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Expression sequence tag library derived from peripheral blood mononuclear cells of the chlorocebus sabaeus.

Tchitchek N, Jacquelin B, Wincker P, Dossat C, Silva CD, Weissenbach J, Blancher A, Müller-Trutwin M, Benecke A - BMC Genomics (2012)

Bottom Line: For 506 transcripts, sequences were quasi-complete.In addition, 6,576 transcript fragments are potentially specific to the C. sabaeus or corresponding to not yet described primate genes.Furthermore, this library, which particularly well represents immunological and hematological gene expression, will be an important resource for the comparative analysis of gene expression in clinically relevant nonhuman primate and human research.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut des Hautes Études Scientifiques-Centre National de la Recherche Scientifique, Bures-sur-Yvette, France.

ABSTRACT

Background: African Green Monkeys (AGM) are amongst the most frequently used nonhuman primate models in clinical and biomedical research, nevertheless only few genomic resources exist for this species. Such information would be essential for the development of dedicated new generation technologies in fundamental and pre-clinical research using this model, and would deliver new insights into primate evolution.

Results: We have exhaustively sequenced an Expression Sequence Tag (EST) library made from a pool of Peripheral Blood Mononuclear Cells from sixteen Chlorocebus sabaeus monkeys. Twelve of them were infected with the Simian Immunodeficiency Virus. The mononuclear cells were or not stimulated in vitro with Concanavalin A, with lipopolysacharrides, or through mixed lymphocyte reaction in order to generate a representative and broad library of expressed sequences in immune cells. We report here 37,787 sequences, which were assembled into 14,410 contigs representing an estimated 12% of the C. sabaeus transcriptome. Using data from primate genome databases, 9,029 assembled sequences from C. sabaeus could be annotated. Sequences have been systematically aligned with ten cDNA references of primate species including Homo sapiens, Pan troglodytes, and Macaca mulatta to identify ortholog transcripts. For 506 transcripts, sequences were quasi-complete. In addition, 6,576 transcript fragments are potentially specific to the C. sabaeus or corresponding to not yet described primate genes.

Conclusions: The EST library we provide here will prove useful in gene annotation efforts for future sequencing of the African Green Monkey genomes. Furthermore, this library, which particularly well represents immunological and hematological gene expression, will be an important resource for the comparative analysis of gene expression in clinically relevant nonhuman primate and human research.

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Composition and alignment distribution of the EST library and the assembled distinct transcripts. (A) Distribution of the length of the 37,787 original ESTs. The median EST length is equal to 618 nucleotides, the mean EST length is 563 nucleotides, and the standard deviation of the distribution is 167 nucleotides. (B) Distribution of the number of ESTs per contig in the ESTs assembly. The median number of ESTs per contig is equal to 3 ESTs, the mean number of ESTs per contig is 7 ESTs, and the standard deviation of the distribution is 27 ESTs. (C) Distribution of the length of the 14,410 distinct transcripts. The median sequence length is equal to 847 nucleotides, the mean sequence length is 943 nucleotides, and the standard deviation of the distribution is 388 nucleotides. The contribution of the assembled ESTs is shown in red while the contribution of singleton ESTs is shown in blue. (D) Distribution of the number of matched cDNA reference-mapped for both the 37,787 original ESTs (shown in yellow) and the 14,410 distinct transcripts (shown in green).
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Figure 1: Composition and alignment distribution of the EST library and the assembled distinct transcripts. (A) Distribution of the length of the 37,787 original ESTs. The median EST length is equal to 618 nucleotides, the mean EST length is 563 nucleotides, and the standard deviation of the distribution is 167 nucleotides. (B) Distribution of the number of ESTs per contig in the ESTs assembly. The median number of ESTs per contig is equal to 3 ESTs, the mean number of ESTs per contig is 7 ESTs, and the standard deviation of the distribution is 27 ESTs. (C) Distribution of the length of the 14,410 distinct transcripts. The median sequence length is equal to 847 nucleotides, the mean sequence length is 943 nucleotides, and the standard deviation of the distribution is 388 nucleotides. The contribution of the assembled ESTs is shown in red while the contribution of singleton ESTs is shown in blue. (D) Distribution of the number of matched cDNA reference-mapped for both the 37,787 original ESTs (shown in yellow) and the 14,410 distinct transcripts (shown in green).

Mentions: Our aim was to obtain the sequence information for the genes expressed in C. sabaeus immune cells. In order to be representative, we collected fresh PBMC from twelve SIV-infected and four non infected animals. In order to identify as many distinct transcripts as possible, we in vitro stimulated these cells or not with Concanavalin A (ConA), lipopolysaccharides (LPS) and by mixed lymphocyte reactions (MLR), as these stimuli upregulate mRNA expression of many genes. The different stimuli were chosen to activate distinct cellular receptors (T cell receptor, Toll-like receptors) and stimulate distinct immune cells (lymphocytes and antigen-presenting cells). Total RNA preparations from the stimulated and unstimulated cells were pooled and a cDNA library constructed. Sequences were obtained and sequence quality filtering showed that 37,787 ESTs were present in the library. They had a mean length of 563 nucleotides per EST with a standard deviation of 167 nucleotides (Figure 1A). The 37,787 ESTs have been assembled into 3,853 contigs (overlapping or embedded ESTs) and 10,557 singletons (not assembled ESTs). The median number of ESTs per contig was 3 with some outlier contigs being composed of up to 941 ESTs (Figure 1B). The mean length of the 14,410 assembled and singletons ESTs averages at 943 nucleotides (Figure 1C). The total length represented by our AGM EST library is about 21.106 nucleotides and the total length of the assembled distinct transcripts 9.106 nucleotides. Since the total length of the known M. mulatta distinct transcripts corresponds to 72.106 nucleotides in the Ensembl database [38], our AGM sequences represent 12% of the M. mulatta transcriptome and potentialy a similar fraction of the AGM transcriptome.


Expression sequence tag library derived from peripheral blood mononuclear cells of the chlorocebus sabaeus.

Tchitchek N, Jacquelin B, Wincker P, Dossat C, Silva CD, Weissenbach J, Blancher A, Müller-Trutwin M, Benecke A - BMC Genomics (2012)

Composition and alignment distribution of the EST library and the assembled distinct transcripts. (A) Distribution of the length of the 37,787 original ESTs. The median EST length is equal to 618 nucleotides, the mean EST length is 563 nucleotides, and the standard deviation of the distribution is 167 nucleotides. (B) Distribution of the number of ESTs per contig in the ESTs assembly. The median number of ESTs per contig is equal to 3 ESTs, the mean number of ESTs per contig is 7 ESTs, and the standard deviation of the distribution is 27 ESTs. (C) Distribution of the length of the 14,410 distinct transcripts. The median sequence length is equal to 847 nucleotides, the mean sequence length is 943 nucleotides, and the standard deviation of the distribution is 388 nucleotides. The contribution of the assembled ESTs is shown in red while the contribution of singleton ESTs is shown in blue. (D) Distribution of the number of matched cDNA reference-mapped for both the 37,787 original ESTs (shown in yellow) and the 14,410 distinct transcripts (shown in green).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3539953&req=5

Figure 1: Composition and alignment distribution of the EST library and the assembled distinct transcripts. (A) Distribution of the length of the 37,787 original ESTs. The median EST length is equal to 618 nucleotides, the mean EST length is 563 nucleotides, and the standard deviation of the distribution is 167 nucleotides. (B) Distribution of the number of ESTs per contig in the ESTs assembly. The median number of ESTs per contig is equal to 3 ESTs, the mean number of ESTs per contig is 7 ESTs, and the standard deviation of the distribution is 27 ESTs. (C) Distribution of the length of the 14,410 distinct transcripts. The median sequence length is equal to 847 nucleotides, the mean sequence length is 943 nucleotides, and the standard deviation of the distribution is 388 nucleotides. The contribution of the assembled ESTs is shown in red while the contribution of singleton ESTs is shown in blue. (D) Distribution of the number of matched cDNA reference-mapped for both the 37,787 original ESTs (shown in yellow) and the 14,410 distinct transcripts (shown in green).
Mentions: Our aim was to obtain the sequence information for the genes expressed in C. sabaeus immune cells. In order to be representative, we collected fresh PBMC from twelve SIV-infected and four non infected animals. In order to identify as many distinct transcripts as possible, we in vitro stimulated these cells or not with Concanavalin A (ConA), lipopolysaccharides (LPS) and by mixed lymphocyte reactions (MLR), as these stimuli upregulate mRNA expression of many genes. The different stimuli were chosen to activate distinct cellular receptors (T cell receptor, Toll-like receptors) and stimulate distinct immune cells (lymphocytes and antigen-presenting cells). Total RNA preparations from the stimulated and unstimulated cells were pooled and a cDNA library constructed. Sequences were obtained and sequence quality filtering showed that 37,787 ESTs were present in the library. They had a mean length of 563 nucleotides per EST with a standard deviation of 167 nucleotides (Figure 1A). The 37,787 ESTs have been assembled into 3,853 contigs (overlapping or embedded ESTs) and 10,557 singletons (not assembled ESTs). The median number of ESTs per contig was 3 with some outlier contigs being composed of up to 941 ESTs (Figure 1B). The mean length of the 14,410 assembled and singletons ESTs averages at 943 nucleotides (Figure 1C). The total length represented by our AGM EST library is about 21.106 nucleotides and the total length of the assembled distinct transcripts 9.106 nucleotides. Since the total length of the known M. mulatta distinct transcripts corresponds to 72.106 nucleotides in the Ensembl database [38], our AGM sequences represent 12% of the M. mulatta transcriptome and potentialy a similar fraction of the AGM transcriptome.

Bottom Line: For 506 transcripts, sequences were quasi-complete.In addition, 6,576 transcript fragments are potentially specific to the C. sabaeus or corresponding to not yet described primate genes.Furthermore, this library, which particularly well represents immunological and hematological gene expression, will be an important resource for the comparative analysis of gene expression in clinically relevant nonhuman primate and human research.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut des Hautes Études Scientifiques-Centre National de la Recherche Scientifique, Bures-sur-Yvette, France.

ABSTRACT

Background: African Green Monkeys (AGM) are amongst the most frequently used nonhuman primate models in clinical and biomedical research, nevertheless only few genomic resources exist for this species. Such information would be essential for the development of dedicated new generation technologies in fundamental and pre-clinical research using this model, and would deliver new insights into primate evolution.

Results: We have exhaustively sequenced an Expression Sequence Tag (EST) library made from a pool of Peripheral Blood Mononuclear Cells from sixteen Chlorocebus sabaeus monkeys. Twelve of them were infected with the Simian Immunodeficiency Virus. The mononuclear cells were or not stimulated in vitro with Concanavalin A, with lipopolysacharrides, or through mixed lymphocyte reaction in order to generate a representative and broad library of expressed sequences in immune cells. We report here 37,787 sequences, which were assembled into 14,410 contigs representing an estimated 12% of the C. sabaeus transcriptome. Using data from primate genome databases, 9,029 assembled sequences from C. sabaeus could be annotated. Sequences have been systematically aligned with ten cDNA references of primate species including Homo sapiens, Pan troglodytes, and Macaca mulatta to identify ortholog transcripts. For 506 transcripts, sequences were quasi-complete. In addition, 6,576 transcript fragments are potentially specific to the C. sabaeus or corresponding to not yet described primate genes.

Conclusions: The EST library we provide here will prove useful in gene annotation efforts for future sequencing of the African Green Monkey genomes. Furthermore, this library, which particularly well represents immunological and hematological gene expression, will be an important resource for the comparative analysis of gene expression in clinically relevant nonhuman primate and human research.

Show MeSH
Related in: MedlinePlus