cbot System Guide For Research Use Only. Not for use in diagnostic procedures. Document # v03 January 2019 ILLUMINA PROPRIETARY

Transcription

1 cbot System Gide Janary 2019 ILLUMINA PROPRIETARY

2 This docment and its contents are proprietary to Illmina, Inc. and its affiliates ("Illmina"), and are intended solely for the contractal se of its cstomer in connection with the se of the prodct(s) described herein and for no other prpose. This docment and its contents shall not be sed or distribted for any other prpose and/or otherwise commnicated, disclosed, or reprodced in any way whatsoever withot the prior written consent of Illmina. Illmina does not convey any license nder its patent, trademark, copyright, or common-law rights nor similar rights of any third parties by this docment. The instrctions in this docment mst be strictly and explicitly followed by qalified and properly trained personnel in order to ensre the proper and safe se of the prodct(s) described herein. All of the contents of this docment mst be flly read and nderstood prior to sing sch prodct(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, 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) Illmina, Inc. All rights reserved. All trademarks are the property of Illmina, Inc. or their respective owners. For specific trademark information, see ii

3 Revision History Docment Date Description of Change Docment # v03 Docment # v02 Docment # v01 Part # Rev. O Janary 2019 Janary 2016 Agst 2015 Febrary 2015 Updated foil color from red to white for HP5 8-tbe strip. Added denatre and dilte information for Nextera DNA Flex libraries. Removed references to TrSeq v2 GA kits as they are no longer spported. Removed material nmber as this docment is no longer printed. Added PhiX and library volmes to the PhiX spike-in procedre for libraries clstered on a HiSeq 3000/4000 flow cell. Added recommendation for annal preventive maintenance service. Updated instrctions for nloading rn components to inclde flow cell storage options. Listed the cbot System Configration Gide (docment # ) in Additional Resorces. Updated software descriptions to cbot software v3.0, which enables the se of the HiSeq 3000/4000 SR Clster Kit. Added recipes for the following flow cells: HiSeq X v2.5, HiSeq 3000/4000 SR, TrSeq v3, and GAIIx v2. Added the following information: Instrctions by flow cell type for preparing clstering reagents. Instrction to se HiSeq X and HiSeq 3000/4000 flow cells within 4 hors of opening. Clstering dration for HiSeq 3000/4000 SR flow cells. Fisher Scientific catalog # AB-0784 for 8-cap strips. Updated instrctions for resetting the barcode scanner to the defalt configration. Moved trobleshooting information to Appendix A. Removed descriptions of software screens. Descriptions are inclded with clstering steps as needed. Updated spported kits to inclde the HiSeq X Five Reagent Kit v2, single pack and 10 pack, and the HiSeq 3000/4000 PE Clster Kit. Added names of recipes sed with the HiSeq X Five v2 flow cell and the HiSeq 3000/4000 flow cell. Updated clstering workflow to inclde HiSeq X Five v2 flow cells and HiSeq 3000/4000 flow cells. Changed the name of the HiSeq X HD Reagent Kit v2 to HiSeq X Ten Reagent Kit v2, and changed the name of the 20-pack kit to 10-pack kit. (Change in name only; contents have not changed.) Removed Appendix A, which contained configration procedres. For configration procedres, see the cbot System Site Prep Gide (docment # ). Corrected clstering dration for HiSeq v4 and HiSeq X flow cells from approximately 2.5 hors to approximately 3 hors. iii

4 Docment Date Description of Change Part # Rev. N Part # Rev. M November 2014 September 2014 Added the HiSeq Rapid Do cbot Sample Loading Kit, which spports clstering for the HiSeq Rapid Rn v2 mode on the HiSeq 2500 and HiSeq Added a type of rapid flow cell, HiSeq Rapid v2 flow cell, inclding compatible recipes. Added clstering dration for rapid flow cells. Added the HiSeq X HD Reagent Kit v2, single pack and 20 pack. Added information abot cbot reagent plate orientation for HiSeq X kit. Added note stating that it is not possible to confirm reagent delivery from the reagent plate provided in the HiSeq X HD Reagent Kit v2. Removed the HiSeq Mlti-Primer Rehybridization Kit v4 from available cbot kits. The HiSeq Mlti-Primer Rehybridization Kit v4 is sed on the HiSeq only. Removed HiSeq X from the list of workflows that reqire primers in the Primers position. The 8-tbe strip containing the ExAmp reagents and library in the HiSeq X workflow is loaded in the Templates position. Added name of recipe sed with a rapid flow cell and the TrSeq Rapid Do Sample Loading Kit to cbot Recipes and Flow Cell Types. Corrected freqency on periodic maintenance schedle for the monthly maintenance wash. Corrected docmentation titles in Additional Resorces. Updated URL for Safety Data Sheets (SDS) to spport.illmina.com/sds.html. Part # Rev. L April 2014 Updated to cbot software v2.0, which enables the se of HiSeq v4 and HiSeq X kits. Added workflow information for sing HiSeq v4 flow cells and HiSeq X flow cells. Removed the procedre to denatre libraries and prepare a PhiX control. See Denatring and Dilting Libraries for the HiSeq and GAIIx (part # ). Removed reagent preparation instrctions. For reagent preparation instrctions inclding information abot seqencing primers, see the kit docmentation. Removed the site prep and installation information. See the cbot Site Prep and Installation Gide (part # ). Part # Rev. K October 2012 Added information for template hybridization on a TrSeq Rapid flow cell. Part # Rev. J Jly 2012 Added seqencing primers reqirements for dal-indexed TrSeq HT libraries. Part # Rev. H April 2012 Updated information for seqencing dal-indexed libraries. Added the following procedres: Reagent preparation instrctions, inclding instrctions for preparing HP10 Primer rehybridization procedre Part # Rev. G October 2011 Added new section titled Dal-Indexed Seqencing Modifications. iv

5 Docment Date Description of Change Part # Rev. F Jne 2011 Updated procedre for preparing template DNA to inclde instrctions for higher concentrations, and added note abot high NaOH concentration. Part # Rev. E April 2011 Updated software descriptions to cbot software v1.4. Added the following information: TrSeq Clster Kit v3 and catalog nmber Description of keyed corner as visal orientation when loading HiSeq Flow Cell v3 New section titled Version Compatibility of Rn Components that lists compatible software and recipe versions for varios flow cell types Updated recommended DNA template storage to a concentration of 2 nm, and adjsted protocol for preparing DNA sing a 2 nm template. Part # Rev. D October 2010 Updated software descriptions to cbot software v1.3. Added the following information: Recommended clster densities based on version of analysis software Instrctions for pgrading the software Instrctions for recovering a rn Part # Rev. C May 2010 Updated software descriptions to cbot software v1.1. Added flow cell storage recommendation. Increased water wash volme to 12 ml and DECON to 10 ml. Part # Rev. B Part # Rev. A March 2010 October 2009 Corrected centrifge instrctions for thawing the reagent plate. Added the following information: Instrctions for loading the HiSeq flow cell, and associated templates and primers Instrctions for installing the adapter plate HiSeq Clster Generation Kit catalog nmbers and kit descriptions Instrctions for setting local date and time sing the Time tab Monthly maintenance wash procedre Initial release. v

6 Table of Contents Chapter 1 Overview 1 Introdction 1 Additional Resorces 1 cbot Components 2 Illmina Consmables 5 cbot Reagent Plates 6 Chapter 2 Getting Started 8 Start the cbot 8 Version Compatibility of Rn Components 8 User-Spplied Consmables 9 Chapter 3 Preparing Reagents 10 Introdction 10 HiSeq X Flow Cell 10 HiSeq 3000/4000 Flow Cell 14 HiSeq High Otpt Flow Cell 17 HiSeq Rapid Flow Cell 18 Chapter 4 Clstering 19 Introdction 19 Clstering Workflow 19 Perform a Pre-Rn Wash 20 Select a Protocol 21 Load Consmables 21 Perform a Pre-Rn Check 24 Monitor the Rn 25 Unload Rn Components 26 Perform a Post-Rn Wash 27 Confirm Reagent Delivery (Optional) 27 Chapter 5 Maintenance 29 Perform Periodic Maintenance 29 Perform a Monthly Maintenance Wash 29 Change the Adapter Plate 30 Upgrade the Software 32 Upgrade Recipes 32 Sht Down the cbot 33 Chapter A Trobleshooting 35 Pase or Abort a Rn 35 Trobleshoot Flow Check Failre 35 Trobleshoot Rn Problems 37 vi

7 Reset the Barcode Scanner 38 Edit Protocols 38 Index 41 Technical Assistance 44 vii

8 Chapter 1 Overview Introdction 1 Additional Resorces 1 cbot Components 2 Illmina Consmables 5 cbot Reagent Plates 6 Introdction The cbot ses amplification to create hndreds of millions of single-molecle DNA templates simltaneosly. The cbot software dispenses reagents and controls reaction times, flow rates, and temperatres. Setp and operation are performed on-instrment from the cbot software interface sing the toch screen monitor. An on-instrment barcode reader records the reagents and flow cell sed for each experiment. Figre 1 cbot Several clster kits are available for se on the cbot. Use a kit that is compatible with the seqencing instrment and type of seqencing rn to be performed. For a list of available kits, see Illmina Consmables on page 5. Additional Resorces The following docmentation is available for download from the Illmina website. Resorce cbot System Site Prep Gide (docment # ) cbot Safety and Compliance Booklet (part # ) HiSeq Systems Denatre and Dilte Libraries Gide (docment # ) Description Provides specifications for laboratory space, electrical reqirements, and environmental considerations and instrctions for configring the instrment. Provides information abot instrment labeling, compliance certifications, and safety considerations. Provides instrctions for denatring and dilting prepared libraries before seqencing, and preparing a PhiX control. This step applies to most library types and flow cells. Visit the cbot or cbotcbotspport page on the Illmina website for access to docmentation, software downloads, online training, and freqently asked qestions. 1

9 cbot Components The cbot ses sensors to detect the presence of rn components, and provides prompts when a component is missing or installed incorrectly. The thermal stage and reagent stage are located nder the cbot lid. For safety, the software prompts yo to close the lid before proceeding with the rn. Figre 2 cbot Components A B C D E F Thermal stage Holds the flow cell and controls the flow cell temperatre throghot the rn. Reagent stage Holds the cbot reagent plate, library templates, and specialty primers. Waste bottle compartment Holds a sensor-controlled waste bottle that collects spent reagents. Barcode scanner Records the niqe ID of the reagent plate and flow cell sed with each rn. Power switch Trns on the instrment. The start btton, located to the left of the waste bottle compartment, starts the instrment software. Toch screen monitor Provides on-instrment rn setp and visal stats of the clster generation process. Thermal Stage The thermal stage holds the flow cell and manifold, which is seated over the flow cell. The flow cell clamp locks the flow cell and manifold into place. WARNING Do not toch the alminm thermal block on the thermal stage. The heater poses a serios brn hazard dring operation. For more safety information, see the cbot Safety and Compliance Booklet (part # ). 2

10 Figre 3 Thermal Stage A B C D E Otpt clamp Flow cell clamp Flow cell and manifold Thermal stage Sipper comb The manifold is a single-se component that delivers reagents from the reagent plate to the flow cell. The sippers on the sipper comb pierce the foil-sealed reagent tbes seated in the reagent plate. The otlet end of the manifold transfers waste to the waste container. The otlet clamp locks the otlet end of the manifold in place. Flow Cell Adapter Plates The cbot performs clster generation on HiSeq flow cells. When switching flow cell types, change the adapter plate on the flow cell stage. For details, see Change the Adapter Plate on page 30. Reagent Stage The reagent stage holds the cbot reagent plate. The reagent plate is locked into position by the reagent plate lever. Two 8-tbe strip holders in front of the reagent plate hold prepared library templates and additional primers. Figre 4 cbot Reagent Stage A B C cbot reagent plate Reagent plate lever Template row 3

11 D Primer row cbot Software The cbot software interface provides prompts for setting p the instrment and monitoring clstering progress. Dring a clster generation rn, the following screens are sed: Start screen, Rn Setp screens, and the Rn Stats screen. Use the software interface to configre inpt reqirements, wash preferences, notifications, and remote monitoring. Sensor Stats Icons Displayed at the bottom of the screen, sensor stats icons indicate whether a component is properly installed and ready for the rn. Icon Indication GAIIx flow cell adapter plate installed. HiSeq flow cell adapter plate installed. Flow cell adapter plate type nknown. Instrment lid is open. Instrment lid is closed. Waste bottle is present and ready for se. Waste bottle is fll. Waste bottle is missing. Coolant is flowing and coolant level is good. Warning: Coolant is flowing, bt coolant level is low. Error: Coolant is not flowing, bt coolant level is good. Error: Coolant is not flowing and coolant level is low. Manifold is loaded and sipper comb is secre. Manifold is missing or sipper comb is not secre. 4

12 Configration Use the software interface to configre system settings, inpt reqirements, and wash preferences. Using a network connection, yo can enable remote monitoring, alerts, and LIMS spport. Configration settings can be modified as needed before the start of each rn. For configration instrctions, see the cbot System Configration Gide (docment # ). Illmina Consmables cbot reagents are provided in a reagent plate that loads directly onto the instrment after thawing. cbot reagent plates are provided in the following Illmina kits. Descriptions of kit contents and other kit docmentation is available on the cbot spport page on the Illmina website. For reagent preparation instrctions, see Preparing Reagents on page 10. Clster Kits for the HiSeq Each kit contains a HiSeq flow cell, a flow cell-specific manifold, and the reqired reagents for clstering the flow cell on the cbot. Kit Name Kit Catalog # HiSeq 3000/4000 SR Clster Kit HiSeq 3000/4000 PE Clster Kit HiSeq SR Clster Kit v4 HiSeq PE Clster Kit v4 TrSeq SR Clster Kit v3 - HS TrSeq PE Clster Kit v3 - HS HiSeq Rapid Do cbot Sample Loading Kit Catalog # GD Catalog # PE Catalog # GD Catalog # PE Catalog # GD Catalog # PE Catalog # CT Clster Kits for the HiSeq X Each kit contains mltiple HiSeq X flow cells, flow cell-specific manifolds, and the reqired reagents for clstering each flow cell on the cbot. Single-pack kits contain consmables for clstering 2 flow cells and 10- pack kits contain consmables for clstering 20 flow cells. Kit Name Kit Catalog # HiSeq X Ten Reagent Kit v2.5 HiSeq X Ten Reagent Kit v2.5 (10 pack) HiSeq X Five Reagent Kit v2.5 HiSeq X Five Reagent Kit v2.5 (10 pack) Catalog # FC Catalog # FC Catalog # FC Catalog # FC Rehybridization Kits Use a cbot rehybridization kit to perform Read 1 primer rehybridization for rn recovery or after extended flow cell storage. 5

13 Kit Name Catalog # HiSeq X cbot Mlti-Primer Rehybridization Kit v2 HiSeq 3000/4000 cbot Mlti-Primer Rehybridization Kit TrSeq v2 cbot Mlti-Primer Rehybridization Kit HiSeq Mlti-Primer Rehybridization Kit v4 Catalog # GD Catalog # GD Catalog # GD Catalog # GD For more information, see the rehybridization gide for yor flow cell: HiSeq X Read 1 Primer Rehybridization on a HiSeq X Flow Cell (docment # ) HiSeq 3000/4000 Read 1 Primer Rehybridization on a HiSeq 3000/4000 Flow Cell (docment # TrSeq v3 Read 1 Primer Rehybridization on a TrSeq v3 Flow Cell (docment # ) Read 1 Seqencing Primer for Nextera Libraries The Read 1 seqencing primer (HP6) provided in the following kits is not compatible with Nextera libraries: TrSeq Clster Kit v3 - HS If yo are seqencing Nextera libraries, se the Read 1 seqencing primer (HP10), regardless of the type of rn yo are performing. HP10 is provided in the TrSeq Dal Index Seqencing Primer Box. Kit Name Catalog # TrSeq Dal Index Seqencing Primer Box, Single Read TrSeq Dal Index Seqencing Primer Box, Paired End Catalog # FC Catalog # PE All other cbot kits inclde HP10, which is compatible with TrSeq and Nextera libraries. cbot Reagent Plates The configration of the reagent plate differs between kit types, inclding the nmber of rows that contain reagent. Each 8-tbe strip is labeled with the reagent name followed by a nmber. The nmber indicates the row that it occpies on the reagent plate. If an 8-tbe strip becomes dislodged, se the row nmber on the label to retrn the tbe strip to the appropriate position. 6

14 Flow Cell Type HiSeq X and HiSeq 3000/4000 Reagent Plate Description Contains 12 rows with 8 deep wells each. Each reagent occpies a fll row of 8 wells. Not all rows contain reagent. HiSeq High Otpt (HiSeq v4) Contains 8 rows of foil-sealed 8-tbe strips filled with clster generation reagents. Rows 9 throgh 12 are empty. HiSeq High Otpt (TrSeq v3) Contains 11 rows of foil-sealed 8-tbe strips filled with clster generation reagents. Row 12 is empty. HiSeq Rapid Contains 3 rows of foil-sealed 8-tbe strips filled with template hybridization and first extension reagents. Rows 4 throgh 12 are empty. WARNING Except for the HiSeq rapid reagent plate, these sets of reagents contain formamide, an aliphatic amide that is a probable reprodctive toxin. Personal injry can occr throgh inhalation, ingestion, skin contact, and eye contact. Dispose of containers and any nsed contents in accordance with the governmental safety standards for yor region. For more information, see the SDS for this kit at spport.illmina.com/sds.html. 7

15 Chapter 2 Getting Started Start the cbot 8 Version Compatibility of Rn Components 8 User-Spplied Consmables 9 Start the cbot A B Start btton Power switch 1 Switch the power switch on the right side of the instrment to the ON position. 2 Press the start btton to the left of the waste bottle compartment to start the software. When the start-p rotine is complete, the Start screen appears. Version Compatibility of Rn Components For best performance and reslts, always se compatible versions of cbot software and kits. Kit Version Recipe Version Software Version HiSeq 3000/4000 Clster Kit Version 1.0 recipes cbot v3.0.46, or later (SR kit) cbot v2.0.34, or later (PE kit) HiSeq X Ten Reagent Kit v2.5 Version 2.0 recipes cbot v2.0.29, or later HiSeq X Five Reagent Kit v2.5 Version 2.0 recipes cbot v2.0.29, or later HiSeq Clster Kit v4 Version 9.0 recipes cbot v2.0.16, or later HiSeq Rapid Do cbot Sample Loading Kit Version R recipes cbot v1.5, or later TrSeq Rapid Do cbot Sample Loading Kit Version R recipes cbot v1.5, or later TrSeq Dal Index Seqencing Primer Box Version 8.0 recipes (HiSeq) Version 7.0 recipes (GA) cbot v1.4.36, or later TrSeq Clster Kit v3 - HS Version 8.0 recipes cbot v1.4, or later TrSeq Clster Kit v2 - GA* Version 7.0 recipes cbot v1.3, or later *This option is visible bt is no longer spported. cbot Recipes and Flow Cell Types 8

16 Flow Cell HiSeq 3000/4000 Patterned Flow Cell HiSeq X Ten v2.5 Patterned Flow Cell HiSeq X Five v2.5 Patterned Flow Cell HiSeq v4 Flow Cell TrSeq v3 Flow Cell HiSeq Rapid v2 Flow Cell Primary Recipe Name HiSeq_3000_4000_SR_HD_Exclsion_Amp_v1.0 HiSeq_3000_4000_HD_Exclsion_Amp_v1.0 HiSeq_X_HD_Exclsion_Amp_v2.0 HiSeq_X_HD_Exclsion_Amp_v2.0 SR_HiSeq_Clster_Kit_v4_cBot_recipe_v9.0 PE_HiSeq_Clster_Kit_v4_cBot_recipe_v9.0 SR_Amp_Lin_Block_TbeStripHyb_v8.0 PE_Amp_Lin_Block_TbeStripHyb_v8.0 SR_Amp_Lin_Block_Hyb_v8.0 PE_Amp_Lin_Block_Hyb_v8.0 RR_TemplateHyb_FirstExt_vR¹ ¹ Used only with the Rapid Do kits. User-Spplied Consmables The following ser-spplied consmables are sed for preparation of clstering reagents provided in the HiSeq X and HiSeq 3000/4000 kits. The HiSeq X and HiSeq 3000/4000 kits introdce a denatration step before clstering on the cbot. Using these kits, libraries are denatred in the 8-tbe strip before adding the ExAmp reaction mix. Component Spplier Prpose 1 N NaOH General lab spplier Use for library denatration. 200 mm Tris-HCl, ph 8.0 General lab spplier Use for library denatration after dilting to 0.1 N NaOH. Laboratory-grade water Millipore or General lab spplier 8-tbe strips, 0.2 ml Fisher Scientific, catalog # AB cap strip, flat Fisher Scientific, catalog # AB-0784 Microcentrifge tbe, 1.5 ml VWR, catalog # * Use for library denatration. Use for the ExAmp reaction and library mix on the cbot. Use to cap the 8-tbe strips when not loaded onto the cbot. Use for preparation of the ExAmp Reaction master mix. *or eqivalent 9

17 Chapter 3 Preparing Reagents Introdction 10 HiSeq X Flow Cell 10 HiSeq 3000/4000 Flow Cell 14 HiSeq High Otpt Flow Cell 17 HiSeq Rapid Flow Cell 18 Introdction Reagent preparation instrctions depend on yor reagent kit. The instrctions are organized by flow cell type and inclde HiSeq X, HiSeq 3000/4000, HiSeq high otpt, and HiSeq rapid. After preparation, clstering reagents are ready to be loaded onto the cbot when prompted by the software. Best Practices Wear a fresh pair of gloves when preparing clstering reagents. Do not remove the protective clear plastic lid on the reagent plate ntil yo are ready to load reagents onto the cbot. Never pnctre the foil seals. Hold reagent plates that contain 8-tbe strips by the plate base to avoid dislodging reagent tbes. Make sre that the tbes are secrely seated in the reagent plate before and after vortexing or inverting. Loose tbes can damage the cbot manifold. For clster generation on a HiSeq X or HiSeq 3000/4000 flow cell, always prepare freshly dilted NaOH for denatring libraries. This step is essential to the denatration process. To prevent small pipetting errors, prepare at least 1 ml of freshly dilted 0.1 N NaOH. HiSeq X Flow Cell Prepare the HiSeq X patterned flow cell, then prepare clstering reagents. To prepare clstering reagents, thaw the cbot reagent plate and prepare the ExAmp master mix. If yo are sing the 10-pack kit, prepare 4 flow cells and thaw 4 cbot reagent plates. Make sre that 4 cbot instrments are available. Reagents cannot be stored after preparation. Abot Reagents ExAmp reagents are viscos, especially EPX2 and EPX3. Aspirate and dispense reagents slowly to ensre accrate pipetting. EPX3 does not move when inverted de to viscosity. Never vortex ExAmp reagents, and do not refreeze after thawing. The ExAmp master mix can be clody, which is normal. If the soltion separates into a clody portion and a transparent portion, slowly pipette to mix. Prepare the Flow Cell 1 Remove a new flow cell package from 2 C to 8 C storage. 2 Set the flow cell package aside at room temperatre for at least 30 mintes. NOTE 10

18 If the foil packaging is intact, the flow cell can remain at room temperatre p to 12 hors. Yo can retrn the packaged flow cell to 2 C to 8 C storage for later se 1 time only. Avoid repeated cooling and warming of the flow cell. 3 Pt on a new pair of powder-free gloves. 4 Peel open the foil package from the end with the angled seal. Use the flow cell within 4 hors of opening the foil package. Figre 5 Open Flow Cell Package 5 Remove the clamshell package from the foil packaging. Figre 6 Remove From Foil Packaging 6 Open the clamshell package and remove the flow cell. Figre 7 Remove Flow Cell From Clamshell Package 7 Clean the flow cell with a lint-free alcohol wipe. Dry with a lint-free tisse. 11

19 8 Set aside at room temperatre. Thaw the cbot Reagent Plate 1 Remove the cbot reagent plate from -25 C to -15 C storage. 2 Thaw in a room temperatre water bath for 60 mintes. Thaw EPX1, EPX2, EPX3, and RSB 1 Remove 1 tbe each of the following reagents from -25 C to -15 C storage. Single-pack kit EPX1, EPX2, EPX3, and RSB. Each tbe contains enogh reagent for 1 flow cell. 10-pack kit EPX1M, EPX2M, EPX3M, and RSB. Each tbe contains enogh reagent for 4 flow cells. 2 Thaw at room temperatre for 10 mintes. 3 Set aside on ice. Prepare a Fresh Diltion of NaOH 1 Combine the following volmes in a microcentrifge tbe to prepare 1 ml of 0.1 N NaOH: Laboratory-grade water (900 µl) Stock 1 N NaOH (100 µl) 2 Invert to mix. Denatre Libraries and Add Optional PhiX Control The library loading concentration depends on the libraries to be seqenced. The following instrctions apply to either TrSeq Nano DNA (350 bp) or TrSeq DNA PCR-Free (350 bp) libraries. Dilte to a concentration appropriate for the library type. A DNA loading concentration that is too high cases redced %PF. A DNA loading concentration that is too low cases redced %PF and a high percentage of dplicates that negatively affect depth of coverage. Repeat the following instrctions for each flow cell to be seqenced. 1 Dilte the library or pooled libraries to the appropriate concentration: TrSeq Nano DNA libraries Dilte to 2 3 nm in RSB. TrSeq DNA PCR-Free libraries Dilte to 1 2 nm in RSB. 2 [Optional] Spike in 1% nondenatred Illmina PhiX control to nondenatred libraries: TrSeq Nano DNA libraries Add 0.5 µl 2 3 nm PhiX to 50 µl 2 3 nm library. TrSeq DNA PCR-Free libraries Add 0.5 µl 1 2 nm PhiX to 50 µl 1 2 nm library. 3 Label the tbes of an 8-tbe strip #1 throgh #8. 4 Denatre the library in the 8-tbe strip as follows: a b c d Add 5 µl nondenatred library to the bottom of each well. Add 5 µl freshly dilted 0.1 N NaOH. Slowly pipette to mix. Incbate at room temperatre for 8 mintes. Add 5 µl 200 mm Tris-HCl ph 8.0. Slowly pipette to mix. 5 Set aside on ice ntil yo are ready to add the ExAmp master mix. 12

20 CAUTION Prepare and add the ExAmp master mix within 30 mintes. Prepare the cbot Reagent Plate 1 Invert to mix. 2 Vortex to dislodge trapped air bbbles. 3 Tap on a hard srface to collect reagent droplets. Alternatively, plse centrifge. 4 Set aside on ice. Prepare the ExAmp Reaction Prepare the ExAmp reaction master mix immediately before se. Follow the appropriate instrctions for the nmber of flow cells yo are preparing. ExAmp Reaction for 1 Flow Cell (Single-Pack Kit) 1 Invert EPX1 and EPX2 to mix. 2 Briefly centrifge EPX1, EPX2, and EPX3. 3 Prepare the ExAmp master mix in a 1.5 ml tbe as follows. a b c Add 210 μl EPX1. Add 30 μl EPX2. Slowly pipette to mix. Add 110 μl EPX3. Slowly pipette to mix. Make sre that the bottom of the tbe is free of bbbles. 4 Add 35 µl master mix to the bottom of each well of the 8-tbe strip. Slowly pipette to mix. Change tips between samples. 5 Briefly centrifge, and then set aside on ice ntil yo are ready to load the cbot. CAUTION Load the 8-tbe strip onto the cbot within 15 mintes. ExAmp Reaction for 4 Flow Cells (10-Pack Kit) 1 Invert EPX1M and EPX2M to mix. 2 Briefly centrifge EPX1M, EPX2M, and EPX3M. 3 Add the following volmes to a 1.5 ml tbe in the order listed: a b c Add 756 μl EPX1M. Add 108 μl EPX2M. Slowly pipette to mix. Add 396 μl EPX3M. Slowly pipette to mix. Make sre that the bottom of the tbe is free of bbbles. 4 Add 35 µl of the master mix into the bottom of each well of the 8-tbe strips. Slowly pipette to mix. Change tips between samples. 5 Briefly centrifge the 8-tbe strip, and then set aside on ice ntil yo are ready to load the cbot. CAUTION Load the ExAmp master mix and library soltion onto the cbot within 15 mintes. 13

21 HiSeq 3000/4000 Flow Cell Prepare the HiSeq 3000/4000 patterned flow cell, then prepare clstering reagents. To prepare clstering reagents, thaw the cbot reagent plate and prepare the ExAmp reaction master mix. Abot Reagents ExAmp reagents are viscos, especially EPX2 and EPX3. Aspirate and dispense reagents slowly to ensre accrate pipetting. EPX3 does not move when inverted de to viscosity. Never vortex ExAmp reagents, and do not refreeze after thawing. The ExAmp master mix can be clody, which is normal. If the soltion separates into a clody portion and a transparent portion, slowly pipette to mix. Prepare the Flow Cell 1 Remove a new flow cell package from 2 C to 8 C storage. 2 Set the flow cell package aside at room temperatre for at least 30 mintes. NOTE If the foil packaging is intact, the flow cell can remain at room temperatre p to 12 hors. Yo can retrn the packaged flow cell to 2 C to 8 C storage for later se 1 time only. Avoid repeated cooling and warming of the flow cell. 3 Pt on a new pair of powder-free gloves. 4 Peel open the foil package from the end with the angled seal. Use the flow cell within 4 hors of opening the foil package. Figre 8 Open Flow Cell Package 5 Remove the clamshell package from the foil packaging. 14

22 Figre 9 Remove From Foil Packaging 6 Open the clamshell package and remove the flow cell. Figre 10 Remove Flow Cell From Clamshell Package 7 Clean the flow cell with a lint-free alcohol wipe. Dry with a lint-free tisse. 8 Set aside at room temperatre. Thaw the cbot Reagent Plate 1 Remove the cbot reagent plate from -25 C to -15 C storage. 2 Place the reagent plate in a room temperatre water bath for 60 mintes. Thaw EPX1, EPX2, EPX3, and RSB 1 Remove EPX1, EPX2, EPX3, and RSB from -25 C to -15 C storage. 2 Thaw at room temperatre for 10 mintes. 3 Set aside on ice. Prepare a Fresh Diltion of NaOH 1 Combine the following volmes in a microcentrifge tbe to prepare 1 ml of 0.1 N NaOH: Laboratory-grade water (900 µl) Stock 1 N NaOH (100 µl) 2 Invert to mix. 15

23 Denatre Libraries and Add Optional PhiX Control The library loading concentration depends on the libraries to be seqenced. The following instrctions apply to spported Illmina libraries and assme an insert size typical for the associated library type. Make sre that yo dilte to a concentration appropriate for the library type. A DNA loading concentration that is too high cases redced %PF. A DNA loading concentration that is too low cases redced %PF and a high percentage of dplicates that negatively affect depth of coverage. 1 Dilte the library or pooled libraries to the appropriate concentration. Library Type TrSeq DNA PCR-Free Nextera DNA Flex TrSeq Nano DNA Nextera Rapid Captre Exome TrSeq Stranded Total RNA TrSeq Stranded mrna Diltion Dilte to 1 2 nm in RSB. Dilte to 2 3 nm in RSB. Dilte to 2 3 nm in RSB. 2 [Optional] Spike-in 1% nondenatred Illmina PhiX control to nondenatred libraries. Library Type TrSeq DNA PCR-Free Nextera DNA Flex TrSeq Nano DNA Nextera Rapid Captre Exome TrSeq Stranded Total RNA TrSeq Stranded mrna Spike-In Add 5 µl pm PhiX to 45 µl 1 2 nm library. Dilte to 2 3 nm in RSB. Add 5 µl pm PhiX to 45 µl 2 3 nm library. 3 Label a new 8-tbe strip #1 throgh #8. 4 Denatre the library in the 8-tbe strip as follows. a b c d Add 5 µl nondenatred library into the bottom of each well. Add 5 µl freshly dilted 0.1 N NaOH. Slowly pipette to mix. Incbate at room temperatre for 8 mintes. Add 5 µl 200 mm Tris-HCl ph 8.0. Slowly pipette to mix. 5 Set aside on ice for p to 30 mintes, ntil yo are ready to add the ExAmp master mix. Prepare the cbot Reagent Plate 1 Invert to mix. 2 Vortex to dislodge trapped air bbbles. 3 Tap on a hard srface to collect reagent droplets. Alternatively, plse centrifge. 4 Set aside on ice. Prepare the ExAmp Reaction Prepare the ExAmp reaction master mix immediately before se. 16

24 1 Invert EPX1 and EPX2 to mix. 2 Briefly centrifge EPX1, EPX2, and EPX3. 3 Prepare the ExAmp master mix in a 1.5 ml tbe as follows. a b c Add 210 μl EPX1. Add 30 μl EPX2. Slowly pipette to mix. Add 110 μl EPX3. Slowly pipette to mix. Make sre that the bottom of the tbe is free of bbbles. 4 Add 35 µl master mix to the bottom of each well of the 8-tbe strip. Slowly pipette to mix. Change tips between samples. 5 Briefly centrifge, and then set aside on ice ntil yo are ready to load the cbot. CAUTION Load the 8-tbe strip onto the cbot within 15 mintes. HiSeq High Otpt Flow Cell To prepare reagents, thaw and inspect the reagent plate. The reagent plate takes approximately 60 mintes to thaw sing a room temperatre water bath. Alternatively, yo can thaw reagents at 2 C to 8 C overnight, not to exceed 16 hors. Abot Reagents When vortexing or inverting the cbot reagent plate, keep a hand on top of the tbes. Thaw the cbot Reagent Plate 1 Remove the cbot reagent plate from -25 C to -15 C storage. 2 Make sre that the tbe strips are secrely seated in the plate. 3 Thaw in a room temperatre water bath for 60 mintes. Prepare the cbot Reagent Plate 1 Invert the cbot plate to mix reagents. 2 Vortex to dislodge trapped air bbbles. 3 Tap on a hard srface to collect reagent droplets at the bottom of the tbes. Alternatively, plse centrifge. 4 Make sre that the tbes are free of air bbbles, secrely seated, and ordered as nmbered. 5 Promptly proceed to setting p the cbot. 6 If yo are seqencing Nextera libraries on a TrSeq v3 flow cell, proceed to Prepare HP10 (TrSeq v3) before setting p the cbot. Prepare HP10 (TrSeq v3) Prepare HP10 for se on the cbot only when sing Nextera libraries on a TrSeq v3 flow cell. HP10 is also compatible with other Illmina library types. 1 Remove HP10 from -25 C to -15 C storage. 17

25 2 Thaw in a beaker of room temperatre deionized water for 20 mintes. 3 Add 150 µl HP10 to each tbe of an 8-tbe strip. 4 Set aside on ice. 5 Promptly proceed to setting p the cbot. HiSeq Rapid Flow Cell To prepare reagents, thaw and inspect the reagent plate. The reagent plate takes approximately 30 mintes to thaw sing a room temperatre water bath. Alternatively, yo can thaw reagents at 2 C to 8 C overnight, not to exceed 16 hors. Abot Reagents When vortexing or inverting the cbot reagent plate, keep a hand on top of the tbes. Thaw the cbot Reagent Plate 1 Remove the cbot reagent plate from -25 C to -15 C storage. 2 Make sre that the tbe strips are secrely seated in the plate. 3 Thaw in a room temperatre water bath for 60 mintes. Prepare the cbot Reagent Plate 1 Invert the cbot reagent plate to mix reagents. 2 Vortex to dislodge trapped air bbbles. 3 Tap on a hard srface to collect reagent droplets at the bottom of the tbes. Alternatively, plse centrifge. 4 Inspect to make sre that the tbes are free of air bbbles, secrely seated, and ordered as nmbered on the label. 5 Promptly proceed to setting p the cbot. 18

26 Chapter 4 Clstering Introdction 19 Clstering Workflow 19 Perform a Pre-Rn Wash 20 Select a Protocol 21 Load Consmables 21 Perform a Pre-Rn Check 24 Monitor the Rn 25 Unload Rn Components 26 Perform a Post-Rn Wash 27 Confirm Reagent Delivery (Optional) 27 Introdction All clstering steps are performed on the cbot except for preparing libraries for seqencing and preparing the cbot reagent plate. Clstering steps for a rapid flow cell consist only of template hybridization and first extension. The remaining steps are performed on the seqencing instrment. Setting p the cbot for clster generation incldes the steps to select a protocol and then load consmables. Scan reqired inpt, sch as reagent ID and flow cell ID, sing the barcode scanner, or select the keyboard icon and enter the ID manally. Manal inpt and system inpt are shown on the screen. Library Prep Before setting p the cbot for clster generation, prepare libraries for seqencing. The process differs depending on library type and flow cell type. Most libraries on TrSeq flow cells and HiSeq flow cells reqire a denatration and diltion step. For more information, see HiSeq Systems Denatre and Dilte Libraries Gide (docment # ). The denatration protocol differs for HiSeq X and HiSeq 3000/4000 patterned flow cells. Denatre libraries for se with these flow cell types only as described in the reagent preparation instrctions for yor flow cell type. For more information, see Preparing Reagents on page 10. Clstering Workflow Prepare the reagent plate and flow cell. See Preparing Reagents on page 10. Prepare libraries for seqencing and load libraries into an 8-tbe strip. Perform a pre-rn wash. 19

27 Select a protocol, scan and load consmables, and load tbe strips containing prepared libraries. Select Pre-Rn Check to initiate the atomated pre-rn check. Select Start. Monitor rn progress from the Rn Stats screen. Unload rn components and confirm reagent delivery. Perform a post-rn wash. Perform a Pre-Rn Wash A wash is recommended before clstering on the cbot. 1 Select User Name. 2 Using the onscreen keyboard, type yor name and then select Enter. 3 Select Start. 4 If the Manifold removed checkbox is not selected on the Wash screen, remove the manifold. 5 Raise the instrment lid by lifting from the top-right corner. 6 Fill the wash reservoir with abot 12 ml deionized water. 7 Close the instrment lid. 8 Select the Reservoir filled with water checkbox. 9 Select Wash. 10 After the wash is finished, blot excess water from the wash reservoir with a low-lint tisse. 20

28 Figre 11 Dry the Wash Reservoir 11 Select the Wash reservoir dry checkbox. 12 Select Next. Select a Protocol 1 Select Experiment Name. 2 Using the onscreen keyboard, type yor experiment name and then select Enter. 3 Select the appropriate recipe for yor experiment from the list of protocols. Scroll throgh to see all available protocols. 4 Select Next. Load Consmables The software gides yo throgh the steps to load the cbot reagent plate, flow cell, cbot manifold, and the 8- tbe strip containing prepared libraries. Depending on the clstering protocol selected, the software prompts yo to load an 8-tbe strip of additional primers. Load the Reagent Plate 1 Remove the clear plastic lid from the cbot reagent plate. 2 Select Scan Reagent ID to activate the barcode scanner. 3 Raise the instrment lid by lifting from the top-right corner. 4 [For TrSeq v3 reagents] Remove the white foil seal: a b Hold each end of the tbe strip in row 10 and peel the white foil from the 8-tbe strip. Discard the foil appropriately. Select the onscreen checkbox to indicate that the seal is removed. 5 Pll the reagent plate lever toward yo and place the reagent plate onto the reagent stage: All reagent plates except HiSeq X and HiSeq 3000/4000 Position with row 1 directly behind the tbe strip holders. The beveled corner of the plate is positioned in the front-right corner. HiSeq X and HiSeq 3000/4000 reagent plates Position with the barcode label facing toward the back of the instrment. The beveled corners of the plate are positioned directly behind the tbe strip holders. 21

29 Figre 12 Position the Reagent Plate 6 Release the lever to secre the reagent plate. 7 Select the onscreen checkbox to indicate that the reagent plate is loaded, and then select Next. Load the Flow Cell 1 Lift the flow cell clamp. 2 Wash the adapter plate on the thermal stage with a small amont of deionized water, and wipe dry with a lint-free cleaning tisse. 3 Remove the flow cell from storage: All flow cells except HiSeq X and HiSeq 3000/4000 Remove the flow cell from the storage tbe sing plastic forceps. Rinse the flow cell with deionized water and gently dry with a lens cleaning tisse. Save the tbe and bffer for later storage. HiSeq X and HiSeq 3000/4000 flow cells The patterned flow cell is ready to se after flow cell preparation. 4 Select Scan Flow Cell ID to activate the barcode scanner. 5 To scan the flow cell ID, hold the labeled flow cell package or tbe close to the scanner tray with the barcode positioned toward the instrment. 6 Position the flow cell on the thermal stage with flow cell port holes facing pward. Lane 1 is on the right side with the keyed corner. 7 Select the checkbox to indicate that the flow cell is loaded, and then select Next. Load the Manifold Use the manifold from the same clster kit as the flow cell. 1 Inspect the sippers on the sipper comb for damage. Make sre that the black rbber gaskets are evenly seated. 2 Position the manifold over the flow cell with the sipper comb pointing toward the front of the cbot. 3 Align the manifold with the gide pins on the thermal stage and seat the manifold over the flow cell. Seat evenly to form a tight seal. 4 Select the Manifold seated over flow cell checkbox. 5 Close the flow cell clamp to secre the manifold. 22

30 Figre 13 Close the Flow Cell Clamp 6 Select the Flow cell clamp closed checkbox. 7 Connect the otlet end of the manifold to the otlet port in the wash reservoir. Make sre that the otlet is evenly seated. Figre 14 Secre Otlet End A B Otlet port Otpt clamp 8 Snap the otlet clamp closed to secre the otlet end of the manifold. 9 Select the Otlet clamp closed checkbox. 10 Align the sipper comb with the 2 metal gide pins on the front of the thermal stage. Figre 15 Secre the Sipper Comb 23

31 A B Metal gide pins Plastic tabs 11 Snap the sipper comb into place sing the plastic tabs on either side of the comb. 12 Make sre that the sippers are straight and perpendiclar to the reagent plate, and that the Sipper comb in place checkbox is selected. 13 Select the Sipper comb in place checkbox, and then select Next. Load Templates 1 Select Enter Template Name. 2 Using the onscreen keyboard, type the template ID and then select Enter. 3 Load the 8-tbe strip containing prepared libraries into the template row. 4 Select the checkbox to indicate that templates are loaded. 5 If yo are sing additional primers, proceed to Load Primers. Otherwise, close the cbot lid and select Next to proceed to Perform a Pre-Rn Check on page 24. Load Primers The Load Primers screen appears for workflows that allow cstom primers or reqire additional primers. Seqencing Nextera libraries on a TrSeq v3 flow cell reqires that yo load an 8-tbe strip containing HP10. 1 Select Enter Primer Name. 2 Using the onscreen keyboard, type the primer name and then select Enter. 3 Load the 8-tbe strip containing primers into the primer row. Make sre that the order of nmbered tbes aligns with the lane orientation of the flow cell. Tbes are nmbered right to left. HiSeq X, HiSeq 3000/4000, HiSeq v4, and TrSeq v3 (HiSeq) HiSeq Rapid v2, TrSeq Rapid (HiSeq) 4 Select the checkbox to indicate that primers are loaded. 5 Close the instrment lid. 6 Select Next. Perform a Pre-Rn Check The pre-rn check reads the instrment sensors to detect the correct installation of rn components, and then performs a flow check sing bbble sensors to detect air in the tbes. The pre-rn check takes approximately 3 mintes. 1 After sccessfl completion of the pre-rn check, select Start. 24

32 The Rn Stats screen opens and the rn begins. Rn Component Errors If the pre-rn check fails de to errors related to rn components, perform the following steps: 1 Check any rn component with an indicated error to make sre that it is present and loaded correctly. 2 Select Rern Check to repeat the sensor check. 3 If the check contines to fail, select Cancel Rn to end the rn and set p a new one. Flow Check Failre Flow check failre might be the reslt of an improperly loaded flow cell, a falty manifold, or a clog in the lines. Before bypassing the flow check, see Trobleshoot Flow Check Failre on page 35. Monitor the Rn 1 Use the Rn Stats screen to monitor the rn in progress. The Rn Stats screen provides the rn stats and the following details: Start date and time, end date and time, and time remaining Clster generation protocol steps with stats bar for each step Reagent crrently in se Crrent temperatre ( C) Stats of the command in the crrent step Figre 16 Rn Stats Screen 2 Wait for the rn to complete: HiSeq v4, HiSeq 3000/4000 PE, or HiSeq X Allow approximately 3 hors. HiSeq 3000/4000 SR Allow approximately 4 hors. HiSeq Rapid v2 Allow approximately 1 hor. TrSeq v3 Allow approximately 5 hors. 3 After the rn is complete, yo can leave the flow cell on the instrment overnight. Otherwise, proceed to Unload Rn Components. 25

33 The instrment holds the flow cell at 20 C. Rn Data Report The rn data report provides a smmary of the rn in progress. It lists the following information: Protocol name Flow cell ID Reagent ID Template name Start time and finish time Yo can view the report at any time dring the rn. Select Men and then select Rn Data. At the end of the rn, the rn data report atomatically opens to alert yo that the rn is complete. Unload Rn Components 1 When the rn is complete, select Unload to proceed. Figre 17 Rn Complete, Unload Components 2 Raise the instrment lid. 3 Release the otlet clamp secring the otlet end of the manifold. 4 Disconnect the otlet end of the manifold from the otlet port in the wash reservoir. 5 Remove the sipper comb from the metal gide pins sing the plastic tabs on either side of the sipper comb. 6 Release the flow cell clamp. 7 Remove the manifold. Make sre that the flow cell remains on the thermal stage. 8 Lift the flow cell from the thermal stage. 9 Store the flow cell as appropriate: TrSeq v3 and HiSeq v4 flow cells Store in storage bffer in the flow cell tbe at 2 C to 8 C. The flow cell is stable after primer hybridization p to 10 days when properly stored in the flow cell tbe. 26

34 HiSeq Rapid v2 flow cells Perform the seqencing rn on the same day as library loading. HiSeq X and HiSeq 3000/4000 flow cells Store in storage bffer p to 48 hors at 2 C to 8 C. 10 Pll the reagent plate lever toward yo to release the reagent plate. Remove the reagent plate from the reagent stage. WARNING This set of reagents contains formamide, an aliphatic amide that is a probable reprodctive toxin. Personal injry can occr throgh inhalation, ingestion, skin contact, and eye contact. Wear protective eqipment, inclding eye protection, gloves, and laboratory coat. Handle sed reagents as chemical waste and discard in accordance with the governmental safety standards for yor region. For environmental, health, and safety information, see the SDS for this kit at spport.illmina.com/sds.html. 11 Remove the 8-tbe strip containing libraries. 12 Remove the 8-tbe strip containing additional primers, if applicable. 13 Select the checkbox to indicate that yo have nloaded the reagents, templates, and primers. 14 Choose a wash option: Select Wash to proceed to the post-rn wash. Select Exit to bypass the post-rn wash, if the bypass option is available. Perform a Post-Rn Wash 1 Wash the plate on the thermal stage with deionized water to remove any salts. Dry with a lint-free cleaning tisse. 2 Fill the wash reservoir with approximately 12 ml deionized water and close the instrment lid. 3 Select the checkbox to indicate that water is present, and then select Wash. 4 When the wash is complete, blot any excess water remaining in the wash reservoir. Avoid the otlet ports to prevent fibers from entering the holes. 5 Select the checkbox to indicate that the wash reservoir is dry, and then select Exit. The Start screen opens and yor cbot is ready for another rn. Confirm Reagent Delivery (Optional) Yo can confirm delivery of individal reagents from the reagent plate provided in any kit except HiSeq X and HiSeq 3000/ Inspect the foil-sealed tops of each tbe strip to make sre that each seal was pierced. 2 Release each tbe strip from the base of the reagent plate as follows. a b Grasp the reagent plate firmly, with finger tips nder the base. Gently press pward on the center tbes of the tbe strip. 3 Inspect each tbe to confirm that a similar volme remains in each tbe. NOTE Small differences are normal. 27

35 Figre 18 Example of Sccessfl Reagent Delivery (8-Lane Flow Cell) Figre 19 Example of Sccessfl Reagent Delivery (2-Lane Flow Cell) 4 If reagent delivery was not sccessfl and the foil seals on the reagent tbes are pierced, contact Illmina Technical Spport. 5 Inspect the 8-tbe strip containing library template. 6 If yo sed additional primers with yor rn, inspect the 8-tbe strip containing primers. 28

36 Chapter 5 Maintenance Perform Periodic Maintenance 29 Perform a Monthly Maintenance Wash 29 Change the Adapter Plate 30 Upgrade the Software 32 Upgrade Recipes 32 Sht Down the cbot 33 Perform Periodic Maintenance Perform the basic maintenance steps described in this section to ensre optimal performance. Maintenance Freqency Description Instrment wash Empty waste bottle Clean srfaces Clean barcode scanner window Maintenance Wash Check coolant level Between each rn and if the instrment is idle for more than a day. Between each rn. One time per week. One time per week. One time per month. Every 3 months. Always perform an instrment wash after each rn to clear salts and enzymes from the instrment hardware and to prevent clogs. If the instrment has been idle for more than 24 hors, a pre-rn wash is recommended. For more information, see Perform a Pre-Rn Wash on page 20. To make sre that yor rn is not interrpted, empty the waste bottle between rns. Use deionized water and a lint-free cleaning tisse to clean the srface of the thermal stage and the reagent stage. Clean the srface of the template and primer tbe strip holders. Use deionized water and a lint-free cleaning tisse to clean the barcode scanner window. Use 5% DECON (or 100 mm NaOH) to remove traces of reagents from internal cbot components and inhibit the growth of microorganisms. For more information, see Perform a Monthly Maintenance Wash on page 29. Make sre that the green coolant is visible throgh the coolant window on the rear panel of the instrment. If necessary, se a mirror to view the coolant window. If the coolant is low, se a wide coin or standard screwdriver to remove the coolant reservoir cap, and fill the reservoir to jst below the reservoir cap. Use only Illmina coolant (part # ). If yo need additional coolant, contact yor Illmina FAS or FSE. Preventive Maintenance Illmina recommends that yo schedle a preventive maintenance service each year. If yo are not nder a service contract, contact yor Territory Accont Manager or Illmina Technical Spport to arrange for a billable preventive maintenance service. Perform a Monthly Maintenance Wash Perform a monthly maintenance wash sing 5% DECON to remove traces of reagents from internal cbot components and inhibit microbial growth. If DECON is not available, sbstitte 100 mm NaOH. The maintenance wash reqires approximately 10 mintes of hands-on time and consists of 4 wash steps: a water wash, a DECON or NaOH wash, and 2 more water washes. 29

37 Water Wash 1 Confirm that all rn components are removed. 2 From the Start screen, select Men and then Manal Commands to open the Manal Commands screen. 3 Select Commands to open the Commands tab. 4 Fill the wash reservoir with approximately 12 ml deionized water. 5 Select Wash. 6 After the wash is complete, blot excess water from the wash reservoir with a low-lint tisse. Avoid the otlet ports to prevent fibers from entering the holes. DECON (or NaOH) Wash 1 Fill the wash reservoir with 10 ml 5% DECON or 100 mm NaOH. 2 Select Wash. 3 After the wash is complete, pt on a new pair of gloves. 4 Blot the 5% DECON remaining in the wash reservoir with a low-lint tisse. Avoid the otlet ports. CAUTION DECON is highly alkaline. 5 Immediately proceed to the water wash to prevent DECON from drying and clogging the wash reservoir holes. Water Wash (First Rinse) 1 Fill the wash reservoir with approximately 12 ml deionized water. 2 Select Wash. 3 After the wash is complete, blot water remaining in the wash reservoir with a low-lint tisse. Avoid the otlet ports. Water Wash (Final Rinse) 1 Fill the wash reservoir with approximately 12 ml deionized water. 2 Select Wash. 3 After the wash is finished, blot water remaining in the wash reservoir with a low-lint tisse. Avoid the otlet ports. 4 Close the instrment lid. 5 Empty the waste bottle. Yor cbot is ready for the next clster generation rn. Change the Adapter Plate Yo can se a HiSeq flow cell or a GAIIx flow cell on the cbot. Each flow cell type reqires that a specific adapter plate is installed. Icons on the Start screen indicate which adapter plate is installed. 30

38 NOTE The cbot is shipped with the HiSeq adapter plate installed. 1 Open the instrment lid by gently lifting from the top-right corner. 2 Lift the flow cell clamp. 3 Loosen the 2 captive Phillips head screws secring the adapter plate. Figre 20 Flow Cell Adapter Plate A B Captive screws Adapter plate 4 Lift the existing adapter plate from the thermal stage and set aside. 5 If salts are present on the thermal stage, wipe with a lint-free cleaning tisse slightly moistened with water. 6 Position the new adapter plate on the thermal stage. Align the sensor arm with the corresponding slot on the right side of the thermal stage. Figre 21 Sensor Arm Location A B C GAIIx adapter plate HiSeq adapter plate Adapter plate sensor arm 7 Tighten the 2 screws to secre the adapter plate. For optimal heat transfer, make sre that the adapter plate is sitting flat and the screws are tightened evenly. 8 Wipe the installed adapter plate with a lint-free cleaning tisse moistened with water. Dry with a clean tisse. 31

39 Upgrade the Software Using cbot software v1.3, or later, yo can pgrade instrment software sing a USB flash drive. 1 Insert the USB flash drive containing the new software version installer (for example, cbotsetpx86_ exe) into either of the USB ports on the front of the instrment. The installer mst reside in the root directory of the USB flash drive, not in a folder. CAUTION Leave the USB drive in the USB slot dring the pgrade process. Do not interact with the instrment dring the pgrade. 2 Select Men in the pper-left corner of the Start screen, and then select Configre. Figre 22 Start Screen Men 3 Use the onscreen keyboard to type the defalt password, admin, and then select Enter. 4 Select Men, and then select Upgrade. 5 A dialog box opens with a message abot the software version: Message The software installer version is greater than the version crrently installed on the cbot cbot cannot find a valid software installer The software installer version is eqal or lower than the version crrently installed on the cbot Action Select OK to proceed with the installation of the newer version. Yo can either insert a valid cbot pgrade and select OK to try again, or Cancel to abort the pgrade. Select Cancel to abort the pgrade, or OK to proceed with installation of a previos version. 6 When the restart is complete and the login screen opens, yo can remove the USB flash drive. Upgrade Recipes Upgrade recipe versions independently of software pgrades sing a USB flash drive containing the recipe installer. 1 Insert the USB flash drive containing the new recipe installer into either of the USB ports on the front of the instrment. The installer mst reside in the root directory of the USB flash drive, not in a folder. 32

40 2 Select Men in the pper-left corner of the Start screen, and then select Configre. Figre 23 Start Screen Men 3 Use the onscreen keyboard to type the defalt password, admin, and then select Enter. 4 Select Men, and then select Upgrade Recipes. When the pgrade is complete, the cbot restarts atomatically. The restart process takes abot 10 mintes. CAUTION Leave the USB drive in the USB slot dring the pgrade process. Do not interact with the instrment dring the pgrade. 5 When the restart is complete and the login screen opens, remove the USB flash drive. Sht Down the cbot The cbot is designed to rn in an idle state from the Start screen, so shtting it down between rns is not necessary. 1 Select Men in the pper-left corner of the Start screen, and then select Configre. Figre 24 Start Screen Men 33

41 2 Use the onscreen keyboard to type the defalt password, admin, and then select Enter. 3 From the Configration screen, select Men and then select Sht Down Station. The cbot software shts down. Figre 25 Sht Down Station 4 After the software shts down, switch the power switch to the OFF position. Reboot in FSE Mode The option to reboot in FSE Mode is for se by a trained Illmina Field Application Scientist (FAS) or Field Service Engineer (FSE) to pdate software or service the instrment. 34

42 Chapter A Trobleshooting Pase or Abort a Rn 35 Trobleshoot Flow Check Failre 35 Trobleshoot Rn Problems 37 Reset the Barcode Scanner 38 Edit Protocols 38 Pase or Abort a Rn Use commands on the Rn Stats screen to pase or abort a rn. Pase Completes the crrent command in the protocol, and then pases the rn. Allow a few mintes before the rn pases. When the rn is pased, the sippers lift from the reagent tbes, the reagent stage retrns the home position, and the Pase btton changes to the Resme btton. When the rn is active, select Pase to pase the rn. When the rn is pased, select Resme to resme the rn. Abort Rn Ends the rn withot the option of resming. Select Unload to nload rn components. Trobleshoot Flow Check Failre Perform the following procedre to trobleshoot flow check failre. Do not select the option to bypass flow check ntil yo have completed this procedre to determine the following conditions: The flow cell is properly positioned on the instrment. The manifold and hardware are working properly. NOTE Bypassing the flow check can reslt in nsccessfl clstering of some lanes. Becase different flow cell types se different flow checks, make sre that yo se the correct recipe, manifold, and flow cell combination. 1 Make sre that yo have enogh HT1 in row 1 of the reagent plate, and replenish as needed. 2 Note which lanes failed the flow check. This information is provided on the top-left corner of the interface screen. If all 8 lanes failed, the flow cell is probably loaded improperly. Remove the manifold and confirm that the flow cell holes are facing pward, and the flow cell orientation is correct. If only some lanes failed, the flow cell might not be seated. Remove the manifold, reseat the flow cell, and then reinstall the manifold. 3 Select Rern Check to repeat the flow check a second time. 4 If the flow check fails a second time, note which lanes failed the flow check, and do 1 of the following: If all 8 lanes failed, yo probably have a falty manifold. Replace it with a new manifold. If different lanes failed, yo probably do not have a falty manifold. Inspect the volmes of HT1 in row 1 to make sre that the tbes contain eqal volmes. 5 Select Rern Check to repeat the flow check for a third time. If this flow check fails after replacing the manifold, go to step 6. If this check fails and yo did not need to replace the manifold, go to step 7. 6 If the flow check fails a third time after replacing the manifold, yo might have a clog in the hardware. 35

43 a b c d Inspect the volmes of HT1 in row 1 to make sre that the tbes contain eqal volmes. Higher volmes in the tbes that correspond to lanes with repeated flow check failre indicate a hardware clog. Unload the rn components and perform a maintenance wash. After the wash, trn off the instrment sing the power switch. After a few seconds, trn on the power switch and then press the start btton to restart the software. Power cycling the instrment resets the allowable nmber of pre-rn check attempts. Follow the software prompts to reload rn components and set p yor rn. 7 If the flow check fails a third time, yo can safely bypass the flow check: a b Select Bypass Flow Check to proceed with the rn. After the rn, check reagent delivery from all tbes. Trobleshooting Flowchart The following flowchart illstrates the trobleshooting procedre. Steps to repeat the flow check inclde a nmber to indicate how many of the allowed flow checks have been performed at that point in the procedre. 36

44 Figre 26 Trobleshooting Flowchart Trobleshoot Rn Problems Use the following table to trobleshoot problems encontered dring a clster generation rn. Problem Possible Case Action Temperatre ot of range Often indicates that the cbot did not reach the set temperatre in the expected time. Can also indicate a potential control board failre. Illmina Technical Spport. Coolant is flowing and coolant level is low Coolant has slowly evaporated to a low level. Add Illmina coolant (part # ) to the coolant reservoir. 37

45 Problem Possible Case Action Coolant is not flowing and coolant level is low Coolant is not flowing and coolant level is not low Instrment is in a locked state Coolant level might be too low to generate flow. Potential coolant pmp failre. Potential software error. Add Illmina coolant (part # ) to the coolant reservoir. Illmina Technical Spport. Illmina Technical Spport. Reset the Barcode Scanner The barcode scanner is ready for se when yo receive yor cbot. If the scanner is reset to an incorrect configration, se the following instrctions to restore it to the defalt configration. 1 Print the barcode. Figre 27 Restore Defalts Barcode 2 From the start screen, select Men and then Manal Commands. 3 Select the General tab to access the barcode reader manal control inpts. Figre 28 Manal Commands, General Tab 4 Select Trn Off and then select Trn On to activate the barcode scanner. The laser line is visible on the scanner plate nder the LCD screen. 5 Place the barcode nder the barcode scanner. 6 Select Trn Off, and then Trn On to scan the barcode. A beep indicates a sccessfl scan. Edit Protocols Use the Protocol Editor to edit protocols to fit yor needs. Yo might want to repeat steps in a protocol, or change the nmber of amplification cycles in the chemistry section. 38

46 Each protocol consists of 2 main sections: Chemistry section Contains instrctions for pmping reagents, temperatre changes, and wait drations. This section appears in the pper portion of the Protocol Editor screen. Protocol section Contains a series of steps made p of chemistry definitions. This section appears in the lower portion of the Protocol Editor screen. If yo edit an existing protocol, make sre to rename yor protocol. Protocol Editor 1 From the Start screen, select Men, and then select Protocol Editor. 2 From the Protocol Editor, select Men, and then select the appropriate command: Open Opens an existing protocol. Select Load from Library Loads an existing chemistry definition or protocol step stored in the cbot library. Select New Chemistry Definition or New Protocol Step Creates a definition or step and stores it in the cbot library. 3 Use the down arrow to the left of the step to expand the commands in the step. Use the p arrow to collapse the commands. 4 To edit a step in a chemistry definition, highlight the step. Selections to change the pmp, temperatre ramp, or wait commands appear in the right-hand panel. 5 To edit a step in the protocol, highlight the step. Selections to change to the nmber of cycles for the selected chemistry definition appear in the righthand panel. 6 Use the Protocol Editor icons to the right of the step name to rearrange, delete, or copy steps and commands. Figre 29 Protocol Editor, Expanded Steps A B C Chemistry section Expanded chemistry section Protocol section 39