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

Tom Ruttink, Lieven Sterck, Antje Rohde, Christian Bendixen, Pierre Rouzé, Torben Asp, Yves Van de Peer, Isabel Roldan-Ruiz

    Onderzoeksoutput: Bijdrage aan tijdschriftA1: Web of Science-artikel

    Uittreksel

    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.
    TaalEngels
    TijdschriftPlant Biotechnology Journal
    Volume11
    Exemplaarnummer5
    Pagina's (van-tot)605-617
    Aantal pagina's13
    ISSN1467-7644
    StatusGepubliceerd - 2013

    Trefwoorden

    • B110-bioinformatica
    • B225-plantengenetica

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