HiSeq X. System Guide. For Research Use Only. Not for use in diagnostic procedures. Document # v06 Material # October 2017

Transcription

1 HiSeq X System Gide Docment # v06 Material # October 2017 ILLUMINA PROPRIETARY

2 HiSeq X System Gide 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. Illmina, BaseSpace, HiSeq X, and the streaming bases design are registered or pending trademarks of Illmina, Inc. and/or its affiliate(s) in the U.S. and/or other contries. All other names, logos, and other trademarks are the property of their respective owners. Docment # v06 Material # ii

3 HiSeq X System Gide Revision History Docment Date Description of Change Docment # v06 Material # Docment # v05 Material # Docment # v04 Material # Docment # v03 Material # Docment # v02 Material # Docment # v01 October 2017 October 2017 Janary 2017 September 2016 May 2016 December 2015 Updated software descriptions for HiSeq Control Software HD v3.5, which enables dal indexing. Updated reagent information to replace HP12 with HP14 and PLM2 with PLM2 v2. Version not sed. Updated the maintenance wash procedre. Updated the control software name to HiSeq Control Software HD v3.4. Updated Illmina catalog # for HiSeq X HD cbot Mlti-Primer Rehybridization Kit togd Removed Sigma-Aldrich catalog # SRE0076 for SeqClin Wash Soltion. Added Cstom Protocol Selector to Additional Resorces. Added Sigma-Aldrich catalog # SRE0076 for SeqClin Wash Soltion. Noted approximate freqency for renewing wash bottles and tbes. Updated instrctions for starting the instrment: Wait for the system to load before logging on to the operating system, not after. Increased the dration for instrment devices to be configred and DoNotEject to initialize from 1 minte to 3 mintes. Noted that hard drives mst be empty for proper operation. Updated qick format instrctions to inclde the scratch (S:\) drive. Corrected instrctions for accessing the log file. Updated software descriptions for HiSeq Control Software v3.3.76: Added instrctions for BaseSpace Enterprise sbscribers to configre a domain. Added information abot the sensor indicators showing the data transfer stats of BaseSpace Seqence Hb. Updated the instrctions for staggering rns to inclde se of the Pase btton. Renamed BaseSpace to BaseSpace Seqence Hb. Added the cbot 2 System Gide (docment # ) as a reference for clstering. Removed the defalt ser name and password reqired to log on to the operating system. Illmina recommends sing site-specific credentials. Removed the catalog nmber for this gide. Added the folder strctre for otpt files and information abot the rn folder. Added recommendation for annal preventive maintenance service. Corrected expected volmes for the maintenance wash. Docment # v06 Material # iii

4 HiSeq X System Gide Docment Date Description of Change Part # Rev. E Part # Rev. D Jly 2015 Janary 2015 Updated software descriptions for HiSeq X Control Software v3.3: Updated the maintenance wash protocol. A Tween 20 and ProClin 300 wash replaces the three-step NaOH wash. Replaced flow cell type HiSeq X HD v2 with flow cell type HiSeq X on the Flow Cell Setp screen. Added information abot a sensor indicator that shows data transfer stats for the RnCopyService software. Added instrctions for preparing reagents and seqencing primers sed for indexing and paired-end resynthesis. Added section titled Workflow Differences for Illmina SeqLab, which describes where to find reagent preparation and seqencing instrctions if yo are sing Illmina SeqLab. Added catalog nmbers for HiSeq X v2.5 kits and pdated the catalog nmber of the rehybridization kit. Updated instrctions for staggering rns on flow cell A and flow cell B. Replaced descriptions of system settings with instrctions for cstomizing system settings. Moved information abot trobleshooting to Appendix A, and information abot Real-Time Analysis software to Appendix B. Removed offsets files and phasing files from the list of seqencing otpts. RTA2 no longer prodces these files. Removed the HiSeq X Five Lab Setp and Site Prep Gide (part # ) from Additional Resorces. Lab setp and site prep information for HiSeq X Ten, HiSeq X Five, and Illmina SeqLab is available in the HiSeq X System Lab Setp and Site Prep Gide (docment # ). Corrected the nmber of base calls below 0.6 allowed in the first 25 cycles from 2 to 1. Updated the list of HiSeq X reagent kits to inclde HiSeq X Ten reagent kits and HiSeq X Five reagent kits. Changed the name of the 20-pack kit to a 10-pack kit. (Change in name only; contents have not changed.) Added the HiSeq X Five Lab Setp and Site Prep Gide (part # ) to Additional Resorces. Docment # v06 Material # iv

5 HiSeq X System Gide Docment Date Description of Change Part # Rev. C Part # Rev. B Part # Rev. A October 2014 May 2014 March 2014 Updated software descriptions for HiSeq X Control Software v3.1: Added flow cell type HiSeq X HD v2 to the Flow Cell Setp screen. Added option to perform either a maintenance wash or a water wash after each rn. A maintenance wash is recommended after each rn bt no longer reqired. Gasket replacement is no longer reqired with every maintenance; instead replace gaskets before starting the reqired maintenance wash every 10 days. Added option to wash SBS reagent positions only. Removed Indexing Reagent Kit ID from the Reagents screen. Indexing reagents are packaged with paired-end reagents in HiSeq X HD reagent kits. Updated the Seqence command on the Welcome screen. Noted that the HiSeq X HD (v1) flow cell and the HiSeq X HD v2 flow cell can be seqenced simltaneosly as Flow Cell A and Flow Cell B. Noted that specifying an otpt folder is reqired even when connected to BaseSpace for storage and analysis. Added reminder to invert each bottle several times before changing the caps and loading SBS reagents. Updated VWR catalog # for alcohol wipes to Updated URL for Safety Data Sheets (SDS) to spport.illmina.com/sds.html. Updated the following information: Added description of clsters passing filter based on characteristics of a patterned flow cell. Added description of Q-score binning. Updated available disk space reqirements. Noted that wait time for instrment devices to initialize is at least 1 minte. Added best practice to perform a qick format of the O:\ drive after each rn. Corrected part nmber of the HiSeq X HD Reagent Kit Reference Gide on the workflow diagram to Initial release. Docment # v06 Material # v

6 Table of Contents Revision History iii Chapter 1 Overview 1 Introdction 1 Additional Resorces 1 Instrment Components 2 Seqencing Consmables Overview 6 Chapter 2 Getting Started 8 Start the HiSeq X 8 Cstomize System Settings 8 View and Send Instrment Data 9 User-Spplied Consmables 10 Chapter 3 Preparing Reagents 11 Introdction 11 Prepare SBS Reagents 11 Prepare Indexing and Paired-End Reagents 12 Chapter 4 Seqencing 13 Introdction 13 Seqencing Workflow 13 Enter Rn Parameters 14 Load and Prime Reagents 16 Load the Seqencing Flow Cell 20 Monitor the Rn 22 Unload Reagents 22 Perform a Water Wash 23 Qick Format the Otpt and Scratch Drives 24 Chapter 5 Maintenance 25 Introdction 25 Perform a Maintenance Wash 25 Idle the Instrment 29 Sht Down the Instrment 30 Appendix A Trobleshooting 31 Log File 31 Possible Rn Setp Problems 31 Perform a Flidics Check 31 Pase or End a Rn on the HiSeq X 32 Stagger Rns on Flow Cell A and Flow Cell B 33 Docment # v06 Material # vi

7 HiSeq X System Gide Possible Read 1 Primer Rehybridization 33 Appendix B Real-Time Analysis 34 Real-Time Analysis Overview 34 Real-Time Analysis Workflow 35 Appendix C Otpt Files and Folders 39 Seqencing Otpt Files 39 Otpt Folder Strctre 39 Rn Folder Name and Path 40 Tile Nmbering 40 Index 42 Technical Assistance 46 Docment # v06 Material # vii

8 Chapter 1 Overview Introdction 1 Additional Resorces 1 Instrment Components 2 Seqencing Consmables Overview 6 Introdction The HiSeq X system combines innovative engineering with proven SBS technology and the power to enable poplation-scale hman whole-genome seqencing. Featres Dal-srface imaging The HiSeq X ses a two-camera, for-sensor epiflorescence system with ctting-edge scanning technology to enable dal srface imaging. Patterned flow cell A patterned flow cell allows the generation of seqencing clsters in an ordered arrangement, which increases otpt reads and data. High-capacity reagent chiller The reagent compartment is a high-capacity chiller that holds enogh reagents for the entire seqencing rn. Integrated flidics for paired-end rns Integrated paired-end flidics provide reagents from the reagent compartment to the flow cell for Read 2 resynthesis and for indexed seqencing. Interface control options The instrment software interface provides options for setting p a rn and operating the instrment. Use the toch screen monitor or the integrated keyboard to provide inpt. Real-time base calling The instrment software extracts intensities from images and performs qalityscored base calling on the instrment compter. This method allows monitoring of qality metrics dring the rn and saves time dring sbseqent data analysis. Downstream analysis of seqencing data can be performed with Illmina analysis software or third-party software on a cstom infrastrctre. BaseSpace Seqence Hb integration The seqencing workflow is integrated with BaseSpace Seqence Hb, the Illmina genomics compting environment for data analysis, storage, and collaboration. As the rn progresses, otpt files are streamed in real time to BaseSpace Seqence Hb. Workflow Differences for Illmina SeqLab When sing HiSeq X as a component of Illmina SeqLab, Clarity LIMS X Edition introdces workflow differences that are not describes in this gide. All steps from library prep throgh seqencing are affected. Visit the Illmina SeqLab spport page on the Illmina website to generate a cstom workflow gide for yor experiment. Additional Resorces The following docmentation is available for download from the Illmina website. Docment # v06 Material #

9 HiSeq X System Gide Resorce Cstom Protocol Selector HiSeq X System Lab Setp and Site Prep Gide (docment # ) HiSeq X System Safety and Compliance Gide (docment # ) Description A wizard for generating cstomized end-to-end docmentation that is tailored to the library prep method, rn parameters, and analysis method sed for the seqencing rn. Provides specifications for laboratory space, electrical reqirements, and environmental considerations. Provides information abot instrment labeling, compliance certifications, and safety considerations. Visit the HiSeq X spport page on the Illmina website for access to docmentation, software downloads, online training, and freqently asked qestions. For information specific to Illmina SeqLab, visit the Illmina SeqLab spport page. Instrment Components The HiSeq X system comprises the instrment, monitor, instrment control compter, and accessories, sch as a keyboard, mose, and barcode scanner. The instrment incldes for main compartments: the optics modle, flow cell compartment, flidics compartment, and reagents compartment. An illminated stats bar indicates operating stats. Figre 1 External Components A Optics modle Contains optical components that enable dal srface imaging of the flow cell, imaging A, C, G, and T at the same time sing epiflorescence. The excitation laser beam passes throgh the objective and the florescence is simltaneosly collected throgh the same objective. B Flow cell compartment Contains the vacm-controlled flow cell stage, which holds the flow cell in place dring seqencing rns. C Flidics compartment Contains flidics pmps that deliver reagents to the flow cell, and then to the waste container. D Stats bar Uses three colors to indicate instrment stats. Ble indicates that the instrment is rnning, orange indicates that the instrment needs attention, and green indicates that the instrment is ready to begin the next rn. E Reagent compartment Contains reagent racks that hold reagents for seqencing rns and wash soltion for instrment washes. Docment # v06 Material #

10 HiSeq X System Gide Flow Cell Compartment The flow cell compartment hoses the flow cell stage, the thermal stations, the vacm system, and the flidics connections to each flow cell. Figre 2 Flow Cell Stage With Two Flow Cells A B C D Flow cella Flow cellb Flow celllever A Flow celllever B Flow cell A is on the left, and flow cell B is on the right. Each flow cell is seated on the flow cell stage, which moves in and ot of the optics modle as directed by the control software. The flow cell stage mst be in the forward-most position to open the flow cell compartment door and load or remove a flow cell. The flow cell is positioned on the flow cell holder with the inlet and otlet ports facing down. A vacm beneath the flow cell holder holds the flow cell in place. The illminated flow cell lever in front of each flow cell holder controls the vacm. The flow cell lever trns green when the vacm seal is secre. Reagent Compartment The reagent compartment is a high-capacity reagent chiller that holds three reagent racks: two for SBS reagents and 1 for indexing and paired-end reagents. Sipper handles lower the sippers into the reagent bottles. SBS reagent racks Hold 250 ml conical bottles. The reagent rack for flow cell A is in the center position, and the rack for flow cell B is in the far right position. Each reagent rack has nmbered positions that correspond to connections on an internal reagent selector valve. Indexing and paired-end reagent rack Located in the left position. It has two rows of nmbered positions that hold 15 ml conical tbes containing paired-end reagents and indexing reagents. The left row is for flow cell A, and the right row is for flow cell B. Reagent chiller The reagent chiller hoses the reagent racks and maintains an internal temperatre of 2 C to 8 C. Docment # v06 Material #

11 HiSeq X System Gide Figre 3 Reagent Compartment A B C D Sipper handles Reagent rack for indexing and paired-end reagents Reagent rack for SBSreagentsfor flow cella Reagent rack for SBSreagentsfor flow cellb HiSeq X Software Three software applications are installed on the instrment compter: HiSeq X control software The HiSeq Control Software HD interface gides yo throgh the steps to set p a seqencing rn. Dring the rn, the control software operates instrment hardware, controls flidics, sets temperatres, and provides a visal smmary of qality statistics. Real-Time Analysis software Integrated with the control software, Real-Time Analysis performs base calling and assigns a qality score to each base for each cycle. For more information, see Real-Time Analysis on page 34. Seqencing Analysis Viewer software Seqencing Analysis Viewer (SAV) provides detailed qality statistics. Stats Icons A stats icon located in the pper-right corner of each screen shows changes in conditions, errors, or warnings dring rn setp and dring the rn. Stats Icon Stats Name Stats okay Description No change. System is normal. Information Information only. No action is reqired Attention Information that might reqire attention. Docment # v06 Material #

12 HiSeq X System Gide Stats Icon Stats Name Warning Description Warnings do not stop a rn, bt might reqire action before proceeding. Error Errors sally stop a rn and generally reqire action before proceeding with the rn. When a change in condition occrs, the associated icon blinks to alert yo. Select the icon to open the stats window and view a description of the condition. Select Acknowledge to accept the message and Close to close the dialog box. Activity and Sensor Indicators The Welcome screen contains a series of icons in the lower-right corner of the screen. The icons indicate instrment activity and stats of specific components based on instrment sensors. Figre 4 Activity Indicators From left to right, activity indicators represent the X, Y, and Z motors, electronics fnctionality, the camera, the flidics system, and processing fnctions. Figre 5 Sensor Indicators From left to right, sensor indicators represent flow cell A temperatre, reagent chiller temperatre, data transfer stats, BaseSpace Hb clod stats, and flow cell B temperatre. Data Transfer Stats The HiSeq X software site incldes Rn Copy Service, which manages data transfer to the otpt folder. A BaseSpace option sends instrment health and seqencing data to BaseSpace Seqence Hb. Two of the sensor indicators on the software interface show the transfer stats of Rn Copy Service and BaseSpace Seqence Hb. Rn Copy Service The transfer stats of Rn Copy Service affects whether yo can start a new rn or safely format the otpt drive. Stats Icon Description Data are transferring. Do not format the otpt drive ntil transfer is complete. Docment # v06 Material #

13 HiSeq X System Gide Stats Icon Description Data are transferring bt the network connection is slow. Yo can set p a seqencing rn and format the otpt drive when transfer is complete. Rn Copy Service is off. Rn Copy Service is on bt not transferring data. BaseSpace Seqence Hb A BaseSpace sensor indicator shows the stats of BaseSpace Seqence Hb. A ble clod indicates an active connection. A gray clod indicates that the software cannot connect. The following table provides additional details abot each stats icon. Stats Icon Description Not connected to BaseSpace Seqence Hb. Connected to BaseSpace Seqence Hb bt not transferring data. Connected to BaseSpace Seqence Hb and transferring data for for rns or less. Connected to BaseSpace Seqence Hb and transferring data for five or more rns. While this icon is displayed, the control software does not allow any new rns to connect to BaseSpace Seqence Hb. Disconnected from BaseSpace Seqence Hb with data qeed for transfer. Seqencing Consmables Overview Illmina reagent kits are reqired for seqencing on the HiSeq X. Each kit contains clstering reagents sed on the cbot, and SBS reagents, indexing reagents, and paired-end reagents sed on the HiSeq X. Single-pack kit Each kit incldes consmables for seqencing two flow cells, or one dal flow cell rn. Consmables are packaged in two complete sets, each set spports one flow cell. 10-pack kit Each kit incldes consmables for seqencing 20 flow cells, or 10 dal flow cell rns. Consmables are packaged to spport for flow cells at a time. Docment # v06 Material #

14 HiSeq X System Gide HiSeq X Kit Name Catalog # HiSeq X Ten Reagent Kit v2.5 (300 cycles) HiSeq X Ten Reagent Kit v2.5 (300 cycles), 10 pack HiSeq X Five Reagent Kit v2.5 (300 cycles) HiSeq X Five Reagent Kit v2.5 (300 cycles), 10 pack FC FC FC FC Patterned Flow Cell The HiSeq X ses a patterned flow cell with billions of ordered nanowells that are manfactred into the glass of the flow cell. The ordered arrangement increases the nmber of otpt reads and amont of seqencing data generated. The patterned flow cell is provided in the HiSeq X Reagent Kit v2.5. Figre 6 Example of Clsters on a Patterned Flow Cell Docment # v06 Material #

15 Chapter 2 Getting Started Start the HiSeq X 8 Cstomize System Settings 8 View and Send Instrment Data 9 User-Spplied Consmables 10 Start the HiSeq X 1 Start the instrment control compter. 2 Wait for the system to load, and then log on to the operating system. If necessary, conslt yor facility administrator for the ser name and password. 3 Locate the power switch on the left side of the instrment and switch it to the ON position. 4 Wait at least three mintes for the instrment devices to be configred and for the instrment drive called DoNotEject to initialize. 5 Close the window that opens when DoNotEject is initialized. If the window does not open, se MyCompter to check for the DoNotEject drive. NOTE Never eject the DoNotEject flash drive located inside the instrment chassis, or modify the files on it. This drive contains hardware configration files and initializes whenever the instrment is trned on. 6 To ensre adeqate disk space, archive previos rn data on the instrment compter to a network location. Perform a qick reformat of the O:\ and S:\ drives to clear any remaining data. The hard drives mst be empty for proper software operation. 7 Open HCS sing the shortct icon on the desktop. When the software has initialized, the Welcome screen opens and the Initialized icon appears in the bottom-right corner of the screen. Instrment and Control Compter Best Practices Do not trn on the compter while the instrment is rnning. Always trn on the compter before trning on the instrment. Do not trn off the instrment while the instrment control software is rnning. Wait 1 minte after trning off the instrment before trning it on again. Connect the USB cables for the instrment, the monitor, and the keyboard to the back of the compter before trning on the compter. Connect the barcode scanner and mose to the USB ports on the front of the compter. Cstomize System Settings The control software incldes cstomizable system settings for rn folders, LIMS preferences, and domains. The Men Options window has settings to define the rn ID template, defalt folder locations, whether to send instrment health data, LIMS athentication, and BaseSpace Enterprise domains. To cstomize yor view of the interface, select Men View. Yo can choose to view the interface in fll screen or in a window, or to minimize. Docment # v06 Material #

16 HiSeq X System Gide Define Rn Folder Settings 1 From the Welcome screen, select Men Tools Options to open the Men Options window. 2 To cstomize the naming convention for rn folder names, modify the settings in the Rn ID Template field. Select Reset to clear the field. 3 To set defalt otpt locations, enter a location for each of the following folders: Defalt Otpt Folder The defalt otpt folder for rns on flow cell A. Defalt Otpt Folder2 The defalt otpt folder for rns on flow cell B. NOTE Illmina recommends a network location for the otpt folders. However, if the location differs from the HiSeq Temp folder, yo can specify a location on the O:\ drive. Do not se the S:\ drive or the C:\ drive. The S:\ drive is reserved for instrment operations and the C:\ drive is too small. 4 To set a location for LIMS sample forms, enter the location in the Rn Setp Folder field. 5 Select OK to save yor work and close the Men Options window. Select Cancel to close withot saving. Set LIMS Preferences 1 From the Welcome screen, select Men Tools Options to open the Men Options window. 2 Enter the following LIMS settings: LIMS Server The server name for interactions with spported Illmina LIMS. LIMS User Name The ser name sed when athenticating to Illmina LIMS. LIMS Password The password sed when athenticating to Illmina LIMS. 3 Select OK to save yor work and close the Men Options window. Select Cancel to close withot saving. Configre a Domain If yo are a BaseSpace Enterprise sbscriber, se the following instrctions to configre yor domain. 1 From the Welcome screen, select Men Tools Options to open the Options window. 2 Enter the domain for the BaseSpace Seqence Hb server. 3 Select OK to save yor work and close the Options window. Select Cancel to close withot saving. View and Send Instrment Data The Men btton on the Welcome screen and the Men Options window provide options for viewing and sending instrment data. To view information abot instrment hardware, software versions, and technical spport contact information, select Men Abot. To permit the instrment to send information to BaseSpace Seqence Hb for each rn (recommended), select Men Tools Options, and then select the Send instrment health data to Illmina to help Illmina improve its prodcts checkbox. All information remains confidential. Docment # v06 Material #

17 HiSeq X System Gide User-Spplied Consmables For a comprehensive list of ser-spplied consmables, see the HiSeq X System Lab Setp and Site Prep Gide (docment # ). Consmable Spplier Prpose Alcohol wipes, 70% Isopropyl or Ethanol, 70% Carboy, at least 6 liters General lab spplier VWR, catalog # General lab spplier Corning, catalog # Gloves, powder-free General lab spplier General se. Cleaning the flow cell and flow cell stage. Preparing maintenance wash soltion. Lab tisse, low-lint VWR, catalog # Cleaning the flow cell holder. ProClin 300, 50 ml* Sigma-Aldrich, catalog # U Maintenance wash. Tbes, centrifge, 250 ml Tbes, conical, 15 ml General lab spplier Corning, catalog # General lab spplier Corning, catalog # Instrment wash. SBS reagent rack, positions containing PW1. Collecting and measring waste volmes. PE reagent rack, positions containing PW1. Tween 20, viscos liqid, 100 ml Sigma-Aldrich, catalog # P7949 Maintenance wash. Tweezers, sqare plastic tip McMaster-Carr, catalog # 7003A22 Removing the flow cell gaskets. Water, laboratory-grade, 18 MΩ Millipore Water wash. SBS and PE reagent racks, positions containing PW1. * ProClin 300 is restricted for IVD se only. Docment # v06 Material #

18 Chapter 3 Preparing Reagents Preparing Reagents Introdction 11 Prepare SBS Reagents 11 Prepare Indexing and Paired-End Reagents 12 Introdction Before setting p the rn, prepare all reagents for seqencing: SBS reagents, indexing reagents, and pairedend reagents. Reagent preparation instrctions are the same regardless of kit version. All reagents are loaded when prompted by the software dring rn setp. Retrning to the instrment dring the rn to reload is not necessary. Seqencing reagents can be prepared dring clster generation. For instrctions on flow cell and clster reagent preparation and clstering, see the cbot 2 System Gide (docment # ) or cbot System Gide (docment # ). Prepare SBS Reagents Use the following instrctions to thaw and inspect SBS reagents: PSM, PIM, and PCM. Use PB1 and PB2 directly from storage. Prepare the appropriate nmber of SBS reagent bottles. Reagent For One Flow Cell (Single Flow Cell Rn) For Two Flow Cells (Dal Flow Cell Rn) For For Flow Cells (Two Dal Flow Cell Rns) PB PB PCM PIM PSM Thaw SBS Reagents 1 Remove for bottles each of PSM, PIM, and PCM from -25 C to -15 C storage. 2 Thaw at 2 C to 8 C for abot 16 hors. Alternatively, thaw PSM and PIM in a room temperatre deionized water bath for abot 90 mintes. Thaw PCM in a separate water bath. NOTE Always replace yor gloves after handling PCM. 3 Invert each bottle to mix. 4 Inspect PSM to make sre that no swirling patterns are visible. 5 Set aside PSM and PIM on ice. 6 Set aside PCM on ice separately to prevent cross-contamination. Docment # v06 Material #

19 HiSeq X System Gide Prepare Indexing and Paired-End Reagents Indexing and paired-end reagents are sed dring the Read 2 resynthesis step of a paired-end seqencing rn. Prepare the appropriate nmber of tbes for each indexing and paired-end reagent. Reagent Qantity for One Flow Cell (Single Flow Cell Rn) Qantity for Two Flow Cells (Dal Flow Cell Rn) Qantity for For Flow Cells (Two Dal Flow Cell Rns) PRM PLM2 v PAM PPM PDR HP HP WARNING This set of reagents contains potentially hazardos chemicals. Personal injry can occr throgh inhalation, ingestion, skin contact, and eye contact. Wear protective eqipment, inclding eye protection, gloves, and laboratory coat appropriate for risk of exposre. Handle sed reagents as chemical waste and discard in accordance with applicable regional, national, and local laws and reglations. For additional environmental, health, and safety information, see the SDS at spport.illmina.com/sds.html. Thaw Indexing and Paired-End Reagents 1 Remove the following reagents from -25 C to -15 C storage: PRM, PLM2 v2, PAM, PPM, PDR, HP11, and HP14. For non-indexed libraries, HP14 is not reqired. 2 Thaw in a room temperatre deionized water bath for approximately 20 mintes. 3 Set aside PRM, PLM2 v2, and PAM on ice. Prepare PRM, PLM2 v2, PAM, PPM, PDR, HP11, and HP14 1 Invert each tbe to mix. 2 Centrifge at 1000 rpm for one minte. 3 Set aside PRM, PLM2 v2, and PAM on ice. 4 Set aside PPM, PDR, HP11, and HP14 at room temperatre. Docment # v06 Material #

20 Chapter 4 Seqencing Introdction 13 Seqencing Workflow 13 Enter Rn Parameters 14 Load and Prime Reagents 16 Load the Seqencing Flow Cell 20 Monitor the Rn 22 Unload Reagents 22 Perform a Water Wash 23 Qick Format the Otpt and Scratch Drives 24 Introdction To perform a rn on the HiSeq X, prepare all reagents and then follow the software prompts to set p the rn. Rn setp steps inclde entering rn parameters, loading and priming reagents, loading the flow cell, and performing a flidics check. Rn setp steps are organized in three tabs: Rn Configration, Pre-Rn Setp, and Initiate Rn. Rn configration screens contain drop-down lists, checkboxes, or text fields for rn parameters. Use the hand-held barcode scanner to scan the flow cell or reagent kit ID, or enter the ID sing the toch screen keyboard. The keyboard icon is located to the right of the text fields. Select Next to move to the next screen, or select Back to retrn to the previos screen. At any time dring the rn setp steps, select Cancel to exit rn setp and retrn to the Welcome screen. For information abot rn dration and other performance specifications, see the HiSeq X specifications page on the Illmina website. Staggering Rns Yo can start a new rn on either flow cell A or flow cell B when a rn on the adjacent flow cell is in progress. For more information, see Stagger Rns on Flow Cell A and Flow Cell B on page 33. Seqencing Workflow Prepare the flow cell and reagents for the rn. Using the prompts on the control software interface, enter rn parameters. Load SBS reagents for Read 1 and Read 2. If applicable, load indexing and paired-end reagents. Docment # v06 Material #

21 HiSeq X System Gide With a sed flow cell, confirm proper flow. Prime SBS reagents and measre priming waste. Load a clstered HiSeq X flow cell, and confirm proper flow. Start the seqencing rn. [Optional] After cycle 2, inspect the First Base Report and then contine Read 1. When the rn is complete, nload reagents. Perform an instrment wash. Enter Rn Parameters Begin rn setp by entering rn parameters from a series of screens on the Rn Configration tab. The software gides yo throgh each screen to specify BaseSpace Seqence Hb connectivity, enter consmable IDs, select indexing options, and record other parameters. Storage Screen 1 From the Welcome screen, select Seqence to open the Storage screen. 2 [Optional] Connect to BaseSpace Seqence Hb as follows. a b c Select Connect to BaseSpace. Select from the following BaseSpace options: Storage and Analysis Sends rn data to BaseSpace Seqence Hb for remote monitoring and data analysis. A sample sheet is reqired with this option. Rn Monitoring Only Sends only InterOp files to BaseSpace Seqence Hb, which allows yo to monitor the rn remotely. Log on to BaseSpace Seqence Hb with yor MyIllmina accont and password. 3 Select Browse to navigate to a preferred otpt folder location. 4 Confirm that the thmbnail setting is Save All Thmbnails. The software atomatically saves all thmbnail images. A thmbnail is a sampling of images from many tiles in each colmn of tiles, or swath, combined in one image. 5 Select Next. Flow Cell Setp Screen The Flow Cell Setp screen records information abot the flow cell sed for the rn. All fields are reqired. 1 Scan or enter the flow cell ID (barcode nmber) of the flow cell to be seqenced. 2 Confirm that the flow cell type is HiSeq X or HiSeq X HD. Docment # v06 Material #

22 HiSeq X System Gide NOTE A HiSeq X flow cell and a HiSeq X HD flow cell can be seqenced simltaneosly as flow cell A and flow cell B. 3 Enter an experiment name to appear on each screen and help identify the rn in progress. 4 Enter a ser name. 5 Select Next. Advanced Screen 1 [Optional] Select the Confirm First Base checkbox. A first base report is generated atomatically for each rn after cycle 2 and placed in the root level of the rn folder. Selecting this option allows yo to confirm the first base report before proceeding with the rn. Otherwise, the rn contines withot showing the confirmation dialog box. 2 [Optional] From the flow cell image, select lanes to remove from the rn. All lanes are inclded by defalt. PhiX alignment is performed atomatically for all lanes. NOTE 3 Select Next. Recipe Screen A dedicated control lane is not reqired or an option. A recipe is generated atomatically from the information entered on the Recipe screen. 1 Select an Index Type option: No Index Performs a non-indexed paired-end rn. Single Index Performs a paired-end rn with an 8 bp indexing read. Dal Index Performs paired-end rn with two 8 bp indexing reads. The remaining fields are atopoplated depending on the selected index type. 2 Confirm the atopoplated settings: Cycles: 151 for Read 1 and Read 2, and 8 or 0 for Index 1 and Index 2 The nmber of cycles in each seqencing and indexing read. SBS: HiSeq X SBS The SBS chemistry sed for Read 1 and Read 2. Index: HiSeq X Seqencing Primer or HiSeq X Dal Index Seqencing Primer If applicable, the chemistry sed for Index Read 1 and Index Read 2. PE trnarond: HiSeq X PE or HiSeq X PE Dal Index The chemistry sed for paired-end resynthesis. Sample Sheet Screen Sample sheets are optional nless yo se BaseSpace Seqence Hb to perform data analysis. 1 Select Browse to locate the sample sheet. 2 Select Next. Reagents Screen The Reagents screen records information abot the reagent kit sed for the rn. 1 In the SBS Reagent Kit ID field, scan or enter the reagent kit barcode ID from any of the following SBS Kit Docment # v06 Material #

23 HiSeq X System Gide boxes: Single-pack kit SBS Kit Box 1 or 2 10-pack kit SBS Kit Boxes A F 2 In the PE Reagent Kit ID field, scan or enter the paired-end reagent kit barcode ID from a PE Clster Kit box: Single-pack kit PE Clster Kit Box 2 10-pack kit PE Clster Kit Box C 3 Select 300 Cycles. The Cycles Remaining field defalts to 325. NOTE The software conts down the nmber of cycles entered from the Cycles Remaining field. When the cycles are low, the software prompts yo to load fresh reagents. 4 Select Prime SBS Reagents to prime reagents. Always prime reagents before loading a clstered flow cell. 5 Select Next. Review Screen 1 Review rn parameters from the Review screen. 2 Select Next to proceed or Back to change parameters. Load and Prime Reagents After entering rn parameters, load SBS, indexing, and paired-end reagents for the rn, and then prime reagents throgh the flidics system. The software gides yo throgh these steps in a series of screens on the Pre-Rn Setp tab. Load SBS Reagents 1 Invert each bottle to mix. CAUTION Mix and load PCM last, after all other reagents are loaded, to prevent cross-contamination. Always discard yor gloves and replace them with a new pair after handling PCM. 2 Replace the cap on each bottle with a fnnel cap. 3 Open the reagent compartment door. 4 Raise the sippers for the SBS reagent rack as follows. a b Pll the sipper handle towards yo and then raise it. Release the handle into the slot on the top end of the groove. Make sre that the handle rests secrely in the slot. 5 Slide the reagent rack ot of the reagent compartment sing the rack handle. 6 Place each bottle into the rack in the associated nmbered position. Make sre that the conical end of the bottle rests in the indentation on the base of the rack. Docment # v06 Material #

24 HiSeq X System Gide Table 1 SBS Reagent Positions Position Reagent Description 1 PIM Patterned Incorporation Mix 2 PW1 25 ml PW1 or laboratory-grade water 3 PSM Patterned Scan Mix 4 PB1 Patterned SBS Bffer 1 5 PB2 Patterned SBS Bffer 2 6 PB2 Patterned SBS Bffer 2 7 PCM Patterned Cleavage Mix 8 PB2 Patterned SBS Bffer 2 7 Pt on a new pair of powder-free latex gloves. 8 Slide the rack into the reagent compartment, aligning the rack with the raised gide on the compartment floor. 9 Lower the sippers into the SBS reagent bottles as follows. a b c Pll the sipper handle towards yo and then lower it. Inspect the sippers to make sre that they do not bend as they lower into the fnnel caps. Release the handle into the slot on the bottom end of the groove. Load Indexing and Paired-End Reagents 1 Raise the sippers for the paired-end reagent rack as follows. a b Pll the handle towards yo and raise it. Release the handle into the slot on the top end of the groove. Make sre that the handle rests secrely in the slot. 2 Slide the reagent rack ot of the reagent compartment sing the rack handle. 3 Load 15 ml conical tbes filled with 10 ml PW1 or laboratory-grade water into positions 12, 18, and 19 of the paired-end rack. 4 Remove reagent tbe caps and place each tbe into the rack in the associated nmbered position or matching label color. Table 2 Paired-End Reagent Positions Position Reagent Description 10 PRM Patterned Resynthesis Mix 11 PLM2 v2 Patterned Linearization Mix 2 12 PW1 10 ml PW1 or laboratory-grade water 13 PAM Patterned Amplification Mix 14 PPM Patterned Amplification Premix 15 PDR Patterned Denatration Mix (contains formamide) 16 HP11 Primer Mix, Read 2 17 HP14* Indexing Primer Mix 18 PW1 10 ml PW1 or laboratory-grade water 19 PW1 10 ml PW1 or laboratory-grade water * HP14 is reqired for indexed rns only. If HP14 is not sed, load a 15 ml conical tbe with 10 ml PW1 or laboratory water. Docment # v06 Material #

25 HiSeq X System Gide 5 Slide the reagent rack into the reagent compartment, aligning the rack with the raised gide on the floor of the compartment. 6 Lower the sippers into the paired-end reagent tbes as follows. a b c Pll the handle towards yo and lower it. Inspect the sippers to make sre that they do not bend as they lower into the tbes. Release the handle into the slot on the bottom end of the groove. 7 Select the PW1 (25 ml) loaded in Position 2 checkbox, and then select Next. Prime Reagents Steps for priming reagents inclde loading a priming flow cell, confirming proper flow, and then starting the prime. CAUTION Always se a sed flow cell to prime reagents. Yo can se the flow cell from a previos rn to prime reagents on a sbseqent rn or for a post-rn wash. Load a Priming Flow Cell 1 Scan or enter the ID (barcode nmber) of the priming flow cell. 2 Rinse the priming flow cell with laboratory-grade water. Dry with a lens cleaning tisse or lint-free tisse. 3 Clean with alcohol wipes and lens cleaning tisse. 4 Place on the flow cell holder with the inlet and otlet ports facing down and the barcode on the right. Make sre that the arrow on the left edge of the flow cell, which indicates flow direction, points towards the instrment. 5 Gently slide the flow cell towards the top and right gide pins ntil it stops. Figre 7 Flow Cell Positioned Against Top and Right Gide Pins A B Top gide pin Right gide pins 6 Remove yor hand from the flow cell to prevent alignment drift. 7 Slowly move the flow cell lever to position 1 to engage the vacm and secre the flow cell. When the flow cell lever is blinking green, the vacm is engaged. If the lever is not green, see Possible Rn Setp Problems on page 31. Docment # v06 Material #

26 HiSeq X System Gide 8 Wait for abot five seconds, and then slowly move the flow cell lever to position 2. When the flow cell lever is solid green, the manifolds are in position and the flow cell is ready. 9 Make sre that the Vacm Engaged checkbox is selected, and then select Next. Confirm Proper Flow Checking for proper flow confirms that the flow cell and gaskets are properly installed and the manifold is engaged. 1 Select Position 2 from the drop-down list. 2 Confirm the following defalt vales: Volme: 125 Aspirate Rate: 250 Dispense Rate: Select Pmp. 4 Inspect the flow cell for bbbles passing throgh the lanes and leaks near the manifolds. 5 If excessive bbbles are present, do as follows. a Check the gaskets for obstrctions. b Redce the aspirate rate to 100. c Pmp another 125 µl of water to the flow cell. d If problems persist, remove the flow cell, repeat the cleaning steps, and reload the flow cell. Position Tbing and Start Prime 1 Remove the waste tbe for each flow cell from the waste container. Figre 8 Position Tbing 2 Place each tbe into a separate empty 15 ml tbe. 3 Select Start Prime. Monitor priming progress from the priming screen. 4 When priming is complete, measre the waste and confirm that the volme in each tbe is 1.75 ml. If yo collected priming volmes in 1 bottle for each flow cell, confirm that the volme is 14 ml. Volmes are calclated as follows: 250 µl for each SBS position except position 2 (250 x 7 = 1.75 ml) Docment # v06 Material #

27 HiSeq X System Gide 1.75 ml for each lane (1.75 x 8 = 14 ml) 5 Retrn the waste tbing to the waste container. 6 Select Next. Load the Seqencing Flow Cell Loading the flow cell for seqencing incldes removing the priming flow cell, cleaning the flow cell holder, loading the clstered flow cell, and confirming proper flow. Remove the Used Flow Cell 1 Slowly move the flow cell lever to position 1 to disengage the manifolds. 2 Slowly move the flow cell lever to position 0 to disengage the vacm seal and release the flow cell. 3 Lift the sed flow cell from the flow cell holder. Clean the Flow Cell Holder 1 Pt on a new pair of powder-free latex gloves. 2 Wipe the srface of the flow cell holder with a lint-free tisse moistened with laboratory-grade water to remove salts. 3 Wipe the srface of the flow cell holder with an alcohol wipe or a lint-free tisse moistened with ethanol or isopropanol. Do not allow alcohol to drip into the vacm holes or arond the manifolds. 4 Dry the stage with a low-lint lab tisse, if necessary. 5 Inspect the flow cell holder to make sre that it is free of lint and the vacm holes are free of obstrctions. Figre 9 Inspect Vacm Holes Load the Seqencing Flow Cell 1 Place the flow cell on the flow cell holder with the inlet and otlet ports facing down and the barcode on the right. Make sre that the arrow on the left edge of the flow cell, which indicates flow direction, points toward the instrment. 2 Gently slide the flow cell toward the top and right gide pins ntil it stops. Docment # v06 Material #

28 HiSeq X System Gide Figre 10 Flow Cell Positioned Against Top and Right Gide Pins A B Top gide pin Right gide pins 3 Remove yor hand from the flow cell to prevent alignment drift over time. 4 Slowly move the flow cell lever to position 1 to engage the vacm and secre the flow cell. When the flow cell lever is blinking green, the vacm is engaged. If the lever is not green, see Possible Rn Setp Problems on page Wait abot five seconds, and then slowly move the flow cell lever to position 2. When the flow cell lever is solid green, the manifolds are in position and the flow cell is ready for se. 6 Make sre that the Vacm Engaged checkbox is selected, and then select Next. Confirm Proper Flow Checking for proper flow confirms that the flow cell and gaskets are properly installed and the manifold is engaged. 1 Select Position 5 from the drop-down list. 2 Enter the following vales: Volme: 250 Aspirate Rate: 250 Dispense Rate: Select Pmp. 4 Inspect the flow cell for bbbles passing throgh the lanes or leaks near the manifolds. 5 If excessive bbbles are present, do as follows. a Check the manifold gaskets for obstrctions. b Repeat the pmp sing position 6 to avoid depleting position 5. c Redce the aspirate rate to 100. d Pmp another 250 µl to the flow cell. 6 Select Next. 7 Make sre that the flow cell lever is green, and then close the flow cell compartment door. 8 Make sre that the Vacm Engaged and Door Closed checkboxes are selected, and then select Next. Docment # v06 Material #

29 HiSeq X System Gide 9 Select Start to begin the seqencing rn. Monitor the Rn 1 Monitor rn metrics from the Rn Overview screen. Figre 11 Rn Overview Screen A B C D E F Progress bar Monitor how many cycles have been completed. Flow cell image Monitor imaged lanes. Flidics graph Expand the flidics section to monitor chemistry steps. Rn Configration Review parameters of crrent rn. Analysis graph Monitor qality scores by cycle. Images graph Monitor intensities by cycle. One thmbnail image is shown for each swath scanned. No other images appear on the software interface. First Base Report If yo opted to confirm first base dring rn setp, the first base confirmation dialog box opens atomatically after imaging of the second cycle is complete. The rn pases at this step. 1 Review the First Base Report from the confirmation dialog box. 2 If the reslts are satisfactory, select Contine. View Rn Metrics When rn metrics are available, Seqencing Analysis Viewer (SAV) opens atomatically and displays them. Metrics appear in the form of plots, graphs, and tables. For more information, see the Seqencing Analysis Viewer User Gide (docment # ). 1 To view pdated metrics, select Refresh at any time dring the rn. Unload Reagents 1 When the rn is complete, open the reagent compartment door. 2 Raise the sippers for the appropriate SBS rack and paired-end rack as follows. Docment # v06 Material #

30 HiSeq X System Gide a b c Pll the sipper handle otward. Raise the sipper handle while plling it otward. Release the sipper handle into the slot on the top end of the groove. Make sre that the sipper handle rests secrely in the slot. 3 Slide each reagent rack ot of the reagent compartment sing the rack handles. 4 Remove each bottle from each reagent rack. WARNING This set of reagents contains potentially hazardos chemicals. Personal injry can occr throgh inhalation, ingestion, skin contact, and eye contact. Wear protective eqipment, inclding eye protection, gloves, and laboratory coat appropriate for risk of exposre. Handle sed reagents as chemical waste and discard in accordance with applicable regional, national, and local laws and reglations. For additional environmental, health, and safety information, see the SDS at spport.illmina.com/sds.html. Perform a Water Wash A water wash is reqired after each seqencing rn to wash the system and check flidics. A maintenance wash is an optional alternative to the post-rn water wash. For instrctions, see Perform a Maintenance Wash on page 25. For instrctions, see HiSeq X System Gide (docment # ). If the instrment has been idle for one day or more, perform a water wash before beginning a new seqencing rn. 1 From the Welcome screen, select Wash Water. 2 Select Yes to wash paired-end reagent positions, and then select Next. 3 Load the instrment with laboratory-grade water: a b Fill eight SBS bottles with 250 ml laboratory-grade water. Fill 10 PE tbes with 12 ml laboratory-grade water. NOTE Wash bottles and tbes are typically replaced every 6 months, althogh the water is replaced abot every week. 4 Make sre that a sed flow cell is loaded. Load a sed flow cell, if necessary. 5 Select Next. 6 Perform a flidics check: a b c d Select soltion 2 from the drop-down list. Accept the defalt pmp vales. Select Pmp. Inspect the flow cell for bbbles passing throgh the lanes and leaks near the manifolds. 7 Remove the waste tbes for the appropriate flow cell from the waste container. 8 Bndle the waste tbes with parafilm. Keep all the ends even. 9 Place the bndled tbe ends into a 250 ml bottle. 10 Select Next to start the water wash. Docment # v06 Material #

31 HiSeq X System Gide Positions Eight SBS positions Eight SBS positions and 10 paired-end positions Approximate Rn Time 20 mintes 60 mintes 11 When the wash is complete, measre the delivered volme. Positions Total Delivered Volme Per Lane Delivered Volme Eight SBS positions 32 ml 4 ml Eight SBS positions and 10 paired-end positions 72 ml 9 ml 12 Unwrap the waste tbes and retrn them to the waste bottle. Qick Format the Otpt and Scratch Drives After data transfer completes, perform a qick format of the otpt (O:\) and scratch (S:\) drives. A qick format clears the drive for a sbseqent rn withot removing important system or instrment maintenance files. Before a rn can begin, a minimm of 2 TB is reqired for a dal flow cell rn. If disk space drops below the safe threshold dring the rn, the software pases the rn and places the flow cell in a safe state. After disk space is made available, the rn resmes atomatically. NOTE Instrment maintenance logs are stored on the C:\ drive. Therefore, it is safe to perform a qick format of the O:\ and S:\ drives dring an instrment wash. 1 From Windows, open Compter to show the list of drives on the compter. 2 Right-click the O:\ drive and select Format. 3 From the Format dialog box, select the Qick Format checkbox. 4 Select Start. 5 Repeat steps 1 4 to clear the S:\ drive. Docment # v06 Material #

32 Chapter 5 Maintenance Introdction 25 Perform a Maintenance Wash 25 Idle the Instrment 29 Sht Down the Instrment 30 Introdction Maintenance procedres ensre contined instrment performance. Sht down or idle the instrment dring periods of inactivity. Spplement the water wash performed at the end of a rn with reglar maintenance washes to maintain flidics. Reglar instrment washes maintain instrment performance by flshing the flidics system and preventing salt accmlation and cross-contamination of reagents. 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 Maintenance Wash Perform a maintenance wash when prompted by the software every 10 days or optionally after a rn. A maintenance wash takes abot 90 mintes and follows one of two workflows, depending on whether ProClin 300 is available: Tween 20 and ProClin 300 wash Washes the system with a ser-prepared soltion of Tween 20 and ProClin 300. SeeTween 20 and ProClin 300 Maintenance Wash on page 25. Tween 20 wash Washes the system with a ser-prepared soltion of Tween 20 and can reqire a water wash. See Tween 20 Maintenance Wash on page 27. When the Load Gasket screen appears before a maintenance wash, the gaskets in the front manifold and back manifold mst be replaced before starting the wash. Tween 20 and ProClin 300 Maintenance Wash Prepare Maintenance Wash Soltion Prepare 5 liters of maintenance wash soltion for se with one instrment. The soltion can be stored for p to 30 days at room temperatre and sed p to three times dring this period. Dispose of the wash soltion in accordance with the governmental safety standards for yor region. 1 Adding the water first, combine the following volmes to dilte Tween 20: Laboratory-grade water (225 ml) Tween 20 (25 ml) These volmes reslt in approximately 10% Tween Place a stir bar in an empty carboy that is at least 6 liters. 3 Adding the water first, combine the following volmes in the carboy: Laboratory-grade water (750 ml) Docment # v06 Material #

33 HiSeq X System Gide 10% Tween 20 (250 ml) ProClin 300 (1.5 ml) These volmes reslt in a soltion that is approximately 2.5% Tween 20 and 0.15% ProClin Mix thoroghly on a stir plate. 5 Add 4 liters laboratory-grade water. These volmes reslt in a soltion that is approximately 0.5% Tween 20 and 0.03% ProClin Contine stirring ntil thoroghly mixed. 7 Set aside in a closed container at room temperatre. Tween 20 and ProClin From the Welcome screen, select Wash Maintenance. 2 If yo are sing fresh maintenance wash soltion, load the instrment with soltion as follows. a b c Fill eight SBS bottles with 250 ml fresh wash soltion. Fill 10 PE tbes with 12 ml fresh wash soltion. Assign each bottle and tbe to a reagent rack position. Maintain these assignments for each sbseqent wash to prevent cross-contamination from reagent present on the sippers. 3 If yo stored maintenance wash soltion from a previos rn, load the instrment with soltion as follows. a b Replenish the stored soltion and invert to mix. Replenish no more than two times after the original se. Load the bottles and tbes into the assigned reagent rack positions. NOTE Monthly replacement of wash bottles and tbes is typically sfficient. 4 Empty the waste bottle. 5 Select Next. 6 Remove the flow cell from the flow cell stage and set it aside. 7 Pt on a new pair of powder-free latex gloves. 8 Apply light pressre to one side of the front gasket ntil the other side lifts. Use tweezers to grasp and remove the gasket. Repeat to remove the rear gasket. Figre 12 Remove Used Manifold Gaskets 9 Place a new gasket in each slot on the front end and back end of the flow cell holder. Press lightly into position. Docment # v06 Material #

34 HiSeq X System Gide 10 Reload the flow cell that yo removed to install the new gaskets. 11 Make sre that the Vacm Engaged checkbox is selected, and then select Next. 12 Perform a flidics check sing the defalt pmp vales: a b c d Select soltion 2 from the drop-down list. Select Pmp. Inspect the flow cell for bbbles passing throgh the lanes and leaks near the manifolds. If yo see a constant stream of bbbles, replace the gasket and repeat the flidics check. 13 Remove the waste tbes for the appropriate flow cell from the waste container. 14 Bndle the eight waste tbes with parafilm. Keep the tbes ends even. 15 Place the bndled tbe ends into a 250 ml bottle. 16 Select Next to start the wash. 17 When the wash is complete, select Retrn to Start. 18 Measre the delivered volme. Positions Eight SBS positions Delivered Volme 74 ml 10 paired-end positions 52 ml All positions NOTE ml per lane All bottles and tbes are filled to capacity to make sre that the sippers are rinsed. However, the delivered volme for each position varies so the bottles and tbes contain different volmes when the wash is complete. 19 Unwrap the waste tbes and retrn them to the waste container. Tween 20 Maintenance Wash Prepare Maintenance Wash Soltion Always prepare fresh wash soltion for a Tween 20 maintenance wash. Prepare 5 liters of maintenance wash soltion. This volme is sfficient to wash both sides of one instrment. Dispose of wash soltion in accordance with the governmental safety standards for yor region. 1 Adding the water first, combine the following volmes to dilte Tween 20: Laboratory-grade water (225 ml) Tween 20 (25 ml) These volmes reslt in approximately 10% Tween Place a stir bar in an empty carboy that is at least 6 liters. 3 Adding the water first, combine the following volmes in the carboy: Laboratory-grade water (750 ml) 10% Tween 20 (250 ml) These volmes reslt in a soltion that is approximately 2.5% Tween Mix thoroghly on a stir plate. Docment # v06 Material #

35 HiSeq X System Gide 5 Add 4 liters laboratory-grade water to reslt in a soltion that is approximately 0.5% Tween Contine stirring ntil thoroghly mixed. 7 Immediately proceed to setting p the wash. Tween 20 Wash 1 From the Welcome screen, select Wash Maintenance. 2 Load the instrment with fresh maintenance wash soltion as follows. a b Fill eight SBS bottles with 250 ml fresh wash soltion. Fill 10 PE tbes with 12 ml fresh wash soltion. 3 Empty the waste bottle. 4 Select Next. 5 Remove the flow cell from the flow cell stage and set it aside. 6 Pt on a new pair of powder-free latex gloves. 7 Apply light pressre to one side of the front gasket ntil the other side lifts. Use tweezers to grasp and remove the gasket. Repeat to remove the rear gasket. Figre 13 Remove Used Manifold Gaskets 8 Place a new gasket in each slot on the front end and back end of the flow cell holder. Press lightly into position. 9 Reload the flow cell that yo removed to install the new gaskets. 10 Make sre that the Vacm Engaged checkbox is selected, and then select Next. 11 Perform a flidics check sing the defalt pmp vales: a b c d Select soltion 2 from the drop-down list. Select Pmp. Inspect the flow cell for bbbles passing throgh the lanes and leaks near the manifolds. If yo see a constant stream of bbbles, replace the gasket and repeat the flidics check. 12 Remove the waste tbes for the appropriate flow cell from the waste container. 13 Bndle the eight waste tbes with parafilm. Keep the tbes ends even. 14 Place the bndled tbe ends into a 250 ml bottle. 15 Select Next to start the wash. Docment # v06 Material #

36 HiSeq X System Gide 16 When the wash is complete, select Retrn to Start. 17 Measre the delivered volme. Positions Eight SBS positions Delivered Volme 74 ml 10 paired-end positions 52 ml All positions NOTE ml per lane All bottles and tbes are filled to capacity to make sre that the sippers are rinsed. However, the delivered volme for each position varies so the bottles and tbes contain different volmes when the wash is complete. 18 Unwrap the waste tbes and retrn them to the waste container. Water Wash If the instrment is going to be idle for more than five days after the Tween 20 wash, perform a water wash. The water wash rinses Tween 20 from the flidics system. 1 From the Welcome screen, select Wash Water Wash. 2 Load the instrment with laboratory-grade water as follows. a b Fill eight SBS bottles with at least 20 ml laboratory-grade water. Fill 10 PE tbes with 10 ml laboratory-grade water. CAUTION Do not rese the water, bottles, or tbes sed for the Tween 20 wash. The water might be contaminated with reagents present on the sippers. 3 Load the bottles and tbes onto the instrment in the appropriate reagent rack. 4 Select Next to start the wash. 5 When the wash is complete, measre the delivered volme. Positions Eight SBS positions Eight SBS positions and 10 paired-end positions Delivered Volme 32 ml 72 ml 6 Unwrap the waste tbes and retrn them to the waste container. Idle the Instrment Use the following instrctions to prepare the instrment to sit idle for p to 10 days. For drations longer than 10 days, sht down the instrment instead. 1 Perform a maintenance wash to flsh the system. 2 Leave the flow cell on the flow cell stage with the flow cell lever in position 2. Leave the manifolds in the raised position. 3 Load 10 ml laboratory-grade water in each position in the reagent racks, and then lower the sippers. 4 Before sing the instrment, perform a water wash. Docment # v06 Material #

37 HiSeq X System Gide Sht Down the Instrment Use the following procedre to prepare flidics safely and sht down the system. Sht down the instrment only if yo do not plan to se it within the next 10 days or more. If yo plan to se the instrment within the next 10 days, idle it instead. 1 Perform a maintenance wash to flsh the system. 2 Remove the flow cell from the flow cell stage. 3 Wipe the srface of the flow cell holder sing an alcohol wipe or a lint-free tisse moistened with ethanol or isopropanol. CAUTION Do not allow alcohol to drip into the vacm holes or arond the manifolds. Use a low-lint lab tisse to dry the stage, if necessary. 4 Load 10 ml laboratory-grade water in each position in the reagent racks, and then lower the sippers. 5 Trn off the instrment. 6 To restart the instrment: a b c Load water in all reagent positions. Trn on the instrment. Perform a water wash. Docment # v06 Material #

38 Appendix A Trobleshooting Log File 31 Possible Rn Setp Problems 31 Perform a Flidics Check 31 Pase or End a Rn on the HiSeq X 32 Stagger Rns on Flow Cell A and Flow Cell B 33 Possible Read 1 Primer Rehybridization 33 Log File The log file lists any errors that occrred in the control software. Use this file for trobleshooting prposes. To access the log file, select Men Tools Show Log from the Welcome screen. Possible Rn Setp Problems Problem Possible Case Action The software did not initialize. Flow cell lever is orange. Flow cell lever is blinking orange. Flow cell lever is blinking green. Poor flid delivery. The software was nable to initialize internal hardware devices. The flow cell did not seat properly. The vacm did not seal. Manifolds did not raise. Vacm is being provided bt is inadeqate. Close the error message and then relanch the instrment software. If the problem persists, restart the instrment compter. If yo are going to restart the compter, first sht down the instrment to make sre the DoNotEject drive is recognized correctly. If the problem persists after restarting the instrment compter, sht down the instrment, wait a minimm of 60 seconds, and then restart the instrment. Remove the flow cell and repeat the cleaning steps. Make sre that the gaskets are present and well-seated. Reload the flow cell. If the preceding steps do not work, try replacing the gaskets, and then reload the flow cell. Remove the flow cell and repeat the cleaning steps. Make sre that the gaskets are present and well-seated. Reload the flow cell. If the preceding steps do not work, try replacing the gaskets, and then reload the flow cell. Vacm pressre is good. Switch flow cell lever to position 2. Potential bbbles in the system. Reposition the flow cell and confirm that the holes are facing down. Look for white precipitate arond the gaskets. If precipitate is present, replace the gaskets. Always replace gaskets before an instrment maintenance wash. Confirm that the sipper assemblies are flly lowered and each sipper is in contact with the reagents. Perform a Flidics Check Perform a flidics check dring instrment installation and when trobleshooting flidics isses. 1 Select Check on the Welcome screen. 2 Scan or enter the wash flow cell ID (barcode nmber) of the priming flow cell. Make sre to se a sed flow cell for this step. Docment # v06material #

39 HiSeq X System Gide 3 Load the sed flow cell onto the instrment. 4 Fill eight SBS bottles with PW1 or laboratory-grade water, and load the bottles onto the SBS reagent rack. 5 Select soltion 2 from the drop-down list. 6 Confirm the following defalt vales: Volme: 250 Aspirate Rate: 250 Dispense Rate: Select Pmp. 8 Inspect the flow cell for bbbles passing throgh the lanes and leaks near the manifolds. 9 If excessive bbbles are present: a Check the manifold gaskets for obstrctions. b Redce the aspirate rate to 100. c Pmp another 250 µl of water to the flow cell. Pase or End a Rn on the HiSeq X Ending a rn does not provide the option to save data or resme the rn. Pasing a rn might be necessary to check rn components or set p a rn on the adjacent flow cell. Pase a Rn Pase a rn to check rn components, sch as reagent volmes, as necessary. Under normal operation, pasing a rn is not necessary. RTA2 resmes atomatically after a pased rn is resmed, so the rn can resme withot loss of data. For more information, see Real-Time Analysis on page From the Rn Overview screen, select Pase Normal Pase. 2 Select Yes to confirm the command. The software completes the crrent chemistry or imaging command and places the flow cell in a safe state. 3 Select Resme to resme the rn. Change Reagents Dring a Rn If yo started the rn with a partial volme of reagents, se the Change Reagents featre to pase the rn and replenish reagents. NOTE Priming is not reqired. 1 From the Rn Overview screen, select Pase to open the pase men. 2 Select Change Reagents. 3 Select Yes to confirm the pase command. The software completes the crrent chemistry or imaging command, places the flow cell in a safe state, and opens the Reagents screen. Docment # v06material #

40 HiSeq X System Gide 4 Enter the following parameters: The reagent kit ID for the new reagents. The nmber of cycles the reagents are expected to last. 5 Select Next to proceed to loading reagents. End a Rn If RTA2 is terminated, the software does not resme processing and rn data are not saved. Therefore, a rn cannot resme after it has been stopped. CAUTION Ending a rn on the HiSeq X is final. 1 To end the rn, select Abort. Confirm or cancel the command. 2 Confirming the commands opens the Welcome screen. 3 Proceed to post-rn procedres. NOTE If a rn is stopped dring Read 1, it is possible to perform primer rehybridization on the cbot. After primer rehybridization, start a new rn on the HiSeq X to seqence the flow cell. Stagger Rns on Flow Cell A and Flow Cell B 1 Select Pase Normal Pase. 2 Wait for the software to complete the crrent chemistry or imaging step. The system is atomatically placed in a safe state. 3 Confirm that the rn is sccessflly pased. When a rn is pased, the Resme btton is present. 4 Set p the new rn. 5 After loading the new flow cell for the new rn, close the compartment door. 6 Select Start to start the new rn. 7 On the adjacent flow cell, select Resme to resme the pased rn. The software atomatically controls chemistry and imaging processes on both flow cells. Possible Read 1 Primer Rehybridization If Read 1 metrics indicate low clster nmbers, low intensities, or other concerns, yo can perform a Read 1 primer rehybridization to resce the flow cell. Read 1 primer rehybridization is performed on the cbot and does not damage clsters on the flow cell. Read 1 primer hybridization on a HiSeq X patterned flow cell reqires the following Illmina consmables: HiSeq X HD cbot Mlti-Primer Rehybridization Kit (catalog # GD ) HiSeq cbot Manifold (catalog # SY ) For more information, see Read 1 Primer Rehyb on a HiSeq X Flow Cell (docment # ). Docment # v06material #

41 Appendix B Real-Time Analysis Real-Time Analysis Overview 34 Real-Time Analysis Workflow 35 Real-Time Analysis Overview The HiSeq X ses an implementation of Real-Time Analysis software called RTA2. RTA2 rns on the instrment compter and extracts intensities from images, performs base calling, and assigns a qality score to the base call. RTA2 and the control software commnicate throgh a web HTTP interface and shared memory files. If RTA2 is terminated, processing does not resme and rn data are not saved. NOTE Inpt Files Demltiplex performance is not calclated, so the Index tab in Seqencing Analysis Viewer (SAV) is not poplated. RTA2 reqires the following inpt files: Tile images contained in local system memory. RnInfo.xml, which the control software generates atomatically at the beginning of the rn. From this file, RTA2 reads the rn name, nmber of cycles, whether a read is indexed, and the nmber of tiles on the flow cell. RTA.exe.config, which is a software configration file in XML format. RTA2 receives commands from the control software that inclde information abot the location of RnInfo.xml file and if an optional otpt folder is specified. Otpt Files Images for each channel are passed in memory to RTA2 as tiles. From these images, RTA2 prodces primary otpt as a set of qality-scored base call files and filter files. Other files spport generation of primary otpt files. Base call files For each tile that is analyzed, one compressed base call (*.bcl) file is generated for each tile per cycle. The base call file contains the base call and associated qality score. Filter files Each tile prodces filter information that is inclded in one filter (*.filter) file for each tile over the whole rn. The filter file specifies whether clsters pass filter. Clster location files One clster location (s.locs) file contains the X,Y coordinates for every clster on the flow cell. Primary otpt files are sed for sbseqent data analysis. Use the bcl2fastq conversion software for demltiplexing and FASTQ conversion. To convert data from the HiSeq X, se bcl2fastq v , or later. For the crrent software version and download information, see the HiSeq X spport page on the Illmina website. RTA2 provides real-time metrics of rn qality stored as InterOp files. InterOp files are binary files containing tile, cycle, and read-level metrics, and reqired to view metrics in Seqencing Analysis Viewer. For viewing metrics generated by RTA2, se SAV v1.8.37, or later. For details abot each otpt file, see Seqencing Otpt Files on page 39. Docment # v06material #

42 HiSeq X System Gide Error Handling RTA2 creates log files and writes them to the RTALogs folder. Errors are recorded in an error file in *.tsv file format. The following log and error files are transferred to the final otpt destination at the end of processing: *GlobalLog*.tsv smmarizes important rn events. *LaneNLog*.tsv lists processing events for each lane. *Error*.tsv lists errors that occrred dring a rn. *WarningLog*.tsv lists warnings that occrred dring a rn. Data Transfer Throghot the rn, RTA2 reqests data transfer from Rn Copy Service, the software that manages the transfer to the specified otpt folder location. If BaseSpace Seqence Hb is sed, the BaseSpace Broker manages the transfer of data to BaseSpace Seqence Hb. If the network connection is interrpted, RTA2 contines processing and writes data locally. Data transfer resmes when the connection is restored. NOTE Make sre that yor network connection meets the minimm reqirements for sending rn data to BaseSpace Seqence Hb. For more information, see the lab setp and site prep gide. After processing is complete, RTA2 creates a marker file called RTAComplete.txt. Data transfer completes after this file is generated. A sensor indicator at the bottom of the screen shows transfer stats. For details, see Activity and Sensor Indicators on page 5. Real-Time Analysis Workflow Template generation Maps clster locations. Registration and intensity extraction Records the location of each clster on the patterned flow cell and determines an intensity vale for each clster. Color matrix correction Corrects cross talk between channels. Empirical phasing correction Corrects the effects of phasing and prephasing. Base calling Determines a base call for every clster. Qality scoring Assigns a qality score to every base call. Template Generation Template generation defines the position of each clster in a tile sing X and Y coordinates. The template is sed as a reference for the sbseqent step of registration and intensity extraction. Becase of the array on the patterned flow cell, clster positions are predetermined per the nmber of rows and colmns, and the distance between nanowells. For more information, see Patterned Flow Cell on page 7. Clster positions are written to one clster location (s.locs) file for the entire rn. Docment # v06material #

43 HiSeq X System Gide Registration and Intensity Extraction Registration and intensity extraction begin after the template of clster positions is generated. Registration transforms the template locations of clsters to the location on the image in each of the for color channels. Intensity extraction determines an intensity vale for each clster in the template for a given image. If registration fails for any images in a cycle, no base calls are generated for that tile in that cycle. Use SAV to examine thmbnail images and identify images that failed registration. Color Matrix Correction After registration and intensity extraction, RTA2 corrects for cross talk between channels. Cross talk occrs when a clster shows intensity in the C channel and some intensity also shows in the A channel, for example. Using a 4 x 4 color matrix, RTA2 generates matrix-corrected intensities with redced or no cross talk, and balances differences in overall intensity between color channels. Empirical Phasing Correction Dring the seqencing reaction, each DNA strand in a clster extends by one base per cycle. Phasing and prephasing occrs when a strand becomes ot of phase with the crrent incorporation cycle. Phasing occrs when a base falls behind. Prephasing occrs when a base jmps ahead. Figre 14 Phasing and Prephasing A B Read with a base that isphasing Read with a base that is prephasing. RTA2 corrects the effects of phasing and prephasing sing the empirical phasing correction algorithm, which maximizes the data qality at every cycle throghot the rn. Base Calling After raw intensities are corrected for cross talk, phasing, and prephasing, the channel with the brightest intensity is the call for that clster in that cycle. Base calling on the HiSeq X sing RTA2 begins after cycle 3. Base calling determines a base (A, C, G, or T) for every clster of a given tile at a specific cycle. Base calls are saved to base call (*.bcl) files, which are binary files with 1 byte per call and qality score. Each base call file contains the base call and the base call qality score. To make a base call, clsters mst first pass the chastity filter. Clsters that do not pass filter or cannot be called becase they are off-image or failed image registration are labeled no-calls. No-calls are represented as (N). Docment # v06material #

44 HiSeq X System Gide Clsters Passing Filter Dring the first 25 cycles of Read 1, the chastity filter removes low-qality clsters from analysis reslts. Clsters pass filter if no more than one base call has a chastity vale below 0.6 in the first 25 cycles. Chastity is defined as the ratio of the brightest base intensity divided by the sm of the brightest and the second brightest base intensities. The percentage of clsters passing filter is represented in analysis reports as %PF. The HiSeq X patterned flow cell has an ordered array of clsters. Empty wells with no clsters and polyclonal wells where more than one seqence exists are inclded in the raw clster cont, bt do not pass filter. Therefore, the ordered array on a patterned flow cell yields a relatively low percentage of clsters passing filter. Figre 15 Empty and Polyclonal Wells (Inclded in Raw Clster Cont) Figre 16 Wells With Non-PF Clsters (Shown in Gray) Qality Scoring A qality score, or Q-score, is a prediction of the probability of an incorrect base call. A higher Q-score implies that a base call is higher qality and more likely to be correct. The Q-score is a compact way to commnicate small error probabilities. Qality scores are represented as Q (X), where X is the score. The following table shows the relationship between the qality score and error probability. Q-Score Q(X) Error Probability Q (1 in 10,000) Q (1 in 1,000) Q (1 in 100) Q (1 in 10) NOTE Qality scoring is based on a modified version of the Phred algorithm. Docment # v06material #