MiniSeq. System Guide. For Research Use Only. Not for use in diagnostic procedures. Document # v02 Material # March 2018

MiniSeq System Gide Docment # 1000000002695 v02 Material # 20014309 March 2018 For Research Use Only. Not for se in diagnostic procedres. ILLUMINA PROPRIETARY

MiniSeq System Gide This docment and its contents are proprietary to Illmina, Inc. and its affiliates ("Illmina"), and are intended solely for the contractal se of its cstomer in connection with the se of the prodct(s) described herein and for no other prpose. This docment and its contents shall not be sed or distribted for any other prpose and/or otherwise commnicated, disclosed, or reprodced in any way whatsoever withot the prior written consent of Illmina. Illmina does not convey any license nder its patent, trademark, copyright, or common-law rights nor similar rights of any third parties by this docment. The instrctions in this docment mst be strictly and explicitly followed by qalified and properly trained personnel in order to ensre the proper and safe se of the prodct(s) described herein. All of the contents of this docment mst be flly read and nderstood prior to sing sch prodct(s). FAILURE TO COMPLETELY READ AND EXPLICITLY FOLLOW ALL OF THE INSTRUCTIONS CONTAINED HEREIN MAY RESULT IN DAMAGE TO THE PRODUCT(S), INJURY TO PERSONS, INCLUDING TO USERS OR OTHERS, AND DAMAGE TO OTHER PROPERTY, AND WILL VOID ANY WARRANTY APPLICABLE TO THE PRODUCT(S). ILLUMINA DOES NOT ASSUME ANY LIABILITY ARISING OUT OF THE IMPROPER USE OF THE PRODUCT(S) DESCRIBED HEREIN (INCLUDING PARTS THEREOF OR SOFTWARE). 2018 Illmina, Inc. All rights reserved. All trademarks are the property of Illmina, Inc. or their respective owners. For specific trademark information, see www.illmina.com/company/legal.html. Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. ii

MiniSeq System Gide Revision History Docment Date Description of Change Material # 20014309 Docment # 1000000002695 v02 Material # 20014309 Docment # 1000000002695 v01 Material # 20002370 Docment # 1000000002695 v00 March 2018 September 2016 Janary 2016 Added information abot the Illmina Proactive monitoring service in the Configre Analysis Settings section. Removed the defalt ser name and password reqired to log on to the operating system. Illmina recommends sing site-specific credentials. Minor text edits. Updated software descriptions to MiniSeq Control Software v1.1.8, which incldes Demo Mode. Updated the atomatic post-rn wash dration to 60 mintes. Added a server configration step to the instrctions for selecting BaseSpace for analysis. Noted that mapped drives are not spported by the Local Rn Manager software. Initial release. Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. iii

Table of Contents Chapter 1 Overview 1 Introdction 1 Additional Resorces 1 Instrment Components 2 Seqencing Consmables Overview 5 Preinstalled Databases and Genomes 7 Chapter 2 Getting Started 8 Starting the Instrment 8 Cstomize System Settings 8 Configre Analysis Settings 10 User-Spplied Consmables and Eqipment 13 Chapter 3 Seqencing 16 Introdction 16 Seqencing Workflow 17 Prepare Consmables 19 Prepare Libraries for Seqencing 20 Set Up a Seqencing Rn 20 Monitor Rn Progress 28 Atomatic Post-Rn Wash 30 Remove Used Reservoir from Position #9 30 Chapter 4 Maintenance 32 Introdction 32 Perform a Manal Instrment Wash 32 Software Updates 35 Appendix A Trobleshooting 38 Trobleshooting Files 38 Atomatic Check Errors 39 RTA Errors 40 Rehybridization Workflow 41 System Check 42 Network Configration Settings 45 Cstom Genomes 46 Sht Down the Instrment 46 Appendix B Real-Time Analysis 48 Real-Time Analysis Overview 48 Inpt and Otpt Files 48 Real-Time Analysis Workflow 49 Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. iv

MiniSeq System Gide Appendix C Otpt Files 52 Seqencing Otpt Files 52 Seqencing Otpt Folder Strctre 52 Analysis Inpt File Reqirements 53 Index 54 Technical Assistance 57 Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. v

Chapter 1 Overview Introdction 1 Additional Resorces 1 Instrment Components 2 Seqencing Consmables Overview 5 Preinstalled Databases and Genomes 7 Introdction The Illmina MiniSeq System provides high-qality, indstry-standard Illmina seqencing technology with the convenience of an easy-to-se cost-effective desktop system. Featres High-qality seqencing The MiniSeq System enables small genome, amplicon, targeted enrichment, and RNA seqencing sing low library volmes. MiniSeq system software The MiniSeq System incldes a site of integrated software that controls instrment operations, processes images, and generates base calls. The site incldes on-instrment data analysis software and data transfer tools for analysis sing other options, sch as BaseSpace. On-instrment data analysis The Local Rn Manager software analyzes rn data according to the analysis modle specified for the rn. A site of analysis modles are inclded in the software. BaseSpace integration The seqencing workflow is integrated with BaseSpace, the Illmina genomics compting environment for rn monitoring, data analysis, storage, and collaboration. Otpt files are streamed in real time to BaseSpace or BaseSpace Onsite for analysis. Convenient consmable loading A clamping mechanism positions the flow cell as it is loaded onto the instrment. A single-se prefilled reagent cartridge provides reagents reqired for a rn and sbseqent instrment wash. The flow cell and reagent cartridge inclde integrated identification to enable accrate tracking. Additional Resorces The MiniSeq System spport pages on the Illmina website provide additional resorces. These resorces inclde software, training, compatible prodcts, and the following docmentation. Always check spport pages for the latest versions. Resorce Cstom Protocol Selector MiniSeq System Site Prep Gide (docment # 1000000002696) MiniSeq System Safety and Compliance Gide (docment # 1000000002698) RFID Reader Compliance Gide (docment # 1000000002699) Description A wizard for generating cstomized end-to-end docmentation that is tailored to the library prep method, rn parameters, and analysis method sed for the seqencing rn. Provides specifications for laboratory space, electrical reqirements, and environmental considerations. Provides information abot operational safety considerations, compliance statements, and instrment labeling. Provides information abot the RFID reader in the instrment, compliance certifications, and safety considerations. Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 1

MiniSeq System Gide Resorce MiniSeq System Denatre and Dilte Libraries Gide (docment # 1000000002697) Local Rn Manager Software Gide (docment # 1000000002702) Description Provides instrctions for denatring and dilting prepared libraries for a seqencing rn, and preparing an optional PhiX control. Provides information abot sing the Local Rn Manager software and available analysis options. Instrment Components The MiniSeq System incldes a toch screen monitor, a stats bar, a flow cell compartment, and a reagent compartment. Figre 1 Instrment Components A B C D E F Toch screen monitor Enables on-instrment configration and setp sing the control software interface. Power btton Trns on the integrated instrment compter and operating system. USB ports Convenient connections for peripheral components. Flow cell compartment Holds the flow cell dring a seqencing rn. Stats bar Indicates instrment stats as processing (ble), reqires attention (orange), ready to seqence (green), or when a wash is de within the next 24 hors (yellow). Reagent compartment Holds the reagent cartridge and spent reagents bottle. Flow Cell Compartment The flow cell stage incldes the flow cell latch, which secres the flow cell when it is closed. As the latch closes, pins near the base of the latch align the flow cell ports with flidics connections. Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 2

MiniSeq System Gide Figre 2 Flow Cell Compartment A B C D E Flow cell compartment door Flow cell latch Flow cell stage Flow cell Flow cell latch release btton The thermal station, located beneath the flow cell stage, controls changes in temperatre reqired for clster generation and seqencing. NOTE Do not place objects on the instrment near the flow cell compartment. Reagent Compartment Setting p a seqencing rn on the MiniSeq System reqires access to the reagent compartment to load rn consmables and empty the spent reagents bottle. Figre 3 Reagent Compartment A B C Spent reagents bottle Incldes a threaded cap to prevent spills when carrying. Reagent cartridge Provides reagents in a single-se prefilled consmable. Reagent compartment door Provides access to the reagent compartment. The reagent compartment door opens otward from hinges at the bottom edge of the instrment. To open the door, gently pll forward from the side edges of the door. Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 3

MiniSeq System Gide NOTE Do not place objects on the reagent compartment door. The compartment door is not designed for se as a shelf. Power Btton The power btton on the front of the instrment trns on power to the instrment and instrment compter. The power btton performs the following actions depending on the state of instrment power. Power State Instrment power is off Instrment power is on Instrment power is on Action Briefly press the btton to trn on the power. Briefly press the btton to trn off the power. A dialog box appears on the screen to confirm a normal instrment shtdown. Press and hold the power btton for 10 seconds to case a hard shtdown of the instrment and instrment compter. Use this method to trn off the instrment only if the instrment is nresponsive. NOTE Under normal conditions, do not trn off the instrment power. Trning off the instrment dring a seqencing rn ends the rn immediately. Ending a rn is final. Rn consmables cannot be resed and seqencing data are not saved. System Software The instrment software site incldes integrated applications that perform seqencing rns and oninstrment analysis. MiniSeq Control Software The control software gides yo throgh the steps to set p a seqencing rn, controls instrment operations, and shows an overview of rn statistics as the rn progresses. Real-Time Analysis (RTA) software RTA performs image analysis and base calling dring the rn. See Real-Time Analysis Overview on page 48. Local Rn Manager Before seqencing, se Local Rn Manager to specify rn parameters and the analysis method. After seqencing, on-instrment data analysis begins atomatically. For more information, see the Local Rn Manager Software Gide (docment # 1000000002702). Stats Icons A stats icon in the top-right corner of the control software interface screen indicates a change in condition dring rn setp or dring the rn. Stats Icon Stats Name Stats OK Description System is normal. Processing System is processing. Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 4

MiniSeq System Gide Stats Icon Stats Name Attention Description Attention is reqired. Warning Error A warning has occrred. Warnings do not stop a rn or reqire action before proceeding. An error has occrred. Errors reqire action before proceeding with the rn. When a change in condition occrs, the icon blinks to alert yo. Select the icon to view a description of the condition. Select Acknowledge to accept the message and Close to close the dialog box. Seqencing Consmables Overview Performing a seqencing rn on the MiniSeq System reqires a single-se MiniSeq Kit. Each kit incldes a flow cell and the reagents reqired for a seqencing rn. The flow cell and reagent cartridge se radio-freqency identification (RFID) for accrate consmable tracking and to ensre compatibility with specified rn parameters. Flow Cell The flow cell is a glass-based sbstrate on which clsters are generated and the seqencing reaction is performed. The flow cell is encased in a flow cell cartridge. Figre 4 Flow Cell Components A B C D Imaging area Flow cell gasket Otlet port Inlet port Reagents enter the flow cell throgh the inlet port, pass throgh the single-lane imaging area, and then exit the flow cell throgh the otlet port. The flow cell is shipped dry in a flow cell tbe that is wrapped in a foil package. Store the flow cell in the sealed foil package at 2 C to 8 C ntil se. For more information, see Prepare the Flow Cell on page 19. Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 5

MiniSeq System Gide Reagent Cartridge Overview The reagent cartridge is a single-se consmable with foil-sealed reservoirs that are prefilled with clstering, seqencing, and wash reagents. Figre 5 Reagent Cartridge The reagent cartridge incldes a designated reservoir for loading prepared libraries. After the rn begins, libraries are transferred atomatically from the reagent cartridge to the flow cell. 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. Store the reagent cartridge at -25 C to -15 C ntil se. For more information, see Prepare the Reagent Cartridge on page 19. Reserved Reservoirs Figre 6 Nmbered Reservoirs Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 6

MiniSeq System Gide Position Description 13, 14, and 15 Reserved for optional cstom primers 16 Load libraries Removable Reservoir in Position #9 The prefilled reagent cartridge incldes a denatration reagent in position #9 that contains formamide. To facilitate safe disposal of any nsed reagent after the seqencing rn, this reservoir is removable. For more information, see Remove Used Reservoir from Position #9 on page 30. Preinstalled Databases and Genomes For most analysis methods, a reference is reqired to perform alignment. Several reference databases and genomes are preinstalled on the instrment compter. Preinstalled Databases Genomes Description mirbase for hman dbsnp for hman RefGene for hman Arabidopsis thaliana cow (Bos tars) E. coli strain DH10B E. coli strain MG1655 frit fly (Drosophila melanogaster) hman (Homo sapiens) bild hg19 mose (Ms mscls) PhiX rat (Ratts norvegics) Rhodobacter sphaeroides 2.4.1 yeast (Saccharomyces cerevisiae S288C) Staphylococcs ares NCTC 8325 Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 7

Chapter 2 Getting Started Starting the Instrment 8 Cstomize System Settings 8 Configre Analysis Settings 10 User-Spplied Consmables and Eqipment 13 Starting the Instrment Make sre that the instrment has been properly installed and initialized, and that instrment setp is complete. Starting the instrment before it is ready can damage the system. 1 Trn on the power toggle switch to the I (on) position. Figre 7 Power Switch Located on Back of Instrment 2 Press the power btton above the reagent compartment. The power btton trns on the integrated instrment compter and operating system. Figre 8 Power Btton Located on Front of Instrment 3 Wait ntil the operating system has finished loading. The MiniSeq Control Software lanches and initializes the system atomatically. The Home screen opens after the initialization step is complete. 4 Wait for the system to load, and then log on to the operating system. If necessary, conslt yor facility administrator for the ser name and password. Cstomize System Settings The control software incldes cstomizable settings for instrment identification and the following workflow preferences: Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 8

MiniSeq System Gide Prging consmables at end of rn. Skipping pre-rn check confirmation to start the rn atomatically. Using the on-screen keyboard for rn setp steps. Enabling adio indicators. Performing a simlated rn. Cstomize Instrment Identification 1 From the Manage Instrment screen, select System Cstomization. 2 To assign an avatar image for the instrment, select Browse and navigate to a preferred image. 3 In the Nick Name field, enter a preferred name for the instrment. 4 Select Save to save settings and advance the screen. The image and name appear in the pper-left corner of each screen. Set Atomatic Prge Option 1 From the Manage Instrment screen, select System Cstomization. 2 Select the Prge consmables at end of rn checkbox. This setting prges nsed reagents from the reagent cartridge to the spent reagents bottle atomatically after each rn. If this setting is disabled, nsed reagents remain in the reagent cartridge. NOTE Prging consmables atomatically adds additional time to the workflow. For example, prging reagents after a 300-cycle rn (2 151), takes approximately 50 mintes. 3 Select Save to save settings and exit the screen. Set Atomatic Start Option 1 From the Manage Instrment screen, select System Cstomization. 2 Select the Skip pre-rn check confirmation checkbox. This setting starts the seqencing rn atomatically after a sccessfl atomatic check. If this setting is disabled, start the rn manally after the pre-rn check. 3 Select Save. Set Atomatic Check for Software Updates 1 From the Manage Instrment screen, select System Cstomization. 2 Select the Atomatically check for software pdates on BaseSpace checkbox. An internet connection is reqired. 3 Select Save to save settings and exit the screen. Set On-Screen Keyboard Option 1 From the Manage Instrment screen, select System Cstomization. 2 Select the Use on-screen keyboard checkbox. This setting enables the on-screen keyboard for inpt dring rn setp steps. Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 9

MiniSeq System Gide 3 Select Save. Enable Adio Indicators 1 From the Manage Instrment screen, select System Cstomization. 2 Select the Play adio checkbox to trn on adio indicators for the following events. Upon instrment initialization When a rn is started When errors occr When ser interaction is reqired When a rn has finished 3 Select Save. Enable Demo Mode Demo Mode simlates a seqencing rn to provide a demonstration of instrment featres and fnction. Use Demo Mode to mimic rn setp withot reagents, a flow cell, or other consmables. 1 From the Manage Instrment screen, select System Cstomization. 2 Select the Enable Demo Mode checkbox. 3 Select Save. 4 [Optional] Enter a 4-digit PIN. When this field is blank, exiting Demo Mode does not reqire a PIN. 5 Select OK to restart the software in Demo Mode. After restart, the MiniSeq Control Software banner reads Demonstration Only. Exit Demo Mode 1 From the Manage Instrment screen, select System Cstomization. 2 Clear the Enable Demo Mode checkbox. 3 Select Save. 4 If a PIN is reqired, enter it when prompted. 5 Select OK to restart the software in the standard mode. Configre Analysis Settings The control software provides the following options for data transfer and sbseqent analysis: BaseSpace Sends data to Illmina BaseSpace for analysis on BaseSpace. BaseSpace Onsite Sends data to the BaseSpace Onsite LT server for analysis on the server. Local Rn Manager Sends data to a specified otpt folder location for analysis sing a Local Rn Manager modle. Standalone Saves data to a specified otpt folder location for later analysis. Select BaseSpace 1 From the Manage Instrment screen, select System Configration. Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 10

MiniSeq System Gide 2 Select Analysis Configration. 3 Select BaseSpace. 4 [Optional] Specify defalt BaseSpace login credentials as follows. a b c Enter a User name and Password. Select the Save the credentials as the defalt checkbox. Select the Bypass Analysis Method login screen checkbox. 5 In the Server Name field, enter the fll path to yor BaseSpace server. For the EU server, replace the US server name with https://api.ec1.sh.basespace.illmina.com. The defalt US server is https://api.basespace.illmina.com. 6 [Optional] Select the Otpt Folder checkbox, select Browse, and navigate to a preferred otpt folder location. This setting saves a copy of base call (BCL) files in the specified location. 7 [Optional] Select Send instrment health information to Illmina to enable the Illmina Proactive monitoring service. The name of the setting in the software interface might be different from the name in this gide, depending on the version of MiniSeq Control Software in se. With this setting trned on, instrment performance data are sent to Illmina. This data helps 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 Illmina Proactive Technical Note (docment # 1000000052503). This service: Does not send seqencing data. Reqires that the instrment be connected to a network with internet access. Is trned on by defalt. To opt ot of this service, disable the Send instrment health information to Illmina setting. 8 Select Save. Select BaseSpace Onsite 1 From the Manage Instrment screen, select System Configration. 2 Select Analysis Configration. 3 Select BaseSpace Onsite. 4 [Optional] Specify defalt BaseSpace Onsite login credentials as follows. a b c Enter a User name and Password. Select the Save the credentials as the defalt checkbox. Select the Bypass Analysis Method login screen checkbox. 5 In the Server Name field, enter the fll path to yor BaseSpace Onsite server. 6 [Optional] Select the Otpt Folder checkbox, select Browse, and navigate to a preferred otpt folder location. This setting saves a copy of base call (BCL) files in the specified location. 7 [Optional] Select Send instrment health information to Illmina to enable the Illmina Proactive monitoring service. The name of the setting in the software interface might be different from the name in this gide, depending on the version of MiniSeq Control Software in se. Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 11

MiniSeq System Gide With this setting trned on, instrment performance data are sent to Illmina. This data helps 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 Illmina Proactive Technical Note (docment # 1000000052503). This service: Does not send seqencing data. Reqires that the instrment be connected to a network with internet access. Is trned on by defalt. To opt ot of this service, disable the Send instrment health information to Illmina setting. 8 Select Save. Select Local Rn Manager 1 From the Manage Instrment screen, select System Configration. 2 Select Analysis Configration. 3 Select Local Rn Manager. 4 [Optional] Specify defalt Local Rn Manager login credentials as follows. a b c Enter a User name and Password. Select the Save credentials as the defalt checkbox. Select the Bypass Analysis Method login screen checkbox. 5 [Optional] Select Use Rn Monitoring to monitor the contents sing visalization tools on BaseSpace. A BaseSpace login and internet connection are reqired. 6 [Optional] Specify defalt BaseSpace login credentials as follows. a b Enter a User name and Password. Select the Save credentials as the defalt checkbox. 7 Select Browse and navigate to a preferred otpt folder location. 8 In the Contents pane of the otpt folder location, create a folder named Rns. NOTE Local Rn Manager does not spport mapped drives. 9 [Optional] Select Send instrment health information to Illmina to enable the Illmina Proactive monitoring service. The name of the setting in the software interface might be different from the name in this gide, depending on the version of MiniSeq Control Software in se. With this setting trned on, instrment performance data are sent to Illmina. This data helps 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 Illmina Proactive Technical Note (docment # 1000000052503). This service: Does not send seqencing data. Reqires that the instrment be connected to a network with internet access. Is trned on by defalt. To opt ot of this service, disable the Send instrment health information to Illmina setting. 10 Select Save. Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 12

MiniSeq System Gide Select Standalone 1 From the Manage Instrment screen, select System Configration. 2 Select Analysis Configration. 3 Select Standalone. 4 [Optional] Select Use Rn Monitoring to monitor the rn sing visalization tools on BaseSpace. A BaseSpace login and internet connection are reqired. 5 [Optional] Specify defalt BaseSpace login credentials as follows. a b Enter a User name and Password. Select the Save credentials as the defalt checkbox. 6 Select Browse and navigate to a preferred otpt folder location. 7 [Optional] Select Send instrment health information to Illmina to enable the Illmina Proactive monitoring service. The name of the setting in the software interface might be different from the name in this gide, depending on the version of MiniSeq Control Software in se. With this setting trned on, instrment performance data are sent to Illmina. This data helps 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 Illmina Proactive Technical Note (docment # 1000000052503). This service: Does not send seqencing data. Reqires that the instrment be connected to a network with internet access. Is trned on by defalt. To opt ot of this service, disable the Send instrment health information to Illmina setting. 8 Select Save. User-Spplied Consmables and Eqipment The following consmables and eqipment are sed for seqencing and system maintenance. User-Spplied Consmables for Seqencing Rns Consmable Spplier Prpose 1 N NaOH (sodim hydroxide) General lab spplier Library denatration, dilted to 0.1 N 200 mm Tris-HCl, ph7 General lab spplier Library netralization after denatration Isopropyl alcohol wipes, 70% or Ethanol, 70% VWR, catalog # 95041-714 (or eqivalent) General lab spplier Disposable gloves, powder-free General lab spplier General prpose Lab tisse, low-lint VWR, catalog # 21905-026 (or eqivalent) Flow cell cleaning and general prpose Flow cell cleaning Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 13

MiniSeq System Gide User-Spplied Consmables for Instrment Maintenance Consmable Spplier Prpose NaOCl, 5% (sodim hypochlorite) Sigma-Aldrich, catalog # 239305 (or laboratory-grade eqivalent) Performing a manal post-rn wash; dilted to 0.12% Tween 20 Sigma-Aldrich, catalog # P7949 Performing a manal instrment wash; dilted to 0.05% Water, laboratory-grade General lab spplier Performing a manal instrment wash Gidelines for Laboratory-Grade Water Always se laboratory-grade water or deionized water to perform instrment procedres. Never se tap water. Use only the following grades of water or eqivalents: Deionized water Illmina PW1 18 Megohms (MΩ) water Milli-Q water Sper-Q water Moleclar biology grade water User-Spplied Eqipment Item Freezer, -25 C to -15 C, frost-free Ice bcket Refrigerator, 2 C to 8 C Sorce General lab spplier General lab spplier General lab spplier Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 14

MiniSeq System Gide Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 15

Chapter 3 Seqencing Introdction 16 Seqencing Workflow 17 Prepare Consmables 19 Prepare Libraries for Seqencing 20 Set Up a Seqencing Rn 20 Monitor Rn Progress 28 Atomatic Post-Rn Wash 30 Remove Used Reservoir from Position #9 30 Introdction To perform a seqencing rn on the MiniSeq System, prepare rn consmables and then follow the software prompts to set p the seqencing rn. Workflow Overview Clster Generation Dring clster generation, single DNA molecles are bond to the srface of the flow cell, and then amplified to form clsters. Seqencing Clsters are imaged sing 2-channel seqencing chemistry and filter combinations specific to each of the florescently labeled chain terminators. After imaging of a tile on the flow cell is complete, the next tile is imaged. The process is repeated for each cycle of seqencing. Following image analysis, the software performs base calling, filtering, and qality scoring. Analysis As the rn progresses, the control software atomatically transfers base call (BCL) files to the specified otpt location for data analysis. Several analysis methods are available depending on yor application and the selected analysis configration for yor system. For more information, see Configre Analysis Settings on page 10. Seqencing Rn Dration Seqencing rn dration depends on the nmber of cycles performed. The maximm rn length is a 150- cycle paired-end rn, pls p to 2 index reads of 8 cycles each. For expected drations and other system specifications, visit the MiniSeq System specifications page on the Illmina website. Nmber of Cycles in a Read In a seqencing rn, the nmber of cycles performed in a read is 1 more cycle than the nmber of cycles analyzed. For example, to perform a 150-cycle paired-end rn, set p the rn to perform 151 cycles per read (2 151) for a total of 302 cycles. At the end of the rn, 2 150 cycles are analyzed. The extra cycle in each read is sed for phasing and prephasing calclations. Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 16

MiniSeq System Gide Seqencing Workflow The rn setp steps vary depending on the analysis configration specified for the rn. Standalone After preparing and loading consmables, all rn parameters are specified sing the MiniSeq Control Software interface. BaseSpace or BaseSpace Onsite, or Local Rn Manager Before setting p the rn on the instrment, assign a rn name and specify rn parameters sing the BaseSpace Prep tab or the Local Rn Manager software. After preparing consmables, select the rn name from the MiniSeq Control Software, and then load consmables for the rn. Workflow (Standalone Configration) Prepare a new reagent cartridge: thaw and inspect. Prepare a new flow cell: bring to room temperatre and then nwrap. Denatre and dilte libraries. See MiniSeq System Denatre and Dilte Libraries Gide (docment # 1000000002697). Load the library diltion onto the reagent cartridge in reservoir #16. From the software interface, select Seqence. Load the flow cell and reagent cartridge. Empty and reload the spent reagents bottle. Specify rn parameters on the Rn Setp screen. After the atomated check, select Start. Monitor the rn on the instrment screen, from BaseSpace if rn monitoring is enabled, or from a networked compter sing Seqencing Analysis Viewer. Data are transferred to the specified otpt folder. An instrment wash begins atomatically when seqencing is complete. Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 17

MiniSeq System Gide Workflow (BaseSpace or Local Rn Manager Configration) Specify rn parameters sing BaseSpace, BaseSpace Onsite, or Local Rn Manager. Prepare a new reagent cartridge: thaw and inspect. Prepare a new flow cell: bring to room temperatre, nwrap, and inspect. Denatre and dilte libraries. See MiniSeq System Denatre and Dilte Libraries Gide (docment # 1000000002697). Load the library diltion onto the reagent cartridge in reservoir #16. From the software interface, select Seqence to begin the rn setp steps. Select the rn name specified on BaseSpace, BaseSpace Onsite, or Local Rn Manager. Load the flow cell. Load the reagent cartridge. Empty and reload the spent reagents bottle. Review rn parameters on the Rn Setp screen. After the atomated check, select Start. Monitor the rn on the instrment screen, from BaseSpace or from a networked compter sing Seqencing Analysis Viewer. Data are transferred to BaseSpace, BaseSpace Onsite, or the specified otpt folder. An instrment wash begins atomatically when seqencing is complete. Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 18

MiniSeq System Gide Prepare Consmables Prepare the Reagent Cartridge 1 Remove the reagent cartridge from -25 C to -15 C storage. 2 Thaw reagents sing the following water bath options. Do not sbmerge the cartridge. When thawed, dry the base before proceeding. Method Time to Thaw Stability Limit 37 C water bath 35 mintes Up to 2 hors Room temperatre water bath (19 C to 25 C) 90 mintes Up to 24 hors If mltiple cartridges are thawing in the same water bath, allow additional thawing time. Alternatively, thaw reagents sing the following options. Method Time to Thaw Stability Limit Room temperatre air (19 C to 25 C) 5 hors Up to 24 hors Chilled at 2 C to 8 C 18 hors Up to 72 hors 3 Invert the cartridge 5 times to mix reagents. 4 Inspect the large reservoirs from the bottom of the cartridge to make sre that reagents have thawed and the reservoirs are free of ice crystals. 5 Gently tap on the bench to redce air bbbles. Prepare the Flow Cell 1 Remove a new flow cell package from 2 C to 8 C storage. 2 Set the nopened flow cell package aside at room temperatre for 30 mintes. NOTE Avoid repeated cooling and warming of the flow cell. 3 Remove the flow cell container from the foil package. 4 Pt on a new pair of powder-free gloves. 5 Grip the flow cell by the plastic cartridge and remove it from the container. Figre 9 Remove Flow Cell 6 Clean the glass srface of the flow cell with a lint-free alcohol wipe. Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 19

MiniSeq System Gide 7 Dry with a lint-free lens cleaning tisse. Use care arond the black flow cell gasket. 8 Inspect the flow cell ports for obstrctions. Make sre that the gasket is well-seated. Prepare Libraries for Seqencing Denatre and Dilte Libraries Before loading libraries onto the reagent cartridge, denatre and dilte libraries and add an optional PhiX control. For more information, see MiniSeq System Denatre and Dilte Libraries Gide (docment # 1000000002697). The loading volme for the MiniSeq System is 500 µl at a loading concentration of 1.8 pm. However, loading concentration can vary depending on library preparation and qantification methods. Load Libraries onto the Reagent Cartridge 1 Clean the foil seal covering reservoir #16 labeled Load library here sing a low-lint tisse. 2 Pierce the seal with a clean 1 ml pipette tip. 3 Add 500 µl prepared 1.8 pm libraries into reservoir #16. Avoid toching the foil seal as yo dispense the libraries. Figre 10 Load Libraries Set Up a Seqencing Rn Rn setp steps differ based on system configration: Standalone configration Yo are prompted to define rn parameters on the control software Rn Setp screen. BaseSpace or Local Rn Manager configration Yo are prompted to log in and select from a list of rns predefined in BaseSpace or Local Rn Manager. Set Up a Rn (Standalone Configration) 1 From the Home screen, select Seqence. The Seqence command releases consmables from a previos rn and opens the series of rn setp screens. Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 20

MiniSeq System Gide Load the Flow Cell 1 Open the flow cell compartment door. 2 Press the release btton to the right of the flow cell latch. Figre 11 Open Flow Cell Latch 3 If present, remove the sed flow cell from a previos rn. 4 Make sre that the flow cell stage is clean. If debris is present, clean the flow cell stage with an alcohol wipe. 5 Place the flow cell on the flow cell stage over the alignment pins. Figre 12 Place Flow Cell on Stage 6 Close the flow cell latch to secre the flow cell. Figre 13 Close Flow Cell Latch 7 Close the flow cell compartment door. Load the Reagent Cartridge 1 Open the reagent compartment door. Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 21

MiniSeq System Gide 2 If present, remove the sed reagent cartridge. NOTE To facilitate safe disposal of nsed reagent containing formamide, the reservoir in position #9 is removable. See Remove Used Reservoir from Position #9 on page 30. 3 Slide the reagent cartridge into the reagent compartment ntil the cartridge stops. Figre 14 Load Reagent Cartridge Empty the Spent Reagents Bottle 1 Remove the spent reagents bottle from the compartment. Figre 15 Remove the Spent Reagents Bottle 2 To prevent spills when carrying the spent reagents bottle, seal the bottle opening with the threaded cap. Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 22

MiniSeq System Gide Figre 16 Seal the Spent Reagents Bottle 3 Discard the contents in accordance with applicable standards. 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. 4 With the threaded cap removed, slide the empty spent reagents bottle into the compartment ntil it stops. 5 Close the compartment door and select Next. Enter Rn Parameters 1 Enter a rn name of yor preference. 2 [Optional] Enter a library ID of yor preference. 3 From the Recipe drop-down list, select a recipe. Only compatible recipes are listed. 4 Select a read type, either Single read or Paired end. 5 Enter the nmber of cycles for each read in the seqencing rn. Read 1 Enter a vale p to 151 cycles. Index 1 Enter the nmber of cycles reqired for the Index 1 (i7) primer. Index 2 Enter the nmber of cycles reqired for the Index 2 (i5) primer. Read 2 Enter a vale p to 151 cycles. This vale is typically the same nmber of cycles as Read 1. The control software confirms the nmber of specified cycles sing the following criteria: Total cycles do not exceed the maximm cycles allowed based on the reagent cartridge loaded for the rn. Cycles for Read 1 are greater than the 5 cycles reqired for template generation. Index Read cycles do not exceed Read 1 and Read 2 cycles. NOTE Make sre that yo specify the appropriate nmber of Index Read cycles for the libraries that yo are seqencing. For more information, see the library prep docmentation. Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 23

MiniSeq System Gide 6 [Optional] If yo are sing cstom primers, select the checkbox for the primers sed. Read 1 Cstom primer for Read 1. Index 1 Cstom primer for Index 1. Index 2 Cstom primer for Index 2. Read 2 Cstom primer for Read 2. 7 [Optional] Select Advanced Settings to change settings for the crrent rn. Prge consmables for this rn Change the setting to prge consmables atomatically after the crrent rn. Use rn monitoring for this rn Change the setting to se rn monitoring in BaseSpace. When enabled, a BaseSpace login and internet connection are reqired. Recipe Select a cstom recipe for this rn. Otpt folder Change the otpt folder location for the crrent rn. Select Browse to navigate to a folder location. 8 Select Next. NOTE Do not open the reagent compartment door or the flow cell compartment door dring the atomated check or dring the seqencing rn. Review Atomated Check 1 Review the atomated check reslts. To stop a check in progress, select Cancel. For any items that do not pass, an action is reqired before yo can proceed. See Atomatic Check Errors on page 39. To restart the check, select Retry. The check resmes at the first incomplete or failed check. 2 To start the rn, select from the following options. If the system is not configred to start atomatically after a sccessfl check, select Start. If the system is configred to start atomatically after a sccessfl check, the seqencing rn begins atomatically. Yo do not have to be present. However, if any errors occr dring the check, the rn does not begin atomatically. Set Up a Rn (BaseSpace or Local Rn Manager Configration) 1 From the Home screen, select Seqence. The Seqence command releases consmables from a previos rn and opens the series of rn setp screens. Log in to BaseSpace or Local Rn Manager 1 Enter yor ser name and password. 2 Select Next. Select Available Rn 1 Select a rn name from the list of available rns. Use the p and down arrows to scroll throgh the list or enter a rn name in the Search field. 2 Select Next. Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 24

MiniSeq System Gide Load the Flow Cell 1 Open the flow cell compartment door. 2 Press the release btton to the right of the flow cell latch. Figre 17 Open Flow Cell Latch 3 If present, remove the sed flow cell from a previos rn. 4 Make sre that the flow cell stage is clean. If debris is present, clean the flow cell stage with an alcohol wipe. 5 Place the flow cell on the flow cell stage over the alignment pins. Figre 18 Place Flow Cell on Stage 6 Close the flow cell latch to secre the flow cell. Figre 19 Close Flow Cell Latch 7 Close the flow cell compartment door. Load the Reagent Cartridge 1 Open the reagent compartment door. Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 25

MiniSeq System Gide 2 If present, remove the sed reagent cartridge. NOTE To facilitate safe disposal of nsed reagent containing formamide, the reservoir in position #9 is removable. See Remove Used Reservoir from Position #9 on page 30. 3 Slide the reagent cartridge into the reagent compartment ntil the cartridge stops. Figre 20 Load Reagent Cartridge Empty the Spent Reagents Bottle 1 Remove the spent reagents bottle from the compartment. Figre 21 Remove the Spent Reagents Bottle 2 To prevent spills when carrying the spent reagents bottle, seal the bottle opening with the threaded cap. Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 26

MiniSeq System Gide Figre 22 Seal the Spent Reagents Bottle 3 Discard the contents in accordance with applicable standards. 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. 4 With the threaded cap removed, slide the empty spent reagents bottle into the compartment ntil it stops. 5 Close the compartment door and select Next. Confirm Rn Parameters 1 Confirm rn parameters. The control software confirms the nmber of specified cycles sing the following criteria: Total cycles do not exceed the maximm cycles allowed based on the reagent cartridge loaded for the rn. Cycles for Read 1 are greater than the 5 cycles reqired for template generation. Index Read cycles do not exceed Read 1 and Read 2 cycles. NOTE Make sre that yo specify the appropriate nmber of Index Read cycles for the libraries that yo are seqencing. For more information, see the library prep docmentation. 2 [Optional] Select Edit to change rn parameters. When finished, select Save. Prge consmables for this rn Change the setting to prge consmables atomatically after the crrent rn. Rn parameters Change the read type or nmber of cycles per read. Cstom primers Change the settings for cstom primers. 3 Select Next. Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 27

MiniSeq System Gide NOTE Do not open the reagent compartment door or the flow cell compartment door dring the atomated check or dring the seqencing rn. Review Atomated Check 1 Review the atomated check reslts. To stop a check in progress, select Cancel. For any items that do not pass, an action is reqired before yo can proceed. See Atomatic Check Errors on page 39. To restart the check, select Retry. The check resmes at the first incomplete or failed check. 2 To start the rn, select from the following options. If the system is not configred to start atomatically after a sccessfl check, select Start. If the system is configred to start atomatically after a sccessfl check, the seqencing rn begins atomatically. Yo do not have to be present. However, if any errors occr dring the check, the rn does not begin atomatically. Monitor Rn Progress 1 Monitor rn progress, intensities, and qality scores as metrics appear on the screen. Figre 23 Seqencing Rn Progress and Metrics A Rn progress Shows the crrent step and nmber of cycles completed for each read. The progress bar is not proportional to the rn rate of each step. B Q-Score Shows the distribtion of qality scores (Q-scores). See Qality Scoring on page 51. C Intensity Shows the vale of clster intensities of the 90 th percentile for each tile. Plot colors indicate each base: red is A, green is C, ble is G, and black is T. D Clster density (K/mm²) Shows the nmber of clsters detected for the rn. E Clsters passing filter (%) Shows the percentage of clsters passing filter. See Clsters Passing Filter on page 50. F Estimated yield (Gb) Shows the nmber of bases projected for the rn. G Data transfer stats Shows the stats of data transfer based on analysis configration. H Time to completion Shows the rn completion date and time (yyyy-mm-dd hh:mm). Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 28

MiniSeq System Gide NOTE After yo select Home, it is not possible to retrn to view rn metrics. However, rn metrics are accessible on BaseSpace, from a networked compter sing Seqencing Analysis Viewer, or from a networked compter sing Local Rn Manager. Cycles for Rn Metrics Rn metrics appear at different points in a rn. Dring the clster generation steps, no metrics appear. The first 5 cycles are reserved for template generation. At cycle 6, raw clster density and cycle 1 intensities are available. After cycle 25, clsters passing filter, yield, and qality scores are available. Seqencing Analysis Viewer The Seqencing Analysis Viewer software shows seqencing metrics generated dring the rn. Metrics appear in the form of plots, graphs, and tables based on data generated by RTA and written to InterOp files. Metrics are pdated as the rn progresses. Select Refresh at any time dring the rn to view pdated metrics. For more information, see the Seqencing Analysis Viewer User Gide (part # 15020619). The Seqencing Analysis Viewer is inclded in the software installed on the instrment compter. Yo can also install Seqencing Analysis Viewer on another compter linked to the same network as the instrment to monitor rn metrics remotely. Data Transfer Stats Depending on the analysis configration selected, an icon appears on the screen dring the rn to indicate connection stats. Stats BaseSpace BaseSpace Onsite Local Rn Manager Standalone Connected Transferring data Disconnected Disabled It is possible that mltiple icons appear on the screen. For example, if rn data are being transferred to BaseSpace and an additional otpt folder location, a BaseSpace icon and a standalone icon appear. Rn Copy Service The MiniSeq System Software Site incldes a Rn Copy Service. As RTA generates files, the service copies files to the specified otpt folder location. Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 29

MiniSeq System Gide If data transfer is interrpted dring the rn, data are stored temporarily on the instrment compter. When the connection is restored, data transfer resmes atomatically dring the rn. If the connection is not restored before the rn ends, manally move data to the preferred location. Transfer to BaseSpace The BaseSpace Broker transfers rn data to BaseSpace. If the connection to BaseSpace is interrpted, the broker attempts to pload data for 7 days. If the connection is not restored after 7 days, rn data are deleted. If yo specify an additional location for rn data, data are transferred to that location regardless of broker stats. Atomatic Post-Rn Wash When the seqencing rn is complete, the software initiates an atomatic post-rn wash sing the wash soltion and NaOCl provided in the reagent cartridge. The atomatic post-rn wash takes approximately 60 mintes. When the wash is complete, the Home btton becomes active. Seqencing reslts remain visible on the screen dring the wash. After the Wash After the wash, the sippers remain in the down position to prevent air from entering the system. Leave the cartridge in place ntil the next rn. Remove Used Reservoir from Position #9 The reservoir in position #9 of the reagent cartridge contains formamide. Before discarding the sed reagent cartridge, yo can remove the reservoir from position #9 to discard it separately. 1 Wearing gloves, press downwards on the white breakaway tab at position #9 to break the 3 connection points. Figre 24 Breakaway Tab at Position #9 A B Breakaway tab shown intact Reservoir clip 2 Slide the breakaway tab sideways towards the left edge of the cartridge, so that the breakaway tab slides nder the cartridge cover. Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 30

MiniSeq System Gide Figre 25 Breakaway Tab Removed, Reservoir Clip Exposed A B Breakaway tab shown nder cartridge cover Reservoir clip 3 Press the clear plastic reservoir clip down and towards the right. The reservoir releases from position nder the reagent cartridge. 4 Dispose of the reservoir in accordance with applicable standards. 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. Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 31

Chapter 4 Maintenance Introdction 32 Perform a Manal Instrment Wash 32 Software Updates 35 Introdction Maintenance procedres inclde manal instrment washes and system software pdates when available. No other periodic maintenance is reqired. Instrment washes An atomatic post-rn wash after each seqencing rn maintains instrment performance. However, a manal instrment wash is reqired nder certain conditions. See Perform a Manal Instrment Wash on page 32. Software pdates When a new version of the system software site is available, yo can pdate the software atomatically throgh a connection to BaseSpace or manally after downloading the installer from the Illmina website. See Software Updates on page 35. Preventive Maintenance Illmina recommends that yo schedle a preventive maintenance service each year. If yo are not nder a service contract, contact yor Territory Accont Manager or Illmina Technical Spport to arrange for a billable preventive maintenance service. Perform a Manal Instrment Wash Manal instrment wash options inclde the Qick Wash and the Manal Post-Rn Wash. Wash Types Qick Wash Dration: 20 mintes Manal Post-Rn Wash Dration: 90 mintes Description A qick wash is reqired every 7 days that the instrment is idle or after an instrment shtdown. The wash flshes the system with a ser-spplied wash soltion of laboratory-grade water and Tween 20. A manal post-rn wash is reqired when the atomatic post-rn wash is not performed, sch as when a rn is ended early and the flow cell is saved for later rehybridization. The wash flshes the system with ser-spplied 0.12% sodim hypochlorite and a wash soltion of laboratory-grade water and Tween 20. NOTE Always se a fresh diltion of NaOCl prepared within the last 24 hors. If yo make a volme larger than 1 ml, store the remaining diltion at 2 C to 8 C for se within the next 24 hors. Otherwise, discard the remaining diltion of NaOCl. A manal instrment wash reqires the wash cartridge and a wash flow cell provided with the instrment. Alternatively, yo can se a sed flow cell for the wash. Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 32

MiniSeq System Gide Figre 26 Wash Cartridge Prepare for a Manal Post-Rn Wash 1 Combine the following volmes to reslt in 0.12% NaOCl. 5% NaOCl (31 µl) Laboratory-grade water (1269 µl) 2 Add 1.3 ml of 0.12% NaOCl to the wash cartridge. The correct reservoir is eqivalent to position #31 on the prefilled reagent cartridge. Figre 27 Positions for NaOCl and Wash Soltion A B 0.12% NaOCl Wash soltion 3 Combine the following volmes to reslt in a 0.05% Tween 20 wash soltion. 100% Tween 20 (40 µl) Laboratory-grade water (80 ml) 4 Add 80 ml wash soltion to the wash cartridge. The correct reservoir is eqivalent to position #40 on the prefilled reagent cartridge. 5 From the Home screen, select Perform Wash, and then select Manal post-rn wash. Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 33

MiniSeq System Gide Prepare for a Qick Wash 1 Combine the following volmes to reslt in a 0.05% Tween 20 wash soltion. 100% Tween 20 (20 µl) Laboratory-grade water (40 ml) 2 Add 40 ml wash soltion to the wash cartridge. The correct reservoir is eqivalent to position #40 on the prefilled reagent cartridge. Figre 28 Position for Wash Soltion A Wash soltion 3 From the Home screen, select Perform wash, and then select Qick Wash. Load the Wash Flow Cell and Wash Cartridge 1 Load the wash flow cell. Close the flow cell clamp and flow cell door. NOTE Alternatively, yo can load a sed flow cell. 2 Remove the sed reagent cartridge from the previos rn, if present. 3 Load the prepared wash cartridge. 4 Remove the spent reagents bottle and discard the contents in accordance with applicable standards. 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. 5 Slide the empty spent reagents bottle into the compartment ntil it stops. 6 Close the reagent compartment door. Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 34

MiniSeq System Gide 7 Select Next. Start the Wash 1 After the check is complete, select Start. 2 When the wash is complete, select Home. After the Wash After the wash, the sippers remain in the down position to prevent air from entering the system. Leave the cartridge in place ntil the next rn. Software Updates Software pdates are packaged in a software bndle called the System Site, which incldes the following software: MiniSeq Control Software MiniSeq recipes RTA2 Local Rn Manager MiniSeq Service Software Seqencing Analysis Viewer BaseSpace Broker Software release notes are available on the MiniSeq System spport page on the Illmina website. Yo can install software pdates atomatically sing an internet connection or manally from a network or USB location. Atomatic pdates For instrments connected to a network with internet access, an alert icon appears on the Manage Instrment btton on the Home screen when an pdate is available. Manal pdates Download the System Site installer from the MiniSeq System spport page on the Illmina website. NOTE Cancelling an pdate before installation is complete stops the pdate at the crrent point in the installation. Any changes made p to the point of cancellation are not ninstalled or reverted to the previos version. Atomatic Software Update 1 Select Manage Instrment. 2 Select Software Update. 3 Select Install the pdate already downloaded from BaseSpace. 4 Select Update to begin the pdate. A dialog box opens to confirm the command. 5 Follow the prompts in the installation wizard: a b Accept the licensing agreement. Review the list of software inclded in the pdate. Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 35

MiniSeq System Gide When the pdate is complete, the control software restarts atomatically. NOTE If a firmware pdate is inclded, an atomatic restart of the system is reqired after the firmware is pdated. Manal Software Update 1 Download the System Site installer from the Illmina website and save it to a network location. Alternatively, copy the software installation file to a portable USB drive. 2 Select Manage Instrment. 3 Select Software Update. 4 Select Manally install the pdate from the following location. 5 Select Browse to navigate to the location of the software installation file, and then select Update. 6 Follow the prompts in the installation wizard: a b Accept the licensing agreement. Review the list of software inclded in the pdate. When the pdate is complete, the control software restarts atomatically. NOTE If a firmware pdate is inclded, an atomatic restart of the system is reqired after the firmware is pdated. Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 36

MiniSeq System Gide Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 37

Appendix A Trobleshooting Trobleshooting Files 38 Atomatic Check Errors 39 RTA Errors 40 Rehybridization Workflow 41 System Check 42 Network Configration Settings 45 Cstom Genomes 46 Sht Down the Instrment 46 Trobleshooting Files Key File Folder Description Rn information file (RnInfo.xml) Rn parameters file (RnParameters.xml) RTA configration file (RTAConfigration.xml) Root folder Root folder Data\Intensities Contains the following information: Rn name Nmber of cycles in the rn Nmber of cycles in each read Whether the read is an indexed read Nmber of swaths and tiles on the flow cell Contains information abot rn parameters and rn components. Information incldes the RFID, serial nmber, lot nmber, and expiration date. Contains the RTA configration settings for the rn. The RTAConfigration.xml file is created at the beginning of the rn. InterOp files (*.bin) InterOp Binary reporting files sed for Seqencing Analysis Viewer. InterOp files are pdated throghot the rn. Log files Logs Log files describe each step performed by the instrment for each cycle, and list software and firmware versions sed with the rn. The file named [InstrmentName]_CrrentHardware.csv lists the serial nmbers of instrment components. Error log files (*ErrorLog*.txt) Global log files (*GlobalLog*.tsv) Lane log files (*LaneLog*.txt) RTA Logs RTA Logs RTA Logs Trobleshooting Resorces Log of RTA errors. Error log files are pdated whenever an error occrs. Log of all RTA events. Global log files are pdated throghot the rn. Log RTA processing events. Lane log files are pdated throghot the rn. For technical qestions, visit the MiniSeq System spport pages on the Illmina website. The spport pages provide access to docmentation, downloads, and freqently asked qestions. Log in to yor MyIllmina accont for access to spport blletins. For rn qality or performance problems, contact Illmina Technical Spport. See Technical Assistance on page 57. Consider sharing a link to the rn smmary in BaseSpace with Illmina Technical Spport to facilitate trobleshooting. Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 38

MiniSeq System Gide Process Stats The MiniSeq Control Software lists the stats of at least 3 rns in the system Temp folder. From the Manage Instrment screen, select Process stats. For each rn name, the system lists the stats of the following components: Real-Time Analysis (RTA) Based on the processing of BCL files Local Rn Manager If Local Rn Manager was sed for the rn File Copy Based on file transfer sing the Rn Copy Service BaseSpace If BaseSpace was sed for the rn Seqencing Archive Folder The MiniSeq Control Software saves rn smmary files on the system compter at D:\Illmina\MiniSeq Seqencing Archive for each rn performed on the instrment. In this folder, a sbfolder exists for each rn performed on the instrment contains the following files: RnCompletionStats.xml Contains the completion stats, rn folder name, nmber of cycles planned and performed, clster density, clsters passing filter, and estimated yield for the rn. RnParameters.xml Contains information abot rn parameters and rn components. Information incldes the RFID, serial nmber, lot nmber, and expiration date. Atomatic Check Errors If errors occr dring the atomatic pre-rn check, se the following recommended actions to resolve the error. If a pre-rn check fails, the reagent cartridge RFID is not locked and can be sed for a sbseqent rn. However, the RFID is locked after the foil seals have been pierced. System Checks Doors closed Consmables loaded Reqired software Instrment disk space Network connection Network disk space Recommended Action Make sre that the compartment doors are closed. Consmable sensors do not register. Make sre that each consmable is properly loaded. On the rn setp screens, select Back to retrn to the loading step, and repeat rn setp. Critical components of the software are missing. Perform a manal software pdate to restore all software components. The instrment hard drive does not have sfficient disk space to perform a rn. Clear rn data from the instrment hard drive. The connection to the specified location of the otpt folder has been interrpted. Althogh the check is labeled network connection, the system checks the connection to any specified location of the otpt folder on a server, an external hard drive, or the local drive. Check the stats of the connection to the specified otpt folder location. The specified location of the otpt folder is fll. Althogh the check is labeled network disk space, the system checks any specified location of the otpt folder on a server, an external hard drive, or the local hard drive. Clear disk space from the specified otpt folder location. Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 39

MiniSeq System Gide Temperatre Temperatre ramp Temperatre sensors Fans Recommended Action Contact Illmina Technical Spport. Contact Illmina Technical Spport. Contact Illmina Technical Spport. Imaging System Imaging limits Z step-and-settle Bit error rate Flow cell registration Recommended Action Contact Illmina Technical Spport. Contact Illmina Technical Spport. Contact Illmina Technical Spport. It is possible that the flow cell is not properly seated. On the rn setp screens, select Back to retrn to the flow cell step. Unload and reload the flow cell to make sre that it is seated properly. Reagent Delivery Valve response Pmp Recommended Action Contact Illmina Technical Spport. Contact Illmina Technical Spport. Hard Drive Space The instrment compter hard drive can hold approximately 45 rns based on data generated from a rn sing the following rn parameters: Approximately 5 6 GB of space is reqired for 150-cycle paired-end rn. An additional 10 GB of space is reqired for analysis files when sing the Local Rn Manager Reseqencing analysis modle. For each rn performed, a temporary rn folder is created as part of the software operation. As files are written to the temporary rn folder, the files are copied to the otpt folder. Therefore, if yo specify an otpt folder location on the instrment hard drive, 2 copies of that rn are written to the hard drive. The software saves the most recent 3 temporary rn folders. When sing the Local Rn Manager software for analysis, temporary files are not deleted, by defalt. The retention policy is set manally from the Local Rn Manager System Settings screen. Eventally, temporary files can fill the hard drive space. Consider sing a network location for rn data and setting a reasonable Local Rn Manager retention policy based on the nmber of rns that yo perform. RTA Errors To trobleshoot RTA errors, first check the RTA error log, which is stored in the RTALogs folder. This file is not present for sccessfl rns. Inclde the error log when reporting isses to Illmina Technical Spport. Error Handling RTA2 creates log files and writes them to the RTALogs folder. Errors are recorded in an error file in *.tsv file format. The following log and error files are transferred to the final otpt destination at the end of processing: *GlobalLog*.tsv smmarizes important rn events. *LaneNLog*.tsv lists processing events. N is always 1 on a MiniSeq flow cell. *Error*.tsv lists errors that occrred dring a rn. Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 40

MiniSeq System Gide *WarningLog*.tsv lists warnings that occrred dring a rn. Rehybridization Workflow A rehybridization rn might be necessary if metrics generated dring the first few cycles show intensities below 2500. Some low-diversity libraries can show intensities below 1000, which is expected and cannot be resolved with rehybridization. NOTE The End Rn command is final. The rn cannot be resmed, rn consmables cannot be resed, and seqencing data from the rn are not saved. When yo end a rn and save the flow cell, the software performs the following steps before the rn ends: Places the flow cell in a safe state. Unlocks the flow cell RFID for a later rn. Assigns a rehybridization expiration date to the flow cell. Writes the rn logs for completed cycles. A delay is normal. Bypasses the atomatic post-rn wash. When yo start a rehybridization rn, the software performs the following steps to perform the rn: Creates a rn folder based on a niqe rn name. Checks that the flow cell rehybridization date has not expired. Primes reagents. A delay is normal. Skips the clstering step. Removes the previos Read 1 primer. Hybridizes a fresh Read 1 primer. Contines throgh Read 1 and the remainder of the rn based on specified rn parameters. Points to End a Rn for Rehybridization Later rehybridization is possible only if yo end the rn at the following points: After cycle 5 Intensities appear after template registration, which reqires the first 5 cycles of seqencing. Althogh it is safe to end a rn after cycle 1, ending after cycle 5 is recommended. Do not end a rn dring clster generation. Read 1 or Index 1 Read End the rn before paired-end resynthesis begins. The flow cell cannot be saved for later rehybridization after paired-end resynthesis begins. Reqired Consmables A rehybridization rn reqires a new MiniSeq reagent cartridge regardless of when the rn was stopped. End the Crrent Rn 1 Select End Rn. When prompted to confirm the command, select Yes. 2 When prompted to save the flow cell, select Yes. Note the expiration date for rehybridization. 3 Remove the saved flow cell and set aside at 2 C to 8 C ntil yo are ready to set p the rehybridization rn. Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 41

MiniSeq System Gide NOTE Yo can store the flow cell p to 7 days at 2 C to 8 C in the flow cell container with the cap closed. For best reslts, rehybridize the saved flow cell within 3 days. Perform a Manal Wash 1 From the Home screen, select Perform Wash. 2 From the Wash Selection screen, select Manal Post-Rn Wash. See Perform a Manal Instrment Wash on page 32. NOTE If yo have not yet removed the reagent cartridge from the stopped rn, yo can se them for the manal wash. Otherwise, perform the manal wash with the wash cartridge. Set Up a New Rn on the BaseSpace Prep Tab 1 If the instrment is configred for BaseSpace or BaseSpace Onsite, set p a new rn on the Prep tab sing the same parameters as the original rn. TIP Click the Pools tab, select the appropriate Pool ID to retain the previos rn settings, and then assign a niqe name for the new rn. Set Up a Rn on the Instrment 1 Prepare a new reagent cartridge. 2 If the saved flow cell was stored, allow it to reach room temperatre (15 30 mintes). 3 Clean and load the saved flow cell. The system reads the flow cell RFID as a saved flow cell and confirms a valid rehybridization date. 4 Remove the spent reagents bottle and discard the contents appropriately, and then reload the empty container. 5 Load the new reagent cartridge. 6 From the Rn Setp screen, select from the following options: BaseSpace, BaseSpace Onsite, or Local Rn Manager configration Select the rn and confirm rn parameters. Standalone configration Enter the name of the rn and specify the same parameters as the original rn. 7 Select Next to proceed to the pre-rn check and start the rn. System Check A system check is not reqired for normal operation or instrment maintenance. However, an Illmina Technical Spport representative might ask yo to perform a system check for trobleshooting prposes. NOTE If an instrment wash is de, perform the wash before starting a system check. Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 42

MiniSeq System Gide Starting a system check atomatically closes the control software and lanches the MiniSeq Service Software. The service software lanches and opens to the Load screen, which is configred to se the advanced loading option. Figre 29 Available System Checks After loading consmables, the Select screen opens, which lists available system checks. Inactive checkboxes on the Select screen indicate tests that reqire assistance from an Illmina field representative. Perform a System Check 1 From the Manage Instrment screen, select System Check. When prompted to close the control software, select Yes. 2 Add 40 ml deionized water to the wash cartridge. The correct reservoir is eqivalent to position #40 on the prefilled reagent cartridge. Figre 30 Position for Wash Soltion A Wash soltion 3 Load the consmables as follows: Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 43

MiniSeq System Gide a b c If a sed flow cell is not already on the instrment, load a sed flow cell. Empty the spent reagents bottle and retrn it to the instrment. Load the wash cartridge. 4 Select Load. The software moves the flow cell and wash cartridge into position. 5 Select Next. The system check begins. 6 [Optional] When the system check is complete, select View next to the check name to view the vales associated with each check. 7 Select Next. The system check report opens. 8 Select Save to save the report to a zipped file. Navigate to a network location to save the file. 9 When finished, select Exit. 10 When prompted to close the service software and restart the control software, select Yes. The control software restarts atomatically. Motion Checks System Check FCLM & FAM Stage Tests Description Checks the gain and distance of the Flow Cell Load Mechanism (FCLM) and Flidics Atomation Modle (FAM) to confirm that the modles are working properly. Checks the travel limits and performance of the XY-stage and Z-stage. Optics Check System Check Flow Cell Registration Description Measres flow cell tilt on an optical plane, tests camera fnctionality, tests the imaging modle, and verifies registration of the flow cell in the correct imaging position. Flidics Checks System Check Valve Response Pressre Decay Flow Rate Description Checks the accracy of the valve and pmp movements, and tests the pmp syringe range of movement. Checks the leak rate of a sealed flidics system, which confirms that the flow cell is properly monted in the seqencing position. Checks the fnctionality of the bbble sensors, which are sed to detect the presence of air in the reagent lines. Measres the flow rates to check for occlsions or leaks. Thermal Checks System Check Fans Thermal Probes Description Checks the speed of system fans in plse per minte (PPM) to confirm that fans are fnctioning. Fans that are not fnctioning retrn a negative vale. Checks the average temperatre of each thermal sensor. Thermal sensors that are not fnctioning retrn a negative vale. Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 44

MiniSeq System Gide Network Configration Settings Network settings are configred dring installation. If the system has to be reconfigred, yo can change settings or reset settings on the Network Configration screen. Configration settings inclde the IP address, domain name server (DNS) address, and domain name. Set Network Configration 1 From the Manage Instrment screen, select System configration. 2 Select Network configration. 3 Select Obtain an IP address atomatically to obtain the IP address sing DHCP server. NOTE Dynamic Host Configration Protocol (DHCP) is a standard network protocol sed on IP networks for dynamically distribting network configration parameters. Alternatively, select Use the following IP address to connect the instrment to another server manally as follows. Contact yor network administrator for the addresses specific to yor facility. Enter IP address. The IP address is a series of 4 nmbers separated by a dot, similar to 168.62.20.37, for example. Enter the sbnet mask, which is a sbdivision of the IP network. Enter the defalt gateway, which is the roter on the network that connects to the internet. 4 Select Obtain a DNS server address atomatically to connect the instrment to the domain name server associated with IP address. Alternatively, select Use the following DNS server addresses to connect the instrment to the domain name server manally as follows. Enter the preferred DNS address. The DNS address is the server name sed to translate domain names into IP addresses. Enter the alternate DNS address. The alternate is sed if the preferred DNS cannot translate a particlar domain name to an IP address. 5 Select Save. Configre Compter Domain NOTE The instrment compter name is assigned to the instrment compter at the time of manfactre. Any changes to the compter name can affect connectivity and reqire a network administrator. 1 Connect the instrment compter to a domain or a workgrop as follows. For instrments connected to the internet Select Member of domain, and then enter domain name associated with the internet connection at yor facility. NOTE Domain changes reqire an administrator ser name and password. For instrments not connected to the internet Select Member of work grop, and then enter a work grop name. The work grop name is niqe to yor facility. 2 Select Save. Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 45

MiniSeq System Gide Cstom Genomes Yo can pload yor own reference in FASTA format to the instrment compter. Yo can pload several single FASTA files or a single mlti-fasta file (recommended), bt not a combination of both. To trobleshoot isses with a cstom genome file, confirm the following reqirements. 1 Make sre that the file ses a *.fa or *.fasta extension and that it is stored in a dedicated folder for the reference. 2 Make sre that the chromosome name does not contain any of following characters: # -? ( ) [ ] / \ = + < > : ; " ', * ^ & For best reslts, se only alpha-nmeric characters as chromosome names. Sht Down the Instrment Under normal conditions, there is no reason to sht down the instrment. 1 Select Manage Instrment. 2 Select Shtdown options. 3 Select Sht down. The command safely shts down the software and trns off instrment power. Wait at least 60 seconds before trning on the instrment again. A wash is reqired before the next seqencing rn. CAUTION Do not relocate the instrment. Moving the instrment improperly can affect the optical alignment and compromise data integrity. If yo have to relocate the instrment, contact yor Illmina representative. Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 46

MiniSeq System Gide Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 47

Appendix B Real-Time Analysis Real-Time Analysis Overview 48 Inpt and Otpt Files 48 Real-Time Analysis Workflow 49 Real-Time Analysis Overview Real-Time Analysis is a software that rns on the instrment compter, extracts intensities from images to perform base calling, and then assigns a qality score to the base call. The MiniSeq System ses an implementation of Real-Time Analysis called RTA2. The system control software and RTA2 commnicate throgh a web HTTP interface and shared memory files. If RTA2 is terminated, processing does not resme and rn data are not saved. Inpt and Otpt Files Inpt Files Real-Time Analysis software reqires the following inpt for processing: Tile images contained in local system memory. RnInfo.xml, which is generated atomatically at the beginning of the rn and provides the rn name, nmber of cycles, whether a read is indexed, and the nmber of tiles on the flow cell. Real-Time Analysis software receives commands from the control software abot the location of RnInfo.xml and whether an optional otpt folder is specified. Otpt Files Images for each channel are passed in memory as tiles. Tiles are small imaging areas on the flow cell defined as the field of view by the camera. From these images, the software prodces otpt as a set of qalityscored base call files and filter files. Otpt files are sed for downstream analysis on BaseSpace or by Local Rn Manager analysis modles. File Type Base call files Filter files Clster location files Base call index files Description Each tile that is analyzed is inclded in an aggregated base call (*.bcl) file for each lane and for each cycle. The aggregated base call file contains the base call and associated qality score for every clster in that lane. Each tile prodces filter information that is aggregated into 1 filter (*.filter) file for each lane. The filter file specifies whether a clster passes filters. Clster location (*.locs) files contain the X,Y coordinates for every clster in a tile. A clster location file is generated for each lane dring template generation. A base call index (*.bci) file is prodced for each lane to preserve the original tile information. The index file contains a pair of vales for each tile, which are tile nmber and nmber of clsters for that tile. RTA2 provides real-time metrics of rn qality stored as InterOp files. InterOp files are a binary otpt containing tile, cycle, and read-level metrics, and are sed to view real-time metrics sing the Seqencing Analysis Viewer software. Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 48

MiniSeq System Gide Real-Time Analysis Workflow Template generation Maps clster locations. Registration and intensity extraction Records the location of each clster on the flow cell and 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. Template Generation The first step in the RTA workflow is template generation, which defines the position of each clster in a tile sing X and Y coordinates. Template generation reqires image data from the first 5 cycles of the rn. After the last template cycle for a tile is imaged, the template is generated. NOTE To detect a clster dring template generation, there mst be at least 1 base other than G in the first 5 cycles. The template is sed as a reference for the sbseqent step of registration and intensity extraction. Clster positions for the entire flow cell are written to clster location (*.locs) files, 1 file for each lane. Registration and Intensity Extraction Registration and intensity extraction begin after template generation. Registration aligns images prodced over every sbseqent cycle of imaging against the template. Intensity extraction determines an intensity vale for each clster in the template for a given image. If registration fails for any images in a cycle, no intensities are extracted and all bases are called as N for that tile in that cycle. Use the Seqencing Analysis Viewer software to identify tiles and cycles that have failed registration. Registration failres are easily identified as tiles and cycles that have 0 in the P90 colmn on the Imaging tab. 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. Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 49

MiniSeq System Gide Figre 31 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 MiniSeq System ses 2-channel seqencing, which reqires only 2 images to encode the data for 4 DNA bases, 1 from the red channel and 1 from the green channel. Intensities extracted from an image compared to another image reslt in 4 distinct poplations, each corresponding to a ncleotide. The base calling process determines to which poplation each clster belongs. Figre 32 Visalization of Clster Intensities Table 1 Base Base Calls in 2-Channel Seqencing Red Channel Green Channel Reslt A 1 (on) 1 (on) Clsters that show intensity in both the red and green channels. C 1 (on) 0 (off) Clsters that show intensity in the red channel only. G 0 (off) 0 (off) Clsters that show no intensity at a known location. T 0 (off) 1 (on) Clsters that show intensity in the green channel only. Clsters Passing Filter Dring the rn, RTA2 filters raw data to remove clsters that do not meet the data qality threshold. Docment # 1000000002695 v02 Material # 20014309 For Research Use Only. Not for se in diagnostic procedres. 50