Illumina NextSeq 1000 sequencer

Instruction and user charge for NextSeq 1000 sequencer

The Integrated Microscopy Core

UW Sensory Biology Center

 

The IMCore of the UW Sensory Biology Center is pleased to announce the availability of an Illumina NextSeq 1000 sequencer. This equipment is purchased exclusively via an administrative supplement awarded to Dr. Qian-Quan Sun (3P20GM121310-05S3).  More information about the sequencer can be found at the Illumina website.

DNA sequencing follows three basic steps: library preparation, sequencing and sequence analysis, whereas library preparation is the most important step for any successful sequencing.

Please read the following instruction carefully before using the IMCore sequencer:

1. Library preparation: PIs/users are required to prepare their own DNA libraries for sequencing. The libraries must meet all requirements for the Illumina sequencer. For single cell RNA-seq, visit Illumina’s website about the library kits compatibility. More information about Illumina’s library prep kit can be found here.
2. Quality control: The quality and quantity of the library DNA shall be measured by a TapeStation and Qubit fluorometer. An Agilent TapeStation 4200 and a ThermoFisher Qubit 3 fluorometer are available at the IMCore. Please contact the IMCore staff for assistance.
3. Find the right flow-cells: PIs/users need to determine what type of reagents/flow cells they plan to use for their sequencing. The available reagents for the NextSeq 1000 include P1 (300 cycles) and P2 (100 cycles, 200 cycles or 300 cycles). IMCore may not have the right reagents/flow cells in stock. Contact the IMCore staff as early as possible, so that they can be ordered on time. More information about different reagents/flow cells can be found here. Some basic guidelines/considerations for RNA-Seq read length and read depth can be found here.
4. Complete the form: Once you are ready for sequencing, please complete the sequencing request form, and email the form to IMCore staff.
5. Deliver your libraries: Once your form is received and approved, you will be asked to send the libraries to the core. Your libraries will be sequenced by the core staff.
6. Sequencing data: Instructions on how to download the sequencing data will be available upon completion of the sequencing.
7. Data accessibility: because the instrument is funded in-full by NIH, users must comply with NIH’s data sharing policies, as detailed here. In accordance with the Public Law 110-161 enacted by US congress, compliance with the NIH Public Access Policy is mandatory. More information about the Public Access Policy can be found here. The PI is responsible for complying with this policy. Non-compliance will result in refusal of service.
8. Acknowledgement: Any publications and presentations using the data obtained from this instrument is required to acknowledge the parent award of SBC center (P20GM121310), and the administrative supplement (3P20GM121310-05S3).
9. Waiting period: Please consult with Core staff regarding the waiting period.

Cost for sequencing

The cost for sequencing includes two parts:

1. The cost for supplies. PIs/users will pay the actual cost for the reagents/flow cells, which is around $2,500 per flow cell, depending on the type of flow cells.
2. The cost for sequencing. The IMCore charges a $270 fee for each sequencing run. This fee primarily helps the core to recover part of cost for maintaining the instrument and software subscription (~$30,000/year). SBC subsidize the remaining cost, including personnel.

For each sequencing run, a user fee will be charged to the user, with the composition of the user fee as following: actual cost of the flow cell (~$2,500) + $270.  PIs will be billed after each sequencing run.

 

Cost for quality control

Agilent TapeStation 4200 (operated by IMCore staff)

The fee includes two portions:

1. The actual cost for supplies (tapes, DNA/RNA ladders, and buffer). Currently is about $30 per tape.
2. Instrument user fee: $10.00 per sample if you have 1-4 per run; $8.00 per sample if you have more than 5-15 samples per run.

*This user fee is collected to partially recover the annual maintenance cost of the instrument (~$3,000/year).

ThermoFisher Qubit 3 fluorometer (operated by IMCore staff)

• $15.00 per run.