HiSeq 2000 FAQs

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  • Libraries

  • 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.

    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.

  • Workflow and Washing

  • 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.

    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).

    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 two rapid runs on side A and B, and want to set up only one 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.

    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.

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

    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.

    • The TruSeq controls were not designed to be a qualitative metric of the efficiency of the various steps in the library 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.

    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.

    The recommended maximum cluster density is 750,000–850,000 clusters/mm² when using TruSeq v3 clustering and sequencing reagents.

    Flow cells are designed for a single use. All eight lanes must be used at the same time, either 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.

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

    Yes. Illumina recommends using a PhiX control lane when sequencing ChIP-Seq libraries. Samples that contain genomes with high AT or GC content (less than 40% or greater than 60%) require a dedicated PhiX control lane for cross-talk and phasing calculations. For more information, see the  Using a PhiX Control for HiSeq Sequencing Runs technical note.

    A PhiX spike-in employs a small amount of PhiX control library in the same lane as an experimental library. The spike-in allows real-time quality metrics as the PhiX is analyzed during the run.

    A PhiX spike-in is not recommended for sequencing a genome with high similarity to the PhiX genome. It does not allow for normalization of data in that lane as per a control lane.

    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.

    Illumina provides 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. 

    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

    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.

    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 Illumina Technical Support if you need assistance creating custom recipes.

    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.

    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.

  • Software

  • 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.

    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.

    The instrument control computer is a computational engine performing real-time analysis of data. To avoid loss of data and other adverse effects, do not install any additional software except anti-virus software.

    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.

    The ARM9BoardSerialPort (ARM9CHEM): timed out waiting error 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 System 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.

    The HiSeq control computer employs 64-bit Windows Vista.

    The error message “Must have at least one valid ETF to normalize/correct the failed ones” indicates a lack of fluorescence from the flow cell. To find focus at the start of a run, the software uses ETF. ETF is a focusing method that reads fluorescence from clusters on the flow cell. Before a run can start, ETF must find fluorescence in at least one lane of the flow cell.

    To correct the problem, perform a primer rehybridization. Reannealing 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 the library type. When you reload the flow cell onto the HiSeq system, confirm that the fluidics system is functioning correctly.

    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.

  • Analysis

  • Image analysis occurs in real time, phasing estimates and base calling start after cycle 12, and base calling and quality scoring starts after cycle 25.

    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.

    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:

    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.

    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, 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.

    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.

    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.

    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. 

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

    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.

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

    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 is a compact way to communicate 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.

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

    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.