Application of the targeted sequencing approach reveals the single nucleotide polymorphism (SNP) repertoire in microRNA genes in the pig genome

Abstract

MicroRNAs (miRNAs) are recognized as gene expression regulators, indirectly orchestrating a plethora of biological processes. Single nucleotide polymorphism (SNP), one of the most common genetic variations in the genome, is established to affect miRNA functioning and influence complex traits and diseases. SNPs in miRNAs have also been associated with important production traits in livestock. Thus, the aim of our study was to reveal the SNP variability of miRNA genes in the genome of the pig, which is a significant farm animal and large-mammal human model. To this end, we applied the targeted sequencing approach, enabling deep sequencing of specified genomic regions. As a result, 73 SNPs localized in 50 distinct pre-miRNAs were identified. In silico analysis revealed that many of the identified SNPs influenced the structure and energy of the hairpin precursors. Moreover, SNPs localized in the seed regions were shown to alter targeted genes and, as a result, enrich different biological pathways. The obtained results corroborate a significant impact of SNPs on the miRNA processing and broaden the state of knowledge in the field of animal genomics. We also report the targeted sequencing approach to be a promising alternative for the whole genome sequencing in miRNA genes focused studies.

Figure 1

Distribution of the localization of all SNPs identified in mature miRNA sequences in this study. The upper row of the table contains data on the number of SNPs identified in a given position of the mature miRNA sequence, while the lower row indicates a nucleotide position of the mature miRNA sequence. The light green coloured cells stand for the positions of the seed nucleotides (2–8).

Figure 2

Data on the number of SNPs identified in the seed sequence (blue triangle), in the mature miRNA sequence beyond the seed (green traingle), and in the pre-miRNA sequence beyond the mature miRNA (grey triangle). Numbers in brackets denote the number of miRNAs with such SNPs.

Figure 3

Details on the frequency of SNP substitution types identified in the whole pre-miRNA sequences (pre-miR) as well as mature miRNA sequences (miR). Lighter-coloured bars denote transitions, while darker-coloured bars stand for transversions.

Figure 4

Distribution of minimal free energy changes (MFE; ΔΔG, kcal/mol) of pre-miRNA hairpin secondary structures induced by single SNPs. The blue bars denote the decrease of MFE, while the orange bars denote the increase of MFE.

Figure 5

Graphical visualization of an SNP (C>A; rs334271387) identified in the sequence of ssc-miR-7141-5p and its impact on the hairpin secondary structure and its minimal free energy (MFE; ΔG). The seed region is highlighted in blue, while the SNP is framed. The yellow arrow shows the SNP localization and a bulge which it created.

Figure 6

Graphical visualization of an SNP (G>A) identified in the seed region of ssc-miR-9819-5p and its impact on the hairpin secondary structure and its minimal free energy (MFE; ΔG). The seed region is highlighted in blue, while the SNP is framed.

Figure 7

Venn diagram of target genes predicted for wild type and SNP-altered miRNA seeds: (A) data on the number of predicted target genes of miR-9819-5p for the reference seed (the blue circle), and the SNP-altered seed (the green circle); (B) data on the number of predicted target genes of miR-9791-1-3p for the reference seed (the blue circle), and the SNP-altered seed (the green circle).

Figure 8

(A) statistically significant (p value ≤ 0.05) GO terms enriched in target genes of miR-9819-5p reference sequence (the blue bars) and miR-9819-5p with the SNP (the orange bars); (B) statistically significant (p value ≤ 0.06) GO terms enriched in target genes of miR-9791-1-3p reference sequence (the blue bars) and miR-9791-1-3p with the SNP (the orange bars).

Figure 9

Exemplary Sanger sequencing chromatograms: (A) region 3, ssc-miR-9831, chr1 4108477 A>G; (B) region 6, ssc-miR-9853-1, chr12 57502421 C>T; (C) region 8, ssc-miR-9819, C-1: chr5 8348872 G>A (GG homozygote), 8348891 T>C; C-2: chr5 8348943, C>T; (D) region 17, ssc-miR-146b, chr 14 123301846 T>C; (E) region 20, ssc-miR-2483, chr X 117608224 C>G. The red arrow points to the given SNP.