Questions & Answers

• Libraries |
• Reagents and Flow Cells |
• Workflow |
• Software |
• Analysis

Libraries

• 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.

• 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.

• Can I use only one of the indices of a dual-indexed library?

  Sample prep for dual-indexed libraries requires that both indices 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 eight-base single-indexed sequencing run.

• 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.

Reagents and Flow Cells

• 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.

• 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.

• 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.

• 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).

• 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.

• 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.

• 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.

• 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.

• 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.
  

Workflow

• 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.

• 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 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.

• 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.

• 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.

• 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.

• 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.

• 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

• 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.

• 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.

• 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.

• 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.

• 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.

• 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.

• What is the workflow for dual indexing?

  Please 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 eight 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 seven chemistry-only cycles (no images are taken) followed by eight 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 eight 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 many cycles should be used during the Index Read for single-indexed libraries?

  Index reads for single-read libraries use seven cycles reads. Illumina does not support six cycle index reads for single-indexed libraries.

• 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.

• 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 sample prep in order to confirm appropriate index combinations.

• Is a PhiX control lane necessary with ChIP-Seq libraries?

  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 Using a PhiX Control for HiSeq Sequencing Runs.

• 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.

Software

• 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.

• 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 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.

• 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.

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

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

• 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.

• 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.

• 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.

• Can I designate a control lane using HiSeq Control Software (HCS)?

  HCS allows you to designate a control lane during the run setup steps. Generally, you do not need to designate a control lane if the sequence you are analyzing has a balanced genome. In the case of an unbalanced or skewed base composition (e.g., bisulfite-treated samples) a control lane is recommended. This is not equivalent to a PhiX spike-in.

• What operating system is used on the instrument computer?

  The HiSeq instrument computer employs 64-bit Windows Vista.

• 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.

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

  The first time the HCS 2.x (or later) is launched, you will see a notification regarding instrument health data. This notification will appear 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. If the customer is not present to view this notification, Illumina staff will refer the customer to the Options menu and ensure that they are aware of this setting.

• Exactly what files are uploaded as instrument health data?

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

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

  HCS software allows HiSeq instruments connected to the internet to send instrument health information to Illumina. This information will be anonymous and will include only generic run metrics. This information will be used by Illumina to help improve Illumina’s products. If you wish to turn off this option, or would like further information, please go to the Options menu in the HiSeq control software. You can find the Options menu under Menu|Tools.

Analysis

• How do I use BaseSpace for run monitoring?

  The BaseSpace run monitoring option enables you to monitor an ongoing run’s progress remotely by logging into your BaseSpace account. You will need to select the Run Monitoring option during run setup. Then, you can log into your BaseSpace account from anywhere and view your run in the BaseSpace version of Sequence Analysis Viewer.

• If I use BaseSpace for HiSeq Run Monitoring only, what files are sent to BaseSpace?

  The HiSeq will send only the InterOp folder, RunInfo.xml file, and RunParameters.xml file.

• 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.

• 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 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 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 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 I save cifs with HCS 2.x?

  Yes. HCS 2.x enables you to save cifs.
  

• How do the HCS 2.x data compression options change data analysis or data handling?

  If a run has zipped bcls, you will need to use a new software tool, Bcl2Fastq, 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 zipped or non-zipped bcl files.

  If you send your data to BaseSpace, bcl to fastq conversion and demultiplexing will be performed automatically following the completion of the data upload.

• What do I need to do to make sure my HiSeq is ready to send data to BaseSpace?

  If you want to upload data from a HiSeq to BaseSpace, you will need a minimum upstream connection bandwidth of 10Mbit/second per instrument. You can assess network speed using free online tools, such as www.speedtest.net.

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

  Please see the Illumina whitepaper, Reducing Whole-Genome Data Storage Footprint, which is available on MyIllumina. You can find it on the HiSeq Documentation and Literature support pages.
  

• What data compression options are available for HiSeq runs using HCS 2.x?

  HCS 2.x features the two data compression options: bcl file zipping and Qscores binning. These options are available during run setup in HCS 2.x. If you are using BaseSpace for data storage and analysis, bcl files will be zipped by default. If you are using BaseSpace for only run monitoring, bcl zipping is not required. Other run folder files are unchanged from previous versions of HCS.

  If you are not using BaseSpace, the data compression options are as follows:
  1. No compression
  2. Zip bcls + bin Qscores
  3. Zip bcls only

  If you are using BaseSpace, the data compression options are as follows:
  1. Zip bcls only (required for BaseSpace)
  2. Zip bcls + bin Qscores

• How many swaths and tiles on a Rapid two-lane flow cell?

  Scanning and analysis of a Rapid two-lane flow cell creates two swaths per surface on two surfaces per lane. Each swath is divided into 16 tiles; for a two lane flow cell there are a total of 128 tiles per flow cell.

• 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.

• What is deconvolution?

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

• What criteria determine clusters passing filter for HiSeq runs?

  To remove the least reliable data from the analysis results, often derived from overlapping clusters, raw data is 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 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 is 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 the majority of cases, throttling of the BaseSpace Broker data upload should not be necessary. Throttling may be needed if you need to apply greater control over network bandwidth usage (such as at sites at which instruments share the network with other users or sites with limited upload speed). Throttling may be necessary in certain scenarios, such as when local network connectivity is temporarily lost and then restored, causing BaseSpace Broker to suddenly consume more network bandwidth as it tries to “catch up” with the upload of accumulated data. In these types of scenarios, if no throttling is applied, BaseSpace Broker could consume all available bandwidth on the network until the backlog of data is cleared. If throttling is to be applied and if the local network permits, Illumina suggests throttling to higher than the 10Mbit/sec minimum specification. 20Mbit/s (approximately 3Mbyte/s = 24Mbit/s) is recommended. This will provide BaseSpace Broker with enough bandwidth to recover in the case of data upload delays.

  If you need to throttle BaseSpace Broker, please supply the following instructions to your local IT administrator:

  Throttling of BaseSpace is done on the HiSeq-PC by application rather than by IP address.

  1. In windows, open the Local Policy Editor by opening a cmd window and running gpedit.msc.

  2. Expand the Computer Configuration / Windows Settings nodes.

  3. Select Policy-based QoS.

  4. Right-click and then choose Create new policy. Then, perform the following steps:

  a. Enter a name.

  b. Uncheck Specify DSCP value.

  c. Select Specify Outbound Throttle Rate, and then enter 3 Mbytes/s (or 24Mbit/s). This should enable the system to catch-up if necessary.

  d. Select Next.

  e. Select Only applications with this executable name, and then enter Illumina.BaseSpace.Broker.exe.

  f. Click Next (this policy applies to any source IP and target IP addresses).

  g. Click Finish (this policy applies to all ports and protocols).

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

  You can use OLB 1.9.4. However, please note that OLB 1.9.4 will be replaced by RTAOLB, which is a means of running RTA in a Linux environment. An RPM of RTA plus needed libraries and instructions will be released on My Illumina in November of 2012.

• How do I merge data from two HiSeq flow cells?

  With Casava, you can merge data from different HiSeq flow cells (different runs) with Casava 1.8.2’s configureBuild script.

  First, align the data (samples) from each flow cell with Casava 1.8.2 separately, using configureAlignment. Then, include each Sample Directory as an input directory in the configureBuild.pl command line. You can specify input directories by using the –id option, as detailed on page 100 of the Casava 1.8.2 User Guide Rev C.

  With BaseSpace, a sample merge application is available, which enables you to merge data for a single sample that originated from different flow cells. Merging is performed prior to alignment analysis of the sample data.

• What is bcl2fastq?

  bcl2fastq is a separate piece of stand-alone software that is run on a Linux scientific computing system. You can get the installer from MyIllumina. Dependencies are equivalent to those for Casava 1.8.2 and are outlined in the bcl2fastq user guide. They are also listed on pages 114 to 117 of the Casava 1.8.2 User Guide Rev C. If the option to compress bcl files was selected during run setup, then conversion of bcl files will require the use of the bcl2fastq.

• Is the output of bcl2fastq compatible with Casava 1.8.2?

  Yes. bcl2fastq is used in place of the configureBcl2fastq step in Casava. The output of bcl2fastq is fastq.gz files organized into Project and sample directories as specified in the sample sheet. This output will be compatible with the configureAlignment and configureBuild components of Casava 1.8.2. The sample sheet format required for bcl2fastq is equivalent to Casava 1.8.2 sample sheet format, and it is described in the Bcl Conversion with bcl2fastq User Guide.

• Do I need the new bcl2fastq software package?

  You will need bcl2fastq to handle zipped bcl files. It can also handle regular (non zipped) bcl files.

• How do I use BaseSpace for Run Monitoring only (for example, for SAV functionality in BaseSpace)?

  You must select run monitoring on the Storage tab of HCS during Run Configuration.

• Does BaseSpace require a Sample Sheet?

  If you choose to use BaseSpace for Run Monitoring only, a sample sheet is not required. If you want to use BaseSpace for data storage and analysis, a sample sheet is required. It must be in Casava format, and all samples listed in BaseSpace must be indexed or all must not be indexed (for example, you cannot have a mix of indexed and unindexed samples in a single flow cell when using BaseSpace).

• How do I send HiSeq run data to BaseSpace?

  HiSeq Run data can be uploaded to BaseSpace only during a sequencing run and only if you have selected the BaseSpace option in HCS. Please see the HiSeq User Guide for information on setting up a run for upload to BaseSpace.

  For more information on BaseSpace, or to set up a free BaseSpace account, please go to https://basespace.illumina.com/home/index.

• Will local proxies affect BaseSpace?

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

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

  Please contact your local IT administrator if local security policies will need to be modified to allow access to BaseSpace. BaseSpace uses SSL/https port 443 and the domains api.basespace.illumina.com and basespace.illumina.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/

• Can I analyze cifs with BaseSpace?

  No. Even if you do choose to save cifs, they will be saved only locally and will not be transferred to BaseSpace.