Large-scale detection of rare variants via pooled multiplexed next-generation sequencing: Towards next-generation Ecotilling

Common variants, such as those identified by genome-wide association scans, explain only a small proportionof trait variation. Growing evidence suggests that rare functional variants, which are usually missed bygenome-wide association scans, play an important role in determining the phenotype. We used pooledmultiplexed next-generation sequencing and a customized analysis workflow to detect mutations in fivecandidate 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 mostcommon variant was estimated to be present in 672 of the 1536 tested chromosomes, while the rarest wasestimated to occur only once in 1536 chromosomes. Comparison with individual Sanger sequencing in aselected sub-sample confirmed that variants are identified with high sensitivity and specificity, and that thevariant frequency was estimated accurately. This proposed method for identification of rare polymorphismsallows accurate detection of variation in many individuals, and is cost-effective compared to individualsequencing.