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Characterizing Benzo[a]pyrene-induced lacZ mutation spectrum in transgenic mice using next-generation sequencing.

ABSTRACT: BACKGROUND:The transgenic rodent mutation reporter assay provides an efficient approach to identify mutagenic agents in vivo. A major advantage of this assay is that mutant reporter transgenes can be sequenced to provide information on the mode of action of a mutagen and to identify clonally expanded mutations. However, conventional DNA sequence analysis is laborious and expensive for long transgenes, such as lacZ (3096 bp), and is not normally implemented in routine screening. METHODS:We developed a high-throughput next-generation sequencing (NGS) approach to simultaneously sequence large numbers of barcoded mutant lacZ transgenes from different animals. We collected 3872 mutants derived from the bone marrow DNA of six Muta™Mouse males exposed to the well-established mutagen benzo[a]pyrene (BaP) and six solvent-exposed controls. Mutants within animal samples were pooled, barcoded, and then sequenced using NGS. RESULTS:We identified 1652 mutant sequences from 1006 independent mutations that underwent clonal expansion. This deep sequencing analysis of mutation spectrum demonstrated that BaP causes primarily guanine transversions (e.g. G:C ? T:A), which is highly consistent with previous studies employing Sanger sequencing. Furthermore, we identified novel mutational hotspots in the lacZ transgene that were previously uncharacterized by Sanger sequencing. Deep sequencing also allowed for an unprecedented ability to correct for clonal expansion events, improving the sensitivity of the mutation reporter assay by 50 %. CONCLUSION:These results demonstrate that the high-throughput nature and reduced costs offered by NGS provide a sensitive and fast approach for elucidating and comparing mutagenic mechanisms of various agents among tissues and enabling improved evaluation of genotoxins.

SUBMITTER: Beal MA

PROVIDER: S-EPMC4617527 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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Characterizing Benzo[a]pyrene-induced lacZ mutation spectrum in transgenic mice using next-generation sequencing.

Beal Marc A MA Gagné Rémi R Williams Andrew A Marchetti Francesco F Yauk Carole L CL

BMC genomics 20151019

<h4>Background</h4>The transgenic rodent mutation reporter assay provides an efficient approach to identify mutagenic agents in vivo. A major advantage of this assay is that mutant reporter transgenes can be sequenced to provide information on the mode of action of a mutagen and to identify clonally expanded mutations. However, conventional DNA sequence analysis is laborious and expensive for long transgenes, such as lacZ (3096 bp), and is not normally implemented in routine screening.<h4>Method ...[more]

PMID: 26481219

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Project description:Purpose: This study aimed to identify differentially expressed genes and transcripts in zebrafish embryos and larvae following benzo[a]pyrene (BaP) exposure. Methods: Adult zebrafish (2 males × 4 females, N=6 replicate tanks for each treatment) were acclimated for 7 days in an 818 Low Temp Illuminated Incubator (Precision Scientific, Chennai, India) at 28.5°C. Next, adult fish were waterborne exposed to control or 50 μg/L (ppb) BaP for 7 days; ethanol was used as vehicle solvent, and final ethanol concentration was 0.1 mL/L (100 ppm) in all treatment groups. This dose of ethanol is not teratogenic to zebrafish. Water was changed and/or re-dosed daily. From day 7 to 11 of the parental exposure, eggs were collected, counted, and raised in normal conditions (control) or continuously exposed to 50 μg/L BaP until 3.3 and 96 hours post fertilization (hpf). At 3.3 or 96 hpf, embryos (200/pool) or larvae (10/pool) were collected and pooled. Total RNA was isolated for transcriptomic RNA sequencing with Illumina HiSeq2000 (2X100bp). RNA-seq reads were uploaded to the galaxy platform https://main.g2.bx.psu.edu/. RNA-seq reads were trimmed, filtered, and aligned to the zebrafish genome (Danio_rerio.Zv9.68) with the Tophat for Illumina tool. Counting and annotation of RNA-seq reads were performed with Partek Genomics Suite version 6.11. Refseq Transcripts (2013-04-10) and Ensembl Transcripts release 70 databases were used for gene and transcript annotation. Differential expression of gene and transcript reads between treatments was analyzed with R package EdgeR. Genes/transcripts with false discovery rate (FDR) less than 0.05 and absolute fold change greater than 1.5 were considered as significant. Differentially expressed genes were defined as genes with altered expression at either gene or transcript level. Results: Differential expression analysis with EdgeR revealed that gene expression was vastly different between 3.3 hpf zebrafish embryos and 96 hpf larvae. Using Refseq annotation, we found that 10644 out of 13950 transcribed zebrafish genes were differentially expressed between the two developmental time-points, with 5961 up-regulated genes and 4683 down-regulated genes in 96 hpf larvae compared with 3.3 hpf embryos. Similarly, using Ensembl annotation, 16529 out of 19886 transcribed zebrafish genes were differentially expressed, with 9318 up-regulated genes and 7211 down-regulated genes in 96 hpf larvae compared with 3.3 hpf embryos. In 3.3 hpf embryos, four genes and seven transcripts were differentially expressed after BaP exposure. In 96 hpf larvae, 447 and 484 zebrafish genes were significantly up- and down-regulated, respectively, by BaP exposure. Conclusions: Parental and developmental BaP exposure caused gene expression changes in zebrafish embryos and larvae. Illumina HiSeq2000 deep sequencing was used to generate transcriptomic profiles for BaP-exposed 3.3 hpf zebrafish embryos (n=3 for control, n=3 for BaP) and 96 hpf larvae (n=2 for control, n=2 for BaP).

Dataset Information

Characterizing Benzo[a]pyrene-induced lacZ mutation spectrum in transgenic mice using next-generation sequencing.

Publications

Characterizing Benzo[a]pyrene-induced lacZ mutation spectrum in transgenic mice using next-generation sequencing.

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