Directed Electronics equalizer 2500 Guide de l'utilisateur

Customize a short end-to-end workow guide with the Custom Protocol Selectorsupport.illumina.com/custom-protocol-selector.htmlFOR RESEARCH USE ONLYIL

xPart # 15035786 Rev. DIntroduction 70Rapid Run Sequencing Workflow 72Run Types for Rapid Run Chemistry 73Pre-Run Volume Check 75Enter Run Parameters

Perform a Rapid Run90Part # 15035786 Rev. DLoad a Flow CellThe next step is to remove the used flow cell and load the flow cell that you want tosequen

Load a Flow CellHiSeq 2500 System User Guide91Figure 43 Flow Cell Lever in Position 04 Lift the used flow cell from the flow cell holder.Clean the Flo

Perform a Rapid Run92Part # 15035786 Rev. DClean the Flow Cell1 Remove the flow cell from the flow cell container using a pair of plastistats.2 Rinse

Load a Flow CellHiSeq 2500 System User Guide93Figure 45 Flow Cell Positioned Against Top and Right Guide PinsA Top Guide PinB Right Guide PinsNOTERemo

Perform a Rapid Run94Part # 15035786 Rev. DFigure 47 Flow Cell Lever in Position 25 Make sure that the Vacuum Engaged checkbox is selected on the load

Load a Flow CellHiSeq 2500 System User Guide958 Confirm that the Vacuum Engaged and Door Closed checkboxes are selected, and thenselect Next.9 Select

Perform a Rapid Run96Part # 15035786 Rev. DMonitor the RunMonitor run metrics on the run overview screen, fluidics, and imaging.Figure 48 Run Overview

Chapter 5HiSeq 2500 System User Guide97Chapter 5 Post-Run ProceduresPost-Run ProceduresIntroduction 98Unload and Weigh Reagents 99Perform a Mainten

Post-Run Procedures98Part # 15035786 Rev. DIntroductionPost-run procedures include the removal and weighing of reagents, and an instrumentwash. A wate

Unload and Weigh ReagentsHiSeq 2500 System User Guide99Unload and Weigh Reagents1 Open the reagent compartment door.2 Raise the sippers for the approp

Chapter 1HiSeq 2500 System User Guide1Chapter 1 OverviewOverviewIntroduction 2HiSeq 2500 Components 3Start the HiSeq 2500 6HiSeq 2500 Software 7Ava

Post-Run Procedures100Part # 15035786 Rev. DPerform a Maintenance WashA maintenance wash is required every 10 days or when switching between high outp

Perform a Maintenance WashHiSeq 2500 System User Guide101• Laboratory-grade water (750 ml)• 10% Tween 20 (250 ml)• ProClin 300 (1.5 ml)These volumes r

Post-Run Procedures102Part # 15035786 Rev. D9 Wearing a new pair of gloves, apply light pressure to 1 side of the front gasket until theother side lif

Perform a Maintenance WashHiSeq 2500 System User Guide10317 [For Rapid Run mode] Place the ends of tubes 4 and 5 into an empty container. Placethe end

Post-Run Procedures104Part # 15035786 Rev. DPerform a Water WashA water wash is required after each sequencing run. After a high output run, you canpe

Perform a Water WashHiSeq 2500 System User Guide1056 Remove the waste tubing for the appropriate flow cell from the waste container. Do notinclude the

Post-Run Procedures106Part # 15035786 Rev. DSwitch Sequencing ModesUse the Mode Select command from the Welcome screen to switch between high outputmo

Switch Sequencing ModesHiSeq 2500 System User Guide107SpecificationRapid MaintenanceWashHigh OutputMaintenance WashFlow Cell TypeRapid flow cell (2 la

Post-Run Procedures108Part # 15035786 Rev. DIdle the InstrumentUse the following instructions to prepare the instrument to sit idle for up to 10 days.

Shut Down the InstrumentHiSeq 2500 System User Guide109Shut Down the InstrumentShut down the instrument only if you do not plan to use it within the n

Overview2Part # 15035786 Rev. DIntroductionThe HiSeq®system combines innovative engineering with proven SBS technology to setnew standards in output,

110Part # 15035786 Rev. D

Chapter 6HiSeq 2500 System User Guide111Chapter 6 Real-Time AnalysisReal-Time AnalysisIntroduction 112Real-Time Analysis Overview 113Monitor Run Me

Real-Time Analysis112Part # 15035786 Rev. DIntroductionReal-Time Analysis (RTA) software performs on-instrument image analysis and basecalling during

Real-Time Analysis OverviewHiSeq 2500 System User Guide113Real-Time Analysis OverviewReal-Time Analysis runs on the instrument computer, performing ba

Real-Time Analysis114Part # 15035786 Rev. D} Statistics files—For each cycle, 1 statistics file (*.stats) is produced. The statistics filecontains agg

Monitor Run MetricsHiSeq 2500 System User Guide115Monitor Run MetricsReal-TimeAnalysis automatically generates quality metrics when image analysis be

Real-Time Analysis116Part # 15035786 Rev. DSequencing Analysis ViewerThe Sequencing Analysis Viewer software shows metrics generated during the sequen

Real-Time Analysis WorkflowHiSeq 2500 System User Guide117Real-Time Analysis WorkflowReal-Time Analysis and the control software perform the Real-Time

Real-Time Analysis118Part # 15035786 Rev. DColor Matrix CorrectionAfter registration and intensity extraction, Real-Time Analysis corrects for cross t

Real-Time Analysis WorkflowHiSeq 2500 System User Guide119Base CallingAfter raw intensities have been color corrected and phasing and prephasing corre

HiSeq 2500 ComponentsHiSeq 2500 System User Guide3HiSeq 2500 ComponentsThe HiSeq 2500 system comprises the instrument, monitor, instrument control com

Real-Time Analysis120Part # 15035786 Rev. DNOTEQuality scoring is based on a modified version of the Phred algorithm. For moreinformation, see en.wiki

Sequencing Output FilesHiSeq 2500 System User Guide121Sequencing Output FilesFile Type File Description, Location, and NameBase call files Each tile a

Real-Time Analysis122Part # 15035786 Rev. DFile Type File Description, Location, and NamePhasing files Contains empirical phasing information by tile.

Output Folder StructureHiSeq 2500 System User Guide123Output Folder StructureConfig—Configuration settings for the run.DataIntensitiesBaseCallsL00[X]—

Real-Time Analysis124Part # 15035786 Rev. DThe run folder is written to the output path specified on the Scan screen during run setup.The temporary ru

Tile NumberingHiSeq 2500 System User Guide125Tile NumberingThe HiSeq high output flow cell is imaged in 96 tiles on each lane, top and bottom, for eac

Real-Time Analysis126Part # 15035786 Rev. DThumbnail ImagesYou can configure the control software to generate thumbnail images in *.jpg file format.Th

Chapter 7HiSeq 2500 System User Guide127Chapter 7 TroubleshootingTroubleshootingIntroduction 128Possible Run Setup Problems 129Stagger Runs on Flow

Troubleshooting128Part # 15035786 Rev. DIntroductionThis section describes what to do if a problem occurs during a sequencing run and how toperform a

Possible Run Setup ProblemsHiSeq 2500 System User Guide129Possible Run Setup ProblemsProblem Possible Cause ActionThe software did notinitialize.The s

Overview4Part # 15035786 Rev. DE Reagent compartment—Contains reagent racks that hold reagents for sequencing runsand wash solution for instrument was

Troubleshooting130Part # 15035786 Rev. DStagger Runs on Flow Cell A and Flow Cell B1 Wait for the run on the adjacent flow cell to begin a chemistry s

Perform a Fluidics CheckHiSeq 2500 System User Guide131Perform a Fluidics CheckThe Check button on the Welcome screen performs a fluidics check. Use t

Troubleshooting132Part # 15035786 Rev. DBaseSpace is UnavailableIf BaseSpace is not available, open Windows Services to make sure that the BaseSpaceBr

Stop and Resume a RunHiSeq 2500 System User Guide133Stop and Resume a RunStopping a run might be necessary if the run was set up incorrectly, if the d

Troubleshooting134Part # 15035786 Rev. D• Keep As Is—The run is stopped without any modifications to Real-Time Analysis.The run can resume where it wa

Stop and Resume a RunHiSeq 2500 System User Guide1354 Confirm the settings on the Resume screen or select the imaging and chemistrycommands to resume.

Troubleshooting136Part # 15035786 Rev. DPause a RunCAUTIONDo not pause a run during imaging. Use the Normal stop, end of cycle or end of lane feature,

Primer RehybridizationHiSeq 2500 System User Guide137Primer RehybridizationA rehybridization run repeats the sequencing primer hybridization step. If

Troubleshooting138Part # 15035786 Rev. DRehyb Kit Name Workflow InstructionsHiSeq Rapid Rehyb KitCatalog # GD-404-1001HiSeq Rapid Run Primer Rehybridi

IndexHiSeq 2500 System User Guide139Index%%PF 119Aactivity indicators 9Bbase call files 113, 121base callingcycle 12 119quality score 119BaseSpacesamp

HiSeq 2500 ComponentsHiSeq 2500 System User Guide5Flow Cell CompartmentThe flow cell compartment houses the flow cell stage, the thermal stations, the

Index140Part # 15035786 Rev. Dvacuum hole locations 30, 54, 85flow cell leverposition 0 30, 54, 85position 1 31, 37, 55, 61position 2 31, 37, 55, 61fl

IndexHiSeq 2500 System User Guide141SBS 26reagentsloading 27, 29, 52, 66loading, ICB 67loading, SBS 81paired end 27, 29, 66priming 34, 58, 89Reagents

Index142Part # 15035786 Rev. Dduration 104expected volumes 104weighing reagentsafter run 99before run 26Welcome screen 7commands 8menu 10workflowstagg

Technical AssistanceHiSeq 2500 System User Guide143Technical AssistanceFor technical assistance, contact Illumina Technical Support.Address5200 Illumi

IlluminaSan Diego, California 92122 U.S.A.+1.800.809.ILMN (4566)+1.858.202.4566 (outside North America) [email protected]

Overview6Part # 15035786 Rev. DStart the HiSeq 25001 Start the instrument control computer.2 Log on to the operating system using the default user nam

HiSeq 2500 SoftwareHiSeq 2500 System User Guide7HiSeq 2500 SoftwareThree software applications are installed on the instrument computer:} HiSeq 2500 c

Overview8Part # 15035786 Rev. DFigure 4 Welcome ScreenA Welcome screen menu buttonB Interface panel for flow cell AC Interface panel for flow cell BD

HiSeq 2500 SoftwareHiSeq 2500 System User Guide9} Wash—Select Wash to initiate an instrument water wash or maintenance wash.Figure 6 Wash Command Opti

iiPart # 15035786 Rev. DThis document and its contents are proprietary to Illumina, Inc. and its affiliates ("Illumina"), and are intended s

Overview10Part # 15035786 Rev. DStatus IconsA status icon located in the upper-right corner of each screen shows changes in conditions,errors, or warn

HiSeq 2500 SoftwareHiSeq 2500 System User Guide11} Exit—Closes the control software interface.Menu Options WindowThe Menu Options window provides sett

Overview12Part # 15035786 Rev. DAvailable Disk SpaceThe HiSeq instrument computer has a storage capacity of over 2.7TB per flow cell. Datafrom flow c

Sample Sheet OverviewHiSeq 2500 System User Guide13Sample Sheet OverviewThe sample sheet is a user-generated file in *.csv format that stores informat

Overview14Part # 15035786 Rev. DSequencing ConsumablesSequencing on the HiSeq 2500 requires reagents and other consumables provided inIllumina kits. R

Sequencing ConsumablesHiSeq 2500 System User Guide15Consumable Supplier PurposeTweezers, square plastic tip McMaster-Carr, catalog #7003A22Removing th

Overview16Part # 15035786 Rev. DAdditional ResourcesThe following documentation is available for download from the Illumina website.Resource Descripti

Chapter 2HiSeq 2500 System User Guide17Chapter 2 Perform a HiSeq v4 RunPerform a HiSeq v4 RunIntroduction 18HiSeq v4 Sequencing Workflow 19Run Type

Perform a HiSeq v4 Run18Part # 15035786 Rev. DIntroductionTo perform a HiSeq v4 run on the HiSeq 2500, prepare all reagents for the run and thenfollow

HiSeq v4 Sequencing WorkflowHiSeq 2500 System User Guide19HiSeq v4 Sequencing WorkflowPrepare reagents for the run. Weigh reagents after preparation.F

HiSeq 2500 System User Guideiii1 Definitions. "Application Specific IP" means Illumina owned or controlled intellectual property rights that

Perform a HiSeq v4 Run20Part # 15035786 Rev. DRun Types for HiSeq v4 ChemistryThe following table shows types of sequencing runs and the number of pos

Enter Run ParametersHiSeq 2500 System User Guide21Enter Run ParametersFrom the Welcome screen, select Sequence | New Run.The control software interfac

Perform a HiSeq v4 Run22Part # 15035786 Rev. Danalysis, this field is optional.2 Select Zip BCL files to reduce required storage space. If the run is

Enter Run ParametersHiSeq 2500 System User Guide23A first base report is generated automatically for each run. Selecting this option opensthe first ba

Perform a HiSeq v4 Run24Part # 15035786 Rev. Dallow the software to create the recipe from the run parameters entered.Sample Sheet ScreenSample sheets

Enter Run ParametersHiSeq 2500 System User Guide252 Select Next to proceed or select Back to change parameters.

Perform a HiSeq v4 Run26Part # 15035786 Rev. DLoad and Prime ReagentsAfter entering run parameters, load SBS, indexing, and paired-end reagents for th

Load and Prime ReagentsHiSeq 2500 System User Guide277 Place each reagent bottle onto the rack in the associated numbered position. Make surethat the

Perform a HiSeq v4 Run28Part # 15035786 Rev. D4 Slide the reagent rack out of the reagent compartment using the rack handle.5 Remove the caps from eac

Load and Prime ReagentsHiSeq 2500 System User Guide29Load Paired-End Reagents1 Record the weight of each reagent on the lab tracking form.2 Raise the

ivPart # 15035786 Rev. DProduct, or (iv) transfer to a third-party, or grant a sublicense, to any Software or any third-party software. Purchaserfurth

Perform a HiSeq v4 Run30Part # 15035786 Rev. DPrime ReagentsSteps for priming reagents include cleaning the flow cell holder, loading a priming flowce

Load and Prime ReagentsHiSeq 2500 System User Guide31Figure 11 Vacuum Hole LocationsLoad a Priming Flow CellFrom the Load Priming Flow Cell screen, lo

Perform a HiSeq v4 Run32Part # 15035786 Rev. DFigure 12 Flow Cell Positioned Against Top and Right Guide PinsA Top guide pinB Right guide pins5 Slowly

Load and Prime ReagentsHiSeq 2500 System User Guide33Figure 14 Flow Cell Lever in Position 27 Make sure that the Vacuum Engaged checkbox is selected o

Perform a HiSeq v4 Run34Part # 15035786 Rev. DPosition Tubing and Start Prime1 Remove the 8 waste tubes for the appropriate flow cell from the waste c

Load a Flow CellHiSeq 2500 System User Guide35Load a Flow CellSteps to load the clustered flow cell include removing the priming flow cell, cleaning t

Perform a HiSeq v4 Run36Part # 15035786 Rev. DFigure 17 Flow Cell Lever in Position 03 Lift the used flow cell from the flow cell holder.Clean the Flo

Load a Flow CellHiSeq 2500 System User Guide37Clean the Flow Cell1 Remove the flow cell from the flow cell container using a pair of plastistats.2 Rin

Perform a HiSeq v4 Run38Part # 15035786 Rev. DFigure 19 Flow Cell Positioned Against Top and Right Guide PinsA Top guide pinB Right guide pinsNOTERemo

Load a Flow CellHiSeq 2500 System User Guide39Figure 21 Flow Cell Lever in Position 25 Make sure that the Vacuum Engaged checkbox is selected on the L

HiSeq 2500 System User Guidevc Exclusions from Warranty Coverage. The foregoing warranties do not apply to the extent a non-conformance isdue to (i) a

Perform a HiSeq v4 Run40Part # 15035786 Rev. DMonitor the RunMonitor run metrics on the run overview screen, fluidics, and imaging.Figure 22 Run Overv

Chapter 3HiSeq 2500 System User Guide41Chapter 3 Perform a TruSeq v3 RunPerform a TruSeq v3 RunIntroduction 42TruSeq v3 Sequencing Workflow 43Run T

Perform a TruSeq v3 Run42Part # 15035786 Rev. DIntroductionTo perform a TruSeq v3 run on the HiSeq 2500, prepare SBS reagents for Read 1 andindexing r

TruSeq v3 Sequencing WorkflowHiSeq 2500 System User Guide43TruSeq v3 Sequencing WorkflowPrepare SBS reagents for Read 1 and indexing reagents. Weigh r

Perform a TruSeq v3 Run44Part # 15035786 Rev. DLoad paired-end reagents and fresh ICB for Read 2.Continue the run. The software automatically primes p

Run Types for TruSeq v3 ChemistryHiSeq 2500 System User Guide45Run Types for TruSeq v3 ChemistryThe following table shows types of sequencing runs and

Perform a TruSeq v3 Run46Part # 15035786 Rev. DEnter Run ParametersFrom the Welcome screen, select Sequence | New Run.The control software interface g

Enter Run ParametersHiSeq 2500 System User Guide47analysis, this field is optional.2 Select Zip BCL files to reduce required storage space. If the run

Perform a TruSeq v3 Run48Part # 15035786 Rev. D2 [Optional] From the Align to PhiX checkboxes, clear the checkbox for lanes that do notcontain PhiX.By

Enter Run ParametersHiSeq 2500 System User Guide49a SBS: TruSeq SBS Kit v3b Index: TruSeq Multiplex Sequencing Primer Box or TruSeq Dual IndexSequenci

viPart # 15035786 Rev. Dc Indemnification by Purchaser. Purchaser shall defend, indemnify and hold harmless Illumina, its affiliates, theirnon-affilia

Perform a TruSeq v3 Run50Part # 15035786 Rev. DReview Screen1 Review the run parameters on the Review screen.2 Select Next to proceed or select Back t

Load and Prime ReagentsHiSeq 2500 System User Guide51Load and Prime ReagentsAfter entering run parameters, load SBS and indexing reagents for the run,

Perform a TruSeq v3 Run52Part # 15035786 Rev. D6 Place each reagent bottle onto the rack in the associated numbered position. Make surethat the conica

Load and Prime ReagentsHiSeq 2500 System User Guide534 Slide the reagent rack out of the reagent compartment using the rack handle.5 Remove the caps f

Perform a TruSeq v3 Run54Part # 15035786 Rev. D10 Select Next.Prime ReagentsSteps for priming reagents include cleaning the flow cell holder, loading

Load and Prime ReagentsHiSeq 2500 System User Guide556 Visually inspect the flow cell holder to make sure that it is free of lint and the vacuumholes

Perform a TruSeq v3 Run56Part # 15035786 Rev. DFigure 25 Flow Cell Positioned Against Top and Right Guide PinsA Top guide pinB Right guide pins5 Slowl

Load and Prime ReagentsHiSeq 2500 System User Guide57Figure 27 Flow Cell Lever in Position 27 Make sure that the Vacuum Engaged checkbox is selected o

Perform a TruSeq v3 Run58Part # 15035786 Rev. DPosition Tubing and Start Prime1 Remove the 8 waste tubes for the appropriate flow cell from the waste

Load a Flow CellHiSeq 2500 System User Guide59Load a Flow CellSteps to load the clustered flow cell include removing the priming flow cell, cleaning t

HiSeq 2500 System User GuideviiRevision HistoryPart # Rev. DateDescription of Change15035786D November2014Updated Rapid Run mode workflow for compatib

Perform a TruSeq v3 Run60Part # 15035786 Rev. DFigure 30 Flow Cell Lever in Position 03 Lift the used flow cell from the flow cell holder.Clean the Fl

Load a Flow CellHiSeq 2500 System User Guide61Clean the Flow Cell1 Remove the flow cell from the flow cell container using a pair of plastistats.2 Rin

Perform a TruSeq v3 Run62Part # 15035786 Rev. DFigure 32 Flow Cell Positioned Against Top and Right Guide PinsA Top guide pinB Right guide pinsNOTERem

Load a Flow CellHiSeq 2500 System User Guide63Figure 34 Flow Cell Lever in Position 25 Make sure that the Vacuum Engaged checkbox is selected on the L

Perform a TruSeq v3 Run64Part # 15035786 Rev. DMonitor the RunMonitor run metrics on the run overview screen, fluidics, and imaging.Figure 35 Run Over

Prepare Reagents for Read 2HiSeq 2500 System User Guide65Prepare Reagents for Read 2Before the completion of Read 1 and any index reads, prepare reage

Perform a TruSeq v3 Run66Part # 15035786 Rev. DLoad Reagents for Read 2After completion of Read 1 and any index reads, load paired-end reagents for Re

Load Reagents for Read 2HiSeq 2500 System User Guide677 Slide the reagent rack into the reagent compartment, aligning the rack with the raisedguide on

68Part # 15035786 Rev. D

Chapter 4HiSeq 2500 System User Guide69Chapter 4 Perform a Rapid RunPerform a Rapid RunIntroduction 70Rapid Run Sequencing Workflow 72Run Types for

viiiPart # 15035786 Rev. D

Perform a Rapid Run70Part # 15035786 Rev. DIntroductionRapid Run mode provides 2 options for the cluster generation step, either on the cBot or onthe

IntroductionHiSeq 2500 System User Guide71• TruSeq Rapid PE Cluster Kit Reagent Prep Guide (part # 15038861)• TruSeq Rapid SR Cluster Kit Reagent Prep

Perform a Rapid Run72Part # 15035786 Rev. DRapid Run Sequencing WorkflowPrepare all reagents for the run and prepare the library template.For reagent

Run Types for Rapid Run ChemistryHiSeq 2500 System User Guide73Run Types for Rapid Run ChemistryThe following tables show types of sequencing runs and

Perform a Rapid Run74Part # 15035786 Rev. D¹ Number of cycles for single-indexed libraries² Number of cycles for dual-indexed libraries³ Index 2 Read

Pre-Run Volume CheckHiSeq 2500 System User Guide75Pre-Run Volume CheckFrom the Welcome screen, select Sequence | New Run. The Volume Check screen open

Perform a Rapid Run76Part # 15035786 Rev. DEnter Run ParametersFrom the Welcome screen, select Sequence | New Run.The control software interface guide

Enter Run ParametersHiSeq 2500 System User Guide77analysis, this field is optional.2 Select Zip BCL files to reduce required storage space. If the run

Perform a Rapid Run78Part # 15035786 Rev. DA first base report is generated automatically for each run. Selecting this option opensthe first base repo

Enter Run ParametersHiSeq 2500 System User Guide794 Confirm the following chemistry settings. These fields are auto-populated dependingon the selected

HiSeq 2500 System User GuideixTable of ContentsRevision History viiTable of Contents ixChapter 1 Overview 1Introduction 2HiSeq 2500 Components 3Start

Perform a Rapid Run80Part # 15035786 Rev. D• Select Custom for a partial kit or multiple 50-cycle kits. In the Cycles Remainingfield, enter the number

Load and Prime ReagentsHiSeq 2500 System User Guide81Load and Prime ReagentsAfter entering run parameters, load SBS, clustering, indexing, and paired-

Perform a Rapid Run82Part # 15035786 Rev. Da Pull the handle towards you and then raise the handle.b Release the sipper handle into the slot on the to

Load and Prime ReagentsHiSeq 2500 System User Guide839 Select the PW1 (25 ml) loaded checkbox.10 Slide the PEreagent rack out of the reagent compartm

Perform a Rapid Run84Part # 15035786 Rev. Dpositions:• Paired-end run—Position 19Non-indexed—Position 17• Single-read run—Positions 10, 11, and 12Non-

Load and Prime ReagentsHiSeq 2500 System User Guide85Figure 36 Loading StationNOTEThe liquid remaining in the tube after the run is highly diluted and

Perform a Rapid Run86Part # 15035786 Rev. DFigure 37 Flow Cell Lever in Position 03 Put on a new pair of powder-free latex gloves.4 If the flow cell f

Load and Prime ReagentsHiSeq 2500 System User Guide87Load a Priming Flow CellNOTEUse a used flow cell to prime reagents. Do not use the flow cell that

Perform a Rapid Run88Part # 15035786 Rev. DFigure 40 Flow Cell Lever in Position 16 Wait for about 5 seconds, and then slowly move the flow cell lever

Load and Prime ReagentsHiSeq 2500 System User Guide89CAUTIONUse water to confirm proper flow on a used flow cell only. Never use water to confirmprope