HiSeq 3000 Sequencing

HiSeq3000 Technology

The HiSeq 3000 sequencer represents the very latest sequencer generation and enables significantly faster sequencing and reduced per-base-costs. Please note that for some samples the library preps need to be adjusted for the HiSeq 3000. The new sequencer is more than three times faster than the HiSeq 2500 and yields about 65% more reads per lane. For example the run-time of a paired-end 100 bp run is only 3 days on the HiSeq 3000 compared to 11 days on the HiSeq 2500. The number of reads per lane has improved from between 150-190 million (HiSeq 2500) to 260-310 million reads (official figures for the HiSeq 3000). Our first data indicate averages of more than 370 million clusters passing filter per lane are possible.  Please see this post for more details, quality scores, and for a link to example data.  The maximum HiSeq 3000 read length is 150 bp compared to 100 with the HiSeq 2500 High Output mode. Thus, the maximum yield per lane has more than doubled using the HiSeq 3000 sequencers (2.5x).  The HiSeq 2500 is still of great interest since it generates paired-end 250 bp reads in Rapid Mode.

The progress in speed and output is enabled by two new technologies: patterned flow cells and kinetic exclusion amplification (please see the video). In contrast to the random clustering employed in previous HiSeqs, the clusters are now generated in ordered nano-wells to allow for higher cluster densities and unambiguous cluster identification.

 Libraries for HiSeq3000 Sequencing
The majority of existing Illumina sequencing libraries can be sequenced as is on the HiSeq 3000.  Other sequencing libraries can be made compatible by size-selection (removing both adapter-dimer traces and fragments with inserts longer than 550 bases if the latter are numerous). Any library with Truseq-style adapters (e.g,  Nugen, Bioo, Nimblegen, Agilent adapters) will work just fine as long as they do not fall under the exclusion criteria below.
Illumina does not provide a lot of information on the suitability criteria of libraries for the new sequencers other than recommending only a limited selection of their own library prep kits (TruSeq Nano DNA (350 bp inserts), TruSeq DNA PCR Free (350 bp inserts), TruSeq Stranded mRNA, TruSeq Stranded Total RNA, TruSeq RNA Access, Nextera Rapid Capture Exome).
At the moment we are operating under the assumption to avoid:
  • low complexity libraries
  • bisulfite sequencing libraries
  • even traces of adapter dimers
  • any libraries with a considerable percentage of longer fragments (insert sizes over 550bp)
  • libraries with single-end adapters (these adapters should have been dropped years ago, though)
  • Nextera libraries likely will require size selection
 Other important considerations when loading the libraries likely will be:
  • The new exclusion amplification on the flowcells more strongly advantages short library molecules (especially the even shorter adapter dimers) as compared to the previous bridge amplification protocol. According to Illumina 1% adapter dimer content can result in more than 6% of adapter dimer reads;  10% adapter dimer content  resulted in 84% being unusable adapter dimer reads.
  • The minimum library concentration has increased to 2 nM. Please submit libraries with concentrations of 5 nM or higher if possible.


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