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

Stephen Tsoi,John Dobrinsky,J Pasternak,Jason R Grant,Chi Zhou,Julie Nieminen,Claude Robert,Philippe Rigault,Michael K Dyck,George R Foxcroft,Marc-André Sirard

doi:10.1186/1471-2164-13-370

Stephen Tsoi, John Dobrinsky + Show 9 more

Open Access

https://doi.org/10.1186/1471-2164-13-370

Copy DOI

Journal: BMC Genomics	Publication Date: Jan 1, 2012
Citations: 80	License type: cc-by

Affiliation: University of Alberta, Université Laval

Abstract

BackgroundThe domestic pig is an important livestock species and there is strong interest in the factors that affect the development of viable embryos and offspring in this species. A limited understanding of the molecular mechanisms involved in early embryonic development has inhibited our ability to fully elucidate these factors. Next generation deep sequencing and microarray technologies are powerful tools for delineation of molecular pathways involved in the developing embryo.ResultsHere we present the development 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. Two cDNA libraries constructed from in vitro and in vivo produced preimplantation porcine embryos were normalized and sequenced using 454 Titanium pyrosequencing technology. Over one million high-quality EST sequences were obtained and used to develop the EMbryogene Porcine Version 1 (EMPV1) microarray composed of 43,795 probes. Based on an initial probe sequence annotation, the EMPV1 features 17,409 protein-coding, 473 pseudogenes, 46 retrotransposed, 2,359 non-coding RNA, 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 on the EMPV1 was performed and revealed an even distribution of ten clusters of spiked-in control spots and array to array (dye-swap) correlation was 0.97.ConclusionsUsing next-generation deep sequencing we have produced a large EST dataset to allow for the selection of probe sequences for the development of the EMPV1 microarray platform. The quality of this embryo-specific array was confirmed with a high-level of reproducibility using current Agilent microarray technology. With more than an estimated 20,000 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 porcine embryos, as well as the effects of these factors on the live offspring that result from these embryos.

Highlights

The domestic pig is an important livestock species and there is strong interest in the factors that affect the development of viable embryos and offspring in this species
These probe sequences have not been generated from preimplantation embryonic tissues and there has been limited extensive deep sequencing projects related to porcine preimplantation embryonic development
In order to facilitate characterization of the porcine embryonic transcriptome, Complementary Deoxyribonucleic Acid (cDNA) normalization using duplexspecific nuclease (DSN) and 454 deep sequencing were conducted to account for low abundance of mRNA transcripts in developing embryos produced using both in vitro and in vivo procedures

Summary

Introduction

The domestic pig is an important livestock species and there is strong interest in the factors that affect the development of viable embryos and offspring in this species. A limited understanding of the molecular mechanisms involved in early embryonic development has inhibited our ability to fully elucidate these factors. Studies to determine the effects of various factors on embryonic development and competence have been limited to morphological and phenotypic evaluations [9,10,11]. Current understanding of the molecular events during the development of porcine preimplantation embryos is limited. Increased knowledge in this area will contribute to our understanding of basic reproductive biology. It will allow us to identify the molecular markers related to embryonic quality, and facilitate improved maternal management as well as in vitro production and manipulation of embryos

Objectives

Methods

Results

Conclusion