Illumina MiSeq and NextSeq 500 Walk-up Sequencing Service for internal Users

OGC have two platforms available on a walk-up basis for users from Wellcome Centre for Human Genetics (WHG): Illumina MiSeq and NextSeq 500.

How does it work?

If you are based in WHG, 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.

If you are unsure of the application, unfamiliar with library preparation and/or the sequencing technology then please talk to one of OGC’s project managers before registering your interest in the walk-up service. Contact OGC.

 

 

Step 1

Register and Training

All users will require basic training before they may run either the MiSeq or the NextSeq 500 unaided. Training will entail an overview of the system requirements, the provision of relevant user guides, and that the user’s first run will be performed with a member of OGC. You can request training or information by contacting ogc-walkup@well.ox.ac.uk. Please note, due to specific considerations, it is advisable to contact OGC before preparing Illumina libraries for either platform (e.g. please see the Index Adaptors Pooling Guide and the support Bulletin-Library pooling guidelines for the NextSeq and MiniSeq systems, which can be downloaded from the Illumina website).

You will also need an Illumina BaseSpace account as data will be sent directly to BaseSpace. No external drives will be connected to either system.
Setup a BaseSpace account.

In addition, you will need to download the Illumina Experiment Manager (IEM) from the Illumina website. This program is used to generate the sample sheet for the run.

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 by following the step by step instructions 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 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, as detailed in the table below.
150PE or less  250PE 300PE All mid or 75PE high output 150PE high ouput
MiSeq £100, 1 slot £200, 2 slots £300, 3 slots N/A N/A
NextSeq N/A N/A N/A £200, 1 run slot £400, 2 slots
  • Each booked run slot will be billed 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 1 week 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 2 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 1 working day that your run slot is approved or if there is a problem. If you need to book a run less than 1 day in advance, please e-mail ogc-walkup@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 MiSeq and NextSeq 500 platform can be found in the MiSeq and NextSeq denature and dilute libraries 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 (details on performance parameters can be found below).
  • 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 available for you to set up your run, but please be advised that you will not have access to OGC consumables or reagent

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 either system fail, all bookings will be adjusted in line with the number of days the platform is not operational.

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.

 

 

 

MiSeq System Performance parameters
Flow Cell Configuration Reda Length (bp) Output (Gb)* Run Time (Hrs) Data Quality Kit Catalogue No.
Standard MiSeq v2 1 x 36 0.54-0.61 4 >90% bases higher than Q30 MS-102-2001
2 x 25 0.75-0.85 5.5 >90% bases higher than Q30 MS-102-2001
2 x 150 4.5-5.1 24 >80% bases higher than Q30 MS-102-2002
2 x 250 7.5-8.5 39 >75% bases higher than Q30 MS-102-2003
Standard MiSeq v3 2 x 75 3.3-3.8 21 >85% bases higher than Q30 MS-102-3001
2 x 300 13.2-15 56 >70% bases higher than Q30 MS-102-3003
Micro v2 2 x 150 1.2 19 >80% bases higher than Q30 MS-103-1002
Nano v2 2 x 150 0.3 17 >80% bases higher than Q30 MS-103-1001
2 x 250 0.5 28 >75% bases higher than Q30 MS-103-1003

* Yields are optimised PhiX runs.

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. Read more about our in-house data processing.

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 these platforms. 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

All relevant Illumina documentation can be downloaded from the following website:

https://www.illumina.com/