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

Abstract

BackgroundReduced 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.ResultsWe 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.ConclusionsIn 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.

DNA methylation patterns vary in boar sperm cells with different levels of DNA fragmentation

“…The average percentage of bisulphite conversion rate was 99.8% (Table 1), which is higher than the previously reported results for similar libraries in pigs [13, 18] and slightly higher compared to results for bovine RRBS libraries [19]. Bisulphite treatment converts non-methylated Cs into Us and a higher conversion rate indicates more efficient and stable bisulphite conversion process.…”

Dynamic DNA methylation of ovaries during pubertal transition in gilts

“…The library constructions and sequencing services were provided by RiboBio Co., Ltd. (Guangzhou, China) as previously described in our studies [16, 33, 44]. The genomic DNA of these ovarian tissues was extracted using a DNeasy Blood & Tissue Kit (Qiagen, Beijing), and then, after checking on the quality of the extracted DNA, one library was built for each pigs based on previously published RRBS studies [16, 33, 44]. The processes and procedures of RRBS libraries were briefed as follows.…”

“…Secondly, methylated Illumina sequencing adapters with 3′ T overhangs were ligated to the digested segments, and the products obtained were purified. Then 110–220 bp fragments were selected [44] and converted by bisulfite using an EZ DNA Methylation Gold Kit (Zymo Research, USA). Lastly, libraries of 110–220 bp fragments were PCR amplified and each library was sequenced using one lane of an Illumina HiSeq 2500 and 100 bp paired-end reads.…”

Genome-wide DNA methylation analysis of pituitaries during the initiation of puberty in gilts

“…The library constructions and sequencing services were provided by RiboBio Co., Ltd. (Guangzhou, China), which were previously described in our studies [32, 33]. The genomic DNA of these pituitary tissues was extracted using a DNeasy Blood & Tissue Kit (Qiagen, Beijing), and then, after checking on the quality of the extracted DNA, one library was built for each gilt based on previously published RRBS studies [17, 32, 33]. The processes and procedures of RRBS libraries were briefed as follows.…”

“…Secondly, methylated Illumina sequencing adapters with 3′ T overhangs were ligated to the digested segments, and the products obtained were purified. Then 110–220 bp fragments were selected [32] and converted by bisulfite using an EZ DNA Methylation Gold Kit (Zymo Research, USA). Lastly, libraries of 110–220 bp fragments were PCR amplified and each library was sequenced using one lane of an Illumina HiSeq 2500 and 100 bp paired-end reads.…”