Illumina_MiSeqTM_CopyRight2011
Illumina MiSeq

Next Generation Sequencing Facility

Use of the Illumina MiSeq and NextSeq500 for Next Generation Sequencing

Introduction

The WIMM Next Generation Sequencing Facility is located in the laboratories in the basement of the Women’s Centre, together with the NanoString nCounter and the Droplet Digital PCR instrumentation. If you would like to use the MiSeq or NextSeq, please get in contact. If you intend to do the setup yourself, you will need card access – please see Allan Hayle  about this.  If you are new to Illumina and their technology please refer to this quick start protocol for using the Nextseq 500. A more detailed manual for use of the Nextseq 500 is available here.

MiSeq and NextSeq500

Kits
We do not stock any kits – you will have to purchase the required kit before your allotted time slot and this can be done via R12. I can provide catalogue numbers and current pricing information – please email for details. As with the MiSeq, we currently do not offer a library preparation service.

Runs
WIMM Sequencing Facility will require a Purchase Order (PO) to be raised in R12 for all MiSeq and NextSeq work (Form Download). The supplier in R12 is 'Weatherall Institute of Molecular Medicine'. The current prices are as follows:

NextSeq: £200 per run.

MiSeq:

£50 for 50 cycles
£125 for 150 cycles
£150 for 300 cycles
£250 for 500 cycles
£350 for 600 cycles

Accounts required

Prior to using the MiSeq or NextSeq500 you will require various accounts:

An Agendo account

If you are already using the Agendo system for FACS/microscopy bookings, you do not need to register again. If you haven’t got an Agendo account, go to the Agendo booking system and click [New User] and fill in the details.  The Resource is either Illumina MiSeq (Sequencing Facility) or Illumina NextSeq (Sequencing Facility).

A CBRG account

If you intend to use the CBRG facilities for analysing your data, you must have a CBRG account and it is imperative that you discuss your project with CBRG (Steve Taylor/Simon McGowan) prior to using the sequencing facility.

MiSeq data will be transferred to your folder once the run is completed. NextSeq run data will be automatically uploaded to your BaseSpace account. There will be an option in the future for local CBRG storage, once the infrastructure is in place.

An Illumina BaseSpace Account

Having a BaseSpace account will enable you to monitor runs from the MiSeq and NextSeq500 within any browser. You must have a BaseSpace account before you can use the NextSeq500 so that you can prepare a valid sample sheet. Accounts can be created from the Illumina website.

 

Bookings

To book a time slot on the MiSeq or NextSeq500, check the availability of the instruments using the Agendo MiSeq calendar or Agendo NextSeq calendar, then ring or email and I will reserve a day/days for your experiment. Please note MiSeq 500 and 600 cycle runs will have to be performed over a weekend.

 

Submitting Samples and Quality Control

Ideally we would like a prepared library of either 4nM or 2nM concentration.  This applies to both the MiSeq and NextSeq500. Please pool libraries into one tube. When submitting samples we need to know the average molecular weight of your fragments (done by a bioanalyser or equivalent machine) and the concentration in ng/ul (done using a Qubit or similar fluorescence assay, NOT Nanodrop). It is your responsibility to provide accurate information – failed runs will still be chargeable.

Illumina’s guide to library dilution is available here.
A helpful spreadsheet for calculations is here.
The run request form for the MiSeq and NextSeq500 is available here.

 

Creating Sample Sheets for the MiSeq and NextSeq

MiSeq:  For your sample to be successfully queued, the MiSeq requires a sample sheet in csv format that can either be created in Excel or by using Illumina Experiment Manager (IEM – for PC only). IEM can be downloaded from the Illumina website. If you only have a few indices, the sample sheet can be created locally on the MiSeq during run setup.

NextSeq: Sample sheets must be made using the Prep tab in BaseSpace, they can be edited locally however on the NextSeq.

N.B. Any special or custom sequencing primers that your kit requires must also be supplied when submitting your samples. If in doubt, please ask – it could mean success rather than failure!

 

Hands on Training

First time users will be shown how to use the system by Tim Rostron who will take them through the set up of a run. The next time they use it they will be supervised by Tim while they go through the set up. There after they can use the machines independently. There are however many experienced users in the WIMM and questions about the machines can be addressed to the Illumina Users mailing group.

Cost

The cost of each run will be the University discounted price for the kit and a nominal charge for machine time. This will help us to cover the service contract for the MiSeq. Prices of kits are variable – we have up to date price lists from Illumina here and the additional charge is based on the length of the run.

MiSeq

Kit

Read Length

Total Time

Output

Charge

50 v2

2 × 25 bp

~5.5 hrs

750-850 Mb

£50

150 v3

2 x 75 bp

~20 hrs

3.3-3.8 Gb

£125

300 v2

2 × 150 bp

~24 hrs

4.5-5.1 Gb

£150

500 v2

2 × 250 bp

~39 hrs

7.5-8.5 Gb

£250

600 v3

2 x 300 bp

~55 hrs

13.2-15 Gb

£350

NextSeq500

The cost of a NextSeq500 run will be £200 as of 2015-11-11, regardless of which kit is used. Users must supply their own kits.

Summary of Workflow

  1. Prepare library of DNA/RNA using whichever method is compatible with the Illumina platform (e.g Fluidigm Access Array, NEBNext, Nextera, TruSeq etc)
  2. QC the library – and try to get either a 2nM or 4nM pool
  1. Use Agendo system to choose a free time slot and email Tim Rostron so he can book it for you.
  2. Make a sample sheet
    Use IEM/Excel for MiSeq, save csv file to USB stick
    BaseSpace Prep tab for NextSeq
  3. Bring sample/custom primers/kit components/QC data/USB stick to the laboratory when advised.

Illumina Users Group

A users mailing list has been created and queries about the machines, protocols or problems should be addressed to this group.