NextSeq System Denature and Dilute Libraries Guide …

1 NextSeqSystemDenature andDilute LibrariesGuideOverview3 Consumables and Equipment4 ProtocolA: StandardNormalizationMethod4 ProtocolB: Bead-BasedNormalizationMethod6 ProtocolC: AmpliSeqfor IlluminaPanelsNormalizationMethod7 ProtocolD: AmpliSeqLibraryEqualizerfor Illumina NormalizationMethod9 ProtocolE: TruSight Tumor170 LibraryDenaturationand DilutionMethod11 Protocol F: TruSight Oncology500 LibraryDenaturationand DilutionMethod12 Denature andDilute PhiXControl (ProtocolsA D)13 Denature andDilute PhiXControl (ProtocolE)15 Denature andDilute PhiXControl (Protocol F)16 Next Steps18 PreparePhiX for a TroubleshootingRun18 RevisionHistory20 TechnicalAssistance21 Document#15048776v09 December2018 ILLUMINAPROPRIETARYFor Research UseOnly. Not for use in diagnostic document and its contents are proprietary to Illumina, Inc. and its affiliates ("Illumina"), and are intended solely forthe contractual use of its customer in connection with the use of the product(s) described herein and for no otherpurpose.

2 This document and its contents shall not be used or distributed for any other purpose and/or otherwisecommunicated, disclosed, or reproduced in any way whatsoever without the prior written consent of Illumina. Illuminadoes not convey any license under its patent, trademark, copyright, or common-law rights nor similar rights of any thirdparties by this instructions in this document must be strictly and explicitly followed by qualified and properly trained personnel inorder to ensure the proper and safe use of the product(s) described herein. All of the contents of this document mustbe fully read and understood prior to using such product(s).FAILURE TO COMPLETELY READ AND EXPLICITLY FOLLOW ALL OF THE INSTRUCTIONS CONTAINED HEREINMAY RESULT IN DAMAGE TO THE PRODUCT(S), INJURY TO PERSONS, INCLUDING TO USERS OR OTHERS,AND DAMAGE TO OTHER PROPERTY, AND WILL VOID ANY WARRANTY APPLICABLE TO THE PRODUCT(S).ILLUMINA DOES NOT ASSUME ANY LIABILITY ARISING OUT OF THE IMPROPER USE OF THE PRODUCT(S)DESCRIBED HEREIN (INCLUDING PARTS THEREOF OR SOFTWARE).

3 2018 Illumina, Inc. All rights trademarks are the property of Illumina, Inc. or their respective owners. For specific trademark information, #15048776v09 For Research Use Only. Not for use in diagnostic Denature andDilute Libraries GuideOverviewThis Guide explains how to Denature and Dilute prepared Libraries for sequencing on the Illumina NextSeq Guide includes instructions for preparing a PhiX library for the following purposes:uFor a control Prepare a PhiX library to combine with prepared Libraries for use as a sequencingcontrol. PhiX control protocols starton page troubleshooting Prepare a PhiX library for a PhiX-only sequencing run for troubleshootingpurposes. SeePrepare PhiX for a Troubleshooting Runon page procedure denatures and dilutes Libraries to a final loading volume of ml at a recommendedconcentration of pM for high output kits and pM for mid output kits. In practice, loadingconcentration can vary depending on library preparation and quantification the appropriate Denature and Dilute protocol depending on the procedure used during library normalization Libraries are normalized using standard library quantification and qualitycontrol procedures recommended in the library prep documentation.

4 For these Libraries , followProtocol A. SeeProtocol A: Standard Normalization Methodon page normalization Libraries are normalized using a bead-based procedure described in thelibrary prep documentation for methods that support bead-based normalization. For these Libraries ,followProtocol B. SeeProtocol B: Bead-Based Normalization Methodon page TruSight Tumor 170 Libraries , followProtocol E: TruSight Tumor 170 Library Denaturationand Dilution Methodon page TruSight Oncology 500 Libraries , followProtocol F: TruSight Oncology 500 LibraryDenaturation and Dilution Methodon page 12.)uAmpliSeq for Illumina normalization For all Libraries prepared using the standard AmpliSeq forIllumina workflow, followProtocol C. SeeProtocol C: AmpliSeq for Illumina Panels NormalizationMethodon page Library Equalizer for Illumina normalization For all Libraries prepared using theAmpliSeq Library Equalizer for Illumina workflow, followProtocol D. SeeProtocol D: AmpliSeqLibrary Equalizer for Illumina Normalization Methodon page Tumor 170 library denaturation and dilution protocol For TruSight Tumor 170 Libraries ,followProtocol E.

5 SeeProtocol E: TruSight Tumor 170 Library Denaturation and Dilution Methodonpage Oncology 500 library denaturation and dilution protocol For TruSight Oncology 500libraries, followProtocol F. SeeProtocol F: TruSight Oncology 500 Library Denaturation andDilution Methodon page freshly diluted NaOH for denaturing Libraries for cluster generation. This step isessential to the denaturation #15048776v09 For Research Use Only. Not for use in diagnostic Denature andDilute Libraries GuideuTo prevent small pipetting errors from affecting the final NaOH concentration, prepare at least 1 mlof freshly diluted best results, begin thawing reagents before denaturing and diluting Libraries . For instructions,see the System Guide for your following consumables are required to Denature and Dilute Libraries and prepare a PhiX (Hybridization Buffer)Component of the NextSeq 500/550 Kit[Protocol C]Low TEIllumina, Provided in the AmpliSeq Library PLUS kitUser-Supplied ConsumablesSupplier1 N NaOH, molecular biology-gradeGeneral lab supplier[Protocol A D]200 mM Tris-HCl, pH lab supplierTris-HCl, pH lab supplierThe following additional consumables are required to prepare a PhiX NamePhiX, 10 nMRSB (Resuspension Buffer)Illumina, catalog # FC-110-3002[Protocol E and F]HP3 (2 N NaOH)Illumina, provided in library prep kit contentsEquipmentThe following equipment is used to Denature Libraries that have been normalized using a Microsample IncubatorSciGene, catalog # 1057-30-O (115 V), or equivalentSciGene, catalog # 1057-30-2 (230 V), or equivalentBlock for ml microcentrifuge tubesSciGene, catalog # 1057-34-0, or equivalentProtocolA.

6 StandardNormalizationMethodUse protocol A to Denature and Dilute Libraries that have been normalized using standard libraryquantification and quality control procedures recommended in the library prep , it is important that not more than 1 mM NaOH is in the final solution after diluting with , introducing 200 mM Tris-HCl ensures that the NaOH is fully hydrolyzed in the final a result, template hybridization is not affected even if the final NaOH concentration after dilutingwith HT1 is greater than 1 #15048776v09 For Research Use Only. Not for use in diagnostic Denature andDilute Libraries GuidePrepareReagentsPrepareaFreshDilutio nofNaOH1 Combine the following volumes in a microcentrifuge tube:uLaboratory-grade water (800 l)uStock N NaOH (200 l)The result is 1 ml of N Invert the tube several times to the fresh dilution within12 Remove HT1 from -25 C to -15 C storage and thaw at room Store at 2 C to 8 C until you are ready to Dilute denatured place of RSB, you can use 10 mM Tris-HCl, pH with Tween Remove the tube of RSB from -25 C to -15 C storage and thaw at room Store thawed RSB at 2 C to 8 C until you are ready to Dilute Combine the following volumes of library and freshly diluted N NaOH in a microcentrifuge Library N NaOH4 nM5 l5 l2 nM10 l10 l1 nM20 l20 nM40 l40 l2 Vortex briefly and then centrifuge at 280 g for 1 Incubate at room temperature for 5 Add the following volume of 200 mM Tris-HCl, pH Library Concentration200 mM Tris-HCl, pH 74 nM5 l2 nM10 l1 nM20 nM40 l5 Vortex briefly and then centrifuge at 280 g for 1 #15048776v09 For Research Use Only.

7 Not for use in diagnostic Denature andDilute Libraries GuideDiluteDenaturedLibrariesto20pM1 Add the following volume of prechilled HT1 to the tube of denatured Library ConcentrationPrechilled HT14 nM985 l2 nM970 l1 nM940 nM880 lThe result is a 20 pM denatured Vortex briefly and then centrifuge at 280 g for 1 Place the 20 pM Libraries on ice until you are ready to proceed to final Dilute the denatured 20 pM library solution to pM as library solution (117 l)uPrechilled HT1 (1183 l)The total volume is ml at Invert to mix and then pulse If you plan to add a PhiX control, proceed toDenature and Dilute PhiX Control (Protocols A D)onpage 13. Otherwise, seeNext Stepson page Dilute the denatured 20 pM library solution to pM as library solution (97 l)uPrechilled HT1 (1203 l)The total volume is ml at Invert to mix and then pulse If you plan to add a PhiX control, proceed toDenature and Dilute PhiX Control (Protocols A D)onpage 13. Otherwise, seeNext Stepson page :Bead-BasedNormalizationMethodUse protocol B to Denature and Dilute Libraries that have been normalized and pooled using a bead-basedprocedure described in the library prep documentation for methods that support TruSight Tumor 170 Libraries , followProtocol E: TruSight Tumor 170 Library Denaturation andDilution Methodon page TruSight Oncology 500 Libraries , followProtocol F: TruSight Oncology 500 Library Denaturation andDilution Methodon page #15048776v09 For Research Use Only.

8 Not for use in diagnostic Denature andDilute Libraries GuideBead-based normalization procedures can be variable. Depending upon library type and experience, 2 5 l of library produces optimal Remove HT1 from -25 C to -15 C storage and thaw at room Store at 2 C to 8 C until you are ready to Dilute denatured Preheat the incubator to 98 Combine the following volumes of pooled Libraries and prechilled Hybridization Buffer in amicrocentrifuge PoolPrechilled Hybridization Buffer2 l998 l3 l997 l4 l996 l5 l995 lThe total volume is 1 Vortex briefly and then centrifuge at 280 g for 1 Transfer 750 l diluted library to a new microcentrifuge Add 750 l prechilled Hybridization Vortex briefly and then centrifuge at 280 g for 1 Place the tube on the preheated incubator for 2 Immediately cool on Leave on ice for 5 If you plan to add a PhiX control, proceed toDenature and Dilute PhiX Control (Protocols A D)onpage 13. Otherwise, seeNext Stepson page :AmpliSeqforIlluminaPanelsNormalizationM ethodUse protocol C to Denature and Dilute Libraries prepared using the standard AmpliSeq for Illuminaworkflow.

9 Final loading concentration and volume vary depending on library preparation andquantification methods. For information about the number of Libraries supported per sequencing run, usethe Illumina support websiteto refer to the AmpliSeq for Illumina support page for your #15048776v09 For Research Use Only. Not for use in diagnostic Denature andDilute Libraries GuidePrepareReagentsPrepareaFreshDilutio nofNaOH1 Combine the following volumes in a microcentrifuge tube:uLaboratory-grade water (800 l)uStock N NaOH (200 l)The result is 1 ml of N Invert the tube several times to the fresh dilution within12 Remove HT1 from -25 C to -15 C storage and thaw at room Store at 2 C to 8 C until you are ready to Dilute denatured If frozen, remove Low TE from -25 C to -15 C storage and thaw at room Store thawed Low TE at room temperature until you are ready to Dilute In a new 96-well LoBind PCR plate, Dilute each library to 2 nM using Low Transfer equal volumes of each 2 nM library from the plate to a mL LoBind applicable, make sure to use separate tubes for DNA and RNA Vortex each tube to Centrifuge each tube If DNA and RNA Libraries are to be grouped in a single sequencing run, combine the DNA andRNA library pools at the following ratio of DNA to RNA.

10 PanelDNA to RNA ratioAmpliSeq for Illumina Myeloid Panel8:1 AmpliSeq for Illumina Childhood Cancer Panel5:1 AmpliSeq for Illumina Comprehensive Panel v325:15 After combining the pools, vortex tube to mix and then centrifuge Combine the following volumes of library and freshly diluted N NaOH in a microcentrifuge #15048776v09 For Research Use Only. Not for use in diagnostic Denature andDilute Libraries GuideReagentVolume ( l)Pooled N NaOH102 Vortex briefly and then centrifuge Incubate at room temperature for 5 Add 10 l 200 mM Tris-HCl, pH to the tube containing 2 nM pooled Vortex briefly and then centrifuge Add 970 l prechilled HT1 to the tube of 2 nM denatured library result is a 20 pM denatured Vortex briefly and then centrifuge Place the 20 pM Libraries on ice until you are ready to proceed to final Use prechilled HT1 to Dilute the denatured 20 pM library solution to the desired final loadingconcentration at a final volume of systemSuggested Final Loading Concentration (pM)AmpliSeq for Illumina Exome PanelOther AmpliSeq for Illumina PanelsBioanalyzer~ ~ Analyzer~ ~ ~ ~ ~ ~ HS~ ~ (non-size adjusted)~ ~ (size adjusted)~ ~ 1 Suggested Final Loading ConcentrationsuDenatured library solution (117 l)uPrechilled HT1 (1183 l)