Equipment
Successful applicants to the facility are provided with training in how to prepare samples and how to use the equipment needed for their project.
If you are interested in gaining access to our facilities, expertise and equipment here at NEOF, please contact the Facility Manager Dr Deborah Dawson.
If you are a member of the University of Sheffield and wish to use the equipment, please contact senior technician Rachel Tucker.
The NERC Environmental Omics Visitor Facility is based in a very well-equipped large molecular genetics laboratory. We also have two separate clean rooms - one is used for eDNA extraction and typing, and the second room is used for the pre-PCR stages during ancient DNA work and SNP typing.
We have numerous multi-block PCR machines. For sample storage, we have a walk-in refrigerated 4°C room, two -18°C freezer rooms, 14 standalone -18°C freezers and three -80°C freezers.
A suite of PC computers is available along with a managed computing cluster that makes available commonly used software and scripts. We also provide training in how to analyse data (your own data, or that obtained in our facility).
Some of the equipment available at the facility includes:
MiSeq Sequencer (Illumina) : Next generation sequencing, metabarcoding, MHC-typing.
We commonly use the MiSeq for obtaining sequences from mixed samples where multiple species are present - for example, DNA-based studies of diet from faecal material. It is also used for investigations of mixed bacterial communities, such as those found in water pipes. We use enrichment techniques to isolate microsatellites and the repeat-enriched fragments produced are sequenced on the MiSeq.
PacBio Sequel System: Whole genome sequencing.
The long reads produced allow resequencing to improve existing genome assemblies, haplotyping of diploid data and epigenome sequencing.
ABI 3730 48-capillary DNA analyzer (Applied Biosystems): High-throughput automated genotyping of microsatellite markers and Sanger sequencing.
Common uses include studies of parentage and population structure. The ABI is also used for the DNA sex-typing of birds, and regularly used for the Sanger sequencing of reference species expected to be detected in diet studies.
During microsatellite genotyping, we use a 4-dye ROX size standard set (with 6-FAM, NED, and HEX fluoro-labels available for markers) and the 5-dye LIZ size standard set (with 6-FAM, NED, VIC and PET).
KASP SNP genotyping platform (LGC): example uses include linkage mapping, fine scale mapping and parentage studies.
We have several years' experience in SNP typing obtained using the Illumina Beadxpress platform. In 2014 we became an Illumina Certified Service Provider (CSPro) for Veracode SNP genotyping on the BeadXpress platform; the first in Europe.
In 2016 we replaced our Illumina BeadXpress platform (the equipment and chemistry have been discontinued by the manufacturer) with the LGC SNPline LITE system (more information here). This platform performs KASP genotyping (more information here). It is rapid, accurate, cost-effective and flexible.
Our platform is equally well suited to projects that require a few SNPs being typed in thousands of individuals, to those where several hundred SNPs need to be typed in perhaps a few hundred samples. For example, it was recently used to fine map and identify the red locus, responsible for carotenoid-based pigmentation in birds (Mundy et al. 2016 Current Biology 26:1435-1440; NBAF project NBAF961).
The platform is ideal for small-medium SNP typing projects such as building low-density linkage maps, parentage analysis, candidate-gene association studies or confirming results of a GWAS experiment.
Very high-throughput methods (ie where thousands or millions of SNPs are typed) are better served by other methodologies such as the Illumina Infinium or Affymetrix Axiom high density SNP chip arrays; we do not provide these services.
Meridian Dispenser liquid handing system (LGC)
This liquid handling robot is used with the KASP SNP typing system.
QuantStudio 12K Flex Real-time PCR System - the all-in-one qPCR instrument.
The QuantStudio platform is a high-throughput platform that can be used to perform a very wide variety of applications.
It is capable of generating 12,000 datapoints in a single run, and upto 2 million datapoints in a single day, which means qPCR / gene expression studies can be scaled up from typing a few genes in a few individuals to typing tens or hundreds of genes in population-level sampling schemes. Other ongoing applications include telomere length assays in studies of aging and environmental DNA (eDNA) typing.
The QuantStudio is also very effective for quantifying the amount of DNA in libraries that require normalisation prior to next-generation sequencing. For example, prior to pooling samples or libraries that will be run in a single cell of a sequencer.
Qubit Fluorometer (ThermoFisher Scientific)
The Qubit is an excellent piece of kit for quantifying the amount of DNA or RNA in a sample. It is rapid and accurate, even for low concentration samples. This makes it especially useful for studies working on small amounts of DNA – for example where samples were collected from old, degraded or low yielding tissues.
TapeStation 4200 (Agilent)
The TapeStation is primarily designed for assaying the quantity and quality of DNA and RNA.
It is possible to analyse the quality of DNA and RNA libraries, and analyse the amount of DNA/RNA degradation in up to 96 samples at a time. It is ideal in obtaining the size of PCR products prior to next-generation sequencing, for example, to identify which amplicons to select and sequence on the MiSeq sequencer.
Blue Pippin (Sage Science)
The pippin is used to check the quantity and quality of DNA and RNA.
It is used to assess samples before they are sequenced on the PacBio.
Biomek 2000 Robotic Liquid-handling Workstation with peripherals including a plate-reading fluorometer.
This robot is used for aliquoting DNA samples and setting up PCR reactions for large scale projects involving hundreds to thousands of samples.
It has been used on our research projects studying badger populations and is useful for genome mapping work.
Bioanalyzer (Agilent)
The bioanalyzer has similar uses to the TapeStation and Pippin. It has been used to check the quantity and quality of DNA and RNA, such as assessing libraries before they are sequenced on the MiSeq.
The Bioanalyzer is used for assessing samples before they are sequenced on the PacBio.
Nanodrop 8000 (Thermo Scientific)
Used for obtaining the concentration of genomic DNA and RNA, following extractions from various sample types.
We also have a fluorimeter and spectrophometer for assessing DNA and RNA concentrations.
We use these three pieces of kit for checking the concentration of genomic DNA extracted from various sources, such as mouth swabs, plants, museum specimens, blood, feathers and faecal material.