High resolution profiling of human exon methylation by liquid hybridization capture-based bisulfite sequencing

Abstract

BackgroundDNA methylation plays important roles in gene regulation during both normal developmental and disease states. In the past decade, a number of methods have been developed and applied to characterize the genome-wide distribution of DNA methylation. Most of these methods endeavored to screen whole genome and turned to be enormously costly and time consuming for studies of the complex mammalian genome. Thus, they are not practical for researchers to study multiple clinical samples in biomarker research.ResultsHere, we display a novel strategy that relies on the selective capture of target regions by liquid hybridization followed by bisulfite conversion and deep sequencing, which is referred to as liquid hybridization capture-based bisulfite sequencing (LHC-BS). To estimate this method, we utilized about 2 μg of native genomic DNA from YanHuang (YH) whole blood samples and a mature dendritic cell (mDC) line, respectively, to evaluate their methylation statuses of target regions of exome. The results indicated that the LHC-BS system was able to cover more than 97% of the exome regions and detect their methylation statuses with acceptable allele dropouts. Most of the regions that couldn't provide accurate methylation information were distributed in chromosomes 6 and Y because of multiple mapping to those regions. The accuracy of this strategy was evaluated by pair-wise comparisons using the results from whole genome bisulfite sequencing and validated by bisulfite specific PCR sequencing.ConclusionsIn the present study, we employed a liquid hybridisation capture system to enrich for exon regions and then combined with bisulfite sequencing to examine the methylation statuses for the first time. This technique is highly sensitive and flexible and can be applied to identify differentially methylated regions (DMRs) at specific genomic locations of interest, such as regulatory elements or promoters.

Global Analysis of DNA Methylation in Hepatocellular Carcinoma by a Liquid Hybridization Capture-Based Bisulfite Sequencing Approach

“…A LHC-BS approach [14,15] was subsequently applied to profile the promoter methylome of the 8 sample pairs. Promoters were denoted as regions from −2200 bp to +500 bp of the transcriptional start sites (TSS) [16].…”

“…Although this has been extensively studied in colon cancer, with many genes identified as harboring altered methylation in their promoter CGIs [21], there is currently far less information regarding HCC. Our previous study showed that the novel LHC-BS approach is a reliable and efficient analytical platform for generating a single-base-pair resolution methylome map of promoter regions in cancer and normal cell lines [14,15]. In the current study, we further optimized the technology to profile the promoter methylome in 8 paired HCC samples.…”

“…We demonstrated that this approach could be used to examine the human exome [14] as well as the promoter methylome [15]. In the present study, we initially performed promoter-targeted LHC-BS on eight paired HCC tissues to analyze 1.86 million CpG sites located at the promoter regions of 31,372 (91.8 %) genes.…”

Analysis of DNA modifications in aging research

“…We term these approaches as bisulfite oligonucleotide capture sequencing (BOCS). Several very similar approaches have been developed for assessing the human (Wang et al 2011;Ivanov et al 2013;Allum et al 2015;Li et al 2015), mouse (Hing et al 2015;Li et al 2015), and rat (Masser et al 2016) genomes. In all of these approaches, capture of targeted regions is by complimentary oligonucleotide probes.…”

Accurate CpG and non-CpG cytosine methylation analysis by high-throughput locus-specific pyrosequencing in plants

“…Moreover, as an alternative to WGBS, other next generation sequencing methods have been developed in mammalian genomes for the analysis of medium to high number (100-140,000) of candidate regions as cost, genome complexity and DNA quantities can be an obstacle to the use of WGBS (Lee et al 2013). These methods are based on the capture of targeted regions of interest to enrich target sequences prior to or after bisulfite conversion using bisulfite padlock probes (Ball et al 2009), solution hybrid selection (Wang et al 2011), array capture (Hodges et al 2009) or microdroplet PCR (Komori et al 2011), but often require a significant amount of input DNA. Further, PCR amplification products of bisulfite treated DNA can also be sequenced on next generation sequencing instruments (Nones et al 2014), and which might circumvent some of the inherent limitations of pyrosequencing such as quantification in hompolymers.…”