Next Generation Sequencing (NGS)

Ion S5 prime DNA sequencer machine

Our Next Generation Sequencing (NGS) facility provides sequencing for diagnostics, research, and clinical trials.

The facility is supported by partners across Sheffield and has three NGS systems: an Illumina MiSeq and HiSeq 2500, and a Life Technologies Ion GeneStudio S5 Prime. We provide NGS diagnostic services to the NHS in a cost effective and quality driven way within a UKAS (ISO15189:2012) accredited laboratory, ensuring a better service for patients within Yorkshire and Humber and beyond.

We are also open to researchers undertaking either basic research or clinical trials. The NGS facility serves many users at the University of Sheffield but can also receive samples from further afield. Whether you require NGS for diagnostics or research, for small or large projects, we welcome discussions about how the laboratory can support your needs.

NGS services – diagnostic

We currently have NGS services that cover connective tissue disorders, inborn errors of metabolism, hereditary cancers, neurodegenerative disorders and bone marrow failure/Fanconi anaemia. Details of these services are given in the document below:

If you wish to explore any further clinical developments please contact us to discuss how we could help.

NGS services – research

We offer a complete sequencing service including library preparation, sequencing, and bioinformatics, or we can provide standalone sequencing of user-prepared libraries. Please see below for details of our Illumina sequencing service. We can make a wide variety of RNA and DNA libraries, and should be able to accommodate most library preparation requests, including:

  • Whole genome
  • RNA-Seq
  • Capture enrichment e.g. whole exome, custom gene panels
  • Amplicon e.g. 16S metagenomics, custom gene panels

Note that we can prepare capture or amplicon libraries for custom panels designed around your genes of interest, which can be very cost-effective compared to e.g. whole exome sequencing. Capture libraries for exomes or gene panels can also be prepared and sequenced to diagnostic standards.

Bioinformatics support is also available. We can provide a range of services including (but not limited to):

  • Data management, data sharing (including confidential data)
  • Analysis of NGS data, statistical analysis of large data sets, data visualisation

Please contact us if you require a quote. For more information on clinical trials, please see the relevant section of our Laboratory Services webpage.

Illumina sequencing for research projects

We currently offer Next Generation Sequencing on the Illumina HiSeq2500 and MiSeq platforms, although researchers interested in using our Ion GeneStudio S5 Prime are also welcome to get in touch.

HiSeq and MiSeq sequencing runs can be set up as single read or paired end, with a minimum read length of 25 bp. Refer to the table below for maximum read lengths by platform. We usually require our users to submit enough sample for a full flowcell (2 lanes for Rapid Run, 8 lanes for High Output; MiSeq flowcells contain only 1 lane). If you require less sequencing than this, we may be able to include your sample on a mixed run, but waiting times will be unpredictable. See the table for a guide of how many reads to expect from each run type. Quotes are available upon request.

You are welcome to consult us if you are unsure how much sequencing you require, or for advice on how to get the most out of your sequencing budget. For instance, single reads of 50-100bp are sufficient for most RNA-Seq experiments, and offer substantial savings over longer and paired-end reads.

Approximate number of clusters passing filter, by platform and run type

Type of runApprox. number of clusters
passing filter per lane (per run)*
Maximum read length
HiSeq2500
Rapid Run (2 lanes)120-150 Million (240-300 Million)2 x 250 bp
High Output (8 lanes)160-270 Million (1.28-2.16 Billion)2 x 100 bp
MiSeq
v2 chemistry
Standard
Micro
Nano

10-15 Million
4 Million
1 Million
2 x 250 bp
v3 chemistry20-25 Million2 x 300 bp

* Single read runs return one read per cluster. Paired end runs return two reads per cluster.

Sequencing costs include quality control (QC) on your pooled sample (Tapestation and Qubit analysis). Additional or standalone QC requests are charged at £5 per sample, and we can quantify by qPCR at additional cost.

Please try to provide at least 20 µl of your library at a concentration of 4 nM or above. We may be able to work with less than this , but please get in touch to confirm this. We will also need a list of your sample/index combinations.

By default we include a 1% PhiX spike-in to monitor run quality. Please notify us if you are submitting low diversity libraries, in which case we will load a lower amount and spike in a higher % PhiX. Failure to do so may result in a low number of reads passing filter.Hi Seq 2500 DNA sequencing machine

Data will be provided in fastq format and transferred to you using Amazon S3 public storage. The link to your data will only be shared with you. On receipt your data can be checked by the md5 checksum provided to ensure fidelity. Once you have received your data it will be deleted from Amazon S3 but will remain on our private servers for six months. After six months the data will be deleted to ensure efficient use of storage.

Please note that we cannot accept responsibility for failed sequencing due to library failure if the libraries have been prepared and pooled outside of the Sheffield Genetics Service.

For quotes/enquiries relating to clinical trials, please contact Sara Morosini, and for all other research projects please contact Richard Kirk.

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