Questions & Answers

See Denaturing and Diluting Libraries for the HiSeq and GAIIx (part # 15050107).

See the Cluster Densities Specifications technical bulletin on MyIllumina for recommendations.

See system specifications on the HiSeq 2500 Specifications page.

No. The HiScanSQ runs HCS 1.5.15 and is not upgradeable.

Any Hiseq system that has Rapid Run mode can run HiSeq Rapid Run v2 chemistry.

System	Serial Number
HiSeq 2500	SN# D00101 or higher
HiSeq 1500	SN# C00101 or higher
HiSeq 2000	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 Overview tab.

All validated Illumina library prep methods are supported to run on the HiSeq. However, make sure that you match samples with the desired output to maximize workflow efficiency.

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 new HiSeq v4 reagent kits now 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 v4 SBS kits:

• The 250-cycle kit contains enough reagents for 275 cycles.
• The 50-cycle kit contains enough reagents for 75 cycles.

TruSeq v3 SBS kits:

• The 200-cycle kit contains reagents for 209 total cycles.
• The 50-cycle kit contains reagents for 58 total cycles.

HiSeq Rapid v2 SBS kits:

• The 500-cycle kit contains reagents for 525 total cycles.
• The 200-cycle kit contains reagents for 225 total cycles.
• The 50-cycle kit contains reagents for 74 total cycles.

TruSeq Rapid SBS kits:

• The 200-cycle kit contains reagents for 225 total cycles.
• The 50-cycle kit contains reagents for 74 total cycles.

No. Rapid run SBS reagents and high output 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 combined.

Yes. See “Preparing SBS Reagents” in the HiSeq 2500 and HiSeq 1500 user guides for a list of how many SBS kits can be combined for supported run lengths.

• HiSeq Rapid SBS Kit v2 - HS (50 cycle) - Cat # FC-402-4022
• HiSeq Rapid SBS Kit v2 - HS (200 cycle) - Cat # FC-402-4021
• HiSeq Rapid SBS Kit v2 - HS (500 cycle) - Cat # FC-402-4023
• HiSeq Rapid PE Cluster Kit v2 - Cat # PE-402-4002
• HiSeq Rapid SR Cluster Kit v2 - Cat # GD-402-4002
• HiSeq Rapid Duo cBot Sample Loading Kit - Cat # CT-403-2001
• HiSeq Rapid Rehybridization Kit - Cat # GD-404-1001

No. HiSeq 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 v4 and TruSeq v3 high output 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. 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 previously used flow cell that has been stored properly in buffer can be used for the instrument wash. It is easiest to use the flow cell from the recently completed run to perform post-run and maintenance washes. 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, which means that all reagents are loaded before starting the run. Splitting incorporation buffer for Read 2 is no longer necessary.

Yes. The incorporation reagent included in the HiSeq v4 SBS kit appears bluer, instead of the purple color of incorporation reagents included in the TruSeq v3 SBS kit.

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.

Yes. You can continue to use TruSeq v3 reagents as well as HiSeq v4 reagents. The chemistry type is specified when you select the mode before starting the run.

The HiSeq v4 chemistry includes a pre-mixed incorporation reagent similar to the TruSeq Rapid kit.

No. All eligible HiSeq 1500 and HiSeq 2000 systems only need the FPGA and software upgrades to use the HiSeq v4 reagents.

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 v4 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.

Maintenance wash solution can be stored for up to 30 days at room temperature. In that 30-day period, the solution can be used up to three times. For the first use, assign each bottle and tube to a reagent rack position. Maintain those positions for the second and third uses to prevent cross-contamination.

Yes, a cBot 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.

No. Priming is integrated into the HCS workflow for Rapid Run mode.

Yes. Priming SBS reagents is required. The control software prompts for this step, which takes about 15 minutes.

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 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.

See the appropriate HiSeq instrument user guide for details on the loading of reagents with different workflows and which primers you need to use for your library type.

• Read 1: An indexed Read 1 follows the standard Read 1 protocol using reagents provided in the TruSeq SBS Kit. The Read 1 sequencing primer is annealed to the template strand during the cluster generation process on the cBot (HP6 or HP10).
• Index Read 1 (i7): Following the completion of Read 1, the run proceeds to Index Read preparation. The Read 1 product is removed and the Index 1 (i7) sequencing primer (HP8 or HP12) is annealed to the same template strand. The run proceeds through 8 cycles of sequencing to read the Index 1 (i7).
• Index Read 2 (i5): For paired-end flow cells, the Index 1 (i7) Read product is removed and the template anneals to the grafted P5 primer on the surface of the flow cell. The run proceeds through an additional 7 chemistry-only cycles (no images are taken) followed by 8 cycles of sequencing to read Index 2 (i5).
  For single-read flow cells, the Index 1 (i7) Read product is removed and the Index 2 (i5) sequencing primer (HP9) is annealed to the same template strand. The run proceeds through 8 cycles of sequencing to read the Index 2 (i5).
• Paired End Resynthesis: Read 2 re-synthesis for dual-indexed paired-end sequencing uses reagents provided in the TruSeq PE Cluster Kit.
• Read 2: Read 2 follows the standard paired-end sequencing protocol using standard SBS reagents.

Dual-indexed runs on the HiSeq comprise 8 bp of index sequence rather than 6 bp plus a seventh for phasing calculations. For more information, see the user guide for your sequencing instrument.

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, load the 8-port gasket in the back manifold position and load the 10-port gasket in the front manifold position. For a run on a HiSeq 2000, load 8-port gaskets in both the front and back positions.

If you use 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 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, making sure to position wells 1 and 2 towards the right side.

A water wash and maintenance wash each take approximately 1 hour. The maintenance wash protocol has changed to consist of only one step, washing the system with Tween 20 and ProClin 300, so there is no need to return to the instrument to reload wash solution or water.

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 6 months, although the water is replaced about every week.

For HiSeq v4 runs, there might be a noticeable increase in the reported intensity by cycle in SAV after template generation has completed after cycle 5. 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, you will see a notification regarding instrument health data. This notification appears only once during the first initialization of the HCS, and will not appear again. Note that in pre-release, early access versions of HCS 2.0, this notification does not appear. However, 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 has been removed in 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.

The files uploaded as instrument health data are RunInfo.xml, RunParameters.xml, RTAComplete.txt, InterOp files, and RTAConfiguration.xml.

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.

Yes. HCS 2.2 is available for all customers.

Saving the standard set of files without thumbnails results in a run folder that is approximately 200–500 GB. If standard thumbnails are saved, approximately an additional 300 GB is required. Approximately 1 TB is storage used by the alignment folder and related folders.

This amount of free space 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.

In HiSeq v4 mode .cif files cannot be saved. The option to save .cif files is available in other modes.

If you currently use CASAVA, you can analyze HiSeq v4 data with CASAVA. You need to use the bcl2fastq v1.8.4 conversion software in place of the configureBclToFastq component of CASAVA.

For HiSeq v4 runs, perform alignment of data from each separately, and then merge the data at the configureBuild step.

If you are not 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.

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.

Two data compression options are zipping of BCL files and binning of Q-scores. Other run folder files are unchanged. These options are available during run setup in HCS v2.2. If you are using BaseSpace for data storage and analysis, BCL files are zipped automatically. Due to the size of the run folder with the extra cycles and shorter run durations, zipped BCL files are required for HiSeq v4 runs. This setting cannot be turned off. You can select or deselect Q-score binning depending on your preference.

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, 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 access.

Contact your local IT administrator if local security policies have to be modified to allow access to BaseSpace. BaseSpace uses SSL/https port 443 and the domains *.basespace.illumina.com and *.s3.amazonaws.com. Data streaming to BaseSpace is encrypted using the AES256 standard and uses SSL for protection. More information on encryption can be found at http://blog.basespace.illumina.com/2011/12/13/basespace-security/

If you are using CASAVA, you can analyze rapid run data with CASAVA. If the zip BCL files option was chosen during run set up, you will need to use the bcl2fastq converter in place of the configureBclToFastq component of CASAVA. For rapid runs, you will align data from each flow cell separately and then merge the data at the configureBuild step.

If you are not 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.

The files that are sent to BaseSpace 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 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 only for run monitoring and your samples are not indexed, a sample sheet is not required. If you want to use BaseSpace 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, 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://basespace.illumina.com/home/index.

Yes. To convert zipped .bcl files, use bcl2fastq v1.8.4. This version can also convert non-zipped .bcl files.

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:

1. In Windows, open a cmd window and open the Local Policy Editor. Run the program gpedit.msc.
2. Expand the Computer Configuration / Windows Settings nodes.
3. Select Policy-based QoS.
4. Right-click Create new policy.
5. Enter a name, such as Limit BaseSpace upload.
6. Clear the Specify DSCP value.
7. Select Specify Outbound Throttle Rate and enter 3 MBps (3 Mbytes/sec, or 24Mbit/sec), which is sufficient to allow data transfer to catch up.
8. Click Next.
9. Select Only applications with this executable name and enter Illumina.BaseSpace.Broker.exe.
10. This policy applies to any source IP and target IP addresses. Click Next.
11. This policy applies to all ports and protocols. Click Finish.

If you are using CASAVA, it is compatible. However, bcl2fastq v1.8.4 must be used in place of the configureBcl2fastq step in CASAVA. The output of bcl2fastq v1.8.4 is in the fastq.gz file format organized into project and sample directories as specified in the sample sheet. This output is compatible with the configureAlignment and configureBuild components of CASAVA v1.8.2. The sample sheet format required for bcl2fastq v1.8.4 is equivalent to CASAVA v1.8.2 sample sheet format, and is described in the bcl2fastq v1.8.4 User Guide (part # 15038058).

If you are not 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.

To remove the least reliable data from the analysis results, often derived from overlapping clusters, raw data are filtered to remove any reads that do not meet the overall quality as measured by the Illumina chastity filter. The chastity of a base call is calculated as the ratio of the brightest intensity divided by the sum of the brightest and second brightest intensities.

Clusters passing filter are represented by PF in analysis reports. Clusters pass filter if no more than one base call in the first 25 cycles has a chastity of < 0.6.

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. 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 on page 100 of the CASAVA v1.8.2 User Guide (Rev C).

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 3 swaths per surface on 2 surfaces per lane. Each swath is divided into 16 tiles. Therefore, an 8-lane flow cell contains 768 tiles per flow cell.

See the Illumina whitepaper, Reducing Whole-Genome Data Storage Footprint, which is available on the Illumina website.

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 Illumina’s BaseSpace Core Apps to analysis data in BaseSpace. Available apps include BWA and Isaac WGS, RNA Seq, Enrichment, and Tumor-Normal analysis. Please see https://basespace.illumina.com/apps for more details, including descriptions of each app.

Where .cif files can be generated, you can use OLB 1.9.4.

No. File directory structures from a HiSeq system are incompatible with MiSeq Reporter software.

However, the TruSeq Amplicon App is available in BaseSpace and can be used to analyze the this kit.

Scanning and analysis of a 2-lane rapid run flow cell creates 2 swaths per surface on 2 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.