iseq 100 Sequencing System Guide For Research Use Only. Not for use in diagnostic procedures. Document # v04 October 2018

Transcription

1 iseq 100 Seqencing System Gide October 2018 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 # v04 Docment # v03 Docment # v02 October 2018 Agst 2018 Jne 2018 Added recommended loading concentrations and diltion instrctions for Nextera DNA Flex for Enrichment, TrSeq DNA Nano, and TrSeq DNA PCR-Free libraries. Added information on sing a normalization method that does not reslt in single-stranded libraries. Added descriptions of the two rn modes, Local Rn Manager and Manal. Added a 5% PhiX spike-in option and defined the prpose of each spikein percentage. Added the following steps: Switching to the sbsadmin operating system accont when installing the control software, analysis modles, and other software. Power cycling the instrment when restoring to factory settings. Referenced Illmina Adapter Seqences (docment # ) to determine Index 2 (i5) orientations for a sample sheet. Clarified the following points: Cartridges mst be sed immediately after thawing. The loading concentrations listed for Nextera DNA Flex and Nextera Flex for Enrichment libraries are not applicable to other Nextera library types. SreCell WTA 3 is not a compatible library. Updated software descriptions to iseq Control Software v1.3: Added configration instrctions for Universal Copy Service. Renamed the Network Configration tab to Network Access. Added instrctions on opening Local Rn Manager from the control software. Updated the defalt otpt folder location to D:\SeqencingRns. Added instrctions on connecting the system to a proxy server. Added reqirement to specify a UNC path for otpt folder and sample sheet locations on the network. Indicated the niqe reqirements for configring an otpt folder location on an internal drive, external drive, or network location. Made instrctions on creating a sample sheet for Manal mode the first step of rn setp. Corrected instrctions on sing the system site installation wizard. Corrected the description of otpt thmbnail files. Updated tbes sed for dilting libraries to Fisher Scientific catalog # or eqivalent low-bind tbes. Added a section describing the regional availability of Advanced Exchange. Clarified that libraries dilted to the loading concentration mst be seqenced that day. Clarified that the reagent cartridge mst be removed from the box for thawing. iii

4 Docment Date Description of Change Docment # v01 Docment # v01 Docment # v00 May 2018 May 2018 Febrary 2018 Updated software descriptions to iseq Control Software v1.2: Added option to browse to a downloaded software installer from the control software. Added instrctions for saving thmbnails. Moved network settings to the Network Configration tab. Increased maximm ses of resable test components to 36 and noted that the remaining nmber of ses appear onscreen. Updated Local Rn Manager information: Added steps for opening Local Rn Manager and setting p the rn. Added RNA Amplicon as a preinstalled analysis modle, and DNA Enrichment and Reseqencing as other spported modles. Updated docmentation references to Local Rn Manager Software Gide (docment # ). Updated cartridge thawing instrctions: Added a room-temperatre thawing option. Provided more detailed water bath instrctions, inclding storing before thawing. Updated instrctions on preparing libraries for seqencing: Updated the Nextera DNA Flex loading concentration to 200 pm. Added starting loading concentration for nlisted library types. Added information on the %Occpied metric. Increased volme of 1 nm PhiX for a spike-in to 50 µl. Updated Illmina catalog nmbers for: iseq 100 Spare Drip Tray Pad to iseq 100 Spare Air Filter to Updated pipette and pipette tip recommendations. Added the following miscellaneos instrctions: Performing validation rns. Creating a sample sheet when seqencing in Manal mode. Minimizing the control software to access other applications. Added the following steps to the system check procedre: Unloading and storing resable test components. Cleaning visible debris from the resable test flow cell. Reorganized the following content to improve continity: Merged instrctions for performing a PhiX-only rn with the standard seqencing instrctions. Merged flow cell preparation instrctions with library diltion instrctions. Consolidated PhiX spike-in instrctions. Moved information on nmber of cycles in a read. Moved Real-Time Analysis and renamed to Seqencing Otpt. Simplified the error messages workflow diagram. Removed information on Tablet and Desktop modes. The operating system rns in Desktop mode by defalt and Tablet mode is not necessary. Removed reqirement to complete and retrn a decontamination certificate for advanced exchange. Corrected average rn size to 2 GB. Initial release. iv

5 Table of Contents Chapter 1 Overview 1 Introdction 1 Additional Resorces 2 System Components 3 iseq 100 i1 Reagents 7 Chapter 2 Getting Started 11 First Time Setp 11 Validation Rns 11 Minimize the Control Software 11 Configre Rn Settings 12 Cstomize System Settings 14 Configre Network Settings 15 User-Spplied Consmables and Eqipment 16 Chapter 3 Seqencing 18 Introdction 18 Nmber of Cycles in a Read 18 Best Practices 19 Thaw the Bagged Cartridge 19 Prepare the Flow Cell and Libraries 20 Load Consmables Into the Cartridge 22 Set Up a Seqencing Rn (Local Rn Manager Mode) 24 Set Up a Seqencing Rn (Manal Mode) 27 Chapter 4 Maintenance 31 Clear Hard Drive Space 31 Software Updates 31 Replace the Air Filter 32 Relocate the Instrment 34 Software Restriction Policies 35 Appendix A Seqencing Otpt 38 Real-Time Analysis Overview 38 Real-Time Analysis Workflow 40 Appendix B Trobleshooting 44 Error Message Resoltion 44 Cancel a Rn 44 Power Cycle the Instrment 45 Perform a System Check 46 Leak Mitigation 48 Restore to Factory Settings 50 v

6 Appendix C Advanced Exchange 51 Exchanging the iseq 100 System 51 Receive a Replacement System 51 Prepare the Original System for Retrn 51 Retrn the Original System 54 Index 58 Technical Assistance 63 vi

7 Chapter 1 Overview Introdction 1 Additional Resorces 2 System Components 3 iseq 100 i1 Reagents 7 Introdction The Illmina iseq 100 Seqencing System provides a targeted approach to next-generation seqencing (NGS). This applications-focsed system packages Illmina seqencing technology into a cost-effective desktop instrment. Featres Accessibility and reliability The iseq 100 System has a small footprint and is easy to install and se. Flidics and imaging components are bilt into the consmable, simplifying instrment maintenance. Single-step consmable loading A single-se cartridge is prefilled with all reagents reqired for a rn. Library and a sensor-eqipped flow cell load directly into the cartridge, which is then loaded onto the instrment. Integrated identification enables accrate tracking. iseq 100 System software A site of integrated software controls instrment operations, processes images, and generates base calls. This site featres on-instrment data analysis and data transfer tools for external analysis. On-instrment analysis Local Rn Manager inpts sample information and then analyzes rn data sing the analysis modle specified for the rn. The software incldes a site of analysis modles. Clod-based analysis The seqencing workflow is integrated with BaseSpace Seqence Hb, the Illmina clod compting environment for rn monitoring, data analysis, storage, and collaboration. Otpt files are streamed in real time to BaseSpace Seqence Hb for analysis. 1

8 Sample to Analysis The following diagram illstrates the complete seqencing workflow, from sample to analysis. Tools and docmentation are inclded with each step. This gide covers the Seqence Libraries step. For other docmentation, visit spport.illmina.com. Figre 1 Sample to Analysis Workflow Additional Resorces The iseq 100 Seqencing System spport pages on the Illmina website provide additional system resorces.these resorces inclde software, training, compatible prodcts, and the following docmentation. Always check spport pages for the latest versions. Resorce Cstom Protocol Selector iseq 100 Seqencing System Setp Poster (docment # ) iseq 100 Seqencing System Site Prep Gide (docment # ) Description A tool for generating end-to-end instrctions tailored to yor library prep method, rn parameters, and analysis method, with options to refine the level of detail. Provides instrctions for instrment installation and initiating first time setp. Provides specifications for laboratory space, electrical reqirements, and environmental and network considerations. 2

9 Resorce iseq 100 Seqencing System Safety and Compliance Gide (docment # ) RFID Reader Compliance Gide (docment # ) Description Provides information abot operational safety considerations, compliance statements, and instrment labeling. Provides information abot the RFID reader in the instrment, inclding compliance certifications and safety considerations. System Components iseq 100 Seqencing System comprises a power btton, monitor, stats bar, consmables compartment, and drip tray. Figre 2 External System Components A B C D E Power btton Controls instrment power and indicates whether the system is on (glows), off (dark), or off bt with AC power (plses). Toch-screen monitor Enables on-instrment configration and setp sing the control software interface. Stats bar Indicates system stats as ready to seqence (green), processing (ble), or needs attention (orange). Consmables compartment Contains consmables dring a rn. Drip tray door Provides access to the drip tray, which catches leaked flids. Power and Axiliary Connections Yo can move the instrment to access USB ports and other rear panel components. The back of the instrment has the switch and inlet that controls power to the instrment, and an Ethernet port for an optional Ethernet connection. Two USB ports provide the option to connect a mose and keyboard, or to pload and download data sing a portable device. NOTE Connecting the system to a keyboard and mose overrides the onscreen keyboard. 3

10 Figre 3 Rear Panel Components A B C D Ethernet port Optional Ethernet cable connection. USB ports Two ports for connecting axiliary components. Toggle switch Trns instrment power on and off. AC power inlet Power cord connection. Consmables Compartment The consmables compartment contains the cartridge for a seqencing rn. Figre 4 Loaded Consmables Compartment A B C Cartridge Contains the flow cell, library, and reagents, and collects sed reagents dring the rn. Tray Holds the cartridge dring seqencing. Door Opens to a 60 degree angle to provide access to the consmables compartment. The software opens and closes the compartment door and positions the cartridge for imaging. The door opens down from hinges toward the base of the instrment. Do not place objects on the open door, which is not designed for se as a shelf. Resable Test Cartridge and Flow Cell The instrment ships with a resable test cartridge and flow cell to se for system checks. 4

11 Store in the original packaging at room temperatre. Rese p to 36 times or for 5 years from the date of manfactre, whichever comes first. Dring a system check, the software displays the remaining nmber of ses. Replace expired test components with the iseq 100 System Test Kit. Figre 5 Resable Test Components A B Resable test flow cell Resable test cartridge The test components look similar to the seqencing components provided in iseq 100 i1 Reagents, and the loading orientations are the same. However, the test cartridge does not have a library reservoir and neither test component has the chemistry needed for a rn. System Software The system software site incldes integrated applications that perform seqencing rns and on-instrment analysis. iseq Control Software Controls instrment operations and provides an interface for configring the system, setting p a seqencing rn, and monitoring rn statistics as seqencing progresses. Local Rn Manager Defines rn parameters and the analysis method before seqencing. After seqencing, on-instrment data analysis begins atomatically. The system is shipped with the DNA Amplicon, RNA Amplicon, and Generate FASTQ analysis modles installed. The system also spports the DNA Enrichment and Reseqencing analysis modles, which are available on the Local Rn Manager spport pages. For more information on Local Rn Manager and the analysis modles, see the Local Rn Manager Software Gide (docment # ). Real-Time Analysis (RTA2) Performs image analysis and base calling dring the rn. For more information, see Seqencing Otpt on page 38. Universal Copy Service Copies seqencing otpt files from the rn folder to BaseSpace Seqence Hb (if applicable) and the otpt folder, where yo can access them. Real-Time Analysis and Universal Copy Service rn backgrond processes only. Local Rn Manager and the control software can reqire ser inpt. Notifications and Alerts An icon appears next to the instrment name to indicate notifications. Select the icon to view a list of the notifications, which inclde warnings and errors. 5

12 Warnings reqire attention, bt do not stop a rn or reqire action other than acknowledgment. Errors reqire action before starting or proceeding with a rn. A panel on the left side of rn setp screens displays alerts specific to cartridge loading and pre-rn checks. Figre 6 Onscreen Locations A B Rn setp alerts Other notifications System Information The control software men has an Abot section where yo can view Illmina contact information and the following system information: Serial nmber Compter name and IP address Recipe fragment version Rn cont Process Management The Process Management screen displays hard drive (D:\) space and rn stats, identifying each rn by name, ID, and date. The screen is atomatically refreshed every three mintes. The Stats colmn indicates whether a rn is progressing or complete, based on the processing of BCL files. For each rn, Process Management also displays the stats of backgrond processes Universal Copy Service, BaseSpace Seqence Hb, and Local Rn Manager. Nonapplicable processes do not appear on the screen. For example, if a rn is not connected to BaseSpace Seqence Hb, Process Management does not display the BaseSpace stats for that rn. To trobleshoot stats isses, see Process Management Stats on page 44. To delete rns and clear space, see Clear Hard Drive Space on page 31. Stats of Universal Copy Service Universal Copy Service shows the stats of files being copied to the otpt folder: In Progress Universal Copy Service is copying files to the otpt folder. Complete Universal Copy Services sccessflly copied all files to the otpt folder. 6

13 Stats of BaseSpace Seqence Hb BaseSpace Seqence Hb shows the pload stats: In Progress The control software is ploading files to BaseSpace Seqence Hb. Complete All files are sccessflly ploaded to BaseSpace Seqence Hb. Stats of Local Rn Manager Local Rn Manager shows the analysis stats in control software: Not Started The analysis is qeed to start or Local Rn Manager is waiting for Real-Time Analysis to finish. In Progress Local Rn Manager is analyzing files. Check the Local Rn Manager software for a more detailed stats. Stopped The analysis has stopped, bt is incomplete. Complete Local Rn Manager has sccessflly completed the analysis. For more information on analysis stats, check the Local Rn Manager software. iseq 100 i1 Reagents Performing a rn on the iseq 100 System reqires one single-se iseq 100 i1 Reagents kit. The kit is available in one size (300-cycle) and two bndles: Single Incldes consmables for one rn. For-pack Incldes consmables for for rns. Contents and Storage iseq 100 i1 Reagents provide the cartridge and flow cell for seqencing. When yo receive iseq 100 i1 Reagents: Table 1 Do not open the white foil packages ntil instrcted to do so dring the cartridge thawing procedre. The cartridge is thawed in the bag. Promptly store components at the indicated temperatres to ensre proper performance. Store cartridges so that the package labels face p. Store cartridges for at least one day before thawing in a water bath. Kit Components Bndle Consmable Qantity Storage Temperatre Single Cartridge 1-25 C to -15 C Flow cell 1 2 C to 8 C* For-pack Cartridge 4-25 C to -15 C Flow cell 4 2 C to 8 C* *Shipped at room temperatre. Both consmables have identifiers for tracking and ensring compatibility. The cartridge ses radiofreqency identification (RFID). The flow cell ses an electrical interface: electrically erasable programmable read-only memory (EEPROM). 7

14 Flow Cell The flow cell is a patterned, single-lane flow cell bilt over a complementary metal oxide semicondctor (CMOS) optical sensor. A plastic cartridge encases the glass-based flow cell. Raised grip points on the plastic ensre safe handling. A B C D E F Grip points CMOS sensor (top) Imaging area Gasket (one of two) CMOS sensor (bottom) Electrical interface Millions of nanowells cover the srface of the flow cell. Clsters are generated in the nanowells, from which the seqencing reaction is then performed. The ordered arrangement of the nanowells increases otpt reads and data. Dring seqencing, the CMOS sensor captres images for analysis. Cartridge The seqencing reagent cartridge is prefilled with clstering, seqencing, paired-end, and indexing reagents. A foil-sealed reservoir is reserved for libraries, and a slot on the front is reserved for the flow cell. Illminator light reaches the flow cell throgh an access window on top of the cartridge. A B C Access window Flow cell slot Library reservoir 8

15 The cartridge contains all consmables for a rn: reagents, library, and flow cell. The library and flow cell are loaded into the thawed cartridge, which is then loaded onto the instrment. After the rn begins, reagents and library are atomatically transferred from the cartridge to the flow cell. The cartridge contains pmps, valves, and all flidics for the system, inclding an nderside reservoir to collect sed reagents. The cartridge is discarded after a rn, so instrment washes are not necessary. Spported Nmber of Cycles The 300-cycle label on the cartridge indicates how many cycles are analyzed, not how many cycles are performed. The flow cell is compatible with any nmber of cycles and any read type. The cartridge provides sfficient reagent for p to 318 cycles of seqencing. The 318 cycles inclde 151 cycles each for Read 1 and Read 2, pls eight cycles each for Index 1 and Index 2. For information on how many cycles to seqence, see Nmber of Cycles in a Read on page 18. Symbol Descriptions The following table describes the symbols on the consmable or consmable packaging. Symbol Description The date the consmable expires. For best reslts, se the consmable before this date. Indicates the manfactrer (Illmina). The date when the consmable was manfactred. The intended se is Research Use Only (RUO). Indicates the part nmber so that the consmable can be identified.¹ 9

16 Symbol Description Indicates the batch code to identify the batch or lot that the consmable was manfactred in.¹ Indicates that cation is necessary. Indicates a health hazard. Storage temperatre range in degrees Celsis. Store the consmable within the indicated range.² ¹ REF identifies the individal component, while LOT identifies the lot or batch the component belongs to. ² Storage temperatre can differ from shipping temperatre. 10

17 Chapter 2 Getting Started First Time Setp 11 Validation Rns 11 Minimize the Control Software 11 Configre Rn Settings 12 Cstomize System Settings 14 Configre Network Settings 15 User-Spplied Consmables and Eqipment 16 First Time Setp The first time the system is trned on, the control software is lanched with a series of screens to gide yo throgh first time setp. First time setp incldes performing a system check to confirm instrment performance, configring rn settings, and cstomizing system settings. After first time setp, edit the initial settings at any time from the System Settings option on the control software men. Operating System Acconts The Windows operating system has two acconts: administrator (sbsadmin) and ser (sbsser). The administrator accont is intended for IT se, system pdates, and installing the control software, Local Rn Manager analysis modles, and other software. Perform all other fnctions, inclding seqencing, from the ser accont. Password Reqirements The operating system reqires a password change for both acconts (sbsadmin and sbsser) at the first logon. Update each password every 180 days, when prompted. Table 2 Defalt Password Policies Policy Enforce password history Lockot threshold Minimm password length Password mst meet complexity reqirements Store passwords sing reversible encryption Setting Five passwords remembered Ten invalid logon attempts Ten characters Disabled Disabled Validation Rns Optionally perform a validation rn before seqencing experimental libraries for the first time. A validation rn seqences 100% PhiX, which fnctions as a control library, to confirm system operation. For instrctions, see Seqencing on page 18. Minimize the Control Software Minimize the control software to access other applications. For example, to browse to the otpt folder in File Explorer or find a sample sheet. 1 Swipe p on the toch screen to open the Windows taskbar. 11

18 2 Select the iseq 100 System icon or another application. The control software is minimized. 3 [Optional] Attach a keyboard and mose to the instrment to facilitate navigation and typing otside of the control software. 4 To maximize the control software, swipe p and select iseq 100 System. Configre Rn Settings Rn settings apply to all rns and determine the otpt folder location, rn mode, and analysis method. Specify the Otpt Folder Location This setting determines the defalt location of the otpt folder, which yo can change on a per-rn basis dring rn setp. The software saves BCL files and other rn data to the otpt folder. An otpt folder is reqired nless BaseSpace Seqence Hb is configred for Rn Monitoring and Storage. The defalt location is D:\SeqencingRns. 1 From the control software men, select System Settings, and then select the Rn Mode tab. 2 In the Otpt Folder field, enter a location for the otpt folder or select Browse to navigate to a location. Internal drive Enter an existing location on drive D. Drive C lacks sfficient space. External drive Enter the location of a USB drive connected to the instrment. Network location Enter the UNC path (\\servername\directory) to a network location. Starting a path with a mapped network drive letter, sch as T:\, is not spported. 3 Proceed as follows. If yo specified an internal or external drive location, select Save to finish. If yo specified a network location, contine throgh step 7 to configre Universal Copy Service to connect to an accont that has access to the location. 4 Select the Network Access tab. 5 Under Universal Copy Service, select an accont type: Local System Accont The otpt folder resides in a directory that is accessible with a local accont, which has access to most local locations. Windows Accont The otpt folder resides in a directory that reqires logon credentials. This setting applies to the defalt otpt folder location and any location specified dring rn setp. 6 If yo selected Windows Accont, enter the ser name and password for the accont. 7 Select Save. Configre the Rn Mode The rn mode determines where to enter rn parameters and how to analyze data: Rn Mode Rn Parameters Data Analysis Local Rn Manager Entered in Local Rn Manager. The software saves data to the specified otpt folder for atomatic analysis in Local Rn Manager. Manal Entered in the control software. The software saves data to a specified otpt folder for later analysis off the instrment. 12

19 Local Rn Manager is the defalt rn mode, and it provides the most streamlined workflow: create and save rns in the Local Rn Manager software, which then appear in the control software. After seqencing, Local Rn Manager atomatically performs data analysis. Separate sample sheets and analysis applications are not needed. NOTE Local Rn Manager is not a featre of the control software. It is integrated software for recording samples for seqencing, specifying rn parameters, and analyzing data. For analysis prposes, BaseSpace Seqence Hb can pair with either rn mode. When the rn mode is Local Rn Manager and BaseSpace Seqence Hb is configred, both applications analyze the data. Select Local Rn Manager Mode 1 From the control software men, select System Settings, and then select the Rn Mode tab. 2 For Rn Setp Mode, select Local Rn Manager. Before seqencing in Local Rn Manager mode, see the Local Rn Manager Software Gide (docment # ) for important information on sing the software. 3 Select Save. Select Manal Mode 1 From the control software men, select System Settings, and then select the Rn Mode tab. 2 For Rn Setp Mode, select Manal. 3 Select Save. Configre BaseSpace Seqence Hb Use the following instrctions to configre BaseSpace Seqence Hb on yor system. For accont setp instrctions, see the BaseSpace Seqence Hb Online Help (docment # ). 1 From the control software men, select System Settings, and then select the Rn Mode tab. 2 For BaseSpace Settings, select one of the following options for connecting to BaseSpace Seqence Hb: Option Rn Monitoring and Storage Rn Monitoring Only Instrment Performance Data Only* None Description and Reqirements Send InterOp files, log files, and rn data to BaseSpace Seqence Hb for remote monitoring and analysis. Reqires a BaseSpace Seqence Hb accont, an internet connection, and a sample sheet or rn setp in Local Rn Manager. Send InterOp and log files to BaseSpace Seqence Hb for remote rn monitoring. This option is the defalt. Reqires a BaseSpace Seqence Hb accont and an internet connection. Send operational data to Illmina withot connecting rns to BaseSpace Seqence Hb. Reqires an internet connection. Disconnect rns from BaseSpace Seqence Hb acconts and do not send instrment performance data. * Depending on the control software version, the name of this setting on the software interface might differ from the name in this gide. 13

20 When any option except None is selected, instrment performance data are sent to Illmina. These data help Illmina trobleshoot more easily and detect potential failres, enabling proactive maintenance and maximizing instrment ptime. For more information on the benefits of this service, see the Illmina Proactive Technical Note (docment # ). This service: Does not send seqencing data nless Rn Monitoring and Storage is selected. Is trned on by defalt. To opt ot of this service, select None. 3 If yo selected None at step 2, select Save to finish. Otherwise, contine throgh step 6. 4 From the Hosting Location list, select the location of the server where data are ploaded to: USA (N. Virginia), EU (Frankfrt), or China (Beijing). 5 If yo have an Enterprise sbscription, configre yor domain as follows. a b Select the Private Domain checkbox. Enter the domain name (URL) sed for single sign-on to BaseSpace Seqence Hb. For example: 6 Select Save. Sample Sheet Considerations When the system is configred for Manal mode with Rn Monitoring and Storage in BaseSpace Seqence Hb, each rn reqires a sample sheet. Create a sample sheet by editing the iseq 100 System Sample Sheet Template for Manal Mode, then importing it into the control software dring rn setp. Download the template from the iseq 100 Seqencing System spport pages. CAUTION Make sre that yo enter the Index 2 (i5) adapter seqences in the correct orientation for the iseq 100 Seqencing System. For index orientation, see Illmina Adapter Seqences (docment # ). A sample sheet is also reqired when the system is configred for Local Rn Manager mode. However, Local Rn Manager creates the sample sheet for yo and saves it to the appropriate location. For all other circmstances, a sample sheet is optional. Cstomize System Settings System settings control the instrment name, adio and thmbnail saving preferences, and the software pdate method. Name the Instrment 1 From the control software men, select System Settings, and then select the System Cstomization tab. 2 In the Instrment Nickname field, enter a preferred name for the instrment. The name appears along the top of each screen. 3 Select Save. Trn Adio On or Off 1 From the control software men, select System Settings, and then select the System Cstomization tab. 14

21 2 Choose whether to mte the system: To trn on the adio, select On. To trn off the adio, select Off. 3 Select Save. Save Thmbnails 1 From the control software men, select System Settings, and then select the System Cstomization tab. 2 Choose whether to save thmbnail images: To save all thmbnails, select the Save additional thmbnail images checkbox. To save no thmbnails, deselect the Save additional thmbnail images checkbox. The defalt is to save all thmbnails. Saving thmbnails minimally increases rn size, bt helps with trobleshooting. 3 Select Save. Configre Software Updates 1 From the control software men, select System Settings, and then select the System Cstomization tab. 2 Choose whether the system atomatically checks for software pdates: To check atomatically, select the Atocheck for software pdates checkbox. To check manally, deselect the Atocheck for software pdates checkbox. Checking for software pdates atomatically reqires an internet connection. For more information on installing software pdates, see Software Updates on page Select Save. Configre Network Settings Operating the system and transferring data reqires only a WiFi or Ethernet connection with the defalt network settings. These settings do not need to be changed nless yor organization has cstom network reqirements. If it does, conslt yor IT representative for help changing the defalt network settings. The iseq 100 Seqencing System Site Prep Gide (docment # ) provides gidelines on network settings and control compter secrity. Connect to the Internet Configre a WiFi or Ethernet internet connection in Windows Network & Internet settings, which yo can access from the control software. The defalt Ethernet transfers data more reliably. 1 From the control software men, select System Settings, and then select the Network Access tab. 2 Select Network Configration, which minimizes the control software and opens Windows Network & Internet settings. 3 Configre a WiFi or Ethernet connection. If configring WiFi, change the adapter option to Wi-Fi. For detailed configration instrctions, see the Windows 10 help on the Microsoft website. 4 When configration is complete, close Windows settings and maximize the control software. 5 From the Network Access tab, select Save. 15

22 Connect to a Proxy Server 1 Minimize the control software. 2 From Windows Start, open the Rn dialog box. 3 Enter cmd, and then select OK. 4 Enter the following command. Replace port> with yor proxy server address and proxy port, and NULL with any bypasses. C:\windows\System32\bitsadmin.exe /Util /SetIEProxy LocalSystem Manal_ proxy port> NULL 5 Power cycle the instrment. For instrctions, see Power Cycle the Instrment on page 45. User-Spplied Consmables and Eqipment The following ser-spplied consmables and eqipment are sed for seqencing, maintenance, and trobleshooting. Consmables for Seqencing Consmable Spplier Prpose Disposable gloves, powder-free General lab spplier General prpose. iseq 100 i1 Reagents Illmina, catalog # (single) or # (for-pack) Microtbes, 1.5 ml Fisher Scientific, catalog # , or eqivalent low-bind tbes Provides the reagents and flow cell for a rn. Dilting libraries to the loading concentration. Paper towels General lab spplier Drying the cartridge after a water bath. Pipette tips, 20 µl General lab spplier Dilting and loading libraries. Pipette tips, 100 µl General lab spplier Dilting and loading libraries. Resspension Bffer (RSB) Illmina, provided with library prep kits Dilting libraries to the loading concentration. [Optional] 10 mm Tris-HCl, ph 8.5 General lab spplier Sbstitte for RSB to dilte libraries to the loading concentration. [Optional] PhiX Control v3 Illmina, catalog # FC Performing a PhiX-only rn or spiking in a PhiX control. Consmables for Maintenance and Trobleshooting Consmable Spplier Prpose Bleach wipes, 10% VWR, catalog # , or eqivalent Disposable gloves, powder-free General lab spplier General prpose. Decontaminating the instrment and cleaning work srfaces. iseq 100 Spare Drip Tray Pad¹ Illmina, catalog # Lining the drip tray to absorb any leaked flids. iseq 100 Spare Air Filter¹ Illmina, catalog # Replacing the air filter every six months. 16

23 Consmable Spplier Prpose iseq 100 System Test Kit² Illmina, catalog # Performing a system check. Isopropyl alcohol wipes, 70% Lab tisse, low-lint VWR, catalog # , or eqivalent VWR, catalog # , or eqivalent Cleaning the instrment and resable test flow cell. Drying the drip tray and resable test flow cell. Paper towels General lab spplier Drying flid arond the instrment. [Optional] Bleach soltion, 10% VWR, catalog # (32 oz), (16 oz), or eqivalent [Optional] Ethanol wipes, 70% Fisher Scientific, catalog # , or eqivalent Cleaning work srfaces after decontamination. Sbstitte for isopropyl alcohol wipes to clean the instrment and resable test flow cell. ¹ The instrment ships with one installed and one spare. When not nder warranty, replacements are ser-spplied. Keep packaged ntil se. ² Replaces the resable test components shipped with the instrment when they expire after 5 years or 36 ses. Eqipment Item Sorce Prpose Freezer, -25 C to -15 C General lab spplier Storing the cartridge. Ice bcket General lab spplier Setting aside libraries. Pipette, 10 µl General lab spplier Dilting libraries to the loading concentration. Pipette, 20 µl General lab spplier Dilting libraries to the loading concentration. Pipette, 100 µl General lab spplier Dilting libraries to the loading concentration. Refrigerator, 2 C to 8 C General lab spplier Storing the flow cell. [Optional] Keyboard General lab spplier Spplementing the onscreen keyboard. [Optional] Mose General lab spplier Spplementing the toch screen interface. [Optional] Water bath General lab spplier Thawing the cartridge. 17

24 Chapter 3 Seqencing Introdction 18 Nmber of Cycles in a Read 18 Best Practices 19 Thaw the Bagged Cartridge 19 Prepare the Flow Cell and Libraries 20 Load Consmables Into the Cartridge 22 Set Up a Seqencing Rn (Local Rn Manager Mode) 24 Set Up a Seqencing Rn (Manal Mode) 27 Introdction Clster generation, seqencing, and analysis comprise seqencing on the iseq 100 Seqencing System. Each step occrs atomatically dring a seqencing rn. Depending on system configration, frther analysis is performed off-instrment after the rn is complete. Clster Generation Library is atomatically denatred into single strands and frther dilted onboard the instrment. Dring clster generation, single DNA molecles are bond to the srface of the flow cell and amplified to form clsters. Seqencing Clsters are imaged sing one-dye chemistry, which ses one florescent label and two imaging cycles to encode data for the for ncleotides. The first imaging cycle detects adenine (A) and thymine (T). A chemistry cycle then cleaves the dye from A and simltaneosly adds a similar dye to cytosine (C). The second imaging cycle detects C and T. After the second imaging cycle, Real-Time Analysis software performs base calling, filtering, and qality scoring. This process is repeated for each cycle of seqencing. For more information on one-dye chemistry, see Base Calling on page 41. Analysis As the rn progresses, the control software atomatically transfers base call files (*.bcl) to the specified otpt location for data analysis. The method of data analysis depends on yor application and system configration. Nmber of Cycles in a Read For each read, entering a minimm of 26 cycles and a maximm of 151 cycles helps ensre data qality. The exact nmber of cycles depends on yor experiment. The minimm and maximm cycle nmbers inclde an extra cycle. Always add one cycle to the desired read length to correct the effects of phasing and prephasing. Read length is the nmber of seqencing cycles in Read 1 and Read 2, which excldes extra cycles and index cycles. Example rn setp: For a read length of 36 (single-read), enter 37 in the Read 1 field. For a read length of 150 per read (paired-end), enter 151 in the Read 1 field and 151 in the Read 2 field. 18

25 Best Practices When handling reagents and other chemicals, wear safety glasses, a lab coat, and powder-free gloves. Switch gloves when prompted to prevent cross-contamination. Make sre that yo have the reqired consmables and eqipment before starting a protocol. See User- Spplied Consmables and Eqipment on page 16. Follow the protocols in the order shown, sing the specified volmes, temperatres, and drations. Unless a stopping point is specified, proceed immediately to the next step. Thaw the Bagged Cartridge 1 Pt on a new pair of powder-free gloves. 2 Remove the cartridge from -25 C to -15 C storage. If thawing in a water bath, which is the fastest of three thaw methods, store at least 1 day before se. CAUTION Thawing a cartridge in a water bath directly from shipment, when it has been stored on dry ice, can adversely affect cartridge performance. 3 If the cartridge is boxed, remove it from the box bt do not open the white foil bag. 4 Thaw the bagged cartridge sing one of the following methods. Use immediately after thawing, withot refreezing or otherwise storing. Method Thaw Time Instrction 20 C to 25 C water bath 6 hors, not exceeding 18 hors 2 C to 8 C refrigerator 36 hors, not exceeding 72 hors Room-temperatre air (20 C to 25 C) 9 hors, not exceeding 18 hors Use 6 L (1.5 gal) water per cartridge. Set a temperatre-controlled water bath to 25 C or mix hot and cold water to achieve 20 C to 25 C. Face the bag label p, sbmerge the cartridge completely, and apply ~2 kg (4.5 lbs) weight to prevent floating. Do not stack cartridges in the water bath nless it is temperatre-controlled. Position the cartridge so that the label faces p and air can circlate on all sides, inclding the bottom. Position the cartridge so that the label faces p and air can circlate on all sides, inclding the bottom. 5 If wet from a water bath, dry with paper towels. 19

26 Prepare the Flow Cell and Libraries Before loading the flow cell and libraries into the cartridge, bring the flow cell to room temperatre and dilte libraries with an optional PhiX spike-in. Libraries are denatred atomatically onboard the instrment. The following diltion instrctions apply to spported Illmina libraries that are doble-stranded. Always perform a qality control analysis, optimize the loading concentration for yor library, and se a normalization method that generates doble-strand libraries. Bead-based normalization that generates single-stranded libraries is not compatible with onboard denatration. Loading Volme and Concentrations Loading volme is 20 µl. Loading concentration varies by library type: Library Type Loading Concentration (pm) 100 % PhiX 60 AmpliSeq for Illmina Library PLUS 50 Nextera DNA Flex 200 Nextera Flex for Enrichment 100 TrSeq DNA Nano 100 TrSeq DNA PCR-Free 100 For other library types, 50 pm is the recommended starting loading concentration. Optimize this concentration over sbseqent rns to identify a loading concentration that consistently yields data that meets specifications. A loading concentration that is too low reslts in a low percent of clsters passing filter (%PF) and a low percent occpied (%Occpancy) rate. A loading concentration that is too high reslts in sboptimal %PF and very high %Occpancy. NOTE The %Occpancy rate is visible in BaseSpace Seqence Hb and indicates the percentage of wells containing clsters. Dilte Library to 1 nm 1 Prepare the flow cell as follows. a b Remove a new flow cell from 2 C to 8 C storage. Set aside the nopened package at room temperatre for mintes to prevent condensation. Preparing the flow cell now ensres that it reaches room temperatre on time. 2 Remove Resspension Bffer (RSB) from -25 C to -15 C storage. Alternatively, se 10 mm Tris-HCl, ph 8.5 in place of RSB. 3 [Optional] Remove 10 nm PhiX stock from -25 C to -15 C storage. PhiX is needed only for an optional spike-in or a PhiX-only rn. 4 Thaw RSB and optional PhiX at room temperatre for 10 mintes. 20

27 5 Using RSB as dilent, prepare the applicable volme of 1 nm library in low-bind microtbes: Library Type Volme of 1 nm Library to Prepare (µl) Nextera DNA Flex 25 Nextera Flex for Enrichment 12 TrSeq DNA Nano 12 TrSeq DNA PCR-Free 12 All other libraries 10 6 Vortex briefly, and then centrifge at 280 g for 1 minte. 7 [Optional] Store 1 nm library at -25 C to -15 C for p to 1 month. Dilte 1 nm Library to Loading Concentration 1 Combine the following volmes in a low-bind microtbe to prepare 100 µl library dilted to the appropriate loading concentration: Library Type* Loading Concentration (pm) 1 nm Library Volme (µl) RSB Volme (µl) 100% PhiX (for a PhiX-only rn) AmpliSeq for Illmina Library PLUS Nextera DNA Flex Nextera Flex for Enrichment TrSeq DNA Nano TrSeq DNA PCR-Free * For nlisted library types, inclding other Nextera libraries, start with a loading concentration of 50 pm and optimize over sbseqent rns. This table provides example loading concentrations. The iseq 100 System is compatible with all Illmina library prep kits except SreCell WTA 3, bt the optimal loading concentration can vary. 2 Vortex briefly, and then centrifge at 280 g for 1 minte. 3 Set aside dilted library on ice for seqencing. Seqence libraries dilted to the loading concentration the same day they are dilted. 4 If yo are not adding PhiX or are performing a PhiX-only rn, skip the next section and proceed to Load Consmables Into the Cartridge on page 22. Add a PhiX Control (Optional) PhiX is a small, ready-to-se Illmina library with balanced ncleotide representation. Adding a 2% PhiX spike-in to yor library provides additional metrics. For low-diversity libraries, a 5% spike-in is recommended to increase base diversity. NOTE A spike-in as low as 1% is effective for providing additional metrics, bt makes pipetting difficlt. 1 Combine the following volmes in a low-bind microtbe to prepare 50 µl 1 nm PhiX: 10 nm PhiX (5 µl) RSB (45 µl) 2 Vortex briefly, and then centrifge at 280 g for 1 minte. 3 [Optional] Store 1 nm PhiX at -25 C to -15 C for p to 1 month. 21

28 4 Combine the following volmes in a low-bind microtbe to dilte PhiX to the same loading concentration as the library, for example: PhiX Loading Concentration (pm) 1 nm PhiX Volme (µl) RSB Volme (µl) Combine PhiX and library: For a 2% spike-in, add 2 µl dilted PhiX to 100 µl dilted library. For a 5% spike-in, add 5 µl dilted PhiX to 100 µl dilted library. Actal PhiX percentage varies depending on library qality and qantity. 6 Vortex briefly, and then centrifge at 280 g for 1 minte. 7 Set aside the library with PhiX spike-in on ice. Load Consmables Into the Cartridge 1 [Optional] To see an instrctional video on cartridge preparation and loading, select Seqence. 2 Open the cartridge bag from the notches. 3 Avoiding the access window on top of the cartridge, remove the cartridge from the bag. Discard the bag. 4 Invert the cartridge five times to mix reagents. Internal components can rattle dring inversion, which is normal. 5 Tap the cartridge (label facing p) on the bench or other hard srface five times to ensre reagent aspiration. Load Library 1 Using a new pipette tip, pierce the Library reservoir and psh the foil to the edges to enlarge the hole. 2 Discard the pipette tip to prevent contamination. 22

29 3 Add 20 µl dilted library to the bottom of the reservoir. Avoid toching the foil. Load the Flow Cell 1 Open the white foil package from the notches. Use within 24 hors of opening. 2 Pll the flow cell ot of the package. Toch only the plastic when handling the flow cell. Avoid toching the electrical interface, CMOS sensor, glass, and gaskets on either side of the glass. 3 Hold the flow cell by the grip points with the label facing p. 4 Insert the flow cell into the slot on the front of the cartridge. An adible click indicates that the flow cell is in place. When properly loaded, the grip protrdes from the cartridge and the glass is visible from the access window. A B Loading the flow cell Loaded flow cell 23

30 5 Dispose of the packaging as follows. a b c Remove the clamshell case from the foil package. Remove the desiccant from the clamshell case. Recycle the clamshell case, and discard the foil package and desiccant. 6 Proceed depending on rn mode: For Local Rn Manager rn mode, follow Set Up a Seqencing Rn (Local Rn Manager Mode) on page 24. For manal rn mode, follow Set Up a Seqencing Rn (Manal Mode) on page 27. Set Up a Seqencing Rn (Local Rn Manager Mode) Local Rn Manager mode is the defalt rn mode. Rn parameters are specified in the Local Rn Manager software and sent to the control software. After seqencing, analysis starts atomatically. 1 Open Local Rn Manager locally on the instrment monitor or remotely from another compter: Access Type Local Remote Open Local Rn Manager From the control software men, select Local Rn Manager, and then select Open Local Rn Manager. From the control software men, select Abot to obtain the system IP address. From a compter on the same network as the instrment, open Local Rn Manager in Chromim. Use the system IP address to connect. 2 If Chromim is blank on the instrment monitor, power cycle the instrment and restart rn setp. For instrctions, see Power Cycle the Instrment on page In Local Rn Manager, create and save the rn. See the Local Rn Manager Software Gide (docment # ) for instrctions. Set p a PhiX-only rn to be non-indexed. Local Rn Manager atomatically sends saved rns to the control software. 4 In the control software, select Seqence from the Home screen. The software opens the door at an angle, ejects the tray, and initiates the series of rn setp screens. 5 [Optional] Select Help to view an onscreen prompt. These prompts are available at each rn setp screen to provide additional gidance. 24

31 Load the Cartridge Onto the Instrment 1 Make sre that the cartridge is thawed and contains the flow cell and dilted library. 2 Place the cartridge onto the tray so that the access window faces p and the flow cell is inside the instrment. Do not psh the cartridge or tray into the instrment. 3 Select Close Door to retract the cartridge and close the door. A panel appears on the left side of the screen to show information from the scanned consmables. Dring scanning, yo can contine with rn setp. Sign In to BaseSpace Seqence Hb The BaseSpace Seqence Hb screen appears when the system is configred for Rn Monitoring or Rn Monitoring and Storage. 1 To disconnect the crrent rn from BaseSpace Seqence Hb, select Skip BaseSpace Seqence Hb Sign In. Instrment performance data are still sent to Illmina. 2 To change the BaseSpace Seqence Hb setting for the crrent rn, select a Configration option: Rn Monitoring Only Send InterOp files only to BaseSpace Seqence Hb to allow remote monitoring. Rn Monitoring and Storage Send rn data to BaseSpace Seqence Hb to allow remote monitoring and analysis. 3 Enter yor address and BaseSpace Seqence Hb password, and then select Sign In. 4 If the Available Workgrops list appears, select a workgrop to pload rn data to. This list appears when yo belong to mltiple workgrops. 5 Select Rn Setp. Select a Rn 1 If the Local Rn Manager Log In screen appears, enter yor ser name and password, and then select Log In. This screen appears when Local Rn Manager is configred to reqire signing in. By defalt, yo do not sign in. 2 Select a rn from the Rn Name list, which lists rns saved in Local Rn Manager. To view an pdated list, select Refresh. If the list is empty, select Open Local Rn Manager to create a rn. 25

32 Selecting Open Local Rn Manager minimizes the control software and opens Local Rn Manager in Chromim. 3 If yo left the control software to create a rn, retrn and select the rn. 4 [Optional] Select Edit and modify rn parameters: a b c d To change Read Type, select Single Read or Paired End. To change Read Cycle, enter cycles each for Read 1 and Read 2. Add one cycle to the desired nmber of cycles. To change the otpt folder location for the crrent rn, enter the UNC path for the location or select Browse and navigate to it. Select Save, which pdates the rn in both the control software and Local Rn Manager. NOTE A UNC path incldes backslashes, the server name, and directory (\\servername\directory), not a letter for a mapped network drive (T:\). 5 Select Start Rn to start the pre-rn check. Review Pre-Rn Checks Pre-rn checks inclde an instrment check and a flow check. The flow check pierces the cartridge seals and passes reagent throgh the flow cell, so consmables cannot be resed after the flow check starts. 1 Wait abot 15 mintes for pre-rn checks to complete. The rn starts atomatically after sccessfl completion. Unless the system is mted, a chime sond indicates that the rn has started. CAUTION Opening the door dring the pre-rn check or the rn can case rn failre. 2 If an error occrs dring the instrment check, select Retry to redo the check. The instrment check precedes the flow check. When a check is in progress, the bar for that check is animated. 3 To trobleshoot recrring errors, see Error Message Resoltion on page 44. Monitor Rn Progress 1 Monitor rn progress and metrics as they appear on the Seqencing screen. Estimated rn completion The approximate date and time of rn completion. %Q30 Read 1 The percentage of Read 1 base calls with a Q-score 30. %Q30 Read 2 The percentage of Read 2 base calls with a Q-score 30. Projected Total Yield The expected nmber of bases called for the rn. Q30 and yield metrics appear after cycle To monitor rn processes, select the control software men, and then select Process Management. Unload Consmables 1 When seqencing is complete, select Eject Cartridge. The software ejects the sed cartridge from the instrment. 2 Remove the cartridge from the tray. 26

33 3 Remove the flow cell from the cartridge. 4 Dispose of the flow cell, which has electronic components, in accordance with applicable standards for yor region. 5 Dispose of the cartridge, which contains sed reagents, in accordance with applicable standards for yor region. A post-rn wash is not necessary becase flidics are discarded with the cartridge. 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. 6 Select Close Door to reload the tray and retrn to the Home screen. The software atomatically reloads the tray and sensors confirm cartridge removal. Set Up a Seqencing Rn (Manal Mode) When setting p a rn in Manal mode, rn parameters are specified in the control software. Data are saved to the specified otpt folder for off-instrment analysis. Generating FASTQ files reqires an additional analysis step. 1 If necessary, create a sample sheet for the rn: a Download the iseq 100 System Sample Sheet Template for Manal Mode from the iseq 100 Seqencing System spport pages. b Modify the template as needed and save it in *.csv file format. Make sre that: The Index 2 (i5) adapter seqences are in the correct orientation. For index orientation, see Illmina Adapter Seqences (docment # ). Sample sheet vales match control software vales. For example, enter 151 in the Read 1 field of both the sample sheet and the Rn Setp screen. Systems configred for BaseSpace Seqence Hb with Rn Monitoring and Storage reqire a sample sheet for each rn. 2 In the control software, select Seqence from the Home screen. The software opens the door at an angle, ejects the tray, and initiates the series of rn setp screens. 3 [Optional] Select Help to view an onscreen prompt. These prompts are available at each rn setp screen to provide additional gidance. 27

34 Load the Cartridge Onto the Instrment 1 Make sre that the cartridge is thawed and contains the flow cell and dilted library. 2 Place the cartridge onto the tray so that the access window faces p and the flow cell is inside the instrment. Do not psh the cartridge or tray into the instrment. 3 Select Close Door to retract the cartridge and close the door. A panel appears on the left side of the screen to show information from the scanned consmables. Dring scanning, yo can contine with rn setp. Sign In to BaseSpace Seqence Hb The BaseSpace Seqence Hb screen appears when the system is configred for Rn Monitoring or Rn Monitoring and Storage. 1 To disconnect the crrent rn from BaseSpace Seqence Hb, select Skip BaseSpace Seqence Hb Sign In. Instrment performance data are still sent to Illmina. 2 To change the BaseSpace Seqence Hb setting for the crrent rn, select a Configration option: Rn Monitoring Only Send InterOp files only to BaseSpace Seqence Hb to allow remote monitoring. Rn Monitoring and Storage Send rn data to BaseSpace Seqence Hb to allow remote monitoring and analysis (reqires a sample sheet). 3 Enter yor address and BaseSpace Seqence Hb password, and then select Sign In. 4 If the Available Workgrops list appears, select a workgrop to pload rn data to. This list appears when yo belong to mltiple workgrops. 5 Select Rn Setp. Enter Rn Parameters 1 In the Rn Name field, enter a niqe name of yor preference to identify the crrent rn. The rn name can contain alphanmeric characters, hyphens, and nderscores. 2 For Read Type, select how many seqencing reads to perform: Single Read Perform one read, which is the simpler, faster option. Paired End Perform two reads, which generates higher-qality data and provides more accrate alignment. 3 For Read Cycle, enter the nmber of cycles performed in each read: 28

35 Read 1 Enter cycles, adding one cycle to the desired nmber of cycles. Index 1 Enter p to 8 cycles for the Index 1 (i7) primer. For a PhiX-only rn, enter 0 in both index fields. Index 2 Enter p to 8 cycles for the Index 2 (i5) primer. Read 2 Enter cycles (typically the same vale as Read 1, inclding the extra cycle). 4 To specify an otpt folder for the crrent rn or pload a sample sheet, expand Advanced: In the Otpt Folder field, enter the UNC path for the otpt folder location or select Browse and navigate to it. In the Sample Sheet field, enter the UNC path for the sample sheet location or select Browse and navigate to it. NOTE A UNC path incldes backslashes, the server name, and directory (\\servername\directory), not a letter for a mapped network drive (T:\). 5 Select Start Rn to start the pre-rn check. Review Pre-Rn Checks Pre-rn checks inclde an instrment check and a flow check. The flow check pierces the cartridge seals and passes reagent throgh the flow cell, so consmables cannot be resed after the flow check starts. 1 Wait abot 15 mintes for pre-rn checks to complete. The rn starts atomatically after sccessfl completion. Unless the system is mted, a chime sond indicates that the rn has started. CAUTION Opening the door dring the pre-rn check or the rn can case rn failre. 2 If an error occrs dring the instrment check, select Retry to redo the check. The instrment check precedes the flow check. When a check is in progress, the bar for that check is animated. 3 To trobleshoot recrring errors, see Error Message Resoltion on page 44. Monitor Rn Progress 1 Monitor rn progress and metrics as they appear on the Seqencing screen. Estimated rn completion The approximate date and time of rn completion. %Q30 Read 1 The percentage of Read 1 base calls with a Q-score 30. %Q30 Read 2 The percentage of Read 2 base calls with a Q-score 30. Projected Total Yield The expected nmber of bases called for the rn. Q30 and yield metrics appear after cycle To monitor rn processes, select the control software men, and then select Process Management. Unload Consmables 1 When seqencing is complete, select Eject Cartridge. The software ejects the sed cartridge from the instrment. 2 Remove the cartridge from the tray. 3 Remove the flow cell from the cartridge. 29

36 4 Dispose of the flow cell, which has electronic components, in accordance with applicable standards for yor region. 5 Dispose of the cartridge, which contains sed reagents, in accordance with applicable standards for yor region. A post-rn wash is not necessary becase flidics are discarded with the cartridge. 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. 6 Select Close Door to reload the tray and retrn to the Home screen. The software atomatically reloads the tray and sensors confirm cartridge removal. 30

37 Chapter 4 Maintenance Clear Hard Drive Space 31 Software Updates 31 Replace the Air Filter 32 Relocate the Instrment 34 Software Restriction Policies 35 Clear Hard Drive Space A seqencing rn reqires abot 2 GB of hard drive space. When space is low, se the following steps to delete completed rns and clear space. 1 From the control software men, select Process Management. The Process Management screen appears with a list of rns saved to the hard drive. 2 For the rn yo want to delete, select Delete. Deleting a rn deletes the local rn folder. The otpt folder, which is a copy of the rn folder, is retained. 3 In the dialog box, select Yes to confirm deleting the rn. 4 Repeat steps 2 and 3 for each rn yo want to delete. 5 When yo are finished, close Process Management to retrn to the Home screen. Software Updates Updating the software ensres that yor system has the latest featres and fixes. Software pdates are bndled into a system site, which incldes the following software: iseq Control Software iseq 100 System recipes Universal Copy Service Real-Time Analysis Local Rn Manager (framework only) NOTE Althogh Local Rn Manager is inclded in the system site, the analysis modles are not. Install them separately as needed sing the sbsadmin accont. Access the analysis modle software from the Local Rn Manager spport pages. The system is configred to download software pdates atomatically or manally: Atomatic pdates Updates are atomatically downloaded from BaseSpace Seqence Hb for yo to install. This option reqires an internet connection, bt not a BaseSpace Seqence Hb accont. Manal pdates Updates are manally downloaded from the web, saved locally or to a portable device, and installed from the saved location. This option does not reqire an internet connection. Install an Atomatic Software Update 1 Switch to the sbsadmin operating system accont. 2 Select the control software men, and then select Software Update to open the Software Update dialog box. Systems configred for atomatic pdates display an alert when a software pdate is available. 31

38 3 To check for an pdate, select one of the following options: Check for Update Check for a software pdate. Atocheck for Updates Check for a software pdate and configre the system to atomatically check for ftre pdates. These options are visible on systems that are connected to the internet bt not configred for atomatic pdates. 4 Select Update to download the new version of software. When the download is complete, the control software is closed and the installation wizard appears. 5 In the installation wizard, select Install. 6 When the installation is complete, select Close. The control software atomatically restarts. Any firmware pdate occrs atomatically after the restart. NOTE Canceling an pdate before installation is complete stops the pdate at the crrent point. Any changes made p to the cancellation point are reverted to the previos version or not installed. Install a Manal Software Update 1 Switch to the sbsadmin operating system accont. 2 When a software pdate is available, download the site installer (*.exe) from the iseq 100 Seqencing System spport page. Save the installer to a local or portable drive. 3 If yo saved the installer to a portable drive, plg the drive into a USB port on the back of the instrment. Move the instrment as needed to access the back. 4 In the control software, select Software Update from the control software men. 5 In the Software Update dialog box, expand Install from local or portable drive. 6 Select Browse to navigate to the installer. 7 Select Update to start installation. The control software is closed and the installation wizard appears. 8 In the installation wizard, select Install. 9 When the installation is complete, select Close. The control software atomatically restarts. Any firmware pdate occrs atomatically after the restart. NOTE Canceling an pdate before installation is complete stops the pdate at the crrent point. Any changes made p to the cancellation point are reverted to the previos version or not installed. Replace the Air Filter The software prompts for an air filter change every six months from when first time setp was started. Use the following instrctions to replace an expired air filter. The air filter is a single-se foam piece that covers the two fans on the back of the instrment. It ensres proper cooling and prevents debris from entering the system.the instrment is shipped with one air filter installed and one spare. Additional spares are inclded with the warranty, or they can be prchased from Illmina. 32

39 1 Position the instrment so that yo can easily access the back. 2 On the back of the instrment, press the right side of the top panel to disengage as shown in the following illstration. 3 Remove the panel from the instrment. 4 Remove the foam air filter from the center of the panel and discard. 5 Place a new air filter into the panel, and press to secre. 33

40 6 Insert the two panel hooks into the holes in the instrment and press the panel into place. 7 Retrn the instrment to the original placement. 8 Select Filter Changed to proceed. Relocate the Instrment Use the following instrctions to safely relocate an instrment. Make sre that the new location meets the reqirements described in the iseq 100 Seqencing System Site Prep Gide (docment # ). If yo are retrning an instrment, skip this section and see Advanced Exchange on page From the men, select Sht Down System. 2 If the system does not sht down, hold the power btton on the left side of the instrment ntil the lights fade. 3 When the power btton plses, press the power off (Ο) side of the toggle switch on the rear panel. The power btton might contine to plse after the power is trned off. Figre 7 Toggle Switch Location 4 Disconnect the power cord from the wall otlet, and then from the AC power otlet on the rear panel. 5 If applicable, disconnect the Ethernet cable from the wall otlet, and then from the Ethernet port on the rear panel. 6 Lower the monitor. 34

41 7 Relocate the instrment to the desired location. The instrment weighs 15.9 kg (35 lbs) and reqires a two-person lift. 8 Raise the monitor. 9 If the instrment is connected to a network, connect the Ethernet cable to an Ethernet port. 10 Connect the power cord to the AC inlet on the rear panel, and then to a wall otlet. 11 Press the power on (I) side of the toggle switch. 12 When the power btton plses, press it. Figre 8 Power Btton Location 13 When the operating system is loaded, log on to Windows. The control software is lanched and initializes the system. The Home screen appears when initialization is complete. Software Restriction Policies Windows Software Restriction Policies (SRP) se rles to allow only specified software to rn. For the iseq 100 System, SRP rles are based on certificates, file names and extensions, and directories. By defalt, SRP is trned on to prevent nwanted software from rnning on the control compter. An IT representative or system administrator can add and remove rles to cstomize the secrity level. If the system is added to a domain, the local Grop Policy Object (GPO) might atomatically modify the rles and trn off SRP. Allowed SRP Rles On the iseq 100 Seqencing System, SRP defalts to allowing the following rles. Certificates iseq Application Certificate 35

42 Exectable Files Portmon.exe Procmon.exe Procmon64.exe Tcpview.exe File Extensions *.cfg *.lnk *.png *.tif *.txt *.xml Directories %HKEY_LOCAL_MACHINE\SOFTWARE\Microsoft\Windows\CrrentVersion\ProgramFilesDir% %HKEY_LOCAL_MACHINE\SOFTWARE\Microsoft\Windows NT\CrrentVersion\SystemRoot% C:\CrashDmps\* C:\Illmina Maintenance Logs\* C:\Illmina Manfactring Test Reslts\* C:\Illmina\* C:\Program Files (x86)\chromim\application\* C:\Program Files (x86)\illmina\* C:\Program Files (x86)\internet Explorer\* C:\Program Files\Illmina\* C:\Program Files\Internet Explorer\* C:\Program Files\Jenoptik\* C:\Program Files\Ophir Optronics\* C:\ProgramData\Illmina\* C:\ProgramData\Package Cache\* C:\ProgramData\webex\* C:\Users\*\AppData\Local\GoToAssist Corporate\* C:\Users\*\AppData\Local\Temp\Citrix\* C:\Users\*\AppData\Local\Temp\CitrixLogs\* C:\Users\*\AppData\Local\Temp\LogMeIn\* C:\Users\*\AppData\Local\Temp\LogMeInLogs\* D:\Recovery\* GoToAssist Corporate Opener*.exe Trnover to cstomer.bat Add and Remove SRP Rles Add and remove SRP rles to cstomize system secrity. Modifying the rles reqires temporarily trning off SRP. CAUTION Trning off SRP overrides the defalt protections. 1 Log in to the operating system with the administrator accont. The administrator accont has the privileges necessary to modify SRP rles. 2 Trn off SRP: a b c Navigate to the directory C:\Illmina\Secrity. Doble-click Disable.reg. Select Yes to confirm the changes. 36

43 NOTE When sing the toch-screen interface, tapping and holding for abot 2 seconds is eqivalent to a rightclick. 3 Select Start, and then select Rn. 4 In the Open field, enter secpol.msc. 5 In the Local Secrity Policy dialog box, expand Software Restriction Policies, and then select Additional Rles. 6 To add a rle: a b c d e On the Action men, select New Path Rle. In the Path field, enter the certificate, file name, file extension, or directory that yo want to allow. In the Secrity level list, select Unrestricted. [Optional] In the Description field, enter a reason for creating the rle. Select OK to add the rle. 7 To delete a rle: a b Select the rle yo want to delete, and then select Delete. Select Yes to confirm the deletion. 8 Close the Local Secrity Policy dialog box. 9 Immediately reinstate SRP: a b Navigate to the directory C:\Illmina\Secrity. Doble-click Enable.reg. 10 If SRP rles were modified for the first time, log off and then log on again for the rles to take effect. 37

44 Appendix A Seqencing Otpt Real-Time Analysis Overview 38 Real-Time Analysis Workflow 40 Real-Time Analysis Overview The Real-Time Analysis software rns on the instrment control compter. Dring a seqencing rn, it extracts intensities from images to perform base calling, and then assigns a qality score to the base call. iseq 100 Seqencing System ses the RTA2 implementation of Real-Time Analysis. 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 is not poplated. RTA2 receives commands from the control software that inclde information abot the location of RnInfo.xml file and whether an otpt folder is specified. RTA2 reqires the following inpt files for processing: Tile images contained in local system memory. Real-Time Analysis configration file in XML format. 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. Otpt Files Images are passed in memory to RTA2 as tiles, which are small imaging areas on the flow cell defined by one camera view. The iseq 100 flow cell has 16 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. File Type Base call files Base call index files Clster locations file File Description, Location, and Name Each tile analyzed is inclded in a base call file, aggregated in one file per cycle. The aggregated file contains the base call and associated qality score for every clster. Data\Intensities\BaseCalls\L001 [Cycle].bcl.bgzf, where [Cycle] represents the cycle nmber in for digits. Base call files are compressed sing block gzip compression. A base call index file preserves the original tile information. For each tile, the index file contains the tile nmber and nmber of clsters. Data\Intensities\BaseCalls\L001 [Cycle].bcl.bgzf.bci One clster location (s.locs) file contains the X,Y coordinates for every clster on the flow cell. Data\Intensities s.locs 38

45 File Type Filter files InterOp files RTA configration file Rn information file* Thmbnail files File Description, Location, and Name Filter files specify whether clsters pass filter. For each tile, one filter file is generated. Filter files are generated at cycle 26 sing 25 cycles of data. Data\Intensities\BaseCalls\L001 s_[lane].filter Real-time metrics of rn qality that are pdated throghot the rn. These binary files contain tile, cycle, and read-level metrics, and are reqired to view metrics in Seqencing Analysis Viewer. InterOp folder Created at the beginning of the rn and lists parameters for the rn. This file combines vales from the inpt configration file and vales that RTA2 defines. [Root folder], RTAConfigration.xml Created at the beginning of the rn. Lists the rn name, nmber of cycles per read, whether the read is an Index Read, and the nmber of swaths and tiles. [Root folder], RnInfo.xml Thmbnail images of flow cell tiles. Images\L001\C[X.1] Files are stored in one folder for each lane and one sbfolder for each cycle. s_[lane]_[tile].jpg The thmbnail image incldes the tile nmber. *Created by the control software. RTA2 creates all other files listed in this table. Local Rn Manager and BaseSpace Seqence Hb se the base call files for data analysis. For FASTQ conversion and third-party analysis soltions, se the bcl2fastq conversion software. To download the latest version, see the iseq 100 Seqencing System spport pages on the Illmina website. Otpt Folder Name and Path For each rn, the control software atomatically generates an otpt folder and a rn folder. Access the rn data from the otpt folder, which is a copy of the rn folder. The rn folder is for system se. The path to the otpt folder is ser-defined, bt defalts to D:\. The control software names the otpt folder sing the following format. Format <YYYYMMDD>_<Instrment ID>_<Rn Nmber>_<Flow Cell ID> Example _FFSP247_4_BNS The rn nmber increments by one each time the system performs a rn. Serial nmbers identify the instrment and flow cell. 39

46 Otpt Folder Strctre Recipe Rn-specific recipe file. Logs Log files describing instrment analytics, operational steps, and other events. Config Configration settings for the rn. RnParameters.xml RnInfo.xml CopyComplete.txt RnCompletionStats.txt RTAComplete.txt RTAConfigration.xml Data InterOp Images Intensities BaseCalls s.locs L001 SampleSheet.csv Sample sheet or the sample manifest. RTALogs Log files describing RTA2 events. 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. *Error*.tsv lists errors that occrred dring a rn. *WarningLog*.tsv lists warnings that occrred dring a rn. Real-Time Analysis Workflow Intensity Extraction Determines an intensity vale for each clster. 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. Intensity Extraction Intensity extraction calclates an intensity vale for each nanowell in a given image. 40

47 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 9 Phasing and Prephasing A B Read with a base that is phasing Read with a base that is prephasing RTA2 corrects the effects of phasing and prephasing, which maximizes the data qality at every cycle throghot the rn. Base Calling Base calling determines a base (A, C, G, or T) for every clster of a given tile at a specific cycle. The iseq 100 System ses one-dye seqencing, which reqires one dye and two images to encode data for the for bases. Intensities extracted from one image and compared to a second image reslt in for distinct poplations, each corresponding to a ncleotide. Base calling determines which poplation each clster belongs to. Figre 10 Visalization of Clster Intensities 41

48 Table 3 Base Base Calls in One-Dye Seqencing Dye in First Image Dye in Second Image Conclsion From Compared Images T On On Clsters that show intensity in both images are T bases. A On Off Clsters that show intensity in the first image only are A bases. C Off On Clsters that show intensity in the second image only are C bases. G Off Off Clsters that show intensity in neither image are G bases. Clsters Passing Filter Dring the rn, RTA2 filters raw data to remove reads that do not meet the data qality threshold. Overlapping and low-qality clsters are removed. For one-dye seqencing, RTA2 ses a poplation-based system to determine the chastity (intensity prity measrement) of a base call. Clsters pass filter (PF) when no more than one base call in the first 25 cycles has a chastity below a fixed threshold. PhiX alignment is performed at cycle 26 on a sbset of tiles for clsters that passed filter. Clsters that do not pass filter are not base called and not aligned. Index Reads The process for base calling index reads differs from base calling seqencing reads. The first two cycles of an Index Read cannot start with two G bases, otherwise intensity is not generated. To ensre demltiplexing performance, intensity mst be present in either of the first two cycles. Make sre that at least one index seqence in a library pool does not start with two G bases. Select balanced index seqences so that signal is present in at least one image (preferably both images) for every cycle. The plate layot and seqences provided in IDT for Illmina TrSeq UD Indexes are designed to have the proper balance. For more information on indexing and pooling, see the Index Adapter Pooling Gide (docment # ). 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. Q(X) represents qality scores, 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. 42

49 Qality scoring calclates a set of predictors for each base call, and then ses the predictor vales to look p the Q-score in a qality table. Qality tables are created to provide optimally accrate qality predictions for rns generated by a specific configration of seqencing platform and version of chemistry. After the Q-score is determined, reslts are recorded in the base call files. 43

50 Appendix B Trobleshooting Error Message Resoltion 44 Cancel a Rn 44 Power Cycle the Instrment 45 Perform a System Check 46 Leak Mitigation 48 Restore to Factory Settings 50 Error Message Resoltion This appendix provides detailed instrctions for varios trobleshooting steps. The following flowchart provides an overview on trobleshooting error messages that appear dring initialization, rn setp, pre-rn checks, or seqencing, and are not resolved with a retry. Many errors can be resolved with a power cycle: trning off the instrment, and then restarting it. Others reqire a system check to diagnose and resolve. Figre 11 Error Messages Overview Process Management Stats To trobleshoot an stats isse on the Process Management screen: If the rn is in progress, close the Process Management screen, wait abot five mintes, and then reopen. If the rn is not in progress, power cycle the instrment, and then reopen the Process Management screen. See Power Cycle the Instrment on page 45. Cancel a Rn 1 Select Stop Rn, and then select Yes, Cancel. The Seqencing Canceled screen appears with timestamps for the date and time the rn stopped. 44

51 CAUTION Canceling a rn is final. The software cannot resme the rn and consmables cannot be resed after the instrment check portion of pre-rn checks. 2 Select Eject Cartridge to open the door and eject the tray. 3 Remove the cartridge from the tray. 4 Store or dispose of the cartridge, depending on when the cancellation occrred: Circmstance Yo canceled before or dring the instrment check and want to rese consmables. All other circmstances. Instrction Leave the flow cell and library inside the cartridge, and set aside at room temperatre for p to 1 hor. Remove the flow cell from the cartridge. Dispose of both components in accordance with applicable regional standards. The flow cell has electronic components The cartridge contains sed reagents and library. 5 Select Close Door to reload the tray and retrn to the Home screen. Sensors confirm cartridge removal. Power Cycle the Instrment Power cycling the instrment safely shts down and restarts the system to restore a lost connection, align a specification, or resolve an initialization failre. Software messages indicate when to power cycle to resolve an error or warning. 1 From the men, select Sht Down System. 2 If the system does not sht down, hold the power btton on the left side of the instrment ntil the lights fade. 3 When the power btton plses, press the power off (Ο) side of the toggle switch on the rear panel. The power btton might contine to plse after the power is trned off. Figre 12 Toggle Switch Location 4 Wait 30 seconds. 5 Press the power on (I) side of the toggle switch. 45

52 6 When the power btton plses, press it. Figre 13 Power Btton Location 7 When the operating system is loaded, log on to Windows. The control software is lanched and initializes the system. The Home screen appears when initialization is complete. Perform a System Check A system check takes abot 45 mintes and ses the resable test flow cell and resable test cartridge to trobleshoot pre-rn check errors and other isses. For sbsystem tests confirm whether components are properly aligned and fnctional. Normal operation and maintenance do not reqire a system check. 1 Remove the resable test flow cell and resable test cartridge from room temperatre storage. 2 From the control software men, select System Check. The System Check dialog box appears with the mechanical, thermal, optical, and sensor tests selected. 3 Select Unload to open cartridge compartment door and eject the tray. 4 If present, remove the sed cartridge from the tray. 5 Inspect the glass srface of the resable flow cell for visible debris. If debris is present, clean as follows. a b c Clean the glass srface with an alcohol wipe. Dry with a low-lint lab tisse. Make sre that the flow cell is free of lint or fibers. Under normal circmstances, the resable test flow cell does not reqire cleaning. 6 Hold the resable test flow cell by the grip points with the label facing p. 46

53 7 Insert the resable test flow cell into the slot on the front of the resable test cartridge. An adible click indicates that the flow cell is in place. When properly loaded, the grip protrdes from the cartridge and the glass is visible from the access window. a b Loading the resable test flow cell Loaded resable test flow cell 8 Place the resable test cartridge onto the tray so that the access window faces p and the flow cell is inside the instrment. 9 Select Load to load the resable test cartridge and close the door. 10 Select Start to begin the system check. Dring the system check, the software ejects and retracts the cartridge one time. NOTE The resable test flow cell and cartridge are valid for 36 ses or 5 years from the date of manfactre, whichever comes first. The remaining nmber of ses are displayed onscreen. 11 When the system check is complete, review whether each test passed or failed. Reslt Indication Action All for tests passed At least one test failed The instrment is fnctioning properly and the isse is likely consmable- or library-related. The instrment might have a hardware isse. Set p a new rn. If consmables from the previos rn were stored, se them for the new rn. Contact Illmina Technical Spport. 12 Select Unload to eject the resable test cartridge. 13 Remove the resable test cartridge from the tray. 47

54 14 Remove the resable test flow cell from the cartridge. 15 Retrn the resable test components to the original packaging and store at room temperatre. 16 Close the System Check dialog box. Leak Mitigation If a poor flidics connection, cartridge isse, or leak is detected dring a pre-rn check or seqencing, the software ends the rn and notifies yo. After assessing the leak and cleaning the instrment, a system check confirms that normal operation can contine. A drip tray at the base of the instrment catches flids leaking from the cartridge. However, leaked flids can reach other areas of the system. Under normal circmstances, the drip tray is dry. Assess the Leak 1 Pt on a new pair of powder-free gloves. 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. 2 Follow the onscreen prompts to eject the cartridge. 3 Inspect the cartridge for visible flid. A small amont of flid (< 500 µl) on the glass srface of the flow cell is acceptable. 4 If no flid (or an acceptable amont of flid) is visible, proceed to Clean the Instrment. After cleaning, a system check confirms normal operation. 5 If a significant amont of flid is visible on the flow cell, cartridge, or instrment, shtdown and disconnect as follows and contact Illmina Technical Spport. a b c d e f From the men, select Sht Down System. If the shtdown command is nresponsive, press and hold the power btton on the left side of the instrment ntil the lights fade. When the power btton plses, press the power off (Ο) side of the toggle switch on the back of the instrment. Wait 30 seconds. Disconnect the power cord from the wall otlet, and then from the AC power otlet on the rear panel. If applicable, disconnect the Ethernet cable from the wall otlet, and then from the Ethernet port on the rear panel. Clean the Instrment 1 For safety, trn off and disconnect the instrment: a From the men, select Sht Down System. 48

55 b c d e f If the shtdown command is nresponsive, press and hold the power btton on the left side of the instrment ntil the lights fade. When the power btton plses, press the power off (Ο) side of the toggle switch on the back of the instrment. Wait 30 seconds. Disconnect the power cord from the wall otlet, and then from the AC power otlet on the rear panel. If applicable, disconnect the Ethernet cable from the wall otlet, and then from the Ethernet port on the rear panel. 2 Locate the drip tray door below the cartridge compartment on the front of the instrment, and then lower the door. 3 Open the drip tray and remove the drip tray pad. 4 Wipe any residal flid from the bottom of the tray sing paper towels. 5 Dispose of the pad and other consmables in accordance with applicable standards, which vary by region. For more information, see the Safety Data Sheet (SDS) at spport.illmina.com/sds.html. 6 Place a new pad onto the drip tray. 7 Close the drip tray, and then close the drip tray door. 8 Dry any visible flid on or arond the instrment sing paper towels. 49

56 9 Trn on and reconnect the instrment as follows. a b c d e If applicable, connect the Ethernet cable to an Ethernet port. Connect the power cord to the AC inlet on the rear panel, and then to a wall otlet. Press the power on (I) side of the toggle switch on the rear panel. When the power btton plses, press it. When the operating system is loaded, log on to Windows. The control software is lanched and initializes the system. The Home screen appears when initialization is complete. 10 Perform a system check to confirm that the system is fnctioning normally. Passing the system check indicates that the instrment can resme normal operation. For instrctions, see Perform a System Check on page 46. Restore to Factory Settings Restore the system to factory defalts to downgrade the software, recover from an ndesirable configration, or clear ser data before retrning an instrment to Illmina. Restoring the system ninstalls control software and clears C:\. 1 Restart Windows. 2 When prompted to choose an operating system, select Restore to Factory Settings. The operating system options appear briefly before atomatically proceeding with iseq Control Software. 3 Wait abot 30 mintes for the restoration to complete. The restoration can inclde several reboots. When complete, the system reboots with the original factory settings, less the control software. 4 Install the control software: a Download the software installer from the iseq 100 Seqencing System spport pages. Save the installer to a network location or portable USB drive. b Copy the installer to C:\Illmina. c Open iseqsiteinstaller.exe, and then follow the prompts to perform the install. d When the pdate is complete, select Finish. e Power cycle the instrment. For instrctions, see Power Cycle the Instrment on page Follow the onscreen prompts to perform first time setp, inclding the system check with the resable test cartridge and resable test flow cell. 6 Install any Local Rn Manager analysis modles: a b c d e Switch to the sbsadmin operating system accont. Download the software installers from the Local Rn Manager spport pages. Save the installers to a network location or portable USB drive. Copy the installer to C:\Illmina. Open the installer (*.exe), and then follow the prompts to perform the install. When the pdate is complete, select Finish. 50

57 Appendix C Advanced Exchange Exchanging the iseq 100 System 51 Receive a Replacement System 51 Prepare the Original System for Retrn 51 Retrn the Original System 54 Exchanging the iseq 100 System Becase the air filter and drip tray pad are the only serviceable parts the iseq 100 System has, Illmina ses advanced exchange to fix isses that cannot be resolved remotely. Advanced exchange replaces a damaged or defective system with a refrbished system. To minimize downtime, yo receive the replacement system before shipping back the original. Regional Availability Advanced Exchange is available in most regions. Other regions can contine to rely on field service engineers. Ask Illmina Technical Spport which spport models are available in yor region. Receive a Replacement System 1 After a system check and other trobleshooting efforts have failed, contact Illmina Technical Spport. If possible, perform another system check with a different resable test cartridge and resable test flow cell. Make system check reslts available to Technical Spport. If Technical Spport cannot resolve the isse remotely, a retrn is initiated and an order is placed for a replacement system. 2 When yo receive the replacement system: Unpack and install it per the iseq 100 Seqencing System Setp Poster (docment # ). Save all the packaging, which is sed to package the original system and resable test components for retrn. Set aside the retrn docments, which inclde a UPS retrn label and commercial invoice (for international shipments). Prepare the Original System for Retrn Retrn the original system, resable test cartridge, and resable test flow cell to Illmina within 30 days of receiving the replacement. Sht Down and Disconnect If the system is on, follow steps 1 3 to save and clear data before safely shtting down the system. Follow the remaining steps to remove a cartridge (if necessary) and disconnect cords and cables. 1 From File Explorer, copy any files and folders that yo want to save to a portable USB drive. The location of seqencing data is ser-defined, bt drive D is the defalt location. 2 Delete any files and folders yo do not want to share with Illmina. 3 Sht down the system as follows. 51

58 a b c From the men, select Sht Down System. If the shtdown command is nresponsive, press and hold the power btton on the left side of the instrment ntil the lights fade. When the power btton plses, press the power off (Ο) side of the toggle switch on the back of the instrment. 4 If a cartridge is inside the instrment, restart the system and remove the cartridge as follows. a b c d e f g h Press the power on (I) side of the toggle switch on the rear panel. When the power btton plses, press it. When the operating system is loaded, log on to Windows. From the control software men, select System Check. Select Unload to eject the cartridge, and then remove the cartridge from the tray. If ejection fails, contact Illmina Technical Spport for frther instrctions. Select Load to retract the empty tray and close the door. Close the System Check dialog box, and then sht down the system. Shtting down and restarting the system is necessary to position the cartridge for removal. 5 Disconnect the power cord from the wall otlet, and then from the AC power otlet on the rear panel. 6 If applicable, disconnect: The Ethernet cable from the wall otlet, and then from the Ethernet port on the rear panel. The keyboard and mose from the USB ports on the rear panel. Decontaminate the Instrment Shipping an instrment reqires the following decontamination procedre, which Illmina confirms is complete. Site-specific hazards can reqire additional decontamination. Decontaminate With Bleach 1 Pt on a new pair of powder-free gloves. 2 Lower the instrment monitor. 52

59 3 Gently pll the cartridge compartment door from the side edges to open. 4 Clean the entire compartment door with a bleach wipe: Door interior Door exterior Door hinges 5 Close the cartridge compartment door. 6 Locate the drip tray door below the cartridge compartment on the front of the instrment, and then lower the door. 7 Open the drip tray and remove the drip tray pad. 53

60 8 Wipe any residal flid from the bottom of the tray sing paper towels. 9 Dispose of the pad and other consmables in accordance with applicable standards, which vary by region. For more information, see the Safety Data Sheet (SDS) at spport.illmina.com/sds.html. 10 Clean the drip tray with a bleach wipe. 11 Wait 15 mintes for the bleach to take effect. Netralize With Alcohol 1 Moisten a cloth or paper towels with water. Any grade of water is acceptable, inclding tap water. 2 Wipe the following components with the moistened cloth or paper towels: Drip tray Cartridge compartment door (interior and exterior, inclding hinges) The water prevents bleach and alcohol from mixing. 3 Reclean the following components with an alcohol wipe: Drip tray Cartridge compartment door (interior and exterior, inclding hinges) The alcohol removes residal bleach, which can case corrosion. 4 Make sre that the drip tray door and cartridge compartment door are closed. 5 Clean the lab bench arond the instrment with bleach wipes or bleach soltion. Retrn the Original System Package the Instrment 1 Clear adeqate space in the lab for the instrment and packaging. 2 Insert the small foam pad between the lowered monitor and the instrment. 3 Place the gray plastic bag over the instrment. 4 Lower the front flap of the white box. 5 Place the instrment into the white box so that the front of the instrment faces yo. 54

61 6 Place the sqare foam over the instrment so that the thinner sides of the foam rest on the front and back of the instrment. Make sre that the foam is flsh with the top of the box. 7 Close the front flap, and then close the top of the box. Package the Resable Test Components 1 Pt the iseq 100 Resable Test Cartridge into the larger resealable bag and seal. 2 Pt the iseq 100 Resable Test Flow Cell into the clamshell case. 3 Pt the clamshell case into the smaller resealable bag and seal. 4 Place both resealable bags into the iseq 100 Seqencing System Accessories box. 5 Close the accessories box. 55

62 Ship the System 1 If it was removed, place the protective foam base into the bottom of the brown shipping box. 2 Lift the white box by the handles (a two-person lift is recommended), and then lower the white box into the brown box. Any orientation is acceptable. CAUTION The white box mst be shipped inside the brown box. The white box is not designed or labeled for shipping. 3 Place the protective foam cover over the top of the white box. 4 Place the accessories box into the center of the foam cover. 5 Place the black foam pad on top of the accessories box. 6 [Optional] Place the Ethernet cable and power cord anywhere in the brown box. 7 Close the brown box and secre with shipping tape. 8 Place the retrn label over the original shipping label, or remove the original shipping label. 56