title: Large-scale detection of rare variants via pooled multiplexed next-generation sequencing: towards next-generation Ecotilling.
abstract: Common variants, such as those identified by genome-wide association scans, explain only a small proportion of trait variation. Growing evidence suggests that rare functional variants, which are usually missed by genome-wide association scans, play an important role in determining the phenotype. We used pooled multiplexed next-generation sequencing and a customized analysis workflow to detect mutations in five candidate genes for lignin biosynthesis in 768 pooled Populus nigra accessions. We identified a total of 36 non-synonymous single nucleotide polymorphisms, one of which causes a premature stop codon. The most common variant was estimated to be present in 672 of the 1536 tested chromosomes, while the rarest was estimated to occur only once in 1536 chromosomes. Comparison with individual Sanger sequencing in a selected sub-sample confirmed that variants are identified with high sensitivity and specificity, and that the variant frequency was estimated accurately. This proposed method for identification of rare polymorphisms allows accurate detection of variation in many individuals, and is cost-effective compared to individual sequencing.
authors.affiliation: Istituto di Genomica Applicata, 33100 Udine, Italy. marroni@appliedgenomics.org
authors.forename: Fabio
authors.initials: F
authors.lastname: Marroni
authors.forename: Sara
authors.initials: S
authors.lastname: Pinosio
authors.forename: Eleonora
authors.initials: E
authors.lastname: Di Centa
authors.forename: Irena
authors.initials: I
authors.lastname: Jurman
authors.forename: Wout
authors.initials: W
authors.lastname: Boerjan
authors.forename: Nicoletta
authors.initials: N
authors.lastname: Felice
authors.forename: Federica
authors.initials: F
authors.lastname: Cattonaro
authors.forename: Michele
authors.initials: M
authors.lastname: Morgante
mesh_terms: D000483:Alleles; D001483:Base Sequence; D002874:Chromosome Mapping / Q000191:economics / Q000379:methods*; D032461:Chromosomes, Plant / Q000235:genetics; D014644:Genetic Variation / Q000235:genetics*; D018745:Genome, Plant / Q000235:genetics*; D055106:Genome-Wide Association Study / Q000191:economics / Q000379:methods*; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D008031:Lignin / Q000096:biosynthesis; D009154:Mutation; D010641:Phenotype; D010940:Plant Proteins / Q000235:genetics; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D032107:Populus / Q000235:genetics*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
chemical_list: D010940:Plant Proteins; D008031:Lignin