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.
If possible, start the sample prep with 1–5 μg of DNA, although 1–2 μg is enough for many flow cells.
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).
cDNA libraries should be considered an equivalent to gDNA for clustering and sequencing steps, assuming an equivalent complexity of the sample.
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.
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.
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.
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.
No. The Illumina PhiX Control v3 is a separate product. The catalog number is: FC-110-3001.
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.