HiSeq® 2500 System User Guide FOR RESEARCH USE ONLY ILLUMINA PROPRIETARY Part # 15035786 Rev. D November 2014 Catalog # SY-401-9001DOC Customize a short end-to-end workflow guide with the Custom Protocol Selector support.illumina.com/custom-protocol-selector.
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Revision History Part # Rev. Date 15035786 D November 2014 15035786 C April 2014 Updated software descriptions to HiSeq Control Software v2.2, which includes the HiSeq v4 high output mode, removal of the control lane option, default Q-score binning, and the option to use different indexing schemes in each lane. Added the HiSeq v4 workflow for use with HiSeq v4 chemistry. Added calculation for total SBS priming volume. 15035786 B November 2013 Removed reagent preparation instructions.
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Table of Contents Revision History Table of Contents Chapter 1 Overview Introduction HiSeq 2500 Components Start the HiSeq 2500 HiSeq 2500 Software Available Disk Space Sample Sheet Overview Sequencing Consumables Additional Resources Chapter 2 Perform a HiSeq v4 Run vii ix 1 2 3 6 7 12 13 14 16 17 Introduction HiSeq v4 Sequencing Workflow Run Types for HiSeq v4 Chemistry Enter Run Parameters Load and Prime Reagents Load a Flow Cell Monitor the Run 18 19 20 21 26 35 40 Chapter 3 Perform a TruSeq v3
Introduction Rapid Run Sequencing Workflow Run Types for Rapid Run Chemistry Pre-Run Volume Check Enter Run Parameters Load and Prime Reagents Load a Flow Cell Monitor the Run Chapter 5 Post-Run Procedures Introduction Unload and Weigh Reagents Perform a Maintenance Wash Perform a Water Wash Switch Sequencing Modes Idle the Instrument Shut Down the Instrument Chapter 6 Real-Time Analysis Introduction Real-Time Analysis Overview Monitor Run Metrics Real-Time Analysis Workflow Sequencing Output Files Output
Chapter 1 Overview Introduction HiSeq 2500 Components Start the HiSeq 2500 HiSeq 2500 Software Available Disk Space Sample Sheet Overview Sequencing Consumables Additional Resources HiSeq 2500 System User Guide 2 3 6 7 12 13 14 16 1 Chapter 1 Overview
Overview Introduction The HiSeq® system combines innovative engineering with proven SBS technology to set new standards in output, simplicity, and cost-effectiveness. The HiSeq 2500 includes the following features: } Dual-surface imaging—The HiSeq 2500 uses a 4-camera epifluorescence system with cutting-edge scanning technology to enable dual surface imaging.
The HiSeq 2500 system comprises the instrument, monitor, instrument control computer, and accessories, such as a keyboard, mouse, and barcode scanner. The instrument includes 4 main compartments: the optics module, flow cell compartment, fluidics compartment, and reagents compartment. Instrument operating status is indicated on an illuminated status bar.
Overview E Reagent compartment—Contains reagent racks that hold reagents for sequencing runs and wash solution for instrument washes. Reagent Compartment The reagent compartment is a high-capacity reagent chiller that holds 3 reagent racks: 2 for SBS reagents and 1 for clustering, indexing, and paired-end reagents. Sipper handles lower the sippers into the reagent bottles. } SBS reagent racks—Hold 250 ml conical bottles.
HiSeq 2500 Components Flow Cell Compartment The flow cell compartment houses the flow cell stage, the thermal stations, the vacuum system, and the fluidics connections to each flow cell. Figure 3 Flow Cell Stage With 2 Flow Cells A B C D Flow cell A Flow cell B Flow cell lever A Flow cell lever B The flow cell on the left is flow cell A, and the flow cell on the right is flow cell B.
Overview Start the HiSeq 2500 1 Start the instrument control computer. 2 Log on to the operating system using the default user name and password. • User name: sbsuser • Password: sbs123 Wait until it has loaded. If the default values do not work, consult your facility administrator for the site-specific user name and password. 3 Turn on the main power switch to the ON position. When facing the front of the instrument, the power switch is on the left side.
Three software applications are installed on the instrument computer: } HiSeq 2500 control software—The HiSeq Control Software (HCS) interface guides you through the steps to set up a sequencing run. During the run, the control software operates instrument hardware, controls fluidics, sets temperatures, and provides a visual summary of quality statistics.
Overview Figure 4 Welcome Screen A B C D Welcome screen menu button Interface panel for flow cell A Interface panel for flow cell B Activity indicators Welcome Screen Commands The Welcome screen commands include Sequence, Wash, Check, and Mode Select. } Sequence—Select Sequence to begin the steps to set up a new sequencing run or resume an existing run.
Figure 6 Wash Command Options • Water Wash—The water wash flushes water through the system. This wash is required after a sequencing run or after the instrument has been idle for 1 day or more. See Perform a Water Wash on page 104. • Maintenance Wash—The maintenance wash flushes Tween 20 and ProClin 300 through the system. This wash is required before switching modes or every 10 days, and is a recommended option after a high output run. See Perform a Maintenance Wash on page 100.
Overview Status Icons A status icon located in the upper-right corner of each screen shows changes in conditions, errors, or warnings during run setup steps and during the run. Status Icon Status Name Status OK Description No change. System is normal. Information Information only. No action is required Attention Information that might require attention. Warning Warnings do not stop a run, but might require action before proceeding.
Menu Options Window The Menu Options window provides settings to define the run ID template, default folder locations, a LIMS server, user name, and password, and whether to send instrument health information to Illumina. Figure 9 Menu Options Window } Run ID Template—The naming convention used to generate run folder names. } Default Output Folder—The default output folder for runs on flow cell A. This location can be changed on a per run basis.
Overview Available Disk Space The HiSeq instrument computer has a storage capacity of over 2.7 TB per flow cell. Data from flow cell A is stored on the D: drive, and data from flow cell B is stored on the E: drive. At the end of each imaging cycle for each lane, the software checks available disk space on the local D: and E: drives. The software does not check the network location during the run.
The sample sheet is a user-generated file in *.csv format that stores information about the sequencing run. When the run begins, the software copies the sample sheet to the run folder where it is later used for analysis. Sample sheets are optional unless you are using BaseSpace to perform data analysis, performing an indexing run, or planning to monitor demultiplexing performance using Sequencing Analysis Viewer. Use the Illumina Experiment Manager (IEM) to create a sample sheet before you start the run.
Overview Sequencing Consumables Sequencing on the HiSeq 2500 requires reagents and other consumables provided in Illumina kits. Required kits depend on the type of run to be performed. } For HiSeq v4 high output runs, see HiSeq v4 Sequencing Consumables on page 18. } For TruSeq v3 high output runs, see TruSeq v3 Sequencing Consumables on page 42. } For Rapid Run, see Rapid Run Sequencing Consumables on page 70.
Water, laboratory-grade, 18 M Ohm Supplier McMaster-Carr, catalog # 7003A22 Millipore Purpose Removing the flow cell gaskets. SBS reagent rack, position 2. Instrument wash. Microcentrifuge Tubes for Rapid Run Mode Consumable Microcentrifuge tube, 1.5 ml Microcentrifuge tube, 1.
Overview Additional Resources The following documentation is available for download from the Illumina website. Resource Description HiSeq 2500, 1500, and 2000 Site Prep Guide (part # 15006407) Provides specifications for laboratory space, electrical requirements, and environmental considerations. HiSeq Safety and Compliance Guide (part # 15012614) Provides information about instrument labeling, compliance certifications, and safety considerations.
Chapter 2 Perform a HiSeq v4 Run Introduction HiSeq v4 Sequencing Workflow Run Types for HiSeq v4 Chemistry Enter Run Parameters Load and Prime Reagents Load a Flow Cell Monitor the Run HiSeq 2500 System User Guide 18 19 20 21 26 35 40 17 Chapter 2 Perform a HiSeq v4 Run
Perform a HiSeq v4 Run Introduction To perform a HiSeq v4 run on the HiSeq 2500, prepare all reagents for the run and then follow the software prompts to set up the run. Run setup steps include entering run parameters, loading and priming reagents, loading the flow cell, and performing a fluidics check. Visit the HiSeq 2500 specifications page on the Illumina website for information about run duration and other performance specifications.
Prepare reagents for the run. Weigh reagents after preparation. For reagent preparation information, see Reagent Preparation Steps on page 18. Using the control software, enter run parameters. When prompted, load all reagents for the run: • Load SBS reagents for Read 1 and Read 2. • For indexed runs, load indexing reagents. • For paired-end runs, load paired-end reagents. With a used flow cell on the instrument, confirm proper flow. Prime SBS reagents and measure priming waste.
Perform a HiSeq v4 Run Run Types for HiSeq v4 Chemistry The following table shows types of sequencing runs and the number of possible cycles for each read when using HiSeq v4 chemistry. Use this information as a reference when setting up the run.
From the Welcome screen, select Sequence | New Run. The control software interface guides you through the steps to set up the run. Run setup steps are organized in 3 tabs: Run Configuration, Pre-Run Setup, and Initiate Run. } Run configuration screens contain drop-down lists, checkboxes, or text fields for run parameters. Use the hand-held barcode scanner to scan the flow cell or reagent kit ID, or enter the ID using the touch screen keyboard. The keyboard icon is located to the right of the text fields.
Perform a HiSeq v4 Run analysis, this field is optional. 2 Select Zip BCL files to reduce required storage space. If the run is connected to BaseSpace, the Zip BCL files option is selected by default. NOTE The Bin Q-Scores setting is enabled by default to reduce required storage space. This setting groups quality scores over a wider range of values without affecting accuracy or performance. 3 Select from the following Save Auxiliary Files options: • Save All Thumbnails—Saves all thumbnails images.
2 [Optional] From the Align to PhiX checkboxes, clear the checkbox for lanes that do not contain PhiX. By default, all lanes are selected for alignment by Real-Time Analysis software. Alternatively, select lanes on the flow cell image to add or remove lanes for PhiX alignment. NOTE A dedicated control lane is not required with HCS v2.2 and RTA v1.18. Therefore, the option to assign a control lane is not available with this software configuration. 3 Select Next.
Perform a HiSeq v4 Run allow the software to create the recipe from the run parameters entered. Sample Sheet Screen Sample sheets are optional unless you use BaseSpace to perform data analysis or perform an indexed run. 1 Select Browse to navigate to the sample sheet location. 2 Select Next. NOTE HiSeq Control Software v2.2 allows a different indexing scheme in each lane. Reagents Screen The Reagents screen records information about reagent kits used for the run.
Enter Run Parameters 2 Select Next to proceed or select Back to change parameters.
Perform a HiSeq v4 Run Load and Prime Reagents After entering run parameters, load SBS, indexing, and paired-end reagents for the run, and then prime reagents through the fluidics system. The software guides you through these steps in a series of screens on the Pre-Run Setup tab.
Place each reagent bottle onto the rack in the associated numbered position. Make sure that the conical end of the bottle rests in the indentation on the base of the rack.
Perform a HiSeq v4 Run 4 Slide the reagent rack out of the reagent compartment using the rack handle. 5 Remove the caps from each reagent tube and place the tube onto the rack in the associated numbered position or matching label color. Table 2 Single-Read Flow Cells Position Reagent 15 16 17 FDR HP9 * HP12 Description Fast Denaturation Reagent (contains formamide) Index Sequencing Primer i5 Index Sequencing Primer i7 * HP9 is required for dual-indexed runs only.
1 Record the weight of each reagent on the lab tracking form. 2 Raise the sippers for the paired-end reagent rack using the following motion: a Pull the handle towards you and then raise the handle. b Release the handle into the slot on the top end of the groove. Make sure that the handle rests securely in the slot. 3 Slide the reagent rack out of the reagent compartment using the rack handle.
Perform a HiSeq v4 Run Prime Reagents Steps for priming reagents include cleaning the flow cell holder, loading a priming flow cell, confirming proper flow, and then starting the prime. Clean the Flow Cell Holder 1 Open the flow cell compartment door. CAUTION Do not place fluids on the flow cell compartment door or on the flow cell stage when the door is open. Spills in this area can damage the instrument. 2 Make sure that the flow cell lever is in the OFF position.
Load a Priming Flow Cell From the Load Priming Flow Cell screen, load a used flow cell for the priming step. After loading a used flow cell, confirm that the vacuum is engaged. NOTE Illumina recommends using the flow cell from a previous run for priming reagents on a subsequent run or for a post-run instrument wash. 1 Rinse the used flow cell with laboratory-grade water. Dry the flow cell with a lens cleaning tissue or lint-free tissue. 2 Clean the flow cell using alcohol wipes and lens cleaning tissue.
Perform a HiSeq v4 Run Figure 12 Flow Cell Positioned Against Top and Right Guide Pins A B 5 Top guide pin Right guide pins Slowly move the flow cell lever to position 1, which engages the vacuum and secures the flow cell into position. When the flow cell lever is blinking green, the vacuum is engaged. If the lever is not green, see Possible Run Setup Problems on page 129.
7 Make sure that the Vacuum Engaged checkbox is selected on the load prime flow cell screen, and then select Next. Confirm Proper Flow Checking for proper flow confirms that the flow cell and gaskets are properly installed and the manifold is engaged. 1 Select solution 2 (laboratory-grade water) from the drop-down list. CAUTION Use water to confirm proper flow on a used flow cell only. Never use water to confirm proper flow on a clustered flow cell.
Perform a HiSeq v4 Run Position Tubing and Start Prime 1 Remove the 8 waste tubes for the appropriate flow cell from the waste container. Do not include the 8 tubes for the opposite flow cell or the tube for the condensation pump. Figure 15 Position Tubing A B 34 Flow cell waste tubes for reagent positions 1–8 Condensation pump tubing (do not remove) 2 Place waste tubing into an empty 15 ml tube, 1 waste tube per 15 ml tube. Priming waste is collected and measured after the priming step.
Steps to load the clustered flow cell include removing the priming flow cell, cleaning the flow cell holder, cleaning the flow cell, loading the flow cell, and confirming proper flow. User-Supplied Consumables } } } } Lens cleaning tissue 70% ethanol or alcohol wipes Low-lint lab tissue One pair of plastistats Remove the Used Flow Cell 1 Slowly move the flow cell lever to position 1 to disengage the manifolds.
Perform a HiSeq v4 Run Figure 17 Flow Cell Lever in Position 0 3 Lift the used flow cell from the flow cell holder. Clean the Flow Cell Holder 1 Put on a new pair of powder-free latex gloves. 2 Using an alcohol wipe or a lint-free tissue moistened with ethanol or isopropanol, carefully wipe the surface of the flow cell holder until it is clean. CAUTION Do not allow alcohol to drip into the vacuum holes or around the manifolds. Use a low-lint lab tissue to dry the stage, if necessary.
1 Remove the flow cell from the flow cell container using a pair of plastistats. 2 Rinse the flow cell with laboratory-grade water and dry it with a lens cleaning tissue. 3 Fold an alcohol wipe to approximately the size of the flow cell. 4 Hold the edges of the clustered flow cell with 2 fingers. Make sure that the inlet and outlet ports are facing up. 5 Wipe each side of the flow cell with a single sweeping motion. Repeat, refolding the alcohol wipe with each pass, until the flow cell is clean.
Perform a HiSeq v4 Run Figure 19 Flow Cell Positioned Against Top and Right Guide Pins A B Top guide pin Right guide pins NOTE Remove your hand from the flow cell before engaging the vacuum switch to prevent possible alignment drift over time. 3 Slowly move the flow cell lever to position 1, which engages the vacuum and secures the flow cell into position. When the flow cell lever is blinking green, the vacuum is engaged. If the lever is not green, see Possible Run Setup Problems on page 129.
Load a Flow Cell Figure 21 Flow Cell Lever in Position 2 5 Make sure that the Vacuum Engaged checkbox is selected on the Load Sequencing Flow Cell screen. Confirm Proper Flow Checking for proper flow confirms that the flow cell and gaskets are properly installed and the manifold is engaged. 1 Select solution 5 from the drop-down list. 2 Enter the following default values: • Volume: 250 • Aspirate Rate: 250 • Dispense Rate: 2000 3 Select Pump.
Perform a HiSeq v4 Run Monitor the Run Monitor run metrics on the run overview screen, fluidics, and imaging. Figure 22 Run Overview Screen A B C D E Progress bar—Use the progress bar to monitor how many cycles have been completed. Fluidics graph—Expand the fluidics section to monitor chemistry steps. Run Configuration—Review parameters of current run. Analysis graph—Use the analysis graph to monitor quality scores by cycle. Images graph—Use the images graph to monitor intensities by cycle.
Chapter 3 Perform a TruSeq v3 Run Introduction TruSeq v3 Sequencing Workflow Run Types for TruSeq v3 Chemistry Enter Run Parameters Load and Prime Reagents Load a Flow Cell Monitor the Run Prepare Reagents for Read 2 Load Reagents for Read 2 HiSeq 2500 System User Guide 42 43 45 46 51 59 64 65 66 41 Chapter 3 Perform a TruSeq v3 Run
Perform a TruSeq v3 Run Introduction To perform a TruSeq v3 run on the HiSeq 2500, prepare SBS reagents for Read 1 and indexing reagents before setting up the run. Follow the software prompts to set up the run, which includes entering run parameters, loading and priming reagents, loading the flow cell, and performing a fluidics check. Prepare and load paired-end reagents and SBS reagents for Read 2 after the completion of Read 1 and any index reads.
Prepare SBS reagents for Read 1 and indexing reagents. Weigh reagents after preparation. For reagent preparation information, see Reagent Preparation Steps on page 42. Using the control software, enter run parameters. When prompted, load all SBS reagents for Read 1. Load SBS reagents for Read 2, except ICB. Load indexing reagents. With a used flow cell on the instrument, confirm proper flow. Prime SBS reagents and measure priming waste. Load the clustered flow cell for sequencing. Confirm proper flow.
Perform a TruSeq v3 Run Load paired-end reagents and fresh ICB for Read 2. Continue the run. The software automatically primes paired-end reagents and performs Read 2 resynthesis and Read 2. When the run is complete, unload and weigh reagents. Perform an instrument wash. 44 Part # 15035786 Rev.
The following table shows types of sequencing runs and the number of possible cycles for each read when using TruSeq v3 chemistry. Use this information as a reference when setting up the run.
Perform a TruSeq v3 Run Enter Run Parameters From the Welcome screen, select Sequence | New Run. The control software interface guides you through the steps to set up the run. Run setup steps are organized in 3 tabs: Run Configuration, Pre-Run Setup, and Initiate Run. } Run configuration screens contain drop-down lists, checkboxes, or text fields for run parameters. Use the hand-held barcode scanner to scan the flow cell or reagent kit ID, or enter the ID using the touch screen keyboard.
2 Select Zip BCL files to reduce required storage space. If the run is connected to BaseSpace, the Zip BCL files option is selected by default. NOTE The Bin Q-Scores setting is enabled by default to reduce required storage space. This setting groups quality scores over a wider range of values without affecting accuracy or performance. 3 Select from the following Save Auxiliary Files options: • Save All Thumbnails—Saves all thumbnails images.
Perform a TruSeq v3 Run 2 [Optional] From the Align to PhiX checkboxes, clear the checkbox for lanes that do not contain PhiX. By default, all lanes are selected for alignment by Real-Time Analysis (RTA). Alternatively, select lanes on the flow cell image to add or remove lanes for PhiX alignment. NOTE A dedicated control lane is not required with HCS v2.2 and RTA v1.18. Therefore, the option to assign a control lane is not available with this software configuration.
c 5 SBS: TruSeq SBS Kit v3 Index: TruSeq Multiplex Sequencing Primer Box or TruSeq Dual Index Sequencing Primer Box PE turnaround: TruSeq PE Cluster Kit v3 [Optional] Select the Use Existing Recipe to use a custom recipe. Otherwise, allow the software to create the recipe from run parameters entered. Sample Sheet Screen Sample sheets are optional unless you use BaseSpace to perform data analysis or perform an indexed run. 1 Select Browse to navigate to the sample sheet location. 2 Select Next.
Perform a TruSeq v3 Run Review Screen 50 1 Review the run parameters on the Review screen. 2 Select Next to proceed or select Back to change parameters. Part # 15035786 Rev.
After entering run parameters, load SBS and indexing reagents for the run, and then prime reagents through the fluidics system. The software guides you through these steps in a series of screens on the Pre-Run Setup tab.
Perform a TruSeq v3 Run 6 Place each reagent bottle onto the rack in the associated numbered position. Make sure that the conical end of the bottle rests in the indentation on the base of the rack.
Slide the reagent rack out of the reagent compartment using the rack handle. 5 Remove the caps from each reagent tube and place the tube onto the rack in the associated numbered position or matching label color.
Perform a TruSeq v3 Run 10 Select Next. Prime Reagents Steps for priming reagents include cleaning the flow cell holder, loading a priming flow cell, confirming proper flow, and then starting the prime. Clean the Flow Cell Holder 1 Open the flow cell compartment door. CAUTION Do not place fluids on the flow cell compartment door or on the flow cell stage when the door is open. Spills in this area can damage the instrument. 2 Make sure that the flow cell lever is in the OFF position.
Visually inspect the flow cell holder to make sure that it is free of lint and the vacuum holes are free of obstructions. Figure 24 Vacuum Hole Locations Load a Priming Flow Cell From the Load Priming Flow Cell screen, load a used flow cell for the priming step. After loading a used flow cell, confirm that the vacuum is engaged. NOTE Illumina recommends using the flow cell from a previous run for priming reagents on a subsequent run or for a post-run instrument wash.
Perform a TruSeq v3 Run Figure 25 Flow Cell Positioned Against Top and Right Guide Pins A B 5 Top guide pin Right guide pins Slowly move the flow cell lever to position 1, which engages the vacuum and secures the flow cell into position. When the flow cell lever is blinking green, the vacuum is engaged. If the lever is not green, see Possible Run Setup Problems on page 129.
7 Make sure that the Vacuum Engaged checkbox is selected on the load prime flow cell screen, and then select Next. Confirm Proper Flow Checking for proper flow confirms that the flow cell and gaskets are properly installed and the manifold is engaged. 1 Select solution 2 (laboratory-grade water) from the drop-down list. CAUTION Use water to confirm proper flow on a used flow cell only. Never use water to confirm proper flow on a clustered flow cell.
Perform a TruSeq v3 Run Position Tubing and Start Prime 1 Remove the 8 waste tubes for the appropriate flow cell from the waste container. Do not include the 8 tubes for the opposite flow cell or the tube for the condensation pump. Figure 28 Position Tubing A B 58 Flow cell waste tubes for reagent positions 1–8 Condensation pump tubing (do not remove) 2 Place waste tubing into an empty 15 ml tube, 1 waste tube per 15 ml tube. Priming waste is collected and measured after the priming step.
Steps to load the clustered flow cell include removing the priming flow cell, cleaning the flow cell holder, cleaning the flow cell, loading the flow cell, and confirming proper flow. User-Supplied Consumables } } } } Lens cleaning tissue 70% ethanol or alcohol wipes Low-lint lab tissue One pair of plastistats Remove the Used Flow Cell 1 Slowly move the flow cell lever to position 1 to disengage the manifolds.
Perform a TruSeq v3 Run Figure 30 Flow Cell Lever in Position 0 3 Lift the used flow cell from the flow cell holder. Clean the Flow Cell Holder 1 Put on a new pair of powder-free latex gloves. 2 Using an alcohol wipe or a lint-free tissue moistened with ethanol or isopropanol, carefully wipe the surface of the flow cell holder until it is clean. CAUTION Do not allow alcohol to drip into the vacuum holes or around the manifolds. Use a low-lint lab tissue to dry the stage, if necessary.
1 Remove the flow cell from the flow cell container using a pair of plastistats. 2 Rinse the flow cell with laboratory-grade water and dry it with a lens cleaning tissue. 3 Fold an alcohol wipe to approximately the size of the flow cell. 4 Hold the edges of the clustered flow cell with 2 fingers. Make sure that the inlet and outlet ports are facing up. 5 Wipe each side of the flow cell with a single sweeping motion. Repeat, refolding the alcohol wipe with each pass, until the flow cell is clean.
Perform a TruSeq v3 Run Figure 32 Flow Cell Positioned Against Top and Right Guide Pins A B Top guide pin Right guide pins NOTE Remove your hand from the flow cell before engaging the vacuum switch to prevent possible alignment drift over time. 3 Slowly move the flow cell lever to position 1, which engages the vacuum and secures the flow cell into position. When the flow cell lever is blinking green, the vacuum is engaged. If the lever is not green, see Possible Run Setup Problems on page 129.
Load a Flow Cell Figure 34 Flow Cell Lever in Position 2 5 Make sure that the Vacuum Engaged checkbox is selected on the Load Sequencing Flow Cell screen. Confirm Proper Flow Checking for proper flow confirms that the flow cell and gaskets are properly installed and the manifold is engaged. 1 Select solution 5 from the drop-down list. 2 Enter the following default values: • Volume: 250 • Aspirate Rate: 250 • Dispense Rate: 2000 3 Select Pump.
Perform a TruSeq v3 Run Monitor the Run Monitor run metrics on the run overview screen, fluidics, and imaging. Figure 35 Run Overview Screen A B C D E Progress bar—Use the progress bar to monitor how many cycles have been completed. Fluidics graph—Expand the fluidics section to monitor chemistry steps. Run Configuration—Review parameters of current run. Analysis graph—Use the analysis graph to monitor quality scores by cycle. Images graph—Use the images graph to monitor intensities by cycle.
Before the completion of Read 1 and any index reads, prepare reagents for Read 2 resynthesis and fresh ICB for Read 2. For reagent preparation instructions, see the TruSeq PE Cluster Kit v3 Reagent Prep Guide (part # 15023336). This guide includes instructions for preparing sequencing reagents provided in the TruSeq Dual Index Sequencing Primer Box. NOTE For optimal performance, Illumina recommends preparing fresh ICB (Incorporation Mix) for Read 2.
Perform a TruSeq v3 Run Load Reagents for Read 2 After completion of Read 1 and any index reads, load paired-end reagents for Read 2 resynthesis and freshly prepared ICB for Read 2. Load Paired-End Reagents 1 Record the weight of each reagent on the lab tracking form. 2 Make sure that the paired-end rack is not in use on the opposite flow cell for Read 2 resynthesis, Index 1 (i7) Read preparation, or Index 2 (i5) Read preparation.
Slide the reagent rack into the reagent compartment, aligning the rack with the raised guide on the floor of the compartment. 8 Lower the sippers into the paired-end reagent tubes as follows: a Pull the handle towards you and lower the handle. b Visually inspect the sippers to make sure that they do not bend as they lower into the tubes. c Release the handle into the slot on the bottom end of the groove. Load ICB for Read 2 1 Record the weight of the reagent on the lab tracking form.
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Chapter 4 Perform a Rapid Run Introduction Rapid Run Sequencing Workflow Run Types for Rapid Run Chemistry Pre-Run Volume Check Enter Run Parameters Load and Prime Reagents Load a Flow Cell Monitor the Run HiSeq 2500 System User Guide 70 72 73 75 76 81 90 96 69 Chapter 4 Perform a Rapid Run
Perform a Rapid Run Introduction Rapid Run mode provides 2 options for the cluster generation step, either on the cBot or on the HiSeq 2500. Clustering on the cBot allows 2 libraries, 1 each lane, on the 2-lane rapid flow cell. After template hybridization and first extension on the cBot, the remainder of the clustering process is performed on the HiSeq.
Prepare all reagents before setting up the run. When prompted by the control software, load all reagents. When using rapid run chemistry, there is no need to return to the instrument during the run to load reagents.
Perform a Rapid Run Rapid Run Sequencing Workflow Prepare all reagents for the run and prepare the library template. For reagent preparation information, see Reagent Preparation Steps on page 70. Using the control software, perform a volume check and enter run parameters. For on-instrument clustering—Load all reagents for the run and the prepared library template. For cluster generation on the cBot—Load all reagents for the run. With a used flow cell on the instrument, confirm proper flow.
The following tables show types of sequencing runs and the number of possible cycles for each read when using rapid run chemistry. Use this information as a reference when setting up the run.
Perform a Rapid Run ¹ Number of cycles for single-indexed libraries ² Number of cycles for dual-indexed libraries ³ Index 2 Read of a paired-end dual-indexed run includes 7 additional chemistry-only cycles 74 Part # 15035786 Rev.
From the Welcome screen, select Sequence | New Run. The Volume Check screen opens. Volume Check Screen 1 When prompted by the software to perform a volume check, select Yes. 2 Place waste tubes 1, 2, 3, 6, 7, and 8 for the current flow cell in a 1 liter bottle filled with deionized water. Placing the tubes in deionized water prevents damage to the reagent pumps.
Perform a Rapid Run Enter Run Parameters From the Welcome screen, select Sequence | New Run. The control software interface guides you through the steps to set up the run. Run setup steps are organized in 3 tabs: Run Configuration, Pre-Run Setup, and Initiate Run. } Run configuration screens contain drop-down lists, checkboxes, or text fields for run parameters. Use the hand-held barcode scanner to scan the flow cell or reagent kit ID, or enter the ID using the touch screen keyboard.
2 Select Zip BCL files to reduce required storage space. If the run is connected to BaseSpace, the Zip BCL files option is selected by default. NOTE The Bin Q-Scores setting is enabled by default to reduce required storage space. This setting groups quality scores over a wider range of values without affecting accuracy or performance. 3 Select from the following Save Auxiliary Files options: • Save All Thumbnails—Saves all thumbnails images.
Perform a Rapid Run A first base report is generated automatically for each run. Selecting this option opens the first base report before proceeding with the run. 2 [Optional] From the Align to PhiX checkboxes, clear the checkbox for lanes that do not contain PhiX. By default, all lanes are selected for alignment by Real-Time Analysis. Alternatively, select lanes on the flow cell image to add or remove lanes for PhiX alignment. NOTE A dedicated control lane is not required with HCS v2.2 and RTA v1.18.
Confirm the following chemistry settings. These fields are auto-populated depending on the selected reagent kit type and flow cell format option. a SBS: TruSeq Rapid SBS Kit v1 or HiSeq Rapid SBS Kit v2 b Cluster Kit: TruSeq Rapid PE Cluster Kit v1, TruSeq Rapid SR Cluster Kit v1, HiSeq Rapid PE Cluster Kit v2, or HiSeq Rapid SR Cluster Kit v2 5 [Optional] Select the Use Existing Recipe checkbox to use a custom recipe. Otherwise, allow the software to create the recipe from run parameters entered.
Perform a Rapid Run • Select Custom for a partial kit or multiple 50-cycle kits. In the Cycles Remaining field, enter the number of SBS cycles that reagents are expected to last. NOTE For partial kits, the software counts down the number of cycles entered. When the cycles are low, the software prompts you to load fresh reagents. 4 Select Next. Review Screen 80 1 Review the run parameters on the Review screen. 2 Select Next to proceed or select Back to change parameters. Part # 15035786 Rev.
After entering run parameters, load SBS, clustering, indexing, and paired-end reagents for the run, and then prime reagents through the fluidics system. The software guides you through these steps in a series of screens on the Pre-Run Setup tab.
Perform a Rapid Run a b Pull the handle towards you and then raise the handle. Release the sipper handle into the slot on the top end of the groove. Make sure that the sipper handle rests securely in the slot. 4 Slide the SBS reagent rack out of the reagent compartment. 5 Remove the cap from each reagent bottle and replace it with a funnel cap. Replace the cap on the bottle of CRM last, and then replace your gloves. 6 Place each SBS reagent bottle onto the rack in the associated numbered position.
Select the PW1 (25 ml) loaded checkbox. 10 Slide the PE reagent rack out of the reagent compartment. 11 Remove the caps from each reagent tube and place the tube onto the rack in the associated numbered position.
Perform a Rapid Run positions: • Paired-end run—Position 19 Non-indexed—Position 17 • Single-read run—Positions 10, 11, and 12 Non-indexed—Positions 16 and 17 13 Slide the PE rack into the reagent compartment, aligning the racks with the raised guide on the floor of the compartment. 14 Lower the sippers into the sequencing reagent bottles as follows: a Pull the sipper handle towards you and then lower the sipper handle.
NOTE The liquid remaining in the tube after the run is highly diluted and not suitable for further use. d Slowly close the loading station door, making sure that the sippers are properly aligned with the Eppendorf tubes when the lid is closed. 3 Select the Template loaded and template loading station closed checkbox. 4 Select Next. Prime Reagents NOTE Prime reagents only if the HiSeq or TruSeq Rapid Duo Sample Loading Kit was used to perform template hybridization on the cBot.
Perform a Rapid Run Figure 37 Flow Cell Lever in Position 0 3 Put on a new pair of powder-free latex gloves. 4 If the flow cell from a previous run is present, remove it and set aside in a tube of storage buffer or laboratory-grade water to keep it from drying out. It can be used to confirm proper flow before loading the clustered flow cell. 5 Using an alcohol wipe or a lint-free tissue moistened with ethanol or isopropanol, carefully wipe the surface of the flow cell holder until it is clean.
NOTE Use a used flow cell to prime reagents. Do not use the flow cell that you want to sequence. 1 Rinse a used flow cell with laboratory-grade water. Dry it with lens cleaning tissue or lint-free tissue. 2 Clean the flow cell using alcohol wipes and lens cleaning tissue. NOTE Do not remove or replace the manifold gaskets. 3 Place the used flow cell on the flow cell holder with the inlet and outlet ports facing down and the barcode on the right.
Perform a Rapid Run Figure 40 Flow Cell Lever in Position 1 6 Wait for about 5 seconds, and then slowly move the flow cell lever to position 2 (farright). When the flow cell lever is solid green, the manifolds are in position and the flow cell is ready for use. Figure 41 Flow Cell Lever in Position 2 7 Enter the flow cell ID. NOTE You can use a TruSeq Rapid flow cell or a HiSeq Rapid v2 flow cell for priming.
2 Confirm the following default values: • Volume: 250 • Aspirate Rate: 1500 • Dispense Rate: 2000 3 Select Pump. 4 Visually inspect the flow cell for bubbles passing through the lanes and leaks near the manifolds. If excessive bubbles are present, check the gaskets for obstructions, reduce the aspirate rate to 1000, and pump another 250 µl of water to the flow cell. If problems persist, remove the flow cell, repeat the cleaning steps, and reload the flow cell.
Perform a Rapid Run Load a Flow Cell The next step is to remove the used flow cell and load the flow cell that you want to sequence. NOTE If the cBot was used for clustering, load the clustered flow cell. For on-instrument clustering, load a new flow cell. Remove the Used Flow Cell 1 Open the flow cell compartment door. Figure 42 Flow Cell Lever in Position 1 90 2 Slowly move the flow cell lever to position 1 to disengage the manifolds.
Load a Flow Cell Figure 43 Flow Cell Lever in Position 0 4 Lift the used flow cell from the flow cell holder. Clean the Flow Cell Holder 1 Put on a new pair of powder-free latex gloves. 2 Using an alcohol wipe or a lint-free tissue moistened with ethanol or isopropanol, carefully wipe the surface of the flow cell holder until it is clean. CAUTION Do not allow alcohol to drip into the vacuum holes or around the manifolds. Use a low-lint lab tissue to dry the stage, if necessary.
Perform a Rapid Run Clean the Flow Cell 1 Remove the flow cell from the flow cell container using a pair of plastistats. 2 Rinse the flow cell with laboratory-grade water and dry it with a lens cleaning tissue. 3 Fold an alcohol wipe to approximately the size of the flow cell. 4 Hold the edges of the clustered flow cell with 2 fingers. Make sure that the inlet and outlet ports are facing up. 5 Wipe each side of the flow cell with a single sweeping motion.
Load a Flow Cell Figure 45 Flow Cell Positioned Against Top and Right Guide Pins A B Top Guide Pin Right Guide Pins NOTE Remove your hand from the flow cell before engaging the vacuum switch to prevent possible alignment drift over time. 3 Slowly move the flow cell lever to position 1 to engage the vacuum and secure the flow cell into position. When the flow cell lever is green, the vacuum is engaged.
Perform a Rapid Run Figure 47 Flow Cell Lever in Position 2 5 Make sure that the Vacuum Engaged checkbox is selected on the load sequencing flow cell screen. Confirm Proper Flow 94 1 Select solution 5 (USB) from the drop-down list. 2 Make sure that the following default values are entered: • Volume: 250 • Aspirate Rate: 1500 • Dispense Rate: 2000 3 Make sure that waste outlet tubes 1, 2, 3, 6, 7, and 8 are in a bottle of clean water and that tubes 4 and 5 are in the waste container.
Confirm that the Vacuum Engaged and Door Closed checkboxes are selected, and then select Next. 9 Select Start to start the sequencing run.
Perform a Rapid Run Monitor the Run Monitor run metrics on the run overview screen, fluidics, and imaging. Figure 48 Run Overview Screen A B C D E Progress bar—Use the progress bar to monitor how many cycles have been completed. Fluidics graph—Expand the fluidics section to monitor chemistry steps. Run Configuration—Review parameters of current run. Analysis graph—Use the analysis graph to monitor quality scores by cycle. Images graph—Use the images graph to monitor intensities by cycle.
Chapter 5 Post-Run Procedures Introduction Unload and Weigh Reagents Perform a Maintenance Wash Perform a Water Wash Switch Sequencing Modes Idle the Instrument Shut Down the Instrument HiSeq 2500 System User Guide 98 99 100 104 106 108 109 97 Chapter 5 Post-Run Procedures
Post-Run Procedures Introduction Post-run procedures include the removal and weighing of reagents, and an instrument wash. A water wash is required after each run, with the option after a high output run to perform a maintenance wash instead. Illumina recommends a maintenance wash. Regular instrument washes ensure continued performance in the following ways: } Flush any remaining reagents and sample from the fluidics tubing and sippers.
1 Open the reagent compartment door. 2 Raise the sippers for the appropriate SBS rack and paired-end rack using the following motion: a Pull the sipper handle outward. b Raise the sipper handle while pulling it outward. c Release the sipper handle into the slot on the top end of the groove. Make sure that the sipper handle rests securely in the slot. 3 Slide the reagent rack out of the reagent compartment using the rack handle.
Post-Run Procedures Perform a Maintenance Wash A maintenance wash is required every 10 days or when switching between high output and rapid modes, and is an option after a high output run. Illumina recommends performing a maintenance wash after a high output run. The Load Gasket screen opens with a maintenance wash every 10 days and when switching from rapid mode to a high output mode.
4 Place the carboy onto a stir plate and stir until the solution is thoroughly mixed. 5 Add 4 liters laboratory-grade water to the solution. These volumes result in approximately 0.5% Tween 20 and 0.03% ProClin 300 wash solution. 6 Continue stirring until the solution is thoroughly mixed. 7 Set aside in a closed container at room temperature until you are ready to fill or replenish reagent bottles and tubes with wash solution.
Post-Run Procedures 9 Wearing a new pair of gloves, apply light pressure to 1 side of the front gasket until the other side lifts. Use tweezers to grasp and remove the gasket. Repeat to remove the rear gasket. Figure 49 Remove Used Manifold Gaskets 10 Position a new 10-port gasket in the front manifold and a new 8-port gasket in the rear manifold. 11 Reload the flow cell. 12 Make sure that the Vacuum Engaged checkbox is selected on the Load Wash Flow Cell screen. 13 Select Next.
18 Select Next to start the wash. 19 When the wash is complete, select Return to Start. 20 Measure the delivered volume. NOTE All bottles and tubes are filled to capacity to make sure that the sippers are rinsed. However, the delivered volume for each position varies so the bottles and tubes contain different volumes when the wash is complete. Positions 8 SBS positions 10 PE positions 1 library position All positions High Output Flow Cell Delivered Volume 82 ml 76 ml Empty 19.
Post-Run Procedures Perform a Water Wash A water wash is required after each sequencing run. After a high output run, you can perform a maintenance wash instead. If the instrument has been idle for 1 day or more, perform a water wash before beginning a new sequencing run.
Remove the waste tubing for the appropriate flow cell from the waste container. Do not include the waste tubing for the opposite flow cell, or the tubing from the condensation pump. 7 [For high output modes] Bundle the waste tubing with parafilm, making sure to keep all of the ends even. Place the bundled tubing ends into a 250 ml bottle. 8 [For Rapid Run mode] Place the ends of tubes 4 and 5 into an empty container.
Post-Run Procedures Switch Sequencing Modes Use the Mode Select command from the Welcome screen to switch between high output modes and Rapid Run mode. Only runs of the same mode can be performed simultaneously. Therefore, mode changes are applied to both flow cell A and flow cell B. If either flow cell is in progress, a mode change is not possible. A maintenance wash and gasket change are required when switching between run modes. For more information, see Perform a Maintenance Wash on page 100.
Rapid Maintenance Wash High Output Maintenance Wash Flow Cell Type Rapid flow cell (2 lanes) High output flow cell (8 lanes) Flow Cell Gasket 10-port gasket and 8port gasket 10-port gasket and 8port gasket Expected Volumes (ml) 60.
Post-Run Procedures Idle the Instrument Use the following instructions to prepare the instrument to sit idle for up to 10 days. For durations longer than 10 days, see Shut Down the Instrument on page 109. 108 1 Perform a complete maintenance wash to flush the system fully. For more information, see Perform a Maintenance Wash on page 100. 2 Leave the flow cell on the flow cell stage with the flow cell lever in position 2. The manifolds remain in the raised position.
Shut down the instrument only if you do not plan to use it within the next 10 days or more. If you plan to use the instrument within the next 10 days, see Idle the Instrument on page 108. Use the following procedure to prepare fluidics safely and shut down the system. 1 Perform a maintenance wash to flush the system. For more information, see Perform a Maintenance Wash on page 100. 2 Remove the flow cell from the flow cell stage.
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Chapter 6 Real-Time Analysis Introduction Real-Time Analysis Overview Monitor Run Metrics Real-Time Analysis Workflow Sequencing Output Files Output Folder Structure Tile Numbering Thumbnail Images HiSeq 2500 System User Guide 112 113 115 117 121 123 125 126 111 Chapter 6 Real-Time Analysis
Real-Time Analysis Introduction Real-Time Analysis (RTA) software performs on-instrument image analysis and base calling during a sequencing run on the HiSeq 2500, which saves valuable time during subsequent data analysis. 112 Part # 15035786 Rev.
Real-Time Analysis runs on the instrument computer, performing base calling and assigning a quality score to each base call. The software tracks the status of each tile and determines when to advance it to the next process step. When advancing a tile, Real-Time Analysis outputs a file for the completed step and then starts the next step. Thus, the software can determine the status of each tile based on which files exist. If Real-Time Analysis is terminated, it saves run data and can resume processing.
Real-Time Analysis } Statistics files—For each cycle, 1 statistics file (*.stats) is produced. The statistics file contains aggregate statistics for the cycle. Primary output files are used for subsequent data analysis. Use bcl2fastq for demultiplexing and conversion of .bcl files into FASTQ files, which can be used as input for alignment. To convert data from the HiSeq, use bcl2fastq 1.8.4, or later. Real-Time Analysis provides real-time metrics of run quality stored as InterOp files.
Real-Time Analysis automatically generates quality metrics when image analysis begins. However, not all metrics are available at the early cycles because some processes need multiple cycles to generate data. Data Cycle Image analysis After cycle 5. During the first 5 cycles of the run, the software generates a template of cluster locations. Base calls After cycle 12. Base calling begins after the color matrix is estimated at cycle 12. Phasing estimates After cycle 25.
Real-Time Analysis Sequencing Analysis Viewer The Sequencing Analysis Viewer software shows metrics generated during the sequencing run. Metrics appear in the form of plots, graphs, and tables. Sequencing Analysis Viewer opens automatically after run metrics are available. Select Refresh at any time during the run to view updated metrics. For more information, see the Sequencing Analysis Viewer User Guide (part # 15020619). 116 Part # 15035786 Rev.
Real-Time Analysis and the control software perform the Real-Time Analysis workflow. The workflow includes the following steps: } Template generation—Maps cluster locations. } Registration and intensity extraction—Records the location of each image on the flow cell and determines an intensity value for each cluster. } Color matrix correction—Corrects cross talk between channels. } Empirical phasing correction—Corrects the effects of phasing and prephasing.
Real-Time Analysis Color Matrix Correction After registration and intensity extraction, Real-Time Analysis corrects for cross talk between channels. Cross talk occurs when a cluster shows intensity in the C channel and some intensity also shows in the A channel, for example. Using a 4 x 4 color matrix, RealTime Analysis generates matrix-corrected intensities with reduced or no cross talk, and balances differences in overall intensity between color channels.
After raw intensities have been color corrected and phasing and prephasing corrected, the color channel with the brightest intensity is the base call for that cluster in that cycle. Base calling on the HiSeq 2500 using Real-Time Analysis begins after cycle 12. Base calling determines a base (A, C, G, or T) for every cluster of a given tile at a specific cycle. Base calls are saved to base call (*.bcl) files, which are binary files with 1 byte per call and quality score.
Real-Time Analysis NOTE Quality scoring is based on a modified version of the Phred algorithm. For more information, see en.wikipedia.org/wiki/Phred_quality_score. 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.
File Type File Description, Location, and Name Base call files Each tile analyzed is included in a base call file that contains the base call and encoded quality score. Data\Intensities\BaseCalls\L00[X]—Files are stored in per cycle folders for each lane. s_[Lane]_[Tile].bcl.gz, where lane is the single-digit lane number and tile is the 4-digit tile number. Base call files are compressed using gzip compression.
Real-Time Analysis File Type File Description, Location, and Name Phasing files Contains empirical phasing information by tile. Phasing files are created at the first cycle base called and updated after each cycle base called. Data\Intensities\BaseCalls\Phasing EmpiricalPhasing_[lane]_[read]_[tile].txt—Tile is represented with a 4digit number that indicates surface, swath, and tile.
Config—Configuration settings for the run. Data Intensities BaseCalls L00[X]—Base call files for each lane, aggregated in 1 file per cycle. Phasing—Empirical phasing files, 1 file per tile at every cycle. L00[X]—Aggregated cluster location files for each lane. Offsets—Two offsets file for the run. RTAConfiguration.xml Images Focus L00[X]—Focus images for each lane. InterOp—Binary files used by Sequencing Analysis Viewer. Logs—Log files describing operational events.
Real-Time Analysis The run folder is written to the output path specified on the Scan screen during run setup. The temporary run folder for flow cell A is written to the D: drive and the temporary run folder for flow cell B is written to the E: drive. 124 Part # 15035786 Rev.
The HiSeq high output flow cell is imaged in 96 tiles on each lane, top and bottom, for each cycle. Each lane has 3 swaths with 16 tiles per swath. The rapid flow cell is imaged in 64 tiles. Each lane has 2 swaths with 16 tiles per swath. The tile name is a 4-digit number that represents the position on the flow cell.
Real-Time Analysis Thumbnail Images You can configure the control software to generate thumbnail images in *.jpg file format. Thumbnail images are generated for each cycle and base. The control software collects images from 9 sections of a tile. The 9 images are combined into 1 thumbnail image and can be used to troubleshoot a run. Thumbnail images are not suitable for image analysis, but can be used for troubleshooting. Figure 52 Thumbnail Image 126 Part # 15035786 Rev.
Chapter 7 Troubleshooting Introduction Possible Run Setup Problems Stagger Runs on Flow Cell A and Flow Cell B Perform a Fluidics Check BaseSpace is Unavailable Stop and Resume a Run Pause a Run Primer Rehybridization HiSeq 2500 System User Guide 128 129 130 131 132 133 136 137 127 Chapter 7 Troubleshooting
Troubleshooting Introduction This section describes what to do if a problem occurs during a sequencing run and how to perform a fluidics check from the Welcome screen. 128 Part # 15035786 Rev.
Problem The software did not initialize. Flow cell lever is orange. Flow cell lever is blinking orange. Flow cell lever is blinking green. Poor fluid delivery. HiSeq 2500 System User Guide Possible Cause The software was unable to initialize internal hardware devices. Action Close the error message and then relaunch the instrument software. If the problem persists, restart the instrument computer.
Troubleshooting Stagger Runs on Flow Cell A and Flow Cell B 1 Wait for the run on the adjacent flow cell to begin a chemistry step, and then select Pause. The Pause menu opens. NOTE Always pause the current run during a chemistry step opposed to an imaging step. 130 2 Select Normal Pause. 3 Wait for the software to complete the current chemistry step. The system is placed in a safe state automatically. 4 After the adjacent run is paused, set up the new run.
The Check button on the Welcome screen performs a fluidics check. Use this option during instrument installation and when troubleshooting fluidics issues. 1 Load a used flow cell onto the instrument. 2 Load 8 SBS bottles with PW1 or laboratory-grade water, and load the bottles onto the corresponding reagent rack. Load the rack onto the instrument. 3 Select Check on the Welcome screen. 4 Select solution 5 (SB2) from the drop-down list.
Troubleshooting BaseSpace is Unavailable If BaseSpace is not available, open Windows Services to make sure that the BaseSpace Broker has started. If it has not started, restart it. If services are running and BaseSpace is still unavailable, contact Illumina Technical Support. 132 Part # 15035786 Rev.
Stopping a run might be necessary if the run was set up incorrectly, if the data quality is bad, or if there is a hardware error. To resume a stopped run, make sure that you select the appropriate normal stop options that allow the run to resume. Stop Option Normal Stop (End of Lane\Chemistry) Real-Time Analysis Option Keep As Is Complete For Run Complete For Read Normal Stop (End of Cycle) Keep As Is Complete For Run Complete For Read Immediate Stop No option 1 Able to Resume? Yes.
Troubleshooting • Keep As Is—The run is stopped without any modifications to Real-Time Analysis. The run can resume where it was stopped. • Complete For Run—Real-Time Analysis is stopped. The run info, run parameters, and recipe files are updated to reflect total cycles as the last cycle completed. Then Real-Time Analysis restarts to complete base calling for the run up to the point the run was stopped. The run cannot resume. • Complete For Read—Real-Time Analysis is stopped.
Confirm the settings on the Resume screen or select the imaging and chemistry commands to resume. For more information, see Example Settings for Resuming a Run on page 135. 5 Select Next to proceed. The software guides you through the remaining run setup steps. Example Settings for Resuming a Run If the run was stopped after imaging lane 1 at cycle 23, the software automatically sets up the resume run settings for Read 1 at cycle 23.
Troubleshooting Pause a Run CAUTION Do not pause a run during imaging. Use the Normal stop, end of cycle or end of lane feature, to stop and resume a run. Pause a run from the Run Overview screen. Pausing a run might be necessary to check run components, such as reagent volumes, before proceeding with the run. Under normal operation, pausing a run is not necessary. 1 From the Run Overview screen, select Pause. The pause menu opens. Figure 55 Pause Options 2 Select Normal Pause.
A rehybridization run repeats the sequencing primer hybridization step. If run metrics indicate low cluster numbers, low cluster intensities, or other concerns, perform primer rehybridization to rescue the flow cell. Primer rehybridization does not damage clusters on the flow cell. HiSeq v4 Flow Cell All rehyb steps are performed on the HiSeq 2500. The kit includes primers for Read 1, Index 1 Read, Index 2 Read for single-read flow cells, and Read 2.
Troubleshooting 138 Rehyb Kit Name Workflow Instructions HiSeq Rapid Rehyb Kit Catalog # GD-404-1001 HiSeq Rapid Run Primer Rehybridization (part # 15059379) TruSeq Rapid Rehyb Kit Catalog # GD-402-2001 TruSeq Rapid Run Primer Rehybridization (part # 15039627) Part # 15035786 Rev.
% Index Index cycles, monitoring 40, 64, 96 %PF 119 D A data compression 120 disk space, checking 12 documentation 16, 143 DoNotEject drive 6 dual indexing on paired-end flow cell 23, 48, 78 settings on recipe screen 23, 48, 78 activity indicators 9 B base call files 113, 121 base calling cycle 12 119 quality score 119 BaseSpace sample sheet 24, 49 sample sheets 13 bcl2fastq software 113 C chastity filter 119 chemistry steps, monitoring 40, 64, 96 cluster intensities 40, 64, 96, 118 cluster locati
Index vacuum hole locations 30, 54, 85 flow cell lever position 0 30, 54, 85 position 1 31, 37, 55, 61 position 2 31, 37, 55, 61 flow cell loading bubbles, leaks 33, 39, 57, 63 priming flow cell 31, 55 sequencing flow cell 37, 61 Flow Cell Set-up Screen 47, 77 Flow Cell Setup Screen 22 fluidics compartment 3 fluidics, troubleshooting 129, 131 folder locations, default settings 11 locs files 121 log files, Real-Time Analysis 121 H no-calls 119 normal stop 133 help documentation 16 help, technical 143 Hi
S sample sheet BaseSpace 24, 49 overview 13 SBS reagent positions 26 scan screen 75 sensor indicators 9 Sequencing Analysis Viewer 7 InterOp files 113 overview 116 sequencing consumables 18, 42, 70 HiSeq 2500 System User Guide sequencing modes switching 106 show log file 10 software about, version 10 Experiment Manager 13 HiSeq Control 12 HiSeq Control Software 7 initialization 6 Real-Time Analysis 7 Sequencing Analysis Viewer 7 show log file 10 troubleshooting 129 view full screen 10 Welcome screen 7 sta
Index duration 104 expected volumes 104 weighing reagents after run 99 before run 26 Welcome screen 7 commands 8 menu 10 workflow staggered runs 130 142 Part # 15035786 Rev.
For technical assistance, contact Illumina Technical Support. Table 16 Illumina General Contact Information Address 5200 Illumina Way San Diego, CA 92122 USA Website www.illumina.com Email techsupport@illumina.com Table 17 Illumina Customer Support Telephone Numbers Region North America Austria Belgium Denmark Finland France Germany Ireland Contact Number 1.800.809.4566 0800.296575 0800.81102 80882346 0800.918363 0800.911850 0800.180.8994 1.800.
Illumina San Diego, California 92122 U.S.A. +1.800.809.ILMN (4566) +1.858.202.4566 (outside North America) techsupport@illumina.com www.illumina.
