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.
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 (requires a MyIllumina login) or the HiSeq Systems Denature and Dilute Libraries Guide.
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.
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 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.