Questions & Answers

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  • Where can I find information about library input for the various HiSeq chemistries?

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

  • How much time does a standard HiSeq run take?

    See system specifications on the HiSeq 2500 Specifications page.

  • Is the new HCS compatible with HiScanSQ?

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

  • Which systems are eligible for the HiSeq v4 upgrade?


    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.

  • Can I upgrade my HiSeq 2500 or earlier model to a HiSeq 4000?

    No. An upgrade package (catalog # SY-401-4002) is available for HiSeq 3000 only.


  • Can I use only 1 of the indexes of a dual-indexed library?

    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.

  • Are TruSeq Custom Amplicon, TruSeq Amplicon Cancer Panel, and Nextera XT applications supported to run on the HiSeq?

    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.

  • Can I process a sample with low sequence diversity?

    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.

  • Are my existing Illumina sequencing libraries compatible with the HiSeq?

    Yes. Single read and paired end libraries for RNA and DNA applications are compatible with the HiSeq.

  • Do homopolymers and repetitive DNA regions impact sequencing efficiency?

    Homopolymers do not impact sequencing. The number of uniquely alignable reads is a function of the repeat content, so this will have an impact on productivity. With longer reads and paired-end sequencing, this may be less of an issue.

  • Do I need to re-titrate my samples for use with flow cell v3?

    Yes. The new cluster protocol and wider lanes on flow cell v3 make it imperative to re-titrate your samples to optimize cluster densities for higher throughput. qPCR is highly recommended.

Reagents and Flow Cells

  • How many cycles of sequencing are possible with SBS kits?

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

  • Can HiSeq v4 and TruSeq v3 high output SBS reagents be combined?

    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.

  • Can I combine HiSeq v4 SBS kits to achieve read lengths longer than what is provided in a kit?

    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.

  • Can I split my 200-cycle kit for shorter runs?

    Yes. You can split the 200-cycle SBS Kit into two equal volumes suitable for up to 101 cycles each. See the TruSeq SBS Kit Reagent Preparation Guide (200 Cycles) for instructions and storage requirements. If you need smaller volumes for shorter runs, Illumina recommends using the TruSeq SBS Kit (50 Cycles).

  • Which flow cell can I use to perform an instrument wash on my HiSeq system?

    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.

  • How long can maintenance wash solution be stored after initial preparation?

    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.

  • Will TruSeq v3 reagents continue to be sold?

    Yes. There are currently no plans to discontinue the sale of TruSeq v3 kits.

  • When using HiSeq v4 reagents, do you have to change reagents during the run as with TruSeq v3 reagents?

    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.

  • How many additional cycles of SBS reagents do I need to calculate into the run for sequencing dual-indexed libraries?

    For dual index paired-end runs, there are 23 additional cycles (index & chemistry only).
    For dual-index single-read runs, there are 16 additional cycles of indexing.
    For information about the number of SBS kits required on the HiSeq, HiScanSQ, or GAIIx, see the user guide for your instrument guide.

  • Why is my SBS v3 reagent waste brown?

    Due to an interaction with one of the v3 reagents, SRE, waste appears dark brown in color and has a stronger odor. This is normal. The change in waste color does not impact performance and is not toxic. You might see a discoloration on the funnel caps and SRE sipper line. Any spills will be dark brown in color as well.

  • Is the incorporation reagent supposed to be a different color?

    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.

  • Are there any special requirements for the master-mixed HiSeq v4 incorporation reagent?

    Store the reagent at the recommended temperature labeled on the box and protect the reagent from natural and fluorescent light.

  • Can TruSeq v3 reagents be used on a system that has been upgraded for HiSeq v4?

    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.

  • Why are the polymerase and FFNs missing from the HiSeq SBS Kit v4?

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

  • Does a HiSeq 1500 or HiSeq 2000 need to be upgraded to a HiSeq 2500 to run the HiSeq v4 chemistry?

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

  • How long can HiSeq v4 reagents be stored after initial preparation?

    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.

  • What types of SBS kits are available for the HiSeq?

    Illumina currently offers two SBS kits for the HiSeq: a 200-cycle kit and a 50-cycle kit. Both kits contain the same formulations and only differ in volume.

  • Are HiSeq/HiScanSQ flow cells compatible with the Cluster Station?

    No. The HiSeq flow cell, also used on the HiScanSQ, must be clustered on a cBot equipped with an adapter plate suitable for the larger format of the flow cell.

  • Will I have enough reagents in my 200-Cycle SBS Kit to perform a 2x100 cycle run with indexing?

    Yes. The TruSeq SBS Kit (200 Cycles) contains sufficient reagents for 209 cycles of sequencing, which covers 101 cycles for Read 1, 7 cycles for the Index Read, and 101 cycles for Read 2.

  • Can I use the same reagents on the HiSeq that I use on my Genome Analyzer?

    No. Cluster Kits and SBS Kits for the HiSeq are not equivalent or compatible with the Genome Analyzer. Likewise, Cluster Kits and SBS Kits for the Genome Analyzer are not equivalent or compatible with the HiSeq.

  • Has the tile layout or numbering changed due the wider lanes on Flow Cell v3?

    No, the tile numbering is unchanged from the format introduced in HCS v1.3. However, when using Flow Cell v3, the tile numbering reflects the three-swath imaging pattern, where a 3 in the tile number represents the third swath.

  • Do I need oil to load a flow cell on the HiSeq or HiScanSQ?

    No. The HiSeq and HiScanSQ do not require immersion oil to properly load a flow cell in the way that the Genome Analyzer does. Instead, the flow cell is held in place by a vacuum, which removes air and replaces the need for immersion oil.

Workflow and Washing

  • Do I need a cBot instrument to run sequencing v4 flow cells on my HiSeq?

    Yes, a cBot is needed for cluster generation on any 8-lane high output flow cell, including HiSeq v4 and TruSeq v3.

  • How long does the resynthesis step take on a HiSeq?

    The resynthesis step takes approximately 3 hours.

  • Is there a rehyb protocol available for HiSeq v4 runs?

    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).

  • Can I switch modes on only 1 side of my HiSeq system?

    A single side of the instrument can be switched from one mode to the other. However, after a run begins on one side, a run cannot be started on the other side unless it is the same mode. Perform mode switching procedures on the side that you intend to sequence on in the new mode. For example, if you have completed 2 rapid runs on side A and B, and want to set up only 1 high output flow cell on side A, change the mode for only side A.

    A high output run on side B cannot be performed until a mode change is complete on side B. For efficiency and the most run flexibility, perform mode switching on both sides of the system at the same time.

  • Can I run my HiSeq system with different run modes on each side?

    No, only runs of the same mode can be performed simultaneously. If you run TruSeq v3 mode on side A, then you must run TruSeq v3 mode on side B. The same is true for running HiSeq v4 mode. For Rapid Run mode, you can perform a rapid run on both sides using TruSeq Rapid kits on one side and HiSeq Rapid v2 kits on the other.

  • Do I have to run two flow cells at the same time on a HiSeq 2000?

    No. You can choose to run only one flow cell at a time.

  • How many cycles should be used during the Index Read for single-indexed libraries?

    Index reads for single-read libraries use 7 cycle reads. Illumina does not support 6 cycle index reads for single-indexed libraries.

  • What is the workflow for dual indexing?

    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.

  • How much additional time will be added to the current sequencing run time on HiSeq to support dual indexing?

    When using v3 SBS reagents and the v3 flow cell on HiSeq, each additional index cycle is approximately 53 minutes per cycle (with 16 total indexing cycles) plus ~2 hours for the seven chemistry-only cycles for the PE workflow, resulting in ~16 hours of additional time for dual indexing on HiSeq.

  • For dual-indexed libraries, how many cycles are performed for index reads?

    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.

  • Do I need to prime SBS reagents in HiSeq v4 run mode?

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

  • What are the workflow changes for analysis/demultiplexing on the MiSeq, HiSeq, and GA for dual-indexed libraries compared to single-indexed libraries?

    There are no changes for MiSeq analysis. HiSeq and GA data require an upgrade to CASAVA 1.8.2 to demultiplex dual-indexed libraries. It is also recommended to upgrade to SAV 1.8.4 or higher to use the new Index tab for real time demultiplexing information.

  • How do I know if the TruSeq controls have worked?

    • The TruSeq controls were not designed to be a qualitative metric of the efficiency of the various steps in the sample prep but rather an indicator of whether or not the step worked or not. Actual counts of the controls will vary based on sample type, input, etc.
    • In SAV (Sequencing Analysis Viewer), if you see counts for a particular control, this mean that this step has worked whereas if you see no counts (dark blue color), this step has likely failed. If the band has been cut across multiple insert sizes, you may see counts at different size increments.

  • What is the alternative SBS workflow?

    TruSeq SBS v3 reagents enable an alternative workflow for loading all SBS reagents at the start of a 2x101-cycle sequencing run for both Read 1 and Read 2. Using this workflow might result in a slight increase in phasing in Read 2, which should not result in a decrease in quality.

  • What is the optimal cluster density on the HiSeq?

    The recommended maximum cluster density is 750,000-850,000 clusters/mm² when using Illumina's v3 cluster generation and sequencing reagents in combination with HCS v1.4.

  • Is a sample sheet/library sheet optional or mandatory for sequencing runs and analysis?

    For runs on the HiSeq, HiScanSQ, or GAIIx, creating and loading a sample sheet at the start of the run is optional. However, using a sample sheet allows you to view data shown on the indexing tab in the Sequencing Analysis Viewer (SAV) during the run. If you do not load a sample sheet at the start of a run in HCS, you will not be able to view indexing data in SAV. When analyzing indexed samples using CASAVA v1.8.2, a sample sheet is required. MiSeq runs require a sample sheet when setting up the run in MCS.

    Illumina recommends that you create the sample sheet using the Illumina Experiment Manager (IEM) prior to performing library prep in order to confirm appropriate index combinations.

  • How many samples can be run on one flow cell?

    Flow cells are designed for single-use. All eight lanes must be used at the same time. They can be used for the same sample or for dif­ferent samples. You can run eight samples at a time without multiplexing. With multiplexing, you can increase throughput to up to 12 samples per lane or up to 96 samples per flow cell.

  • Do I need to use a cBot to cluster a HiSeq/HiScanSQ flow cell?

    Yes. The HiSeq flow cell, also used on the HiScanSQ, requires the use of a cBot for clustering on the flow cell prior to sequencing.

  • How long does it take to complete a water wash and maintenance wash?

    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.

  • What is a PhiX spike-in?

    A PhiX spike-in employs a small amount of PhiX control in the same lane as a sample. This allows real time quality metrics as the PhiX is analyzed during the run. This is not recommended for sequencing a genome with high similarity to the PhiX genome, and does not allow for normalization of data in that lane as per a control lane.

  • How many flow cells can I run on the HiSeq?

    The HiSeq 2000 is a dual flow cell system, which allows you to run two flow cells simultaneously. The HiSeq 1000 is a single flow cell system.

  • Can I monitor my HiSeq run remotely?

    Illumina provides the Sequencing Analysis Viewer (SAV) software that can be run on a Windows PC to remotely monitor your run. The software does not allow any control over the run and requires that the PC is connected to the analysis server over the network. Another application you can use to monitor your run is SeqMonitor, which allows you to monitor your run using your iPhone or iPad.

  • What are the expected wash volumes following a HiSeq maintenance wash?

    The HiSeq maintenance wash has three steps: a water wash, followed by a NaOH wash, and then a final water wash. You can expect the following delivered volumes from the eight lines of waste tubing:

    • First water wash-SBS positions only, 32 ml; SBS and PE positions, 72 ml
    • NaOH wash-SBS positions only, 16 ml; SBS and PE positions, 36 ml
    • Final water wash-SBS positions only, 32 ml; SBS and PE positions, 72 ml

  • If I run two flow cells at the same time, do they need to be identical in setup?

    No. You can start each flow cell independently from the other. Each flow cell can have a different number of reads and cycles.

  • Is initial cycle indexing possible on the HiSeq?

    No, the system does not support an initial cycle indexing method. To ensure the highest quality data, Illumina recommends and supports a separate indexing read for multiplexed samples.

  • How long does the cBot take to perform clustering on a HiSeq v4 flow cell?

    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.

  • Can I customize a HiSeq recipe?

    With HCS v1.3 and later, you can customize a recipe to contain any number of reads. Reads can be indexed or non-indexed. However, Illumina does not guarantee the performance of custom recipes. Contact your Illumina Technical Support if you need assistance creating custom recipes.

  • How often do I need to wash my HiSeq system?

    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.

  • How often do wash bottles and tubes need to be renewed?

    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.

Is a sample sheet/library sheet optional or mandatory for sequencing runs and analysis?

For runs on the HiSeq, HiScanSQ, or GAIIx, creating and loading a sample sheet at the start of the run is optional. However, using a sample sheet allows you to view data shown on the indexing tab in the Sequencing Analysis Viewer (SAV) during the run. If you do not load a sample sheet at the start of a run in HCS, you will not be able to view indexing data in SAV. When analyzing indexed samples using CASAVA v1.8.2, a sample sheet is required. MiSeq runs require a sample sheet when setting up the run in MCS.

Illumina recommends that you create the sample sheet using the Illumina Experiment Manager (IEM) prior to performing library prep in order to confirm appropriate index combinations.


  • What is the intensity spike after template generation?

    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.

  • How is the instrument health upload option communicated to customers?

    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.

  • Where is the control lane feature?

    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.

  • Exactly which files are uploaded as instrument health data?

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

  • How do I change the instrument health upload option?

    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.

  • What is instrument health data, and where can I find more information about the option to send instrument health data to Illumina?

    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.

  • Is HCS v2.2 available for non-upgradeable systems?

    Yes. HCS 2.2 is available for all customers.

  • How large is the run folder?

    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.

  • How do I select the right number of index cycles and chemistry for sequencing dual-indexed libraries in HCS 1.5?

    In order to perform dual-index sequencing in HCS 1.5, select the TruSeq Dual Index Sequencing Primer Box from the Index chemistry drop down menu on the recipe screen. This selection enables the use of the required chemistry for sequencing dual-indexed libraries, and must be used for sequencing any dual-indexed libraries (Nextera or TruSeq HT) regardless of which sequencing primers you will use for your run. Selecting any other setting will result in less than an eight-cycle index read.

  • What programs can I install on the HiSeq instrument computer?

    The instrument computer is a computational engine performing real time analysis of data. To avoid loss of data and other adverse effects, Illumina does not recommend installing any additional software with the exception of anti-virus software.

  • Can I expect to see any changes in intensity with HCS v1.4 or HCS v1.5?

    Intensity for the G channel is expected to be lower, but the rate of decay is much slower due to one of the new reagents in the TruSeq SBS Kit v3 - HS called SRE. Therefore, your data quality will not be impacted.

  • Can I save images from a run on the HiSeq or HiScanSQ?

    No. Images are deleted automatically after they have been processed.

  • How are images taken on the HiSeq or HiScanSQ?

    Images are taken using a time delayed integration (TDI) line scanning optical system with four CCD sensors. The TDI line scanning system greatly increases throughput by maximizing camera utilization.

  • I got a warning message in HCS about the ARM9BoardSerialPort. What does this mean and what should I do?

    The warning message "ARM9BoardSerialPort (ARM9CHEM): timed out waiting" indicates that an ARM9 communication time out has occurred. The ARM9 board is one of many components that communicate between the HiSeq and instrument computer.  Messages related to an ARM9 time out are not necessarily indicative of a hardware issue, and do not impact the run or data quality.

    If this message appears repeatedly, perform a normal stop on the current run, shut down the HCS/RTA software, and then power cycle the HiSeq and instrument computer to reestablish communication between the systems. Launch HCS and resume your run.  Continue to monitor your run to make sure that the issue is resolved. If it appears that the run data is affected, contact Illumina Technical Support for further assistance.

  • I got a warning message in HCS about the Tdi Scan. What does this mean and what should I do?

    TDI Scan warning messages indicate an issue with image acquisition and storage; however, the system will automatically retry image capture to self-correct. TdiScan messages usually have no effect on the run other than slightly extended cycle times, and do not affect the run data as images are re-captured before continuing.

    In the rare event that the retry threshold is exceeded, one imaging swath is skipped for one cycle. If this message occurs frequently, contact Illumina Technical Support for assistance.

  • Can I save intensities from a run on the HiSeq or HiScanSQ?

    Yes. The *.cif files can be saved and transferred to the analysis server over the network.

  • What operating system is used on the instrument computer?

    The HiSeq instrument computer employs 64-bit Windows Vista.

  • When I started my run, I got the HCS error message, “Must have at least one valid ETF to normalize/correct the failed ones.” What does this mean and what should I do?

    This error message indicates a lack of fluorescence on the flow cell. To find focus at the start of a run, the software uses ETF, which is a focusing method that reads fluorescence from clusters on the flow cell. ETF must find fluorescence in at least one lane of the flow cell before the run can begin.

    To correct this problem, perform a primer rehybridization. Re-annealing the Read 1 sequencing primer usually increases the fluorescence if clusters are present on the flow cell. Additionally, check the cBot plate to make sure that all reagents were delivered correctly and that the sequencing primer was appropriate for your library types. When you reload the flow cell on the HiSeq, confirm that the fluidics system is functioning correctly. If a primer rehybridization does not resolve the issue, contact Illumina Technical Support for further assistance.

  • I received a warning message “need at least 750 GB space in local server” when starting a run. Is the run folder on the local computer 750 GB in size?

    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.


  • When is analysis of a sequencing run performed?

    Image analysis occurs in real time, phasing estimates and base calling begin occur after cycle 12, and base call quality scoring occurs after cycle 25.

  • Can I save .cif files in HiSeq v4 mode?

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

  • Can I analyze v4 data with CASAVA?

    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.

  • How do I make sure that my HiSeq is ready to send data to BaseSpace?

    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

  • What data compression options are available for high output runs using HCS v2.2?

    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.

  • How do data compression options in HCS v2.2 change data analysis or data handling?

    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 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.

  • Do local proxies affect BaseSpace?

    No testing has been performed on the effects of local proxies on BaseSpace access.

  • What ports, domains, and encryption does BaseSpace use?

    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 * and * Data streaming to BaseSpace is encrypted using the AES256 standard and uses SSL for protection. More information on encryption can be found at

  • If I use BaseSpace for run monitoring only, what files are sent to BaseSpace?

    The files that are sent to BaseSpace are the InterOp folder, RunInfo.xml file, and RunParameters.xml file.

  • Does BaseSpace require a sample sheet?

    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.

  • What is bcl2fastq?

    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.

  • How do I send run data to BaseSpace?

    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

  • Do I need a specific version of the bcl2fastq software package?

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

  • How do I know if I need to throttle BaseSpace, and how do I apply throttling?

    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.

  • Is the output of bcl2fastq compatible with CASAVA v1.8.2?

    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.

  • What criteria determine clusters passing filter on Illumina sequencing systems?

    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.

  • How do I use BaseSpace for run monitoring?

    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).

  • How do I use BaseSpace for run monitoring only, such as SAV functionality in BaseSpace?

    Run monitoring with BaseSpace is selected during run setup.

  • Can I use MiSeq Reporter software to analyze TruSight DNA Amplicon data from a NextSeq or HiSeq system?

    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.

  • Can I analyze .cif files with BaseSpace?

    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.

  • How do I merge data from 2 flow cells?

    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 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.

  • How many swaths and tiles are on an 8-lane high output flow cell?

    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.

  • How many swaths and tiles are on a 2-lane rapid run flow cell?

    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.

  • Where can I find technical information describing HiSeq v4 data compression options?

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

  • How do I format my sample sheet?

    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.

  • If I send my data to BaseSpace, what analysis options do I have?

    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 for more details, including descriptions of each app.

  • What are the network requirements for data transfer from the HiSeq to a server?

    You need a one gigabit connection per instrument between the instrument computer and the server. For more information, see the HiSeq System Site Preparation Guide.

  • What are the storage requirements using HCS v1.4 and Flow Cell v3?

    Storage requirements for raw data are approximately 60% greater than current runs based on additional swath data and increased cluster density.

  • What are the sizes of data files from the HiSeq?

    For a dual flow cell 2x101 cycle run (200 Gb) on the HiSeq 2000 using HCS v1.3 and prior, you can expect 2 TB of intensity data (optionally transferred to a server), 250 GB of base call and quality score information, and 1.2 TB of space for alignment output not including 6 TB of disk space used for temporary files removed before completion of alignment. Using HCS v1.4 and Flow Cell v3, storage requirements for raw data are approximately 60% greater than current runs based on additional swath data and increased cluster density.

  • Can thumbnail images be reanalyzed on the HiSeq or HiScanSQ?

    No. Thumbnail images are for visual inspection only to help diagnose problems with a run. They are not suitable for reanalysis.

  • What kind of quality scoring method does Illumina use?

    A quality score (or Q-score) is a  prediction of the probability of an incorrect base call. Based on the Phred scale, the Q-score serves as a compact way to communicate very small error probabilities. Given a base call, X, the probability that X is not true, P(~X), is expressed by a quality score, Q(X), according to the relationship:
    Q(X) = -10 log10(P(~X))
    where P(~X) is the estimated probability of the base call being wrong.

    A quality score of 10 indicates an error probability of 0.1, a quality score of 20  indicates an error probability of 0.01, a quality score of 30 indicates an error probability of 0.001, and so on.

    During analysis, base call quality scores are written to FASTQ files in an encoded compact form, which uses only one byte per quality value. This method represents the quality score with an ASCII code equal to the value + 33.

  • What is deconvolution?

    It is the ability to distinguish between two or more clusters that are in close proximity to each other.

  • What software do I use to analyze .cif files generated with HCS 2.x?

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

  • Can I use MiSeq Reporter software to analyze data from a HiSeq system?

    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.