Orthology Guided Assembly in highly heterozygous crops: creating a reference transcriptome to uncover genetic diversity in Lolium perenne

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaperJournal articleResearchpeer-review

  • Tom Ruttink, Institute for Agricultural and Fisheries Research (ILVO), Plant Sciences Unit, Growth and Development Research Area, Belgium
  • Lieven Sterck, VIB, Department of Plant Systems Biology; Ghent University, Department of Plant Biotechnology and Bioinformatics, Belgium
  • Antje Rohde, Institute for Agricultural and Fisheries Research (ILVO), Plant Sciences Unit, Growth and Development Research Area, Belgium
  • Christian Bendixen
  • Pierre Rouzé, VIB, Department of Plant Systems Biology; Ghent University, Department of Plant Biotechnology and Bioinformatics, Belgium
  • Torben Asp
  • Yves Van de Peer, VIB, Department of Plant Systems Biology; Ghent University, Department of Plant Biotechnology and Bioinformatics, Belgium
  • Isabel Roldán-Ruiz, Institute for Agricultural and Fisheries Research (ILVO), Plant Sciences Unit, Growth and Development Research Area, Belgium
Despite current advances in next-generation sequencing data analysis procedures, de novo assembly of a reference sequence required for SNP discovery and expression analysis is still a major challenge in genetically uncharacterized, highly heterozygous species. High levels of polymorphism inherent to outbreeding crop species hamper De Bruijn Graph-based de novo assembly algorithms, causing transcript fragmentation and the redundant assembly of allelic contigs. If multiple genotypes are sequenced to study genetic diversity, primary de novo assembly is best performed per genotype to limit the level of polymorphism and avoid transcript fragmentation. Here, we propose an Orthology Guided Assembly procedure that first uses sequence similarity (tBLASTn) to proteins of a model species to select allelic and fragmented contigs from all genotypes and then performs CAP3 clustering on a gene-by-gene basis. Thus, we simultaneously annotate putative orthologues for each protein of the model species, resolve allelic redundancy and fragmentation and create a de novo transcript sequence representing the consensus of all alleles present in the sequenced genotypes. We demonstrate the procedure using RNA-seq data from 14 genotypes of Lolium perenne to generate a reference transcriptome for gene discovery and translational research, to reveal the transcriptome-wide distribution and density of SNPs in an outbreeding crop and to illustrate the effect of polymorphisms on the assembly procedure. The results presented here illustrate that constructing a non-redundant reference sequence is essential for comparative genomics, orthology-based annotation and candidate gene selection but also for read mapping and subsequent polymorphism discovery and/or read count-based gene expression analysis
Original languageEnglish
JournalPlant Biotechnology Journal
Volume11
Issue5
Pages (from-to)605-607
Number of pages3
ISSN1467-7644
DOIs
Publication statusPublished - Jun 2013

    Research areas

  • de novo transcriptome assembly, genetic diversity, Lolium perenne, heterozygosity, crops, SNP

See relations at Aarhus University Citationformats

ID: 52775876