EpiNext™ DNA Library Preparation Kit (Illumina)
Contents
1. EpiNext DNA Library Preparation Kit Illumina Base Catalog P 1051 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE Uses The EpiNext DNA Library Preparation Kit Illumina is suitable for preparing a DNA library for next generation sequencing applications using an Illumina sequencer which includes genomic DNA seq ChlP seg MeDIP hMeDIP seq bisulfite seg and targeted re sequencing The optimized protocol and components of the kit allow both non barcoded singleplexed and barcoded multiplexed DNA libraries to be constructed quickly with reduced bias Starting Material and Input Amount Starting materials can include fragmented dsDNA isolated from various tissue or cell samples dsDNA enriched from ChIP reaction MeDIP hMeDIP reaction or exon capture DNA should be relatively free of RNA since large fractions of RNA will impair end repair and dA tailing resulting in reduced ligation capabilities Input amount of DNA can be from 5 ng to 1 ug For optimal preparation the input amount should be 100 ng to 200 ng For amplification free 500 ng or more is needed Precautions To avoid cross contamination carefully pipette the sample or solution into the tube vials Use aerosol barrier pipette tips and always change pipette tips between liguid transfers Wear gloves throughout the entire procedure In case of contact between gloves and sample change gloves immediately 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page l Tel 1 872. Water 0 5 1 5 pl Total Volume 15 ul Mix and incubate for 30 min at 37 C followed by 10 min at 75 C in a thermocycler without heated lid 4 Adaptor Ligation a Prepare a reaction mix for adaptor ligation according to Table 3 Add the following reagents to a 0 2 ml PCR tube containing end repaired dA Tailing DNA from Step 3 Table 3 Adaptor Ligation Component Volume End repaired dA Tailing DNA from step 3 15 ul 2X Ligation Buffer 17 ul T4 DNA Ligase Tul Adaptors 1 ul Total volume 34 ul Mix and incubate for 10 min at 25 C in a thermocycler without heated lid Note 1 The pre annealed adapters included in the kit are suitable for both non barcoded singleplexed and barcoded multiplexed DNA library preparation and are fully compatible with Illumina platforms such as MiSeq or HiSeq sequencers 2 If using adaptors from other suppliers both single end and barcode adaptors make sure they are compatible with Illumina platforms and add the correct amount final concentration 1 5 2 uM or according to the supplier s instruction 5 Size Selection Clean up 5 1 Size Selection of Ligated DNA Note If the starting DNA amount is less than 50 ng the size selection is not recommended and alternatively clean up of ligated DNA can be performed prior to PCR amplification according to section 5 2 of the user guide a Resuspend MQ Binding Beads by vortex b Add 14 ul of resuspe
3. can be assessed using an Agilent Bioanalyzer or a comparable method Library fragments should have the correct size distribution ex 300 bps at peak size without adaptors or adaptor dimers To check the size distribution dilute library 5 fold with water and apply it to an Agilent high sensitivity chip If there is presence of lt 150 bp adaptor dimers or of larger fragments than expected they should be removed To remove fragments below 150 bps use 0 8X MQ Binding Beads Beads ex add 16 ul of MQ Binding Beads to 20 ul of sample according to sub steps a through i of Step 5 2 Clean up of Ligated DNA To remove fragments above 500 bps follow sub steps a through I of Step 5 1 Size Selection of Ligated DNA The prepared DNA library can be guantified using various DNA library guantification methods 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 9 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2013 12 31 Epigentek Group Inc All rights reserved Products are for research use only P 1051 The prepared library DNA can be stored at 20 C until ready to use for seguencing TROUBLESHOOTING Problem Possible Cause Suggestion Low yield of library Insufficient amount of starting DNA To obtain the best results the amount of input DNA should be 100 200 ng For library directly used for seguencing without amplification 500 ng or more is
4. needed Insufficient purity of starting DNA Ensure that RNA is removed by RNase treatment before starting library preparation protocol Improper reaction conditions at each reaction step Check if the reagents are properly added and incubation temperature and time are correct at each reaction step including End Repair dA Tailing Adaptor Ligation Size Selection and Amplification Improper kit storage Ensure that the kit has not exceeded the expiration date Standard shelf life when stored properly is 6 months from date of receipt Unexpected peak size of Agilent Bioanalyzer6 trace Presence of lt 150 bp adaptor dimers or presence of larger fragments than expected Improper ratio of MQ beads to DNA volume during size selection Check if the correct volume of MQ beads is added to the DNA solution accordingly Proper ratios should remove the fragments of unexpected peak size See Step 7 for more details Insufficient ligation Too much or too little input DNA may cause insufficient ligation which can shift the peak size of the fragment population to be shorter or larger than expected Make sure that the ligation reaction is properly processed using the proper amount of input DNA Over amplification of library PCR artifacts from over amplification of the library may cause the fragment population to shift higher than expected Make sure to use proper PCR cycles to avoid
5. stand until the solution is clear about 4 minutes Carefully remove and discard the supernatant Caution Be careful not to disturb or discard the beads that contain DNA Keep the PCR tube in the magnetic stand and add 200 ul of freshly prepared 80 ethanol to the tube Incubate at room temperature for 1 min and then carefully remove and discard the ethanol Repeat Step 2e for a total of two washes Open the cap of the PCR tube and air dry beads for 10 minutes while the tube is on the magnetic stand Resuspend the beads in 12 ul Elution Buffer and incubate at room temperature for 2 minutes to release the DNA from the beads Capture the beads by placing the tube in the magnetic stand for 4 minutes or until the solution is completely clear Transfer 11 12 ul of the supernatant to a new 0 2 ml PCR tube for the dA tailing reaction 3 DNA dA Tailing a Prepare the reaction mix for dA tailing according to Table 2 Add the following reagents to 0 2 ml PCR tube containing end repaired DNA from Step 1 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 6 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2013 12 31 Epigentek Group Inc All rights reserved Products are for research use only P 1051 Table 2 dA Tailing Reaction Component Volume End repaired DNA from step 2 11 12 ul 10X dA Tailing Buffer 1 5 ul Klenow Fragment 3 5 exo 1 ul Distilled
6. subject to change at any time without notice Thus only use the User Guide that was supplied with the kit when using that kit Usage Limitation The EpiNext DNA Library Preparation Kit Illumina is for research use only and is not intended for diagnostic or therapeutic application Intellectual Property The EpiNext DNA Library Preparation Kit Illumina and methods of use contain proprietary technologies by Epigentek A BRIEF OVERVIEW DNA library preparation is a critical step for next generation sequencing NGS For generating accurate sequencing data for NGS the prepared library DNA should be sufficient in yield and of high quality Also as NGS technology is continuously improving DNA library preparation is required to be optimized accordingly For example most of the currently used methods are time consuming expensive and inconvenient Some of the methods are relatively quick by combining end repair and dA tailing or even ligation in one step However these methods have been shown to generate significant G tailing or form concatemers at the ligation step or to have high insertion bias These side reactions eventually result in the prepared DNA library being less efficient and inaccurate An ideal DNA library preparation method should be balanced in speed convenience small sample suitability cost effectiveness and accuracy To address this issue Epigentek offers the EpiNext DNA Library Preparation Kit Illumina This kit ha
7. this problem RELATED PRODUCTS DNA Isolation and Cleanup P 1003 P 1004 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only FitAmp General Tissue Section DNA Isolation Kit FitAmp Plasma Serum DNA Isolation Kit Page 10 Printed 2013 12 31 P 1051 P 1006 DNA Concentrator Kit P 1007 FitAmp M Gel DNA Isolation Kit P 1009 FitAmp Paraffin Tissue Section DNA Isolation Kit P 1017 FitAmp Urine DNA Isolation Kit P 1018 FitAmp Blood and Cultured Cell DNA Extraction Kit Sonication Instruments EQC 1100 EpiSonic Multi Functional Bioprocessor 1100 DNA Enrichment Reaction P 1015 Methylamp Methylated DNA Capture MeDip Kit P 1038 EpiQuik Hydroxymethylated DNA Immunoprecipitation hMeDlP Kit P 1052 EpiQuik MeDIP Ultra Kit P 2002 EpiQuik Chromatin Immunoprecipitation ChIP Kit P 2003 EpiQuik Tissue Chromatin Immunoprecipitation ChIP Kit P 2014 EpiQuik Plant ChIP Kit P 2025 ChromaFlash One Step ChIP Kit P 2026 ChromaFlash One Step Magnetic ChIP kit P 2027 ChromaFlash High Sensitivity ChIP Kit DNA Bisulfite Conversion P 1001 Methylamp DNA Modification Kit P 1026 BisulFlash DNA Modification Kit DNA Library Prep P 1053 EpiNext High Sensitivity DNA Library Preparation Kit Illumina P 1055 EpiNext Post B
8. 7 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2013 12 31 Epigentek Group Inc All rights reserved Products are for research use only P 1051 KIT CONTENTS Component 12 reactions 24 reactions Storage Cat P 1051 12 Cat P 1051 24 Upon Receipt 10X End Repair Buffer 40 ul 80 ul 20 C End Repair Enzyme Mix 25 ul 50 ul 20 C 10X dA Tailing Buffer 40 ul 80 ul 20 C Klenow Fragment 3 5 exo 15 ul 30 ul 20 C 2X Ligation Buffer 250 ul 500 ul 20 C T4 DNA Ligase 15 pl 30 ul 20 C Adaptors 50 HM 15 ul 30 ul 20 C MQ Binding Beads 1 6 ml 3 2 ml 4 C 2X HiFi PCR Master Mix 160 ul 320 ul 20 C Primer U 10 uM 15 ul 30 ul 209C Primer I 10 HM 15 pl 30 ul 20 C Elution Buffer 1000 ul 2000 ul 20 C User Guide 1 1 RT Spin the solution down to the bottom prior to use SHIPPING amp STORAGE The kit is shipped on frozen ice packs at 4 C Upon receipt Store the following components at 20 C immediately 10X End Repair Buffer End Repair Enzyme Mix 10X dA Tailing Buffer Klenow Fragment 3 5 exo 2X Ligation Buffer T4 DNA Ligase Adaptors 2X HiFi PCR Master Mix Primer U Primer I and Elution Buffer Store the following components at 4 C MQ Binding Beads Store all other components at room temperature O Vortex mixer MATERIALS REQUIRED BUT NOT SUPPLIED Sonicator or enzymes for DNA fragme
9. beads Capture the beads by placing the tube in the magnetic stand for 4 minutes or until the solution is completely clear Transfer 11 ul of supernatant to a new 0 2 ml PCR tube for PCR amplification 5 2 Clean up of Ligated DNA Optional j Resuspend MQ Binding Beads by vortex Add 34 ul of resuspended beads to the PCR tube of ligation reaction Mix thoroughly on a vortex mixer or by pipetting up and down at least 10 times Incubate for 5 minutes at room temperature to allow DNA to bind to beads Put the PCR tube on an appropriate magnetic stand until the solution is clear about 4 minutes Carefully remove and discard the supernatant Caution Be careful not to disturb or discard the beads that contain DNA Keep the PCR tube in the magnetic stand and add 200 ul of freshly prepared 80 ethanol to the tube Incubate at room temperature for 1 min and then carefully remove and discard the ethanol Repeat Step 5 2e two times for a total of three washes Open the PCR tube cap and air dry beads for 10 minutes while the tube is on the magnetic stand Resuspend the beads in 12 ul Elution Buffer and incubate at room temperature for 2 minutes to release the DNA from the beads Capture the beads by placing the tube in the magnetic stand for 4 minutes or until the solution is completely clear Transfer 11 ul of supernatant to a new 0 2 ml PCR tube for PCR amplification If the library is prepared using 500 ng or more input DNA the librar
10. c Final Extension 72 C 1 min 1 PCR cycles may vary depending on the input DNA amount In general use 8 PCR cycles for 500 ng 12 cycles for 50 ng and 16 cycles for 5 ng DNA input Further optimization of PCR cycle number may be reguired 7 Clean up of Amplified Library DNA j Resuspend MG Binding Beads by vortex Add 25 ul of resuspended beads to the PCR tube of amplification reaction Mix thoroughly on a vortex mixer or by pipetting up and down at least 10 times Incubate for 5 minutes at room temperature to allow DNA to bind to beads Put the PCR tube on an appropriate magnetic stand until the solution is clear about 4 minutes Carefully remove and discard the supernatant Caution Be careful not to disturb or discard the beads that contain DNA Keep the PCR tube in the magnetic stand and add 200 ul of freshly prepared 80 ethanol to the tube Incubate at room temperature for 1 min and then carefully remove and discard the ethanol Repeat Step 7e two times for total of three washes Open the PCR tube cap and air dry beads for 10 minutes while the tube is on the magnetic stand Resuspend the beads in 22 ul Elution Buffer and incubate at room temperature for 2 minutes to release the DNA from the beads Capture the beads by placing the tube in the magnetic stand for 4 minutes or until the solution is completely clear Transfer 20 ul of supernatant to a new 0 2 ml PCR tube Quality of the prepared library
11. d with use of 500 ng or more input DNA PRINCIPLE amp PROCEDURE The EpiNext DNA Library Preparation Kit Illumina contains all reagents required at each step for carrying out successful DNA library preparation In the library preparation DNA is first fragmented to the appropriate size about 300 bp peak size The end repair of the DNA fragments is performed and an A overhang is added at the 3 end of each strand Adaptors are then ligated to both ends of the end repaired dA tailed DNA fragments for amplification and seguencing Fragments are then size selected and purified with MO beads which allows guick and precise size selection of DNA Size selected DNA fragments are then amplified with a high fidelity PCR Mix which ensures maximum yields from minimum amounts of starting material and provides highly accurate amplification of library DNA with low error rates and minimum bias Next Generation eguencing Fig 1 Workflow of the EpiNext DNA Library Preparation Kit Illumina 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 4 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2013 12 31 Epigentek Group Inc All rights reserved Products are for research use only P 1051 404 30 204 bi eaea aan a 50 300 500 700 1500 10380 bp Fig2 Size distribution of library fragments Human placenta DNA was sheared to 210 bps in peak size and 20
12. isulfite DNA Library Preparation Kit Illumina P 1056 EpiNext Bisulfite Seq High Sensitivity Kit Illumina NGS Barcode P 1060 EpiNext NGS Barcode Index Set 12 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 11 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2013 12 31 Epigentek Group Inc All rights reserved Products are for research use only P 1051
13. nded MQ Binding Beads to the tube of ligation reaction Mix well by pipetting up and down at least 10 times Cc Incubate for 5 minutes at room temperature d Put the tube on an appropriate magnetic stand until the solution is clear about 4 minutes Carefully transfer the supernatant containing DNA to a new tube Caution Do not discard the supernatant Discard the beads that contain the unwanted large fragments e Add 10 ul resuspended beads to the supernatant mix well and incubate for 5 minutes at room temperature 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 7 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2013 12 31 Epigentek Group Inc All rights reserved Products are for research use only P 1051 Put the PCR tube on an appropriate magnetic stand until the solution is clear about 4 minutes Carefully remove and discard the supernatant Caution Be careful not to disturb or discard the beads that contain DNA Keep the PCR tube in the magnetic stand and add 200 ul of freshly prepared 80 ethanol to the tube Incubate at room temperature for 1 min and then carefully remove and discard the ethanol Repeat Step 5 1g for a total of two washes Open the PCR tube cap and air dry beads for 10 minutes while the tube is on the magnetic stand Resuspend the beads in 12 ul Elution Buffer and incubate at room temperature for 2 minutes to release the DNA from the
14. ng of sheared DNA was used for DNA library preparation using EpiNext DNA Library Preparation Kit Illumina ASSAY PROTOCOL For the best results please read the protocol in its entirety prior to starting your experiment Starting Materials Fragmented dsDNA that is isolated from various tissues or cell samples 5 ng 1 yg optimal 100 200 ng per preparation For amplification free 500 ng or more of DNA is needed dsDNA enriched from ChIP reactions MeDIP hMeDIP reactions or exon capture 5 ng 500 ng DNA should be high quality relatively free of RNA RNAse can be used to remove RNA and DNA should be eluted in DNase RNase free water DNA Fragmentation dsDNA enriched from ChIP reactions MeDIP hMeDIP reactions or exon capture should be fragmented already DNA isolated from various tissue or cell samples can be fragmented using one of the following methods For the best results we highly recommend to use waterbath based sonication device The peak size of fragmented DNA should be compatible with the read length of the Illumina sequencing platform to be used In general the peak size of fragments should be about 300 bps Waterbath Sonication Epigentek s EpiSonic 1100 Epigentek Cat No EQC 1100 For a target peak size of 300 bps use 20 ul of DNA solution 50 500 ng per 0 2 ml tube or per PCR plate well Shear 60 cycles under cooling conditions 30 seconds ON 15 seconds OFF each at 120 130 watts For more detailed information of u
15. ntation Agilent Bioanalyzer or comparable method to assess the quality of DNA library Thermocycler Centrifuge including desktop centrifuge up to 14 000 rpm Ll Ll Ll Ll O Magnetic stand 96 well format O Pipettes and pipette tips O PCR tubes or plates O 1 5 ml microcentrifuge tubes Ll 10095 ethanol 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Page 2 Printed 2013 12 31 P 1051 O Distilled water O DNA sample GENERAL PRODUCT INFORMATION Quality Control Each lot of EpiNext DNA Library Preparation Kit Illumina is tested against predetermined specifications to ensure consistent product quality Epigentek guarantees the performance of all products in the manner described in our product instructions Product Warranty If this product does not meet your expectations simply contact our technical support unit or your regional distributor We also encourage you to contact us if you have any suggestions about product performance or new applications and techniques Safety Suitable lab coat disposable gloves and proper eye protection are required when working with this product Product Updates Epigentek reserves the right to change or modify any product to enhance its performance and design The information in this User Guide is
16. s the following features e Fast and streamlined procedure The procedure from fragmented DNA to size selection is less than 2 h 30 min Only one clean up between each step thereby saving time and preventing handling errors as well as loss of valuable samples Gel free size selection further reduces the preparation time e The most convenient for use The kit contains all required components for each step of DNA library preparation which are sufficient for end repair dA tailing ligation clean up size selection and library amplification thereby allowing the library preparation to be convenient with reliable and consistent results e Minimized bias Ultra HiFi amplification and an optional PCR free step enable the user to achieve reproducibly high yields of DNA library with minimal sequence bias and low error rates 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 3 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2013 12 31 Epigentek Group Inc All rights reserved Products are for research use only P 1051 e Flexibility Can be used for both non barcoded singleplexed and barcoded multiplexed DNA library preparation Uses various dsDNA including fragmented dsDNA isolated from various tissue or cell samples dsDNA enriched from ChIP reactions MeDIP hMeDIP reactions or exon capture Broad range of input DNA from 5 ng to 1 ug PCR free library preparation can be performe
17. se please see the DNA Shearing Protocol for EpiSonic 1100 If using other waterbath sonicators please follow the supplier s recommended instructions 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 5 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2013 12 31 Epigentek Group Inc All rights reserved Products are for research use only P 1051 Enzymatic Shearing The DNA can also be sheared using various enzyme based methods Optimization of the shearing conditions for example enzyme concentration and incubation time is needed in order to use enzyme based methods 1 DNA End Repairing a b Prepare end repair reaction in a 0 2 ml PCR tube according to Table 1 Table 1 End Repair Reaction Component Volume Fragmented DNA 5 500 ng 2 10 ul 10X End Repair Buffer 2 ul End Repair Enzyme Mix 1 ul Distilled Water 7 15 ul Total volume 20 ul Note The optimized amount of fragmented DNA is 100 200 ng Mix and incubate for 30 min at 20 C in a thermocycler without a heated lid 2 Clean up of End Repaired DNA j Resuspend MG Binding Beads by vortex Add 36 ul of resuspended beads to the PCR tube of end repair reaction Mix thoroughly on a vortex mixer or by pipetting up and down at least 10 times Incubate for 10 minutes at room temperature to allow DNA to bind to beads Put the PCR tube on an appropriate magnetic
18. y DNA can be directly used for sequencing application after size selection Otherwise go to Step 6 for PCR amplification 6 Library Amplification a Prepare the PCR Reactions Thaw all reaction components including 2X HiFi PCR Master Mix primers and DNA template Mix well by vortexing briefly Keep components on ice while in use and return to 20 C immediately following use Add components into each PCR tube well according to the following table Component Size pl 2X HiFi PCR Master Mix 12 5 ul Primer U 1 ul Primer I or barcode 1 ul Adaptor Ligated DNA 10 5 ul Total Volume 25 ul 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 8 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2013 12 31 Epigentek Group Inc All rights reserved Products are for research use only P 1051 Important Note Use of Primer I included in the kit will generate a singleplexed library For multiplexed library preparation replace Primer I with one of the12 different barcodes indexes contained in the EpiNext NGS Barcode Index Set 12 Cat No P 1060 You can also add user defined barcodes Illumina compatible instead of Primer I Program the PCR Reactions Place the reaction plate in the instrument and set the PCR conditions as follow Cycle Step Temp Time Cycle Activation 98 C 30 sec 1 98 C 10 sec Cycling 55 C 15 sec Variable 72 20 se
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