The Sequencing and Microarray Facility arose from the recent merger of SCRI’s Sequencing and Genotyping Service (Clare Booth and Louise Donnelly) and the Microarray Facility (Pete Hedley and Jenny Morris). The Facility is managed by Pete Hedley and provides access for all SCRI staff and external collaborating groups to high-throughput genomics technologies and expertise.

Exploitation of genomics resources

Major investments have been made in the last few years at SCRI and by research groups around the world to establish extensive catalogues of genes, as expressed sequence tags (ESTs) in crop plants and through whole genome sequencing of plant microbial pathogens. In order for these resources to be fully exploited in crop improvement and research programmes, high-throughput monitoring of gene expression is essential.

Determination of co-ordinated changes in gene expression over time, throughout development, or in response to manipulation, stress or pathogens, will allow novel insights into how they respond to environmental challenges. The Microarray Facility was developed by Pete Hedley and Jenny Morris and has led to the exploitation of parallel gene expression profiling by many collaborative groups at SCRI.

Utilisation of state-of-the-art technology

Parallel gene expression technologies currently being developed and utilised at SCRI include microarrays. These tools enable the simultaneous analysis of the activities of many thousands of genes, providing targets for detailed downstream studies.

There are three microarray platforms currently being utilised at SCRI:

• Agilent IJISS oligonucleotide arrays, composed of 60mer probes representing genes from target organisms;
• spotted arrays, which consist of long probes (as PCR products or oligonucleotides) deposited onto modified glass slides
• Affymetrix GeneChip technology, where each gene is represented by a set of short oligonucleotide probes.

The majority of microarrays used on-site are custom designed Agilent arrays, which allow flexible design at low cost, yet generate very high-quality expression data.

RNA is isolated from target tissues and QC’d using an Agilent Bioanalyzer, prior to being labeled as cDNA, and hybridised against the arrayed gene probes. SCRI uses a Genetix QArray Mini spotting robot for in-house microarray fabrication, along with an Agilent Microarray G2565B scanner for data acquisition.

Following data extraction using Agilent FE software, analysis and exploitation of data is subsequently performed using GeneSpring GX (Agilent) software, which allows identification of differentially expressed genes, comparison of expression profiles, clustering analysis and links to metabolic pathways. The Bioinformatics group and BioSS are providing support for data storage and analysis, respectively.

Collaborative projects

One of the primary aims of the facility at SCRI is to provide optimised, low-cost access to high-throughput gene expression technology on-site through collaborative projects.

Microarrays have been successfully applied to both plant pathogen (Pectobacterium) and crop (potato, barley, raspberry and blackcurrant) species. Studies include determination of pathogenicity factors, analysis of host-pathogen interactions, dissection of food quality traits, expression QTL (eQTL) isolation, characterisation of bud dormancy, microRNA expression and single-nucleotide polymorphism (SNP) discovery.

Sequencing and genotyping services

DNA sequencing and genotyping services are run in the Facility at SCRI by Clare Booth, with assistance from Louise Donnelly and Malcolm Macaulay. The majority of fluorescent sequencing and genotyping (SSRs and AFLPs) are analysed using a capillary-based Applied Biosystems ABI 3730. The Facility routinely processes around 120k runs per annum and provides a full template-to-sequence service to SCRI users and external customers (please contact Pete Hedley for further information).

Recently we have also acquired a BeadXpress (Illumina) system which utilises GoldenGate technology for high-throughput single-nucleotide polymorphism (SNP) genotyping. This is currently being applied to barley and, in the near future, to other crop species.

Next generation (non-Sanger) sequencing, including 454 (Roche) and Solexa (Illumina) are being utilised by SCRI research groups for deep transcriptional analysis in crop plant species. Such technologies are exploited through collaboration with Mark Blaxter at The Gene Pool, Edinburgh University.