Abstract
BackgroundTargeting Induced Local Lesions IN Genomes (TILLING) is a reverse genetics approach to directly identify point mutations in specific genes of interest in genomic DNA from a large chemically mutagenized population. Classical TILLING processes, based on enzymatic detection of mutations in heteroduplex PCR amplicons, are slow and labor intensive.ResultsHere we describe a new TILLING strategy in zebrafish using direct next generation sequencing (NGS) of 250bp amplicons followed by Paired-End Low-Error (PELE) sequence analysis. By pooling a genomic DNA library made from over 9,000 N-ethyl-N-nitrosourea (ENU) mutagenized F1 fish into 32 equal pools of 288 fish, each with a unique Illumina barcode, we reduce the complexity of the template to a level at which we can detect mutations that occur in a single heterozygous fish in the entire library. MiSeq sequencing generates 250 base-pair overlapping paired-end reads, and PELE analysis aligns the overlapping sequences to each other and filters out any imperfect matches, thereby eliminating variants introduced during the sequencing process. We find that this filtering step reduces the number of false positive calls 50-fold without loss of true variant calls. After PELE we were able to validate 61.5% of the mutant calls that occurred at a frequency between 1 mutant call:100 wildtype calls and 1 mutant call:1000 wildtype calls in a pool of 288 fish. We then use high-resolution melt analysis to identify the single heterozygous mutation carrier in the 288-fish pool in which the mutation was identified.ConclusionsUsing this NGS-TILLING protocol we validated 28 nonsense or splice site mutations in 20 genes, at a two-fold higher efficiency than using traditional Cel1 screening. We conclude that this approach significantly increases screening efficiency and accuracy at reduced cost and can be applied in a wide range of organisms.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1263-4) contains supplementary material, which is available to authorized users.
Highlights
Targeting Induced Local Lesions IN Genomes (TILLING) is a reverse genetics approach to directly identify point mutations in specific genes of interest in genomic DNA from a large chemically mutagenized population
Using this next generation sequencing (NGS)-TILLING protocol we validated 28 nonsense or splice site mutations in 20 genes, at a two-fold higher efficiency than using traditional Cel1 screening. We conclude that this approach significantly increases screening efficiency and accuracy at reduced cost and can be applied in a wide range of organisms
We generated a library of 9024 ENU-mutagenized F1 male fish by treating wild type (WT) adult male fish with ENU to mutagenize their spermatogonia, crossing them with WT females, and raising F1 male progeny to adulthood [16]
Summary
Targeting Induced Local Lesions IN Genomes (TILLING) is a reverse genetics approach to directly identify point mutations in specific genes of interest in genomic DNA from a large chemically mutagenized population. The classical TILLING process involves PCR amplification of a specific target from the entire mutagenized library with fluorescent labeled primers and CEL1 enzyme digestion of the resulting PCR amplicons to cut any heteroduplexes caused by the presence of induced mutations that occur only once in the entire library. Full-length and rare cleaved fragments are detected by Li-Cor gel analysis, and point mutations are confirmed by Sanger sequencing [17,18,19] This process has proven effective, it is limited to screening a single target at a time
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