Dataset Information

Characterization of squamate olfactory receptor genes and their transcripts by the high-throughput sequencing approach.

ABSTRACT: The olfactory receptor (OR) genes represent the largest multigene family in the genome of terrestrial vertebrates. Here, the high-throughput next-generation sequencing (NGS) approach was applied to characterization of OR gene repertoires in the green anole lizard Anolis carolinensis and the Japanese four-lined ratsnake Elaphe quadrivirgata. Tagged polymerase chain reaction (PCR) products amplified from either genomic DNA or cDNA of the two species were used for parallel pyrosequencing, assembling, and screening for errors in PCR and pyrosequencing. Starting from the lizard genomic DNA, we accurately identified 56 of 136 OR genes that were identified from its draft genome sequence. These recovered genes were broadly distributed in the phylogenetic tree of vertebrate OR genes without severe biases toward particular OR families. Ninety-six OR genes were identified from the ratsnake genomic DNA, implying that the snake has more OR gene loci than the anole lizard in response to an increased need for the acuity of olfaction. This view is supported by the estimated number of OR genes in the Burmese python's draft genome (?280), although squamates may generally have fewer OR genes than terrestrial mammals and amphibians. The OR gene repertoire of the python seems unique in that many class I OR genes are retained. The NGS approach also allowed us to identify candidates of highly expressed and silent OR gene copies in the lizard's olfactory epithelium. The approach will facilitate efficient and parallel characterization of considerable unbiased proportions of multigene family members and their transcripts from nonmodel organisms.

SUBMITTER: Dehara Y

PROVIDER: S-EPMC3342882 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Characterization of squamate olfactory receptor genes and their transcripts by the high-throughput sequencing approach.

Dehara Yuki Y Hashiguchi Yasuyuki Y Matsubara Kazumi K Yanai Tokuma T Kubo Masahito M Kumazawa Yoshinori Y

Genome biology and evolution 20120417 4

The olfactory receptor (OR) genes represent the largest multigene family in the genome of terrestrial vertebrates. Here, the high-throughput next-generation sequencing (NGS) approach was applied to characterization of OR gene repertoires in the green anole lizard Anolis carolinensis and the Japanese four-lined ratsnake Elaphe quadrivirgata. Tagged polymerase chain reaction (PCR) products amplified from either genomic DNA or cDNA of the two species were used for parallel pyrosequencing, assemblin ...[more]

PMID: 22511035

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Project description:Comprehensive microbial risk assessment requires high-throughput quantification of diverse microbial risks in the environment. Current metagenomic next-generation sequencing approaches can achieve high-throughput detection of genes indicative of microbial risks but lack quantitative capabilities. This study developed and tested a quantitative metagenomic next-generation sequencing (qmNGS) approach. Numerous xenobiotic synthetic internal DNA standards were used to determine the sequencing yield (Yseq) of the qmNGS approach, which can then be used to calculate absolute concentration of target genes in environmental samples based on metagenomic sequencing results. The qmNGS approach exhibited excellent linearity as indicated by a strong linear correlation (r2 = 0.98) between spiked and detected concentrations of internal standards. High-throughput capability of the qmNGS approach was demonstrated with artificial Escherichia coli mixtures and cattle manure samples, for which 95 ± 3 and 208 ± 4 types of antibiotic resistance genes (ARGs) were detected and quantified simultaneously. The qmNGS approach was further compared with quantitative real-time PCR (qPCR) and demonstrated comparable levels of accuracy and less variation for the quantification of six target genes (16S, tetO, sulI, tetM, ermB, and qnrS). IMPORTANCE Monitoring and comprehensive assessment of microbial risks in the environment require high-throughput gene quantification. The quantitative metagenomic NGS (qmNGS) approach developed in this study incorporated numerous xenobiotic and synthetic DNA internal standard fragments into metagenomic NGS workflow, which are used to determine a new parameter called sequencing yield that relates sequence base reads to absolute concentration of target genes in the environmental samples. The qmNGS approach demonstrated excellent method linearity and comparable performance as the qPCR approach with high-throughput capability. This new qmNGS approach can achieve high-throughput and accurate gene quantification in environmental samples and has the potential to become a useful tool in monitoring and comprehensively assessing microbial risks in the environment.

Dataset Information

Characterization of squamate olfactory receptor genes and their transcripts by the high-throughput sequencing approach.

Publications

Characterization of squamate olfactory receptor genes and their transcripts by the high-throughput sequencing approach.

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