Dataset Information

Multiplex exome sequencing reveals genome-wide frequency and distribution of mutations in the 'Micro-Tom' Targeting Induced Local Lesions in Genomes (TILLING) mutant library.

ABSTRACT: While the 'Micro-Tom' TILLING mutant library is used for a wide range of purposes, including both basic research of gene function and breeding of commercial cultivars, genome-wide distribution and frequency of mutations have not yet been thoroughly elucidated on a population scale. In this study, we developed a 96-plex exome sequencing method to identify and analyze mutations within the TILLING mutants that were developed in the University of Tsukuba. First, an Illumina paired-end sequencing coupled with 96-plex exome capture resulted in the acquisition of an exome sequence dataset with an average read count of 5.6 million for the 95 mutants. Over 98% of the capture target region could be covered by the short reads with an averaged read depth of 12.8, which enabled us to identify single nucleotide polymorphisms and Indels in a genome-wide manner. By subtracting intra-cultivar DNA variations that are present between wild-type 'Micro-Tom' lines, we identified 241,391 mutation candidates in 95 mutant individuals. Of these, 64,319 and 6,480 mutations were expected to cause protein amino acid substitutions or premature stop codon, respectively. Based on the exome mutation dataset, a mutant line designated 'TOMJPW601' was found to carry a premature stop codon mutation (W261*) in a putative auxin influx carrier gene SlLAX1 (Solyc09G014380), consistent with our previous report of its curly leaf phenotype. Our results suggested that a population-scale mutation database developed by multiplexed exome sequencing could be used for in silico mutant screening, which in turn could contribute to both gene function research and breeding programs.

SUBMITTER: Yano R

PROVIDER: S-EPMC6978505 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Multiplex exome sequencing reveals genome-wide frequency and distribution of mutations in the 'Micro-Tom' Targeting Induced Local Lesions in Genomes (TILLING) mutant library.

Yano Ryoichi R Hoshikawa Ken K Okabe Yoshihiro Y Wang Ning N Dung Pham Thi PT Imriani Pulungan Sri PS Shiba Hiroshi H Ariizumi Tohru T Ezura Hiroshi H

Plant biotechnology (Tokyo, Japan) 20191201 4

While the 'Micro-Tom' TILLING mutant library is used for a wide range of purposes, including both basic research of gene function and breeding of commercial cultivars, genome-wide distribution and frequency of mutations have not yet been thoroughly elucidated on a population scale. In this study, we developed a 96-plex exome sequencing method to identify and analyze mutations within the TILLING mutants that were developed in the University of Tsukuba. First, an Illumina paired-end sequencing cou ...[more]

PMID: 31983876

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Project description:BackgroundMycoplasma hominis is a human urogenital pathogen involved in gynaecological, neonatal and extra-genital infections. However, no versatile genetic tools are currently available to study the pathogenicity of this bacterium. Targeting-Induced Local Lesions IN Genomes (TILLING) is a reverse-genetic method that combines point mutations induced by chemical mutagenesis with a DNA screening technique. We used ethyl methanesulfonate (EMS) that introduces C-G to T-A transition mutations to generate a library of M. hominis mutants. As a proof of concept, mutagenized organisms were screened for mutations in two target genes previously associated with the mycoplasma pathogenicity, the vaa gene encoding an adhesin lipoprotein and the oppA gene encoding the main ectoATPase of the bacterium. The resulting mutants were evaluated using functional assays, an adhesion to HeLa cell assay for vaa-mutants and an ATPase activity test for oppA-mutants.ResultsA 1200-clone library was generated by exposing M. hominis PG21 to 9 mg/mL EMS for 3 h. To identify mutants of interest, targeted gene fragments were amplified, heat-denatured, slowly reannealed and digested with the mismatch-specific endonuclease ENDO1. If multiple alleles were present in the PCR amplicons, these alleles formed heteroduplexes during reannealing that were specifically cleaved by ENDO1 at mismatching positions. A total of four vaa-mutants and two oppA-mutants harbouring missense mutations were obtained and fully sequenced. Zero to eight additional mutations were identified in the genomes of each mutant. The vaa-mutants were tested for adhesion to immobilized HeLa cells but their adhesion was not significantly different from the adhesion of M. hominis PG21. One of the two oppA-mutants that were tested for ATPase activity presented a higher affinity for its ATP substrate than the parental strain.ConclusionFor the first time, we demonstrated that M. hominis gene-targeted mutants could be successfully obtained using this TILLING strategy. In the absence of robust genetic tools for studying M. hominis, the TILLING strategy that can target any gene of the genome could help to elucidate gene functions and to better understand the pathogenesis of this human pathogenic species.

Dataset Information

Multiplex exome sequencing reveals genome-wide frequency and distribution of mutations in the 'Micro-Tom' Targeting Induced Local Lesions in Genomes (TILLING) mutant library.

Publications

Multiplex exome sequencing reveals genome-wide frequency and distribution of mutations in the 'Micro-Tom' Targeting Induced Local Lesions in Genomes (TILLING) mutant library.

Similar Datasets

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