HiSeq 2500 System Guide (15035786 v01)

HiSeq 2500 System Guide (15035786 v01)
HiSeq® 2500 System Guide
For Research Use Only. Not for use in diagnostic procedures.
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Material # 20000451
Document # 15035786 v01
October 2015
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Revision History
Document
Date
Material # 20000451
Document # 15035786
v01
October
2015
Part # 15035786 Rev. D
November
2014
Part # 15035786 Rev. C
April 2014
Part # 15035786 Rev. B
November
2013
Part # 15035786 Rev. A
October
2012
HiSeq 2500 System Guide
Description of Change
Added instructions for preparing SBS, indexing, paired-end, and
clustering reagents.
Added information about BaseSpace Onsite.
Added recommendation for annual preventive maintenance
service.
Updated software descriptions to HiSeq Control Software
v2.2.58.
Updated Rapid Run mode workflow for compatibility with HiSeq
Rapid v2 chemistry.
Replaced NaOH maintenance wash with Tween 20 and ProClin
300 maintenance wash including information about preparing,
storing, and disposing of maintenance wash solution.
Updated descriptions of maintenance wash and water wash to
specify that a water wash is required after a run.
Added workflow, input and output files, error handling, and
quality scoring descriptions to the Real-Time Analysis chapter.
Updated VWR catalog # for alcohol wipes to 95041-714.
Updated URL for Safety Data Sheets (SDS) to
support.illumina.com/sds.html.
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.
Removed reagent preparation instructions. For reagent prep
instructions including information about various sequencing
primers, see the documentation for the associated kit.
Replaced the following reagents:
• RMR for RMX
Initial release.
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Table of Contents
Revision History
Table of Contents
Chapter 1 Overview
Introduction
Additional Resources
Instrument Components
Sequencing Consumables Overview
Chapter 2 Getting Started
Start the HiSeq 2500
Customize System Settings
View and Send Instrument Data
User-Supplied Consumables
Chapter 3 Sequencing in HiSeq v4 Mode
Introduction
HiSeq v4 Sequencing Workflow
Prepare Reagents
Enter Run Parameters
Load and Prime Reagents
Load the Sequencing Flow Cell
Monitor the Run
Unload Reagents
Perform a Water Wash
Chapter 4 Sequencing in TruSeq v3 Mode
Introduction
TruSeq v3 Sequencing Workflow
Prepare Reagents for Read 1
Enter Run Parameters
Load and Prime Reagents
Load the Sequencing Flow Cell
Monitor the Run
Prepare Reagents for Read 2
Unload Reagents
Perform a Water Wash
Chapter 5 Sequencing in Rapid Run Mode
Introduction
Rapid Run Sequencing Workflow
Prepare Reagents
Perform a Volume Check
Enter Run Parameters
Load and Prime Reagents
Load the Sequencing Flow Cell
Monitor the Run
Unload Reagents
Perform a Water Wash
HiSeq 2500 System Guide
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Chapter 6 Maintenance
Introduction
Perform a Maintenance Wash
Switch Sequencing Modes
Idle the Instrument
Shut Down the Instrument
Appendix A Troubleshooting
Possible Run Setup Problems
Perform a Fluidics Check
BaseSpace is Unavailable
Stop and Resume a Run
Pause a Run
Stagger Runs on Flow Cell A and Flow Cell B
Split SBS Kits
Primer Rehybridization
Appendix B Real-Time Analysis
Real-Time Analysis Overview
Real-Time Analysis Workflow
Monitor Run Metrics
Appendix C Output Files and Folders
Sequencing Output Files
Output Folder Structure
Tile Numbering
Thumbnail Images
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Index
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Technical Assistance
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Chapter 1 Overview
Introduction
Additional Resources
Instrument Components
Sequencing Consumables Overview
HiSeq 2500 System Guide
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Chapter 1
Overview
Overview
Introduction
The HiSeq® 2500 system provides power and efficiency for large-scale genomics.
Innovative engineering combines with proven SBS technology to set new standards in
output, simplicity, and cost-effectiveness. Two high output options offer greatest depth of
coverage, and a rapid run provides quick results.
Features
}
}
}
}
}
}
}
}
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Dual-surface imaging—The HiSeq 2500 uses a 4-camera epifluorescence system with
cutting-edge scanning technology to enable dual surface imaging.
Dual flow cells—The HiSeq 2500 is a dual flow cell system, which allows sequencing
of a single flow cell or 2 flow cells with different read lengths simultaneously.
On-instrument cluster generation—The HiSeq 2500 provides the option of Rapid Run
mode, which includes on-instrument cluster generation.
High-capacity reagent chiller—The reagent compartment is a high-capacity chiller that
holds enough reagents for the entire sequencing run.
Integrated fluidics for paired-end runs—Integrated paired-end fluidics provide
reagents from the reagent compartment to the flow cell for Read 2 resynthesis and for
indexed sequencing.
Interface control options—The instrument software interface provides options for
setting up a run and operating the instrument using the touch screen monitor or the
integrated keyboard.
Real-time base calling—The instrument software extracts intensities from images and
performs quality-scored base calling on the instrument computer, which allows
monitoring of quality metrics during the run and saves time during subsequent data
analysis.
Downstream analysis of sequencing data can be performed with Illumina analysis
software or third-party software on IlluminaCompute, Illumina BaseSpace, or a custom
infrastructure.
BaseSpace® integration—The sequencing workflow is integrated with BaseSpace, the
Illumina genomics computing environment for data analysis, storage, and
collaboration. As the run progresses, output files are streamed in real time to BaseSpace
or BaseSpace Onsite.
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The following documentation is available for download from the Illumina website.
Resource
Description
HiSeq 2500, 1500, and 2000 Site Prep
Guide (document # 15006407)
Provides specifications for laboratory space, electrical
requirements, and environmental considerations.
HiSeq 2500 System Safety and
Compliance Guide (document #
1000000000651)
Provides information about instrument labeling,
compliance certifications, and safety considerations.
HiSeq and GAIIx Systems Denature
and Dilute Libraries Guide
(document # 15050107)
Provides instructions for denaturing and diluting prepared
libraries for a sequencing run, and preparing a PhiX
control. This step applies to most library types.
Visit the HiSeq 2500 support page on the Illumina website for access to documentation,
software downloads, online training, and frequently asked questions.
HiSeq 2500 System Guide
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Additional Resources
Additional Resources
Overview
Instrument Components
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. An illuminated status bar indicates operating status.
Figure 1 External Components
A
B
C
D
E
Optics module—Contains optical components that enable dual surface imaging of the flow
cell, imaging A, C, G, and T at the same time using epifluorescence. The excitation laser beam
passes through the objective and the fluorescence is simultaneously collected through the
same objective.
Flow cell compartment and template loading station—Contains the vacuum-controlled
flow cell stage, which holds the flow cell in place during sequencing runs. Using Rapid Run
mode, the template loading station transfers libraries to the flow cell for on-instrument
cluster generation.
Fluidics compartment—Contains fluidics pumps that deliver reagents to the flow cell, and
then to the waste container.
Status bar—Uses 3 colors to indicate instrument status. Blue indicates that the instrument is
running, orange indicates that the instrument needs attention, and green indicates that the
instrument is ready to begin the next run.
Reagent compartment—Contains reagent racks that hold reagents for sequencing runs and
wash solution for instrument washes.
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.
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Instrument Components
Figure 2 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
Flow cell A is on the left, and flow cell B is on the right. Each flow cell is seated on the flow
cell stage, which moves in and out of the optics module as directed by the control software.
The flow cell stage must be in the forward-most position to open the flow cell compartment
door and load or remove a flow cell.
The flow cell is positioned on the flow cell holder with the inlet and outlet ports facing
down, and is held in place by a vacuum beneath each flow cell holder. The illuminated
flow cell lever in front of each flow cell holder controls the vacuum. The flow cell lever
turns green when the vacuum seal is secure.
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. The reagent rack for flow cell A is
located in the center position, and the rack for flow cell B is located in the far right
position. Each reagent rack has numbered positions that correspond to connections on
an internal reagent selector valve.
} Clustering, indexing, and paired-end reagent rack—Located in the left position. It has
2 rows of numbered positions that hold 15 ml conical tubes containing paired-end
reagents and indexing reagents. The left row is for flow cell A, and the right row is for
flow cell B.
} Reagent chiller—The reagent chiller houses the reagent racks and maintains an
internal temperature of 2°C to 8°C.
HiSeq 2500 System Guide
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Overview
Figure 3 Reagent Compartment
A
B
C
D
Sipper handles
Reagent rack for clustering, indexing, and paired-end reagents
Reagent rack for SBS reagents for flow cell A
Reagent rack for SBS reagents for flow cell B
HiSeq 2500 Software
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.
} Real-Time Analysis software—Integrated with the control software, Real-Time
Analysis (RTA) performs base calling and assigns a quality score to each base for each
cycle. For more information, see Real-Time Analysis on page 105.
} Sequencing Analysis Viewer software—Sequencing Analysis Viewer (SAV) provides
detailed quality statistics.
Status Icons
A status icon located in the upper-right corner of each screen shows changes in conditions,
errors, or warnings during run setup and during the run.
Status Icon
6
Status Name
Status okay
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.
Error
Errors usually stop a run and generally require action before
proceeding with the run.
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Activity and Sensor Indicators
The Welcome screen contains a series of icons in the lower-right corner of the screen that
indicate instrument activity and status of specific components based on instrument
sensors.
Figure 4 Activity Indicators
From left to right, activity indicators represent the X, Y, and Z motors, electronics
functionality, the camera, the fluidics system, and processing functions.
Figure 5 Sensor Indicators
From left to right, sensor indicators represent flow cell A temperature, reagent chiller
temperature, data transfer status, BaseSpace cloud status, and flow cell B temperature.
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. If disk space becomes low, the software pauses the run and
places the flow cell in a safe state. Create more space to continue the run, which resumes
automatically when sufficient space is available.
HiSeq 2500 System Guide
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Instrument Components
When a change in condition occurs, the associated icon blinks to alert you.
} Select the icon to open the status window and view a description of the condition.
} Select Acknowledge to accept the message and Close to close the dialog box.
Overview
Sequencing Consumables Overview
Performing a run on the HiSeq 2500 requires 1 SBS kit and 1 cluster kit.
SBS kits include sequencing reagents used on the HiSeq, with sufficient reagents for
sequencing 1 flow cell. Rapid SBS kits include a set of funnel caps. Sequencing reagents are
provided in 250 ml bottles that load directly onto reagent racks. The reagent labels are
color-coded to reduce loading errors.
Cluster kits for high output modes contain clustering reagents used on the cBot, indexing
and paired-end reagents used on the HiSeq 2500, and funnel caps for SBS reagent bottles.
Rapid cluster kits contain clustering, indexing, and paired-end reagents. All reagents are
used on the HiSeq, and are sufficient for clustering 1 flow cell. All cluster kits contain flow
cell gaskets and are available in paired-end (PE) and single-read (SR) versions.
Reagent Kits for HiSeq v4 Mode
Kit Name
Catalog #
HiSeq SBS Kit v4 (250 cycles)
FC-401-4003
HiSeq SBS Kit v4 (50 cycles)
FC-410-4002
HiSeq SR Cluster Kit v4
GD-401-4001
HiSeq PE Cluster Kit v4
PE-401-4001
Reagent Kits for TruSeq v3 Mode
Kit Name
Catalog #
TruSeq SBS Kit v3 (200 cycles)
FC-401-3001
TruSeq SBS Kit v3 (50 cycles)
FC-401-3002
TruSeq SR Cluster Kit v3
GD-401-3001
TruSeq PE Cluster Kit v4
PE-401-3001
Sequencing Primers for Nextera Libraries
The Index 1 sequencing primer (HP8) and Read 2 sequencing primer (HP7) provided in the
TruSeq v3 cluster kits are not compatible with Nextera libraries. When sequencing Nextera
libraries, use the Index 1 sequencing primer (HP12) and Read 2 sequencing primer (HP11)
provided in the TruSeq Dual Index Sequencing Primer Box.
Performing a dual-index run on a single-read flow cell requires HP9, which is provided in
the SR version of the kit.
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Kit Name
Catalog #
TruSeq Dual Index Sequencing Primer Box, SR
FC-121-1003
TruSeq Dual Index Sequencing Primer Box, PE
PE-121-1003
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Sequencing Consumables Overview
Reagent Kits for Rapid Run
Kit Name
Catalog #
HiSeq Rapid SBS Kit v2 (500 cycles)
FC-402-4023
HiSeq Rapid SBS Kit v2 (200 cycles)
FC-402-4021
HiSeq Rapid SBS Kit v2 (50 cycles)
FC-402-4022
HiSeq Rapid SR Cluster Kit v2
GD-402-4002
HiSeq Rapid PE Cluster Kit v2
PE-402-4002
HiSeq 2500 System Guide
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Chapter 2 Getting Started
Start the HiSeq 2500
Customize System Settings
View and Send Instrument Data
User-Supplied Consumables
HiSeq 2500 System Guide
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Chapter 2
Getting Started
Getting Started
Start the HiSeq 2500
1
Start the instrument control computer.
2
Log on to the operating system using the default user name and password and wait for
it to load.
} User name: sbsuser
} Password: sbs123
If the default values do not work, consult your facility administrator for the site-specific
user name and password.
3
Locate the power switch on the left side of the instrument and switch it to the
ON position.
Wait at least 1 minute for the instrument devices to be properly configured and for the
instrument drive called DoNotEject to initialize.
4
Close the window that opens when DoNotEject is initialized. If the window does not
open, use MyComputer to check for the DoNotEject drive.
NOTE
Never eject the DoNotEject flash drive located inside the instrument chassis, or modify
the files on it. This drive contains hardware configuration files and initializes whenever
the instrument is turned on.
5
To ensure adequate disk space, archive data on the instrument computer from previous
runs to a network location.
6
Open HCS using the shortcut icon on the computer desktop.
When the software has initialized, the Mode Select screen opens and the Initialized
icon appears on the bottom-right corner of the screen.
Instrument and Control Computer Best Practices
}
}
}
}
}
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Do not turn on the computer while the instrument is running. Always turn on the
computer before turning on the instrument.
Do not turn off the instrument while the instrument control software is running.
Wait 1 minute after turning off the instrument before turning it on again.
Connect the USB cables for the instrument, the monitor, and the keyboard to the back of
the computer before turning on the computer.
Connect the barcode scanner and mouse to the USB ports on the front of the computer.
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The control software includes customizable system settings for run folders and LIMS
preferences. The Menu Options window provides settings to define the run ID template,
default folder locations, whether to send instrument health information to Illumina, and
LIMS authentication.
To customize your view of the interface, select Menu | View. You can choose to view the
interface in full screen or in a window, or to minimize.
To activate the command to initialize the software manually, select Menu | Scanner.
Define Run Folder Settings
1
From the Welcome screen, select Menu | Tools | Options to open the Menu Options
window.
2
To customize the naming convention for run folder names, modify the settings in the
Run ID Template field. Select Reset to clear the field.
3
To set default output locations, enter a location for each of the following folders:
} Default Output Folder—The default output folder for runs on flow cell A.
} Default Output Folder2—The default output folder for runs on flow cell B.
NOTE
These locations can be changed on a per run basis.
4
To set the location where temporary files are written during a run, enter a location in
the Default Temp Folder 1 field.
5
To set a location for LIMS sample forms, enter the location in the Run Setup Folder
field.
6
Select OK to save your work and close the Menu Options window. Select Cancel to
close without saving.
Set LIMS Preferences
1
From the Welcome screen, select Menu | Tools | Options to open the Menu Options
window.
2
Enter the following LIMS settings:
} LIMS Server—The server name for interactions with supported Illumina LIMS.
} LIMS User Name—The user name used when authenticating to Illumina LIMS.
} LIMS Password—The password used when authenticating to Illumina LIMS.
3
Select OK to save your work and close the Menu Options window. Select Cancel to
close without saving.
HiSeq 2500 System Guide
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Customize System Settings
Customize System Settings
Getting Started
View and Send Instrument Data
The Menu button on the Welcome screen and the Menu Options window provide options
for viewing and sending instrument data.
} To view information about instrument hardware, software versions, and technical
support contact information, select Menu | About.
} To permit the instrument to send information to BaseSpace for each run
(recommended), select Menu | Tools | Options, and then select the Send instrument
health information to Illumina to aid technical support checkbox.
All information remains confidential.
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Consumable
Alcohol wipes,
70% Isopropyl
or
Ethanol, 70%
Carboy, at least 6 liters
Supplier
VWR, catalog # 95041-714
General lab supplier
Purpose
Cleaning the flow cell and
flow cell stage.
General lab supplier
Centrifuge tubes, 250 ml
Corning, catalog # 430776
Conical tubes, 15 ml
Corning, catalog # 430052
Conical tubes, 50 ml,
self-standing (optional)
Disposable gloves, powder-free
Lab tissue, low-lint
Corning, catalog # 430921
Preparing maintenance
wash solution.
SBS reagent racks, positions
containing PW1.
Instrument wash.
PE reagent racks, positions
containing PW1.
Instrument wash.
Collecting and measuring
waste volumes.
Storing flow cells.
Lens paper, 4 x 6 in
Pipette tips, 200 µl
Pipette tips, 1000 µl
ProClin 300, 50 ml
Tween 20, viscous liquid, 100 ml
Tweezers, square plastic tip
Water, laboratory-grade,
18 M Ohm
General lab supplier
VWR, catalog # 21905-026
VWR, catalog # 52846-001
General lab supplier
General lab supplier
Sigma-Aldrich, catalog # 48912-U
Sigma-Aldrich, catalog # P7949
McMaster-Carr, catalog #
7003A22
Millipore
General use.
Cleaning the flow cell
holder.
Cleaning the flow cell.
Splitting reagent volumes.
Splitting reagent volumes.
Maintenance wash.
Maintenance wash.
Removing the flow cell
gaskets.
SBS and PE reagent racks,
positions containing PW1.
Instrument wash.
Microcentrifuge Tubes for Rapid Run Mode
Consumable
Microcentrifuge tube, 1.5 ml
Microcentrifuge tube, 1.7 ml
HiSeq 2500 System Guide
Supplier
VWR, catalog # 20170-038, catalog # 20170-650, or catalog #
89000-028
Axygen, catalog # MCT-150-C
VWR, catalog # 20170-575
Axygen, catalog # MCT-175-C
Sorenson BioScience, catalog # 16070
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User-Supplied Consumables
User-Supplied Consumables
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Chapter 3 Sequencing in HiSeq v4 Mode
Introduction
HiSeq v4 Sequencing Workflow
Prepare Reagents
Enter Run Parameters
Load and Prime Reagents
Load the Sequencing Flow Cell
Monitor the Run
Unload Reagents
Perform a Water Wash
HiSeq 2500 System Guide
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Chapter 3
Sequencing in HiSeq v4 Mode
Sequencing in HiSeq v4 Mode
Introduction
To perform a sequencing run in HiSeq v4 mode on the HiSeq 2500, prepare all reagents
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.
Stagger Runs
It is possible to start a new run on either flow cell A or flow cell B when a run on the
adjacent flow cell is in progress. For more information, see Stagger Runs on Flow Cell A and
Flow Cell B on page 100.
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. Reference this information when setting up the
run.
Run Type
Read 1
Cycles
Index 1
(i7) Read
Cycles
--
Index 2
(i5) Read
Cycles
--
Read 2
Cycles
Total
Cycles
Single-Read,
Non-Indexed
Single-Read,
Single-Indexed
≤ 126
--
≤ 126
--
--
≤ 126
6 or 7 ¹
8²
8
8
--
≤ 133 ¹
≤ 134 ²
≤ 142
Single-Read,
Dual-Indexed
Paired-End,
Non-Indexed
Paired-End,
Single-Indexed
≤ 126
--
--
≤ 126
≤ 252
≤ 126
7¹
8²
8
--
≤ 126
7+8³
≤ 126
≤ 259 ¹
≤ 260 ²
≤ 275
Paired-End,
Dual-Indexed
≤ 126
≤ 126
¹ Number of cycles for single-indexed libraries
² Number of cycles for dual-indexed libraries
³ The Index 2 Read of a paired-end dual-indexed run includes 7 additional chemistry-only cycles
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HiSeq v4 Sequencing Workflow
HiSeq v4 Sequencing Workflow
Prepare reagents for the run.
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.
Load the clustered flow cell for sequencing. Confirm proper flow.
Start the sequencing run.
[Optional] After cycle 1, inspect the first base report, and then continue
Read 1.
The run continues as specified in run parameters.
When the run is complete, unload reagents.
Perform an instrument wash.
HiSeq 2500 System Guide
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Sequencing in HiSeq v4 Mode
Prepare Reagents
Before setting up the run, prepare all reagents for sequencing: SBS reagents, indexing
reagents, and paired-end reagents, if applicable. Load all reagents when prompted by the
control software. When using HiSeq v4 chemistry, returning to the instrument during the
run to load reagents is not necessary.
Reagents can be prepared during cluster generation. For instructions on preparing
clustering reagents and performing cluster generation, see the cBot System Guide (document #
15006165).
Split Reagents
It is possible to split a 250-cycle kit into 2 sets of reagents to perform shorter runs. For more
information, see Split SBS Kits on page 101.
Prepare SBS Reagents
SBS reagents are loaded onto the instrument at the beginning of the run. To prepare
reagents, use the following instructions to thaw SBS reagents.
Thaw SBS Reagents
1
Remove CRM, IRM, and USM from -25°C to -15°C storage and thaw at 2°C to 8°C for
about 16 hours.
Alternatively, thaw IRM and USM in a room temperature deionized water bath for
about 90 minutes, protecting IRM from light. Thaw CRM in a separate water bath.
NOTE
Always replace your gloves after handling CRM.
2
Invert each bottle to mix.
3
Set IRM and USM aside on ice. Set CRM aside on ice separately to prevent crosscontamination.
4
Use PW1, SB1, SB2, and SB3 directly from storage.
Prepare Indexing Reagents
Indexing reagents are used during the indexing reads of an indexed sequencing run.
The PE and SR cluster kits include HP12, which is used for Index 1 (i7) Read 1, regardless
of flow cell type. Only the SR cluster kit includes HP9, which is required for a dual-indexed
run on a single-read flow cell.
Run Type
Single-indexed paired-end
Single-indexed single-read
Dual-indexed paired-end*
Dual-indexed single-read
Flow Cell Type
PE
SR
PE
SR
Index 1 (i7)
HP12
HP12
HP12
HP12
Index 2 (i5)
---HP9
* Dual-indexed paired-end runs use FRM, a paired-end reagent, for Index 2.
20
Material # 20000451
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1
Remove the following reagents from -25°C to -15°C storage:
} For all indexed runs on a paired-end flow cell—FDR and HP12
} For single-indexed runs on a single-read flow cell—FDR and HP12
} For dual-indexed runs on a single-read flow cell—FDR, HP9, and HP12
2
Thaw reagents in a room temperature deionized water bath for about 20 minutes.
Prepare FDR and HP12
1
Invert tubes to mix.
2
Centrifuge at 1000 rpm for 1 minute.
3
Set aside at room temperature.
Prepare HP9
1
Invert to mix.
2
Briefly pulse centrifuge to collect droplets.
3
Set aside at room temperature.
Prepare Paired-End Reagents
Paired-end reagents are used during the Read 2 resynthesis step of a paired-end sequencing
run.
WARNING
This set of reagents contains formamide, an aliphatic amide that is a probable
reproductive toxin. Personal injury can occur through inhalation, ingestion, skin contact,
and eye contact. Wear protective equipment, including eye protection, gloves, and
laboratory coat. Handle used reagents as chemical waste and discard in accordance with
the governmental safety standards for your region. For environmental, health, and safety
information, see the SDS for this kit at support.illumina.com/sds.html.
Thaw Paired-End Reagents
1
Remove the following reagents from -25°C to -15°C storage:
} For non-indexed runs—AMS, FDR, FLM2, FPM, FRM, and HP11
} For indexed runs—AMS, FLM2, FPM, FRM, and HP11
2
Thaw reagents in a beaker filled with room temperature water for about 20 minutes.
3
Place AMS, FLM2, and FRM on ice.
Prepare AMS, FDR, FLM2, FPM, FRM, and HP11
1
Invert to mix.
2
Centrifuge at 1000 rpm for 1 minute.
3
Set aside AMS, FLM2, and FRM on ice.
4
Set aside FDR, FPM, and HP11 at room temperature.
HiSeq 2500 System Guide
21
Prepare Reagents
Thaw Indexing Reagents
Sequencing in HiSeq v4 Mode
Enter Run Parameters
Begin run setup by entering run parameters from a series of screens on the Run
Configuration tab. The software guides you through each screen as you specify BaseSpace
connectivity, enter consumable IDs, select indexing options, and record other parameters
for the run.
Integration Screen
The Integration screen provides the option to connect the run to BaseSpace.
1
From the Welcome screen, select Sequence | New Run to open the Integration screen.
2
[Optional] Connect to BaseSpace or BaseSpace Onsite as follows.
a
b
c
Select BaseSpace or BaseSpace Onsite.
If you selected BaseSpace, select from the following options:
} Storage and Analysis—Sends run data to BaseSpace for remote monitoring and
data analysis. A sample sheet is required with this option.
} Run Monitoring Only—Sends only InterOp files to BaseSpace, which allows
remote monitoring of the run.
Log on to BaseSpace or BaseSpace Onsite using your MyIllumina account email
and password.
3
[Optional] To proceed without connecting to BaseSpace, select None.
4
Select Next.
Storage Screen
1
Select the Save to an output folder checkbox, and then select Browse to navigate to a
preferred network location.
If the run is connected to BaseSpace for storage and analysis, this step 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. A thumbnail is a sampling of
images from many tiles in each column of tiles, or swath, combined in 1 thumbnail
image.
} Save Tile Thumbnails—Saves tile thumbnails. Tile thumbnails represent a single
tile rather than a sampling of tiles in a swath.
4
Select Next.
Flow Cell Setup Screen
The Flow Cell Setup screen records information about the flow cell used for the run. All
fields are required.
22
Material # 20000451
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Scan or enter the flow cell ID (barcode number) of the flow cell to be sequenced.
The flow cell ID is used to determine flow cell type and reagent compatibility.
2
Confirm that the flow cell type is HiSeq Flow Cell v4.
The flow cell type is selected automatically based on the flow cell ID.
3
Enter an experiment name to appear on each screen and help identify the run in
progress.
4
Enter a user name and then select Next.
Advanced Screen
1
[Optional] Select the Confirm First Base checkbox.
A first base report is generated automatically for each run. Selecting this option opens
the first base report before the run proceeds.
2
[Optional] From the Align to PhiX checkboxes, clear the checkbox for lanes that do not
contain PhiX.
Alternatively, select lanes on the flow cell image to add or remove lanes for PhiX
alignment.
By default, all lanes are selected for 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.
Recipe Screen
1
Select from the following Index Type options:
} No Index—Performs a non-indexed single-read or paired-end run.
} Single Index—Performs a single-read or paired-end run with 1 indexing read.
} Dual Index—Performs a single-read or paired-end run with 2 indexing reads.
} Custom—Performs a single-read or paired-end run with a custom number of cycles
for index reads.
2
If the Dual Index or Custom option is specified, select a Flow Cell Format, either Single
Read or Paired End.
3
Enter the number of cycles for Read 1 and Read 2, if applicable.
The number of cycles performed in a read is 1 more cycle than the number of cycles
analyzed. For example, to perform 125 cycles for Read 1, enter 126.
4
For the Custom indexing option, enter the number of cycles for index reads.
NOTE
Read lengths do not need to be identical.
5
Confirm the following autopopulated chemistry settings:
} SBS: HiSeq SBS Kit v4
} Index: HiSeq v4 Single Index or HiSeq v4 Dual Index
} PE turnaround: HiSeq PE Cluster Kit v4
6
[Optional] Select the Use Existing Recipe checkbox to use a custom recipe.
HiSeq 2500 System Guide
23
Enter Run Parameters
1
Sequencing in HiSeq v4 Mode
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
HCS v2.2 allows a different indexing scheme in each lane.
Reagents Screen
The Reagents screen records information about reagent kits used for the run. The reagent kit
ID (barcode number beginning with RGT) determines reagent kit type and run mode
compatibility.
1
Scan or enter the SBS reagent kit ID.
2
For paired-end runs, scan or enter the reagent kit ID for the paired-end cluster kit.
3
Select the SBS reagent kit for the run:
} Select 250 Cycles for a 250 cycle kit. Cycles remaining defaults to 275.
} Select 50 Cycles for a 50 cycle kit. Cycles remaining defaults to 74.
} 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 for fresh reagents.
4
Select Prime SBS Reagents to prime reagents before starting the run.
5
Select Next.
Review Screen
24
1
Review the run parameters on the Review screen.
2
Select Next to proceed or select Back to change parameters.
Material # 20000451
Document # 15035786 v01
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.
Load SBS Reagents
1
Invert each reagent bottle several times to mix.
CAUTION
Handle the bottle of CRM last, after you have loaded all other reagents, to prevent
cross-contamination. Always replace your gloves after handling CRM.
2
Replace the cap on each bottle with a funnel cap.
3
Open the reagent compartment door.
4
Raise the sippers for the sequencing reagent rack as follows.
a
b
Pull the sipper handle towards you and then raise it.
Release the handle into the slot on the top end of the groove. Make sure that the
handle rests securely in the slot.
5
Slide the reagent rack out of the reagent compartment.
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.
Table 1 SBS Reagent Positions
Position Reagent
1
2
3
4
5
6
7
8
IRM
PW1
USM
SBS Buffer 1 (SB1)
SBS Buffer 2 (SB2)
SBS Buffer 2 (SB2)
CRM
SBS Buffer 3 (SB3)
Description
Incorporation Reagent Master Mix
25 ml of PW1 or laboratory-grade water
Universal Scan Mix
High Salt Buffer
Incorporation Wash Buffer
Incorporation Wash Buffer
Cleavage Reagent Mix
Cleavage Buffer
7
Put on a new pair of powder-free latex gloves.
8
Slide the reagent rack into the reagent compartment. Align the rack with the raised
guide on the floor of the compartment.
9
Lower the sippers into the sequencing reagent bottles as follows.
a
b
c
Pull the sipper handle towards you and then lower it.
Inspect the sippers to make sure that they do not bend as they lower into the funnel
caps.
Release the handle into the slot on the bottom end of the groove.
10 Select the PW1 (25 ml) loaded checkbox.
HiSeq 2500 System Guide
25
Load and Prime Reagents
Load and Prime Reagents
Sequencing in HiSeq v4 Mode
Load Indexing Reagents
1
Make sure that the paired-end rack is not in use on the adjacent flow cell for Read 2
resynthesis, Index 1 (i7) Read preparation, and Index 2 (i5) Read preparation.
2
Raise the sippers for the paired-end reagent rack as follows.
a
b
Pull the handle towards you and raise it.
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.
4
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. If HP9 is not used, load a 15 ml conical tube with 10
ml laboratory-grade water in position 16.
Table 3 Paired-End Flow Cells
Position
Reagent
10
15
17
FRM*
FDR
HP12
Description
Fast Resynthesis Mix
Fast Denaturation Reagent (contains formamide)
Index Sequencing Primer i7
* Load FRM in position 10 for dual-indexed runs on a paired-end flow cell. FRM is required in
position 10 for all paired-end runs regardless of indexing option.
5
If you are performing a paired-end run, skip steps 6 through 9.
6
Place 15 ml conical tubes filled with 10 ml laboratory-grade water in unused positions
on the paired-end rack.
7
Slide the reagent rack into the reagent compartment. Align 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
b
c
9
Pull the handle towards you and lower it.
Inspect the sippers to make sure that they do not bend as they lower into the tubes.
Release the handle into the slot on the bottom end of the groove.
Select Next.
Load Paired-End Reagents
1
Raise the sippers for the paired-end reagent rack as follows.
a
b
26
Pull the handle towards you and raise it.
Release the handle into the slot on the top end of the groove. Make sure that the
handle rests securely in the slot.
Material # 20000451
Document # 15035786 v01
Slide the reagent rack out of the reagent compartment using the rack handle.
3
Remove the caps from each reagent tube and place the tube onto the rack in the
associated numbered position or matching label color.
Table 4 Paired-End Flow Cell
Position
Reagent
10
11
13
14
15
16
FRM*
FLM2
AMS
FPM
FDR*
HP11
Description
Fast Resynthesis Mix
Fast Linearization Mix 2
Fast Amplification Mix
Fast Amplification Premix
Fast Denaturation Reagent (contains formamide)
Read 2 Sequencing Primer
* If you loaded indexing reagents for a single-index run, FRM is already loaded in position 10. If
you loaded indexing reagents for a dual-index run, FRM and FDR are already loaded.
4
Place 15 ml conical tubes filled with 10 ml laboratory-grade water in unused positions
on the paired-end rack.
5
Slide the reagent rack into the reagent compartment. Align the rack with the raised
guide on the floor of the compartment.
6
Lower the sippers into the paired-end reagent tubes as follows.
a
b
c
7
Pull the handle towards you and lower it.
Inspect the sippers to make sure that they do not bend as they lower into the tubes.
Release the handle into the slot on the bottom end of the groove.
Select Next.
Prime Reagents
Steps for priming reagents include loading a priming flow cell, confirming proper flow,
and then starting the prime.
CAUTION
Always use a used flow cell to prime reagents. You can use the flow cell from a previous run
to prime reagents on a subsequent run or for a post-run wash.
Load a Priming Flow Cell
1
Rinse the priming flow cell with laboratory-grade water. Dry with a lens cleaning
tissue or lint-free tissue.
2
Clean the flow cell with alcohol wipes and lens cleaning tissue.
3
Place the flow cell on the flow cell holder with the inlet and outlet ports facing down
and the barcode on the right. Make sure that the arrow on the left edge of the flow cell,
which indicates flow direction, points towards the instrument.
4
Gently slide the flow cell towards the top and right guide pins until it stops.
HiSeq 2500 System Guide
27
Load and Prime Reagents
2
Sequencing in HiSeq v4 Mode
Figure 6 Flow Cell Positioned Against Top and Right Guide Pins
A
B
Top guide pin
Right guide pins
5
Remove your hand from the flow cell to prevent alignment drift over time.
6
Slowly move the flow cell lever to position 1 to engage the vacuum and secure the flow
cell.
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 94.
Figure 7 Flow Cell Lever in Position 1
7
Wait for about 5 seconds, and then slowly move the flow cell lever to position 2.
When the flow cell lever is solid green, the manifolds are in position and the flow cell
is ready.
Figure 8 Flow Cell Lever in Position 2
8
28
Make sure that the Vacuum Engaged checkbox is selected and then select Next.
Material # 20000451
Document # 15035786 v01
Checking for proper flow confirms that the flow cell and gaskets are properly installed and
the manifold is engaged.
1
Scan or enter the priming flow cell ID (barcode number) of the priming flow cell.
2
Select solution 2 from the drop-down list.
3
Confirm the following default values:
} Volume: 125
} Aspirate Rate: 250
} Dispense Rate: 2000
4
Select Pump.
5
Inspect the flow cell for bubbles passing through the lanes and leaks near the
manifolds.
6
If excessive bubbles are present, check the gaskets for obstructions, reduce the aspirate
rate to 100, and pump another 125 µl of water to the flow cell. If problems persist,
remove the flow cell, repeat the cleaning steps, and reload the flow cell.
Position Tubing and Start Prime
1
Remove the 8 waste tubes for the appropriate flow cell from the waste container.
Figure 9 Position Tubing
A
B
Flow cell waste tubes for reagent positions 1–8
Condensation pump tubing
2
Place each waste tubing into a separate empty 15 ml tube.
Waste is collected and measured when priming is complete.
3
Select Start Prime. Monitor priming progress from the Prime screen.
4
When priming is complete, measure the waste and confirm that the volume in each
tube is 1.75 ml for a total of 14 ml.
The total is calculated as follows:
} 250 µl for each SBS position except position 2 (250 x 7 = 1.75 ml)
} 1.75 ml for each lane (1.75 x 8 = 14 ml)
5
Return the waste tubing to the waste container.
6
Select Next.
HiSeq 2500 System Guide
29
Load and Prime Reagents
Confirm Proper Flow
Sequencing in HiSeq v4 Mode
Load the Sequencing Flow Cell
Steps to load the clustered flow cell for sequencing include removing the priming flow cell,
cleaning the flow cell holder, loading the clustered flow cell, and confirming proper flow.
Remove the Used Flow Cell
1
Slowly move the flow cell lever to position 1 to disengage the manifolds.
Figure 10 Flow Cell Lever in Position 1
2
Slowly move the flow cell lever to position 0 to disengage the vacuum seal and release
the flow cell.
Figure 11 Flow Cell Lever in Position 0
3
Lift the used flow cell from the flow cell holder.
Clean the Flow Cell Holder
30
1
Put on a new pair of powder-free latex gloves.
2
Wipe the surface of the flow cell holder with a lint-free tissue moistened with
laboratory-grade water to remove salts.
3
Wipe the surface of the flow cell holder with an alcohol wipe or a lint-free tissue
moistened with ethanol or isopropanol. Do not allow alcohol to drip into the vacuum
holes or around the manifolds.
4
Dry the stage with a low-lint lab tissue, if necessary.
5
Inspect the flow cell holder to make sure that it is free of lint and the vacuum holes are
free of obstructions.
Material # 20000451
Document # 15035786 v01
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. Repeat, refolding the
alcohol wipe with each pass, until the flow cell is clean.
6
Dry the flow cell using a dry lens cleaning tissue.
7
Protect the flow cell from dust until you are ready to load it onto the instrument.
Load the Sequencing Flow Cell
1
Place the flow cell on the flow cell holder with the inlet and outlet ports facing down
and the barcode on the right. Make sure that the arrow on the left edge of the flow cell,
which indicates flow direction, points towards the instrument.
2
Gently slide the flow cell towards the top and right guide pins until it stops.
Figure 13 Flow Cell Positioned Against Top and Right Guide Pins
A
B
3
Top guide pin
Right guide pins
Remove your hand from the flow cell to prevent alignment drift over time.
HiSeq 2500 System Guide
31
Load the Sequencing Flow Cell
Figure 12 Inspect Vacuum Holes
Sequencing in HiSeq v4 Mode
4
Slowly move the flow cell lever to position 1 to engage the vacuum and secure the flow
cell.
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 94.
Figure 14 Flow Cell Lever in Position 1
5
Wait for about 5 seconds, and then slowly move the flow cell lever to position 2.
When the flow cell lever is solid green, the manifolds are in position and the flow cell
is ready for use.
Figure 15 Flow Cell Lever in Position 2
6
Make sure that the Vacuum Engaged checkbox is selected 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.
32
1
Select solution 5 from the drop-down list.
2
Confirm the following default values:
} Volume: 250
} Aspirate Rate: 250
} Dispense Rate: 2000
3
Select Pump.
4
Inspect the flow cell for bubbles passing through the lanes or leaks near the manifolds.
5
If excessive bubbles are present, check the manifold gaskets for obstructions and repeat
the process using solution 6 to avoid depleting position 5. Reduce the aspirate rate to
100, and pump another 250 µl to the flow cell.
6
Select Next.
Material # 20000451
Document # 15035786 v01
Make sure that the flow cell lever is green, and then close the flow cell compartment
door.
8
Confirm that the Vacuum Engaged and Door Closed checkboxes are selected, and then
select Next.
9
Select Start to begin the sequencing run.
HiSeq 2500 System Guide
33
Load the Sequencing Flow Cell
7
Sequencing in HiSeq v4 Mode
Monitor the Run
Monitor run metrics from the run overview screen.
Figure 16 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.
First Base Report
If you selected the Confirm First Base option during run setup, the first base confirmation
dialog box opens automatically after imaging of the first cycle is complete. The run pauses
at this step.
1
Review the First Base Report from the confirmation dialog box.
2
If the results are satisfactory, select Continue.
Sequencing Analysis Viewer
When run metrics are available, SAV opens automatically and displays metrics generated
during the sequencing run. Metrics appear in the form of plots, graphs, and tables. To view
updated metrics, select Refresh at any time during the run.
For more information, see the Sequencing Analysis Viewer User Guide (part # 15020619).
34
Material # 20000451
Document # 15035786 v01
1
When the run is complete, open the reagent compartment door.
2
Raise the sippers for the appropriate SBS rack and paired-end rack as follows.
a
b
c
Pull the sipper handle outward.
Raise the sipper handle while pulling it outward.
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 each reagent rack out of the reagent compartment using the rack handles.
4
Remove each bottle from each reagent rack.
WARNING
This set of reagents contains formamide, an aliphatic amide that is a probable
reproductive toxin. Personal injury can occur through inhalation, ingestion, skin
contact, and eye contact. Wear protective equipment, including eye protection,
gloves, and laboratory coat. Handle used reagents as chemical waste and discard in
accordance with the governmental safety standards for your region. For
environmental, health, and safety information, see the SDS for this kit at
support.illumina.com/sds.html.
HiSeq 2500 System Guide
35
Unload Reagents
Unload Reagents
Sequencing in HiSeq v4 Mode
Perform a Water Wash
A water wash is required after each sequencing run, with the option to perform a
maintenance wash instead (recommended). For instructions, see Perform a Maintenance
Wash on page 87.
If the instrument has been idle for 1 day or more, perform a water wash before beginning a
new sequencing run.
1
From the Welcome screen, select Wash | Water.
2
Select Yes to wash paired-end reagent positions, and then select Next.
3
Load the instrument with laboratory-grade water:
a
b
Fill 8 SBS bottles with 250 ml laboratory-grade water.
Fill 10 PE tubes with 12 ml laboratory-grade water.
4
Make sure that a used flow cell is loaded. Load a used flow cell, if necessary.
5
Select Next.
6
Perform a fluidics check:
a
b
c
Select solution 2 from the drop-down list. Accept the default pump values.
Select Pump.
Inspect the flow cell for bubbles passing through the lanes and leaks near the
manifolds.
7
Remove the waste tubing for the appropriate flow cell from the waste container.
8
Bundle the waste tubing with parafilm. Keep all of the ends even.
9
Place the bundled tubing ends into a 250 ml bottle.
10 Select Next to start the water wash.
Positions
8 SBS positions
8 SBS positions and 10 paired-end positions
Approximate Run Time
20 minutes
60 minutes
11 When the wash is complete, measure the delivered volume.
Positions
8 SBS positions
8 SBS positions and 10 paired-end positions
Total Delivered
Volume
32 ml
72 ml
Per Lane Delivered
Volume
4 ml
9 ml
12 Unwrap the waste tubing and return it to the waste bottle.
36
Material # 20000451
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Chapter 4 Sequencing in TruSeq v3 Mode
Introduction
TruSeq v3 Sequencing Workflow
Prepare Reagents for Read 1
Enter Run Parameters
Load and Prime Reagents
Load the Sequencing Flow Cell
Monitor the Run
Prepare Reagents for Read 2
Unload Reagents
Perform a Water Wash
HiSeq 2500 System Guide
Chapter 4
Sequencing in TruSeq v3
Mode
38
39
40
44
47
52
56
57
60
61
37
Sequencing in TruSeq v3 Mode
Introduction
To perform a sequencing run in TruSeq v3 mode 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. Run setup includes entering run parameters, loading and priming reagents,
loading the flow cell, and checking fluidics. After the completion of Read 1 and any index
reads, prepare and load paired-end reagents and SBS reagents for Read 2.
Visit the HiSeq 2500 specifications page on the Illumina website for information about run
duration and other performance specifications.
Stagger Runs
It is possible to start a new run on either flow cell A or flow cell B when a run on the
adjacent flow cell is in progress. For more information, see Stagger Runs on Flow Cell A and
Flow Cell B on page 100.
Run Types for TruSeq v3 Chemistry
The following table shows types of sequencing runs and the number of possible cycles for
each read when using TruSeq v3 chemistry. Reference this information when setting up the
run.
Run Type
Read 1
Cycles
Index 1
(i7) Read
Cycles
--
Index 2
(i5) Read
Cycles
--
Read 2
Cycles
Total
Cycles
Single-Read,
Non-Indexed
Single-Read,
Single-Indexed
≤ 101
--
≤ 101
--
--
≤ 101
6 or 7 ¹
8²
8
8
--
≤ 108 ¹
≤ 109 ²
≤ 117
Single-Read,
Dual-Indexed
Paired-End,
Non-Indexed
Paired-End,
Single-Indexed
≤ 101
--
--
≤ 101
≤ 202
≤ 101
7¹
8²
8
--
≤ 101
7+8³
≤ 101
≤ 209 ¹
≤ 210 ²
≤ 225
Paired-End,
Dual-Indexed
≤ 101
≤ 101
¹ Number of cycles for single-indexed libraries
² Number of cycles for dual-indexed libraries
³ The Index 2 Read of a paired-end dual-indexed run includes 7 additional chemistry-only cycles
38
Material # 20000451
Document # 15035786 v01
TruSeq v3 Sequencing Workflow
TruSeq v3 Sequencing Workflow
Prepare SBS reagents for Read 1 and indexing reagents.
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.
Start the sequencing run.
[Optional] After cycle 1, inspect the first base report, and then continue
Read 1.
The run continues as specified in run parameters.
Prepare paired-end reagents and fresh ICB for Read 2.
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 reagents.
Perform an instrument wash.
HiSeq 2500 System Guide
39
Sequencing in TruSeq v3 Mode
Prepare Reagents for Read 1
Before setting up the run, prepare reagents for Read 1 and any index reads. Load prepared
reagents when prompted by the control software.
Reagents for Read 1 can be prepared during cluster generation. For instructions on
preparing clustering reagents and performing cluster generation, see the cBot System Guide
(document # 15006165).
NOTE
Reagents for Read 2 are prepared during Read 1.
Split Reagents
It is possible to split a 200-cycle kit into 2 sets of reagents to perform shorter runs. For more
information, see Split SBS Kits on page 101.
Prepare SBS Reagents
The steps to prepare SBS reagents include thawing CMR, LFN, and SRE and then
preparing ICB with LFN and EDP. Using the 200-cycle SBS kit requires splitting ICB into 2
portions, 1 each for Read 1 and Read 2, during preparation. For dual-indexing, the ICB
preparation step includes calculating the appropriate reagent volumes.
Figure 17 SBS Reagent Preparation Workflow
Thaw SBS Reagents
1
Remove CMR and SRE from -25°C to -15°C storage and thaw at 2°C to 8°C for about 16
hours.
Alternatively, thaw SRE in a room temperature deionized water bath for about
90 minutes. Thaw CMR in a separate water bath.
NOTE
Always replace your gloves after handling CMR.
40
2
Invert reagents to mix.
3
Set SRE aside on ice. Set CMR aside on ice separately to prevent cross-contamination.
4
Remove LFN from -25°C to -15°C storage:
} 50-cycle SBS kit—Remove 1 tube.
} 200-cycle SBS kit—Remove 2 tubes.
Material # 20000451
Document # 15035786 v01
Thaw LFN in a beaker filled with room temperature deionized water for about 20
minutes.
6
Use ICB, SB1, SB2, and SB3 directly from 2°C to 8°C storage.
Prepare ICB for Read 1 (Non-Index or Single-Index) (50-Cycle Kit)
1
Add the contents of the tube of LFN to the bottle of ICB.
2
Rinse the tube of LFN with ICB to make sure that all LFN is transferred.
3
Add the contents of the tube of EDP to the ICB and LFN solution.
4
Rinse the tube of EDP with the ICB and LFN solution to make sure that all EDP is
transferred.
5
Cap the bottle containing EDP, ICB, and LFN and invert to mix.
6
Set aside on ice.
Prepare ICB for Read 1 (Non-Index or Single-Index) (200-Cycle Kit)
1
Add 47 ml ICB to an empty 250 ml bottle to result in 2 bottles of ICB.
2
Set aside the bottle containing 47 ml ICB at 2°C to 8°C for Read 2.
3
Add the contents of 2 tubes of LFN to the original bottle of ICB.
4
Rinse each LFN tube with ICB to make sure that all LFN is transferred.
5
Add 1.1 ml EDP to the ICB and LFN solution.
6
Return the unused portion of EDP to -25°C to -15°C storage.
7
Cap the bottle containing EDP, ICB, and LFN and invert to mix.
8
Set aside on ice.
Prepare ICB for Read 1 (Dual-Index)
Use the following instructions to calculate and prepare the volume of ICB required for Read
1 of a dual-indexed run.
1
Measure the following volumes of EDP, ICB, and LFN for every 10 cycles of sequencing
to be performed in Read 1.
Reagent
ICB
LFN
EDP
Volume per 10 Cycles
4.57 ml
0.6 ml
0.11 ml
Storage
2°C to 8°C
-25°C to -15°C
-25°C to -15°C
2
Transfer the measured volume of ICB to an empty 250 ml bottle.
3
Add the measured volume of LFN to the new bottle of ICB.
4
Rinse each tube of LFN with ICB to make sure that all LFN is transferred.
5
Add the measured volume of EDP to the ICB and LFN solution.
6
Rinse the tube of EDP with the ICB and LFN solution to make sure that all EDP is
transferred.
7
Cap the bottle containing EDP, ICB, and LFN and invert to mix.
8
Set aside on ice.
HiSeq 2500 System Guide
41
Prepare Reagents for Read 1
5
Sequencing in TruSeq v3 Mode
Example Calculation
A 101-cycle dual-indexed paired-end run has a total of 225 cycles:
} 124 cycles (101 + 8 + 7 + 8) for Read 1 and the Index Reads
} 101 cycles for Read 2
Read 1 and Index Reads (124 Cycles)
124 cycles / 10 cycles = 12.4
• ICB: 12.4 x 4.57 ml = 56.9 ml
• LFN: 12.4 x 0.6 ml = 7.44 ml
• EDP: 12.4 x 0.11 ml = 1.36 ml
Read 2 (101 Cycles)
101 cycles / 10 cycles = 10
• ICB: 10 x 4.57 ml = 45.7 ml
• LFN: 10 x 0.6 ml = 6 ml
• EDP: 10 x 0.11 ml = 1.1 ml
Use the same calculation for any dual-indexed run. Base the calculation on the total
number of cycles in Read 1 and the Index Reads plus the total number of cycles in Read 2.
For dual-indexed paired-end runs, include the 7 chemistry-only cycles in the Index 2 Read.
Prepare Indexing Reagents
Indexing reagents are used during the indexing reads of an indexed sequencing run.
Prepare the reagents that are appropriate for the run type and library type.
Run Type
Single-indexed
Dual-indexed paired-end
Dual-indexed single-read
Library Type
All libraries except Nextera
Nextera libraries
All libraries except Nextera
Nextera libraries
All libraries except Nextera
Nextera libraries
Index 1 (i7)
HP8
HP12*
HP8
HP12*
HP8
HP12*
Index 2 (i5)
--RMR
RMR
HP9*
HP9*
* Provided in the TruSeq Dual Index Sequencing Primer Box.
Thaw Indexing Reagents
1
Remove the following reagents from -25°C to -15°C storage:
} For all indexed runs—HP3, HP12 or HP8, and HT2
} For dual-indexed runs on a single-read flow cell—HP9
} For dual-indexed runs on a paired-end flow cell—RMR
2
Thaw reagents in a beaker filled with room temperature deionized water for about
20 minutes.
Prepare HT2
1
Invert to mix.
2
Centrifuge at 1000 rpm for 1 minute.
3
Set aside at room temperature.
Prepare HP3 for Indexed Runs
42
1
Invert to mix, and then pulse centrifuge.
2
Combine the following volumes of HP3 and PW1:
Material # 20000451
Document # 15035786 v01
3
Invert to mix.
4
Centrifuge at 1000 rpm for 1 minute.
5
Set aside at room temperature.
Prepare HP8 or HP12
1
Invert to mix.
2
Centrifuge at 1000 rpm for 1 minute.
3
Set aside at room temperature.
Prepare RMR
1
Invert to mix.
2
Centrifuge at 1000 rpm for 1 minute. Do not vortex.
3
Set aside on ice.
Prepare HP9
1
Invert to mix.
2
Briefly pulse centrifuge to collect droplets.
3
Set aside at room temperature.
HiSeq 2500 System Guide
43
Prepare Reagents for Read 1
} For single-indexed runs—Transfer 3325 µl PW1 to an empty 15 ml conical tube, and
then add 175 µl HP3.
} For dual-indexed runs—Transfer 3800 µl PW1 to an empty 15 ml conical tube, and
then add 200 µl HP3.
Sequencing in TruSeq v3 Mode
Enter Run Parameters
Begin run setup by entering run parameters from a series of screens on the Run
Configuration tab. The software guides you through each screen as you specify BaseSpace
connectivity, enter consumable IDs, select indexing options, and record other parameters
for the run.
Integration Screen
The Integration screen provides the option to connect the run to BaseSpace.
1
From the Welcome screen, select Sequence | New Run to open the Integration screen.
2
[Optional] Connect to BaseSpace or BaseSpace Onsite as follows.
a
b
c
Select BaseSpace or BaseSpace Onsite.
If you selected BaseSpace, select from the following options:
} Storage and Analysis—Sends run data to BaseSpace for remote monitoring and
data analysis. A sample sheet is required with this option.
} Run Monitoring Only—Sends only InterOp files to BaseSpace, which allows
remote monitoring of the run.
Log on to BaseSpace or BaseSpace Onsite using your MyIllumina account email
and password.
3
[Optional] To proceed without connecting to BaseSpace, select None.
4
Select Next.
Storage Screen
1
Select the Save to an output folder checkbox, and then select Browse to navigate to a
preferred network location.
If the run is connected to BaseSpace for storage and analysis, this step 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. A thumbnail is a sampling of
images from many tiles in each column of tiles, or swath, combined in 1 thumbnail
image.
} Save Tile Thumbnails—Saves tile thumbnails. Tile thumbnails represent a single
tile rather than a sampling of tiles in a swath.
4
Select Next.
Flow Cell Setup Screen
The Flow Cell Setup screen records information about the flow cell used for the run.
44
Material # 20000451
Document # 15035786 v01
Scan the flow cell barcode or enter the flow cell ID (barcode number) of the flow cell to
be sequenced.
The flow cell ID is used to determine flow cell type and reagent compatibility.
2
Confirm that the flow cell type is HiSeq Flow Cell v3.
The flow cell type is selected automatically based on the flow cell ID.
3
Enter an experiment name to appear on each screen and help identify the run in
progress.
4
Enter a user name and then select Next.
Advanced Screen
1
[Optional] Select the Confirm First Base checkbox.
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.
Alternatively, select lanes on the flow cell image to add or remove lanes for PhiX
alignment.
By default, all lanes are selected for 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
[Optional] Select Keep Intensity Files for later reanalysis or custom processing.
NOTE
Selecting this option significantly increases the size of the data output folder. Saving
intensity files is not required for on-instrument analysis.
4
Select Next.
Recipe Screen
A recipe is generated automatically from the information entered on the Recipe screen.
1
Select from the following Index Type options:
} No Index—Performs a non-indexed single-read or paired-end run.
} Single Index—Performs a single-read or paired-end run with 1 indexing read.
} Dual Index—Performs a single-read or paired-end run with 2 indexing reads.
} Custom—Performs a single-read or paired-end run with a custom number of cycles
for index reads.
2
If the Dual Index or Custom option is specified, select a Flow Cell Format, either Single
Read or Paired End.
3
Enter the number of cycles for Read 1 and Read 2, if applicable.
The number of cycles performed in a read is 1 more cycle than the number of cycles
analyzed. For example, to perform 125 cycles for Read 1, enter 126.
4
For the Custom indexing option, enter the number of cycles for index reads.
NOTE
Read lengths do not need to be identical.
5
Confirm the following autopopulated chemistry settings:
HiSeq 2500 System Guide
45
Enter Run Parameters
1
Sequencing in TruSeq v3 Mode
a
b
c
6
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 checkbox to use a custom recipe.
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
HCS v2.2 allows a different indexing scheme in each lane.
Reagents Screen
The Reagents screen records information about reagent kits used for the run. The reagent kit
ID (barcode number beginning with RGT) is used to determine reagent kit type and run
mode compatibility.
1
Scan or enter the SBS reagent kit ID.
2
For paired-end runs, scan or enter the reagent kit ID for paired-end cluster kit.
3
Select the SBS reagent kit for the run:
} Select 200 Cycles for a 200 cycle kit. Cycles remaining defaults to 209.
} Select 50 Cycles for a 50 cycle kit. Cycles remaining defaults to 59.
} 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 for fresh reagents.
4
Select Prime SBS Reagents to prime reagents before starting the run.
5
Select Next.
Review Screen
46
1
Review the run parameters on the Review screen.
2
Select Next to proceed or select Back to change parameters.
Material # 20000451
Document # 15035786 v01
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.
Load SBS Reagents
1
Remove the cap from each reagent bottle and replace it with a funnel cap.
CAUTION
Handle the bottle of CMR last, after you have loaded all other reagents, to prevent
cross-contamination. Always replace your gloves after handling CMR.
2
Open the reagent compartment door.
3
Raise the sippers for the sequencing reagent rack as follows.
a
b
Pull the sipper handle towards you and then raise it.
Release the handle into the slot on the top end of the groove. Make sure that the
handle rests securely in the slot.
4
Slide the reagent rack out of the reagent compartment.
5
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.
Table 5 SBS Reagent Positions
Position Reagent
1
2
3
4
5
6
7
8
ICB
PW1
SRE
SBS Buffer 1 (SB1)
SBS Buffer 2 (SB2)
SBS Buffer 2 (SB2)
CMR
SBS Buffer 3 (SB3)
Description
Incorporation Mix
25 ml of PW1 or laboratory-grade water
Scan Mix Reagent
High Salt Buffer
Incorporation Wash Buffer
Incorporation Wash Buffer
Cleavage Mix Reagent
Cleavage Buffer
6
Slide the reagent rack into the reagent compartment. Align the rack with the raised
guide on the floor of the compartment.
7
Lower the sippers into the sequencing reagent bottles as follows.
a
b
c
8
Pull the sipper handle towards you and then lower it.
Inspect the sippers to make sure that they do not bend as they lower into the funnel
caps.
Release the handle into the slot on the bottom end of the groove.
Select the PW1 (25 ml) loaded checkbox.
Load Indexing Reagents
1
Make sure that the paired-end rack is not in use on the adjacent flow cell for Read 2
resynthesis, Index 1 (i7) Read preparation, and Index 2 (i5) Read preparation.
2
Raise the sippers for the paired-end reagent rack as follows.
HiSeq 2500 System Guide
47
Load and Prime Reagents
Load and Prime Reagents
Sequencing in TruSeq v3 Mode
a
b
Pull the handle towards you and raise it.
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.
4
Remove the caps from each reagent tube and place the tube onto the rack in the
associated numbered position or matching label color.
Table 6 Single-Indexed Run
Position
Reagent
17
18
19
HP8 or HP12
HP3
HT2
Description
Index 1 (i7) Sequencing Primer Mix
Denaturation Solution
Wash Buffer
Table 7 Dual-Indexed Run on a Single-Read Flow Cell
Position
Reagent
Description
16
17
18
19
HP9
HP8 or HP12
HP3
HT2
Index 2 (i5) SR Sequencing Primer Mix
Index 1 (i7) Sequencing Primer Mix
Denaturation Solution
Wash Buffer
Table 8 Dual-Indexed Run on a Paired-End Flow Cell
Position
Reagent
Description
10
17
18
19
RMR
HP8 or HP12
HP3
HT2
Resynthesis Mix
Index 1 (i7) Sequencing Primer Mix
Denaturation Solution
Wash Buffer
5
Place 15 ml conical tubes filled with 10 ml laboratory-grade water in unused rack
positions.
6
Slide the reagent rack into the reagent compartment. Align the rack with the raised
guide on the floor of the compartment.
7
Lower the sippers into the paired-end reagent tubes as follows.
a
b
c
8
Pull the handle towards you and lower it.
Inspect the sippers to make sure that they do not bend as they lower into the tubes.
Release the handle into the slot on the bottom end of the groove.
Select Next.
Prime Reagents
Steps for priming reagents include loading a priming flow cell, confirming proper flow,
and then starting the prime.
CAUTION
Always use a used flow cell to prime reagents. You can use the flow cell from a previous run
to prime reagents on a subsequent run or for a post-run wash.
Load a Priming Flow Cell
48
1
Rinse the priming flow cell with laboratory-grade water. Dry with a lens cleaning
tissue or lint-free tissue.
2
Clean the flow cell with alcohol wipes and lens cleaning tissue.
Material # 20000451
Document # 15035786 v01
Place the flow cell on the flow cell holder with the inlet and outlet ports facing down
and the barcode on the right. Make sure that the arrow on the left edge of the flow cell,
which indicates flow direction, points towards the instrument.
4
Gently slide the flow cell towards the top and right guide pins until it stops.
Figure 18 Flow Cell Positioned Against Top and Right Guide Pins
A
B
Top guide pin
Right guide pins
5
Remove your hand from the flow cell to prevent alignment drift over time.
6
Slowly move the flow cell lever to position 1 to engage the vacuum and secure the flow
cell.
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 94.
Figure 19 Flow Cell Lever in Position 1
7
Wait for about 5 seconds, and then slowly move the flow cell lever to position 2.
When the flow cell lever is solid green, the manifolds are in position and the flow cell
is ready.
HiSeq 2500 System Guide
49
Load and Prime Reagents
3
Sequencing in TruSeq v3 Mode
Figure 20 Flow Cell Lever in Position 2
8
Make sure that the Vacuum Engaged checkbox is selected 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
Scan or enter the priming flow cell ID (barcode number) of the priming flow cell.
2
Select solution 2 from the drop-down list.
3
Confirm the following default values:
} Volume: 125
} Aspirate Rate: 250
} Dispense Rate: 2000
4
Select Pump.
5
Inspect the flow cell for bubbles passing through the lanes and leaks near the
manifolds.
6
If excessive bubbles are present, check the gaskets for obstructions, reduce the aspirate
rate to 100, and pump another 125 µl of water to the flow cell. If problems persist,
remove the flow cell, repeat the cleaning steps, and reload the flow cell.
Position Tubing and Start Prime
1
Remove the 8 waste tubes for the appropriate flow cell from the waste container.
Figure 21 Position Tubing
A
B
2
50
Flow cell waste tubes for reagent positions 1–8
Condensation pump tubing
Place each waste tubing into a separate empty 15 ml tube.
Material # 20000451
Document # 15035786 v01
3
Select Start Prime. Monitor priming progress from the Prime screen.
4
When priming is complete, measure the waste and confirm that the volume in each
tube is 1.75 ml for a total of 14 ml.
The total is calculated as follows:
} 250 µl for each SBS position except position 2 (250 x 7 = 1.75 ml)
} 1.75 ml for each lane (1.75 x 8 = 14 ml)
5
Return the waste tubing to the waste container.
6
Select Next.
HiSeq 2500 System Guide
51
Load and Prime Reagents
Waste is collected and measured when priming is complete.
Sequencing in TruSeq v3 Mode
Load the Sequencing Flow Cell
Steps to load the clustered flow cell for sequencing include removing the priming flow cell,
cleaning the flow cell holder, loading the clustered flow cell, and confirming proper flow.
Remove the Used Flow Cell
1
Slowly move the flow cell lever to position 1 to disengage the manifolds.
Figure 22 Flow Cell Lever in Position 1
2
Slowly move the flow cell lever to position 0 to disengage the vacuum seal and release
the flow cell.
Figure 23 Flow Cell Lever in Position 0
3
Lift the used flow cell from the flow cell holder.
Clean the Flow Cell Holder
52
1
Put on a new pair of powder-free latex gloves.
2
Wipe the surface of the flow cell holder with a lint-free tissue moistened with
laboratory-grade water to remove salts.
3
Wipe the surface of the flow cell holder with an alcohol wipe or a lint-free tissue
moistened with ethanol or isopropanol. Do not allow alcohol to drip into the vacuum
holes or around the manifolds.
4
Dry the stage with a low-lint lab tissue, if necessary.
5
Inspect the flow cell holder to make sure that it is free of lint and the vacuum holes are
free of obstructions.
Material # 20000451
Document # 15035786 v01
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. Repeat, refolding the
alcohol wipe with each pass, until the flow cell is clean.
6
Dry the flow cell using a dry lens cleaning tissue.
7
Protect the flow cell from dust until you are ready to load it onto the instrument.
Load the Sequencing Flow Cell
1
Place the flow cell on the flow cell holder with the inlet and outlet ports facing down
and the barcode on the right. Make sure that the arrow on the left edge of the flow cell,
which indicates flow direction, points towards the instrument.
2
Gently slide the flow cell towards the top and right guide pins until it stops.
Figure 25 Flow Cell Positioned Against Top and Right Guide Pins
A
B
3
Top guide pin
Right guide pins
Remove your hand from the flow cell to prevent alignment drift over time.
HiSeq 2500 System Guide
53
Load the Sequencing Flow Cell
Figure 24 Inspect Vacuum Holes
Sequencing in TruSeq v3 Mode
4
Slowly move the flow cell lever to position 1 to engage the vacuum and secure the flow
cell.
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 94.
Figure 26 Flow Cell Lever in Position 1
5
Wait for about 5 seconds, and then slowly move the flow cell lever to position 2.
When the flow cell lever is solid green, the manifolds are in position and the flow cell
is ready for use.
Figure 27 Flow Cell Lever in Position 2
6
Make sure that the Vacuum Engaged checkbox is selected 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.
54
1
Select solution 5 from the drop-down list.
2
Confirm the following default values:
} Volume: 250
} Aspirate Rate: 250
} Dispense Rate: 2000
3
Select Pump.
4
Inspect the flow cell for bubbles passing through the lanes or leaks near the manifolds.
5
If excessive bubbles are present, check the manifold gaskets for obstructions and repeat
the process using solution 6 to avoid depleting position 5. Reduce the aspirate rate to
100, and pump another 250 µl to the flow cell.
6
Select Next.
Material # 20000451
Document # 15035786 v01
Make sure that the flow cell lever is green, and then close the flow cell compartment
door.
8
Confirm that the Vacuum Engaged and Door Closed checkboxes are selected, and then
select Next.
9
Select Start to begin the sequencing run.
HiSeq 2500 System Guide
55
Load the Sequencing Flow Cell
7
Sequencing in TruSeq v3 Mode
Monitor the Run
Monitor run metrics from the run overview screen.
Figure 28 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.
First Base Report
If you selected the Confirm First Base option during run setup, the first base confirmation
dialog box opens automatically after imaging of the first cycle is complete. The run pauses
at this step.
1
Review the First Base Report from the confirmation dialog box.
2
If the results are satisfactory, select Continue.
Sequencing Analysis Viewer
When run metrics are available, SAV opens automatically and displays metrics generated
during the sequencing run. Metrics appear in the form of plots, graphs, and tables. To view
updated metrics, select Refresh at any time during the run.
For more information, see the Sequencing Analysis Viewer User Guide (part # 15020619).
56
Material # 20000451
Document # 15035786 v01
Before the completion of Read 1 and any index reads, prepare reagents for Read 2
resynthesis and fresh ICB for Read 2. Load reagents when prompted by the control
software.
Prepare Paired-End Reagents
Paired-end reagents are used during the Read 2 resynthesis step of a paired-end sequencing
run.
NOTE
Nextera libraries require HP11, a sequencing primer provided in the TruSeq Dual Index
Sequencing Primer Box. All other libraries use HP7.
Thaw Paired-End Reagents
1
Remove the following reagents from -25°C to -15°C storage:
} For non-indexed or single-indexed runs—AMX2, APM2, AT2, BMX, HP3, HP7 or
HP11, HT2, LMX2, and RMR
} For dual-indexed runs—AMX2, APM2, AT2, BMX, HP3, HP7 or HP11, HT2, and
LMX2
} For paired-end runs—HP3
2
Thaw reagents in a beaker filled with room temperature deionized water for about
20 minutes.
3
Set aside AMX2, BMX, LMX2, and RMR on ice.
Prepare AMX2, APM2, AT2, BMX, HP3, HP7, HP11, HT2, and LMX2
1
Invert each tube to mix.
2
Centrifuge at 1000 rpm for 1 minute.
3
Set aside AMX2, BMX, and LMX2 on ice.
4
Set aside APM2, AT2, HP3, HP7, HP11, and HT2 at room temperature.
Prepare HP3 for Paired-End Runs
1
Invert to mix, and then pulse centrifuge.
2
Transfer 2.85 ml PW1 to an empty 15 ml conical tube, and then add 150 µl HP3.
3
Invert to mix.
4
Centrifuge at 1000 rpm for 1 minute.
5
Set aside at room temperature.
Prepare RMR
1
Invert to mix.
2
Centrifuge at 1000 rpm for 1 minute. Do not vortex.
3
Set aside on ice.
HiSeq 2500 System Guide
57
Prepare Reagents for Read 2
Prepare Reagents for Read 2
Sequencing in TruSeq v3 Mode
Prepare ICB for Read 2 (Non-Index or Single-Index)
1
Remove 2 tubes of LFN from -25°C to -15°C storage and thaw in a beaker filled with
room temperature deionized water for about 20 minutes.
2
When thawed, add the contents of the 2 tubes of LFN to the bottle containing 47 ml
ICB.
3
Rinse each LFN tube with ICB to make sure that all LFN is transferred.
4
Add 1.1 ml EDP to the ICB and LFN solution.
5
Return any unused portion of EDP to -25°C to -15°C storage.
6
Cap the bottle containing EDP, ICB, and LFN and invert to mix.
7
Set aside on ice.
Prepare ICB for Read 2 (Dual-Index)
Use the following instructions to calculate and prepare the volume of ICB required for Read
2 of a dual-indexed run.
1
Measure the following volumes of EDP, ICB, and LFN for every 10 cycles of sequencing
to be performed in Read 2.
Reagent
ICB
LFN
EDP
Volume per 10 Cycles
4.57 ml
0.6 ml
0.11 ml
Storage
2°C to 8°C
-25°C to -15°C
-25°C to -15°C
For more information on calculating volumes, see Example Calculation on page 42.
2
Transfer the measured volume of ICB to an empty 250 ml bottle.
3
Add the measured volume of LFN to the new bottle of ICB.
4
Rinse each tube of LFN with ICB to make sure that all LFN is transferred.
5
Add the measured volume of EDP to the ICB and LFN solution.
6
Rinse the tube of EDP with the ICB and LFN solution to make sure that all EDP is
transferred.
7
Cap the bottle containing EDP, ICB, and LFN and invert to mix.
8
Set aside on ice.
Load Paired-End Reagents
1
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.
2
Raise the sippers for the paired-end reagent rack as follows.
a
b
58
Pull the handle towards you and raise it.
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.
4
Remove the caps from each reagent tube.
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Place each of the reagent tubes onto the rack in the associated numbered positions.
Table 9 Paired-End Reagent Positions
Position
Reagent
10
11
12
13
14
15
16
18
19
RMR
LMX2
BMX
AMX2
APM2
AT2
HP7 or HP11
HP3
HT2
Description
Resynthesis Mix
Linearization Mix 2
Blocking Mix
Amplification Mix 2
AMX2 Premix
100% Formamide
Read 2 Sequencing Primer
Denaturation Solution
Wash Buffer
6
Slide the reagent rack into the reagent compartment. Align the rack with the raised
guide on the floor of the compartment.
7
Lower the sippers into the paired-end reagent tubes as follows.
a
b
c
Pull the handle towards you and lower it.
Inspect the sippers to make sure that they do not bend as they lower into the tubes.
Release the handle into the slot on the bottom end of the groove.
Load ICB for Read 2
1
Raise the sippers for the sequencing reagent rack as follows.
a
b
Pull the sipper handle towards you and then raise it.
Release the handle into the slot on the top end of the groove. Make sure that the
handle rests securely in the slot.
2
Slide the reagent rack out of the reagent compartment.
3
Remove the ICB reagent bottle from position 1 and remove the funnel cap from the
bottle.
4
Place the funnel cap on the new bottle of ICB and load the bottle into position 1. Make
sure that the conical end of the bottle rests in the indentation on the base of the rack.
5
Slide the reagent rack into the reagent compartment. Align the rack with the raised
guide on the floor of the compartment.
6
Lower the sippers into the sequencing reagent bottles as follows.
a
b
c
Pull the sipper handle towards you and then lower it.
Inspect the sippers to make sure that they do not bend as they lower into the funnel
caps.
Release the handle into the slot on the bottom end of the groove.
7
Close the reagent compartment door.
8
Select Next to resume the run.
HiSeq 2500 System Guide
59
Prepare Reagents for Read 2
5
Sequencing in TruSeq v3 Mode
Unload Reagents
1
When the run is complete, open the reagent compartment door.
2
Raise the sippers for the appropriate SBS rack and paired-end rack as follows.
a
b
c
Pull the sipper handle outward.
Raise the sipper handle while pulling it outward.
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 each reagent rack out of the reagent compartment using the rack handles.
4
Remove each bottle from each reagent rack.
WARNING
This set of reagents contains formamide, an aliphatic amide that is a probable
reproductive toxin. Personal injury can occur through inhalation, ingestion, skin
contact, and eye contact. Wear protective equipment, including eye protection,
gloves, and laboratory coat. Handle used reagents as chemical waste and discard in
accordance with the governmental safety standards for your region. For
environmental, health, and safety information, see the SDS for this kit at
support.illumina.com/sds.html.
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A water wash is required after each sequencing run, with the option to perform a
maintenance wash instead (recommended). For instructions, see Perform a Maintenance
Wash on page 87.
If the instrument has been idle for 1 day or more, perform a water wash before beginning a
new sequencing run.
1
From the Welcome screen, select Wash | Water.
2
Select Yes to wash paired-end reagent positions, and then select Next.
3
Load the instrument with laboratory-grade water:
a
b
Fill 8 SBS bottles with 250 ml laboratory-grade water.
Fill 10 PE tubes with 12 ml laboratory-grade water.
4
Make sure that a used flow cell is loaded. Load a used flow cell, if necessary.
5
Select Next.
6
Perform a fluidics check:
a
b
c
Select solution 2 from the drop-down list. Accept the default pump values.
Select Pump.
Inspect the flow cell for bubbles passing through the lanes and leaks near the
manifolds.
7
Remove the waste tubing for the appropriate flow cell from the waste container.
8
Bundle the waste tubing with parafilm. Keep all of the ends even.
9
Place the bundled tubing ends into a 250 ml bottle.
10 Select Next to start the water wash.
Positions
8 SBS positions
8 SBS positions and 10 paired-end positions
Approximate Run Time
20 minutes
60 minutes
11 When the wash is complete, measure the delivered volume.
Positions
8 SBS positions
8 SBS positions and 10 paired-end positions
Total Delivered
Volume
32 ml
72 ml
Per Lane Delivered
Volume
4 ml
9 ml
12 Unwrap the waste tubing and return it to the waste bottle.
HiSeq 2500 System Guide
61
Perform a Water Wash
Perform a Water Wash
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Chapter 5 Sequencing in Rapid Run Mode
Introduction
Rapid Run Sequencing Workflow
Prepare Reagents
Perform a Volume Check
Enter Run Parameters
Load and Prime Reagents
Load the Sequencing Flow Cell
Monitor the Run
Unload Reagents
Perform a Water Wash
HiSeq 2500 System Guide
Chapter 5
Sequencing in Rapid Run
Mode
64
66
67
69
70
73
78
82
83
84
63
Sequencing in Rapid Run Mode
Introduction
Sequencing in Rapid Run mode provides the option to perform cluster generation on the
HiSeq 2500 or the cBot. Cluster on the cBot to sequence a different library in each lane of a
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.
After reagent preparation, run setup steps include entering run parameters, loading and
priming reagents, loading the flow cell, and performing a fluidics check. If clustering is
performed on the HiSeq 2500, the step to prime reagents is skipped.
Visit the HiSeq 2500 specifications page on the Illumina website for information about run
duration and other performance specifications.
Stagger Runs
It is possible to start a new run on either flow cell A or flow cell B when a run on the
adjacent flow cell is in progress. For more information, see Stagger Runs on Flow Cell A and
Flow Cell B on page 100.
Run Types for Rapid Chemistry
The following tables show types of sequencing runs and the number of possible cycles for
each read when using rapid chemistry. Reference this information when setting up the run.
Table 10 HiSeq Rapid SBS Kit v2
Run Type
Read 1
Cycles
Index 2
(i5) Read
Cycles
--
Read 2
Cycles
Total
Cycles
--
≤ 251
--
--
8
--
≤ 258 ¹
≤ 259 ²
≤ 267
Single-Read,
Non-Indexed
Single-Read,
Single-Indexed
≤ 251
Single-Read,
Dual-Indexed
Paired-End,
Non-Indexed
Paired-End,
Single-Indexed
≤ 251
7¹
8²
8
≤ 251
--
--
≤ 251
≤ 502
≤ 251
--
≤ 251
Paired-End,
Dual-Indexed
≤ 251
7¹
8²
8
7+8³
≤ 251
≤ 509 ¹
≤ 510 ²
≤ 525
Index 1
(i7) Read
Cycles
--
Index 2
(i5) Read
Cycles
--
Read 2
Cycles
Total
Cycles
--
≤ 101
7¹
8²
8
--
--
8
--
≤ 108 ¹
≤ 109 ²
≤ 117
≤ 251
Table 11 TruSeq Rapid SBS Kit (v1)
Run Type
Read
1Cycles
64
Index 1
(i7) Read
Cycles
--
Single-Read,
Non-Indexed
Single-Read,
Single-Indexed
≤ 101
Single-Read,
Dual-Indexed
≤ 101
≤ 101
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Read
1Cycles
Paired-End,
Non-Indexed
Paired-End,
Single-Indexed
≤ 101
Paired-End,
Dual-Indexed
≤ 101
≤ 101
Index 1
(i7) Read
Cycles
--
Index 2
(i5) Read
Cycles
--
Read 2
Cycles
Total
Cycles
≤ 101
≤ 202
7¹
8²
8
--
≤ 101
7+8³
≤ 101
≤ 209 ¹
≤ 210 ²
≤ 225
Introduction
Run Type
¹ Number of cycles for single-indexed libraries
² Number of cycles for dual-indexed libraries
³ The Index 2 Read of a paired-end dual-indexed run includes 7 additional chemistry-only cycles
HiSeq 2500 System Guide
65
Sequencing in Rapid Run Mode
Rapid Run Sequencing Workflow
Prepare all reagents for the run and prepare the library template.
For library preparation instructions, see the HiSeq and GAIIx Systems
Denature and Dilute Libraries Guide (document # 15050107).
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.
For cluster generation on the cBot—Prime SBS reagents and measure
priming waste.
Start the sequencing run. After cycle 1, inspect the first base report
(optional setting), and then continue Read 1.
The sequencing run continues through the PE turn and Read 2 with no
intervention necessary.
When the run is complete, unload reagents.
Perform a post-run water wash.
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Before setting up the run, prepare SBS and clustering reagents. Load all reagents when
prompted by the control software. When using rapid chemistry, returning to the instrument
during the run to load reagents is not necessary.
NOTE
HT1 is used to dilute libraries before sequencing. For preparation instructions, see the HiSeq
and GAIIx Systems Denature and Dilute Libraries Guide (document # 15050107).
Prepare SBS Reagents
Use the following instructions to thaw reagents provided in the HiSeq Rapid SBS Kit v2 or
the TruSeq Rapid SBS Kit (v1). Instructions are organized by kit version.
Thaw v2 Reagents
1
Remove CRM, IMT, and USM from -25°C to -15°C storage and thaw at 2°C to 8°C for
up to 16 hours.
Alternatively, thaw IMT and USM in a room temperature deionized water bath for
about 90 minutes. Thaw CRM in a separate water bath.
NOTE
Always replace your gloves after handling CRM.
2
Invert reagents to mix. Make sure that any precipitate in the USM bottle is cleared.
3
Set CRM, IMT, and USM aside at 2°C to 8°C.
4
Use CWM, PW1, and USB directly from storage.
Thaw v1 Reagents
1
Remove all reagents from -25°C to -15°C storage and thaw at 2°C to 8°C for up to 16
hours.
Alternatively, thaw IMM, PW1, SRM, and USB in a room temperature deionized water
bath for about 90 minutes. Thaw CRM in a separate water bath.
NOTE
Always replace your gloves after handling CRM.
2
Invert reagents to mix. Make sure that any precipitate in the USM bottle is cleared.
3
Set CRM, IMM, SRM, and USB aside at 2°C to 8°C.
4
Set aside PW1 at room temperature.
Prepare Clustering Reagents (PE Kit)
Use the following instructions to thaw and prepare clustering, indexing, and paired-end
reagents provided in the HiSeq Rapid PE Cluster Kit v2 or TruSeq Rapid PE Cluster Kit.
Thaw Reagents
1
Remove the following reagents from -25°C to -15°C storage:
} For all runs—AMS, FDR, FLM1, FLM2, FPM, FRM, HP10, and HP11
} For indexed runs—HP12
2
Thaw reagents in a beaker filled with room temperature water for about 20 minutes.
HiSeq 2500 System Guide
67
Prepare Reagents
Prepare Reagents
Sequencing in Rapid Run Mode
Prepare AMS, FDR, FLM1, FLM2, FPM, FRM, HP10, HP11, and HP12
1
Invert each tube to mix.
2
Set aside AMS, FLM1, FLM2, and FRM on ice.
3
Set aside FDR, FPM, HP10, HP11, and HP12 at room temperature.
Prepare Clustering Reagents (SR Kit)
Use the following instructions to thaw and prepare clustering and indexing reagents
provided in the HiSeq Rapid SR Cluster Kit v2 or TruSeq Rapid SR Cluster Kit.
Thaw Reagents
1
Remove the following reagents from -25°C to -15°C storage:
} For all runs—AMS, FDR, FLS, FPM, and HP10
} For single-indexed runs—HP12
} For dual-indexed runs—HP9 and HP12
2
Place reagents in a beaker filled with room temperature water for 20 minutes.
Prepare AMS, FDR, FLS, FPM, HP9, HP10, and HP12
68
1
Invert each tube to mix.
2
Set aside AMS and FLS on ice.
3
Set aside FDR, FPM, HP9, HP10, and HP12 at room temperature.
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From the Welcome screen, select Sequence | New Run.
2
When prompted to perform a volume check, select Yes.
3
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.
4
Load laboratory-grade water into all 8 SBS positions, 10 positions on the paired-end
rack, and the library position for the current flow cell.
5
Close the template loading station.
6
Select the Water loaded and template loading station closed checkbox, and then select
Next.
7
Make sure that a used rapid flow cell is loaded on the instrument.
8
Enter the ID of the used flow cell and select Next.
9
Select Pump to confirm flow.
10 Place tubes 4 and 5 into separate empty 15 ml conical tubes.
11 Select Next to begin the volume check.
When the volume check is complete, the expected volume is 9.5 ml ±10% for each tube.
12 Return all tubes to the waste bottle.
13 Select Next.
HiSeq 2500 System Guide
69
Perform a Volume Check
Perform a Volume Check
Sequencing in Rapid Run Mode
Enter Run Parameters
Begin run setup by entering run parameters from a series of screens on the Run
Configuration tab. The software guides you through each screen as you specify BaseSpace
connectivity, enter consumable IDs, select indexing options, and record other parameters
for the run.
Integration Screen
The Integration screen provides the option to connect the run to BaseSpace.
1
[Optional] Connect to BaseSpace or BaseSpace Onsite as follows.
a
b
c
Select BaseSpace or BaseSpace Onsite.
If you selected BaseSpace, select from the following options:
} Storage and Analysis—Sends run data to BaseSpace for remote monitoring and
data analysis. A sample sheet is required with this option.
} Run Monitoring Only—Sends only InterOp files to BaseSpace, which allows
remote monitoring of the run.
Log on to BaseSpace or BaseSpace Onsite using your MyIllumina account email
and password.
2
[Optional] To proceed without connecting to BaseSpace, select None.
3
Select Next.
Storage Screen
1
Select the Save to an output folder checkbox, and then select Browse to navigate to a
preferred network location.
If the run is connected to BaseSpace for storage and analysis, this step 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. A thumbnail is a sampling of
images from many tiles in each column of tiles, or swath, combined in 1 thumbnail
image.
} Save Tile Thumbnails—Saves tile thumbnails. Tile thumbnails represent a single
tile rather than a sampling of tiles in a swath.
4
Select Next.
Flow Cell Setup Screen
The Flow Cell Setup screen records information about the flow cell used for the run.
1
70
Select a Reagent Kit Type, either TruSeq Rapid v1 or HiSeq Rapid v2.
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Scan the flow cell barcode or enter the flow cell ID (barcode number) of the flow cell to
be sequenced.
The flow cell ID is used to determine flow cell type and reagent compatibility.
3
Confirm that the flow cell type is correct, either TruSeq Rapid Flow Cell v1 or HiSeq
Rapid Flow Cell v2.
The flow cell type is automatically selected based on the flow cell ID.
4
Enter an experiment name to appear on each screen and help identify the run in
progress.
5
Enter a user name and then select Next.
Advanced Screen
1
[Optional] Select the Confirm First Base checkbox.
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.
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
[Optional][TruSeq Rapid v1] Select Keep Intensity Files for later reanalysis or custom
processing.
NOTE
Selecting this option significantly increases the size of the data output folder. Saving
intensity files is not required for on-instrument analysis.
4
Select Next.
Recipe Screen
A recipe is generated automatically from the information entered on the Recipe screen.
1
Select from the following Index Type options:
} No Index—Performs a non-indexed single-read or paired-end run.
} Single Index—Performs a single-read or paired-end run with 1 indexing read.
} Dual Index—Performs a single-read or paired-end run with 2 indexing reads.
} Custom—Performs a single-read or paired-end run with a custom number of cycles
for index reads.
2
If the Dual Index or Custom option is specified, select a Flow Cell Format, either Single
Read or Paired End.
3
Enter the number of cycles for Read 1 and Read 2, if applicable.
The number of cycles performed in a read is 1 more cycle than the number of cycles
analyzed. For example, to perform 100 cycles for Read 1, enter 101.
4
For the Custom indexing option, enter the number of cycles for index reads.
HiSeq 2500 System Guide
71
Enter Run Parameters
2
Sequencing in Rapid Run Mode
NOTE
Read lengths do not need to be identical.
5
Confirm the following autopopulated chemistry settings.
a
b
6
SBS: TruSeq Rapid SBS Kit v1 or HiSeq Rapid SBS Kit v2
Cluster Kit: One of the following:
} TruSeq Rapid PE Cluster Kit v1
} TruSeq Rapid SR Cluster Kit v1
} HiSeq Rapid PE Cluster Kit v2
} HiSeq Rapid SR Cluster Kit v2
[Optional] Select the Use Existing Recipe checkbox to use a custom recipe.
Sample Sheet Screen
A sample sheet is optional unless you are using BaseSpace to analyze data, performing
indexing, or planning to monitor demultiplexing performance using SAV.
1
Select from the following options to specify the clustering method:
} On-Board Cluster Generation—Cluster on the HiSeq only.
} Template Hybridization on cBot—Clustering started on the cBot.
2
Select Next.
3
In the Sample Sheet field, select Browse and navigate to the sample sheet location.
4
Select Next.
Reagents Screen
The Reagents screen records information about reagent kits used for the run.
1
Scan or enter the SBS reagent kit ID (barcode number beginning with RGT).
2
For paired-end runs, scan or enter the reagent kit ID for the cluster kit.
3
Select the SBS reagent kit for the run:
} [HiSeq Rapid SBS Kit v2] Select 500 Cycles for a 500 cycle kit. Cycles remaining
defaults to 525.
} Select 200 Cycles for a 200 cycle kit. Cycles remaining defaults to 225.
} Select 50 Cycles for a 50 cycle kit. Cycles remaining defaults to 74.
} 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 for fresh reagents.
4
Select Next.
Review Screen
72
1
Review the run parameters on the Review screen.
2
Select Next to proceed or select Back to change parameters.
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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.
NOTE
The reagent priming step is not necessary for flow cells clustered on the HiSeq.
Load SBS Reagents
1
Open the reagent compartment door.
2
Raise the sippers for the sequencing reagent rack as follows.
a
b
Pull the sipper handle towards you and then raise it.
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 SBS reagent rack out of the reagent compartment.
4
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.
5
Place each SBS 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.
Table 12 HiSeq Rapid v2 SBS Reagent Positions
Position
Reagent
Description
IMT
Incorporation Master Mix
1
PW1
25 ml PW1 or laboratory-grade water
2
USM
Universal Scan Mix
3
PW1
25 ml PW1 or laboratory-grade water
4
USB
Universal Sequencing Buffer
5
USB
Universal Sequencing Buffer
6
CRM
Cleavage Reagent Master Mix
7
CWM
Cleavage Wash Mix
8
Table 13 TruSeq Rapid (v1) SBS Reagent Positions
Position
Reagent
Description
IMM
Incorporation Master Mix
1
PW1
25 ml PW1 or laboratory-grade water
2
SRM
Scan Reagent Master Mix
3
PW1
25 ml PW1 or laboratory-grade water
4
USB
Universal Sequencing Buffer
5
USB
Universal Sequencing Buffer
6
CRM
Cleavage Reagent Master Mix
7
PW1
25 ml PW1 or laboratory-grade water
8
6
Slide the SBS rack into the reagent compartment. Align the rack with the raised guide
on the floor of the compartment.
7
Select the PW1 (25 ml) loaded checkbox.
8
Lower the sippers into the sequencing reagent bottles as follows.
HiSeq 2500 System Guide
73
Load and Prime Reagents
Load and Prime Reagents
Sequencing in Rapid Run Mode
a
b
c
Pull the sipper handle towards you and then lower it.
Inspect the sippers to make sure that they do not bend as they lower into the funnel
caps.
Release the handle into the slot on the bottom end of the groove.
Load Clustering Reagents
1
Raise the sippers for the paired-end reagent rack as follows.
a
b
Pull the handle towards you and raise it.
Release the handle into the slot on the top end of the groove. Make sure that the
handle rests securely in the slot.
2
Slide the paired-end reagent rack out of the reagent compartment using the rack
handle.
3
Remove the caps from each reagent tube and place the tube onto the rack in the
associated numbered position or matching label color.
Table 14 Single-Read Flow Cell
Position
Reagent
PW1
10
PW1
11
PW1
12
AMS
13
FPM
14
FDR
15
16
17
18
19
HP9*
HP12*
HP10
FLS
Description
10 ml PW1 or laboratory-grade water
10 ml PW1 or laboratory-grade water
10 ml PW1 or laboratory-grade water
Fast Amplification Mix
Fast Premix
Fast Denaturation Reagent (contains
formamide)
i5 Index Primer
i7 Index Primer
Read 1 Primer
Fast Linearization Solution
* HP9 is required only for dual-indexed runs. HP12 is required for all indexing options. If
HP9 and HP12 are not used, load a 15 ml conical tube filled with 10 ml PW1 or laboratorygrade water in each unused position.
Table 15 Paired-End Flow Cell
Position Reagent
FRM
10
FLM2
11
FLM1
12
AMS
13
FPM
14
FDR
15
HP11
16
HP12*
17
HP10
18
PW1
19
Description
Fast Resynthesis Mix
Fast Linearization Mix 2 (Read 2)
Fast Linearization Mix 1 (Read 1)
Fast Amplification Mix
Fast Premix
Fast Denaturation Reagent (contains formamide)
Read 2 Primer
i7 Index Primer
Read 1 Primer
10 ml PW1 or laboratory-grade water
* HP12 is required only for indexed runs. If HP12 is not used, load a 15 ml conical tube filled
with 10 ml PW1 or laboratory-grade water in position 17.
4
74
Slide the paired-end rack into the reagent compartment. Align the racks with the raised
guide on the floor of the compartment.
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Lower the sippers into the paired-end reagent tubes as follows.
a
b
c
Pull the handle towards you and lower it.
Inspect the sippers to make sure that they do not bend as they lower into the tubes.
Release the handle into the slot on the bottom end of the groove.
Load Template
Load the library template for clustering on the instrument. If the flow cell was clustered on
the cBot, place 2 Eppendorf tubes filled with 1 ml deionized water into the loading station
instead of completing the following instructions.
Figure 29 Template Loading Station
1
Load the Eppendorf tube containing 420 µl of prepared library template into the
appropriate side of the loading station as follows.
a
b
c
d
2
Lift the door of the loading station.
Remove the Eppendorf tube containing water and replace it with the Eppendorf
tube containing template.
Secure the lids under the bar behind the tubes to avoid interference with the
sippers.
Slowly close the loading station door. Make sure that the sippers are aligned with
the Eppendorf tubes when the lid is closed.
Select the Template loaded and template loading station closed checkbox, and then
select Next.
Prime Reagents
Prime reagents only if a Rapid Duo Sample Loading Kit was used to perform template
hybridization on the cBot. Otherwise, skip reagent priming and proceed to Load the
Sequencing Flow Cell on page 78.
Steps for priming reagents include cleaning the flow cell holder, loading a used flow cell,
confirming proper flow, and then starting the prime.
Load a Priming Flow Cell
1
Rinse a used flow cell with laboratory-grade water. Dry it with lens cleaning tissue or
lint-free tissue.
NOTE
Always use a used flow cell to prime reagents. You can use the flow cell from a previous
run to prime reagents on a subsequent run or for a post-run wash.
2
Clean the flow cell with alcohol wipes and lens cleaning tissue.
HiSeq 2500 System Guide
75
Load and Prime Reagents
5
Sequencing in Rapid Run Mode
3
Place the flow cell on the flow cell holder with the inlet and outlet ports facing down
and the barcode on the right. Make sure that the arrow on the left edge of the flow cell,
which indicates flow direction, points towards the instrument.
4
Gently slide the flow cell towards the top and right guide pins until it stops.
Figure 30 Flow Cell Positioned Against Top and Right Guide Pins
A
B
Top guide pin
Right guide pins
5
Remove your hand from the flow cell to prevent alignment drift over time.
6
Slowly move the flow cell lever to position 1 to engage the vacuum and secure the flow
cell.
When the flow cell lever is green, the vacuum is engaged. If the lever is not green, see
Possible Run Setup Problems on page 94.
Figure 31 Flow Cell Lever in Position 1
7
76
Wait for about 5 seconds, and then slowly move the flow cell lever to position 2.
When the flow cell lever is solid green, the manifolds are in position and the flow cell
is ready.
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From the Load Priming Flow Cell screen, enter the flow cell ID.
9
Make sure that the Vacuum Engaged checkbox is selected 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.
2
Confirm the following default values:
} Volume: 250
} Aspirate Rate: 1500
} Dispense Rate: 2000
3
Select Pump.
4
Inspect the flow cell for bubbles passing through the lanes and leaks near the
manifolds.
5
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.
Position Tubing and Start Prime
1
Loosen and remove the 8 waste tubes for the appropriate flow cell from the waste
container.
2
Place waste tubes 4 and tube 5 into separate 15 ml tubes.
3
Place waste tubes 1, 2, 3, 6, 7, and 8 into a bottle containing laboratory-grade water.
4
Select Next, and then select Start Prime. Monitor priming progress from the Prime
screen.
5
When priming is complete, measure the waste and confirm that the volume is 2.5 ml
±10%, which is 500 µl per reagent, per lane.
6
Return tubes 4 and 5 to the waste container.
7
Leave tubes 1, 2, 3, 6, 7, and 8 in the bottle containing laboratory-grade water.
8
Select Next.
HiSeq 2500 System Guide
77
Load and Prime Reagents
Figure 32 Flow Cell Lever in Position 2
Sequencing in Rapid Run Mode
Load the Sequencing Flow Cell
Steps to load a flow cell for sequencing include removing the priming flow cell, cleaning
the flow cell holder, loading a clustered or new flow cell, and confirming proper flow. If
cluster generation started on the cBot, load the clustered flow cell. For on-instrument
clustering, load a new flow cell.
Remove the Used Flow Cell
1
Slowly move the flow cell lever to position 1 to disengage the manifolds.
Figure 33 Flow Cell Lever in Position 1
2
Slowly move the flow cell lever to position 0 to disengage the vacuum seal and release
the flow cell.
Figure 34 Flow Cell Lever in Position 0
3
Lift the used flow cell from the flow cell holder.
Clean the Flow Cell Holder
78
1
Put on a new pair of powder-free latex gloves.
2
Wipe the surface of the flow cell holder with a lint-free tissue moistened with
laboratory-grade water to remove salts.
3
Wipe the surface of the flow cell holder with an alcohol wipe or a lint-free tissue
moistened with ethanol or isopropanol. Do not allow alcohol to drip into the vacuum
holes or around the manifolds.
4
Dry the stage with a low-lint lab tissue, if necessary.
5
Inspect the flow cell holder to make sure that it is free of lint and the vacuum holes are
free of obstructions.
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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. Repeat, refolding the
alcohol wipe with each pass, until the flow cell is clean.
6
Dry the flow cell using a dry lens cleaning tissue.
7
Protect the flow cell from dust until you are ready to load it onto the instrument.
Load the Sequencing Flow Cell
1
Place the flow cell on the flow cell holder with the inlet and outlet ports facing down
and the barcode on the right. Make sure that the arrow on the left edge of the flow cell,
which indicates flow direction, points towards the instrument.
2
Gently slide the flow cell towards the top and right guide pins until it stops.
Figure 36 Flow Cell Positioned Against Top and Right Guide Pins
A
B
3
Top Guide Pin
Right Guide Pins
Remove your hand from the flow cell before engaging the vacuum switch to prevent
alignment drift over time.
HiSeq 2500 System Guide
79
Load the Sequencing Flow Cell
Figure 35 Inspect Vacuum Holes
Sequencing in Rapid Run Mode
4
Slowly move the flow cell lever to position 1 to engage the vacuum and secure the flow
cell.
When the flow cell lever is green, the vacuum is engaged. If the lever is not green, see
Possible Run Setup Problems on page 94.
Figure 37 Flow Cell Lever in Position 1
5
Wait for about 5 seconds, and then slowly move the flow cell lever to position 2.
When the flow cell lever is solid green, the manifolds are in position and the flow cell
is ready for use.
Figure 38 Flow Cell Lever in Position 2
6
Make sure that the Vacuum Engaged checkbox is selected.
Confirm Proper Flow
Checking for proper flow confirms that the flow cell and gaskets are properly installed and
the manifold is engaged.
80
1
Select solution 5 from the drop-down list.
2
Confirm the following default values:
} Volume: 250
} Aspirate Rate: 1500
} Dispense Rate: 2000
3
Make sure that waste tubes 4 and 5 are in the waste container, and waste tubes 1, 2, 3,
6, 7, and 8 are in a bottle containing laboratory-grade water.
4
Select Pump.
5
Inspect the flow cell for bubbles passing through the lanes and leaks near the
manifolds.
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If excessive bubbles are present, check the manifold gaskets for obstructions and repeat
the process using solution 6 to avoid depleting position 5. Reduce the aspirate rate to
1000, and pump another 250 µl to the flow cell.
7
Select Next.
8
Make sure that the flow cell lever is green, and then close the flow cell compartment
door.
9
Confirm that the Vacuum Engaged and Door Closed checkboxes are selected, and then
select Next.
10 Select Start to begin the sequencing run.
HiSeq 2500 System Guide
81
Load the Sequencing Flow Cell
6
Sequencing in Rapid Run Mode
Monitor the Run
Monitor run metrics from the run overview screen.
Figure 39 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.
First Base Report
If you selected the Confirm First Base option during run setup, the first base confirmation
dialog box opens automatically after imaging of the first cycle is complete. The run pauses
at this step.
1
Review the First Base Report from the confirmation dialog box.
2
If the results are satisfactory, select Continue.
Sequencing Analysis Viewer
When run metrics are available, SAV opens automatically and displays metrics generated
during the sequencing run. Metrics appear in the form of plots, graphs, and tables. To view
updated metrics, select Refresh at any time during the run.
For more information, see the Sequencing Analysis Viewer User Guide (part # 15020619).
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1
When the run is complete, open the reagent compartment door.
2
Raise the sippers for the appropriate SBS rack and paired-end rack as follows.
a
b
c
Pull the sipper handle outward.
Raise the sipper handle while pulling it outward.
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 each reagent rack out of the reagent compartment using the rack handles.
4
Remove each bottle from each reagent rack.
WARNING
This set of reagents contains formamide, an aliphatic amide that is a probable
reproductive toxin. Personal injury can occur through inhalation, ingestion, skin
contact, and eye contact. Wear protective equipment, including eye protection,
gloves, and laboratory coat. Handle used reagents as chemical waste and discard in
accordance with the governmental safety standards for your region. For
environmental, health, and safety information, see the SDS for this kit at
support.illumina.com/sds.html.
5
Remove the Eppendorf tube from the template loading station.
NOTE
The liquid remaining in the tube after the run is highly diluted and not suitable for
further use.
HiSeq 2500 System Guide
83
Unload Reagents
Unload Reagents
Sequencing in Rapid Run Mode
Perform a Water Wash
A water wash is required after each rapid run.
If the instrument has been idle for 1 day or more, perform a water wash before beginning a
new sequencing run.
1
From the Welcome screen, select Wash | Water.
2
Select Yes to wash paired-end reagent positions, and then select Next.
3
Load the instrument with laboratory-grade water:
a
b
c
Fill 8 SBS bottles with 250 ml laboratory-grade water.
Fill 10 PE tubes with 12 ml laboratory-grade water.
Fill the Eppendorf tube with 1 ml laboratory-grade water.
4
Make sure that a used flow cell is loaded. Load a used flow cell, if necessary.
5
Select Next.
6
Perform a fluidics check:
a
b
c
Select solution 2 from the drop-down list. Accept the default pump values.
Select Pump.
Inspect the flow cell for bubbles passing through the lanes and leaks near the
manifolds.
7
Remove the waste tubing for the appropriate flow cell from the waste container.
8
Place the ends of tubes 4 and 5 into an empty container. Place remaining tube ends
into a bottle of clean water to prevent air from entering the syringe pumps.
9
Select Next to start the water wash.
Positions
8 SBS positions
8 SBS positions and 10 paired-end positions
8 SBS positions, 10 paired-end positions, and 1 template loading position
Approximate Run
Time
20 minutes
60 minutes
10 minutes
10 When the wash is complete, measure the delivered volume.
Positions
8 SBS positions
8 SBS positions and 10 paired-end positions
8 SBS positions, 10 paired-end positions, and 1 template loading position
Delivered Volume
32 ml
72 ml
9.5 ml per lane
11 Unwrap the waste tubing and return it to the waste bottle.
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Chapter 6 Maintenance
Introduction
Perform a Maintenance Wash
Switch Sequencing Modes
Idle the Instrument
Shut Down the Instrument
HiSeq 2500 System Guide
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87
90
91
92
85
Chapter 6
Maintenance
Maintenance
Introduction
Maintenance procedures ensure continued instrument performance. Shut down or idle the
instrument during periods of inactivity. Supplement water washes performed at the end of
a run with regular maintenance washes to switch sequencing modes properly and
maintain fluidics.
The water wash performed at the end of a run washes the system and checks fluidics. A
maintenance wash consists of washing the system with a prepared solution of Tween 20
and ProClin 300. Regular instrument washes maintain instrument performance by flushing
the fluidics system and preventing salt accumulation and cross-contamination of reagents
and libraries.
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.
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Perform a maintenance wash when prompted by the software every 10 days or when
switching between high output and rapid modes. After a high output run, a maintenance
wash is a recommended alternative to a water wash.
The Load Gasket screen opens with a maintenance wash every 10 days and when
switching from rapid mode to a high output mode. Replace the 10-port gasket in the front
manifold and the 8-port gasket in the back manifold before proceeding to the wash, even if
this screen does not appear.
Prepare Maintenance Wash Solution
Prepare 5 liters of maintenance wash solution for use with 1 instrument. The solution can
be stored for up to 30 days at room temperature and used up to 3 times during this period.
Dispose of wash solution in accordance with the governmental safety standards for your
region.
1
Prepare 250 ml of 10% Tween 20 by combining the following volumes. Add the water
first.
} Laboratory-grade water (225 ml)
} Tween 20 (25 ml)
2
Place a stir bar in an empty carboy that is at least 6 liters.
3
Combine the following volumes in the carboy. Add the water first.
} Laboratory-grade water (750 ml)
} 10% Tween 20 (250 ml)
} ProClin 300 (1.5 ml)
These volumes result in approximately 2.5% Tween 20 and 0.15% ProClin 300 solution.
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.
Tween 20 and ProClin 300 Wash
1
From the Welcome screen, select Wash | Maintenance.
2
[For high output modes] If a sequencing run included an indexing read or paired-end
turn, select Yes to wash PE reagent positions. Otherwise, select No.
3
Select Next.
4
If you stored maintenance wash solution from a previous run, prepare the wash
components as follows.
a
b
Replenish the stored solution and invert to mix.
Load the bottles and tubes onto the instrument in the assigned reagent rack
positions.
HiSeq 2500 System Guide
87
Perform a Maintenance Wash
Perform a Maintenance Wash
Maintenance
5
If you are using fresh maintenance wash solution, prepare the wash components as
follows.
a
b
c
[For Rapid Run mode] Fill 2 Eppendorf tubes with 1.6 ml wash solution and load
them into the loading station.
Fill 8 SBS bottles with 250 ml maintenance wash solution.
Fill 10 PE tubes with 12 ml maintenance wash solution.
Assign each bottle and tube to a reagent rack position. Maintain those assignments
for each subsequent wash to prevent cross-contamination from reagent present on
the sippers.
6
Empty the waste bottle.
7
Select the Wash solution loaded and template loading station closed checkbox, and
then select Next.
8
Remove the flow cell from the flow cell stage and set it aside.
9
Put on a new pair of powder-free latex gloves.
10 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 40 Remove Used Manifold Gaskets
11 Place a new 10-port gasket in the front slot of the flow cell holder, and a new 8-port
gasket in the back slot. Press lightly into position.
12 Reload the flow cell that you removed to install the new gaskets.
13 Make sure that the Vacuum Engaged checkbox is selected and select Next.
14 Perform a fluidics check:
a
b
c
d
Select solution 2 from the drop-down list.
Accept the default pump values and select Pump.
Inspect the flow cell for bubbles passing through the lanes and leaks near the
manifolds.
If you see a constant stream of bubbles, replace the gasket and repeat the fluidics
check.
15 Remove the waste tubing for the appropriate flow cell from the waste container.
16 [For high output modes] Bundle the 8 waste tubes with parafilm. Keep the bundled
tube ends tubes even and place them into a 250 ml bottle.
17 [For Rapid Run mode] Place the ends of tubes 4 and 5 into an empty container. Place
the ends of the other tubes into a bottle of laboratory-grade water to avoid introducing
air into the syringe pumps.
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19 When the wash is complete, select Return to Start.
20 Measure the delivered volume.
Positions
8 SBS positions
10 paired-end positions
1 template position
All positions
High Output
82 ml
76 ml
-19.75 ml per lane
Rapid
29 ml
30 ml
1.2 ml
30.1 ml per lane
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.
21 Unwrap the waste tubes and return them to the waste container.
HiSeq 2500 System Guide
89
Perform a Maintenance Wash
18 Select Next to start the wash.
Maintenance
Switch Sequencing Modes
Use the Mode Select command from the Welcome screen to switch between high output
and rapid modes.
Only runs of the same mode can be performed simultaneously. Therefore, a mode change is
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 with a gasket change is required when switching run modes. For
more information, see Perform a Maintenance Wash on page 87.
High Output to Rapid Run
Switching from a high output mode (HiSeq v4 or TruSeq v3) to Rapid Run mode requires a
rapid-mode maintenance wash.
Specification
Rapid Maintenance Wash
Flow Cell Type
Rapid flow cell (2 lanes)
Flow Cell Gasket
10-port gasket and 8-port gasket
Reagents
Tween 20 and ProClin 300
Expected Volumes (ml)
60.2 ml
Approximate Wash Time
60 minutes
Rapid Run to High Output
Switching from Rapid Run mode to a high output mode (HiSeq v4 or TruSeq v3) requires a
rapid-mode maintenance wash followed by a maintenance wash in high output mode.
Rapid Maintenance Wash
Flow Cell Type
Rapid flow cell (2 lanes)
High output flow cell (8
lanes)
Flow Cell Gasket
10-port gasket and 8-port
gasket
10-port gasket and 8-port
gasket
Reagents
Tween 20 and ProClin 300
Tween 20 and ProClin 300
Expected Volumes (ml)
60.2 ml
158 ml
Approximate Wash Time
60 minutes
130 minutes
Total Mode Change Time
90
High Output Maintenance
Wash
Specification
~ 3 hours
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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 92.
1
Perform a maintenance wash to flush the system. For more information, see Perform a
Maintenance Wash on page 87.
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.
3
Load 10 ml laboratory-grade water in each position in the reagent racks, and then
lower the sippers.
4
Load 1 ml laboratory-grade water in the loading station position.
5
Before using the instrument again, perform a water wash. For more information, see
Perform a Water Wash on page 84.
HiSeq 2500 System Guide
91
Idle the Instrument
Idle the Instrument
Maintenance
Shut Down the Instrument
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 91.
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 87.
2
Remove the flow cell from the flow cell stage.
3
Using an alcohol wipe or a lint-free tissue moistened with ethanol or isopropanol,
carefully wipe the surface of the flow cell holder.
CAUTION
Do not allow alcohol to drip into the vacuum holes or around the manifolds. Use a lowlint lab tissue to dry the stage, if necessary.
92
4
Load 10 ml of laboratory-grade water in each position in the reagent racks, and then
lower the sippers.
5
Load 1 ml laboratory-grade water in each loading station position.
6
Turn off the instrument.
7
To restart the instrument, load water in all reagent positions, turn on the instrument,
and perform a water wash. For more information, see Perform a Water Wash on page 84.
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Appendix A Troubleshooting
Possible Run Setup Problems
Perform a Fluidics Check
BaseSpace is Unavailable
Stop and Resume a Run
Pause a Run
Stagger Runs on Flow Cell A and Flow Cell B
Split SBS Kits
Primer Rehybridization
HiSeq 2500 System Guide
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95
96
97
99
100
101
103
93
Appendix A
Troubleshooting
Troubleshooting
Possible Run Setup Problems
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.
94
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. If you are going to restart the
computer, first shut down the instrument to
make sure the DoNotEject drive is recognized
correctly.
If the problem persists after restarting the
instrument computer, shut down the instrument,
wait a minimum of 60 seconds, and then restart
the instrument.
The flow cell did not
Remove the flow cell and repeat the cleaning
seat properly.
steps.
The vacuum did not
Make sure that the gaskets are present and wellseal.
seated.
Manifolds did not raise. Reload the flow cell.
If the preceding steps do not work, try replacing
the gaskets, and then reload the flow cell.
Vacuum is being
Remove the flow cell and repeat the cleaning
provided but is
steps.
inadequate.
Make sure that the gaskets are present and wellseated.
Reload the flow cell.
If the preceding steps do not work, try replacing
the gaskets, and then reload the flow cell.
Vacuum pressure is
Switch flow cell lever to position 2.
good.
Potential bubbles in the Reposition the flow cell and confirm that the
system.
holes are facing down.
Look for white precipitate around the gaskets. If
precipitate is present, replace the gaskets.
Always replace gaskets before an instrument
maintenance wash.
Confirm that the sipper assemblies are fully
lowered and each sipper is in contact with the
reagents.
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Perform a fluidics check during instrument installation and when troubleshooting fluidics
issues.
1
Select Check on the Welcome screen.
2
Scan or enter the wash flow cell ID (barcode number) of the priming flow cell. Make
sure to use a used flow cell for this step.
3
Load the used flow cell onto the instrument.
4
Fill 8 SBS bottles with PW1 or laboratory-grade water, and load the bottles onto the
SBS reagent rack.
5
Select solution 2 from the drop-down list.
6
Enter the following values:
} Volume: 250
} [For high output modes] Aspirate Rate: 250
} [For Rapid Run mode] Aspirate Rate: 1500
} Dispense Rate: 2000
7
Select Pump.
8
Inspect the flow cell for bubbles passing through the lanes and leaks near the
manifolds.
9
If excessive bubbles are present, check the manifold gaskets for obstructions, reduce the
aspirate rate to 100, and pump another 250 µl of water to the flow cell.
HiSeq 2500 System Guide
95
Perform a Fluidics Check
Perform a Fluidics Check
Troubleshooting
BaseSpace is Unavailable
If BaseSpace is not available, open Windows Services to confirm 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.
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Stopping a run might be necessary if the run was set up incorrectly, if the data quality is
bad, or for a hardware error. When resuming a stopped run, select the appropriate normal
stop option that allows the run to resume.
Stop Option
Real-Time Analysis
(RTA) Option
Able to Resume?
Normal Stop
(End of Lane\Chemistry)
Keep As Is
Yes. The run resumes at the next chemistry
or imaging command.
Complete For Run
No. The run cannot be resumed.
Complete For Read
Yes. The run resumes at the beginning of
the next read.
Keep As Is
Yes. The run resumes at the next cycle.
Complete For Run
No. The run cannot be resumed.
Complete For Read
Yes. The run resumes at the beginning of
the next read.
No option
No.
Normal Stop
(End of Cycle)
Immediate Stop
Stop a Run
1
From the Run Overview screen, select Stop to open the Stop Menu.
Figure 41 Stop Menu
2
Select from the following stop options:
} Normal Stop (End of Lane\Chemistry)—Stops the run only after the current
chemistry or imaging command is complete, and then places the flow cell in a safe
state.
} Normal Stop (End of Cycle)—Stops the run after the current cycle is complete, and
then places the flow cell in a safe state.
} Immediate Stop—Stops the run without completing the current operation and does
not place the flow cell in safe state. You cannot resume a run that was stopped with
this option.
3
Select from the following RTA options:
} Keep As Is—The run is stopped without any modifications to RTA. The run can
resume where it was stopped.
} Complete For Run—RTA is stopped. The run info, run parameters, and recipe files
are updated to reflect total cycles as the last cycle completed. Then RTA restarts to
complete base calling for the run up to the point the run was stopped. The run
cannot be resumed.
} Complete For Read—RTA is stopped. The run info, run parameters, and recipe files
are updated to trim the length of the current read to the last cycle completed.
HiSeq 2500 System Guide
97
Stop and Resume a Run
Stop and Resume a Run
Troubleshooting
Subsequent reads are not affected. Then RTA restarts to complete analysis for the
current read. The run can resume at the beginning of the next read.
4
When the run is stopped, select Return to Start on the Run Overview screen.
The Welcome screen opens.
Resume a Stopped Run
Use the following steps to resume a run that was stopped using a normal stop option with
an RTA option that allows the run to be resumed.
NOTE
If the adjacent side is performing cluster generation or paired-end chemistry, the run
resumes when the ongoing process is complete.
1
From the Welcome screen, select Sequence, and then select Resume Run.
2
From the Resume screen, select the appropriate run folder from the drop-down list.
The software resumes a run at the point where it was stopped, and defaults to the
correct setup on the Resume screen.
3
Confirm the following default settings or select the appropriate point at which to
resume the run. For more information, see Example Settings for Resuming a Run on page
98.
} Resume At—The read or point in the run to resume.
} Start At Cycle—The cycle to resume.
CAUTION
Do not select the point of paired-end turnaround, other than for Read 2 Primer
rehybridization.
4
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 the
resume run settings for Read 1 at cycle 23. The Resume screen displays these settings as
follows.
} Resume At: Read 1
} Start At Cycle: 23
Figure 42 Example of Resuming at Cycle 23
Because the run in this example was stopped during an imaging step, Imaging (no
chemistry) is selected automatically.
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Pausing a run might be necessary to check run components, such as reagent volumes.
Under normal operation, pausing a run is not necessary.
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.
1
From the Run Overview screen, select Pause to see pause options.
Figure 43 Pause Options
2
Select Normal Pause.
3
Select Yes to confirm.
The software completes the current chemistry or imaging command and places the
flow cell in a safe state.
4
Select Resume to resume the run.
Change Reagents During a Run
If you started the run with a partial volume of reagents, use the Change Reagents feature to
pause the run and replenish reagents.
NOTE
Priming is not required.
1
From the Run Overview screen, select Pause to open the pause menu.
2
Select Change Reagents.
3
Select Yes to confirm the pause command.
The software completes the current chemistry or imaging command, places the flow
cell in a safe state, and opens the Reagents screen.
4
Enter the following parameters:
} The reagent kit ID for the new reagents.
} The number of cycles the reagents are expected to last.
5
Select Next to proceed to loading reagents.
HiSeq 2500 System Guide
99
Pause a Run
Pause a Run
Troubleshooting
Stagger Runs on Flow Cell A and Flow Cell B
When staggering runs on flow cell A and flow cell B, set up a new run and the software
automatically pauses and resumes the run on the adjacent flow cell as needed. The system
is placed in a safe state automatically.
1
Set up a new run.
NOTE
If the adjacent run is completing an imaging step, the software pauses the adjacent flow
cell before you can load and prime reagents.
100
2
After loading the sequencing flow cell for the new run, close the compartment door.
3
Select Start to start the new sequencing run.
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To perform a paired-end run that is shorter than 125 cycles per read, split a 250-cycle
HiSeq v4 kit into 2 sets of reagents. One set provides reagents for a 126-cycle run with dual
indexing and 1 prime. For a run that is shorter than 67 cycles, use a 50-cycle kit.
Splitting a 200-cycle TruSeq v3 kit provides enough reagents to perform up to 101 cycles
with 1 prime. For a run that is shorter than 101 cycles, use a 50-cycle kit.
Split a 250-Cycle Kit
1
Thaw reagents from the HiSeq v4 SBS kit (250 cycles). See Thaw SBS Reagents on page
20.
2
Label 7 250 ml bottles with the position number and reagent name, as follows.
} #1 IRM
} #3 USM
} #4 SB1
} #5 SB2
} #6 SB2
} #7 CRM
} #8 SB3
3
Add the following volume of each reagent into the appropriate 250 ml bottle to result
in a second set of reagents. Prepare CRM last and then replace your gloves.
Number
1
3
4
5
6
7
8
4
Reagent Name
IRM
USM
SB1
SB2 (for position #5)
SB2 (for position #6)
CRM
SB3
Volume
72 ml
82 ml
100 ml
62 ml
62 ml
72 ml
110 ml
Use both sets of reagents for 2 concurrent runs or store 1 set at the following
temperatures:
} Store IRM, USM, and CRM at -25°C to -15°C.
} Store SB1, SB2, and SB3 at 15°C to 30°C.
Split a 200-Cycle Kit
1
Thaw reagents from the TruSeq v3 SBS kit (200 cycles). See Thaw SBS Reagents on page
40.
2
Label 7 250 ml bottles with the position number and reagent name, as follows.
} #1 ICB
} #3 SRE
} #4 SB1
} #5 SB2
} #6 SB2
} #7 CMR
} #8 SB3
HiSeq 2500 System Guide
101
Split SBS Kits
Split SBS Kits
Troubleshooting
3
Add the following volume of each reagent to the appropriate 250 ml bottle to result in a
second set of reagents. Prepare CMR last and then replace your gloves.
Number
1
3
4
5
6
7
8
4
Reagent Name
ICB
SRE
SB1
SB2 (for position #5)
SB2 (for position #6)
CMR
SB3
Volume
47 ml
107 ml
100 ml
62 ml
62 ml
107 ml
110 ml
Use both sets of reagents for 2 concurrent runs or store 1 set at the following
temperatures:
} Store CMR, SRE, and EDP at -25°C to -15°C.
} Store ICB, SB1, SB2, and SB3 at 2°C to 8°C.
Prepare ICB
102
1
Add the contents of 2 tubes of LFN to 1 bottle of ICB.
2
Rinse each tube of LFN with ICB to make sure that all LFN is transferred.
3
Add 1.1 ml EDP to the ICB and LFN solution.
4
Return the unused portion of EDP to -25°C to -15°C storage.
5
Cap the bottle containing EDP, ICB, and LFN and invert to mix.
6
Set aside on ice.
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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.
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.
Rehyb Kit Name
Workflow Instructions
HiSeq Multi-Primer Rehyb Kit v4
Catalog # GD-403-4001
HiSeq High Output Primer Rehybridization Guide
(part # 15050105)
TruSeq v3 Flow Cell
Read 1 primer rehyb is performed on the cBot. The rehyb kit includes a cBot reagent plate
containing the Read 1 sequencing primer HP6. For Nextera libraries, use HP10 from the
TruSeq Dual Index Sequencing Primer Box.
Rehyb Kit Name
Workflow Instructions
TruSeq v2 cBot Multi-Primer Rehyb Kit
Catalog # GD-304-2001
Read 1 Primer Rehyb on a TruSeq v3 or TruSeq v2
Flow Cell (part # 15018149)
Rapid 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.
Rehyb Kit Name
Workflow Instructions
HiSeq Rapid Rehyb Kit
Catalog # GD-404-1001
HiSeq Rapid Run Primer Rehybridization (part #
15059379)
HiSeq 2500 System Guide
103
Primer Rehybridization
Primer Rehybridization
104
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Appendix B Real-Time Analysis
Real-Time Analysis Overview
Real-Time Analysis Workflow
Monitor Run Metrics
HiSeq 2500 System Guide
106
108
111
105
Appendix B
Real-Time Analysis
Real-Time Analysis
Real-Time Analysis Overview
Real-Time Analysis (RTA) 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, RTA 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 RTA is terminated, it saves run data and can resume processing.
Input Files
RTA requires the following input files:
} Cluster intensity files, which contain image analysis results.
} RunInfo.xml, which the control software generates automatically at the beginning of the
run. From this file, RTA reads the run name, number of cycles, whether a read is
indexed, and the number of tiles on the flow cell.
} HiSeq.Configuration.xml, which is an instrument configuration file in XML format.
} RTA.exe.config, which is a software configuration file in XML format.
RTA uses run parameters entered during run setup, and receives commands from the
control software that include information about when to initiate and the location of
RunInfo.xml.
Output Files
Tiles are small imaging areas on the flow cell defined as 1 field of view by the camera. For
each tile that is analyzed, RTA produces a set of quality-scored base call files and filter files
as primary output. Other files support generation of primary output files.
} Base call files—For each tile that is analyzed, 1 compressed base call (*.bcl) file is
generated for each tile per cycle. The base call file contains the base call and associated
quality score.
} Filter files—Each tile produces filter information that is included in 1 filter (*.filter) file
for each tile over the whole run. The filter file specifies whether clusters pass filter.
} Cluster location files—One cluster location (*.locs) file contains the X,Y coordinates for
every cluster on the flow cell.
} 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.
RTA provides real-time metrics of run quality stored as InterOp files. InterOp files are
binary files containing tile, cycle, and read-level metrics, and are required for viewing
metrics in Sequencing Analysis Viewer (SAV). For viewing metrics generated by RTA, use
SAV v1.8.20 or later.
For details about each output file, see Sequencing Output Files on page 114.
Error Handling
RTA stores log files in the RTALogs folder. If an error occurs, RTA creates an error log file
called *Error.txt and records the error.
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Throughout the run, RTA automatically copies data generated from raw image files to the
output folder. If image analysis lags, RTA stops processing and places the flow cell in a
safe state. Processing resumes when image data are available.
If RTA stops functioning, processing is automatically resumed during the next cycle at the
appropriate point on the flow cell. Do not restart RTA manually.
Data transfer is complete when a marker file called Basecalling_Netcopy_complete.txt is
generated. One of these files is generated for each read, and 1 is generated for the entire run.
HiSeq 2500 System Guide
107
Real-Time Analysis Overview
Data Transfer
Real-Time Analysis
Real-Time Analysis Workflow
Template generation
Registration and
intensity extraction
Color matrix correction
Empirical phasing
correction
Maps cluster locations.
Records the location of each cluster on the flow cell and determines
an intensity value for each cluster.
Corrects cross talk between channels.
Corrects the effects of phasing and prephasing.
Base calling
Determines a base call for every cluster.
Quality scoring
Assigns a quality score to every base call.
Template Generation
Template generation locates and defines the position of each cluster in a tile using X and Y
coordinates. The template is used as a reference for the subsequent step of registration and
intensity extraction.
Because of the random array of clusters on the flow cell, template generation requires image
data from the first 5 cycles of the run. After the last template cycle for a tile is imaged, the
template is generated.
Cluster positions are written to 1 cluster location (*.locs) file or compressed cluster location
(*.clocs) file for each tile. For more information, see Sequencing Output Files on page 114.
Registration and Intensity Extraction
Registration and intensity extraction begin after the template of cluster positions is
generated.
} Registration aligns the images produced over every cycle subsequent to template
generation against the template.
} 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 base calls are generated for that tile in that
cycle. Use SAV to examine thumbnail images and identify images that failed registration.
Color Matrix Correction
After registration and intensity extraction, RTA 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, RTA generates matrixcorrected intensities with reduced or no cross talk, and balances differences in overall
intensity between color channels.
Empirical Phasing Correction
During the sequencing reaction, each DNA strand in a cluster extends by 1 base per cycle.
Phasing and prephasing occurs when a strand becomes out of phase with the current
incorporation cycle.
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Phasing occurs when a base falls behind.
Prephasing occurs when a base jumps ahead.
Figure 44 Phasing and Prephasing
A
B
Read with a base that is phasing
Read with a base that is prephasing.
RTA corrects the effects of phasing and prephasing using the empirical phasing correction
algorithm, which maximizes the data quality at every cycle throughout the run.
Base Calling
After raw intensities have been corrected for cross talk, phasing, and prephasing, the color
channel with the brightest intensity is the base call for that cluster in that cycle. Base
calling on the HiSeq 2500 using RTA 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. Each base call file contains the base call and the base call quality score.
To make a base call, clusters must first pass the chastity filter. Clusters that do not pass
filter or cannot be called because they are off-image or failed image registration are labeled
no-calls. No-calls are represented as (N).
Clusters Passing Filter
During the first 25 cycles of Read 1, the chastity filter removes the least reliable clusters
from analysis results. Clusters pass filter when no more than 2 base calls have a chastity
value below 0.6 in the first 25 cycles. Chastity is the ratio of the brightest base intensity
divided by the sum of the brightest and the second brightest base intensities. Analysis
reports represent the percentage of clusters passing filter as %PF.
Low quality clusters are removed from the raw cluster count during template generation,
which yields a relatively high percentage of clusters passing filter.
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 serves as a compact way to communicate small error probabilities. Quality
scores are represented as Q(X), where X is the score. The following table shows the
relationship between the quality score and error probability.
Q-Score Q(X)
Q40
HiSeq 2500 System Guide
Error Probability
0.0001 (1 in 10,000)
109
Real-Time Analysis Workflow
}
}
Real-Time Analysis
Q-Score Q(X)
Q30
Q20
Q10
Error Probability
0.001 (1 in 1,000)
0.01 (1 in 100)
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 base call (*.bcl) files.
Q-Score Binning
RTA groups quality scores into specific ranges, or bins, and assigns a value to each range.
Q-score binning significantly reduces storage space requirements without affecting accuracy
or performance of downstream applications.
Q-score binning contributes to the efficiency of analysis processes and data transfer
requirements associated with the high throughput of the HiSeq 2500. The resulting *.bcl file
is smaller because the compression algorithms are able to compress the file more
effectively. Less data are written to the instrument computer and transferred to a network
location, making the file copy faster.
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RTA automatically generates quality metrics when image analysis begins. However, not all
metrics are available at the early cycles because some processes require multiple cycles to
generate data.
Data
Cycle
Image analysis
After cycle 5.
During the first 5 cycles of the run, RTA 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.
The phasing corrections for the first 25 cycles determine the
phasing estimate.
Quality scores
After cycle 25.
A quality score is generated for reads that pass the quality filter.
Because quality scores require corrected intensities from future
cycles, quality scoring always follows base calling.
Error rates
After cycle 25.
Error rates are generated only when PhiX clusters are present
and the Align to PhiX option is selected during run setup.
Inline controls
At cycle 52 of each read, or at the end of the run for runs with
fewer than 52 cycles.
Inline controls are generated only for TruSeq library preparation
methods.*
Index count
After the index reads are complete.
The index count per lane is generated when a sample sheet is
provided.
* Sequencing Analysis Viewer (SAV) v1.8.44 and later no longer includes the TruSeq Controls tab,
where SAV reports results for the analysis of the inline controls.
HiSeq 2500 System Guide
111
Monitor Run Metrics
Monitor Run Metrics
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Appendix C Output Files and Folders
Sequencing Output Files
Output Folder Structure
Tile Numbering
Thumbnail Images
HiSeq 2500 System Guide
114
116
117
118
113
Appendix C
Output Files and Folders
Output Files and Folders
Sequencing Output Files
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.
Cluster location files
For each tile, 1 cluster location file contains the XY coordinates for
every cluster. Cluster location files are the result of template
generation.
Data\Intensities
Filter files
The filter file specifies whether a cluster passed filters. Filter files are
generated at cycle 26 using 25 cycles of data.
Data\Intensities\BaseCalls\L00[X]—Files are stored in 1 folder for
each lane and tile.
s_[lane]_[tile].filter
InterOp files
Binary reporting files used for Sequencing Analysis Viewer. InterOp
files are updated throughout the run.
InterOp folder
Log files
Record events and are updated throughout the run.
Data\RTALogs
Offsets files
Two offsets files are created for each run:
• offsets.txt—Contains tile offsets for every cycle and channel relative
to the template.
• SubTileOffsets.txt—Contains the measured shift for each quadrant
of each image relative to the frame of reference.
Data\Intensities\Offsets
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.
Real-Time Analysis
configuration file
Statistics files
114
Created at the beginning of the run, the Real-Time Analysis
configuration file lists settings for the run.
Data\Intensities
RTAConfiguration.xml
Statistics created at base calling for each cycle.
Data\Intensities\Basecalls\L00[X]\C[X.1]—Files are stored in 1 folder
for each lane and 1 subfolder for each cycle.
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File Description, Location, and Name
Run information file
Lists the run name, number of cycles in each read, whether the read is
an indexed read, and the number of swaths and tiles on the flow cell.
The run info file is created at the beginning of the run.
[Root folder]
RunInfo.xml
Thumbnail files
A thumbnail image for each channel and tile in each swath at every
cycle during imaging.
Thumbnail_Images\L00[X]\C[X.1]—Files are stored in 1 folder for
each lane and 1 subfolder for each cycle.
s_[lane]_[tile]_[channel].jpg—Tile is represented with a 4-digit number
that indicates surface, swath, and tile. See Tile Numbering on page 117.
HiSeq 2500 System Guide
115
Sequencing Output Files
File Type
Output Files and Folders
Output Folder Structure
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.
Recipe—Run-specific recipe file named with reagent cartridge ID.
RTALogs—Log files describing RTA events.
Thumbnail_Images—Thumbnail images of 9 locations from each tile, generated for
each cycle and base.
RunInfo.xml
RunParameters.xml
Run Folder Name and Path
The run folder is the root folder for output from a sequencing run. During run setup, the
software prompts you to enter the path for the run folder. By default, the folder is named
using the following format:
YYMMDD_<Computer Name>_<Run Number>_<Flow Cell ID>
Example: 110114_SN106_0716_A90095ACXX
The run number increments by 1 each time the instrument performs a sequencing run. The
flow cell ID entered during the run setup steps appends to the run folder name.
The run folder is written to the output path specified 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.
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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 HiSeq rapid flow cell is imaged
in 64 tiles. Each lane has 2 swaths with 16 tiles per swath.
NOTE
A swath is a column of tiles within a lane of the flow cell.
The tile name is a 4-digit number that represents the position on the flow cell.
} The first digit represents the surface:
} 1 is for top
} 2 is for bottom
} The second digit represents the swath:
} 1 is for the first swath
} 2 is for the second swath
} 3 is for the third swath (if applicable)
} The last 2 digits represent the tile, 01 through 16. Tile numbering starts with 01 at the
output end of the flow cell through 16 at the input end.
Figure 45 Tile Numbering
This example indicates a tile from the top surface of the flow cell, the second swath,
and the seventh tile.
HiSeq 2500 System Guide
117
Tile Numbering
Tile Numbering
Output Files and Folders
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 46 Thumbnail Image
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%
%PF 109
A
additional documentation, HiSeq 3
alerts
descriptions 6
resolving 7
aligning to PhiX 23, 45, 71
applications, installed 6
B
base call files 109, 111
BaseSpace
connecting a run 22, 44, 70
integration 2
sample sheets 24, 46, 72
troubleshooting 96
BaseSpace Onsite
connecting a run 22, 44, 70
integration 2
bcl2fastq, version 106
blinking flow cell lever 94
bubbles 29, 32, 50, 54, 77, 80
C
calculating ICB volumes 41-42, 58
catalog numbers
Illumina reagent kits 8
Illumina rehyb kits 103
user-supplied consumables 15
cBot
cluster kits 8
documentation 20, 40
Rapid Duo Sample Loading Kit 75
Read 1 rehyb 103
changing modes 90
changing reagents mid-run 99
chastity filter 109
chemistry steps, monitoring 34, 56, 82
cluster locations 108, 111
cluster quality 109
clustering
cBot vs. HiSeq 64
instructions 20, 40
rapid flow cell 75
clustering options, rapid run 64, 72
clustering reagent positions 74
colors, status bar 4
compartments 4
configuration file 114
connecting USB cables 12
consumables
Illumina 8
user-supplied 15
control lane 23, 45, 71
HiSeq 2500 System Guide
Index
Index
cross talk 108
custom recipes 23, 46, 72
customer support 123
cycles
HiSeq v4 defaults 24
Rapid Run defaults 72
TruSeq v3 defaults 46
D
data
by cycle 111
compressing 110
sending to Illumina 14
data storage 7, 22, 44, 70
default cycles 24, 46, 72
default folder locations 13
delivered volumes
maintenance washes 89-90
priming 29, 51, 77
volume checks 69
water washes 36, 61, 84
denaturing and diluting libraries 66
documentation 123
E
error logs 106
error probability 109
errors 106, 111
expected volumes
maintenance washes 89-90
priming 29, 51, 77
volume checks 69
water washes 36, 61, 84
experiment name 23, 45, 71
F
file locations 114, 116
first base incorporation 34, 56, 82
first base report 23, 45, 71
flow cell ID, recording 23, 45, 71
flow cell lever 4
blinking 94
orange 94
flow cells
cluster locations 108
imaging 117-118
inspecting 29, 32, 50, 54, 77, 80
positioning 5, 27, 31, 49, 53, 76, 79
priming 27, 48, 75
fluidics system 4
accessing 4
maintaining 86
troubleshooting 94-95
folder locations 13, 116
folder structure 116
119
Index
G
gaskets 8, 87, 90
gaskets, troubleshooting 94
guide pins 27, 31, 49, 53, 76, 79
H
hardware features 2
HCS 6
opening 12
view options 13
help
clustering 20, 40
denaturing and diluting libraries 3,
66
HiSeq documentation 3
primer rehyb 103
SAV 34, 56, 82
help, technical 123
HP3 preparation, SI vs. DI 42
HP7 vs. HP11 57
HP8 vs. HP12 42
HP9 vs. HP12 20
HP9 vs. RMR 42
HT1, preparing 67
I
ICB
calculating volumes 41-42, 58
example preparation 42
splitting 40
icons 6-7
idling vs. shutting down 92
idling, acceptable duration 91
Illumina reagent kits, catalog
numbers 8
image alignment 108
images, saving 22, 44, 70
indexing
number of cycles 18, 38, 64
required reagents 20, 42
indexing reagent positions 26, 48
indexing reagents
HiSeq v4 20
TruSeq v3 42
indexing scheme 24, 46, 72
initializing software 12
initializing software, troubleshooting 94
installation, fluidics check 95
intensities, monitoring 34, 56, 82
intensity values 108
InterOp files 106, 114
L
leaks 29, 32, 50, 54, 77, 80
libraries
loading onto instrument 75
preparing 66
LIMS
server 13
settings 13
loading station 75
locating clusters 108, 111
log files 114
logging on 12
120
low cluster intensities,
troubleshooting 103
low cluster numbers,
troubleshooting 103
M
maintenance wash solution 87
maintenance washes 87
delivered volumes 89-90
duration 90
frequency 87
reusing solution 87
maintenance, preventive 86
Menu Options window 13
mode change 90
N
naming
run folders 13, 116
runs 23, 45, 71
tiles 117
Nextera libraries, primers 8, 57
Nextera libraries, rehyb primers 103
no-calls (N) 109
normal stop vs. immediate stop 97
number of cycles
calculating ICB volumes 42
defaults 24, 46, 72
HiSeq v4 chemistry 18
performed vs. entered 23, 45, 71
rapid chemistry 64
split reagent kits 101
TruSeq v3 chemistry 38
O
online support 3
optics module 4
orange flow cell lever 94
output folders
locations 13
structure 116
P
password, default 12
pause options 99
PE reagent positions 27, 59
performance specifications 18, 38, 64
phasing 108, 111
PhiX
alignment 23, 45, 71
control lane 23, 45, 71
positioning flow cells 27, 31, 49, 53, 76,
79
positions, reagents
clustering 74
indexing 26, 48
PE 27, 59
SBS 25, 47, 73
possible cycles
HiSeq v4 chemistry 18
rapid chemistry 64
TruSeq v3 chemistry 38
preparing HT1 67
preparing reagents, TruSeq v3
workflow 40
Material # 20000451
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Q
Q-scores 109, 111
quality scores 109
monitoring 34, 56, 82
Phred algorithm 110
R
racks, reagent 5
Rapid Duo Sample Loading Kit 75
rapid run clustering options 64, 72
reagent chiller, temperature 5
reagent kit ID, recording 24, 46, 72
reagent positions
PE rack 26-27, 48, 59, 74
SBS rack 25, 47, 73
reagent racks 5
reagent volumes, split SBS kits 101-102
reagents
changing mid-run 99
handling post-run 35, 60, 83
shorter runs 101
reagents, SBS
creating 2 sets 101
TruSeq v3 workflow 40
reagents, storing split kits 101-102
recipes, custom 23, 46, 72
recording flow cell IDs 23, 45, 71
recording reagent kit IDs 24, 46, 72
registration, troubleshooting 108
rehybridization 103
remote monitoring 22, 44, 70
reports, first base incorporation 34, 56,
82
restarting the instrument 92
resume options 97
resuming runs, settings 98
reusing maintenance wash solution 87
RTA 6, 106
input files 106
resuming 98
stop options 97
stopped runs 97, 107
run folder location 116
run folder size 7
run information file 114
run metrics
availability by cycle 111
viewing 34, 56, 82
Run Overview screen 34, 56, 82
S
sample sheet 111
sample sheets, requiring 24, 46, 72
SAV 6
documentation 34, 56, 82
InterOp files 114
version 106
saving thumbnail images 22, 44, 70
SBS reagent positions 25, 47, 73
sensors 7
HiSeq 2500 System Guide
sequencing steps, overview
HiSeq v4 mode 19
Rapid Run mode 66
RTA 108
TruSeq v3 mode 39
settings
LIMS 13
resuming a run 98
settings, resuming runs 98
shutting down vs. idling 92
simultaneous runs 100
software
features 2
installed applications 6
troubleshooting 94
specifications, performance 18, 38, 64
splitting ICB 40
staggering runs 100
status bar colors 4
stop options 97
storage capacity 7
optimizing 22, 44, 70, 110
storing maintenance wash solution 87
storing split reagent kits 101-102
swaths 22, 44, 70, 117
T
technical assistance 123
temperature, reagent chiller 5
thumbnails 114, 118
saving 22, 44, 70
tiles 106, 117
TruSeq Controls tab 111
TruSeq Dual Index Sequencing Primer
Box
purpose 8
required reagents 20
turning off the instrument 92
turning on the instrument 12
U
USB cables, connecting 12
user name, default 12
V
vacuum system 4
volume check volumes 69
volumes, split SBS kits 101-102
W
washes
benefits 86
maintenance wash solution 87
system requirements 36, 61, 84, 87
water vs. maintenance 86
waste tubing 29, 50, 69, 77, 88
water washes
delivered volumes 36, 61, 84
duration and frequency 36, 61, 84
121
Index
prephasing 108
preventive maintenance 86
priming flow cell 27, 48, 75
priming waste 29, 51, 77
Index
122
Material # 20000451
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For technical assistance, contact Illumina Technical Support.
Table 16 Illumina General Contact Information
Website
Email
www.illumina.com
techsupport@illumina.com
Table 17 Illumina Customer Support Telephone Numbers
Region
Contact Number
Region
North America
1.800.809.4566
Italy
Australia
1.800.775.688
Netherlands
Austria
0800.296575
New Zealand
Belgium
0800.81102
Norway
Denmark
80882346
Spain
Finland
0800.918363
Sweden
France
0800.911850
Switzerland
Germany
0800.180.8994
United Kingdom
Ireland
1.800.812949
Other countries
Contact Number
800.874909
0800.0223859
0800.451.650
800.16836
900.812168
020790181
0800.563118
0800.917.0041
+44.1799.534000
Safety data sheets (SDSs)—Available on the Illumina website at
support.illumina.com/sds.html.
Product documentation—Available for download in PDF from the Illumina website. Go
to support.illumina.com, select a product, then select Documentation & Literature.
HiSeq 2500 System Guide
123
Technical Assistance
Technical Assistance
Illumina
5200 Illumina Way
San Diego, California 92122 U.S.A.
+1.800.809.ILMN (4566)
+1.858.202.4566 (outside North America)
techsupport@illumina.com
www.illumina.com
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