Project description:Background The domestic pig is an important livestock species for meat production worldwide and is becoming an established biomedical research model. As a result, there is a strong interest in the factors that affect the efficient production of viable embryos and offspring in this species using either in vivo or in vitro production methods. A limited understanding of the molecular mechanisms involved in this critical physiological process has inhibited our ability to fully elucidate these factors. The use of next generation deep sequencing and microarray technology are powerful tools for delineation of molecular pathways during early embryonic development of mammals. Here, we report on the assessment of a porcine-embryo-specific microarray platform created from a large expressed sequence tag (EST) analysis generated by Roche/454 next-generation sequencing of cDNAs constructed from critical stages of in vivo or in vitro porcine preimplantation embryos. Results Two cDNA libraries constructed from in vitro and in vivo produced preimplantation porcine embryos were normalized and sequenced using the 454 Titanium pyrosequencing technology. Treatment of cDNA libraries with BAL 31 nuclease digestion resulted in a 2 fold improvement of sequencing quality compared with untreated libraries. Over one million high quality EST sequences were obtained from this process and used to create an augmented porcine genome catalogue. Using the resulting dataset the EMbryogene Porcine Version 1 (EMPV1) microarray was developed and is composed of 43,795 probes printed onto a 4 × 44 K Agilent array. Based on the initial probe sequences annotation, the EMPV1 featured 17,409 protein-coding, 473 pseudogenes, 46 retrotransposed, 2,359 non-coding RNA (snRNA, snoRNA, etc.), 4,121 splice variants in 2,862 genes and a total of 12,324 Novel Transcript Regions (NTR). After re-annotation, the total unique genes increased from 11,961 to 16,281 and 1.9% of them belonged to a large olfactory receptor (OR) gene family. Quality control of EMPV1 was performed using porcine cumulus–oocyte complexes (COC) as well as early developmental stages of embryos. This revealed an even distribution of ten clusters of spike-in control spots and array to array (dye-swap) correction was 0.97. Further bioinformatics analysis revealed that our microarray probes hybridized with more developmental related transcripts from embryonic labelled targets when compared to COC. Conclusions Using next-generation deep sequencing we have produced a large EST dataset to provide the selection of probe sequences for the development of the EMPV1 microarray platform. The quality of this embryo- specific array was confirmed with the high level of reproducibility using current Agilent microarray technology. Despite the current limitations for full NTR annotation, due to the incomplete porcine genome sequencing project, a significant number of NTR were annotated using Version 10 of porcine genome and human RefSeq RNA database to enrich the orthologous genes with unique gene symbol (GS) for Gene Ontology (GO) search. GO terms confirmed that many are related relevant developmental processes. With more than an estimated 20 thousands unique genes represented on the EMPV1, this platform will provide the foundation for future research into the in vivo and in vitro factors that affect the viability of the porcine embryos, as well as the effects of these factors on the live offspring that result from these embryos.

Project description:Background The domestic pig is an important livestock species for meat production worldwide and is becoming an established biomedical research model. As a result, there is a strong interest in the factors that affect the efficient production of viable embryos and offspring in this species using either in vivo or in vitro production methods. A limited understanding of the molecular mechanisms involved in this critical physiological process has inhibited our ability to fully elucidate these factors. The use of next generation deep sequencing and microarray technology are powerful tools for delineation of molecular pathways during early embryonic development of mammals. Here, we report on the assessment of a porcine-embryo-specific microarray platform created from a large expressed sequence tag (EST) analysis generated by Roche/454 next-generation sequencing of cDNAs constructed from critical stages of in vivo or in vitro porcine preimplantation embryos. Results Two cDNA libraries constructed from in vitro and in vivo produced preimplantation porcine embryos were normalized and sequenced using the 454 Titanium pyrosequencing technology. Treatment of cDNA libraries with BAL 31 nuclease digestion resulted in a 2 fold improvement of sequencing quality compared with untreated libraries. Over one million high quality EST sequences were obtained from this process and used to create an augmented porcine genome catalogue. Using the resulting dataset the EMbryogene Porcine Version 1 (EMPV1) microarray was developed and is composed of 43,795 probes printed onto a 4 × 44 K Agilent array. Based on the initial probe sequences annotation, the EMPV1 featured 17,409 protein-coding, 473 pseudogenes, 46 retrotransposed, 2,359 non-coding RNA (snRNA, snoRNA, etc.), 4,121 splice variants in 2,862 genes and a total of 12,324 Novel Transcript Regions (NTR). After re-annotation, the total unique genes increased from 11,961 to 16,281 and 1.9% of them belonged to a large olfactory receptor (OR) gene family. Quality control of EMPV1 was performed using porcine cumulus–oocyte complexes (COC) as well as early developmental stages of embryos. This revealed an even distribution of ten clusters of spike-in control spots and array to array (dye-swap) correction was 0.97. Further bioinformatics analysis revealed that our microarray probes hybridized with more developmental related transcripts from embryonic labelled targets when compared to COC. Conclusions Using next-generation deep sequencing we have produced a large EST dataset to provide the selection of probe sequences for the development of the EMPV1 microarray platform. The quality of this embryo- specific array was confirmed with the high level of reproducibility using current Agilent microarray technology. Despite the current limitations for full NTR annotation, due to the incomplete porcine genome sequencing project, a significant number of NTR were annotated using Version 10 of porcine genome and human RefSeq RNA database to enrich the orthologous genes with unique gene symbol (GS) for Gene Ontology (GO) search. GO terms confirmed that many are related relevant developmental processes. With more than an estimated 20 thousands unique genes represented on the EMPV1, this platform will provide the foundation for future research into the in vivo and in vitro factors that affect the viability of the porcine embryos, as well as the effects of these factors on the live offspring that result from these embryos. Two biological samples.

Dataset Information

Development of a porcine (Sus scofa) embryo-specific microarray: array annotation and validation.

Publications

Development of a porcine (Sus scofa) embryo-specific microarray: array annotation and validation.

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