Questions & Answers

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  • 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.

  • 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.

  • 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.

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.

  • 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.

  • 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.


  • 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.