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Discovery of rare mutations in populations: TILLING by sequencing

Identifiers: SRA: SRP006801
UCDTILL: TILLING by Sequencing
Study Type: 
Whole Genome Sequencing
Abstract: Discovery of rare mutations in populations requires methods, such as TILLING, for processing and analyzing many individuals in parallel. Previous TILLING protocols employed enzymatic or physical discrimination of heteroduplexed from homoduplexed target DNA. Using mutant populations of rice (Oryza sativa) and wheat (Triticum durum and T. aestivum), we developed a method based on Illumina sequencing of target genes amplified from multi-dimensionally pooled templates representing 768 individuals per experiment. Parallel processing of sequencing libraries was aided by unique tracer sequences and barcodes allowing flexibility in the number and pooling arrangement of targeted genes, species, and pooling scheme. Sequencing reads were processed and aligned to the reference to identify possible single nucleotide changes, which were then evaluated for frequency, sequencing quality, intersection pattern in pools, and statistical relevance to produce a Bayesian score with an associated confidence threshold. Discovery was robust both in rice and wheat using either 2D or 3D pooling schemes. The method compared favorably to other molecular and computational approaches, providing high sensitivity and specificity.
External Link: /pubmed:21531898

Related SRA data

Experiments:
87 ( 4 samples )
Runs:
87 (50.9Gbp; 26.4Gb)