The HiSeq 3000 Control Software (HCS) eliminates the dedicated PhiX control lane (#8) (previously used on HiSeq 2000 runs) that provides quality control for the other seven HiSeq lanes. See Illumina's technote "Low-Diversity Sequencing on the Illumina HiSeq Platform"

The following is now recommended:

  • For lanes containing “balanced samples,” e.g., de novo sequencing, the CQLS will add a 1% PhiX spike-in for quality control; users may opt to eliminate this spike-in, but at the user’s own risk.
  • For lanes containing sequence from organisms with high homology to PhiX, the spike-in will be eliminated.
  • For lanes containing “imbalanced samples,” i.e., lanes containing low nucleotide diversity, at least 10% phiX will be added as a spike-in to aid in base calling. Users with low nucleotide diversity in their samples should consult with the CQLS prior to submitting their sample.
  • Lanes using commercial, multiplexed sample preparation kits, e.g., TruSeq and Nextera, will automatically have the PhiX reads filtered out. Information on barcodes
  • Lanes not using commercial multiplexed sample preparation kits will have the PhiX reads present in their FASTQ files.  The Core offers to filter out the PhiX reads from the original FASTQ files as part of the Illumina HiSeq service.