears homogeneous and consistent in color. Add 90 µL of homogeneous AMPure XP beads to each 50-µL dA-tailed 4Incubate samples for 5 minutes at room temperature. 5Put the plate or tube strip into a magnetic separation device. Wait for the solution to clear (approximately 3 to 5 minutes). 6Keep the plate or tube strip in the magnetic stand. Carefully remove and discard the cleared solution from each well. Do not touch the beads while removing the solution. 200 µL of freshly-prepared 70% ethanol in each sample well. 8Wait for 1 minute to allow any disturbed beads to settle, then remove the ethanol. 9Repeat 7 to step 8 step once. 10Seal the wells with strip caps, then briefly spin the plate or tube strip to collect the residual ethanol. Return the samples to the magnetic 11Dry the samples by placing the unsealed plate or tube strip on the thermal cycler, set to hold samples at 37°C, for 1 to 2 minutes or until the residual ethanol completely evaporates. Add 35 µL nuclease-free water to each sample well. 13Seal the wells with strip caps, then mix well on a vortex mixer and briefly spin the plate or tube strip to collect the liquid. Incubate for 2 minutes at room temperature. 15Put the plate or tube strip in the magnetic stand and leave for 2 to 3 minutes, until the solution is clear. 16Remove 33.5 µL of the cleared supernatant to a fresh well. You can discard the beads at this time. 17Proceed immediately to the next step, adapter” on page 24 . 2Sample Preparation (3 µg DNA Samples)Use the SureSelect Methyl-Seq Library Prep Kit for this step. Table 9Preparation of Ligation master mix containing dA-tailed, purified DNA (approximately 33.5 µL). Table 10Ligation Thermal Cycler Program Step 8. Purify the adapter-ligated DNA using Reagent Volume for 1 reaction 5 µL82.5µL5× T4 DNA Ligase Buffer (green cap)10 µL165 µLT4 DNA Ligase (red cap)1.5 µL24.75 µL Total 16.5 µL 272.25 µL Step Temperature Step 120°C 15 minutesStep 24°C Hold Sample Preparation (3 µg DNA Samples)2 SureSelect Methyl-Seq Target Enrichment System25 beads 1Let the AMPure XP beads come to room temperature for at least 30 minutes. Do not freeze the beads at any time. 2Mix the bead suspension well so that the reagent appears homogeneous and consistent in color. Add 90 µL of homogeneous AMPure XP beads to each 50-µL adapter-ligated DNA sample well. Pipette up and down to mix. 4Incubate samples for 5 minutes at room temperature. 5Put the plate or tube strip into a magnetic separation device. Wait for the solution to clear (approximately 3 to 5 minutes). 6Keep the plate or tube strip in the magnetic stand. Carefully remove and discard the cleared solution from each well. Do not touch the beads while removing the solution. 200 µL of freshly-prepared 70% ethanol in each sample well. 8Wait for 1 minute to allow any disturbed beads to settle, then remove the ethanol. 9Repeat 7 and step 8 step once. 10Seal the wells with strip caps, then briefly spin the samples to collect the residual ethanol. Return the plate or tube strip to the magnetic seconds. Remove the residual ethanol with a P20 pipette. 11Dry the samples by placing the unsealed plate or tube strip on the thermal cycler, set to hold samples at 37°C, for 1 to 2 minutes or until the residual ethanol completely evaporates. Add 22 µL nuclease-free water to each sample well. 13Seal the wells with strip caps, then mix well on a vortex mixer and briefly spin the samples to collect the liquid. Incubate for 2 minutes at room temperature. 15Put the plate or tube strip in the magnetic stand and leave for 2 to 3 minutes, until the solution is clear. 16Remove the cleared supernatant (approximately 22 µL) to a fresh well. You can discard the beads at this time. Stopping Point If you do not continue to the next step, seal the wells and store at –20°C. 2Sample Preparation (3 µg DNA Samples)DNA. If the yield is ligated DNA Sample Preparation (3 µg DNA Samples)2 SureSelect Methyl-Seq Target Enrichment System27end-repaired DNA samples using the 4200 TapeStation. For more DNA. If the yield is ligated DNA .Figure 5Analysis of adapter-ligated DNA using a D1000 ScreenTape. CAUTION Make sure that you thoroughly mix the combined DNA and sample buffer on a vortex mixer for 5 seconds for accurate quantitation. 2Sample Preparation (3 µg DNA Samples) 28SureSelect Methyl-Seq Target Enrichment System 29 XT Methyl-Seq Target Enrichment System for Illumina Multiplexed Sequencing Protocol Sample Preparation (1 µg DNA Samples) enrichment for methyl-C sequence analysis using the Illumina platform. For each sample to be sequenced, an individual methylated adapter-ligated This section contains instructions for the preparation of gDNA libraries from 1 13 3Sample Preparation (1 µg DNA Samples) 5012-8701).quality gDNA with 1X Low TE Buffer in a 1.5-mL series or S-series instrument. NOTEMake sure genomic DNA samples are of high quality with an OD 260/280 ratio ranging NOTE Sample Preparation (1 µg DNA Samples)3 SureSelect Methyl-Seq Target Enrichment System31through the pre-split septa. Table 11Shear settings for Covaris instruments using SonoLab software version 7 or pre-split septa, then slowly remove the sheared DNA. Setting Duty Factor10%Peak Incident Power (PIP)175Cycles per Burst200Treatment Time360 secondsBath Temperature4° to 8° C Setting Duty Cycle 10%Cycles per Burst200Time6 cycles of 60 seconds eachSet ModeFrequency sweepingTemperature4° to 7° C 3Sample Preparation (1 µg DNA Samples) 32SureSelect Methyl-Seq Target Enrichment System Assess sample quality and quantity using the 2100 Bioanalyzer system and DNA 1000 Assay, as described on page 36 , or using the 4200 TapeStation, as described on page 37 Verify that the electropherogram shows a DNA fragment size peak Sample Preparation (1 µg DNA Samples)3 SureSelect Methyl-Seq Target Enrichment System33Use the SureSelect Methyl-Seq Library Prep Kit for this step. Table 13Preparation of End Repair master mix 2Add 52 µL of the master mix to each sample well containing 48 µL of sheared DNA. Mix by vortexing for 5 seconds then spin the samples 3Incubate the samples in the thermal cycler and run the program in 14. Do not use a heated lid. Table 14End Repair Thermal Cycler Program Reagent Volume for 1 reaction Nuclease-free water35.2 µL580.8 µL10× End Repair Buffer (clear cap)10 µL165 µLdNTP Mix (green cap)1.6 µL26.4 µLT4 DNA Polymerase (purple cap)1 µL16.5 µLKlenow DNA Polymerase (yellow cap)2 µL33 µLT4 Polynucleotide Kinase (orange cap)2.2 µL36.3 µL Total 52 µL 858 µL Step Temperature Step 120°C 30 minutesStep 24°C Hold 3Sample Preparation (1 µg DNA Samples) NOTE Sample Preparation (1 µg DNA Samples)3 SureSelect Methyl-Seq Target Enrichment System35 Seal the wells with strip caps, then mix well on a vortex mixer and briefly spin the samples to collect the liquid. Incubate for 2 minutes at room temperature. 16Put the plate or tube strip in the magnetic stand and leave for 2 to 3 minutes, until the solution is clear. 17Remove the cleared supernatant (approximately 42 µL) to a fresh well. You can discard the beads at this time. Stopping Point If you do not continue to the next step, seal the wells and store at –20°C. 3Sample Preparation (1 µg DNA Samples)end-repaired DNA samples. For more information to do this step, see the Sample Preparation (1 µg DNA Samples)3 SureSelect Methyl-Seq Target Enrichment System37end-repaired DNA samples using the 4200 TapeStation. For more Prepare the TapeStation samples as instructed in the instrument user manual. Use 1 µL of each DNA sample diluted with 3 µL of D1000 sample buffer for the analysis. 2Load the sample plate or tube strips from step 1 , the D1000 ScreenTape, and loading tips into the 4200 TapeStation as instructed in the instrument user manual. Start the run. Verify that the electropherogram shows a DNA fragment size peak Figure 7 Figure 7Analysis of end-repaired DNA using a D1000 ScreenTape. mixer for 5 seconds for accurate quantitation. 3Sample Preparation (1 µg DNA Samples)Use the SureSelect Methyl-Seq Library Prep Kit for this step. Table 15Preparation of dA-Tailing master mix Dispense 9 µL of the dA-Tailing master mix into each sample well containing end-repaired, purified DNA (approximately 41 µL). Table 16dA-Tailing Thermal Cycler Program Reagent Volume for 1 reaction 10× Klenow Polymerase Buffer (blue cap)5 µL82.5 µLdATP (green cap)1 µL16.5 µLExo(–) Klenow (red cap)3 µL49.5 µL Total 9 µL 148.5 µL Step Temperature Step 137°C 30 minutesStep 24°C Hold Sample Preparation (1 µg DNA Samples)3 SureSelect Methyl-Seq Target Enrichment System39 1Let the AMPure XP beads come to room temperature for at least 30 minutes. Do not freeze the beads at any time. 2Mix the bead suspension well so that the reagent appears homogeneous and consistent in color. Add 90 µL of homogeneous AMPure XP beads to each 50-µL dA-tailed 4Incubate samples for 5 minutes at room temperature. 5Put the plate or tube strip into a magnetic separation device. Wait for the solution to clear (approximately 3 to 5 minutes). 6Keep the plate or tube strip in the magnetic stand. Carefully remove and discard the cleared solution from each well. Do not touch the beads while removing the solution. 200 µL of freshly-prepared 70% ethanol in each sample well. 8Wait for 1 minute to allow any disturbed beads to settle, then remove the ethanol. 9Repeat 7 to step 8 step once. 10Seal the wells with strip caps, then briefly spin the plate or tube strip to collect the residual ethanol. Return the samples to the magnetic 11Dry the samples by placing the unsealed plate or tube strip on the thermal cycler, set to hold samples at 37°C, for 1 to 2 minutes or until the residual ethanol completely evaporates. Add 35 µL nuclease-free water to each sample well. 13Seal the wells with strip caps, then mix well on a vortex mixer and briefly spin the plate or tube strip to collect the liquid. Incubate for 2 minutes at room temperature. 15Put the plate or tube strip in the magnetic stand and leave for 2 to 3 minutes, until the solution is clear. 16Remove 33.5 µL of the cleared supernatant to a fresh well. You can discard the beads at this time. 17Proceed immediately to the next step, adapter” on page 40 . 3Sample Preparation (1 µg DNA Samples)Use the SureSelect Methyl-Seq Library Prep Kit for this step. Table 17Preparation of Ligation master mix containing dA-tailed, purified DNA (approximately 33.5 µL). Table 18Ligation Thermal Cycler Program Reagent Volume for 1 reaction Nuclease-free water2.5 µL41.25 µL2.5 µL41.25µL5× T4 DNA Ligase Buffer (green cap)10 µL165 µLT4 DNA Ligase (red cap)1.5 µL24.75 µL Total 16.5 µL 272.25 µL Step Temperature Step 120°C 15 minutesStep 24°C Hold Sample Preparation (1 µg DNA Samples)3 SureSelect Methyl-Seq Target Enrichment System41 Do not exceed the 15 minute incubation time. Proceed immediately to free adapter removal in Step 8. Purify the adapter-ligated DNA using AMPure XP beads . 3Sample Preparation (1 µg DNA Samples) 42SureSelect Methyl-Seq Target Enrichment System beads 1Let the AMPure XP beads come to room temperature for at least 30 minutes. Do not freeze the beads at any time. 2Mix the bead suspension well so that the reagent appears homogeneous and consistent in color. Add 90 µL of homogeneous AMPure XP beads to each 50-µL adapter-ligated DNA sample well. Pipette up and down to mix. 4Incubate samples for 5 minutes at room temperature. 5Put the plate or tube strip into a magnetic separation device. Wait for the solution to clear (approximately 3 to 5 minutes). 6Keep the plate or tube strip in the magnetic stand. Carefully remove and discard the cleared solution from each well. Do not touch the beads while removing the solution. 200 µL of freshly-prepared 70% ethanol in each sample well. 8Wait for 1 minute to allow any disturbed beads to settle, then remove the ethanol. 9Repeat 7 and step 8 step once. 10Seal the wells with strip caps, then briefly spin the samples to collect the residual ethanol. Return the plate or tube strip to the magnetic seconds. Remove the residual ethanol with a P20 pipette. 11Dry the samples by placing the unsealed plate or tube strip on the thermal cycler, set to hold samples at 37°C, for 1 to 2 minutes or until the residual ethanol completely evaporates. Add 22 µL nuclease-free water to each sample well. 13Seal the wells with strip caps, then mix well on a vortex mixer and briefly spin the samples to collect the liquid. Incubate for 2 minutes at room temperature. 15Put the plate or tube strip in the magnetic stand and leave for 2 to 3 minutes, until the solution is clear. 16Remove the cleared supernatant (approximately 22 µL) to a fresh well. You can discard the beads at this time. Stopping Point If you do not continue to the next step, seal the wells and store at –20°C. Sample Preparation (1 µg DNA Samples)3 SureSelect Methyl-Seq Target Enrichment System43DNA. If the yield is ligated DNA 3Sample Preparation (1 µg DNA Samples)end-repaired DNA samples using the 4200 TapeStation. For more DNA. If the yield is ligated DNA .Figure 9Analysis of adapter-ligated DNA using a D1000 ScreenTape. CAUTION Make sure that you thoroughly mix the combined DNA and sample buffer on a vortex mixer for 5 seconds for accurate quantitation. 45 XT Methyl-Seq Target Enrichment System for Illumina Multiplexed Sequencing Protocol Hybridization This chapter describes the steps to combine the prepped library with the hybridization reagents, blocking agents and the SureSelect capture library. CAUTION capture.Protocol steps from hybridization through bisulfite conversion (pages 46 through 57) must be completed without stopping points. Plan your experiments accordingly. 4Hybridization46SureSelect Methyl-Seq Target Enrichment System Use all of the methylated adapter-ligated DNA in the hybridization 350 ng of adaptor-ligated DNA in a volume of 3.4 µL, for If you have recovered less than 350 ng of adaptor-ligated DNA, do another Use a vacuum concentrator to concentrate the samples at 45°C. Reduce the volume of each sample from the starting volume of µL. Do not completely dry the samples in the wells. Reconstitute each sample with nuclease-free water to a final volume of µL. 3Mix each sample thoroughly using a vortex mixer and then spin the plate or strip tube in a centrifuge for 1 minute to collect the liquid in each well. CAUTION 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. Hybridization4 SureSelect Methyl-Seq Target Enrichment System47 Table 19Preparation of Hybridization Buffer 5Prepare the SureSelect Block Mix by mixing the components in 20. Keep the mixture on ice until it is used in step 6 Table 20Preparation of SureSelect Block Mix Reagent *Prepare Hybridization Buffer for at least 5 reaction equivalents per run to allow accurate pipetting SureSelect Hyb 1 (orange cap or bottle)6.63 µL116 µLSureSelect Hyb 2 (red cap)0.27 µL4.7 µLSureSelect Hyb 3 (yellow cap or bottle)2.65 µL46.4 µLSureSelect Hyb 4 (black cap or bottle)3.45 µL60.4 µL Total 13 µL 227.5 Reagent Volume for 1 reaction SureSelect Indexing Block 1 (green cap)2.5 µL42.5 µLSureSelect Block 2 (blue cap)2.5 µL42.5 µLSureSelect Methyl-Seq Block 3 (brown cap)0.6 µL10.2 µL Total 5.6 µL 95.2 µL 4Hybridization48SureSelect Methyl-Seq Target Enrichment System Table 21Thermal cycler program for DNA + Block Mix prior to hybridization 8Prepare the required volume of a 1:3 dilution of SureSelect RNase Block (for a final concentration of 25%), on ice, as shown in Table 22 Table 22Preparation of 25% RNase Block solution CAUTIONFor each protocol step that requires removal of tube cap strips, make sure to reseal the tubes with a fresh strip of caps. Reuse of strip caps can cause sample loss, sample Step Temperature Step 195°C 5 minutesStep 265°C 2 minutesStep 365°C Hold CAUTION The lid of the thermal cycler is hot and can cause burns. Use caution when working near the lid. Component Volume for 1 reaction RNase Block (purple cap)0.5 µL8.5 µLNuclease-free water1.5 µL25.5 µL Total 2 µL 34 µL Hybridization4 SureSelect Methyl-Seq Target Enrichment System49 Table 23Preparation of Capture Library Hybridization Mix Use of other SureSelect Capture Libraries is not supported by this protocol and requires optimization of amount of capture library added to the hybridization mix. Begin optimization 49Libraries 3 Mb, and by using 2 µL of Capture Libraries ( µL using nuclease-free water).Additional protocol steps, including post-capture PCR cycling conditions may also require optimization. See the 10Maintain the DNA library + Block Mix plate or strip tube at 65°C while you add 20 µL of the Capture Library Hybridization Mix from step 9 to each sample well. Mix well by pipetting up and down 8 to 10 times.The hybridization reaction wells now contain approximately 27 to 29 µL, depending on the degree of evaporation during the thermal cycler incubation. step of 2 minute duration described in Reagent Volume for 1 reaction 13 µL221 µL2 µL34 µLSureSelect Human Methyl-Seq Capture Library5 µL85 µL Total 20 µL 340 µL NOTE 4Hybridization50SureSelect Methyl-Seq Target Enrichment System Seal the wells with strip caps. Make sure that all wells are completely sealed. 12Incubate the hybridization mixture for 16 hours at 65°C with a heated lid at 105°C. CAUTION negatively impacted. When using the SureCycler 8800 thermal cycler and sealing with strip caps, make sure to use domed strip caps and to place a compression mat over the PCR plate or strip tubes in the thermal cycler. Hybridization4 SureSelect Methyl-Seq Target Enrichment System51 1Vigorously resuspend the Dynabeads MyOne Streptavidin T1 magnetic beads on a vortex mixer. Magnetic beads settle during storage. wells of a 96-well plate or 8-well tube strip. 3Wash the beads: aAdd 200 µL of SureSelect Binding Buffer. bMix the beads by pipetting up and down 10 times. cPut the plate or tube strip into a magnetic separation device and allow the solution to clear (approximately 5 minutes). dRemove and discard the supernatant. eRepeat a through step d for a total of 3 washes. 4Resuspend the beads in 200 µL of SureSelect Binding Buffer. 4Hybridization52SureSelect Methyl-Seq Target Enrichment System µL aliquots of Wash Buffer 2 in wells of a fresh 96-well capture reaction plate or strip tube in a centrifuge or mini-plate NOTE Hybridization4 SureSelect Methyl-Seq Target Enrichment System53 quality 10 M NaOH stock solution with nuclease-free NaOH with 990 µL of nuclease-free water.Add 20 µL of the freshly-prepared 0.1 M NaOH solution to the bead-bound samples from CAUTIONIt is important to maintain bead suspensions at 65°C during the washing procedure below to ensure specificity of capture. Make sure that the SureSelect Wash Buffer 2 is pre-warmed to 65°C before use.fluctuations, for the incubation steps. CAUTION Using high-quality NaOH is critical for optimal DNA sample quality. 4Hybridization54SureSelect Methyl-Seq Target Enrichment System Incubate the samples for 20 minutes at room temperature. During the 20-minute incubation, prepare the EZ DNA Methylation-Gold step 1 and step 2 on page 56 Collect the beads from the elution mixture on a magnetic separator. Use a pipette to transfer the supernatant from each well to wells of a The supernatant contains the captured DNA. The beads can now be discarded. Proceed immediately to “Bisulfite Conversion” on page 55 . 55 XT Methyl-Seq Target Enrichment System for Illumina Multiplexed Sequencing Protocol Bisulfite Conversion This chapter describes the steps for bisulfite treatment of the captured DNA library to differentiate methylated and unmethylated DNA segments. When treated with bisulfite, unmethylated cytosine residues in the library are converted to uracil residues. Methylated cytosine residues remain unmodified, resulting in a primary sequence difference that may be cycles to minimize PCR-based bias. 5Bisulfite ConversionMethylation-Gold Kit to modify unmethylated cytosine residues in the treated DNA is then desulphonated using a Zymo-Spin IC column and additional reagents from the EZ DNA Methylation-Gold Kit.free water, 300 µL of M-Dilution Buffer, and 50 µL of M-Dissolving Buffer to the vial. Table 24Thermal cycler program for bisulfite conversion NOTE Step Temperature Step 164°C 2.5 hoursStep 24°C Hold Bisulfite Conversion5 SureSelect Methyl-Seq Target Enrichment System57 Desulphonate the sample using a Zymo-Spin IC column. Use one column for each 150-µL DNA sample, after recombining the two 75-µL M-Wash buffer provided with the EZ DNA Methylation-Gold Kit has Add 600 µL of M-Binding Buffer to a Zymo-Spin IC column and Load the 150-µL bisulfite-converted DNA sample onto the column. Centrifuge for 60 seconds at 13,000 rpm. Discard the flow-through, Wash the column by adding 100 µL of prepared M-Wash Buffer. Centrifuge for 60 seconds at 13,000 rpm. Discard the flow-through, Add 200 µL of M-Desulphonation Buffer to the column. Incubate at Centrifuge for 60 seconds at 13,000 rpm. Discard the flow-through, Add 200 µL of prepared M-Wash Buffer to the column. Centrifuge for 60 seconds at 13,000 rpm. Discard the flow-through, then place the Add another 200 µL of prepared M-Wash Buffer to the column. Place the column in a fresh 1.5-mL tube. Allow the column to sit at Add 10 µL of M-Elution Buffer to the column and incubate at room kCentrifuge for 60 seconds at 13,000 rpm. While retaining the flow-through, add an additional 10 µL of M-Elution Buffer to the column. Incubate at room temperature for minutes. Centrifuge for 60 seconds at 13,000 rpm. Retain the combined 20-µL flow-through for further processing. 5Bisulfite ConversionIn this step, the SureSelect-enriched and bisulfite-converted libraries are Prepare 1 amplification reaction for each bisulfite-treated library. PCR Reaction Mix Mix well using a vortex mixer and keep on ice.2For each amplification reaction, place 82 µL of the PCR reaction mixture from step 1 in the wells of a PCR plate.3Add 18 µL of each bisulfite-converted library to the appropriate PCR reaction mixture well. Mix thoroughly by pipetting. Reagent Volume for 1 Reaction Nuclease-free water30 µL495 µLSureSelect Methyl-Seq PCR Master Mix50 µL825 µLMethyl-Seq PCR1 Primer F1 µL16.5 µLMethyl-Seq PCR1 Primer R1 µL16.5 µL Total Volume 82 µL 1353 µL Bisulfite Conversion5 SureSelect Methyl-Seq Target Enrichment System59 Table 26Bisulfite-converted library amplification PCR cycling program Segment Number of Cycles Temperature 1 195°C 2 minutes2 895°C 30 seconds60°C30 seconds 72°C 30 seconds 3172°C 7 minutes414°C Hold 5Bisulfite Conversion 60SureSelect Methyl-Seq Target Enrichment System 1Let the AMPure XP beads come to room temperature for at least 30 minutes. Do not freeze the beads at any time. 2Prepare 400 µL of 70% ethanol per sample, plus excess, for use in step 8 3Mix the bead suspension well so that the reagent appears homogeneous and consistent in color. 4Add 180 µL of homogeneous AMPure XP beads to each sample well containing amplified library DNA. Pipette up and down 10 times to mix. 5Incubate samples for 5 minutes at room temperature. 6Put the plate or tube strip into a magnetic separation device. Wait for the solution to clear (approximately 7 to 10 minutes). 7Keep the plate or tube strip in the magnetic stand. Carefully remove and discard the cleared solution from each well. Do not touch the beads while removing the solution. you dispense 200 µL of freshly-prepared 70% ethanol in each sample 9Wait for 1 minute to allow any disturbed beads to settle, then remove the ethanol. 10Repeat 8 to step 9 step once. 11Seal the wells with strip caps, then briefly spin the samples to collect the residual ethanol. Return the plate or tube strip to the magnetic 12Dry the samples by placing the unsealed plate or tube strip on the thermal cycler, set to hold samples at 37°C, for 3 to 5 minutes or until the residual ethanol completely evaporates. Add 21 µL nuclease-free water to each sample well. 14Seal the wells with strip caps, then mix well on a vortex mixer and briefly spin the samples to collect the liquid. Incubate for 2 minutes at room temperature. 16Put the plate or tube strip in the magnetic stand and leave for 2 to 3 minutes, until the solution is clear. 17Remove the cleared supernatant (approximately 19.5 µL) to a fresh well. You can discard the beads at this time. 61 XT Methyl-Seq Target Enrichment System for Illumina Multiplexed Sequencing Protocol Indexing and Sample Pooling for Multiplexed Sequencing This chapter describes the steps to index the captured DNA libraries that were modified by bisulfite conversion and to pool the indexed samples for multiplexed sequence analysis. 6Indexing and Sample Pooling for Multiplexed Sequencingbisulfite-converted library. Table 27Preparation of PCR Indexing Reaction Mix each sample well containing 19.5 µL of amplified, bisulfite-converted CAUTION To avoid cross-contamination of libraries, set up PCR reactions (all components except the library DNA) in a dedicated clean area or PCR hood with UV sterilization and Reagent Volume for 1 Reaction SureSelect Methyl-Seq PCR Master Mix25 µL412.5 µL0.5 µL8.25 µL Total Volume 25.5 µL 420.75 µL Indexing and Sample Pooling for Multiplexed Sequencing6 SureSelect Methyl-Seq Target Enrichment System63 Table 28 PCR indexing cycling program Segment Number of Cycles Temperature 1 195°C 2 minutes2 695°C 30 seconds60°C30 seconds 72°C 30 seconds 3172°C 7 minutes414°C Hold 6Indexing and Sample Pooling for Multiplexed Sequencing 64SureSelect Methyl-Seq Target Enrichment System 1Let the AMPure XP beads come to room temperature for at least 30 minutes. Do not freeze the beads at any time. 2Prepare 400 µL of 70% ethanol per sample, plus excess, for use in step 8 3Mix the bead suspension well so that the reagent appears homogeneous and consistent in color. Add 90 µL of homogeneous AMPure XP beads to each 50-µL amplified 5Incubate samples for 5 minutes at room temperature. 6Put the plate into a magnetic separation device. Wait for the solution to clear (approximately 3 to 5 minutes). cleared solution from each well. Do not touch the beads while removing 200 µL of freshly-prepared 70% ethanol in each sample well. 9Wait for 1 minute to allow any disturbed beads to settle, then remove the ethanol. 10Repeat 8 to step 9 step once. 11Seal the wells with strip caps, then briefly spin the plate to collect the residual ethanol. Return the plate to the magnetic stand for 30 seconds. Remove the residual ethanol with a P20 pipette. 12Dry the samples by placing the unsealed plate on the thermal cycler, set to hold samples at 37°C, for 1 to 2 minutes or until the residual ethanol completely evaporates. Add 24 µL nuclease-free water to each sample well. 14Seal the wells then mix well on a vortex mixer and briefly spin the plate to collect the liquid. Incubate for 2 minutes at room temperature. 16Put the plate in the magnetic stand and leave for 2 to 3 minutes, until the solution is clear. 17Remove the cleared supernatant (approximately 24 µL) to a fresh well. You can discard the beads at this time. Indexing and Sample Pooling for Multiplexed Sequencing6 SureSelect Methyl-Seq Target Enrichment System65 a week, or at -20°C for longer periods. 6Indexing and Sample Pooling for Multiplexed Sequencing 66SureSelect Methyl-Seq Target Enrichment System at for 1Set up the 2100 Bioanalyzer instrument as instructed in the reagent kit guide. 2Prepare the chip, samples and ladder as instructed in the reagent kit guide, using 1 µl of each sample for the analysis. 3Load the prepared chip into the instrument and start the run within five minutes after preparation. Verify the results. Check that the electropherogram shows a distribution sample electropherogram is shown in Figure 10 If a significant primer-dimer peak is observed in one or more of the 5Determine the concentration of each library by integration under the peak in each electropherogram. Use the quantities of indexed libraries determined at this step to pool Indexing and Sample Pooling for Multiplexed Sequencing6 SureSelect Methyl-Seq Target Enrichment System67 6Indexing and Sample Pooling for Multiplexed Sequencing 68SureSelect Methyl-Seq Target Enrichment System Option 2: Analysis using the Agilent 4200 TapeStation and High Sensitivity D1000 ScreenTape Use a High Sensitivity D1000 ScreenTape and reagent kit to analyze the amplified indexed DNA. For more information to do this step, see the TapeStation instrument user manual at www.genomics.agilent.com. 1Prepare the TapeStation samples as instructed in the instrument user manual. Use 2 µL of each indexed DNA sample diluted with 2 µL of 2Load the sample plate or tube strips from step 1 , the High Sensitivity D1000 ScreenTape, and loading tips into the TapeStation as instructed in the instrument user manual. Start the run. 3 Verify the results. Check that the electropherogram shows a distribution sample electropherogram is shown in Figure 11 If a significant primer-dimer peak is observed in one or more of the 4Determine the concentration of each library by integration under the peak in each electropherogram. Use the quantities of indexed libraries determined at this step to pool CAUTION mixer for 5 seconds for accurate quantitation. Indexing and Sample Pooling for Multiplexed Sequencing6 SureSelect Methyl-Seq Target Enrichment System69 6Indexing and Sample Pooling for Multiplexed SequencingCombine the indexed DNA samples such that each index-tagged sample nM for the Methyl-Seq sequencing protocol, 2Adjust the final volume of the pooled library to the desired final concentration.If the final volume of the combined index-tagged samples is less than the desired final volume, V(f), add Low TE to bring the volume to the desired level.If the final volume of the combined index-tagged samples is greater than the final desired volume, V(f), lyophilize and reconstitute to the Table 29 shows an example of the amount of 2 capture pool samples and Low TE needed for a final volume of 25 µL at 10 nM final DNA concentration. Table 29Example of indexed Methyl-Seq sample volume calculations for a 25-µL final sequencing sample pool containing 10 nM DNA Component V(f) C(i) C(f) # Sample 125 µL10 nM10 nM2Sample 225 µL12.5 nM10 nM2 Indexing and Sample Pooling for Multiplexed Sequencing6 SureSelect Methyl-Seq Target Enrichment System71 If a significant primer-dimer peak was observed for any of the indexed “Step 2. Purify the indexed libraries using AMPure XP beads” on page 64 , using 45 µL of AMPure XP bead suspension for each 25-µL sequencing sample. Elute the purified DNA in 25 µL of nuclease-free water. 6Indexing and Sample Pooling for Multiplexed Sequencing 72SureSelect Methyl-Seq Target Enrichment System 1Analyze the final indexed DNA pool using either a Bioanalyzer High Sensitivity DNA Assay kit (see page 66 for instructions) or a High Sensitivity D1000 ScreenTape (see page 68 for instructions). Check that the electropherogram shows a single peak between 3Determine the concentration of the indexed library pool by integration under the peak in the electropherogram. The final concentration of the indexed DNA pool should be approximately 10 nM. 4Proceed to template denaturation and flow cell preparation. Refer to the appropriate Illumina protocol and to “Guidelines for sequencing sample preparation and run setup” on page 73 . Indexing and Sample Pooling for Multiplexed Sequencing6 SureSelect Methyl-Seq Target Enrichment System73 Use the appropriate Illumina Paired-End Cluster Generation Kit to do Illumina Paired-End Cluster Generation Kit. Methyl-Seq libraries 30 for guidelines. Seeding concentration and cluster PhiX control DNA as a low-concentration spike-in for improved Sequencing run setup guidelines for 8-bp indexes For libraries prepared using kits with 8-bp indexes, sequencing runs must be set up to perform an 8-bp index read. For the HiSeq platform, use the settings shown in 31 screen of the instrument control software from the index type selection buttons. For complete 8-bp index sequence information, see 37 on page 80. HiSeq 2500Rapid Runv210–13 pM5%HiSeq 2500High Outputv412–14 pM5%HiSeq 3000/4000 All Runsv1210 pM10% Read 1100Index 1 (i7)8Index 2 (i5)0Read 2100 6Indexing and Sample Pooling for Multiplexed Sequencing 74SureSelect Methyl-Seq Target Enrichment System 75 XT Methyl-Seq Target Enrichment System for Illumina Multiplexed Sequencing Protocol Reference XT Indexes A01 to H12 This chapter contains component kit contents reference information. 7Reference76SureSelect Methyl-Seq Target Enrichment System Methyl-Seq system includes the following component SureSelect Methyl-Seq Library Prep Kit–20°C5500-01285500-01295 x 5500-0129SureSelect Methyl-Seq Target Enrichment Box 1Room Temperature5190-50005190-50025 x 5190-5002SureSelect Methyl-Seq Hybridization Kit Box 2–20°C5190-50015190-50035 x 5190-5003–80°C5190-46615190-46625190-4663 Reference7 SureSelect Methyl-Seq Target Enrichment System77 32 are 10X End Repair Buffertube with clear captube with clear cap10X Klenow Polymerase Buffertube with blue captube with blue cap5X T4 DNA Ligase Buffertube with green captube with green capT4 DNA Ligasetube with red captube with red capExo(–) Klenowtube with red captube with red capT4 DNA Polymerasetube with purple captube with purple capKlenow DNA Polymerasetube with yellow captube with yellow capT4 Polynucleotide Kinasetube with orange captube with orange capdATPtube with green captube with green capdNTP Mixtube with green captube with green capSureSelect Methyl-Seq PCR Master Mixtube with clear capbottleSureSelect Methyl-Seq Methylated Adaptertube with green captube with green capSureSelect Methyl-Seq PCR1 Primer Ftube with brown captube with brown capSureSelect Methyl-Seq PCR1 Primer Rtube with brown captube with brown captube with blue captube with blue cap*See 37 80 for index sequences.†See 36 79 for a plate map. 7Reference78SureSelect Methyl-Seq Target Enrichment System SureSelect Hyb 1tube with orange capbottleSureSelect Hyb 2tube with red captube with red capSureSelect Hyb 4tube with black captube with black capSureSelect Binding BufferbottlebottleSureSelect Wash Buffer 1bottlebottleSureSelect Wash Buffer 2bottlebottle*Kits received prior to May, 2018 may also contain SureSelect Elution Buffer. For optimal results, discard this component and NaOH for the elution step, as directed in the protocol on 53 SureSelect Hyb 3tube with yellow captube with yellow capSureSelect Indexing Block 1tube with green captube with green capSureSelect Block 2tube with blue captube with blue capSureSelect Methyl-Seq Block 3tube with brown captube with brown capSureSelect RNase Blocktube with purple captube with purple cap Reference7 SureSelect Methyl-Seq Target Enrichment System79 A01A02A03A04A05A06A07A08A09A10A11A12 B01B02B03B04B05B06B07B08B09B10B11B12 C01C02C03C04C05C06C07C08C09C10C11C12 D01D02D03D04D05D06D07D08D09D10D11D12 E01E02E03E04E05E06E07E08E09E10E11E12 F01F02F03F04F05F06F07F08F09F10F11F12 G01G02G03G04G05G06G07G08G09G10G11G12 H01H02H03H04H05H06H07H08H09H10H11H12 7Reference80SureSelect Methyl-Seq Target Enrichment System XT Indexes A01 to H12 73 for sequencing run setup requirements for sequencing libraries using 8-bp indexes.Table 37SureSelect A01ATGCCTAAA04AACTCACCA07ACGTATCAA10AATGTTGCB01GAATCTGAB04GCTAACGAB07GTCTGTCAB10TGAAGAGAC01AACGTGATC04CAGATCTGC07CTAAGGTCC10AGATCGCAD01CACTTCGAD04ATCCTGTAD07CGACACACD10AAGAGATCE01GCCAAGACE04CTGTAGCCE07CCGTGAGAE10CAACCACAF01GACTAGTAF04GCTCGGTAF07GTGTTCTAF10TGGAACAAG01ATTGGCTCG04ACACGACCG07CAATGGAAG10CCTCTATCH01GATGAATCH04AGTCACTAH07AGCACCTCH10ACAGATTCA02AGCAGGAAA05AACGCTTAA08CAGCGTTAA11CCAGTTCAB02GAGCTGAAB05GGAGAACAB08TAGGATGAB11TGGCTTCAC02AAACATCGC05CATCAAGTC08AGTGGTCAC11CGACTGGAD02GAGTTAGCD05AAGGTACAD08ACAGCAGAD11CAAGACTAE02CGAACTTAE05CGCTGATCE08CATACCAAE11CCTCCTGAF02GATAGACAF05GGTGCGAAF08TATCAGCAF11TGGTGGTAG02AAGGACACG05CCTAATCCG08ATAGCGACG11AACAACCAH02GACAGTGCH05CTGAGCCAH08ACGCTCGAH11AATCCGTCA03ATCATTCCA06AGCCATGCA09CTCAATGAA12CAAGGAGCB03GCCACATAB06GTACGCAAB09TCCGTCTAB12TTCACGCAC03ACCACTGTC06AGTACAAGC09AGGCTAACC12CACCTTACD03CTGGCATAD06ACATTGGCD09CCATCCTCD12AAGACGGAE03ACCTCCAAE06ATTGAGGAE09AGATGTACE12ACACAGAAF03GCGAGTAAF06GTCGTAGAF09TCTTCACAF12GAACAGGCG03ACTATGCAG06AGAGTCAAG09CCGAAGTAG12AACCGAGAH03CGGATTGCH06CCGACAACH09CGCATACAH12ACAAGCTA www.agilent.com This guide contains information to run the SureSelect XT Methyl-Seq Agilent Technologies, Inc. 2015, 2018Version E0, April 2018*G7530-90002 *G7530-90002 Agilent Technologies XT Methyl-Seq Target SurePrint Technology For Research Use Only. Not for use in diagnostic Hybridization4 SureSelect Methyl-Seq Target Enrichment System49Prepare the Methyl-Seq Capture Library Hybridization Mix according to Table 23Preparation of Capture Library Hybridization Mix Use of other SureSelect Capture Libraries is not supported by this protocol and requires optimization of amount of capture library added to the hybridization mix. Begin optimization of hybridization mixture components by using amounts shown in for Capture Libraries 3 Mb, and by using 2 µL of Capture Libraries ( µL using nuclease-free water).Additional protocol steps, including post-capture PCR cycling conditions may also require optimization. See the 10Maintain the DNA library + Block Mix plate or strip tube at 65°C while you add 20 µL of the Capture Library Hybridization Mix from step 9 to each sample well. Mix well by pipetting up and down 8 to 10 times.The hybridization reaction wells now contain approximately 27 to 29 µL, depending on the degree of evaporation during the thermal cycler incubation. step of 2 minute duration described in Reagent Volume for 1 reaction 13 µL221 µL2 µL34 µLSureSelect Human Methyl-Seq Capture Library5 µL85 µL Total 20 µL 340 µL NOTE Agilent Technologies SureSelectXT Methyl-Seq Target Enrichment System for ProtocolVersion E0, April 2018 SureSelect platform manufactured with Agilent SurePrint TechnologyFor Research Use Only. Not for use in diagnostic procedures.