HiSeq 1500 FAQs

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  • Libraries


  • 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 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.

  • Analysis


  • The option to save CIF files is available for all modes except HiSeq v4.

    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 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.

    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 Sequence Hub are the InterOp folder, RunInfo.xml file, and RunParameters.xml file.

    No, file directory structures are incompatible with MiSeq Reporter software. However, the TruSeq Amplicon App is available in BaseSpace Sequence Hub and can be used to analyze the TruSeq Amplicon Cancer Panel, the TruSight Myeloid Sequencing Panel, and the TruSeq Custom Amplicon panels.

    If you choose to use BaseSpace Sequence Hub for run monitoring only and your samples are not indexed, a sample sheet is not required. If you want to use BaseSpace Sequence Hub for data storage and analysis, a sample sheet is required. The sample sheet can be in either HiSeq Analysis Software format or CASAVA format. When using BaseSpace Sequence Hub, combining indexed and non-indexed samples on a flow cell is not possible.

    The bcl2fastq v1.8.4 conversion software is a separate piece of standalone software that is run on a Linux scientific computing system. The installer can be downloaded from the Illumina website. System requirements are outlined in the bcl2fastq User Guide (part # 15038058). If BCL files are zipped, then the use of the bcl2fastq v1.8.4 is required.

    Run data can only be uploaded to BaseSpace if the BaseSpace option is selected during run setup in the HiSeq Control Software. See the HiSeq 2500 System User Guide (part # 15035786) for information on setting up a run with a connection to BaseSpace.

    For more information on BaseSpace, or to set up a free BaseSpace account, see https://www.illumina.com/products/by-type/informatics-products/basespace-sequence-hub.html.

    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 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.

    When using BaseSpace, sample sheet format can follow either HiSeq Analysis format or CASAVA format. For runs that require demultiplexing with either bcl2fastq 1.8.4 or CASAVA, a CASAVA-formatted sample sheet is required. This format is described in the bcl2fastq 1.8.4 User Guide (part # 15038058) and the CASAVA User Guide (part # 15011196)

    Sample sheets for rapid runs include information for two lanes, as compared to eight lanes included in a sample sheet for a high output run. Sample sheets for rapid runs can be generated manually, using Excel or a text editor.

    If you are using BaseSpace for data storage and analysis, a sample sheet is required for both rapid runs and high output runs. If using BaseSpace only for run monitoring and you are not indexing, a sample sheet is not required.

    You can use BaseSpace Apps to analyze data in BaseSpace Sequence Hub. Select the Apps tab in BaseSpace Sequence Hub to see available apps and descriptions. 

    Where *.cif files can be generated, you can use OLB v1.9.4.

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

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

    Scanning and analysis of a 2-lane rapid run flow cell creates two swaths per surface on two surfaces per lane. Each swath is divided into 16 tiles. For a 2-lane flow cell, there are a total of 128 tiles per flow cell.