A data filtering to utilizing Human Methylation arrays in genome-wide study of bovine DNA methylation

Abstract

DNA methylation is important for regulating gene expression during many biological processes. There are two popular techniques for detecting genome-wide DNA methylation profiles: next-generation sequencing and DNA arrays. We conducted a pilot study for data filtering using Human Methylation arrays to evaluate the genome-wide methylation profiles using bovine DNA samples. Five tissues of two female Japanese Black cattle were analyzed in this study. We focused on the detection P-value as an indicator of the reliability of data. Here, detection P-values were used for examining array performance, which indicate the proportion of 600 negative control probes with higher signal intensity than measured for each probe. We first investigated the distribution of detection P-values using bovine DNA, and next evaluated bovine sequences with high similarity to human sequences on the probes by a BLAST search. A total of 75,207 CpG sites had a detection P-value of 0 in all examined samples (15.5%). The detection P-values for bovine DNA were markedly worse than for humans and monkeys, but better than for mice. The CpG sites with low signal intensity apparently had low quality. The total signal intensity level at each CpG site was also shown to be very important to improve array performance, similarly to the detection P-value. Among the CpG sites that fulfilled the thresholds of both detection P-value and mean total signal intensity, it was determined that approximately 70% had been evaluated properly. Therefore, it was expected that useful methylation data with good quality were obtained for approximately 50,000 CpG sites (about 10% of all probes). These results show that the Human Methylation arrays have potential for evaluating a few tens of thousands of CpG sites in cattle by appropriate filtering to ensure the quality of the data.

Age-related changes in DNA methylation levels at CpG sites in bull spermatozoa and <i>in vitro</i> fertilization-derived blastocyst-stage embryos revealed by combined bisulfite restriction analysis

“…After methylation array analysis, informative CpG sites were selected as previously described [17]. Briefly, the fluorescence signal intensities of the methylated and unmethylated alleles were obtained [20].…”

“…The BLAST program (http://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to identify the sequences with high similarities for each quality-controlled CpG site on bovine genome sequences (UMD3.1.1), using human probes, as previously reported [17, 18]. Then, the CpG sites located at the restriction enzyme sites for Aci I, Bst UI, Hpy CH4IV, and Taq I were selected within the bovine genome sequences identified by the BLAST search.…”

“…Previously, we conducted a pilot study using Human Methylation array to evaluate genome-wide methylation profiles in 37,000 CpG sites using DNA samples extracted from bull spermatozoa or tissues [17, 18]. In the case of non-model organisms, such as pigs and cattle, equivalent tools for studying DNA methylation at a genome-scale are currently unavailable for commercial use.…”

“…In the case of non-model organisms, such as pigs and cattle, equivalent tools for studying DNA methylation at a genome-scale are currently unavailable for commercial use. Methylation profiles of individual and age-related alterations in bull spermatozoa can be analyzed using human microarray, and methylation changes in some CpG sites can be easily visualized using combined bisulfite restriction analysis (COBRA) [17, 18]. Specifically, one CpG site was hypothesized to reflect an age-related increase in methylation levels, which was then confirmed by COBRA and bisulfite sequencing analyses.…”

Differentially methylated CpG sites in bull spermatozoa revealed by human DNA methylation arrays and bisulfite analysis

“…It was found that analyzing DNA methylation in the mouse genome using Infinium HM27 and HM450 platforms [ 11 ] is feasible. Recently, Kobayashi and Takeda determined the utility of Infinium HM450 BeadChips for analyzing bovine genomic DNA, using the benchmark that useful methylation data with good quality are expected to analyze approximately 50,000 CpG sites (about 10% of all probes) [ 12 ]. These results suggest that the technique can advance genome-wide screening of differentially methylated regions (DMRs) in bovine spermatozoa.…”