Performances of Different Fragment Sizes for Reduced Representation Bisulfite Sequencing in Pigs

Abstract

Background: Reduced representation bisulfite sequencing (RRBS) has been widely used to profile genome-scale DNA methylation in mammalian genomes. However, the applications and technical performances of RRBS with different fragment sizes have not been systematically reported in pigs, which serve as one of the important biomedical models for humans. The aims of this study were to evaluate capacities of RRBS libraries with different fragment sizes to characterize the porcine genome.

Results: We found that the MspI-digested segments between 40 and 220 bp harbored a high distribution peak at 74 bp, which were highly overlapped with the repetitive elements and might reduce the unique mapping alignment. The RRBS library of 110-220 bp fragment size had the highest unique mapping alignment and the lowest multiple alignment. The cost-effectiveness of the 40-110 bp, 110-220 bp and 40-220 bp fragment sizes might decrease when the dataset size was more than 70, 50 and 110 million reads for these three fragment sizes, respectively. Given a 50-million dataset size, the average sequencing depth of the detected CpG sites in the 110-220 bp fragment size appeared to be deeper than in the 40-110 bp and 40-220 bp fragment sizes, and these detected CpG sties differently located in gene- and CpG island-related regions.

Conclusions: In this study, our results demonstrated that selections of fragment sizes could affect the numbers and sequencing depth of detected CpG sites as well as the cost-efficiency. No single solution of RRBS is optimal in all circumstances for investigating genome-scale DNA methylation. This work provides the useful knowledge on designing and executing RRBS for investigating the genome-wide DNA methylation in tissues from pigs.

Keywords: DNA methylation; Different fragment sizes; Pigs; RRBS.

Fig. 1

The length distribution of the MspI-digested segments between 40 and 220 bp

Fig. 2

Mapping efficiencies of the 40–110 bp, 110–220 bp and 40–220 bp fragment sizes

Fig. 3

Distributions of detected CpG sites in the differently sub-sampled RRBS data. The number distributions of detected CpG sites with ≥5, 10 and 15 covered reads (5X, 10X and 15X) in the differently sub-sampled RRBS data for 40–110 bp (a), 110–220 bp (c) and 40–220 bp (e) fragment sizes in triplications. The percentages of 5X, 10X and 15X detected CpG sites over the 3× detected CpG sites in the differently sub-sampled RRBS data for 40–110 bp (b), 110–220 bp (d) and 40–220 bp (f) fragment sizes in triplications

Fig. 4

Distributions of detected CpG sites versus the differently covered depth for the three fragment sizes in 50 million reads in triplications

Fig. 5

Coverage of the detected CpG sites of these three fragment sizes across the gene-related and CGI-related regions for the whole porcine genome. The coverages of 5X, 10X and 15X detected CpG sites across the gene-related regions for 40–110 bp (a), 110–220 bp (c) and 40–220 bp (e) fragment sizes in triplications. The coverages of 5X, 10X and 15X detected CpG sites across the CGI-related regions for 40–110 bp (b), 110–220 bp (d) and 40–220 bp (f) fragment sizes in triplications