Illumina Sequencing Services

HiSeq 4000

The Illumina HiSeq 4000 is over three times faster than an identical run length on the HiSeq 2000 while providing over 60% more reads per lane. However, the 4000 has more stringent library requirements and requires a larger sample size than the 2000.

A 2×101 run will take about 2-3 days to complete; a 2×151 will take 3-4. (Time variation can occur depending on the time of day clustering was performed: in the morning or afternoon).

The HiSeq 4000 generally gives 280-330 million reads per lane for a flowcell total of around 2.2 billion reads. While the patterned flow cell used on the 4000 is fixed at 480 million wells/reads per lane, Illumina recommends between 60-70% passed filter (PF) to maximize the number of unique reads in the lane. A higher PF will provide a higher amount of non-unique reads, which means more duplicate data. The HiSeq 4000 is biased towards smaller fragment sizes, so please try to keep your samples close to a consistent fragment length.

More information on the HiSeq 4000 specifications can be found on Illumina's website (PDF).

HiSeq 2000/2500

The Illumina HiSeq 2000/2500 allows more versatility in types of runs than the HiSeq 4000.  As many clients are producing libraries that haven't been optimized or cleared for use on the 4000, the 2000/2500 are good substitutes.  A 2x101 run will take about 11 days to complete; a 1x50 run will take 3 days (plus additional time for clustering).  The HiSeq 2500 generally gives 190 million reads per lane for a flow cell total of around 1.5 billion reads.

More information on the HiSeq 2500/2000 specifications can be found on Illumina’s website (PDF).

Rapid Run Mode

Illumina indicates that the Rapid flowcells can get up to ~150 million paired reads per lane under optimal conditions.
Rapid Run Mode is great for libraries that are not optimized for the 4000, require more data than a MiSeq provides, and need the results quicker than a 2500 high throughput mode will allow.


One of the great advantages to the MiSeq is the quick turnaround time for runs. The MiSeq is capable of long reads, making it great for de novo assembly of small genomes. The MiSeq is also great for QC tests on sequencing workflows before committing to larger batches on more expensive machines.

The MiSeq v2 kit provides 12-15 million single reads or 24-30 million paired-end reads, while the MiSeq v3 kit provides 22-25 million single reads or 44-50 million paired-end reads.

More information on the MiSeq specifications can be found on Illumina’s website (PDF).



We’ve moved our data analysis pipeline to DNAnexus. Please email Paul Billing-Ross ( for information on how to set up an account.

Please note: If you do not provide billing information (grant or credit card) when setting up your account, your data will be free to download for 1 month. If you do provide billing information, you will be charged for data storage after 1 month.

Acknowledging Our Services

Please support the Sequencing Service Center by acknowledging our services in your publications.

If you use the HiSeq 4000, please also acknowledge NIH Grant # 1S10OD020141-01

Thanks for your help!