For information on library library input for the various HiSeq chemistries, see the HiSeq Systems Denature and Dilute Libraries Guide.
See system specifications on the HiSeq 2500 Specifications page.
The HiSeq 2500 and HiSeq 1500 Systems have Rapid Run mode and can run HiSeq Rapid Run v2 chemistry.
SN# D00101 or higher
SN# C00101 or higher
SN# 7001403 or higher
Field-upgraded HiSeq 2500
SN# 7001403 or higher
Field-upgraded HiSeq 1500
SN# L179 or higher
For more information, see the upgrade resources on the Documentation page for your instrument.
Real-Time Analysis (RTA) v1.18 includes optimizations to the algorithms that identify clusters and estimate the color normalization matrix and phasing and prephasing rates. These optimizations improve the ability of Real-Time Analysis to handle low-diversity samples, such as samples with unbalanced genome compositions (AT- or GC-rich genomes) or samples with low sequence diversity (amplicon sequencing). Because of these improvements, it is no longer necessary to designate a control lane in the control software to estimate matrix and phasing. For details, see Low-Diversity Sequencing on the Illumina HiSeq Platform.
The HiSeq v4 reagent kits support dual-indexing workflows without requiring the purchase of additional SBS agents. Sample prep for dual-indexed libraries requires that both indexes be present on the library. However, the second index does not need to be read during sequencing. A single-indexing workflow is supported on Illumina sequencing instruments, where only Index 1 is used. See the instrument user guide for more information about setting up an 8-base single-indexed sequencing run.
HiSeq SBS Kit v4:
TruSeq SBS Kit v3:
HiSeq Rapid SBS Kit v2:
TruSeq Rapid SBS Kits:
Yes. See “Preparing SBS Reagents” in the HiSeq 2500 and HiSeq 1500 system guides for a list of how many SBS kits can be combined for supported run lengths.
No. HiSeq Rapid SBS v2 and TruSeq Rapid SBS reagents cannot be combined. Each set of reagents has been formulated specifically for use only with its respective flow cell and instrument run parameters. The cluster kits and flow cells are also paired with respective kit types and cannot be used interchangeably.
No. HiSeq SBS v4 and TruSeq SBS v3-HS reagents cannot be combined. Each set of reagents has been formulated specifically for use only with its respective flow cell and instrument run parameters. The cluster kits and flow cells are also paired with respective kit types and cannot be used interchangeably.
No. The same sequencing primer is distributed across both lanes of a rapid flow cell as part of the HiSeq 2500 on-instrument cluster generation workflow.
Any used flow cell that has been properly stored in buffer can be used for the instrument wash. Using the flow cell from a recently completed run to perform post-run and maintenance washes is often easiest.
Both v3 and v4 flow cells can be used to wash after high output modes. After Rapid Run mode, both HiSeq v2 and TruSeq flow cells can be used.
Yes. There are currently no plans to discontinue the sale of TruSeq v3 kits.
HiSeq v4 kits are designed for a walk-away workflow, so all reagents are loaded before the run is started. Splitting incorporation buffer for Read 2 is not necessary.
Yes. The incorporation reagent included in the HiSeq SBS Kit v4 appears more blue than the purple shade of the incorporation reagents included in the TruSeq SBS Kit v3.
You can store the rapid flow cell up to 24 hours after template hybridization and first extension. However, Illumina recommends that you start the sequencing run on the same day.
Store the reagent at the recommended temperature labeled on the box and protect the reagent from natural and fluorescent light.
Prepare rapid SBS reagents the night before or on the same day of use only. Do not store reagents longer than overnight. For use on the same day, store prepared reagents on ice in the original bottle with the cap tightened. For use the next day, store prepared reagents at 2°C to 8°C overnight.
Prepare HiSeq SBS Kit v4 reagents the night before or on the same day of use only. Do not store prepared reagents longer than overnight. For use on the same day, store prepared reagents on ice in the original bottle with the cap tightened. For use the next day, store prepared reagents at 2°C to 8°C overnight.
Yes, a cBot System is needed for cluster generation on any 8-lane high output flow cell, including HiSeq v4 and TruSeq v3.
The resynthesis step takes approximately 3 hours.
All HiSeq systems can perform the Tween 20 and ProClin 300 maintenance wash.
At the end of all rapid sequencing runs, the instrument flushes water back into the sample tube to clean out the lines and prevent drying. This flush is an automatic procedure and does not require user intervention. This procedure dilutes sample left in the tube. Therefore, any sample left in the tube should be discarded after the run.
Yes. Primer rehybridization for HiSeq v4 runs can rehyb the Read 1 primer, the Index 1 Read primer, or the Read 2 primer. Rehyb runs are performed on the HiSeq. For more information, see the HiSeq High Output Primer Rehybridization Reference Guide (part # 15050105).
Index reads for single-read libraries use 7-cycle reads. Illumina does not support 6-cycle index reads for single-indexed libraries.
For information on reagent loading and which primers to use for your library type, see the system guide for your instument. See the Indexed Sequencing Overview Guide for details on each indexing workflow.
Dual-indexed runs on HiSeq systems comprise 8 bp of index sequence rather than 6 bp plus a seventh for phasing calculations. For more information, see the system guide for your sequencing system.
It takes about 45 minutes to change from a high output (HiSeq v4 or TruSeq v3) to TruSeq Rapid mode. It takes about 3 hours to change from Rapid Run to a high output mode. Time spent for mode switching is in addition to instrument washing performed at the end of each run.
For setting up either a Rapid Run mode, TruSeq v3 mode, or HiSeq v4 mode on a HiSeq 2500 or HiSeq 1500 System, load the eight-port gasket in the back manifold position and the 10-port gasket in the front manifold position.
For a run on a HiSeq 2000 System, load eight-port gaskets in both the front and back positions.
If you are using the TruSeq Cluster Kit v3 and need 10-port gaskets for the front manifold, contact Illumina Technical Support.
Clustering takes slightly more than 2 hours. Clustering on a HiSeq v4 flow cell requires the updated cBot software (v2.0.16, or later) and requires v9.0 recipes.
Load 135 μl of the denatured and HT1 diluted library into wells 1 and 2 of an 8-tube strip. Then load the 8-tube strip into the cBot tube strip holder with wells 1 and 2 positioned toward the right side.
A maintenance wash is required every 10 days or when switching between high output and rapid modes. A water wash is required after each rapid run. After a high output run, you can choose between a water wash or a maintenance wash. Illumina recommends a maintenance wash.
Monthly replacement of wash bottles and tubes containing maintenance wash solution is typically sufficient. Wash bottles and tubes containing water are typically replaced every six months, although the water is replaced about every week.
After template generation is completed with cycle 5, HiSeq v4 runs might have a noticeable increase in the reported intensity by cycle in Sequencing Analysis Viewer. This increase occurs at this point because the reported intensities include only the clusters included in the final template. Before template generation, total intensity is reported.
The first time the HCS 2.2 is launched, a notification regarding instrument health data appears. This notification appears only once during the first initialization of the HCS, and will not appear again. Instrument health agreement and notification is always available from Menu | Options | Tools, where you can also get more information and turn the option on or off.
The option to designate a control lane was removed from HiSeq Control Software (HCS) v2.2. The software includes optimizations that improve the handling of low-diversity libraries, which eliminates the need for a control lane for matrix and phasing estimates.
From the Welcome screen, select Menu, then Tools. The Options menu includes the checkbox to turn on or off instrument health data. Select View Terms for more information about the instrument health option.
HCS v2.2 allows HiSeq instruments connected to the internet to send instrument health information to Illumina. This information is anonymous and includes only generic run metrics. This information is used by Illumina to help improve Illumina products. If you want to turn off this option or would like further information, see the Options menu in the HiSeq Control Software. You can find the Options menu under Menu, then Tools.
750 GB is required at the beginning of a run. The system assumes that data are transferred to the network copy of the run folder in real time. Therefore, 750 GB is the safe level to start a run. The software assumes that the run copies and deletes the files as they are processed, and that the connection to the network server can keep up with file transfer.
The option to save CIF files is available for all modes except HiSeq v4.
To upload data to BaseSpace from a HiSeq, a minimum upstream connection of 10 Mbit/second per instrument is needed. Network speed can be assessed by using free online tools such as www.speedtest.net.
Because run output has zipped BCL files, you must use the bcl2fastq
v1.8.4 conversion software to perform BCL to FASTQ conversion on your
local Linux analysis system. This tool is run on Linux and has the
same syntax, options, and functions (including demultiplexing) as the
configureBclToFastq.pl script of CASAVA. The only difference is that
it can be used to analyze either zipped or non-zipped BCL files.
If you send your data to BaseSpace Sequence Hub, BCL to FASTQ conversion and demultiplexing are performed automatically following the completion of the data upload.
No testing has been performed on the effects of local proxies on BaseSpace Sequence Hub access.
BaseSpace Sequence Hub uses SSL/https port 443 and the domains *.basespace.illumina.com and *.s3.amazonaws.com. Data streaming to BaseSpace Sequence Hub is encrypted using the AES256 standard. SSL is used for protection. For more information on encryption, see BaseSpace Security.
If local security policies must be modified to allow access to BaseSpace Sequence Hub, contact your IT representative.
The files that are sent to BaseSpace Sequence Hub are the InterOp folder, RunInfo.xml file, and RunParameters.xml file.
No, file directory structures are incompatible with MiSeq Reporter software. However, the TruSeq Amplicon App is available in BaseSpace Sequence Hub and can be used to analyze the TruSeq Amplicon Cancer Panel, the TruSight Myeloid Sequencing Panel, and the TruSeq Custom Amplicon panels.
If you choose to use BaseSpace Sequence Hub for run monitoring only and your samples are not indexed, a sample sheet is not required. If you want to use BaseSpace Sequence Hub for data storage and analysis, a sample sheet is required. The sample sheet can be in either HiSeq Analysis Software format or CASAVA format. When using BaseSpace Sequence Hub, combining indexed and non-indexed samples on a flow cell is not possible.
The bcl2fastq v1.8.4 conversion software is a separate piece of standalone software that is run on a Linux scientific computing system. The installer can be downloaded from the Illumina website. System requirements are outlined in the bcl2fastq User Guide (part # 15038058). If BCL files are zipped, then the use of the bcl2fastq v1.8.4 is required.
Run data can only be uploaded to BaseSpace if the BaseSpace option is selected during run setup in the HiSeq Control Software. See the HiSeq 2500 System User Guide (part # 15035786) for information on setting up a run with a connection to BaseSpace.
For more information on BaseSpace, or to set up a free BaseSpace account, see https://www.illumina.com/products/by-type/informatics-products/basespace-sequence-hub.html.
The BaseSpace Broker is designed to upload data to BaseSpace as soon as the data are generated on the HiSeq local drive. It will use as much bandwidth as is necessary to keep up with the data being produced. Under typical HiSeq run conditions, the upload of run data for storage and analysis will average less than 10Mbit/sec.
In most cases, throttling of the BaseSpace Broker data upload is not necessary. Throttling can be necessary if greater control over network bandwidth usage is required, such as sites where instruments share the network with other users or sites with limited upload speed. Throttling might be necessary in scenarios where the local network connectivity is temporarily lost and then restored. This interruption causes the BaseSpace Broker to suddenly consume more network bandwidth as it attempts to catch up with transfer of accumulated data. If no throttling is applied in such cases, the BaseSpace Broker might consume all available bandwidth on the network until the backlog of data are cleared. If throttling is applied and if the local network allows, Illumina recommends throttling to higher than the 10 Mbit/sec minimum specification. A recommended value of 20 Mbit/sec (approximately 3Mbytes/sec = 24Mbits/sec) allows the BaseSpace Broker enough bandwidth to recover, even if some delays in data transfer occur.
If throttling is needed, provide the following instructions to your local IT administrator:
Throttling of BaseSpace is performed on the HiSeq computer by application, rather than by IP address, as follows:
The Run Monitoring BaseSpace option allows you to remotely monitor a run in progress by logging in to your BaseSpace account. You need to select the Run Monitoring option during run setup. Then, log in to your BaseSpace account from anywhere and view your run in the BaseSpace version of Sequence Analysis Viewer (SAV).
Run monitoring with BaseSpace is selected during run setup.
No, .cif files cannot be analyzed with BaseSpace Sequence Hub. Additionally, it is not possible to output .cif files with HCS v2.2 on HiSeq v4 mode or Rapid Run mode with HiSeq v2 chemistry. The option to output .cif files is available in TruSeq v3 mode and Rapid Run mode with TruSeq chemistry.
Using CASAVA: To merge data from different flow cells (different runs), use the configureBuild script in CASAVA v1.8.2. First, align the data (samples) from each flow cell separately using configureAlignment. Then, include each sample directory as an input directory in the configureBuild.pl command line. Input directories are specified by the -id option, as detailed in the CASAVA v1.8.2 User Guide.
If you are using CASAVA, note that Illumina is discontinuing distribution of CASAVA software to better support new products available on BaseSpace. BaseSpace features analysis options for a large array of NGS applications.
Using BaseSpace: BaseSpace includes a Sample Merge function that allows you to merge data from a single sample originating from different flow cells. This merging is performed before alignment analysis of the sample data.
Scanning and analysis of a high output flow cell is performed in three swaths per surface on two surfaces per lane. Each swath is divided into 16 tiles. An 8-lane flow cell contains 768 tiles per flow cell.
See the Illumina whitepaper, Reducing Whole-Genome Data Storage Footprint.
When using BaseSpace, sample sheet format can follow either HiSeq Analysis format or CASAVA format. For runs that require demultiplexing with either bcl2fastq 1.8.4 or CASAVA, a CASAVA-formatted sample sheet is required. This format is described in the bcl2fastq 1.8.4 User Guide (part # 15038058) and the CASAVA User Guide (part # 15011196).
Sample sheets for rapid runs include information for two lanes, as compared to eight lanes included in a sample sheet for a high output run. Sample sheets for rapid runs can be generated manually, using Excel or a text editor.
If you are using BaseSpace for data storage and analysis, a sample sheet is required for both rapid runs and high output runs. If using BaseSpace only for run monitoring and you are not indexing, a sample sheet is not required.
You can use BaseSpace Apps to analyze data in BaseSpace Sequence Hub. Select the Apps tab in BaseSpace Sequence Hub to see available apps and descriptions.
Where *.cif files can be generated, you can use OLB v1.9.4.
No. File directory structures from a HiSeq System are incompatible with MiSeq Reporter software.
However, the TruSeq Amplicon App is available in BaseSpace Sequence Hub and can be used to analyze the this kit.
Scanning and analysis of a 2-lane rapid run flow cell creates two swaths per surface on two surfaces per lane. Each swath is divided into 16 tiles. For a 2-lane flow cell, there are a total of 128 tiles per flow cell.