Annotation of the goat genome using next generation sequencing of microRNA expressed by the lactating mammary gland: comparison of three approaches

Abstract

Background: MicroRNAs (miRNA) are small endogenous non-coding RNA involved in the post-transcriptional regulation of specific mRNA targets. The first whole goat genome sequence became available in 2013, with few annotations. Our goal was to establish a list of the miRNA expressed in the mammary gland of lactating goats, thus enabling implementation of the goat miRNA repertoire and considerably enriching annotation of the goat genome.

Results: Here, we performed high throughput RNA sequencing on 10 lactating goat mammary glands. The bioinformatic detection of miRNA was carried out using miRDeep2 software. Three different methods were used to predict, quantify and annotate the sequenced reads. The first was a de novo approach based on the prediction of miRNA from the goat genome only. The second approach used bovine miRNA as an external reference whereas the last one used recently available goat miRNA. The three methods enabled the prediction and annotation of hundreds of miRNA, more than 95% were commonly identified. Using bovine miRNA, 1,178 distinct miRNA were detected, together with the annotation of 88 miRNA for which corresponding precursors could not be retrieved in the goat genome, and which were not detected using the de novo approach or with the use of goat miRNA. Each chromosomal coordinate of the precursors determined here were generated and depicted on a reference localisation map. Forty six goat miRNA clusters were also reported. The study revealed 263 precursors located in goat protein-coding genes, amongst which the location of 43 precursors was conserved between human, mouse and bovine, revealing potential new gene regulations in the goat mammary gland. Using the publicly available cattle QTL database, and cow precursors conserved in the goat and expressed in lactating mammary gland, 114 precursors were located within known QTL regions for milk production and composition.

Conclusions: The results reported here represent the first major identification study on miRNA expressed in the goat mammary gland at peak lactation. The elements generated by this study will now be used as references to decipher the regulation of miRNA expression in the goat mammary gland and to clarify their involvement in the lactation process.

Figure 1

Summary of the three approaches used to predict, quantify and annotate sequencing reads. Common processes and results are indicated in red for the prediction step and in blue for the quantification and annotation steps. Specific processes and results are indicated in purple for the de novo approach, in orange and green for the approach using bovine and goat miRNA, respectively.

Figure 2

Comparison of the number of potential precursor and miRNA obtained by the three approaches. Potential precursors are represented in A, known and putative miRNA are represented in B and C, respectively. *: Adding precursors of reference species containing known miRNA not found initially in potential precursors enabled the annotation of 88 and 4 miRNA with the approach using bovine and goat miRNA, respectively.

Figure 3

Map of the CHI 19 location of miRNA precursors. In left, putative (red) and known (green) miRNA localized on the + strand. In right, putative (blue) and known (green) miRNA localized on the - strand.

Figure 4

Number of precursors and clusters in each goat chromosome. Clusters were positioned on chromosomes. Interdistance between precursors of less than 10 kb was considered to be a cluster. The number of precursors per chromosome is indicated at the extremity of each chromosome.

Figure 5

Conserved intragenic precursors in human, mouse, cow and goat. Human, mouse and cattle precursors were obtained from miRBase v21, and the locations of protein-coding genes were downloaded from BioMartEnsembl (http://www.ensembl.org/biomart). The locations of goat mRNA were obtained from Dong et al. [18].