Questions & Answers

Expand All


  • How can I optimize cluster formation?

    Short fragments tend to create smaller clusters, allowing greater data density. The optimal fragment size for a single-read run is 150–300 bp. The optimal fragment size for a paired-end run is 250–500 bp.

  • How much DNA is required to load a flow cell lane for bridge PCR?

    If possible, start the sample prep with 1–5 μg of DNA, although 1–2 μg is enough for many flow cells.

  • What is the recommended DNA concentration for cluster generation?

    The first time you process template DNA, try a concentration titration range to optimize the number of clusters formed. If the concentration is too low, fewer clusters are generated and result in a low sequencing yield. If the concentration is too high, clusters are too dense and can complicate data analysis. For more information, see the Sequencing Library qPCR Quantification Guide (part # 11322363) (a MyIllumina login is required) or the HiSeq and GAIIx Systems Denature and Dilute Libraries Guide (part # 15050107).

  • Does cDNA cluster in the same way as gDNA?

    cDNA libraries should be considered an equivalent to gDNA for clustering and sequencing steps, assuming an equivalent complexity of the sample.

  • When do I use a PhiX spike-in for sequencing?

    For samples with an unbalanced base composition, Illumina recommends using a high-concentration spike-in of PhiX control lane to create a more diverse set of of clusters. Unbalanced samples contain genomes with AT or GC content of less than 40% or greater than 60%. PhiX Control v3, Catalog # FC-110-3001, is a balanced and diverse library with a mean insert size of approximately 375 bp, corresponding to 500 bp library size if visualized on a Bioanalyzer. For more information, see Using a PhiX Control for HiSeq Sequencing Runs.

  • 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.

  • What is the minimum PhiX amount that can be spiked in?

    For most libraries, Illumina recommends using a low-concentration spike-in (1%) of PhiX Control v3 as an in-lane positive control for alignment calculations and quantification efficiency.

Reagents and Flow Cells

  • How long can flow cells be stored?

    After amplification, linearization, blocking, and primer hybridization, you can store the flow cell in storage buffer at 2° to 8°C for up to 10 days. HiSeq X flow cells can be stored up to 48 hours.

    For rapid flow cells, perform the sequencing run on the same day as sample loading.

  • Will cBot plates be updated with the sequencing primers required for dual-indexed Nextera libraries and TruSeq HT libraries?

    Not at this time. If you are sequencing Nextera libraries, you need sequencing primers provided in the TruSeq Dual Index Sequencing Primer Box for any run types. If you are sequencing dual-indexed TruSeq HT libraries on a single-read flow cell, you need sequencing primers provided in the TruSeq Dual Index Sequencing Primer Box, Single Read.

  • Are there any protocol changes when using v3 reagents and flow cell v3?

    You need to increase your starting template concentration. However, the starting template volume (120 µl) is unchanged from previous kits.

  • Is a PhiX control included in the TruSeq Cluster Kit?

    No. The Illumina PhiX Control v3 is a separate product. The catalog number is: FC-110-3001.


  • 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.

  • Can my cBot cluster both HiSeq and Genome Analyzer flow cells?

    Yes. The cBot can cluster both HiSeq and Genome Analyzer flow cells. However, you must use the appropriate flow cell adapter plate to accommodate the different dimensions of the flow cells. Dedicating a cBot to one platform avoids routinely switching adapter plates.

  • How long does it take to generate clusters on a flow cell?

    On the Cluster Station, it takes about 5 hours. On the cBot, clustering duration depends on the flow cell that you are using. HiSeq rapid flow cells take about 1 hour to cluster, and other HiSeq flow cells take about 3 hours. TruSeq v3 and GAIIx v2 flow cells are clustered in about 5 hours.

  • Do I need to use a cBot with the MiSeq?

    No. Cluster generation is performed on the MiSeq instrument as part of the run. No other instruments are required for sequencing on the MiSeq.

  • How do I connect to LIMS?

    In Configuration, select the LIMS tab and then select the Enable LIMS checkbox. Select the LIMS Server field and enter your LIMS server name using the onscreen keyboard. To generate a run data file (*.xml) for use with LIMS, select the Generate File with Experiment Information checkbox and specify a network location for the output. For more details, see the cBot System Configuration Guide (document # 1000000005301).