HiSeq 3000/4000 Cluster Kit Reference Guide
Transcription
HiSeq 3000/4000 Cluster Kit Reference Guide
HiSeq® 3000/4000 Cluster Kit Reference Guide FOR RESEARCH USE ONLY Reagent Kit Overview Kit Contents and Storage Requirements cBot Reagent Plate User-Supplied Consumables Patterned Flow Cell Prepare the Flow Cell Prepare Clustering Reagents Next Steps for Clustering Prepare Indexing and Paired-End Reagents Paired-End and Indexing Reagent Positions Next Steps for Sequencing Technical Assistance ILLUMINA PROPRIETARY Part # 15066495 Rev. A Catalog # PE-410-9001DOC February 2015 3 4 6 7 8 9 11 16 17 18 19 This document and its contents are proprietary to Illumina, Inc. and its affiliates ("Illumina"), and are intended solely for the contractual use of its customer in connection with the use of the product(s) described herein and for no other purpose. This document and its contents shall not be used or distributed for any other purpose and/or otherwise communicated, disclosed, or reproduced in any way whatsoever without the prior written consent of Illumina. Illumina does not convey any license under its patent, trademark, copyright, or commonlaw rights nor similar rights of any third parties by this document. The instructions in this document must be strictly and explicitly followed by qualified and properly trained personnel in order to ensure the proper and safe use of the product(s) described herein. All of the contents of this document must be fully read and understood prior to using such product(s). FAILURE TO COMPLETELY READ AND EXPLICITLY FOLLOW ALL OF THE INSTRUCTIONS CONTAINED HEREIN MAY RESULT IN DAMAGE TO THE PRODUCT(S), INJURY TO PERSONS, INCLUDING TO USERS OR OTHERS, AND DAMAGE TO OTHER PROPERTY. ILLUMINA DOES NOT ASSUME ANY LIABILITY ARISING OUT OF THE IMPROPER USE OF THE PRODUCT(S) DESCRIBED HEREIN (INCLUDING PARTS THEREOF OR SOFTWARE) OR ANY USE OF SUCH PRODUCT(S) OUTSIDE THE SCOPE OF THE EXPRESS WRITTEN LICENSES OR PERMISSIONS GRANTED BY ILLUMINA IN CONNECTION WITH CUSTOMER'S ACQUISITION OF SUCH PRODUCT (S). FOR RESEARCH USE ONLY © 2015 Illumina, Inc. All rights reserved. Illumina, 24sure, BaseSpace, BeadArray, BlueFish, BlueFuse, BlueGnome, cBot, CSPro, CytoChip, DesignStudio, Epicentre, GAIIx, Genetic Energy, Genome Analyzer, GenomeStudio, GoldenGate, HiScan, HiSeq, HiSeq X, Infinium, iScan, iSelect, MiSeq, NeoPrep, Nextera, NextBio, NextSeq, Powered by Illumina, SeqMonitor, SureMDA, TruGenome, TruSeq, TruSight, Understand Your Genome, UYG, VeraCode, verifi, VeriSeq, the pumpkin orange color, and the streaming bases design are trademarks of Illumina, Inc. and/or its affiliate(s) in the U.S. and/or other countries. All other names, logos, and other trademarks are the property of their respective owners. To cluster a flow cell on the cBot for subsequent sequencing on the HiSeq 4000 or HiSeq 3000, you need 1 HiSeq 3000/4000 PE Cluster Kit. Kit Name Catalog # HiSeq 3000/4000 PE Cluster Kit PE-410-1001 NOTE The kit includes a denaturation step before clustering on the cBot. Libraries are denatured in the 8-tube strip before adding the ExAmp reaction mix. HiSeq 3000/4000 Cluster Kit Reference Guide 3 Reagent Kit Overview Reagent Kit Overview Kit Contents and Storage Requirements When you receive your kit, promptly store the kit components at the indicated temperature to ensure proper performance. Reagents are sensitive to light. Store reagents in a dark location away from light. Cluster Kit Box 1 Box 1 contains components used on the cBot. RSB is used to dilute libraries. Reagent Quantity Storage Description cBot Reagent Plate 1 -25°C to -15°C cBot reagent plate, PE RSB 1 -25°C to -15°C Resuspension Buffer EPX1 1 -25°C to -15°C Enhanced Pattern Cluster Mix 1 EPX2 1 -25°C to -15°C Enhanced Pattern Cluster Mix 2 EPX3 1 -25°C to -15°C Enhanced Pattern Cluster Mix 3 4 Part # 15066495 Rev. A Box 2 contains reagents and sequencing primers used for indexing and paired-end resynthesis on the HiSeq. Reagent Quantity Storage Description HRM 1 -25°C to -15°C HT Resynthesis Mix HLM2 1 -25°C to -15°C HT Linearization Mix 2 HAM 1 -25°C to -15°C HT Amplification Mix HPM 1 -25°C to -15°C HT Amp Premix HDR 1 -25°C to -15°C HT Denaturation Mix (contains formamide) HP11 1 -25°C to -15°C Primer Mix – Read 2 HP14 1 -25°C to -15°C Indexing Primer Mix Accessory Kit The accessory kit contains components used for setting up a sequencing run on the HiSeq. Accessory Quantity Purpose Flow cell gaskets (package of 4) 1 Use to replace the flow cell gaskets between runs; replace before a maintenance wash. Flow cell storage tube with storage buffer, 50 ml 1 Use to store the flow cell after clustering and before sequencing. Funnel bottle caps (package of 8) 1 Use to replace SBS reagent bottle caps before loading reagents onto the instrument. HiSeq 3000/4000 Cluster Kit Reference Guide 5 Kit Contents and Storage Requirements Cluster Kit Box 2 cBot Reagent Plate Figure 1 cBot Reagent Plate The cBot reagent plate contains 12 rows with 8 deep wells each. Each reagent occupies a full row of 8 wells. Not all rows of the reagent plate contain reagent. WARNING This set of reagents contains formamide, an aliphatic amide that is a probable reproductive toxin. Personal injury can occur through inhalation, ingestion, skin contact, and eye contact. Dispose of containers and any unused contents in accordance with the governmental safety standards for your region. For more information, see the SDS for this kit at support.illumina.com/sds.html. 6 Part # 15066495 Rev. A The following user-supplied consumables are used for preparation and loading of reagents. Component Supplier Purpose 1 N NaOH General lab supplier Use for library denaturation step. 200 mM Tris-HCl, pH 8.0 General lab supplier Use for library denaturation step after diluting to 0.1 N NaOH. 8-tube strip with caps, 0.2 ml Fisher Scientific, catalog # AB-0264* Use for the ExAmp Reaction and library mix on the cBot. Microcentrifuge tube, 1.5 ml VWR, catalog #20170-038* Use for preparation of the ExAmp Reaction master mix. Laboratory-grade water Millipore or General lab supplier Use for library denaturation step. *or equivalent HiSeq 3000/4000 Cluster Kit Reference Guide 7 User-Supplied Consumables User-Supplied Consumables Patterned Flow Cell The flow cell used on the HiSeq 4000 and HiSeq 3000 is a patterned flow cell with billions of ordered nanowells. The wells are manufactured into the glass of the flow cell allowing for the generation of sequencing clusters in an ordered arrangement. Clusters are aligned closely together, increasing the number of output reads and amount of sequencing data generated. The patterned flow cell contains 8 lanes with 2 swaths per lane, and has the same general dimensions as other HiSeq high-output flow cells. The patterned flow cell is provided in the HiSeq 3000/4000 PE Cluster Kit, and is shipped dry in a foil-wrapped package. Inside the foil package, the flow cell is packaged in a protective plastic clamshell case. Figure 2 Example of Clusters on a Patterned Flow Cell 8 Part # 15066495 Rev. A 1 Remove a new flow cell package from 2°C to 8°C storage. Do not remove the flow cell from the packaging. 2 Set the flow cell package aside at room temperature for at least 30 minutes. NOTE If the foil packaging is intact, the flow cell can remain at room temperature up to 12 hours. You can return the packaged flow cell to 2°C to 8°C storage for later use 1 time only. Avoid repeated cooling and warming of the flow cell. 3 Put on a new pair of powder-free gloves. 4 Peel open the foil package from the end with the angled seal. Figure 3 Open Foil Packaging HiSeq 3000/4000 Cluster Kit Reference Guide 9 Prepare the Flow Cell Prepare the Flow Cell 5 Remove the clear clamshell package from the foil packaging. Figure 4 Remove From Foil Packaging 6 Open the clear plastic clamshell packaging and remove the flow cell. Figure 5 Remove Flow Cell From Clamshell Package 7 Using a lint-free alcohol wipe or a lint-free tissue moistened with ethanol or isopropanol, clean the glass surface of the flow cell. Dry the glass with a lint-free tissue or lens paper. 8 Set aside until you are ready to load the flow cell onto the cBot when prompted by the cBot software. 10 Part # 15066495 Rev. A Clustering reagents provided in the HiSeq 3000/4000 PE Cluster Kit are used on the cBot. To prepare reagents, thaw the cBot reagent plate and prepare the ExAmp master mix. Best Practices } Wear a fresh pair of gloves when preparing clustering reagents. } The clear plastic lid on the reagent plate protects the foil seal from being damaged or punctured during thawing. Remove the lid only when you are ready to load the cBot reagent plate onto the cBot. } Always prepare freshly diluted NaOH for denaturing libraries for cluster generation. This step is essential to the denaturation process. } To prevent small pipetting errors from affecting the final NaOH concentration, prepare at least 1 ml of freshly diluted 0.1 N NaOH. } After completing the reagent preparation steps, proceed to setting up the cBot for clustering and load reagents when prompted. Thaw the cBot Reagent Plate The cBot reagent plate takes approximately 60 minutes to thaw using a room temperature water bath. Alternatively, you can thaw reagents at 2°C to 8°C overnight, not to exceed 16 hours. 1 Remove the cBot reagent plate from -25°C to -15°C storage. 2 Place the reagent plate in a water bath containing enough room temperature deionized water to submerge only the reagent plate base. Allow reagents to thaw for 60 minutes. Make sure that reagents have thawed before proceeding. Thaw EPX1, EPX2, EPX3, and RSB The following reagents are used to create the ExAmp master mix and dilute libraries. 1 Remove EPX1, EPX2, EPX3, and RSB from -25°C to -15°C storage. 2 Thaw each tube at room temperature for approximately 10 minutes. Do not vortex. 3 When thawed, place on ice until you are ready to prepare the ExAmp master mix. HiSeq 3000/4000 Cluster Kit Reference Guide 11 Prepare Clustering Reagents Prepare Clustering Reagents Prepare a Fresh Dilution of NaOH CAUTION Using freshly diluted NaOH is essential in denaturing libraries for cluster generation on the HiSeq. 1 Prepare 1 ml of 0.1 N NaOH by combining the following volumes in a microcentrifuge tube: • Laboratory-grade water (900 µl) • Stock 1 N NaOH (100 µl) 2 Invert the tube several times to mix. NOTE A fresh dilution of 0.1 N NaOH is required for denaturing libraries and a PhiX control. After denaturing libraries, you can set aside the remaining NaOH to prepare a PhiX control within the next 12 hours. Otherwise, discard the remaining dilution of NaOH. Denature Libraries and Add Optional PhiX Control The library loading concentration depends on the libraries to be sequenced. The following instructions apply to supported Illumina libraries and assume an insert size typical for the associated library type. Make sure that you dilute to a concentration appropriate for the library type. } Too high DNA loading concentration leads to reduced %PF. } Too low DNA loading concentration leads to reduced or unacceptable %PF, and high % duplicates that negatively affect depth of coverage. 1 Dilute the library or pooled libraries to the appropriate concentration for the library type. Library Type Dilution TruSeq Nano DNA Dilute to 2–3 nM in RSB. TruSeq DNA PCR-Free Dilute to 1–2 nM in RSB. Nextera Rapid Capture Exome Dilute to 2–3 nM in RSB. TruSeq Stranded Total RNA Dilute to 2–3 nM in RSB. TruSeq Stranded mRNA Dilute to 2–3 nM in RSB. 12 Part # 15066495 Rev. A [Optional] Spike-in 1% nondenaturedIllumina PhiX control to nondenatured libraries. Library Type Spike-In TruSeq Nano DNA Add 200–300 pM PhiX to 2–3 nM library. TruSeq DNA PCR-Free Add 100–200 pM PhiX to 1–2 nM library. Nextera Rapid Capture Exome Add 200–300 pM PhiX to 2–3 nM library. TruSeq Stranded Total RNA Add 200–300 pM PhiX to 2–3 nM library. TruSeq Stranded mRNA Add 200–300 pM PhiX to 2–3 nM library. 3 Label a new 8-tube strip #1 through #8. 4 To denature the library, add the following volumes to the labeled 8-tube strip in the order listed: a Add 5 µl of nondenatured library into the bottom of each well. b Add 5 µl of freshly diluted 0.1 N NaOH. Using a P10 or P20 pipette set to 5 µl, slowly pipette up and down 10 times to mix the reaction. c Incubate for 8 minutes at room temperature. d Add 5 µl of 200 mM Tris-HCl pH 8.0. Using a P10 or P20 pipette set to 5 µl, slowly pipette up and down 10 times to mix the reaction. 5 Cap and set aside the 8-tube strip containing the denatured library on ice until you are ready to add the ExAmp master mix. CAUTION To ensure proper performance, do not exceed 30 minutes before adding the ExAmp master mix. Prepare the cBot Reagent Plate 1 Invert the reagent plate several times to mix the thawed reagents. 2 Vortex the plate for approximately 10 seconds to dislodge trapped air bubbles. 3 Tap the reagent plate on a hard surface 5–10 times to collect reagent droplets at the bottom of the tubes. Alternatively, pulse centrifuge the reagent plate. HiSeq 3000/4000 Cluster Kit Reference Guide 13 Prepare Clustering Reagents 2 4 Set the reagent plate aside on ice until you are ready to load it onto the cBot. Make sure that you remove the clear plastic cover from the cBot reagent plate before loading, and do not puncture the foil seals. Prepare the ExAmp Reaction Prepare the ExAmp reaction master mix immediately before use. CAUTION Do not refreeze ExAmp reagents after thawing. 1 Invert each tube of EPX1 and EPX2 several times each to make sure that the reagents are thoroughly mixed. NOTE EPX3 does not move when inverted. This condition is normal due to the viscosity of EPX3. 2 Briefly centrifuge EPX1, EPX2, and EPX3. Do not vortex. 3 Add the following volumes to a 1.5 ml tube in the order listed: a Add 210 µl EPX1. b Add 30 µl EPX2. Using a P1000 pipette set to 200 µl, slowly pipette up and down 10 times to mix the reaction. Do not vortex. NOTE Due to the viscosity of ExAmp reagents, especially EPX2 and EPX3, aspirate and dispense reagents slowly to ensure accurate pipetting. c d Add 110 µl EPX3. Using a P1000 pipette set to 200 µl, slowly pipette up and down 10 times to mix the reaction. Make sure that you do not introduce air bubbles. Briefly centrifuge the reaction. Do not vortex. NOTE The solution can be cloudy, which is normal. If the mixture separates into a cloudy portion and a transparent portion, repeat the pipette mixing step to make sure that the solution is uniform. 4 Add 35 µl of the master mix into the bottom of each well of the 8-tube strip containing denatured and neutralized libraries. Change tips between samples. 5 Using a multichannel P100 or P200 pipette set to 40 µl, slowly pipette up and down 10 times to mix the reaction. Do not vortex. 14 Part # 15066495 Rev. A Make sure that the tubes are capped, and then briefly centrifuge the 8-tube strip. 7 Set aside the 8-tube strip containing the ExAmp master mix and library solution on ice until you are ready to load it onto the cBot. CAUTION Do not exceed 15 minutes before loading the 8-tube strip containing the ExAmp master mix and library solution onto the cBot. 8 Promptly proceed to the cBot setup steps. When prompted by the cBot software, carefully remove the caps and place the 8-tube strip in the template row on the cBot. HiSeq 3000/4000 Cluster Kit Reference Guide 15 Prepare Clustering Reagents 6 Next Steps for Clustering After you have prepared the flow cell and clustering reagents, you are ready to load them onto the cBot when prompted by the cBot software. For cBot workflow instructions, see the cBot System Guide (part # 15006165). Clustering takes approximately 3 hours. After clustering, the patterned flow cell can be stored in the flow cell storage buffer provided in the kit for up to 48 hours at 2°C to 8°C. During clustering on the cBot, prepare SBS and paired-end reagents for the HiSeq 4000 or HiSeq 3000. For SBS reagent preparation instructions, see the HiSeq 3000/4000 SBS Kit Reference Guide (part # 15066494). 16 Part # 15066495 Rev. A Indexing and paired-end reagents are loaded onto the instrument at the beginning of the run and used during indexing reads and the Read 2 resynthesis step of a sequencing run. WARNING This set of reagents contains formamide, an aliphatic amide that is a probable reproductive toxin. Personal injury can occur through inhalation, ingestion, skin contact, and eye contact. Dispose of containers and any unused contents in accordance with the governmental safety standards for your region. For more information, see the SDS for this kit at support.illumina.com/sds.html. Thaw Indexing and Paired-End Reagents 1 Remove the following reagents from -25°C to -15°C storage: HAM, HDR, HLM2, HP11, HP14, HPM, and HRM. NOTE For non-indexed libraries, HP14 is not required. 2 Thaw reagents in a beaker filled with room temperature deionized water for about 20 minutes, or until reagents are fully thawed. 3 After reagents have thawed, place HAM, HLM2, and HRM on ice. Do not place HDR on ice. Prepare HAM, HDR, HLM2, HP11, HP14, HPM, and HRM 1 Invert each tube several times to mix the reagent. 2 Centrifuge the reagent at 1000 rpm for 1 minute. 3 For HAM, HLM2, and HRM—Set aside on ice. For HDR, HP11, HP14, and HPM—Set aside at room temperature. HiSeq 3000/4000 Cluster Kit Reference Guide 17 Prepare Indexing and Paired-End Reagents Prepare Indexing and Paired-End Reagents Paired-End and Indexing Reagent Positions Use the following information for reference. For reagent loading instructions, see the system guide for the HiSeq 4000 or HiSeq 3000. Figure 6 Paired-End Reagent Rack Table 1 Paired-End Reagent Positions Position Reagent Description 10 ml PW1 or laboratory-grade water 19 PW1 10 ml PW1 or laboratory-grade water 18 PW1 Indexing Primer Mix 17 HP14 Primer Mix – Read 2 16 HP11 HT Denaturation Reagent 15 HDR HT Amplification Premix 14 HPM HT Amplification Mix 13 HAM 10 ml PW1 or laboratory-grade water 12 PW1 HT Linearization Mix 2 11 HLM2 HT Resynthesis Mix 10 HRM 18 Part # 15066495 Rev. A After you have prepared paired-end reagents, you are ready to load them onto the HiSeq. For reagent loading and sequencing workflow instructions, see the HiSeq 4000 System Guide (part # 15066496) or the HiSeq 3000 System Guide (part # 15066493). Make sure that you use the compatible version of SBS kit and HiSeq Control Software (HCS). Cluster Kit Version SBS Kit Version HCS/RTA Version HiSeq 3000/4000 PE Cluster Kit HiSeq 3000/4000 SBS Kit HCS v3.3/RTA v2.3, or later HiSeq 3000/4000 Cluster Kit Reference Guide 19 Next Steps for Sequencing Next Steps for Sequencing Notes For technical assistance, contact Illumina Technical Support. Table 2 Illumina General Contact Information Website Email www.illumina.com [email protected] Table 3 Illumina Customer Support Telephone Numbers Region Contact Number Region North America 1.800.809.4566 Italy Australia 1.800.775.688 Netherlands Austria 0800.296575 New Zealand Belgium 0800.81102 Norway Denmark 80882346 Spain Finland 0800.918363 Sweden France 0800.911850 Switzerland Germany 0800.180.8994 United Kingdom Ireland 1.800.812949 Other countries Contact Number 800.874909 0800.0223859 0800.451.650 800.16836 900.812168 020790181 0800.563118 0800.917.0041 +44.1799.534000 Safety Data Sheets Safety data sheets (SDSs) are available on the Illumina website at support.illumina.com/sds.html. Product Documentation Product documentation in PDF is available for download from the Illumina website. Go to support.illumina.com, select a product, then click Documentation & Literature. HiSeq 3000/4000 Cluster Kit Reference Guide Technical Assistance Technical Assistance *15066495* Part # 15066495 Rev. A Illumina San Diego, California 92122 U.S.A. +1.800.809.ILMN (4566) +1.858.202.4566 (outside North America) [email protected] www.illumina.com