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Measuring differential gene expression by short read sequencing: quantitative comparison to 2-channel gene expression microarrays.

ABSTRACT: High-throughput cDNA synthesis and sequencing of poly(A)-enriched RNA is rapidly emerging as a technology competing to replace microarrays as a quantitative platform for measuring gene expression.Consequently, we compared full length cDNA sequencing to 2-channel gene expression microarrays in the context of measuring differential gene expression. Because of its comparable cost to a gene expression microarray, our study focused on the data obtainable from a single lane of an Illumina 1 G sequencer. We compared sequencing data to a highly replicated microarray experiment profiling two divergent strains of S. cerevisiae.Using a large number of quantitative PCR (qPCR) assays, more than previous studies, we found that neither technology is decisively better at measuring differential gene expression. Further, we report sequencing results from a diploid hybrid of two strains of S. cerevisiae that indicate full length cDNA sequencing can discover heterozygosity and measure quantitative allele-specific expression simultaneously.

SUBMITTER: Bloom JS

PROVIDER: S-EPMC2686739 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Measuring differential gene expression by short read sequencing: quantitative comparison to 2-channel gene expression microarrays.

Bloom Joshua S JS Khan Zia Z Kruglyak Leonid L Singh Mona M Caudy Amy A AA

BMC genomics 20090512

<h4>Background</h4>High-throughput cDNA synthesis and sequencing of poly(A)-enriched RNA is rapidly emerging as a technology competing to replace microarrays as a quantitative platform for measuring gene expression.<h4>Results</h4>Consequently, we compared full length cDNA sequencing to 2-channel gene expression microarrays in the context of measuring differential gene expression. Because of its comparable cost to a gene expression microarray, our study focused on the data obtainable from a sing ...[more]

PMID: 19435513

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Project description:The common marmoset (Callithrix jacchus) is a non-human primate that could prove useful as human pharmacokinetic and biomedical research models. The cytochromes P450 (P450s) are a superfamily of enzymes that have critical roles in drug metabolism and disposition via monooxygenation of a broad range of xenobiotics; however, information on some marmoset P450s is currently limited. Therefore, identification and quantitative analysis of tissue-specific mRNA transcripts, including those of P450s and flavin-containing monooxygenases (FMO, another monooxygenase family), need to be carried out in detail before the marmoset can be used as an animal model in drug development. De novo assembly and expression analysis of marmoset transcripts were conducted with pooled liver, intestine, kidney, and brain samples from three male and three female marmosets. After unique sequences were automatically aligned by assembling software, the mean contig length was 718 bp (with a standard deviation of 457 bp) among a total of 47,883 transcripts. Approximately 30% of the total transcripts were matched to known marmoset sequences. Gene expression in 18 marmoset P450- and 4 FMO-like genes displayed some tissue-specific patterns. Of these, the three most highly expressed in marmoset liver were P450 2D-, 2E-, and 3A-like genes. In extrahepatic tissues, including brain, gene expressions of these monooxygenases were lower than those in liver, although P450 3A4 (previously P450 3A21) in intestine and P450 4A11- and FMO1-like genes in kidney were relatively highly expressed. By means of massive parallel long-read sequencing and short-read technology applied to marmoset liver, intestine, kidney, and brain, the combined next-generation sequencing analyses reported here were able to identify novel marmoset drug-metabolizing P450 transcripts that have until now been little reported. These results provide a foundation for mechanistic studies and pave the way for the use of marmosets as model animals for drug development in the future.

Dataset Information

Measuring differential gene expression by short read sequencing: quantitative comparison to 2-channel gene expression microarrays.

Publications

Measuring differential gene expression by short read sequencing: quantitative comparison to 2-channel gene expression microarrays.

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