Highly sensitive targeted methylome sequencing by post-bisulfite adaptor tagging.
ABSTRACT: The current gold standard method for methylome analysis is whole-genome bisulfite sequencing (WGBS), but its cost is substantial, especially for the purpose of multi-sample comparison of large methylomes. Shotgun bisulfite sequencing of target-enriched DNA, or targeted methylome sequencing (TMS), can be a flexible, cost-effective alternative to WGBS. However, the current TMS protocol requires a considerable amount of input DNA and hence is hardly applicable to samples of limited quantity. Here we report a method to overcome this limitation by using post-bisulfite adaptor tagging (PBAT), in which adaptor tagging is conducted after bisulfite treatment to circumvent bisulfite-induced loss of intact sequencing templates, thereby enabling TMS of a 100-fold smaller amount of input DNA with far fewer cycles of polymerase chain reaction than in the current protocol. We thus expect that the PBAT-mediated TMS will serve as an invaluable method in epigenomics.
Project description:Whole-genome bisulfite sequencing (WGBS) is the current gold standard of methylome analysis. Post-bisulfite adaptor tagging (PBAT) is an increasingly popular WGBS protocol because of high sensitivity and low bias. PBAT originally relied on two rounds of random priming for adaptor-tagging of single-stranded DNA (ssDNA) to attain high efficiency but at a cost of library insert length. To overcome this limitation, we developed terminal deoxyribonucleotidyl transferase (TdT)-assisted adenylate connector-mediated ssDNA (TACS) ligation as an alternative to random priming. In this method, TdT attaches adenylates to the 3'-end of input ssDNA, which are then utilized by RNA ligase as an efficient connector to the ssDNA adaptor. A protocol that uses TACS ligation instead of the second random priming step substantially increased the lengths of PBAT library fragments. Moreover, we devised a dual-library strategy that splits the input DNA to prepare two libraries with reciprocal adaptor polarity, combining them prior to sequencing. This strategy ensured an ideal base-color balance to eliminate the need for DNA spike-in for color compensation, further improving the throughput and quality of WGBS. Adopting the above strategies to the HiSeq X Ten and NovaSeq 6000 platforms, we established a cost-effective, high-quality WGBS, which should accelerate various methylome analyses.
Project description:DNA methylation plays a key role in epigenetic regulation of eukaryotic genomes. Hence the genome-wide distribution of 5-methylcytosine, or the methylome, has been attracting intense attention. In recent years, whole-genome bisulfite sequencing (WGBS) has enabled methylome analysis at single-base resolution. However, WGBS typically requires microgram quantities of DNA as well as global PCR amplification, thereby precluding its application to samples of limited amounts. This is presumably because bisulfite treatment of adaptor-tagged templates, which is inherent to current WGBS methods, leads to substantial DNA fragmentation. To circumvent the bisulfite-induced loss of intact sequencing templates, we conceived an alternative method termed Post-Bisulfite Adaptor Tagging (PBAT) wherein bisulfite treatment precedes adaptor tagging by two rounds of random primer extension. The PBAT method can generate a substantial number of unamplified reads from as little as subnanogram quantities of DNA. It requires only 100 ng of DNA for amplification-free WGBS of mammalian genomes. Thus, the PBAT method will enable various novel applications that would not otherwise be possible, thereby contributing to the rapidly growing field of epigenomics.
Project description:Methylation of cytosine in genomic DNA is a well-characterized epigenetic modification involved in many cellular processes and diseases. Whole-genome bisulfite sequencing (WGBS), such as MethylC-seq and post-bisulfite adaptor tagging sequencing (PBAT-seq), uses the power of high-throughput DNA sequencers and provides genome-wide DNA methylation profiles at single-base resolution. However, the accuracy and consistency of WGBS outputs in relation to the operating conditions of high-throughput sequencers have not been explored.We have used the Illumina HiSeq platform for our PBAT-based WGBS, and found that different versions of HiSeq Control Software (HCS) and Real-Time Analysis (RTA) installed on the system provided different global CpG methylation levels (approximately 5% overall difference) for the same libraries. This problem was reproduced multiple times with different WGBS libraries and likely to be associated with the low sequence diversity of bisulfite-converted DNA. We found that HCS was the major determinant in the observed differences. To determine which version of HCS is most suitable for WGBS, we used substrates with predetermined CpG methylation levels, and found that HCS v2.0.5 is the best among the examined versions. HCS v2.0.12 showed the poorest performance and provided artificially lower CpG methylation levels when 5-methylcytosine is read as guanine (first read of PBAT-seq and second read of MethylC-seq). In addition, paired-end sequencing of low diversity libraries using HCS v2.2.38 or the latest HCS v2.2.58 was greatly affected by cluster densities.Software updates in the Illumina HiSeq platform can affect the outputs from low-diversity sequencing libraries such as WGBS libraries. More recent versions are not necessarily the better, and HCS v2.0.5 is currently the best for WGBS among the examined HCS versions. Thus, together with other experimental conditions, special care has to be taken on this point when CpG methylation levels are to be compared between different samples by WGBS.
Project description:Sodium bisulphite treatment of DNA combined with next generation sequencing (NGS) is a powerful combination for the interrogation of genome-wide DNA methylation profiles. Library preparation for whole genome bisulphite sequencing (WGBS) is challenging due to side effects of the bisulphite treatment, which leads to extensive DNA damage. Recently, a new generation of methods for bisulphite sequencing library preparation have been devised. They are based on initial bisulphite treatment of the DNA, followed by adaptor tagging of single stranded DNA fragments, and enable WGBS using low quantities of input DNA. In this study, we present a novel approach for quick and cost effective WGBS library preparation that is based on splinted adaptor tagging (SPLAT) of bisulphite-converted single-stranded DNA. Moreover, we validate SPLAT against three commercially available WGBS library preparation techniques, two of which are based on bisulphite treatment prior to adaptor tagging and one is a conventional WGBS method.
Project description:Whole-genome bisulfite sequencing (WGBS) has been widely used to quantify cytosine DNA methylation frequency in an expanding array of cell and tissue types. Because of the denaturing conditions used, this method ultimately leads to the measurement of methylation frequencies at single cytosines. Hence, the methylation frequency of CpG dyads (two complementary CG dinucleotides) can be only indirectly inferred by overlaying the methylation frequency of two cytosines measured independently. Furthermore, hemi-methylated CpGs (hemiCpGs) have not been previously analyzed in WGBS studies. We recently developed in silico strand annealing (iSA), a bioinformatics method applicable to WGBS data, to resolve the methylation status of CpG dyads into unmethylated, hemi-methylated, and methylated. HemiCpGs account for 4-20% of the DNA methylome in different cell types, and some can be inherited across cell divisions, suggesting a role as a stable epigenetic mark. Therefore, it is important to resolve hemiCpGs from fully methylated CpGs in WGBS studies. This protocol describes step-by-step commands to accomplish this task, including dividing alignments by strand, pairing alignments between strands, and extracting single-fragment methylation calls. The versatility of iSA enables its application downstream of other WGBS-related methods such as nasBS-seq (nascent DNA bisulfite sequencing), ChIP-BS-seq (ChIP followed by bisulfite sequencing), TAB-seq, oxBS-seq, and fCAB-seq. iSA is also tunable for analyzing the methylation status of cytosines in any sequence context. We exemplify this flexibility by uncovering the single-fragment non-CpG methylome. This protocol provides enough details for users with little experience in bioinformatic analysis and takes 2-7 h.
Project description:Pre-eclampsia is a serious heritable disorder that affects 5-8% of pregnancies worldwide. While classical genetic studies have identified several susceptibility genes they do not fully explain the heritability of pre-eclampsia. An additional contribution to risk can be quantified by examining the epigenome, in particular the methylome, which is a representation of interactions between environmental and genetic influences on the phenotype. Current array-based epigenetic studies only examine 2-5% of the methylome. Here, we used whole-genome bisulfite sequencing (WGBS) to determine the entire methylome of 13 individuals from two multiplex pre-eclampsia families, comprising one woman with eclampsia, six women with pre-eclampsia, four women with uncomplicated normotensive pregnancies and two male relatives. The analysis of WGBS profiles using two bioinformatics platforms, BSmooth and Bismark, revealed 18,909 differentially methylated CpGs and 4157 differentially methylated regions (DMRs) concordant in females. The methylation patterns support the involvement of previously reported candidate genes, including COL4A1, SLC2A4, PER3, FLT1, GPI, LCT, DDAH1, TGFB3, DLX5, and LRP1B. Statistical analysis of DMRs revealed three novel genes significantly correlated with pre-eclampsia: sorbitol dehydrogenase (SORD, p = 9.98 × 10-6), diacylglycerol kinase iota (DGKI, p = 2.52 × 10-5), and islet cell autoantigen 1 (ICA1, 7.54 × 10-3), demonstrating the potential of WGBS in families for elucidating the role of epigenome in pre-eclampsia and other complex diseases.
Project description:Library preparation for whole genome bisulphite sequencing (WGBS) is challenging due to side effects of the bisulphite treatment, which leads to extensive DNA damage. Recently, a new generation of methods for bisulphite sequencing library preparation have been devised. They are based on initial bisulphite treatment of the DNA, followed by adaptor tagging of single stranded DNA fragments, and enable WGBS using low quantities of input DNA. In this study, we present a novel approach for quick and cost effective WGBS library preparation that is based on splinted adaptor tagging (SPLAT) of bisulphite-converted single-stranded DNA. Moreover, we validate SPLAT against three commercially available WGBS library preparation techniques, two of which are based on bisulphite treatment prior to adaptor tagging and one is a conventional WGBS method. Overall design: WGBS libraries were prepared with DNA from human lymphoblastoid cell line NA10860 and the ALL cell line REH
Project description:Background 5? methylation of cytosines in DNA molecules is an important epigenetic mark in eukaryotes. Bisulfite sequencing is the gold standard of DNA methylation detection, and whole-genome bisulfite sequencing (WGBS) has been widely used to detect methylation at single-nucleotide resolution on a genome-wide scale. However, sodium bisulfite is known to severely degrade DNA, which, in combination with biases introduced during PCR amplification, leads to unbalanced base representation in the final sequencing libraries. Enzymatic conversion of unmethylated cytosines to uracils can achieve the same end product for sequencing as does bisulfite treatment and does not affect the integrity of the DNA; enzymatic methylation sequencing may, thus, provide advantages over bisulfite sequencing. Results Using an enzymatic methyl-seq (EM-seq) technique to selectively deaminate unmethylated cytosines to uracils, we generated and sequenced libraries based on different amounts of Arabidopsis input DNA and different numbers of PCR cycles, and compared these data to results from traditional whole-genome bisulfite sequencing. We found that EM-seq libraries were more consistent between replicates and had higher mapping and lower duplication rates, lower background noise, higher average coverage, and higher coverage of total cytosines. Differential methylation region (DMR) analysis showed that WGBS tended to over-estimate methylation levels especially in CHG and CHH contexts, whereas EM-seq detected higher CG methylation levels in certain highly methylated areas. These phenomena can be mostly explained by a correlation of WGBS methylation estimation with GC content and methylated cytosine density. We used EM-seq to compare methylation between leaves and flowers, and found that CHG methylation level is greatly elevated in flowers, especially in pericentromeric regions. Conclusion We suggest that EM-seq is a more accurate and reliable approach than WGBS to detect methylation. Compared to WGBS, the results of EM-seq are less affected by differences in library preparation conditions or by the skewed base composition in the converted DNA. It may therefore be more desirable to use EM-seq in methylation studies.
Project description:Ethylene has long been used to promote flowering in pineapple production. Ethylene-induced flowering is dose dependent, with a critical threshold level of ethylene response factors needed to trigger flowering. The mechanism of ethylene-induced flowering is still unclear. Here, we integrated isoform sequencing (iso-seq), Illumina short-reads sequencing and whole-genome bisulfite sequencing (WGBS) to explore the early changes of transcriptomic and DNA methylation in pineapple following high-concentration ethylene (HE) and low-concentration ethylene (LE) treatment. Iso-seq produced 122,338 transcripts, including 26,893 alternative splicing isoforms, 8,090 novel transcripts and 12,536 candidate long non-coding RNAs. The WGBS results suggested a decrease in CG methylation and increase in CHH methylation following HE treatment. The LE and HE treatments induced drastic changes in transcriptome and DNA methylome, with LE inducing the initial response to flower induction and HE inducing the subsequent response. The dose-dependent induction of FLOWERING LOCUS T-like genes (FTLs) may have contributed to dose-dependent flowering induction in pineapple by ethylene. Alterations in DNA methylation, lncRNAs and multiple genes may be involved in the regulation of FTLs. Our data provided a landscape of the transcriptome and DNA methylome and revealed a candidate network that regulates flowering time in pineapple, which may promote further studies.
Project description:DNA CpG methylation is a widespread epigenetic mark in high eukaryotes including mammals. DNA methylation plays key roles in diverse biological processes such as X chromosome inactivation, transposable element repression, genomic imprinting, and control of gene expression. Recent advancements in sequencing-based DNA methylation profiling methods provide an unprecedented opportunity to measure DNA methylation in a genome-wide fashion, making it possible to comprehensively investigate the role of DNA methylation. Several methods have been developed, such as Whole Genome Bisulfite Sequencing (WGBS), Reduced Representation Bisulfite Sequencing (RRBS), and enrichment-based methods including Methylation Dependent ImmunoPrecipitation followed by sequencing (MeDIP-seq), methyl-CpG binding domain (MBD) protein-enriched genome sequencing (MBD-seq), methyltransferase-directed Transfer of Activated Groups followed by sequencing (mTAG), and Methylation-sensitive Restriction Enzyme digestion followed by sequencing (MRE-seq). These methods differ by their genomic CpG coverage, resolution, quantitative accuracy, cost, and software for analyzing the data. Among these, WGBS is considered the gold standard. However, it is still a cost-prohibitive technology for a typical laboratory due to the required sequencing depth. We found that by integrating two enrichment-based methods that are complementary in nature (i.e., MeDIP-seq and MRE-seq), we can significantly increase the efficiency of whole DNA methylome profiling. By using two recently developed computational algorithms (i.e., M&M and methylCRF), the combination of MeDIP-seq and MRE-seq produces genome-wide CpG methylation measurement at high coverage and high resolution, and robust predictions of differentially methylated regions. Thus, the combination of the two enrichment-based methods provides a cost-effective alternative to WGBS. In this article we describe both the experimental protocols for performing MeDIP-seq and MRE-seq, and the computational protocols for running M&M and methylCRF.