illumina MiniSeq System Manual

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MiniSeq

System Guide

Document # 1000000002695 v02 Material # 20014309

March 2018

For Researc h Use Only. Not for use in diagnostic procedure s.

ILLUMINA P ROP RIETARY

MiniSeq System Guide

This docume nt a nd its c on tents are proprietary to Illumina, Inc. a nd its a ffiliates ("Illumina ") , and are inte nded sole ly for the contrac tual use of its custome r in conne ction with the use of the produc t(s) desc ribed herein a nd for no othe r purpose. This docume nt and its conten ts shall n ot be used or distribu te d f or a ny othe r pu rpose and/or oth erwise communicate d, disc losed, or reproduce d in any way wha tsoever without the prior written consent of Illum ina. Illumin a doe s n ot con ve y any lic ense un de r its pa tent, trademark, copyright, or c ommon -law rights nor similar righ ts of a ny th ird parties by this docume nt.

The instruction s in this documen t must be strictly and explicitly followe d by qualified and properly traine d pe rsonnel in orde r to ensure the proper and safe use of the product( s) described here in . All of th e conte nts of this docume nt must be fully rea d a nd unde rstood prior to using suc h product(s) .

FAILURE TO C OM PL ETELY READ A ND EXPLIC ITLY FOLLOW A LL OF THE INSTRUCTION S CON TAINED HEREIN MAY RESUL T IN DAMAGE TO THE PRODUC T( S), INJURY TO PERS ON S, INC LUDING TO USERS OR OTHERS, AND DAMAGE TO OTHER PROP ERTY , AND WIL L VOID ANY WA RRA NTY APPLICA BLE TO THE PRODUCT(S) .

ILLUMINA DOES N OT ASSUME A NY LIABILITY ARISING O UT O F THE IMPROPER USE OF TH E PRODUC T( S) DESC RIBED HEREIN (IN CLUDIN G PARTS THEREO F OR SOFTWA RE) .

All tra de ma rks a re the property of Illumina, Inc. or their respective own ers. F or specific trademark information , see

www .illumina.com/ company/lega l.h tml.

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MiniSeq System Guide

Revision History

Document Date Descri pti on of Change

Material # 2001 4309 Documen t # 100000000 2695 v 02

Material # 2001 4309 Documen t # 100000000 2695 v 01

Material # 2000 2370 Documen t # 100000000 2695 v 00

March

2018

Se pte mber

2016

Jan ua ry

2016

Added informa tion about the Illu mina Proac tive monitoring service in the Configure An alysis Settings se c tion. Removed the default user name and pa ssword required to log on to th e ope rating system. Illumina recommen ds using site-spe cific credentials. Min or text edits.

Updated software descriptions to MiniSe q C ontrol Software v1.1.8, which inc ludes De mo Mode . Updated the automa tic post-ru n wash duration to 60 minute s. Added a server c onfiguration step to the instruc tions for sele cting BaseS pa ce for a na ly sis. Noted that mapped drives are not supported by th e Loc a l Run Mana ge r so ftw are.

Initial re le ase.

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iii

Table of Contents

Chapter 1 Overview 1

Introduction 1 Additional Resources 1 Instrument Components 2 Sequencing Consumab les Overview 5 Preinstalled Databases and Genomes 7

Chapter 2 Getting Started 8

Starting the Instrument 8 Customize System Settings 8 Configure Analysis Settings 10 User-Supplied Consumables and Equipment 13

Chapter 3 Sequencing 16

Introduction 16 Sequencing Workflow 17 Prepare Consumables 19 Prepare Libraries for Sequencing 20 Set Up a Sequencing Run 20 Monitor Run Progress 28 Automatic Post-Run Wash 30 Remove Used Reservoir from Position #9 30

Chapter 4 Maintenance 32

Introduction 32 Perform a Manual Instrument Wash 32 Software Updates 35

Appendix A Troubleshooting 38

Troubleshooting Files 38 Automatic Check Errors 39 RTA Errors 40 Rehybridization Workflow 41 System Check 42 Network Configuration Settings 45 Custom Genomes 46 Shut Down the Instrument 46

Appendix B Real-Time Analysis 48

Real-Time Analysis Overview 48 Input and Output Files 48 Real-Time Analysis Workflow 49

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MiniSeq System Guide

Appendix C Output Files 52

Sequencing Outp ut Files 52 Sequencing Outp ut Folder Structure 52 Analysis Input File Requirements 53

Index 54

Technical Assistance 57

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Chapter 1 Overview

Introduction 1 Additional Resources 1 Instrument Components 2 Sequencing Consumab les Overview 5 Preinstalled Databases and Genomes 7

Introduction

The Illumina®MiniSeq ™ System provides high-quality, industry-standard Illumina sequencing technology with the convenience of an easy-to-use cost-effective desktop system.

Features

u High-quality sequencing—The MiniSeq System enables small genome, amplicon, targeted enrichment,

and RNA sequencing using low library volumes.

u MiniSeq system software—The MiniSeq System includes a suite of integrated software that controls

instrument operations, processes images, and generates base calls. The suite includes on-instrument data analysis software and data transfer tools for analysis using other options, such as BaseSpace.

u On-instrument data analysis—The Local Run Manager software analyzes run data according to the

analysis module sp ecified for the run. A suite of analysis modules are included in the software.

u BaseSpace

genomics computing environment for run monitoring, data analysis, storage, and collaboration. Output files are streamed in real time to BaseSpace or BaseSpace Onsite for analysis.

u Convenient consumab le loading—A clamp ing mechanism positions the flow cell as it is loaded onto the

instrument. A single-use prefilled reagent cartridge provides reagents required for a run and subsequent instrument wash. The flow cell and reagent cartridge include integrated identification to enable accurate tracking.

integration—The sequencing workflow is integrated with BaseSpace, the Illumina

Additional Resources

The MiniSeq System support pages on the Illumina website provide additional resources. These resources includ e software, training, compatible products, and the following documentation. Always check support pages for the latest versions.

Resource Descri pti on

Cu stom P rotoco l Selec tor

Min iSeq Syste m Site Prep Guide (documen t #

1000000002696)

Min iSeq Syste m Safety and Complianc e Guide (document #

1000000002698)

RFID Rea de r C omplia nce Guide (documen t #

1000000002699)

Document # 1000000002695 v02 Material # 20014309

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A wiza rd for gen erating customize d end-to-en d documen tation that is ta ilored to the library prep meth od, run para me te rs, and analysis me th od used for th e sequenc ing ru n.

Provide s spec ifications for laboratory spac e, e le ctrical requ ire me nts, a nd e nviron me ntal considerations.

Provide s informa tion a bout opera tiona l safety c onside rations, complian ce state me nts, an d instrume nt la be ling.

Provide s informa tion a bout the RFID reader in th e instrumen t, complia nce certifica tions, an d safety c on side ra tions.

MiniSeq System Guide

Resource Descri pti on

Min iSeq Syste m Denatu re an d Dilute Libraries Guide (document #

1000000002697)

Loca l Run Mana ge r Software Gu ide (doc ument # 1000 000002702)

Provide s instructions for denaturin g and diluting prepa red libraries for a seque ncing run, an d prepa rin g an optiona l P hiX c on trol.

Provide s informa tion a bout using the Loca l Run Man ager sof twa re an d a va ilable analysis options.

Instrument Components

The MiniSeq System includes a touch screen monitor, a status bar, a flow cell compartment, and a reagent compartment.

Figure 1 Instr ument Components

A Touch screen monitor—Enables on-instrument configuration and setup using the control software interface. B Power button–Turns on the integrated instrument computer and operating system. C USB ports—Convenient connections for peripheral components. D Flow cell compartment—Holds the flow cell during a sequencing run. E Status bar—Indicates instrument status as processing (blue), requires attention (orange), ready to sequence

(green), or when a wash is due within the next 24 hours (yellow).

F Reagent compartment—Holds the reagent cartridge and spent reagents bottle.

Flow Cell Compartment

The flow cell stage includes the flow cell latch, which secures the flow cell when it is closed. As the latch closes, pins near the base of the latch align the flow cell ports with fluidics connections.

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MiniSeq System Guide

Figure 2 Flow Cell Compartment

A Flow cell compartment door B Flow cell latch C Flow cell stage D Flow cell E Flow cell latch release button

The thermal station, located beneath the flow cell stage, controls changes in temperature req uired for cluster generation and sequencing.

NOTE

Do not place objects on the instrument near the flow cell compartment.

Reagent Compartment

Setting up a sequencing run on the MiniSeq System requires access to the reagent compartment to load run consumables and empty the spent reagents bottle.

Figure 3 Reagent Compartment

A Spent reagents bottle—Includes a threaded cap to prevent spills when carrying. B Reagent cartridge—Provides reagents in a single-use prefilled consumable. C Reagent compartment door—Provides access to the reagent compartment.

The reagent compartment door opens outward from hinges at the bottom edge of the instrument. To open the door, gently pull forward from the side ed ges of the door.

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MiniSeq System Guide

NOTE

Do not place objects on the reagent compartment door. The compartment door is not designed for use as a shelf.

Power Button

The power button on the front of the instrument turns on power to the instrument and instrument computer. The power button performs the following actions depending on the state of instrument power.

Power Stat e Act ion

Instrume nt power is off Brie fly pre ss the button to tu rn on the power.

Instrume nt power is on Briefly press th e button to turn off the pow er. A dialog box appe ars on the screen to

confirm a n ormal instrume nt sh utdown .

Instrume nt power is on Press a nd hold the powe r button for 10 se c onds to c ause a hard shutdown of the

instrumen t a nd in strume nt computer. Use this method to turn off the in strument only if the instrume nt is unre sponsive.

NOTE

Under normal conditions, do not turn off the instrument power.

Turning off the instrument during a sequencing run ends the run immediately. Ending a run is final. Run consumables cannot be reused and sequencing data are not saved.

System Software

The instrument software suite includes integrated applications that perform sequencing runs and oninstrument analysis.

u MiniSeq Control Software—The control software guides you through the step s to set up a sequencing

run, controls instrument operations, and shows an overview of run statistics as the run p rogresses.

u Real-Time Analysis (RTA) software—RTA performs image analysis and base calling during the run. See

Real-Time Analysis Overview

u Local Run Manager—Before sequencing, use Local Run Manager to specify run p arameters and the

on p age 48.

analysis method. After sequencing, on-instrument data analysis begins automatically. For more information, see the

Local Run Manager Software Guide (document # 1000000002702)

Status Icons

A status icon in the top-right corner of the control software interface screen indicates a change in condition during run setup or during the run.

Status Icon

Status Name

Sta tus OK System is norma l.

Proce ssing System is proce ssing.

Descri pti on

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MiniSeq System Guide

Status Icon

Status Name

Attention Attention is re quired.

Wa rning A warning has occurred.

Error An error ha s occ urre d.

Descri pti on

Wa rnings do not stop a run o r require action before proceeding.

Errors re quire ac tio n before procee ding with the run .

When a change in condition occurs, the icon blinks to alert you. Select the icon to view a description of the condition. Select Ack nowledge to accept the message and Close to close the dialog box.

Sequencing Consumables Overview

Performing a sequencing run on the MiniSeq System requires a single-use MiniSeq Kit. Each kit includes a flow cell and the reagents required for a sequencing run.

The flow cell and reagent cartridge use radio-frequency identification (RFID) for accurate consumable tracking and to ensure compatibility with specified run parameters.

Flow Cell

The flow cell is a glass-based substrate on which clusters are generated and the sequencing reaction is performed. The flow cell is encased in a flow cell cartridge.

Figure 4 Flow Cell Compo nents

A Imaging area B Flow cell gasket C Outlet port D Inlet port

Reagents enter the flow cell through the inlet port, pass through the single-lane imaging area, and then exit the flow cell through the outlet port.

The flow cell is shipped dry in a flow cell tube that is wrapped in a foil package. Store the flow cell in the sealed foil package at 2°C to 8°C until use. For more information, see

Prepare the Flow Cell

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MiniSeq System Guide

Reagent Cartridge Overview

The reagent cartridge is a single-use consumable with foil-sealed reservoirs that are prefilled with clustering, sequencing, and wash reagents.

Figure 5 Reagent Cartridge

The reagent cartridge includes a d esignated reservoir for load ing prepared libraries. After the run begins, libraries are transferred automatically from the reagent cartridge to the flow cell.

WARNING

This set of reagents contains potentially hazardous chemicals. Personal injury can occur through inhalation, ingestion, skin contact, and eye contact. Wear protective equipment, including eye protection, gloves, and laboratory coat appropriate for risk of exposure. Handle used reagents as chemical waste and discard in accordance with applicable regional, national, and local laws and regulations. For additional environmental, health, and safety information, see the SDS at support.illumina.com/sds.html.

Store the reagent cartridge at -25°C to -15°C until use. For more information, see

Cartridge

on p age 19.

Reserved Reservoirs

Figure 6 Numbered Reservoirs

Prepare the Reagent

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MiniSeq System Guide

Posit ion Descri pti on

13, 14, and 15 Reserved for optional c ustom prime rs

16 Load libraries

Removable Reservoir in Position #9

The prefilled reagent cartridge includes a denaturation reagent in position #9 that contains formamide. To facilitate safe disposal of any unused reagent after the sequencing run, this reservoir is removable. For more information, see

Remove Used Reservoir from Position #9

on p age 30.

Preinstalled Databases and Genomes

For most analysis methods, a reference is required to perform alignment. Several reference databases and genomes are preinstalled on the instrument computer.

Preinst al led Descri pti on

Databases

Genomes

• miRbase for human

• dbSNP for human

• RefGe ne for h uman

• Ara bidopsis thalia na

• cow (

Bos taurus

•

E. c oli

•

E. c oli

• fruit f ly (

• human (

strain DH10 B strain MG1655

Drosophila melanogaster

)

Homo sapiens

) build hg19

• mou se (

• PhiX

• rat (

•

)

Rhodobac ter sph aeroide s

• yeast (

•

Sta phylo coccu s a ureu s

Mus musc ulus

Rattus norvegic us

Sa cch aromyce s cerevisiae

)

NC TC 8325

2.4.1 S288C )

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Chapter 2 Getting Started

Starting the Instrument 8 Customize System Settings 8 Configure Analysis Settings 10 User-Supplied Consumables and Equipment 13

Starting the Instrument

Make sure that the instrument has been properly installed and initialized, and that instrument setup is complete. Starting the instrument before it is ready can damage the system.

1 Turn on the power toggle switch to the I (on) position.

Figure 7 Po wer Swit ch Located on Back o f Inst rument

2 Press the power button above the reagent compartment.

The power button turns on the integrated instrument computer and operating system.

Figure 8 Po wer Button Located on Front of I nst rument

3 Wait until the operating system has finished loading.

The MiniSeq Control Software launches and initializes the system automatically. The Home screen opens after the initialization step is complete.

4 Wait for the system to load, and then log on to the operating system. If necessary, consult your facility

administrator for the user name and password.

Customize System Settings

The control software includes customizable settings for instrument identification and the following workflow preferences:

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MiniSeq System Guide

u Purging consumables at end of run.

u Skipping pre-run check confirmation to start the run automatically.

u Using the on-screen keyboard for run setup steps.

u Enabling audio indicators.

u Performing a simulated run.

Customize Instrument Identification

1 From the Manage Instrument screen, select System Customization.

2 To assign an avatar image for the instrument, select Browse and navigate to a preferred image.

3 In the Nick Name field, enter a preferred name for the instrument.

4 Select Save to save settings and advance the screen.

The image and name appear in the upper-left corner of each screen.

Set Automatic Purge Option

1 From the Manage Instrument screen, select System Customization.

2 Select the Purge consumables at end of run checkbox.

This setting purges unused reagents from the reagent cartridge to the spent reagents bottle automatically after each run. If this setting is disabled, unused reagents remain in the reagent cartridge.

NOTE

Purging consumables automatically adds additional time to the workflow. For example, purging reagents after a 300-cycle run (2×151), takes approximately 50 minutes.

3 Select Save to save settings and exit the screen.

Set Automatic Start Option

1 From the Manage Instrument screen, select System Customization.

2 Select the Skip pre-run check confirmation checkbox.

This setting starts the sequencing run automatically after a successful automatic check. If this setting is disabled, start the run manually after the pre-run check.

3 Select Save.

Set Automatic Check for Software Updates

1 From the Manage Instrument screen, select System Customization.

2 Select the Automatically check for software updates on BaseSpace checkbox.

An internet connection is required.

3 Select Save to save settings and exit the screen.

Set On-Screen Keyboard Option

1 From the Manage Instrument screen, select System Customization.

2 Select the Use on-screen keyboard checkbox.

This setting enables the on-screen keyb oard for input during run setup steps.

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MiniSeq System Guide

3 Select Save.

Enable Audio Indicators

1 From the Manage Instrument screen, select System Customization.

2 Select the Play audio checkbox to turn on audio indicators for the following events.

u Upon instrument initialization u When a run is started u When errors occur u When user interaction is required u When a run has finished

3 Select Save.

Enable Demo Mode

Demo Mod e simulates a sequencing run to provide a demonstration of instrument features and function. Use Demo Mod e to mimic run setup without reagents, a flow cell, or other consumables.

1 From the Manage Instrument screen, select System Customization.

2 Select the Enable Demo Mode checkbox.

3 Select Save.

4 [Optional] Enter a 4-d igit PIN.

When this field is blank, exiting Demo Mode does not require a PIN.

5 Select OK to restart the software in Demo Mode.

After restart, the MiniSeq Control Software banner reads Demonstration Only.

Exit Demo Mode

1 From the Manage Instrument screen, select System Customization.

2 Clear the Enable Demo Mod e checkbox.

3 Select Save.

4 If a PIN is required, enter it when prompted.

5 Select OK to restart the software in the standard mode.

Configure Analysis Settings

The control software provides the following options for data transfer and subsequent analysis:

u BaseSpace—Sends data to Illumina BaseSpace for analysis on BaseSpace.

u BaseSpace Onsite—Sends d ata to the BaseSpace Onsite LTserver for analysis on the server.

u Local Run Manager—Sends data to a specified output folder location for analysis using a Local Run

Manager module.

u Standalone—Saves data to a specified output folder location for later analysis.

Select BaseSpace

1 From the Manage Instrument screen, select System Configuration.

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MiniSeq System Guide

2 Select Analysis Configuration.

3 Select BaseSpace.

4 [Optional] Specify default BaseSpace login credentials as follows.

a Enter a User name and Password. b Select the Save the credentials as the default checkbox. c Select the Bypass Analysis Method login screen checkbox.

5 In the Server Name field, enter the full path to your BaseSp ace server. For the EUserver, replace the

USserver name with https://api.euc1.sh.basespace.illumina.com. The default US server is https://api.basespace.illumina.com.

6 [Optional] Select the Output Folder checkb ox, select Browse, and navigate to a preferred output folder

location. This setting saves a copy of base call (BCL) files in the specified location.

7 [Optional] Select Send instrument health information to Illumina to enable the Illumina Proactive monitoring

service. The name of the setting in the software interface might be different from the name in this guide, depending on the version of MiniSeq Control Software in use. With this setting turned on, instrument performance data are sent to Illumina. This data helps Illumina troub leshoot more easily and detect potential failures, enabling proactive maintenance and maximizing instrument uptime. For more information on the benefits of this service, see

Note (document # 1000000052503)

Illumina Proactive Technical

This service:

u Does not send sequencing data. u Requires that the instrument be connected to a network with internet access. u Is turned on by default. To opt out of this service, disable the Send instrument health information to

Illumina setting.

8 Select Save.

Select BaseSpace Onsite

1 From the Manage Instrument screen, select System Configuration.

2 Select Analysis Configuration.

3 Select BaseSpace Onsite.

4 [Optional] Specify default BaseSpace Onsite login credentials as follows.

a Enter a User name and Password. b Select the Save the credentials as the default checkbox. c Select the Bypass Analysis Method login screen checkbox.

5 In the Server Name field, enter the full path to your BaseSp ace Onsite server.

6 [Optional] Select the Output Folder checkb ox, select Browse, and navigate to a preferred output folder

location. This setting saves a copy of base call (BCL) files in the specified location.

7 [Optional] Select Send instrument health information to Illumina to enable the Illumina Proactive monitoring

service. The name of the setting in the software interface might be different from the name in this guide, depending on the version of MiniSeq Control Software in use.

Document # 1000000002695 v02 Material # 20014309

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MiniSeq System Guide

With this setting turned on, instrument performance data are sent to Illumina. This data helps Illumina troub leshoot more easily and detect potential failures, enabling proactive maintenance and maximizing instrument uptime. For more information on the benefits of this service, see

Note (document # 1000000052503)

Illumina Proactive Technical

This service:

Illumina setting.

8 Select Save.

Select Local Run Manager

1 From the Manage Instrument screen, select System Configuration.

2 Select Analysis Configuration.

3 Select Local Run Manager.

4 [Optional] Specify default Local Run Manager login credentials as follows.

a Enter a User name and Password. b Select the Save credentials as the default checkbox. c Select the Bypass Analysis Method login screen checkbox.

5 [Optional] Select Use Run Monitoring to monitor the contents using visualization tools on BaseSpace. A

BaseSpace login and internet connection are required.

6 [Optional] Specify default BaseSpace login credentials as follows.

a Enter a User name and Password. b Select the Save credentials as the default checkbox.

7 Select Browse and navigate to a preferred output folder location.

8 In the Contents pane of the output folder location, create a folder named Runs.

NOTE

Local Run Manager does not support mapped drives.

9 [Optional] Select Send instrument health information to Illumina to enable the Illumina Proactive monitoring

Note (document # 1000000052503)

Illumina Proactive Technical

This service:

Illumina setting.

10 Select Save.

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MiniSeq System Guide

Select Standalone

1 From the Manage Instrument screen, select System Configuration.

2 Select Analysis Configuration.

3 Select Standalone.

4 [Optional] Select Use Run Monitoring to monitor the run using visualization tools on BaseSpace. A

BaseSpace login and internet connection are required.

5 [Optional] Specify default BaseSpace login credentials as follows.

a Enter a User name and Password. b Select the Save credentials as the default checkbox.

6 Select Browse and navigate to a preferred output folder location.

7 [Optional] Select Send instrument health information to Illumina to enable the Illumina Proactive monitoring

Note (document # 1000000052503)

This service:

Illumina setting.

Illumina Proactive Technical

8 Select Save.

User-Supplied Consumables and Equipment

The following consumables and equipment are used for sequencing and system maintenance.

User-Supplied Consumables for Sequencing Runs

Cons umabl e Sup pli er Purpos e

1 N NaOH (sodiu m hydroxide )

200 mM Tris-H C l, pH7 General lab supplier L ibra ry neutra liz ation af ter de na turation

Isopropyl alc oh ol wipe s, 70% or Etha nol, 70%

Disposable gloves, powder-free Genera l lab supplier Gene ral pu rpose

La b tissue, low-lint VWR, ca ta log#21905 -026

Gene ral lab supplier Library den atura tion, dilu ted to 0.1N

VWR, ca ta log#95041 -714 (or equivalent) Gene ral lab supplier

(or equivalent)

Flow cell c le a ning and genera l purpose

Flow cell c le a ning

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MiniSeq System Guide

User-Supplied Consumables for Instrument Maintenance

Cons umabl e Sup pli er Purpos e

Na OC l, 5% (sodiu m hypochlorite)

Twe e n 20 Sigma -A ldrich , c a talog # P7949 Pe rformin g a manua l instru me nt wash;

Wa te r, laboratory-grade Gene ral lab supplier Pe rformin g a manua l instru me nt wash

Sigma -A ldrich , c a talog # 239305 (or la boratory-grade equivale nt)

Pe rformin g a manua l post-ru n wash; dilute d to 0.12%

dilute d to 0.05%

Guidelines for Laboratory-Grade Water

Always use laboratory-grade water or deionized water to perform instrument proced ures. Never use tap water. Use only the following grades of water or equivalents:

u Deionized water

u Illumina PW1

u 18Megohms (MΩ) water

u Milli-Q water

u Super-Q water

u Molecular biology grade water

User-Supplied Equipment

Item Source

Fre ezer, -25°C to -15°C , frost-fre e Gene ral lab supplier

Ice bucke t Gene ral lab supplier

Refrigera tor, 2°C to 8°C Gene ral lab supplier

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MiniSeq System Guide

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Chapter 3 Sequencing

Introduction

To perform a seq uencing run on the MiniSeq System, prepare run consumables and then follow the software prompts to set up the sequencing run.

Workflow Overview

Cluster Generation

During cluster generation, single DNA molecules are bound to the surface of the flow cell, and then amplified to form clusters.

Sequencing

Clusters are imaged using 2-channel sequencing chemistry and filter combinations specific to each of the fluorescently 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 sequencing. Following image analysis, the software performs base calling, filtering, and quality scoring.

Analysis

As the run progresses, the control software automatically transfers base call (BCL) files to the specified output location for data analysis. Several analysis methods are available depending on your application and the selected analysis configuration for your system. For more information, see

10.

Configure Analysis Settings

on p age

Sequencing Run Duration

Sequencing run duration depends on the number of cycles performed. The maximum run length is a 150cycle paired-end run, plus up to 2 index reads of 8 cycles each.

For expected durations and other system specifications, visit the MiniSeq System sp ecifications page on the Illumina website.

Number of Cycles in a Read

In a sequencing run, the number of cycles performed in a read is 1 more cycle than the number of cycles analyzed. For example, to perform a 150-cycle paired-end run, set up the run to perform 151 cycles p er read (2×151) for a total of 302 cycles. At the end of the run, 2×150 cycles are analyzed. The extra cycle in each read is used for phasing and prephasing calculations.

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MiniSeq System Guide

Sequencing Workflow

The run setup steps vary depending on the analysis configuration specified for the run.

u Standalone—After preparing and loading consumables, all run parameters are specified using the

MiniSeq Control Software interface.

u BaseSpace or BaseSpace Onsite, or Local Run Manager—Before setting up the run on the instrument,

assign a run name and specify run parameters using the BaseSpace Prep tab or the Local Run Manager software. After preparing consumables, select the run name from the MiniSeq Control Software, and then load consumables for the run.

Workflow (Standalone Configuration)

Pre pare a n ew re agent cartridge : thaw a nd in spe ct. Pre pare a n ew flow ce ll: bring to room tempera ture and the n unwrap.

Den ature and dilute libraries. S ee

1000000002697)

Load the libra ry dilution onto the rea ge nt cartridge in reservoir #16.

From th e software inte rfa ce, se le ct Sequenc e.

Load the flow c e ll and reagen t c artridge. Empty an d re load the spent reagents bottle.

Spe cify run parameters on the Run Setup screen . After the auto ma ted c he c k, selec t S ta rt.

Min iSeq Syste m Denatu re an d Dilute Libra rie s Guide (docume nt #

Mon itor the run on the instrumen t scre en, from Base Space if run monitoring is e nabled, or from a ne tworked computer usin g Sequenc ing A na lysis Viewer. Data are transfe rred to the spe cified output folder.

An instrumen t wash begins automatica lly whe n sequ enc ing is c omplete.

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Workflow (BaseSpace or Local Run Manager Configuration)

Spe cify run parameters using Ba se Spac e, Base Space Onsite , or Loca l Run Manager.

Pre pare a n ew re agent cartridge : thaw a nd in spe ct. Pre pare a n ew flow ce ll: bring to room tempera ture, unwrap, a nd in spe ct.

Den ature and dilute libraries. S ee

1000000002697)

Load the libra ry dilution onto the rea ge nt cartridge in reservoir #16.

From th e software inte rfa ce, se le ct Sequenc e to begin the run setu p steps.

Se le ct the run nam e specifie d o n Base Spac e, Base Space Onsite , or Loca l Run Manage r.

Load the flow c e ll. Load the rea ge nt cartridge . Empty an d re load the spent reagents bottle.

Min iSeq Syste m Denatu re an d Dilute Libra rie s Guide (docume nt #

Revie w run parameters on the Run Setup screen . After the auto ma ted c he c k, selec t S ta rt.

Mon itor the run on the instrumen t scre en, from Base Space or f rom a netw orke d c omputer using Se quen cing Analysis Vie we r. Data are transf erred to Ba se Spac e, Ba se Spac e Onsite , or the spe cified output folder.

An instrumen t wash begins automatica lly whe n sequ enc ing is c omplete.

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Prepare Consumables

Prepare the Reagent Cartridge

1 Remove thereagent cartridge from -25°C to -15°C storage.

2 Thaw reagents using the following water bath options. Do not submerge the cartridge. When thawed, dry

the base before proceed ing.

Method Time to Thaw Stabil i ty Li mit

37°C wa te r ba th 35 minute s Up to 2 hours

Room te mpera ture wate r bath (19°C to 25°C)

If multiple cartridges are thawing in the same water bath, allow additional thawing time. Alternatively, thaw reagents using the following options.

Method Time to Thaw Stabi lit y Limi t

Room te mpera ture air (19°C to 25°C)

Ch illed at 2°C to 8°C 18 hours Up to 72 hours

90 minute s Up to 24 hours

5 h ours Up to 24 hours

3 Invert the cartridge 5 times to mix reagents.

4 Inspect the large reservoirs from the bottom of the cartridge to make sure that reagents have thawed

and the reservoirs are free of ice crystals.

5 Gently tap on the bench to reduce air bubbles.

Prepare the Flow Cell

1 Remove a new flow cell package from 2°C to 8°C storage.

2 Set the unopened flow cell package aside at room temperature for 30 minutes.

NOTE

Avoid repeated cooling and warming of the flow cell.

3 Remove the flow cell container from the foil package.

4 Put on a new pair of powder-free gloves.

5 Grip the flow cell b y the plastic cartridge and remove it from the container.

Figure 9 Remo ve Flo w Cell

6 Clean the glass surface of the flow cell with a lint-free alcohol wipe.

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7 Dry with a lint-free lens cleaning tissue. Use care around the black flow cell gasket.

8 Inspect the flow cell ports for obstructions. Make sure that the gasket is well-seated.

Prepare Libraries for Sequencing

Denature and Dilute Libraries

Before loading libraries onto the reagent cartridge, denature and dilute libraries and add an optional PhiX control. For more information, see

1000000002697)

The loading volume for the MiniSeq System is 500µl at a loading concentration of 1.8pM. However, loading concentration can vary depending on library preparation and quantification method s.

MiniSeq System Denature and Dilute Libraries Guide (document #

Load Libraries onto the Reagent Cartridge

1 Clean the foil seal covering reservoir #16 labeled Load library here using a low-lint tissue.

2 Pierce the seal with a clean 1 ml pipette tip.

3 Add 500 µl prepared 1.8 pM libraries into reservoir #16. Avoid touching the foil seal as you dispense the

libraries.

Figure 10 Load Libraries

Set Up a Sequencing Run

Run setup steps differ based on system configuration:

u Standalone configuration—You are prompted to define run parameters on the control software Run

Setup screen.

u BaseSpace or Local Run Manager configuration—You are prompted to log in and select from a list of runs

predefined in BaseSp ace or Local Run Manager.

Set Up a Run (Standalone Configuration)

1 From the Home screen, select Sequence.

The Sequence command releases consumables from a previous run and opens the series of run setup screens.

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Load the Flow Cell

1 Open the flow cell compartment door.

2 Press the release button to the right of the flow cell latch.

Figure 11 Open Flo w Cell Latch

3 If present, remove the used flow cell from a previous run.

4 Make sure that the flow cell stage is clean. If debris is present, clean the flow cell stage with an alcohol

wipe.

5 Place the flow cell on the flow cell stage over the alignment pins.

Figure 12 Place Flow Cell on Stage

6 Close the flow cell latch to secure the flow cell.

Figure 13 Close Flow Cell Latch

7 Close the flow cell compartment door.

Load the Reagent Cartridge

1 Open the reagent compartment door.

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2 If present, remove the used reagent cartridge.

NOTE

To facilitate safe disposal of unused reagent containing formamide, the reservoir in position #9 is removable. See

Remove Used Reservoir from Position #9

on page 30.

3 Slide the reagent cartridge into the reagent compartment until the cartridge stops.

Figure 14 Load Reagent Cartridg e

Empty the Spent Reagents Bottle

1 Remove the spent reagents bottle from the compartment.

Figure 15 Remov e the S pent Reagents Bott le

2 To prevent spills when carrying the spent reagents bottle, seal the bottle opening with the threaded cap.

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Figure 16 Seal the S pent Reagent s Bott le

3 Discard the contents in accordance with applicable standards.

WARNING

support.illumina.com/sds.html.

4 With the threaded cap removed, slide the empty spent reagents bottle into the compartment until it

stops.

5 Close the compartment door and select Next.

Enter Run Parameters

1 Enter a run name of your preference.

2 [Optional] Enter a library ID of your 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 number of cycles for each read in the sequencing run.

u Read 1—Enter a value up to 151 cycles. u Index 1—Enter the number of cycles required for the Index 1 (i7) primer. u Index 2—Enter the number of cycles required for the Index 2 (i5) primer. u Read 2—Enter a value up to 151 cycles. This value is typically the same number of cycles as Read 1.

The control software confirms the number of specified cycles using the following criteria:

u Total cycles do not exceed the maximum cycles allowed based on the reagent cartridge loaded for

the run.

u Cycles for Read 1 are greater than the 5 cycles required for template generation. u Index Read cycles do not exceed Read 1 and Read 2 cycles.

NOTE

Make sure that you specify the appropriate number of Index Read cycles for the libraries that you are sequencing. For more information, see the library prep documentation.

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6 [Optional] If you are using custom primers, select the checkbox for the primers used.

u Read 1—Custom primer for Read 1. u Index 1—Custom primer for Index 1. u Index 2—Custom primer for Index 2. u Read 2—Custom primer for Read 2.

7 [Optional] Select Advanced Settings to change settings for the current run.

u Purge consumables for this run—Change the setting to purge consumab les automatically after the

current run.

u Use run monitoring for this run—Change the setting to use run monitoring in BaseSpace. When

enabled, a BaseSpace login and internet connection are required.

u Recipe—Select a custom recipe for this run. u Output folder—Change the output folder location for the current run. Select Browse to navigate to a

folder location.

8 Select Next.

NOTE

Do not open the reagent compartment door or the flow cell compartment door during the automated check or during the sequencing run.

Review Automated Check

1 Review the automated check results.

u To stop a check in p rogress, select Cancel. u For any items that do not pass, an action is required before you can proceed. See

Errors

on p age 39.

u To restart the check, select Retry. The check resumes at the first incomplete or failed check.

Automatic Check

2 To start the run, select from the following options.

u If the system is not configured to start automatically after a successful check, select Start. u If the system is configured to start automatically after a successful check, the sequencing run begins

automatically. You d o not have to be present. However, if any errors occur during the check, the run does not b egin automatically.

Set Up a Run (BaseSpace or Local Run Manager Configuration)

1 From the Home screen, select Sequence.

The Sequence command releases consumables from a previous run and opens the series of run setup screens.

1 Enter your user name and password.

2 Select Next.

Select Available Run

1 Select a run name from the list of available runs.

Use the up and down arrows to scroll through the list or enter a run name in the Search field.

2 Select Next.

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Load the Flow Cell

1 Open the flow cell compartment door.

2 Press the release button to the right of the flow cell latch.

Figure 17 Open Flo w Cell Latch

3 If present, remove the used flow cell from a previous run.

4 Make sure that the flow cell stage is clean. If debris is present, clean the flow cell stage with an alcohol

wipe.

5 Place the flow cell on the flow cell stage over the alignment pins.

Figure 18 Place Flow Cell on Stage

6 Close the flow cell latch to secure the flow cell.

Figure 19 Close Flow Cell Latch

7 Close the flow cell compartment door.

Load the Reagent Cartridge

1 Open the reagent compartment door.

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2 If present, remove the used reagent cartridge.

NOTE

To facilitate safe disposal of unused reagent containing formamide, the reservoir in position #9 is removable. See

Remove Used Reservoir from Position #9

on page 30.

3 Slide the reagent cartridge into the reagent compartment until the cartridge stops.

Figure 20 Load Reagent Cartridg e

Empty the Spent Reagents Bottle

1 Remove the spent reagents bottle from the compartment.

Figure 21 Remov e the S pent Reagents Bott le

2 To prevent spills when carrying the spent reagents bottle, seal the bottle opening with the threaded cap.

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Figure 22 Seal the S pent Reagent s Bott le

3 Discard the contents in accordance with applicable standards.

WARNING

support.illumina.com/sds.html.

4 With the threaded cap removed, slide the empty spent reagents bottle into the compartment until it

stops.

5 Close the compartment door and select Next.

Confirm Run Parameters

1 Confirm run parameters.

The control software confirms the number of specified cycles using the following criteria:

u Total cycles do not exceed the maximum cycles allowed based on the reagent cartridge loaded for

the run.

u Cycles for Read 1 are greater than the 5 cycles required for template generation. u Index Read cycles do not exceed Read 1 and Read 2 cycles.

NOTE

Make sure that you specify the appropriate number of Index Read cycles for the libraries that you are sequencing. For more information, see the library prep documentation.

2 [Optional] Select Ed it to change run parameters. When finished, select Save.

u Purge consumables for this run—Change the setting to purge consumab les automatically after the

current run.

u Run parameters—Change the read type or number of cycles per read. u Custom primers—Change the settings for custom primers.

3 Select Next.

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NOTE

Do not open the reagent compartment door or the flow cell compartment door during the automated check or during the sequencing run.

Review Automated Check

1 Review the automated check results.

u To stop a check in p rogress, select Cancel. u For any items that do not pass, an action is required before you can proceed. See

Errors

on p age 39.

u To restart the check, select Retry. The check resumes at the first incomplete or failed check.

Automatic Check

2 To start the run, select from the following options.

automatically. You d o not have to be present. However, if any errors occur during the check, the run does not b egin automatically.

Monitor Run Progress

1 Monitor run progress, intensities, and quality scores as metrics app ear on the screen.

Figure 23 Sequencing Run Pro gress and Metrics

A Run progress—Shows the current step and number of cycles completed for each read. The progress bar is

not proportional to the run rate of each step.

B Q-Score—Shows the distribution of quality scores (Q-scores). See

Quality Scoring

on page 51.

C Intensity—Shows the value of cluster intensities of the 90thpercentile for each tile. Plot colors indicate each

base: red is A, green is C, blue is G, and black is T.

D Cluster density (K/mm²)—Shows the number of clusters detected for the run. E Clusters passing filter (%)—Shows the percentage of clusters passing filter. See

Clusters Passing Filter

page 50.

F Estimated yield (Gb)—Shows the number of bases projected for the run. G Data transfer status—Shows the status of data transfer based on analysis configuration. H Time to completion—Shows the run completion date and time (yyyy-mm-dd hh:mm).

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NOTE

After you select Home, it is not possible to return to view run metrics. However, run metrics are accessible on BaseSpace, from a networked computer using Sequencing Analysis Viewer, or from a networked computer using Local Run Manager.

Cycles for Run Metrics

Run metrics appear at different points in a run.

u During the cluster generation steps, no metrics appear.

u The first 5 cycles are reserved for template generation.

u At cycle 6, raw cluster density and cycle 1 intensities are available.

u After cycle 25, clusters passing filter, yield, and quality scores are available.

Sequencing Analysis Viewer

The Sequencing Analysis Viewer software shows sequencing metrics generated during the run. Metrics appear in the form of plots, graphs, and tables b ased on data generated by RTA and written to InterOp files. Metrics are updated as the run progresses. Select Refresh at any time during the run to view updated metrics. For more information, see the

The Sequencing Analysis Viewer is included in the software installed on the instrument computer. You can also install Sequencing Analysis Viewer on another computer linked to the same network as the instrument to monitor run metrics remotely.

Sequencing Analysis Viewer User Guide (part # 15020619)

Data Transfer Status

Depending on the analysis configuration selected, an icon appears on the screen d uring the run to indicate connection status.

Status Base Sp ace B as eSpa ce Onsi te Local Run Manager Stand al one

Conn ecte d

Transf erring data

Disc onne cted

Disa ble d

It is possible that multiple icons appear on the screen. For example, if run data are being transferred to BaseSpace and an add itional output folder location, a BaseSp ace icon and a standalone icon appear.

Run Copy Service

The MiniSeq System Software Suite includes a Run Copy Service. As RTAgenerates files, the service copies files to the specified outp ut folder location.

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If data transfer is interrupted during the run, data are stored temporarily on the instrument computer. When the connection is restored, data transfer resumes automatically during the run. If the connection is not restored before the run ends, manually move data to the preferred location.

Transfer to BaseSpace

The BaseSpace Broker transfers run data to BaseSpace. If the connection to BaseSpace is interrupted, the broker attempts to upload data for 7 days. If the connection is not restored after 7 days, run data are deleted.

If you specify an additional location for run data, data are transferred to that location regardless of broker status.

Automatic Post-Run Wash

When the sequencing run is complete, the software initiates an automatic post-run wash using the wash solution and NaOCl provided in the reagent cartridge.

The automatic post-run wash tak es approximately 60 minutes. When the wash is complete, the Home b utton becomes active. Seq uencing results remain visible on the screen during the wash.

After the Wash

After the wash, the sippers remain in the down position to prevent air from entering the system. Leave the cartridge in place until the next run.

Remove Used Reservoir from Position #9

The reservoir in position #9 of the reagent cartridge contains formamide. Before discarding the used reagent cartridge, you can remove the reservoir from position #9 to discard it separately.

1 Wearing gloves, press downwards on the white breakaway tab at position #9 to break the 3 connection

points.

Figure 24 Breakaway Tab at Position #9

A Breakaway tab shown intact B Reservoir clip

2 Slide the breakaway tab sideways towards the left edge of the cartridge, so that the breakaway tab

slides under the cartridge cover.

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Figure 25 Breakaway Tab Removed, Reservoir Clip Exposed

A Breakaway tab shown under cartridge cover B Reservoir clip

3 Press the clear plastic reservoir clip down and towards the right.

The reservoir releases from position under the reagent cartridge.

4 Dispose of the reservoir in accordance with applicable standards.

WARNING

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Chapter 4 Maintenance

Introduction 32 Perform a Manual Instrument Wash 32 Software Updates 35

Introduction

Maintenance procedures include manual instrument washes and system software updates when available. No other periodic maintenance is required.

u Instrument washes—An automatic post-run wash after each sequencing run maintains instrument

performance. However, a manual instrument wash is required under certain conditions. See

Manual Instrument Wash

u Software updates—When a new version of the system software suite is available, you can update the

on p age 32.

software automatically through a connection to BaseSp ace or manually after downloading the installer from the Illumina website. See

Software Updates

on p age 35.

Preventive Maintenance

Illumina recommends that you schedule a preventive maintenance service each year. If you are not under a service contract, contact your Territory Account Manager or Illumina Technical Support to arrange for a billable preventive maintenance service.

Perform a

Perform a Manual Instrument Wash

Manual instrument wash options include the Quick Wash and the Manual Post-Run Wash.

Was h Types Descrip tion

Quick Wa sh Dura tion: 20 m inute s

Manu al Post-Run Wa sh Dura tion: 90 m inute s

NOTE

Always use a fresh dilution of NaOCl prepared within the last 24hours. If you make a volume larger than 1ml, store the remaining dilution at 2°C to 8°C for use within the next 24 hours. Otherwise, discard the remaining dilution of NaOCl.

A manual instrument wash requires the wash cartridge and a wash flow cell provided with the instrument. Alternatively, you can use a used flow cell for the wash.

A quic k wash is required every 7 da ys that th e instrument is idle or afte r a n instrumen t shu tdown. The wash flush es the system w ith a user-supplied wash solu tion of laboratory-grade water and Tween 20.

A manua l post-ru n wash is required wh en the automa tic post-ru n wash is not performe d, suc h as whe n a run is ende d early a nd th e flow c ell is save d for later rehybridization . The wash flush es the system w ith use r-supplied 0.12% sodium hypochlo rite and a wa sh solution of laboratory-grade water and Twe en 2 0.

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Figure 26 Wash Car tridge

Prepare for a Manual Post-Run Wash

1 Combine the following volumes to result in 0.12% NaOCl.

u 5% NaOCl (31µl) u Laboratory-grade water (1269 µl)

2 Add 1.3 ml of 0.12% NaOCl to the wash cartridge.

The correct reservoir is equivalent to position #31 on the prefilled reagent cartridge.

Figure 27 Positions for NaOCl and Wash Solution

A 0.12% NaOCl B Wash solution

3 Combine the following volumes to result in a 0.05% Tween 20 wash solution.

u 100% Tween 20 (40 µl) u Laboratory-grade water (80 ml)

4 Add 80 ml wash solution to the wash cartridge.

The correct reservoir is equivalent to position #40 on the prefilled reagent cartridge.

5 From the Home screen, select Perform Wash, and then select Manual post-run wash.

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Prepare for a Quick Wash

1 Combine the following volumes to result in a 0.05% Tween 20 wash solution.

u 100% Tween 20 (20 µl) u Laboratory-grade water (40 ml)

2 Add 40 ml wash solution to the wash cartridge.

The correct reservoir is equivalent to position #40 on the prefilled reagent cartridge.

Figure 28 Position for Wash Solutio n

A Wash solution

3 From the Home screen, select Perform wash, and then select Quick Wash.

Load the Wash Flow Cell and Wash Cartridge

1 Load the wash flow cell. Close the flow cell clamp and flow cell door.

NOTE

Alternatively, you can load a used flow cell.

2 Remove the used reagent cartridge from the previous run, if present.

3 Load the prepared wash cartridge.

4 Remove the spent reagents bottle and discard the contents in accordance with applicable standards.

WARNING

support.illumina.com/sds.html.

5 Slide the empty spent reagents bottle into the compartment until it stop s.

6 Close the reagent compartment door.

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7 Select Next.

Start the Wash

1 After the check is comp lete, select Start.

2 When the wash is complete, select Home.

After the Wash

After the wash, the sippers remain in the down position to prevent air from entering the system. Leave the cartridge in place until the next run.

Software Updates

Software updates are packaged in a software bund le called the System Suite, which includes the following software:

u MiniSeq Control Software

u MiniSeq recipes

u RTA2

u Local Run Manager

u MiniSeq Service Software

u Sequencing Analysis Viewer

u BaseSpace Broker

Software release notes are available on the MiniSeq System supp ort page on the Illumina website.

You can install software updates automatically using an internet connection or manually from a network or USB location.

u Automatic updates—For instruments connected to a network with internet access, an alert icon

appears on the Manage Instrument button on the Home screen when an update is available.

u Manual updates—Download the System Suite installer from the MiniSeq System support page on the

Illumina website.

NOTE

Cancelling an update before installation is complete stops the update at the current point in the installation. Any changes made up to the point of cancellation are not uninstalled or reverted to the previous version.

Automatic Software Update

1 Select Manage Instrument.

2 Select Software Update.

3 Select Install the upd ate already downloaded from BaseSpace.

4 Select Update to begin the update. A dialog box opens to confirm the command.

5 Follow the prompts in the installation wizard:

a Accept the licensing agreement. b Review the list of software included in the update.

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When the upd ate is complete, the control software restarts automatically.

NOTE

If a firmware update is included, an automatic restart of the system is required after the firmware is updated.

Manual Software Update

1 Download the System Suite installer from the Illumina website and save it to a network location.

Alternatively, copy the software installation file to a portable USB drive.

2 Select Manage Instrument.

3 Select Software Update.

4 Select Manually install the update 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 Accept the licensing agreement. b Review the list of software included in the update.

When the upd ate is complete, the control software restarts automatically.

NOTE

If a firmware update is included, an automatic restart of the system is required after the firmware is updated.

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Appendix A Troubleshooting

Troubleshooting Files 38 Automatic Check Errors 39 RTA Errors 40 Rehybridization Workflow 41 System Check 42 Network Configuration Settings 45 Custom Genomes 46 Shut Down the Instrument 46

Troubleshooting Files

Key File Folder D es crip tion

Run information file (RunInfo.xml)

Run parameters file (RunPa rameters.xml)

RTA configu ration file (RTAC onfiguration.xml)

Inte rOp files (*.bin) Inte rOp Binary reporting f iles used for Seque nc in g Ana lysis Viewe r.

Log file s Logs Log file s desc ribe eac h step performed by the instru me nt for e ach cycle,

Error log file s (* ErrorLog* .txt)

Global log file s (* GlobalLog*.tsv)

La ne log f ile s (* La neL og*.txt)

Root folde r Contains the following informa tion:

• Run na me

• Nu mbe r of cyc le s in the run

• Nu mbe r of cyc le s in e a ch re ad

• Whether the read is an indexed read

• Nu mbe r of swaths and tile s on the flow cell

Root folde r Contains informa tio n abou t ru n para me ters a nd run com pon ents.

Informa tion inc ludes the RFID, seria l number, lot number, an d e xpiration date.

Data\ Intensities Contains the RTA configu ration settings for th e run.

The RTAC on figuration.xml file is crea te d a t the beginn in g of the run.

Inte rOp files are upda te d throu gh out the run.

an d list software and firmw are v ersion s u sed with the run. The file named [In strumentN a me ]_Curren tH ardware.csv lists the serial numbers of instrumen t c om pon ents.

RTALogs Log of RTA errors.

Error log file s a re update d whene ve r a n error occ urs.

RTALogs Log of a ll RTA events.

Global log file s a re update d throughout the run.

RTALogs Log RTA proce ssing events.

La ne log f ile s are upda te d throu ghout the run.

Troubleshooting Resources

For technical questions, visit the MiniSeq System support pages on the Illumina website. The support pages provide access to documentation, downloads, and frequently asked questions.

For run quality or performance problems, contact Illumina Technical Support. See

page 57.

Consider sharing a link to the run summary in BaseSp ace with Illumina Technical Support to facilitate troub leshooting.

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Technical Assistance

MiniSeq System Guide

Process Status

The MiniSeq Control Software lists the status of at least 3 runs in the system Temp folder. From the Manage Instrument screen, select Process status.

For each run name, the system lists the status of the following components:

u Real-Time Analysis (RTA)—Based on the processing of BCL files

u Local Run Manager—If Local Run Manager was used for the run

u File Copy—Based on file transfer using the Run Copy Service

u BaseSpace—If BaseSpace was used for the run

Sequencing Archive Folder

The MiniSeq Control Software saves run summary files on the system computer at D:\Illumina\MiniSeq Sequencing Archive for each run performed on the instrument.

In this folder, a subfolder exists for each run performed on the instrument contains the following files:

u RunCompletionStatus.xml—Contains the completion status, run folder name, number of cycles planned

and performed, cluster density, clusters passing filter, and estimated yield for the run.

u RunParameters.xml—Contains information about run parameters and run components. Information

includ es the RFID, serial number, lot number, and expiration date.

Automatic Check Errors

If errors occur during the automatic pre-run check, use the following recommended actions to resolve the error.

If a pre-run check fails, the reagent cartridge RFID is not locked and can be used for a subsequent run. However, the RFID is locked after the foil seals have been pierced.

Syst em C hecks Recommended Acti on

Doors closed Ma ke sure tha t the compartme nt doors are closed.

Consu ma bles loaded Consu ma ble sensors do n ot register. Make sure that ea ch c on sumable is properly loaded.

Required softw are Critic al c omponen ts of the software are missing.

Instrume nt disk spa ce The in strument hard drive does not h ave sufficient disk spac e to pe rform a run.

Ne twork c onne ction The conn ection to the specifie d location of the output folder has bee n inte rrupted.

Ne twork disk spac e The spe cified loca tion of the output folder is full. A lth ough the ch eck is la be le d n etwork

On the run setup screens, selec t Bac k to retu rn to th e loading step, a nd repe at run setup.

Pe rform a ma nua l software update to re store all software componen ts.

Clea r ru n data from the instrumen t h ard drive.

Although the che ck is labeled network con ne ction, the syste m chec ks the connec tion to an y spec ified lo cation of the output folder on a server, a n exte rnal hard drive, or the local drive . Ch eck the status of the connec tion to the spec if ie d output folder loc ation .

disk spac e, th e syste m chec ks a ny spec if ie d location of the output folder on a se rve r, a n extern al hard drive , or the loca l hard drive. Clea r disk spac e from the spe cified ou tput folder loc ation .

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MiniSeq System Guide

Temperature Recommended Acti on

Tempe rature ramp Conta c t Illumina Tec hn ic al S upport.

Tempe rature sensors Conta c t Illumina Tec hn ic al S upport.

Fa ns Contac t Illumina Tec hn ic al Support.

Imagi ng Sy stem Recommended Acti on

Imagin g limits Contac t Illumina T echnica l Support.

Z ste p-an d-settle C onta ct Illumina Te chnical Support.

Bit error ra te Conta c t Illumina Tec hn ic al S upport.

Flow cell re gistration It is possible tha t th e flow c ell is not properly seated.

Reagent Deliv er y Recommend ed Acti on

Valve response Conta c t Illumina Tec hn ic al S upport.

Pump Conta c t Illumina Tec hn ic al S upport.

• On the run setup screen s, selec t Bac k to retu rn to the flow c ell step.

• Unlo ad and reload the flow c e ll to make sure tha t it is sea te d properly.

Hard Drive Space

The instrument computer hard drive can hold approximately 45 runs based on d ata generated from a run using the following run parameters:

u Approximately 5–6GB of space is required for 150-cycle paired-end run.

u An additional 10GB of space is required for analysis files when using the Local Run Manager

Resequencing analysis module.

For each run p erformed, a temporary run folder is created as part of the software operation. As files are written to the temporary run folder, the files are copied to the output folder. Therefore, if you specify an output folder location on the instrument hard drive, 2 copies of that run are written to the hard drive. The software saves the most recent 3 temporary run folders.

When using the Local Run Manager software for analysis, temporary files are not deleted , by default. The retention p olicy is set manually from the Local Run Manager System Settings screen.

Eventually, temporary files can fill the hard drive space. Consider using a network location for run data and setting a reasonable Local Run Manager retention policy based on the number of runs that you perform.

RTA Errors

To troubleshoot RTA errors, first check the RTA error log, which is stored in the RTALogs folder. This file is not present for successful runs. Include the error log when reporting issues to Illumina Technical Sup port.

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 output destination at the end of processing:

u *GlobalLog*.tsv summarizes important run events.

u *LaneNLog*.tsv lists processing events. N is always 1 on a MiniSeq flow cell.

u *Error*.tsv lists errors that occurred during a run.

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u *WarningLog*.tsv lists warnings that occurred during a run.

Rehybridization Workflow

A rehybridization run might be necessary if metrics generated during the first few cycles show intensities below 2500. Some low-diversity libraries can show intensities below 1000, which is expected and cannot be resolved with rehybridization.

NOTE

The End Run command is final. The run cannot be resumed, run consumables cannot be reused, and sequencing data from the run are not saved.

When you end a run and save the flow cell, the software performs the following steps before the run ends:

u Places the flow cell in a safe state.

u Unlocks the flow cell RFID for a later run.

u Assigns a rehybridization expiration d ate to the flow cell.

u Writes the run logs for completed cycles. A delay is normal.

u Bypasses the automatic post-run wash.

When you start a rehybridization run, the software performs the following steps to perform the run:

u Creates a run folder based on a unique run name.

u Checks that the flow cell rehybridization date has not expired.

u Primes reagents. A d elay is normal.

u Skips the clustering step.

u Removes the previous Read 1 primer.

u Hybridizes a fresh Read 1 primer.

u Continues through Read 1 and the remainder of the run based on specified run parameters.

Points to End a Run for Rehybridization

Later rehybridization is possible only if you end the run at the following points:

u After cycle 5—Intensities app ear after template registration, which requires the first 5 cycles of

sequencing. Although it is safe to end a run after cycle 1, ending after cycle 5 is recommended. Do not end a run during cluster generation.

u Read 1 or Index 1 Read—End the run

before

paired-end resynthesis b egins. The flow cell cannot be

saved for later rehybridization after p aired-end resynthesis begins.

Required Consumables

A rehybridization run requires a new MiniSeq reagent cartridge regardless of when the run was stopped.

End the Current Run

1 Select End Run. When prompted to confirm the command, select Yes.

2 When promp ted to save the flow cell, select Yes. Note the expiration date for rehybridization.

3 Remove the saved flow cell and set aside at 2°C to 8°C until you are ready to set up the rehyb ridization

run.

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MiniSeq System Guide

NOTE

You can store the flow cell up to 7 days at 2°C to 8°C in the flow cell container with the cap closed. For best results, rehybridize the saved flow cell within 3 days.

Perform a Manual Wash

1 From the Home screen, select Perform Wash.

2 From the Wash Selection screen, select Manual Post-Run Wash. See

on p age 32.

NOTE

If you have not yet removed the reagent cartridge from the stopped run, you can use them for the manual wash. Otherwise, perform the manual wash with the wash cartridge.

Perform a Manual Instrument Wash

Set Up a New Run on the BaseSpace Prep Tab

1 If the instrument is configured for BaseSpace or BaseSpace Onsite, set up a new run on the Prep tab

using the same parameters as the original run.

TIP

Click the Pools tab, select the appropriate Pool ID to retain the previous run settings, and then assign a unique name for the new run.

Set Up a Run on the Instrument

1 Prepare a new reagent cartridge.

2 If the saved flow cell was stored , allow it to reach room temperature (15–30 minutes).

3 Clean and load the saved flow cell.

The system reads the flow cell RFID as a saved flow cell and confirms a valid rehybridization date.

4 Remove the spent reagents bottle and discard the contents appropriately, and then reload the empty

container.

5 Load the new reagent cartridge.

6 From the Run Setup screen, select from the following options:

u BaseSpace, BaseSpace Onsite, or Local Run Manager configuration—Select the run and confirm run

parameters.

u Standalone configuration—Enter the name of the run and specify the same parameters as the original

run.

7 Select Next to proceed to the pre-run check and start the run.

System Check

A system check is not required for normal operation or instrument maintenance. However, an Illumina Technical Sup port representative might ask you to perform a system check for troubleshooting purposes.

NOTE

If an instrument wash is due, perform the wash before starting a system check.

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MiniSeq System Guide

Starting a system check automatically closes the control software and launches the MiniSeq Service Software. The service software launches and opens to the Load screen, which is configured to use the advanced loading option.

Figure 29 Available Sy stem Checks

After loading consumables, the Select screen opens, which lists available system checks. Inactive checkboxes on the Select screen indicate tests that require assistance from an Illumina field representative.

Perform a System Check

1 From the Manage Instrument screen, select System Check. When prompted to close the control

software, select Yes.

2 Add 40 ml deionized water to the wash cartridge.

The correct reservoir is equivalent to position #40 on the prefilled reagent cartridge.

Figure 30 Position for Wash Solutio n

A Wash solution

3 Load the consumables as follows:

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a If a used flow cell is not already on the instrument, load a used flow cell. b Empty the spent reagents bottle and return it to the instrument. c Load the wash cartridge.

4 Select Load.

The software moves the flow cell and wash cartridge into position.

5 Select Next. The system check begins.

6 [Optional] When the system check is complete, select View next to the check name to view the values

associated with each check.

7 Select Next.

The system check report opens.

8 Select Save to save the report to a zipped file. Navigate to a network location to save the file.

9 When finished, select Exit.

10 When prompted to close the service software and restart the control software, select Yes.

The control software restarts automatically.

Motion Checks

Syst em C heck Descrip tion

FC LM & FAM Chec ks the gain and distanc e of the Flow C ell Loa d Mec han ism (FCL M) and Fluidic s

Automation Modu le (FAM) to c on firm that th e modu le s a re working properly.

Sta ge Te sts Che cks the trave l limits a nd pe rformanc e of the XY-stage an d Z-sta ge .

Optics Check

Syst em C heck Descrip tion

Flow Cell Registration

Measure s flow ce ll tilt on an optic al plan e, te sts ca me ra functiona lity, tests the ima ging modu le , an d verifies registration of the flow cell in th e correc t imagin g position.

Fluidics Checks

Syst em C heck Descrip tion

Valve Response Ch ecks the ac cura cy of the valve and pump move ments, and te sts the pump syringe rang e of

movem ent.

Pre ssure Decay Che cks th e lea k ra te of a sealed fluidic s system, whic h confirms that the flow c ell is properly

mou nted in the sequ enc ing position.

Flow Rate Ch ecks the func tiona lity of the bubble sensors, which are used to de te ct the prese nce of air in

the reagen t line s. Measures th e flow ra tes to ch eck for occ lusions or leaks.

Thermal Checks

Syst em C heck Descrip tion

Fa ns Ch ecks the speed of system f ans in pulse per minu te (P PM ) to c onfirm th at fans a re

func tioning. Fa ns tha t a re not fun ctionin g return a negative va lu e.

Thermal Probes C hec ks the avera ge temperatu re of each the rma l sensor. The rma l sensors that are not

func tioning return a negative value.

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MiniSeq System Guide

Network Configuration Settings

Network settings are configured during installation. If the system has to be reconfigured, you can change settings or reset settings on the Network Configuration screen. Configuration settings include the IP add ress, domain name server (DNS) address, and domain name.

Set Network Configuration

1 From the Manage Instrument screen, select System configuration.

2 Select Network configuration.

3 Select Obtain an IP address automatically to obtain the IP address using DHCP server.

NOTE

Dynamic Host Configuration Protocol (DHCP) is a standard network protocol used on IP networks for dynamically distributing network configuration parameters.

Alternatively, select Use the following IP address to connect the instrument to another server manually as follows. Contact your network administrator for the ad dresses specific to your facility.

u Enter IP address. The IP address is a series of 4 numbers separated by a dot, similar to

168.62.20.37, for example.

u Enter the subnet mask , which is a subdivision of the IP network. u Enter the default gateway, which is the router on the network that connects to the internet.

4 Select Obtain a DNS server address automatically to connect the instrument to the domain name server

associated with IP address. Alternatively, select Use the following DNS server addresses to connect the instrument to the domain name server manually as follows.

u Enter the preferred DNS add ress. The DNS address is the server name used to translate domain

names into IP addresses.

u Enter the alternate DNSaddress. The alternate is used if the preferred DNS cannot translate a

particular domain name to an IP address.

5 Select Save.

Configure Computer Domain

NOTE

The instrument computer name is assigned to the instrument computer at the time of manufacture. Any changes to the computer name can affect connectivity and require a network administrator.

1 Connect the instrument computer to a domain or a workgroup as follows.

u For instruments connected to the internet—Select Member of domain, and then enter domain name

associated with the internet connection at your facility.

NOTE

Domain changes require an administrator user name and password.

u For instruments not connected to the internet—Select Memb er of work group, and then enter a work

group name. The work group name is unique to your facility.

2 Select Save.

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MiniSeq System Guide

Custom Genomes

You can upload your own reference in FASTA format to the instrument computer. You can upload several single FASTA filesora single multi-FASTA file (recommended), but not a combination of both.

To troubleshoot issues with a custom genome file, confirm the following requirements.

1 Make sure that the file uses a *.fa or *.fasta extension and that it is stored in a ded icated folder for the

reference.

2 Make sure that the chromosome name does not contain any of following characters:

# - ? ( ) [ ] / \ = + < > : ; " ' , * ^ | &

For best results, use only alpha-numeric characters as chromosome names.

Shut Down the Instrument

Under normal conditions, there is no reason to shut down the instrument.

1 Select Manage Instrument.

2 Select Shutdown options.

3 Select Shut down.

The command safely shuts down the software and turns off instrument power. Wait at least 60 seconds before turning on the instrument again. A wash is required before the next sequencing run.

CAUTION

Do not

relocate the instrument. Moving the instrument improperly can affect the optical alignment and

compromise data integrity. If you have to relocate the instrument, contact your Illumina representative.

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Appendix B Real-Time Analysis

Real-Time Analysis Overview 48 Input and Output Files 48 Real-Time Analysis Workflow 49

Real-Time Analysis Overview

Real-Time Analysis is a software that runs on the instrument computer, extracts intensities from images to perform base calling, and then assigns a quality score to the base call.

The MiniSeq System uses an implementation of Real-Time Analysis called RTA2. The system control software and RTA2 communicate through a web HTTP interface and shared memory files. If RTA2 is terminated, processing does not resume and run data are not saved.

Input and Output Files

Input Files

Real-Time Analysis software requires the following input for processing:

u Tile images contained in local system memory.

u RunInfo.xml, which is generated automatically at the beginning of the run and provides the run name,

number of cycles, whether a read is ind exed, and the number of tiles on the flow cell.

Real-Time Analysis software receives commands from the control software about the location of RunInfo.xml and whether an optional output folder is specified.

Output 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 produces output as a set of qualityscored base call files and filter files. Output files are used for downstream analysis on BaseSpace or by Local Run Manager analysis modules.

Fil e Ty pe Descri pti on

Base call file s Eac h tile that is ana lyzed is inc lu ded in a n aggregated base call ( *.bcl) file for e a ch lan e

an d f or each cycle. The aggre ga te d base call f ile contains the base call and associated qua lity score for every c lu ste r in that lane.

Filter files Eac h tile produces filter informa tion that is aggregated into 1 f ilter (*.filter) file for eac h

lane . The filter file specifie s whethe r a cluster passes filte rs.

Cluster loca tion files Cluster loca tion (*.locs) files c ontain the X,Y coordinates for e ve ry cluster in a tile. A

cluster loca tion file is generated for e ach la ne during te mplate gen eration.

Base call inde x f iles A base ca ll index (*.bci) file is produc ed for e ach la ne to prese rve the origin al tile

information. The index file conta in s a pa ir of values for e ach tile, which are tile number an d n um ber of clusters for that tile.

RTA2 provides real-time metrics of run quality stored as InterOp files. InterOp files are a binary output containing tile, cycle, and read-level metrics, and are used to view real-time metrics using the Sequencing Analysis Viewer software.

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MiniSeq System Guide

Real-Time Analysis Workflow

Template ge nera tion M aps c luster loca tions.

Registration and in tensity

extrac tion

Phasing correc tion Correc ts the effects of phasing and pre phasing.

Base calling Determine s a ba se call for e ve ry cluster.

Quality scoring A ssigns a qua lity sc ore to eve ry base call.

Records th e loc ation of ea ch c lu ste r on the flo w c e ll and de termin es a n inte nsity value for ea ch cluster.

Template Generation

The first step in the RTAworkflow is template generation, which defines the position of each cluster in a tile using X and Y coordinates.

Template generation requires image data from the first 5 cycles of the run. After the last temp late cycle for a tile is imaged, the template is generated.

NOTE

To detect a cluster during template generation, there must be at least 1 base other than G in the first 5 cycles.

The template is used as a reference for the subsequent step of registration and intensity extraction. Cluster positions for the entire flow cell are written to cluster location (*.locs) files, 1 file for each lane.

Registration and Intensity Extraction

Registration and intensity extraction begin after template generation.

u Registration aligns images produced over every subsequent cycle of imaging against the template.

u Intensity extraction determines an intensity value for each cluster in the template for a given image.

If registration fails for any images in a cycle, no intensities are extracted and all bases are called as N for that tile in that cycle. Use the Sequencing Analysis Viewersoftware to identify tiles and cycles that have failed registration. Registration failures are easily identified as tiles and cycles that have 0 in the P90 column on the Imaging tab.

Phasing Correction

During the sequencing reaction, each DNA strand in a cluster extends by one base per cycle. Phasing and prephasing occurs when a strand becomes out of p hase with the current incorporation cycle.

u Phasing occurs when a base falls behind.

u Prephasing occurs when a base jumps ahead.

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MiniSeq System Guide

Figure 31 Phasing and Prephasing

A Read with a base that is phasing B Read with a base that is prephasing.

RTA2 corrects the effects of phasing and prephasing, which maximizes the data quality at every cycle throughout the run.

Base Calling

Base calling determines a base (A, C, G, or T) for every cluster of a given tile at a specific cycle. The MiniSeq System uses 2-channel sequencing, which req uires 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 result in 4 distinct populations, each corresponding to a nucleotide. The base calling process determines to which population each cluster belongs.

Figure 32 Visualization of Cluster Intensities

Table 1 Base Calls in 2-Channel Sequencing

Base Red C hannel

A 1 (on) 1 ( on) Cluste rs that show inte nsity in both the red and gree n chan ne ls.

C 1 ( on ) 0 ( off) Clusters tha t sh ow inten sity in the red chan ne l only.

G 0 (off ) 0 (off) Clusters that sh ow no inte nsity at a known loc ation.

T 0 (of f) 1 (on) Cluste rs that show inte nsity in the gree n chan ne l only.

Green

Channel

Result

Clusters PassingFilter

During the run, RTA2 filters raw data to remove clusters that do not meet the data quality threshold.

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MiniSeq System Guide

For 2-channel analysis, RTA2 uses a population-based system to determine the chastity of a base call. Clusters pass filter (PF) when no more than 1 base call in the first 25 cycles has an unacceptable chastity value. Clusters that do not pass filter are not base called in future cycles.

Indexing Considerations

The process for base calling index reads differs from base calling during other reads.

Index reads must 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 must be present in either of the first 2 cycles to ensure demultiplexing performance.

To increase demultiplexing robustness, select index sequences that provide signal in at least 1 channel, preferably both channels, for every cycle. Following this guideline avoids index combinations that result in only G bases at any cycle.

u Red channel—A or C

u Green channel—A or T

This base calling p rocess ensures accuracy when analyzing low-plex samples.

Quality Scoring

A quality 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 quality and more likely to be correct.

The Q-score is a compact way to communicate small error probab ilities. Q(X) represents quality scores, where Xis the score. The following table shows the relationship between the quality score and error probability.

Q-Score Q(X) Error Probabi lit y

Q40 0.0001 (1 in 10,000)

Q30 0.001 (1 in 1,000)

Q20 0.01 ( 1 in 100)

Q10 0.1 (1 in 10)

NOTE

Quality scoring is based on a modified version of the Phred algorithm.

Quality scoring calculates a set of predictors for each base call, and then uses the predictor values to look up the Q-score in a quality table. Quality tables are created to provide optimally accurate quality predictions for runs generated by a specific configuration of sequencing platform and version of chemistry.

After the Q-score is determined, results are recorded in the base call files.

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Appendix C Output Files

Output Files

Sequencing Outp ut Files 52 Sequencing Outp ut Folder Structure 52 Analysis Input File Requirements 53

Sequencing Output Files

Fil e Ty pe Fil e Descrip tion, Locat i on, and Name

Base call file s Eac h tile analyzed is inc luded in a base call file , aggregated in a file for eac h cyc le . The

aggregated file contains the base call a nd enc ode d quality score for eve ry c lu ste r. Data\ Intensities\Ba se Calls\ L001 [C yc le ].bc l.bgzf , where [Cycle] represents the cycle number in 4 digits. Base call file s are compre sse d u sin g block gzip compression .

Base call inde x f ile A binary index file lists the original tile information in a pair of values for e ach tile, which

are tile number and number of cluste rs f or the tile . Base call inde x f iles are cre ated the first time a base call file is create d. Data\ Intensities\Ba se Calls\ L001 s_[Lane ].bc i

Cluster loca tion files For eac h tile , the XY coordin ates for eve ry c lu ste r a re aggregated into a cluster location

file. C luster loca tion file s are the resu lt of templa te gene ration . Data\ Intensities\L001 s_[lane].loc s

Filter files The filte r f ile spec ifies wh ethe r a cluster passed filters. Filter information is aggregate d

into a filte r file for eac h rea d. Filter files a re generated at c ycle 26 using 25 c yc le s o f data . Data\ Intensities\Ba se Calls\ L001 s_[lane].filter

Inte rOp files Binary reporting f iles used for Seque nc in g Ana lysis Viewe r. InterOp f iles are upda te d

throughou t the run. Inte rOp folder

RTA configu ration file Cre ated at th e beginn in g o f the run, the RTA conf igura tion f ile lists settings for the run.

Run information file Lists the run name, numbe r of c yc le s in e ach read, wheth er the read is a n Index Re a d,

[Root folder] , RTAC onfigura tion.xml

an d the numbe r of swaths and tiles on the flow ce ll. The run info f ile is crea ted a t the beginning of the run. [Root folder] , RunIn fo.xml

Sequencing Output Folder Structure

The control software generates the output folder name automatically.

Configs

Data

Intensities

BaseCalls

L001—Base call files, aggregated per cycle

L001—An aggregated *.locs file

Images

Focus

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L001—Focus images

InstrumentAnalyticsLogs—Log files describing instrument analytics steps

InterOp—Binary files used by Sequencing Analysis Viewer

Logs—Log files describing operational steps

Recipe—Run-specific recipe file named with reagent cartridge ID

RTALogs—Log files describing analysis steps

RTAComplete.xml

RTAConfiguration.xml

RunInfo.xml

RunNotes.xml

RunParameters.xml

Analysis Input File Requirements

Local Run Manager requires the following files generated during the sequencing run to perform analysis or to requeue analysis. Some analysis modules require additional input files to perform analysis. For more information, see the workflow guide for the analysis module you are using.

Fil e Name / Type Descri pti on

RTACo mplete.txt A ma rker file that indica te s RTA processing is complete. The presen ce of th is file trigge rs

Loca l Run Man ager to queue analysis.

Run Inf o.xml C ontains high-level run inf ormation, su ch a s the num ber of rea ds and cycles in th e

seque ncing run, and whethe r a read is inde xed.

Base call file s (*.bcl) Conta in s the base call and encoded qu ality score for e ve ry clu ste r for e ach tile,

aggregated in a file for e ach cycle.

Filter files ( *.filter) S pe cifies whe ther a c luste r pa sse d f ilters. Filte r informa tion is a ggregate d into a f ilter file

for eac h read.

Cluster loca tion files (*.loc s) Contains the XY coordin ates for eve ry c lu ste r for e ach tile, aggregated into a c luster

loc ation f ile

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Index

2-channel imaging 50

advanced loading option 9 analysis

output files 52

software 4 analysis configuration 20 analysis, primary

signal purity 50

base call files 52 base calling

2-channel 50

indexing considerations 51 BaseSpace 1

transfer icons 29 BaseSpace Broker 30 BaseSpace configuration 24

wash consumables 32-33 control software 4 customer support 57 cycles in a read 16

data transfer

activity icons 29

BaseSpace Broker 30

run copy service 29 databases, preinstalled 7 dbSNP database 7 Demo Mod e 9 documentation 1, 57

empirical p hasing 49 errors

probability 51 errors and warnings 4

in output files 40

chastity filter 50 cluster location

files 52

template generation 49 clusters passing filter 50 compatibility

flow cell, reagent cartridge 5

RFID tracking 5-6 components

flow cell compartment 2

imaging compartment 2

reagent compartment 2

status bar 2 configuration settings 45 consumables 5

flow cell 5

instrument maintenance 14

laboratory-grade water 14

reagent cartridge 6

sequencing runs 13

user-supplied 13

filter files 52 flow cell

overview 5 preparation 19 rehybridization 41

types 1 flow cell clamp 2 flow cell compartment 2 folder location 23 formamide, position 6 30

hard drive space 40 help, technical 57

icons

errors and warnings 4

status 4 Illumina Proactive monitoring service 11-13

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imaging compartment 2 imaging, 2-channel seq uencing 50 indexing considerations 51 instrument

configuration settings 45 power button 4 start up 8

instrument maintenance

consumables 14 instrument wash 32 intensities 50 InterOp files 38, 52

laboratory-grade water guidelines 14 Local Run Manager 4 locs files 52 log files

GlobalLog 40

LaneNLog 40

maintenance, preventive 32 manage instrument

shut down 46 metrics

base calling 50

cluster density cycles 29

intensity cycles 29 miRbase database 7

output files 52 output files, sequencing 52

passing filter (PF) 50 phasing, prephasing 49 Phred algorithm 51 post-run wash 30 power button 4, 8 power switch 8 pre-run check 24, 28 pre-run check errors 39 preventive maintenance 32 primer rehybridization 41

purge consumables 9

Q-scores 51 quality tab les 51

read length 16 reagent cartridge

overview 6 preparation 19

reservoir #28 33 reagent compartment 2 reagents

kitted 5

proper disposal 22, 26 Real-Time Analysis software 1, 4

results 52 reference genomes

custom genomes 46

file format 7

preinstalled 7 RefGene database 7 rehybridization, Read 1 41 RFID tracking 5 RTA v2

overview 48

termination 48 RTA2

error handling 40 run copy service 29 run duration 16 run metrics 28 run parameters

BaseSpace mode 24

edit parameters 24

standalone mode 23 run setup , advanced option 9 RunInfo.xml 38, 52

Sequencing Analysis Viewer 16 sequencing workflow 49 shutting down the instrument 46 sodium hypochlorite, wash 33 software

analysis 4

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automatic update 35 check for update 9 configuration settings 45 image analysis, base calling 4 initialization 8 instrument control 4 manual update 36 run duration 16

spent reagents

disposal 22, 26, 34 standalone configuration 23 status alerts 4 status bar 2 support pages 1 system check 42 system user name and password 8

technical assistance 57 template generation 49 training 1 troub leshooting

contact options 38

hard drive space 40

low quality metrics 41

pre-run check 39

run-specific files 38

system check 42

pre-run check 24, 28 reagent cartridge 19, 22, 26 run duration 16 run metrics 28 sequencing 49 sodium hypochlorite 33 spent reagents 22, 26 standalone mode 23

updating software 35 user-supplied consumables 13-14 user name and password 8

wash

automatic 30

manual wash 32

user-supplied consumables 32

wash components 32 workflow

advanced loading option 9

analysis configuration 20

BaseSpace login 24

BaseSpace mode 24

indexing considerations 51

overview 17-18

Document # 1000000002695 v02 Material # 20014309

For Researc h Use Only. Not for use in diagnostic procedure s.

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Document # 1000000002695 v02 Material # 20014309

For Researc h Use Only. Not for use in diagnostic procedure s.

Document # 1000000002695 v02

Material # 20014309

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For Researc h Use Only. Not for use in diagnostic procedure s.

illumina MiniSeq System Manual

Specifications and Main Features

Frequently Asked Questions

User Manual

Chapter 1 Overview

Introduction

Additional Resources

Instrument Components

Sequencing Consumables Overview

Preinstalled Databases and Genomes

Chapter 2 Getting Started

Starting the Instrument

Customize System Settings

Configure Analysis Settings

User-Supplied Consumables and Equipment

Chapter 3 Sequencing

Introduction

Sequencing Workflow

Prepare Consumables

Prepare Libraries for Sequencing

Set Up a Sequencing Run

Monitor Run Progress

Automatic Post-Run Wash

Remove Used Reservoir from Position #9

Chapter 4 Maintenance

Introduction

Perform a Manual Instrument Wash

Software Updates

Appendix A Troubleshooting

Troubleshooting Files

Automatic Check Errors

RTA Errors

Rehybridization Workflow

System Check

Network Configuration Settings

Custom Genomes

Shut Down the Instrument

Appendix B Real-Time Analysis

Real-Time Analysis Overview

Input and Output Files

Real-Time Analysis Workflow

Sequencing Output Files

Sequencing Output Folder Structure

Analysis Input File Requirements

Index

Technical Assistance