Ultra-Deep Bisulfite Sequencing to Detect Specific DNA Methylation Patterns of Minor Cell Types in Heterogeneous Cell Populations: An Example of the Pituitary Tissue

<div><p>DNA methylation is an epigenetic modification important for cell fate determination and cell type-specific gene expression. Transcriptional regulatory regions of the mammalian genome contain a large number of tissue/cell type-dependent differentially methylated regions (T-DMRs) with DNA methylation patterns crucial for transcription of the corresponding genes. In general, tissues consist of multiple cell types in various proportions, making it difficult to detect T-DMRs of minor cell types in tissues. The present study attempts to detect T-DMRs of minor cell types in tissues by ultra-deep bisulfite sequencing of cell type-restricted genes and to assume proportions of minor cell types based on DNA methylation patterns of sequenced reads. For this purpose, we focused on transcriptionally active hypomethylated alleles (Hypo-alleles), which can be recognized by the high ratio of unmethylated CpGs in each sequenced read (allele). The pituitary gland contains multiple cell types including five hormone-expressing cell types and stem/progenitor cells, each of which is a minor cell type in the pituitary tissue. By ultra-deep sequencing of more than 100 reads for detection of Hypo-alleles in pituitary cell type-specific genes, we identified T-DMRs specific to hormone-expressing cells and stem/progenitor cells and used them to estimate the proportions of each cell type based on the Hypo-allele ratio in pituitary tissue. Therefore, introduction of the novel Hypo-allele concept enabled us to detect T-DMRs of minor cell types with estimation of their proportions in the tissue by ultra-deep bisulfite sequencing.</p></div

Bisulfite analyses of relatively small numbers of clones (reads) for detection of Hypo-alleles by Sanger and MiSeq sequencing.

<p>Cell type-restricted genes (<i>Gh1</i>, <i>Prl</i>, and <i>Tbx19</i>) were analyzed by Sanger sequencing of 15–20 clones. Black and white circles indicate methylated and unmethylated CpGs, respectively. (B) Successive 20 reads were grouped as a trial and analyzed, and five trials were performed from the first read of MiSeq raw data of the <i>Gh1</i>, <i>Prl</i>, and <i>Tbx19</i> T-DMRs to mimic the conventional bisulfite sequencing analyses of 20 clones (reads) five times each. The Hypo-allele ratio of the <i>Gh1</i> T-DMR from the 3,304 total reads was 12.8%, whereas those of five groups of 20 reads were between 0–30% (average: 13.0%, SD: 11.0%). Similarly, the Hypo-allele ratios of the <i>Prl</i> and <i>Tbx19</i> T-DMRs from the 1,107 and 820 total reads were 22.7% and 23.2%, respectively, whereas those of five trials of 20 reads were between 15–30% (average: 22.0%, SD: 7.0%) for the <i>Prl</i> T-DMR and 10–35% (average: 27.0%, SD: 10.0%) for the <i>Tbx19</i> T-DMR. Methylated and unmethylated CpGs are shown as black and white bars, respectively. Asterisks indicate Hypo-alleles.</p

Validation of hypomethylated alleles (Hypo-alleles) by MiSeq ultra-deep sequencing.

<p>≥75% unmethylated CpG sites (3 or 4 out of the 4 CpGs shown) are defined as Hypo-alleles. Among the five reads, reads 1 and 2 are Hypo-alleles, and the Hypo-allele ratio is 40% (2 reads/5 reads). Left panel: conventional CpG methylation analysis data; white and black circles indicate unmethylated and methylated CpGs, respectively. Right panel: MiSeq ultra-deep bisulfite analysis; white and black bars indicate unmethylated and methylated CpGs, respectively. (B) Validation of Hypo-allele ratio analysis by MiSeq ultra-deep bisulfite sequencing at the <i>Sall4</i> T-DMR using mixtures of genomic DNAs from PFF and iPSC. Sequenced reads above the dotted lines are Hypo-alleles. White and black bars indicate unmethylated and methylated CpGs, respectively. Mixtures of genomic DNAs of PFF and iPSC (0:100, 25:75, 50:50, 75:25, or 100:0) would exhibit the expected respective Hypo-allele ratios (100%, 75%, 50%, 25%, or 0%) for the <i>Sall4</i> T-DMR. (C) Hypo-allele ratios of the <i>Sall4</i> T-DMR analyzed by ultra-deep sequencing. Hypo-allele ratios were calculated from the PFF/iPSC mixtures (expected Hypo-allele ratios of 100%, 75%, 50%, 25%, and 0%) shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146498#pone.0146498.g001" target="_blank">Fig 1B</a>. The Hypo-allele ratios from three independent experiments are shown as mean ± SE (n = 3). (D) Accuracy of detecting Hypo-allele ratio (10%) in relation to sequenced read numbers based on the MiSeq data at the <i>Sall4</i> T-DMR. For this analysis, genomic DNAs of PFF and iPSC were mixed at 90:10 for a Hypo-allele ratio of 10%. From two independent MiSeq analyses, 1,109 reads (Exp. 1) and 1,447 reads (Exp. 2) were obtained. Hypo-allele ratios of each trial are plotted (filled circles, n = 5), and mean ± SD for five trials each for 10, 20, 50, 100, or 200 reads are plotted as triangles with lines. (E) Examination of detection accuracy of Hypo-allele (10% or 50%) by conventional analysis of small numbers of sequencing reads based on the MiSeq data at the <i>Sall4</i> T-DMR. Genomic DNAs of PFF and iPSC were mixed at 90:10 or 50:50 for samples exhibiting Hypo-allele ratios of 10% or 50%, respectively. From the raw data, 20 successive reads were grouped from the first through 100th read. The Hypo-allele ratios of 20-read groups were calculated. Asterisks indicate Hypo-alleles. White and black bars indicate unmethylated CpGs and methylated CpGs, respectively.</p

Comparison of DNA methylation profiles of pituitary-related genes in porcine tissues by ultra-deep bisulfite sequencing between the conventional DNA methylation analysis and a novel Hypo-allele ratio analysis.

<p>Hypo-allele ratios of 37 pituitary-related genes were analyzed in pituitary (#1 and #2), liver, brain, and PFF samples using a MiSeq sequencer. The Hypo-allele data for each tissue are shown as a heatmap after hierarchical clustering based on Euclidean distance (left panel). Using the same bisulfite sequencing data, the conventional DNA methylation degrees calculated by methyl-CpGs/total CpGs are also shown as a heatmap with hierarchical clustering (right panel). ND, No data.</p

Schematic diagram of predicted proportions of pituitary cell types in adult porcine pituitary tissue.

<p>Proportions of several pituitary cell types were estimated based on the Hypo-allele ratios of the pituitary cell type-restricted genes (white).</p

Detection of Hypo-alleles of endogenous imprinted genes in porcine pituitary and liver.

<p>Bisulfite ultra-deep sequencing was performed for differentially methylated regions of imprinted <i>Meg3</i> and <i>Peg10</i> genes, and their Hypo-allele ratios were calculated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146498#pone.0146498.g001" target="_blank">Fig 1</a>. Sequenced reads above the dotted lines are Hypo-alleles. White and black bars indicate unmethylated and methylated CpGs, respectively.</p