Next generation sequencing for molecular breeding

The overall objective of WP3 is to provide a common technology platform to provide all relevant partners with access to Next Generation Sequencing (NGS) technologies.   The goal is the employment of the full power of NGS to facilitate and speed up gene discovery and breeding in the three species of interest. This is accomplished by means of a close interaction between the SME IGA Technology Services, which is a leading European provider of NGS services, and all the other WATBIO partners.  The centralised service platform allows standardisation of data and data analysis making integration of data from different partners easier and enabling considerable efficiencies in terms of costs and use of resources. The NGS data are produced using the Illumina HiSeq 2000 sequencing apparatus.

We are using a web interface through which partners submit experiments, describe the samples and the type of analyses required and track the samples during the lab process. The system is linked to the IGATS LIMS (Laboratory Information Management System) to provide a view on the status of the sample analysis.

The research includes re-sequencing in poplar and miscanthus, either at the whole genome level or of selected fractions after enrichment using hybrid capture to identify SNP and structural variants either from single individuals at high coverage or from multiple individuals at low coverage after indexing and pooling. Custom enrichment kits targeting either specific genomic regions (e.g. QTL intervals to discover SNPs for association mapping) or specific genome fractions (e.g. exome) are produced using the Illumina technology.  Alignments and SNP and SV discovery will be performed using the CLCBio Genomic Workbench.

In arundo, we are sequencing mRNA using the Illumina technology for the purpose of either sequencing transcriptomes de novo and building a reference gene set for gene discovery, gene expression and SNP discovery (arundo).

In poplar, we are using PCR amplification from multiple pools of 80-100 diploid heterozygous individuals to search for rare variants affecting protein structure and function.  Wherever a variant of interest is detected we will sequence single individuals in each pool to identify the carriers of the mutations.  Depending on information on protein structure that is available to allow for homology-based modelling, we infer the effects of missense mutations on protein.  The research will result in a specific kit for the genotyping of all exonic regions using sequencing and enrichment through hybrid capture in poplar to scan specifically all genes.