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Large‐scale detection of rare variants via pooled multiplexed next‐generation sequencing: towards next‐generation Ecotilling
Author(s) -
Marroni Fabio,
Pinosio Sara,
Di Centa Eleonora,
Jurman Irena,
Boerjan Wout,
Felice Nicoletta,
Cattonaro Federica,
Morgante Michele
Publication year - 2011
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/j.1365-313x.2011.04627.x
Subject(s) - sanger sequencing , biology , genetics , dna sequencing , single nucleotide polymorphism , genome , computational biology , candidate gene , gene , genome wide association study , genetic association , whole genome sequencing , genotype
Summary 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.