Next-generation sequencing and genotyping

‌IBERS has made substantial recent investments in next-generation sequencing (NGS) and genotyping infrastructure, resulting in the formation of a new Translational Genomics facility based at the Gogerddan campus.

Dr Matt Hegarty, who oversees this facility, is part of the Breeding Methodologies team and is dedicated to using this technology to assist breeding programmes across IBERS research. Based around the Illumina HiScanSQ platform, there are three main areas currently being developed for IBERS research:

RAD sequencing

RAD (Restriction-associated DNA) sequencing or RADseq involves generation of restriction fragments and ligation of Illumina NGS adaptors to sequence a small (50-100bp) region adjacent to a restriction site. These sequences can then be mined for SNPs or other polymorphisms. By selecting an enzyme that cuts infrequently in the genome, each restriction site can thus be sequenced at high coverage using a fraction of the capacity of an Illumina lane.

Therefore, by tagging each sample with a unique barcoded adaptor, multiple (50-100) individuals can be genotyped in a single lane. IBERS is currently pursuing RADseq in mapping families of Lolium and a draft genetic linkage map based on RADseq data from a partnership with Floragenex (Oregon) is under construction.

SNP discovery

NGS technology enables the rapid generation of large sequence datasets, and thus resequencing approaches to identifying putative SNP loci by comparative genomics.

Whilst full genome sequence is not yet available for many IBERS study species, we have been pursuing comparative transcriptomics as a means for SNP discovery. Transcriptome sequencing of five diverse Lolium genotypes has led to identification of a large pool of putative SNPs which can serve as a resource for marker development.

As more genotypes are sequenced, they will be incorporated into this analysis to identify novel polymorphisms. Similar efforts are planned for other systems such as red clover.

Infinium SNP genotyping

The HiScanSQ platform in place at IBERS is not only capable of NGS, but also processing Illumina’s GoldenGate (<3000 loci) or Infinium (>3000 loci) SNP genotyping microarrays.These use allele-specific oligonucleotides as well as locus-specific probes to produce a binary genotype for SNP loci when interrogated with genomic DNA.

Depending upon the number of loci assayed, 4-24 individuals may be genotyped on a single microarray for up to 2.5 million loci (via Infinium) with a standard throughput of 8 arrays scanned per day.

Whilst commercial assays are not available for many of the plant species studied at IBERS, we have recently developed a custom assay for Lolium using ~3800 SNP loci discovered via comparative transcriptome sequencing, as detailed above. Initial experiments to validate this assay are underway.