In response to user demand, the OGC have recently purchased an Illumina NextSeq 500 sequencing system to be set up to run as a walk-up next generation sequencing platform for internal users.
How does it work?
In four easy steps you can gain access to a desktop Next Generation sequencing platform that supports a variety of applications offering simple, rapid, and affordable generation of sequence data for a wide variety of project types.
Step 1
Register and Training
All users will require basic training before they may run the platform NextSeq 500. Request training by contacting nextseq@well.ox.ac.uk. Please note that it is advisable to undertake training before preparing Illumina libraries for the NextSeq 500, because there are some special considerations for this platform (e.g. please see the document MyIllumina_Support Bulletin-Library pooling guidelines for the NextSeq and MiniSeq systems, available via the Illumina website or in the Tech notes and Bulletins section at the bottom of this page).
Training will entail the provision of relevant user guides, presentations, quick reference guides and tech notes from Illumina (links to Illumina documentation can be found here). A user’s first run will be performed with a member of OGC, and their second run will be shadowed.
You will also need an Illumina BaseSpace account. Sample sheets are generated and accessed by the machine through BaseSpace. Data will be sent directly to BaseSpace. Data can also be sent to a designated storage server on the network if this can be provided by the person using the walk-up service. No external drives will be connected to the NextSeq 500 system. A BaseSpace account can be set up here.
Step 2
Booking Run Slot
We use ARIA, developed by the Division of Structural Biology at Oxford University (STRUBI) as the booking platform for this service.
- Create an ARIA user account here: ARIA booking system
- Once you have registered, e-mail nextseq@well.ox.ac.uk to let us know and we will add you to the list of NextSeq 500 users.
- In order to book a run slot, as of January 2019 you will need a purchase order number.
- Run slots are 1 day (24hrs) each. Depending on the run length multiple runs slots may be required.
- Each booked 24hr run slot will be billed at £200 irrespective of whether or not it is used. WHG members can raise a PO without a quote, please raise a PO for the correct number of run slots in advance of booking the run.
- Run slots can be booked up to 2 weeks in advance. Bookings made beyond this period may be subject to cancellation for operational reasons.
- A run slot may be cancelled without charge up to 3 days before the slot.
Logon to ARIA, view the calendar for availability, and request a run slot. Provide a PO number for payment and submit. You will be notified within 2 working days that your run slot is approved or if there is a problem. If you need to book a run less than 2 days in advance, please e-mail nextseq@well.ox.ac.uk to notify us of the booking.
At the end of each month, usage will be recharged to the appropriate PO number. In the first instance, no formal restriction will be placed on the number of run slots that can be booked and it is hope that internal users will work together to ensure fair and optimal usage.
Step 3
Running
To run the machine, you will need to arrive with the following:
- Library diluted to the recommended concentration. Illumina’s recommended concentration for running libraries on the NextSeq 500 platform can be found in the Nextseq 500 system guide. However, different library types and different preparations will have characteristics specific to them. You will need to optimise the amount to load by experience over several runs.
- Flow cell and sequencing consumables specific to your run needs.
| NextSeq 500 System Performance Parameters | |||||
| Flow Cell Configuration | Read Length (bp) | Output (Gb) | Run Time (Hours) | Data Quality | Kit Catalogue # |
| High-Output Flow Cell Up to 400 M paired-end reads* |
2 x 150 | 100-120 | 29 | > 75% > Q30 | FC-404-2004 |
| 2 x 75 | 50-60 | 18 | > 80% > Q30 | FC-404-2002 | |
| 1 x 75 | 25-30 | 11 | > 80% > Q30 | FC-404-2005 | |
| Mid-Output Flow Cell Up to 130 M paired-end reads* |
2 x 150 | 32-39 | 26 | > 75% > Q30 | FC-404-2003 |
| 2 x 75 | 16-19 | 15 | > 80% > Q30 | FC-404-2001 | |
| * Yields are for optimised PhiX runs and therefore represent a maximum possible yield on this platform. | |||||
- Pipette and tips for loading your library onto the sequencing cartridge. You can pre-load the sample onto the cartridge to avoid having to bring a pipette and tips with you. There is space beside the NextSeq 500 for you to set up your run, but please be advised that you will not have access to OGC consumables or reagents.
N.B. It is essential that your start runs at a time that fits within your booked slot. Currently, runs are expected to be on the machine and started by 12:00pm of the first day of the run slot(s) booked. This is to ensure that all runs are finished before the next slot opens, and to allow time in between runs for routine maintenance on the machine.
Should the NextSeq 500 fail, all bookings will be adjusted in line with the number of days the platform is not operational.
Step 4
Data Transfer
A bcl –> fastq conversion of data can be carried out in BaseSpace, prior to download of data to your own file storage system. Please note you will have to make your own arrangements for data storage and downstream processing. Information on in-house data processing can be found here.
If you do not want to make your own libraries OGC can help. OGC-prepared libraries will be QC-ed and provided at 10nM concentration where possible. Please contact OGC for a quotation.
If you are happy to wait a little longer OGC also offers a full-service option using this platform. The advantages to you of this full-service option are decades of combined experience in next-generation sequencing that maximises output and data quality from Illumina technology, access to the full OGC data QC and project-specific analytical pipelines, and access to a group of dedicated project managers to help plan your project – taking the stress out of generating data.
Illumina Documentation
Click on each of the below to download a PDF
- Analysing Sequencing Data on BaseSpace – quick reference card
- BaseSpace Sequence Hub Apps – quick reference
- BaseSpace User Guide
- BaseSpace Datasheet
Click on each of the below to download a PDF (password protected – please contact for access)
- Introduction to the NextSeq System
- NextSeq Sequencing Workflow Overview
- Troubleshooting the NextSeq System
- Image Analysis and Base Calling
- NextSeq Sequencing Run Monitoring
- Bcl2FastQ2 for NextSeq and HiSeq Systems
- Illumina Sequencing Overview
Click on each of the below to download a PDF
- Analyzing runs on BaseSpace
- Planning Exome Sequencing
- Planning RNA Sequencing
- Planning Whole Genome Sequencing
- Running NextSeq 500
Click on each of the below to download a PDF
- Application Note: RNA Sequencing
- Application Note: Sequencing
- Illumina Adapter Sequences
- Best Practices for Low Diversity Sequencing on the NextSeq System
- Cluster density considerations when migrating Illumina libraries between sequencing platforms
- How do I create a custom library prep kit in BaseSpace Sequence Hub
- How to set up a PhiX validation run on the NextSeq system using BaseSpace Hub Prep tab
- Library pooling guidelines for the NextSeq and MiniSeq systems
- Options for downloading run folders from BaseSpace Sequence Hub
- Diagnosing and Preventing Flow Cell Overclustering on the MiSeq
- Technical Note: DNA Analysis
- Technical Note: Informatics
- Technical Note: Illumina Sequencing
- White Paper: Informatics