NextSeq 550. System Guide. For Research Use Only. Not for use in diagnostic procedures. Document # v04 May 2018 ILLUMINA PROPRIETARY

Transcription

1 NextSeq 550 System Gide Docment # v04 May 2018 For Research Use Only. Not for se in diagnostic procedres. ILLUMINA PROPRIETARY

2 NextSeq 550 System Gide This docment and its contents are proprietary to Illmina, Inc. and its affiliates ("Illmina"), and are intended solely for the contractal se of its cstomer in connection with the se of the prodct(s) described herein and for no other prpose. This docment and its contents shall not be sed or distribted for any other prpose and/or otherwise commnicated, disclosed, or reprodced in any way whatsoever withot the prior written consent of Illmina. Illmina does not convey any license nder its patent, trademark, copyright, or common-law rights nor similar rights of any third parties by this docment. The instrctions in this docment mst be strictly and explicitly followed by qalified and properly trained personnel in order to ensre the proper and safe se of the prodct(s) described herein. All of the contents of this docment mst be flly read and nderstood prior to sing sch prodct(s). FAILURE TO COMPLETELY READ AND EXPLICITLY FOLLOW ALL OF THE INSTRUCTIONS CONTAINED HEREIN MAY RESULT IN DAMAGE TO THE PRODUCT(S), INJURY TO PERSONS, INCLUDING TO USERS OR OTHERS, AND DAMAGE TO OTHER PROPERTY, AND WILL VOID ANY WARRANTY APPLICABLE TO THE PRODUCT(S). ILLUMINA DOES NOT ASSUME ANY LIABILITY ARISING OUT OF THE IMPROPER USE OF THE PRODUCT(S) DESCRIBED HEREIN (INCLUDING PARTS THEREOF OR SOFTWARE) Illmina, Inc. All rights reserved. All trademarks are the property of Illmina, Inc. or their respective owners. For specific trademark information, see Docment # v04 For Research Use Only. Not for se in diagnostic procedres. ii

3 NextSeq 550 System Gide Revision History Docment Date Description of Change Material # Docment # v04 Material # Docment # v03 Material # Docment # v02 Material # Docment # v01 May 2018 March 2018 March 2016 October 2015 Added spport for NextSeq v2.5 reagents. Updated storage/shipping information for NextSeq v2.5 Reagent Kits shipping flow cells at ambient temperatres. NextSeq v2.5 flow cells contine to reqire previos storage conditions. Added information abot NextSeq v2.5 Reagent Kits reqiring software pdates to version 2.2. Added note regarding mid otpt kit loading concentration. Added note regarding saving flow cells. Added note recommending that high otpt flow cells are sed for system checks. Removed the defalt ser name and password reqired to log on to the operating system. Illmina recommends sing site-specific credentials. Added information abot the Illmina Proactive monitoring service in the Select BaseSpace Configration section. Updated RTA v2 software references to RTA2. Added section titled Indexing Considerations. Removed steps to inspect the flow cell. Specified loading volme and concentration in the step to Load Libraries onto the Reagent Cartridge. Specified that an eqivalent to the recommended spplier of NaOCl is a laboratory-grade eqivalent. Added recommendation for annal preventive maintenance service. Reorganized information in the Overview and Getting Started chapters. Added instrctions for cstomizing system settings. Removed Live Help instrctions from trobleshooting chapter. This featre was removed from the control software B May A May 2015 Corrected description of reserved reservoirs on the reagent cartridge. Initial release. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. iii

4 Table of Contents Chapter 1 Overview 1 Introdction 1 Additional Resorces 1 Instrment Components 2 Seqencing Consmables Overview 5 Chapter 2 Getting Started 9 Starting the Instrment 9 Cstomize System Settings 9 Cstomize Rn Settings 10 User-Spplied Consmables and Eqipment 11 Chapter 3 Seqencing 13 Introdction 13 Seqencing Workflow 14 Prepare the Reagent Cartridge 14 Prepare the Flow Cell 15 Prepare Libraries for Seqencing 16 Set Up a Seqencing Rn 16 Monitor Rn Progress 22 Atomatic Post-Rn Wash 24 Chapter 4 Scanning 25 Introdction 25 Scanning Workflow 26 Download the DMAP Folder 26 Load the BeadChip Onto the Adapter 27 Set Up a Scan 28 Monitor Scan Progress 29 Chapter 5 Maintenance 31 Introdction 31 Perform a Manal Wash 31 Software Updates 33 Sht Down the Instrment 35 Appendix A Trobleshooting 36 Introdction 36 Trobleshooting Files 36 Resolve Atomatic Check Errors 37 Spent Reagents Container is Fll 39 Rehybridization Workflow 40 BeadChip and Scan Errors 42 Docment # v04 For Research Use Only. Not for se in diagnostic procedres. iv

5 NextSeq 550 System Gide Cstom Recipes and Recipe Folders 43 System Check 43 RAID Error Message 45 Configre System Settings 45 Appendix B Real-Time Analysis 49 Real-Time Analysis Overview 49 Real-Time Analysis Workflow 50 Appendix C Otpt Files and Folders 53 Seqencing Otpt Files 53 Seqencing Otpt Folder Strctre 56 Scanning Otpt Files 57 Scanning Otpt Folder Strctre 57 Index 58 Technical Assistance 61 Docment # v04 For Research Use Only. Not for se in diagnostic procedres. v

6 Chapter 1 Overview Introdction 1 Additional Resorces 1 Instrment Components 2 Seqencing Consmables Overview 5 Introdction The Illmina NextSeq 550 system is a single soltion that provides a seamless transition between highthroghpt seqencing and array scanning. Seqencing Featres High-throghpt seqencing The NextSeq 550 enables seqencing of exomes, whole genomes, and transcriptomes and spports TrSeq and Nextera libraries. Flow cell types Flow cells are available in configrations for high otpt and mid otpt. Each flow cell type is kitted with a compatible prefilled reagent cartridge. Real-Time Analysis (RTA) Integrated analysis software performs on-instrment data analysis, which incldes image analysis and base calling. The NextSeq ses an implementation of RTA called RTA v2, which incldes important architectre and featre differences. For more information, see Real-Time Analysis on page 49. BaseSpace integration The seqencing workflow is integrated with BaseSpace, the Illmina genomics compting environment for data analysis, storage, and collaboration. For instrments configred for BaseSpace, library information and rn parameters are specified on the BaseSpace Prep tab. Rns that were set p in BaseSpace appear on the instrment interface dring rn setp. As the rn progresses, otpt files are streamed in real time to BaseSpace or BaseSpace Onsite. Array Scanning Featres Integrated array scanning in control software The NextSeq 550 allows yo to transition between array scanning and high-throghpt seqencing on the same instrment sing the same control software. Extended imaging capability The imaging system in the NextSeq 550 incldes software and stage modifications that enable imaging of a larger srface area to accommodate BeadChip scanning. BeadChip types Compatible BeadChip types inclde CytoSNP-12, CytoSNP-850K, and Karyomap-12. BeadChip adapter A resable BeadChip adapter enables easy loading of a BeadChip onto the instrment. Data Analysis Use the BleFse Mlti software to analyze array data. Additional Resorces The following docmentation is available for download from the Illmina website. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 1

7 NextSeq 550 System Gide Resorce NextSeq System Site Prep Gide (docment # ) NextSeq System Safety and Compliance Gide (docment # ) RFID Reader - Model # TR User Gide (docment # ) Denatring and Dilting Libraries for the NextSeq System (docment # ) NextSeq Cstom Primers Gide (docment # ) BaseSpace help (help.basespace.illmina.com) Description 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. Provides instrctions for denatring and dilting prepared libraries for a seqencing rn, and preparing an optional PhiX control. This step applies to most library types. Provides information abot sing cstom seqencing primers in place of Illmina seqencing primers. Provides information abot sing BaseSpace and available analysis options. Visit the NextSeq 550 spport page on the Illmina website for access to docmentation, software downloads, online training, and freqently asked qestions. Instrment Components The NextSeq 550 system incldes a toch screen monitor, a stats bar, and 3 compartments. Figre 1 Instrment Components A B C D E Imaging compartment Holds the flow cell for seqencing or the BeadChip adapter for scanning. Toch screen monitor Enables on-instrment configration and setp sing the control software interface. 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). Bffer compartment Holds the bffer cartridge and the spent reagents container. Reagent compartment Holds the reagent cartridge. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 2

8 NextSeq 550 System Gide Imaging Compartment The imaging compartment hoses the stage, which incldes 3 alignment pins for positioning the flow cell for seqencing or the BeadChip adapter for scanning. After loading the flow cell or BeadChip adapter, the imaging compartment door closes atomatically and moves components into position. Reagent and Bffer Compartments Setting p a seqencing rn on the NextSeq 550 reqires access to the reagent compartment and bffer compartment to load rn consmables and empty the spent reagents container. Figre 2 Reagent and Bffer Compartments A B C D E Reagent compartment door Encloses the reagent compartment with a latch nder the lower-right corner of the door. The reagent compartment holds the reagent cartridge. Reagents are pmped throgh the sippers and flidics system, and then to the flow cell. Reagent cartridge The reagent cartridge is a prefilled single-se consmable. Bffer cartridge The bffer cartridge is a prefilled single-se consmable. Spent reagents container Spent reagents are collected for disposal after each rn. Bffer compartment door Encloses the bffer compartment with a latch nder the lower-left corner of the door. NextSeq Software The instrment software incldes integrated applications that perform seqencing rns or array scanning. NextSeq Control Software (NCS) The control software gides yo throgh the steps to set p a seqencing rn or an array scan. Real-Time Analysis (RTA) software For seqencing rns, RTA performs image analysis and base calling dring the rn. The NextSeq 550 ses RTA v2, which incldes important architectre and featre differences from earlier versions. For more information, see Real-Time Analysis on page 49. Stats Icons A stats icon in the top-right corner of the control software interface screen signals any change in conditions dring rn setp or dring the rn. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 3

9 NextSeq 550 System Gide Stats Icon Stats Name Stats OK Description System is normal. Processing System is processing. 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. Power Btton The power btton on the front of the NextSeq 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 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 from the rn is not saved. Resable BeadChip Adapter Overview The resable BeadChip adapter holds the BeadChip dring scanning. The BeadChip is secred in the recessed shelf of the adapter with the retention clip. Then, the BeadChip adapter is loaded onto the stage in the imaging compartment. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 4

10 NextSeq 550 System Gide Figre 3 Resable BeadChip Adapter A B BeadChip adapter Retention clip Seqencing Consmables Overview Contents and Storage The seqencing consmables reqired to rn the NextSeq are provided separately in a single-se kit. Each kit incldes one flow cell, a reagent cartridge, a bffer cartridge, and library diltion bffer. When yo receive NextSeq 500/550 Kit: Do not open the white foil packages ntil instrcted to do so. Promptly store components at the indicated temperatres to ensre proper performance. Store cartridges so that the package labels face p. Consmable Qantity Storage Temperatre Description Reagent Cartridge 1-25 C to -15 C Contains clstering and seqencing reagents Bffer Cartridge 1 15 C to 30 C Contains bffer and wash soltion HT C to -15 C Hybridization Bffer Flow Cell 1 2 C to 8 C* Single-se flow cell *Shipped at room temperatre for NextSeq v2.5 Reagents kits Reagents are sensitive to light. Store the reagent cartridge and bffer cartridge in a dark location away from light. The flow cell, reagent cartridge, and bffer cartridge se radio-freqency identification (RFID) for accrate consmable tracking and compatibility. All other kits inclde dal-index seqencing primers and NaOCI in the prefilled cartridge. No additional steps are reqired. Kit Compatibility Labeling Kit components are labeled with color-coded indicators to show compatibility between flow cells and reagent cartridges. Always se a compatible reagent cartridge and flow cell. The bffer cartridge is niversal. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 5

11 NextSeq 550 System Gide Each flow cell and reagent cartridge is labeled High or Mid. Always check the label when yo prepare consmables for a rn. Kit Type High Otpt Kit Components Marking on Label Mid Otpt Kit Components Flow Cell Overview Figre 4 Flow Cell Cartridge A Lane pair A Lanes 1 and 3 B Lane pair B Lanes 2 and 4 C Flow cell cartridge frame 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. The flow cell contains 4 lanes that are imaged in pairs. Lanes 1 and 3 (lane pair A) are imaged at the same time. Lanes 2 and 4 (lane pair B) are imaged when imaging of lane pair A is complete. Althogh the flow cell has 4 lanes, only a single library or set of pooled libraries is seqenced on the flow cell. Libraries are loaded onto the reagent cartridge from a single reservoir and transferred atomatically to the flow cell to all 4 lanes. Each lane is imaged in small imaging areas called tiles. For more information, see Flow Cell Tiles on page 53. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 6

12 NextSeq 550 System Gide Reagent Cartridge Overview The reagent cartridge is a single-se consmable with RFID tracking and foil-sealed reservoirs that are prefilled with clstering and seqencing 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 reservoir to the flow cell. Several reservoirs are reserved for the atomatic post-rn wash. Wash soltion is pmped from the bffer cartridge to the reserved reservoirs, throgh the system, and then to the spent reagents container. 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. Reserved Reservoirs Figre 6 Nmbered Reservoirs Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 7

13 NextSeq 550 System Gide Position Description 7, 8, and 9 Reserved for optional cstom primers 10 Load libraries For information abot cstom primers, see NextSeq Cstom Primers Gide (docment # ). Removable Reservoir in Position #6 The prefilled reagent cartridge incldes a denatration reagent in position 6 that contains formamide. To facilitate safe disposal of any nsed reagent after the seqencing rn, the reservoir in position 6 is removable. For more information, see Remove Used Reservoir from Position #6 on page 20. Bffer Cartridge Overview The bffer cartridge is a single-se consmable containing three reservoirs that are prefilled with bffers and wash soltion. The contents of the bffer cartridge are sfficient for seqencing one flow cell. Figre 7 Bffer Cartridge Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 8

14 Chapter 2 Getting Started Starting the Instrment 9 Cstomize System Settings 9 Cstomize Rn Settings 10 User-Spplied Consmables and Eqipment 11 Starting the Instrment Trn on the power toggle switch to the I (on) position. Figre 8 Power Switch Located on Back of Instrment 1 Press the power btton above the reagent compartment. The power btton trns on the instrment power and starts the integrated instrment compter and software. Figre 9 Power Btton Located on Front of Instrment 2 Wait ntil the operating system has finished loading. The NextSeq Control Software (NCS) lanches and initializes the system atomatically. After the initialization step is complete, the Home screen opens. 3 If yor system has been configred to reqire login credentials, 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 system settings for start-p options, inpt preferences, adio settings, and instrment name. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 9

15 NextSeq 550 System Gide Select Start-Up Option 1 From the Manage Instrment screen, select System Cstomization. 2 Select from the following start-p options: Select Kiosk Mode to se the control software interface in fll screen. Select Windows Mode to allow access to Windows on the instrment compter. Interaction with the software interface, sch as btton location, is likely to be altered in this mode. 3 Select Save to save settings and advance the screen. Set Inpt Option and Adio Indicator 1 From the Manage Instrment screen, select System Cstomization. 2 Select the Use on-screen keyboard checkbox to activate the on-screen keyboard for inpt to the instrment. 3 Select the Play adio checkbox to trn on adio indicators for the following events. Upon instrment initialization When a rn is started When certain errors occr When ser interaction is reqired When a rn has finished 4 Select Save to save settings and advance the screen. Cstomize Instrment Identification 1 From the Manage Instrment screen, select System Cstomization. 2 To assign a preferred image for yor instrment, select Browse and navigate to the 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 at the pper-left corner of each screen. Cstomize Rn Settings The control software incldes cstomizable settings for rn setp preferences and prging of nsed reagents. Set Rn Setp Options 1 From the Manage Instrment screen, select System Cstomization. 2 Select Save to advance to Rn Cstomization. 3 Select the Use Advanced Load Consmables checkbox to enable the option to load all rn consmables from a single screen. 4 Select the Skip Pre-Rn Check Confirmation checkbox to start seqencing or scanning atomatically after a sccessfl atomatic check. 5 Select Save to save settings and exit the screen. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 10

16 NextSeq 550 System Gide Set Atomatic Prge Option 1 From the Manage Instrment screen, select System Cstomization. 2 Select Save to advance to Rn Cstomization. 3 Select the Prge Consmables at End of Rn checkbox to prge nsed reagents from the reagent cartridge to the spent reagents container atomatically after each rn. NOTE Prging consmables atomatically adds additional time to the workflow. 4 Select Save to save settings and exit the screen. User-Spplied Consmables and Eqipment The following consmables and eqipment are sed on the NextSeq 550. User-Spplied Consmables for Seqencing Rns Consmable Spplier Prpose 1 N NaOH (sodim hydroxide) General lab spplier Library denatration, dilted to 0.2 N 200 mm Tris-HCl, ph7 General lab spplier Library denatration Alcohol wipes, 70% Isopropyl or Ethanol, 70% VWR, catalog # (or eqivalent) General lab spplier Lab tisse, low-lint VWR, catalog # (or eqivalent) Flow cell cleaning and general prpose Flow cell cleaning User-Spplied Consmables for Instrment Maintenance Consmable Spplier Prpose NaOCl, 5% (sodim hypochlorite) Sigma-Aldrich, catalog # (or laboratory-grade eqivalent) Washing the instrment sing the manal post-rn wash; dilted to 0.12% Tween 20 Sigma-Aldrich, catalog # P7949 Washing the instrment sing manal wash options; dilted to 0.05% Water, laboratory-grade General lab spplier Washing the instrment (manal 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 Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 11

17 NextSeq 550 System Gide 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 # v04 For Research Use Only. Not for se in diagnostic procedres. 12

18 Chapter 3 Seqencing Introdction 13 Seqencing Workflow 14 Prepare the Reagent Cartridge 14 Prepare the Flow Cell 15 Prepare Libraries for Seqencing 16 Set Up a Seqencing Rn 16 Monitor Rn Progress 22 Atomatic Post-Rn Wash 24 Introdction To perform a seqencing rn on the NextSeq 550, prepare a reagent cartridge and flow cell, and then follow the software prompts to set p and start the rn. Clster generation and seqencing are performed oninstrment. After the rn, an instrment wash begins atomatically sing components already loaded on the instrment. 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. Monitor rn progress and statistics from the control software interface, from the Rn tab on BaseSpace, or from a networked compter sing the Seqencing Analysis Viewer (SAV) software. See Seqencing Analysis Viewer on page 23. Analysis As the rn progresses, the control software atomatically transfers base call (BCL) files to BaseSpace or the specified otpt location for secondary analysis. Several analysis methods are available depending on yor application. For more information, see the BaseSpace help (help.basespace.illmina.com). Seqencing Rn Dration Seqencing rn dration depends on the nmber of cycles performed. The maximm rn length is a pairedend rn of 150 cycles each read (2 x 150), pls p to 8 cycles each for 2 index reads. For expected drations and other system specifications, visit the NextSeq 550 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, a paired-end 150-cycle rn performs reads of 151-cycles (2 151) for a total of 302 cycles. At the end of the rn, 2 x 150 cycles are analyzed. The extra cycle is reqired for phasing and Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 13

19 NextSeq 550 System Gide prephasing calclations. Seqencing Workflow When sing NextSeq v2.5 Reagents kit, make sre the NextSeq Control Software is version 2.2, or newer, before beginning preparation of samples or consmables. For sbseqent software pdates, make sre the instrment is pdated prior to preparing consmables for the NextSeq 550 workflow. NOTE NextSeq Control Software Version 2.2 is reqired to read NextSeq v2.5 RFID. Make sre that yo complete all steps in the NextSeq 550 workflow in the specified order. For configrations sing Illmina BaseSpace or BaseSpace Onsite: Set p the rn on the BaseSpace Prep tab. See the BaseSpace help (help.basespace.illmina.com). Prepare a new reagent cartridge: thaw and inspect. Prepare a new flow cell: bring to room temperatre, nwrap, and inspect. Denatre and dilte libraries (does not apply to all library types). See Denatring and Dilting Libraries for the NextSeq System (docment # ). Load the library diltion onto the reagent cartridge in reservoir #10. From the software interface, select Seqence to begin the rn setp steps. Load the flow cell. Empty and reload the spent reagents container. Load the bffer cartridge and reagent cartridge. Review rn parameters and atomatic check reslts. Select Start. Monitor the rn from the control software interface, from the Rn tab on BaseSpace, or from a networked compter sing Seqencing Analysis Viewer. An instrment wash begins atomatically when seqencing is complete. Prepare the Reagent Cartridge 1 Remove the reagent cartridge from -25 C to -15 C storage. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 14

20 NextSeq 550 System Gide 2 Thaw in a room temperatre water bath ntil thawed (~60 mintes). Do not sbmerge the cartridge. 3 Gently tap on the bench to dislodge water from the base, and then dry the base. NOTE [Alternate method] Thaw reagents overnight at 2 C to 8 C. Reagents reqire a minimm of 18 hors to thaw. At this temperatre, reagents are stable p to 1 week. 4 Invert the cartridge 5 times to mix reagents. 5 Inspect positions 29, 30, 31, and 32 to make sre that reagents are thawed. 6 Gently tap on the bench to redce air bbbles. 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. Prepare the Flow Cell 1 Remove a new flow cell package from 2 C to 8 C storage. 2 Set the nwrapped flow cell package aside at room temperatre for 30 mintes. NOTE If the foil package is intact, the flow cell can remain at room temperatre p to 12 hors. Avoid repeated cooling and warming of the flow cell. 3 Remove the flow cell from the foil package. Figre 10 Remove from Foil Package 4 Open the clear plastic clamshell package and remove the flow cell. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 15

21 NextSeq 550 System Gide Figre 11 Remove from Clamshell Package 5 Clean the glass srface of the flow cell with a lint-free alcohol wipe. Dry the glass with a low-lint lab tisse. Prepare Libraries for Seqencing The library volme and loading concentration differ depending on the version of NCS yo are rnning. Control Software Version Library Volme Library Concentration NCS v1.3, or later 1.3 ml 1.8 pm NCS v1.2, or earlier 3 ml 3 pm Denatre and Dilte Libraries Denatre and dilte yor libraries to a loading volme of 1.3 ml and a loading concentration of 1.8 pm for high otpt kits and 1.5 pm for mid otpt kits. In practice, loading concentration can vary depending on library preparation and qantification methods. For instrctions, see the NextSeq System Denatre and Dilte Libraries Gide (docment # ). Load Libraries onto the Reagent Cartridge 1 Clean the foil seal covering reservoir #10 labeled Load Library Here sing a low-lint tisse. 2 Pierce the seal with a clean 1 ml pipette tip. 3 Load 1.3 ml of prepared 1.8 pm libraries into reservoir #10 labeled Load Library Here. Avoid toching the foil seal as yo dispense the libraries. Figre 12 Load Libraries Set Up a Seqencing Rn 1 From the Home screen, select Experiment, and then select Seqence. The Seqence command opens the imaging compartment door, releases consmables from a previos rn, and opens the series of rn setp screens. A short delay is normal. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 16

22 NextSeq 550 System Gide If the instrment is configred for BaseSpace, yo are prompted to log in to BaseSpace. If the instrment is configred for standalone mode, the next step is loading the flow cell. Log in to BaseSpace 1 Enter yor BaseSpace ser name and password. 2 Select Next. Load the Flow Cell 1 Remove the sed flow cell from a previos rn. 2 Align the flow cell over the alignment pins and place the flow cell on the stage. Figre 13 Load the Flow Cell 3 Select Load. The door closes atomatically, the flow cell ID appears on the screen, and the sensors are checked. 4 Select Next. Empty the Spent Reagents Container 1 Remove the spent reagents container and discard the contents in accordance with applicable standards. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 17

23 NextSeq 550 System Gide Figre 14 Remove the Spent Reagents Container NOTE As yo remove the container, place yor other hand nderneath for spport. 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 Slide the empty spent reagents container into the bffer compartment ntil it stops. An adible click indicates that the container is in position. Figre 15 Load the Empty Spent Reagents Container Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 18

24 NextSeq 550 System Gide Load the Bffer Cartridge 1 Remove the sed bffer cartridge from the pper compartment. 2 Slide a new bffer cartridge into the bffer compartment ntil it stops. An adible click indicates that the cartridge is in position, the bffer cartridge ID appears on the screen, and the sensor is checked. Figre 16 Load the Bffer Cartridge 3 Close the bffer compartment door, and select Next. Load the Reagent Cartridge 1 Remove the sed reagent cartridge from the reagent compartment. Dispose of nsed 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. NOTE To facilitate safe disposal of nsed reagent, the reservoir in position 6 is removable. For more information, see Remove Used Reservoir from Position #6 on page Slide the reagent cartridge into the reagent compartment ntil the cartridge stops, and then close the reagent compartment door. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 19

25 NextSeq 550 System Gide Figre 17 Load Reagent Cartridge 3 Select Load. The software moves the cartridge into position atomatically (~30 seconds), the reagent cartridge ID appears on the screen, and the sensors are checked. 4 Select Next. Remove Used Reservoir from Position #6 1 After yo have removed the sed reagent cartridge from the instrment, remove the protective rbber cover over the slot next to position #6. Figre 18 Removable Position #6 A Protective rbber cover B Position #6 2 Press down on the clear plastic tab and psh towards the left to eject the reservoir. 3 Dispose of the reservoir in accordance with applicable standards. Specify Rn Parameters The steps on the Rn Setp screen differ based on system configration: BaseSpace or BaseSpace Onsite The Rn Setp screen lists rns that were set p sing the BaseSpace Prep tab. If the intended rn does not appear on the Rn Setp screen, make sre that the rn is marked for seqencing in BaseSpace. Standalone The Rn Setp screen incldes fields for defining rn parameters. Select Available Rn (BaseSpace Configration) 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. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 20

26 NextSeq 550 System Gide 2 Select Next. 3 Confirm rn parameters. Rn Name Name of the rn as assigned in BaseSpace. Library ID Name of the pooled libraries as assigned in BaseSpace. Recipe Name of the recipe, either NextSeq High or NextSeq Mid depending on the reagent cartridge sed for the rn. Read Type Single Read or Paired End. Read Length Nmber of cycles for each read. [Optional] Cstom Primers, if applicable. 4 [Optional] Select the Edit icon to change rn parameters. When finished, select Save. Rn parameters Change the nmber of reads or nmber of cycles per read. Cstom primers Change the settings for cstom primers. For more information, see NextSeq Cstom Primers Gide (docment # ). Prge consmables for this rn Change the setting to prge consmables atomatically after the crrent rn. 5 Select Next. Enter Rn Parameters (Standalone Configration) 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. Read 2 Enter a vale p to 151 cycles. This vale is typically the same nmber of cycles as Read 1. 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. The control software confirms yor entries sing the following criteria: Total cycles do not exceed the maximm cycles allowed Cycles for Read 1 are greater than the 5 cycles sed for template generation Index Read cycles do not exceed Read 1 and Read 2 cycles 6 [Optional] If yo are sing cstom primers, select the checkbox for the primers sed. For more information, see NextSeq Cstom Primers Gide (docment # ). Read 1 Cstom primer for Read 1. Read 2 Cstom primer for Read 2. Index 1 Cstom primer for Index 1. Index 2 Cstom primer for Index 2. 7 [Optional] Select the Edit icon to change rn parameters. When finished, select Save. Otpt folder location Change the otpt folder location for the crrent rn. Select Browse to navigate to a network location. 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. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 21

27 NextSeq 550 System Gide 8 Select Next. Review Atomated Check The software performs an atomated check of the system. Dring the check, the following indicators appear on the screen: Gray checkmark The check has not been performed yet. Progress icon The check is in progress. Green checkmark The check passed. Red X The check did not pass. For any items that do not pass, an action is reqired before yo can proceed. See Resolve Atomatic Check Errors on page 37. To stop an atomated check in progress, select the select the icon. The check resmes at the first incomplete or failed check. To view the reslts of each individal check within a category, select the Start the Rn icon in the lower-right corner. To restart the check, When the atomated check is complete, select Start. The seqencing rn begins. icon to expand the category. To configre the system to start the rn atomatically after a sccessfl check, see Set Rn Setp Options on page 10. Monitor Rn Progress 1 Monitor rn progress, intensities, and qality scores as metrics appear on the screen. Figre 19 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. Use the time remaining in the pper-right corner to determine actal dration. B Q-Score Shows the distribtion of qality scores (Q-scores). See Qality Scoring on page 52. 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. Colors match base indicators sed in the Seqencing Analysis Software (SAV). D Clster Density (K/mm²) Shows the nmber of clsters detected for the rn. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 22

28 NextSeq 550 System Gide E F G Clsters Passing Filter (%) Shows the percentage of clsters passing filter. See Clsters Passing Filter on page 52. Estimated Yield (Gb) Shows the nmber of bases projected for the rn. Flow cell image Shows the crrent process on each lane pair. One lane pair is imaging when the other lane pair is in a chemistry step. NOTE After yo select Home, it is not possible to retrn to view rn metrics. However, rn metrics are accessible on BaseSpace or viewable from a standalone compter sing the Seqencing Analysis Viewer (SAV). 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. Rn metrics appear after cycle 25, inclding clster density, clsters passing filter, yield, and qality scores. Data Transfer Depending on the analysis configration selected, an icon appears on the screen dring the rn to indicate the data transfer stats. Stats Illmina BaseSpace BaseSpace Onsite Standalone Instrment Connected Connected and transferring data Disconnected 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. If the connection is not restored before the rn ends, manally remove data from the instrment compter before a sbseqent rn can begin. Rn Copy Service The NextSeq System Software Site incldes a Rn Copy Service. RTA v2 reqests the service to copy files from a sorce location to a destination location and the service processes copy reqests in the order received. If an exception occrs, the file is reqeed for copy based on the nmber of files in the copy qee. 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 # ). Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 23

29 NextSeq 550 System Gide 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. Atomatic Post-Rn Wash When the seqencing rn is complete, the software initiates an atomatic post-rn wash sing the wash soltion provided in the bffer cartridge and NaOCl provided in the reagent cartridge. The atomatic post-rn wash takes approximately 90 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 cartridges in place ntil the next rn. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 24

30 Chapter 4 Scanning Scanning Introdction 25 Scanning Workflow 26 Download the DMAP Folder 26 Load the BeadChip Onto the Adapter 27 Set Up a Scan 28 Monitor Scan Progress 29 Introdction To perform a scan on the NextSeq 550, yo need the following rn components: A hybridized and stained BeadChip The resable BeadChip adapter Decode Map (DMAP) files for the BeadChip yo are sing A manifest file for the BeadChip yo are sing A clster file for the BeadChip yo are sing Otpt files are generated dring the scan and then qeed for transfer to the specified otpt folder. Perform analysis sing the BleFse Mlti software, which reqires that scanning data are available in a genotype call (GTC) file format. By defalt, the NextSeq 550 generates normalized data and associated genotype calls in the format of a GTC file. Optionally, yo can configre the instrment to generate additional intensity data (IDAT) files. For more information, see BeadChip Scan Configration on page 47. Decode File Client The DMAP folder contains information that identifies bead locations on the BeadChip and qantifies the signal associated with each bead. A DMAP folder is niqe for each BeadChip barcode. The Decode File Client Utility enables yo to download DMAP folders directly from Illmina servers sing standard HTTP protocol. For access to the Decode File Client, go to the Decode File Client spport page on the Illmina website (spport.illmina.com/array/array_software/decode_file_client/downloads.html). Install the Decode File Client on a compter with access to the network location of the DMAP folder. For more information, see Download the DMAP Folder on page 26. Manifest Files and Clster Files For each BeadChip, the software reqires access to a manifest file and clster file. Each manifest and clster file is niqe to a BeadChip type. Make sre that yo se clster files that inclde NS550 in the file name. These files are compatible with the NextSeq system. Manifest file Manifest files describe the SNP or probe content on a BeadChip. Manifest files se the *.bpm file format. Clster files Clster files describe the clster positions for the Illmina genotyping array and are sed when analyzing data to make the genotype call. Clster files se the *.egt file format. The location of the files is specified on the BeadChip Scan Configration screen. From the NCS Home screen, select Manage Instrment, System Configration, and then BeadChip Scan Configration. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 25

31 NextSeq 550 System Gide When the NextSeq 550 instrment is installed, the Illmina representative downloads these files and specifies the path in the control software. There is no need to change these files except in the case of loss or if a new version is available. For more information, see Replace Manifest Files and Clster Files on page 43. Scanning Workflow Download the DMAP information and save it to the specified DMAP folder location. Load the BeadChip onto the BeadChip adapter. Load the BeadChip adapter onto the instrment. Specify scan parameters: DMAP folder location and otpt location. Review atomatic check reslts. Select Start. Monitor the scan from the control software interface. Download the DMAP Folder Yo can access the DMAP folder sing the Decode File Client by accont or by BeadChip (defalt view). Access DMAP Folder by Accont 1 From the main tab of the Decode File Client, select a download option: AtoPilot All BeadChips not yet downloaded All BeadChips BeadChips by Prchase Order BeadChips by barcode 2 Enter the reqired information. 3 Locate the DMAP folder that yo want to download. 4 Make sre that yo have sfficient free space on the download destination. 5 Start the download. View the download stats on the Download Stats and Log tab. 6 Save the DMAP folder to the specified DMAP folder location. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 26

32 NextSeq 550 System Gide Access DMAP Folder by BeadChip 1 Identify BeadChips sing 2 of the following options: BeadChip barcode BeadChips box ID Prchase order nmber Sales order nmber 2 Locate the DMAP folder that yo want to download. 3 Make sre that yo have sfficient free space on the download destination. 4 Start the download. View the download stats on the Download Stats and Log tab. 5 Save the DMAP folder to the specified DMAP folder location. Load the BeadChip Onto the Adapter 1 Press down on the adapter retention clip. The clip tilts back slightly to open. 2 Holding the BeadChip by the edges, position the BeadChip with the barcode near the retention clip and place the BeadChip onto the recessed shelf of the adapter. Figre 20 Load BeadChip Onto Adapter 3 Using the openings on either side of the BeadChip, make sre that the BeadChip is seated in the recessed shelf of the adapter. Figre 21 Seat and Secre BeadChip 4 Gently release the retention clip to secre the BeadChip. 5 Inspect the BeadChip from a side view to make sre that the BeadChip is sitting flat on the adapter. Reposition the BeadChip, if necessary. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 27

33 NextSeq 550 System Gide Figre 22 Inspect BeadChip Position A B Correct position BeadChip is flat on adapter when clip is released. Incorrect position BeadChip is not flat when clip is released. Set Up a Scan 1 From the Home screen, select Experiment, and then select Scan. The Scan command opens the imaging compartment door, releases consmables from a previos rn (if present), and opens the series of scan setp screens. A short delay is normal. Unload Seqencing Consmables If sed seqencing consmables are present when yo are setting p a scan, the software prompts yo to nload the reagent cartridge and bffer cartridge before proceeding to the next step. 1 If prompted, remove sed seqencing consmables from a previos seqencing rn. a b Remove the reagent cartridge from the reagent compartment. Dispose of nsed contents in accordance with applicable standards. Remove the sed bffer cartridge from the bffer compartment. 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 Close the reagent compartment and bffer compartment doors. Load the BeadChip Adapter 1 Use the alignment pins to position the BeadChip adapter on the stage. Figre 23 Load the BeadChip Adapter Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 28

34 NextSeq 550 System Gide 2 Select Load. The door closes atomatically, the BeadChip ID appears on the screen, and the sensors are checked. A short delay is normal. If the BeadChip barcode cannot be read, a dialog box appears that allows yo to enter the barcode manally. See Software Cannot Read the BeadChip Barcode on page Select Next. Scan Setp 1 On the Scan Setp screen, confirm the following information: Barcode The software reads the BeadChip barcode when the BeadChip is loaded. If the barcode was entered manally, the Edit btton appears for frther changes. Type The BeadChip type field is atopoplated based on the BeadChip barcode. DMAP Location The DMAP folder location is specified on the BeadChip Scan Configration screen. To change the location for the crrent scan only, select Browse and navigate to the correct location. Otpt Location The otpt location is specified on the BeadChip Scan Configration screen. To change the location for the crrent scan only, select Browse and navigate to the preferred location. 2 Select Next. Review Atomated Check The software performs an atomated check of the system. Dring the check, the following indicators appear on the screen: Gray checkmark The check has not been performed yet. Progress icon The check is in progress. Green checkmark The check passed. Red X The check did not pass. For any items that do not pass, an action is reqired before yo can proceed. See Resolve Atomatic Check Errors on page 37. To stop an atomated check in progress, select the select the icon. The check resmes at the first incomplete or failed check. To view the reslts of each individal check within a category, select the Start the Scan When the atomated check is complete, select Start. The scan begins. icon in the lower-right corner. To restart the check, icon to expand the category. To configre the system to start the scan atomatically after a sccessfl check, see Set Rn Setp Options on page 10. Monitor Scan Progress 1 Monitor scan progress sing the BeadChip image. Each color on the image indicates the scanning stats. Light gray Not scanned Dark gray Scanned bt not registered. Green Scanned and registered sccessflly. Red Scan and registration failed. If registration fails, yo can rescan samples that contain failed sections. See BeadChip Scan Failre on page 42. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 29

35 NextSeq 550 System Gide 2 Select the BeadChip image to toggle between a fll view and a detail view of a selected sample. The fll view shows the samples on the BeadChip and sections within each sample. The detail view shows each section within the selected sample. Figre 24 BeadChip Image: Fll View and Detail View NOTE Ending a scan is final. If yo end the scan before the scan is complete, scan data are not saved. Data Transfer Data are qeed for transfer to the scanning otpt folder when the scan is complete. Data are temporarily written to the instrment compter. The temporary folder is deleted from the instrment compter atomatically when a sbseqent scan is started. The time reqired to transfer data depends on yor network connection. Before beginning a sbseqent scan, make sre that data have been written to the otpt folder. To check, make sre that GTC files are present in the barcode folder. For more information, see Scanning Otpt Folder Strctre on page 57. If the connection is interrpted, data transfer resmes atomatically when the connection is restored. Each file has a timer of 1 hor after it has been qeed for transfer to the otpt folder. When the timer expires or if the instrment is rebooted before transfer is complete, data are not written to the otpt folder. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 30

36 Chapter 5 Maintenance Introdction 31 Perform a Manal Wash 31 Software Updates 33 Sht Down the Instrment 35 Introdction Maintenance procedres inclde manal instrment washes and system software pdates when available. Instrment washes An atomatic post-rn wash after each seqencing rn maintains instrment performance. However, a manal wash is reqired periodically nder certain conditions. See Perform a Manal Wash on page 31. Software pdates When an pdated version of the system software is available, yo can install the pdate atomatically throgh a connection to BaseSpace or manally after downloading the installer from the Illmina website. See Software Updates on page 33. 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 Wash Manal washes are initiated from the Home screen. 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 Flshes the system with a ser-spplied wash soltion of laboratory-grade water and Tween 20 (bffer wash cartridge). Reqired every 14 days that the instrment is idle with reagent cartridge and bffer cartridge in place. Reqired every 7 days that the instrment is in a dry state (reagent cartridge and bffer cartridge removed). Reqired after a shtdown. Flshes the system with a ser-spplied wash soltion of laboratory-grade water and Tween 20 (bffer wash cartridge) and 0.12% sodim hypochlorite (reagent wash cartridge). Reqired if the atomatic post-rn wash was not performed. A manal wash reqires the reagent wash cartridge and bffer wash cartridge provided with the instrment, and a sed flow cell. A sed flow cell can be sed p to 20 times for instrment washes. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 31

37 NextSeq 550 System Gide Figre 25 Reagent Wash Cartridge and Bffer Wash Cartridge Prepare for a Manal Post-Rn Wash User-Spplied Consmables Volme and Description NaOCl 1 ml, dilted to 0.12% Loaded onto the reagent wash cartridge (position #28) 100% Tween 20 Laboratory-grade water NOTE Used to make 125 ml 0.05% Tween 20 wash soltion Loaded onto the bffer wash cartridge (center reservoir) 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. 1 Combine the following volmes in a microcentrifge tbe to reslt in 1 ml 0.12% NaOCl: 5% NaOCl (24 µl) Laboratory-grade water (976 µl) 2 Invert the tbe to mix. 3 Add 1 ml of 0.12% NaOCl to the reagent wash cartridge. The correct reservoir is eqivalent to position #28 on the prefilled cartridge. Figre 26 Load NaOCl 4 Combine the following volmes to reslt in a 0.05% Tween 20 wash soltion: 100% Tween 20 (62 µl) Laboratory-grade water (125 ml) 5 Add 125 ml wash soltion to the center reservoir of the bffer wash cartridge. 6 Select Perform Wash, and then select Manal Post-Rn Wash. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 32

38 NextSeq 550 System Gide Prepare for a Qick Wash User-Spplied Consmables 100% Tween 20 Laboratory-grade water Volme and Description Used to make 40 ml 0.05% Tween 20 wash soltion Loaded onto bffer wash cartridge (center reservoir) 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 center reservoir of the bffer wash cartridge. 3 Select Perform Wash, and then select Qick Wash. Load a Used Flow Cell and the Wash Cartridges 1 If a sed flow cell is not present, load a sed flow cell. Select Load, and then select Next. 2 Remove the spent reagents container 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. 3 Slide the empty spent reagents container into the bffer compartment ntil it stops. 4 Remove the sed bffer cartridge from the previos rn, if present. 5 Load the bffer wash cartridge containing wash soltion. 6 Remove the sed reagent cartridge from the previos rn, if present. 7 Load the reagent wash cartridge. 8 Select Next. The prewash check begins atomatically. Start the Wash 1 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 cartridges 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: Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 33

39 NextSeq 550 System Gide NextSeq Control Software (NCS) NextSeq recipes RTA2 NextSeq Service Software (NSS) Seqencing Analysis Viewer (SAV) BaseSpace Broker 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 NextSeq 550 spport page on the Illmina website. 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 c Accept the licensing agreement. Review the release notes. 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. 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 c Accept the licensing agreement. Review the release notes. Review the list of software inclded in the pdate. When the pdate is complete, the control software restarts atomatically. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 34

40 NextSeq 550 System Gide NOTE If a firmware pdate is inclded, an atomatic restart of the system is reqired after the firmware is pdated. Sht Down the Instrment 1 Select Manage Instrment. 2 Select System Power Options. 3 Select Sht Down. The Sht Down command safely shts down the software and trns off instrment power. Wait at least 60 seconds before trning on the instrment again. 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 # v04 For Research Use Only. Not for se in diagnostic procedres. 35

41 Appendix A Trobleshooting Introdction 36 Trobleshooting Files 36 Resolve Atomatic Check Errors 37 Spent Reagents Container is Fll 39 Rehybridization Workflow 40 BeadChip and Scan Errors 42 Cstom Recipes and Recipe Folders 43 System Check 43 RAID Error Message 45 Configre System Settings 45 Introdction For technical qestions, visit the NextSeq 550 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 61. Consider sharing a link to the rn smmary in BaseSpace with Illmina Technical Spport to facilitate trobleshooting. Trobleshooting Files An Illmina Technical Spport representative might reqest copies of rn-specific or scan-specific files to trobleshoot isses. Typically, the following files are sed for trobleshooting. Trobleshooting Files for Seqencing Rns 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, part 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) RTA Logs Log of RTA errors. Error log files are pdated whenever an error occrs. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 36

42 NextSeq 550 System Gide Key File Folder Description Global log files (*GlobalLog*.tsv) Lane log files (*LaneLog*.txt) RTA Logs RTA Logs 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. 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. Trobleshooting Files for Array Scans Key File Folder Description Scan parameters file (ScanParameters.xml) Root folder Contains information abot scan parameters. Information incldes scan date, BeadChip barcode, clster file location, and manifest file location. Log files Logs Log files describe each step performed on the instrment dring the scan. Metrics files [Barcode] Metrics are provided as sample metrics and as section metrics. [barcode]_sample_metrics.csv For each sample and channel (red and green), lists Percent Off Image, Percent Otliers, P05, P50, P95, Avg FWHM Avg, FWHM Stddev, and Min Registration Score. [barcode]_section_metrics.csv For each section and tile, lists Laser Z-position, Throgh Focs Z-position, Red FWHM, Green FWHM, Red Avg Pixel Intensity, Green Avg Pixel Intensity, Red Registration Score, and Green Registration Score. Rescan file [Barcode] [barcode]_rescan.flowcell Lists the tile locations adjsted for a rescan, which inclde an increased tile-to-tile overlap. Resolve Atomatic Check Errors If errors occr dring the atomatic check, se the following recommended actions to resolve the error. Atomatic checks differ for seqencing and array scans. 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. It is possible that data from a previos rn did not transfer. Clear rn data from the instrment hard drive. The network connection has been interrpted. Check network stats and the physical network connection. Either the BaseSpace accont is fll or the network server is fll. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 37

43 NextSeq 550 System Gide Temperatre Temperatre Temperatre Sensors Fans Recommended Action Contact Illmina Technical Spport. Contact Illmina Technical Spport. Contact Illmina Technical Spport. Imaging System Imaging Limits Z Steps-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. The imaging compartment door opens. Unload and reload the flow cell to make sre that it is seated properly. Reagent Delivery Valve Response Pmp Bffer Mechanism Spent Reagents Empty Recommended Action Contact Illmina Technical Spport. Contact Illmina Technical Spport. Contact Illmina Technical Spport. Empty the spent reagents container and reload the empty container. Checks for Seqencing Rns 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. It is possible that data from a previos rn did not transfer. Clear rn data from the instrment hard drive. The network connection has been interrpted. Check network stats and the physical network connection. Either the BaseSpace accont is fll or the network server is fll. Temperatre Temperatre Temperatre Sensors Fans Recommended Action Contact Illmina Technical Spport. Contact Illmina Technical Spport. Contact Illmina Technical Spport. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 38

44 NextSeq 550 System Gide Imaging System Imaging Limits Z Steps-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. The imaging compartment door opens. Unload and reload the flow cell to make sre that it is seated properly. Reagent Delivery Valve Response Pmp Bffer Mechanism Spent Reagents Empty Recommended Action Contact Illmina Technical Spport. Contact Illmina Technical Spport. Contact Illmina Technical Spport. Empty the spent reagents container and reload the empty container. Checks for Array Scans System Checks Doors Closed Consmables Loaded Reqired Software Verify Inpt Files 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. Make sre that the path to the clster file and manifest file is correct and the files are present. The instrment hard drive does not have sfficient disk space to perform a rn. It is possible that data from a previos rn did not transfer. Clear rn data from the instrment hard drive. The network connection has been interrpted. Check network stats and the physical network connection. Either the BaseSpace accont is fll or the network server is fll. Imaging System Imaging Limits Z Steps-and- Settle Bit Error Rate Ato-Center Recommended Action Contact Illmina Technical Spport. Contact Illmina Technical Spport. Contact Illmina Technical Spport. Unload the BeadChip adapter. Make sre that the BeadChip is seated in the adapter, and then reload the adapter. Spent Reagents Container is Fll Always begin a rn with an empty spent reagents container. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 39

45 NextSeq 550 System Gide If yo begin a rn withot emptying the spent reagents container, system sensors trigger the software to pase the rn when the container is fll. System sensors cannot pase a rn dring clstering, paired-end resynthesis, or the atomatic post-rn wash. When the rn pases, a dialog box opens with options to raise the sippers and empty the fll container. Empty Spent Reagents Container 1 Select Raise Sippers. 2 Remove the spent reagents container and discard the contents appropriately. 3 Retrn the empty container to the bffer compartment. 4 Select Contine. The rn resmes atomatically. Rehybridization Workflow A rehybridization rn might be necessary if metrics generated dring the first few cycles show intensities below 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, 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. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 40

46 NextSeq 550 System Gide Reqired Consmables A rehybridization rn reqires a new NextSeq reagent cartridge and bffer 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. Saving the flow cell does not ensre that the crrent rn will be salvageable. 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. NOTE Yo can store the flow cell p to 7 days at 2 C to 8 C in the plastic clamshell case withot the desiccant package. 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 Wash on page 31. NOTE If yo have not removed the reagent cartridge and bffer cartridge from the stopped rn, yo can se them for the manal wash. Otherwise, perform the manal wash with the reagent wash cartridge and bffer 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. 4 Remove the spent reagents container and discard the contents appropriately, and then reload the empty container. 5 Load the new bffer cartridge and reagent cartridge. 6 From the Rn Setp screen, select from the following options: BaseSpace or BaseSpace Onsite Select the rn and confirm rn parameters. Standalone Enter the name of the rn and specify the same parameters as the original rn. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 41

47 NextSeq 550 System Gide 7 Select Next to proceed to the pre-rn check and start the rn. BeadChip and Scan Errors Software Cannot Read the BeadChip Barcode When the barcode error dialog box appears, select from the following options: Select Rescan. The software attempts to read the barcode again. Select text field and enter the nmeric barcode as shown in the image. Depending on the BeadChip, barcode nmbers have p to 12 digits. Select Save. The barcode image is stored in the otpt folder. Select Cancel. The imaging compartment door opens to nload the BeadChip adapter. BeadChip Scan Failre Images are registered after they are scanned. Registration identifies beads by correlating locations on the scanned image with information provided in the bead map, or DMAP folder. Sections that fail registration are indicated in red on the BeadChip image. Figre 27 BeadChip Showing Failed Sections After the scan is complete and scanning data are written to the otpt folder, the Rescan btton becomes active. When Rescan is selected, the software performs the following steps: Rescans samples that contain failed sections sing an increased tile-to-tile overlap. Generates otpt files in the original otpt folder. Overwrites previos otpt files for failed sections. Increments the scan conter by 1 for each rescan, bt does so in the backgrond. The software does not rename the otpt folder. Rescan or Start New Scan 1 Select Rescan to scan samples that contain failed sections. 2 If the scan contines to fail, end the scan. 3 Remove the BeadChip and adapter and inspect the BeadChip for dst or debris. Use canned air or other compressed dsting method to clear the debris. 4 Reload the BeadChip and start a new scan. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 42

48 NextSeq 550 System Gide When a new scan is started, the software performs the following steps: Scans the entire BeadChip. Generates otpt files in a new otpt folder. Increments the scan conter by 1 based on the scan cont of the last rescan. Replace Manifest Files and Clster Files 1 Go to the Illmina spport page (spport.illmina.com) for the BeadChip that yo are sing, and click the Downloads tab. 2 Download the files to be replaced or pdated, and copy the files to yor preferred network location. NOTE Make sre that yo select manifest and clster files that are compatible with the NextSeq 550 system. Compatible files inclde NS550 in the file name. 3 Only if the location has changed, pdate the location on the BeadChip Scan Configration screen, as follows: a b c From the NCS Home screen, select Manage Instrment. Select System Configration. Select BeadChip Scan Configration. 4 Select Browse and navigate to the location of the replaced or pdated files. Cstom Recipes and Recipe Folders Do not modify original recipes. Always make a copy of the original recipe with a new name. If an original recipe is modified, the software pdater can no longer recognize the recipe for later pdates, and newer versions are not installed. Store cstom recipes in the appropriate recipe folder. Recipe folders are organized as follows. Cstom High Cstomized recipes sed with a high-otpt kit. Mid Cstomized recipes sed with a mid-otpt kit. High Original recipes sed with a high-otpt kit. Mid Original recipes sed with a mid-otpt kit. Wash Contains the manal wash recipe. 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. Starting a system check atomatically closes the control software and lanches the NextSeq Service Software (NSS). The service software lanches and opens to the Load screen, which is configred to se the advanced loading option. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 43

49 NextSeq 550 System Gide Figre 28 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 Load the consmables as follows: a b c d If a sed flow cell is not already on the instrment, load a sed flow cell. NOTE Illmina recommends sing a high otpt flow cell for system check prposes. Empty the spent reagents container and retrn it to the instrment. Load the bffer wash cartridge containing 120 ml laboratory-grade water in the center reservoir. Load the reagent wash cartridge. Make sre that the reagent wash cartridge is empty and clean. 3 Select Load. The software moves the flow cell and reagent wash cartridge into position. Select Next. 4 Select Next. The system check begins. 5 [Optional] When the system check is complete, select View next to the check name to view the vales associated with each check. 6 Select Next. The system check report opens. 7 Select Save to save the report to a zipped file. Navigate to a network location to save the file. 8 When finished, select Exit. 9 When prompted to close the service software and restart the control software, select Yes. The control software restarts atomatically. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 44

50 NextSeq 550 System Gide Motion Checks System Check BSM FCLM & FAM Stage Tests Description Checks the gain and distance of the Bottle Straw Mechanism (BSM) to confirm that the modle is working properly. Checks the gain and distance of the Flow Cell Load Mechanism (FCLM) and Flid Atomation Modle (FAM) to confirm that the modles are working properly. Checks the travel limits and performance of the XY-stage and 6 Z-stages, 1 for each camera. 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. RAID Error Message The NextSeq compter is eqipped with 2 hard drives. If a hard drive begins to fail, the system generates a RAID error message and sggests that yo contact Illmina Technical Spport. Usally, a hard drive replacement is reqired. Yo can proceed with the rn setp steps and normal operation. The prpose of the message is for schedling service in advance to avoid interrptions in normal instrment operation. To proceed, select Acknowledge and then Close. Configre System Settings The system is configred dring installation. However, if a change is reqired or if the system has to be reconfigred, se the system configration options. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 45

51 NextSeq 550 System Gide Network Configration Provides options for IP address settings, domain name server (DNS) address, compter name, and domain name. BaseSpace Configration Provides options for analysis methods, inclding BaseSpace, BaseSpace Onsite, standalone mode, and rn monitoring in BaseSpace, and settings for a defalt BaseSpace login and instrment health reporting. BeadChip Scan Configration Provides options to specify the defalt DMAP folder location, the otpt folder location, the file format of saved images, and the otpt file type. 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 , 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 to advance to the Compter screen. 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. 6 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. 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. 7 Select Save. Set BaseSpace Configration 1 From the Manage Instrment screen, select System Configration. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 46

52 NextSeq 550 System Gide 2 Select BaseSpace Configration. 3 Select from the following options to specify a location where data are transferred for sbseqent analysis. Select BaseSpace to send seqencing data to Illmina BaseSpace. [Optional] Select the Otpt Folder checkbox, select Browse, and navigate to a secondary network location to save BCL files in addition to BaseSpace. Select BaseSpace Onsite. In the Server Name field, enter the fll path to yor BaseSpace Onsite server. [Optional] Select the Otpt Folder checkbox, select Browse, and navigate to a secondary network location to save BCL files in addition to the BaseSpace Onsite server. Select Standalone instrment to save data to a network location only. Select Browse and navigate to a preferred network location. The control software generates the otpt folder name atomatically. [Optional] Select Use Rn Monitoring to monitor the rn sing visalization tools on BaseSpace. A BaseSpace login and internet connection is reqired. 4 Select Save to advance to the BaseSpace screen. 5 If yo selected BaseSpace or BaseSpace Onsite, set the BaseSpace parameters as follows. Enter a BaseSpace User Name and Password to register the instrment with BaseSpace. Select Use defalt login and bypass the BaseSpace login screen to set the registered ser name and password as the defalt login. This setting bypasses the BaseSpace screen dring rn setp. 6 Select Send instrment health information to Illmina to enable the Illminia 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 NCS 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 # ). This service: Does not send seqencing data. Reqires that the instrment be connected to a network with internet access. Reqires that the instrment be connected to BaseSpace Seqence Hb. NOTE This option is not available for BaseSpace Onsite Seqence Hb. Is trned on by defalt. To opt ot of this service, disable the Send instrment health information to Illmina setting. 7 Select Save. BeadChip Scan Configration 1 From the Manage Instrment screen, select System Configration. 2 Select BeadChip Scan Configration. 3 To specify a defalt DMAP folder location, select Browse and navigate to the preferred folder location on yor facility network. NOTE Before each scan, download and copy the DMAP content to this location. DMAP content is reqired for each BeadChip and is niqe to each BeadChip barcode. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 47

53 NextSeq 550 System Gide 4 To specify a defalt otpt location, select Browse and navigate to the preferred location on yor facility network. 5 Select an image file format for saved images. The defalt image type is JPG. 6 Select an otpt file format for scan data. The defalt otpt file type is GTC only. 7 Select Save. 8 From the Scan Map screen, specify the fll path to the manifest file and clster file for each BeadChip type. Select Browse for each file type and navigate to the folder location that contains these files. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 48

54 Appendix B Real-Time Analysis Real-Time Analysis Overview 49 Real-Time Analysis Workflow 50 Real-Time Analysis Overview The NextSeq 550 ses an implementation of Real-Time Analysis (RTA) software called RTA2. RTA2 rns on the instrment compter and extracts intensities from images, performs base calling, and assigns a qality score to the base call. RTA2 and the control software commnicate throgh a web HTTP interface and shared memory files. If RTA2 is terminated, processing does not resme and rn data are not saved. NOTE Demltiplex performance is not calclated. Therefore, the Index tab in Seqencing Analysis Viewer (SAV) is not poplated. RTA2 Inpts RTA2 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. RTA.exe.config, which is a software configration file in XML format. RTA2 receives commands from the control software abot the location of RnInfo.xml and whether an optional otpt folder is specified. RTA v2 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. All other files are spporting otpt files. 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. Otpt files are sed for downstream analysis in BaseSpace. Alternatively, se bcl2fastq conversion software for FASTQ conversion and third-party analysis soltions. NextSeq files reqire bcl2fastq v2.0, or later. For the latest version of bcl2fastq, visit the NextSeq downloads page on the Illmina website. RTA v2 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 reqired to view real-time metrics sing the Seqencing Analysis Viewer (SAV). For the latest version of SAV, visit the SAV downloads page on the Illmina website. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 49

55 NextSeq 550 System Gide Error Handling RTA2 creates log files and writes them to the RTALogs folder. Errors are recorded in an error file in *.tsv file format. The following log and error files are transferred to the final otpt destination at the end of processing: *GlobalLog*.tsv smmarizes important rn events. *LaneNLog*.tsv lists processing events for each lane. *Error*.tsv lists errors that occrred dring a rn. *WarningLog*.tsv lists warnings that occrred dring a rn. 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. For any index seqences, RTA v2 reqires at least 1 base other than G in the first 2 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 base calls are generated for that tile in that cycle. Use the Seqencing Analysis Viewer (SAV) software to examine thmbnail images and identify images that failed registration. 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. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 50

56 NextSeq 550 System Gide Phasing occrs when a base falls behind. Prephasing occrs when a base jmps ahead. Figre 29 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 NextSeq 550 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 30 Visalization of Clster Intensities Table 1 Base Calls in 2-Channel Seqencing Base 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 clster location. T 0 (off) 1 (on) Clsters that show intensity in the green channel only. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 51

57 NextSeq 550 System Gide 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 2-channel analysis, RTA2 ses a poplation-based system to determine the chastity of a base call. Clsters pass filter (PF) when no more than 1 base call in the first 25 cycles has a chastity of < Clsters that do not pass filter are not base called. Indexing Considerations The process for base calling index reads differs from base calling dring other reads. Index reads mst begin with at least 1 base other than G in either of the first 2 cycles. If an Index Read begins with 2 base calls of G, no signal intensity is generated. Signal mst be present in either of the first 2 cycles to ensre demltiplexing performance. To increase demltiplexing robstness, select index seqences that provide signal in at least 1 channel, preferably both channels, for every cycle. Following this gideline avoids index combinations that reslt in only G bases at any cycle. Red channel A or C Green channel A or T This base calling process ensres accracy when analyzing low-plex samples. 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. 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. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 52

58 Appendix C Otpt Files and Folders Seqencing Otpt Files 53 Seqencing Otpt Folder Strctre 56 Scanning Otpt Files 57 Scanning Otpt Folder Strctre 57 Seqencing Otpt Files File Type Base call files Base call index file Clster location files Filter files InterOp files RTA configration file Rn information file Thmbnail files File Description, Location, and Name Each tile analyzed is inclded in a base call file, aggregated in 1 file for each lane, for each cycle. The aggregated file contains the base call and encoded qality score for every clster for that lane. Data\Intensities\BaseCalls\L00[X] Files are stored in 1 folder for each lane. [Cycle].bcl.bgzf, where [Cycle] represents the cycle nmber in 4 digits. Base call files are compressed sing block gzip compression. For each lane, a binary index file lists the original tile information in a pair of vales for each tile, which are tile nmber and nmber of clsters for the tile. Base call index files are created the first time a base call file is created for that lane. Data\Intensities\BaseCalls\L00[X] Files are stored in 1 folder for each lane. s_[lane].bci For each tile, the XY coordinates for every clster are aggregated into 1 clster location file for each lane. Clster location files are the reslt of template generation. Data\Intensities\L00[X] Files are stored in 1 folder for each lane. s_[lane].locs The filter file specifies whether a clster passed filters. Filter information is aggregated into 1 filter file for each lane and read. Filter files are generated at cycle 26 sing 25 cycles of data. Data\Intensities\BaseCalls\L00[X] Files are stored in 1 folder for each lane. s_[lane].filter Binary reporting files sed for Seqencing Analysis Viewer (SAV). InterOp files are pdated throghot the rn. InterOp folder Created at the beginning of the rn, the RTA configration file lists settings for the rn. [Root folder], RTAConfigration.xml Lists the rn name, nmber of cycles in each read, whether the read is an indexed read, and the nmber of swaths and tiles on the flow cell. The rn info file is created at the beginning of the rn. [Root folder], RnInfo.xml A thmbnail image for each color channel (red and green) for tiles 1, 6, and 12 from all cameras, top and bottom srfaces, at every cycle dring imaging. Thmbnail_Images\L00[X]\C[X.1] Files are stored in 1 folder for each lane and 1 sbfolder for each cycle. s_[lane]_[tile]_[channel].jpg In the file name, the tile is represented with a 5-digit nmber that indicates srface, swath, camera, and tile. For more information, see Tile Nmbering on page 55 and Thmbnail Image Naming on page 56. Flow Cell Tiles Tiles are small imaging areas on the flow cell defined as the field of view by the camera. The total nmber of tiles depends on the nmber of lanes, swaths, and srfaces that are imaged on the flow cell, and how the cameras work together to collect the images. High-otpt flow cells have a total of 864 tiles. Mid-otpt flow cells have a total of 288 tiles. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 53

59 NextSeq 550 System Gide Table 2 Flow Cell Tiles Flow Cell Component High Otpt Mid Otpt Description Lanes 4 4 A lane is a physical channel with dedicated inpt and otpt ports. Srfaces 2 2 The flow cell is imaged on 2 srfaces, the top and bottom. The top srface of 1 tile is imaged, then the bottom srface of the same tile is imaged before moving to the next tile. Swaths per lane 3 1 A swath is a colmn of tiles in a lane. Camera segments 3 3 The instrment ses 6 cameras to image the flow cell in 3 segments for each lane. Tiles per swath per camera segment A tile is the area on the flow cell that the camera sees as 1 image. Total tiles imaged The total nmber of tiles eqals lanes srfaces swaths camera segments tiles per swath per segment. Lane Nmbering Lanes 1 and 3, called lane pair A, are imaged at the same time. Lanes 2 and 4, called lane pair B, are imaged when imaging of lane pair A is complete. Figre 31 Lane Nmbering A Lane Pair A Lanes 1 and 3 B Lane Pair B Lanes 2 and 4 Swath Nmbering Each lane is imaged in 3 swaths. Swaths are nmbered 1 3 for high otpt flow cells. Figre 32 Swath Nmbering Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 54

60 NextSeq 550 System Gide Camera Nmbering The NextSeq 550 ses 6 cameras to image the flow cell. Cameras are nmbered 1 6. Cameras 1 3 image lane 1. Cameras 4 6 image lane 3. After lanes 1 and 3 are imaged, the imaging modle moves on the X-axis to image lanes 2 and 4. Figre 33 Camera and Segment Nmbering (High otpt flow cell shown) Tile Nmbering There are 12 tiles in each swath of each camera segment. Tiles are nmbered 01 12, regardless of swath nmber or camera segment, and represented in 2 digits. Figre 34 Tile Nmbering The complete tile nmber incldes 5 digits to represent the location, as follows: Srface 1 represents the top srface; 2 represents the bottom srface Swath 1, 2, or 3 Camera 1, 2, 3, 4, 5, or 6 Tile 01, 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, or 12 Example: Tile nmber indicates top srface, swath 2, camera 5, and tile 8. The complete 5-digit tile nmber is sed in the file name of thmbnail images and empirical phasing files. For more information, see Seqencing Otpt Files on page 53. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 55