Illumina Sequencing Services

The Illumina NovaSeq provides a massive upgrade in sequencing throughput compared to the HiSeq 4000.  There are more stringent library requirements and requires a larger sample size.  Due to the vast amount of data produced by the NovaSeq and the known issue of index swapping, unique dual-indexed libraries are required.  If you do not have dual-indexes, you will need to do steps of PAGE purification or use purification columns to help ensure your library is clean.

A 2x101 S1 run will take 1 day to complete, an S2 run will take just over 1 day to complete, and an S4 about 2 days.  A 2x151 S1 run will take just over 1 day to complete, an S2 run will take about 2 days to complete, and the S4 2-3 days. (Time estimate does not include additional time for internal QC or if there is a large queue for the NovaSeq, please enquire about S4 wait times if you do not purchase 4 lanes).

The NovaSeq is biased towards smaller fragment sizes, so please try to keep your samples close to a consistent fragment length.  NovaSeq uses two-color chemistry as opposed to the HiSeq 4000's 4-color chemistry, so please keep that in mind if you wish to pool data from both sequencers.

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

*Note: There is a known issue with sequencing on the NovaSeq that may impact some research projects.  Click below for more information.

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).

*Note: There is a known issue with sequencing on the HiSeq 4000 that may impact some research projects.  Click below for more information.

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).

The iSeq 100 Sequencing System makes next-generation sequencing easier and more affordable than ever. Designed for simplicity, it allows labs of all sizes to sequence DNA and RNA at the push of a button. Discover more—without the cost.

The iSeq provides great turn-around time with the instrument run between 9-17.5 hrs per run. The iSeq is able to provide single reads of 4 millions and paired-end reads of 8 millions.

We’ve moved our data analysis pipeline to DNAnexus. Please email Peter Maguire (pmaguire@stanford.edu) 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.

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

If you use the NovaSeq 6000, please also acknowledge NIH Grant # S10OD025212

Thanks for your help!