Whole-genome Bisulfite Sequencing Libraries Research Articles

Generation of Whole-Genome Bisulfite Sequencing Libraries for Comprehensive DNA Methylome Analysis.

Whole-genome bisulfite sequencing (WGBS) enables the detection of DNA methylation at a single base-pair resolution. The treatment of DNA with sodium bisulfite allows the discrimination of methylated and unmethylated cytosines, but the power of this technology can be limited by the input amounts of DNA and the length of DNA fragments due to DNA damage caused by the desulfonation process. Here, we describe a WGBS library preparation protocol that minimizes the loss and damage of DNA, generating high-quality libraries amplified with fewer polymerase chain reaction (PCR) cycles, and hence data with fewer PCR duplicates, from lower amounts of input material. Briefly, genomic DNA is sheared, end-repaired, 3'-adenylated, and ligated to adaptors with fewer clean-up steps in between, minimizing DNA loss. The adapter-ligated DNA is then treated with sodium bisulfite and amplified with a few PCR cycles to reach the yield needed for sequencing.

164 Genome-wide DNA Methylation Profiling in in Utero Heat-stressed Pigs as Measured by Whole-genome Bisulfite Sequencing

Abstract In utero heat stress (IUHS) has several postnatal consequences in pigs that compromise health, increase stress response, and reduce performance. These phenotypes may be caused by epigenetic modifications such as DNA methylation, which are heritable molecular modifications that impact gene expression and phenotypic outcomes without changing the DNA sequence. Therefore, we aimed to compare the DNA methylation profiles between in-utero thermoneutral (IUTN) and IUHS pigs to identify differentially methylated regions. Twenty-four pregnant gilts were evenly assigned to either a thermoneutral (17.5 ± 2.1°C) or heat stress (cycling 26 to 36°C) chamber from d 0 to 59 of gestation, followed by thermoneutral conditions (20.9 ± 2.3°C) for the rest of gestation and until the piglets were weaned. At 105 d of age, 10 IUTN and 10 IUHS piglets were euthanized and Longissimus dorsi muscle samples were collected and used to perform whole-genome bisulfite sequencing (WGBS). Purified genomic DNA was fragmented and bisulfite conversion was performed. Illumina platforms were used to sequence WGBS libraries. All pigs had similar proportions of methylation at CpG sites. Two-hundred-sixty-eight genomic regions were differentially methylated between IUTN and IUHS pigs. These identified regions are located across all pig chromosomes and ranged from 2 (SSC18) to 40 (SSC10). Eighty-five unique differentially-methylated genes were identified. These genes have been reported to be involved in key biological processes such as transcriptional repressor activity and tRNA processing (e.g., SKOR2,TRMT6, TSEN2), cellular response to heat stress (e.g.,CCAR2), placental vascularization (e.g.,FZD5), central nervous system (e.g.,VEPH1), cholesterol biosynthesis (e.g., CYB5R1), insulin receptor substrate (e.g.,IRS2), synaptic transmission (e.g.,RIMBP2), neurotrophic factor receptor activity (e.g.,LIFR), immune response (e.g., CD84), DNA repair (e.g., CHD1L), and cell proliferation and endocrine signaling (e.g., SSTR1, CYB5R1). These findings contribute to a better understanding of the epigenomic mechanisms underlying postnatal consequences of IUHS in pigs.

Low-Input Whole-Genome Bisulfite Sequencing.

DNA methylation can regulate gene expression by modulating chromatin accessibility and transcription factor binding on promoter and enhancer regions. Whole-genome bisulfite sequencing (WGBS) represents the most informative and comprehensive analysis to profile the DNA methylation status of all the cytosines at single-base resolution. However, most of the available protocols recommend an amount of input DNA (50ng-5μg) that makes the WGBS unsuitable for limited samples and cell populations. In this chapter, we provide complete protocol to perform WGBS libraries from very low-input DNA. This protocol is recommended for the analysis of the whole-genome DNA methylation pattern in rare cell populations, like a defined stem cell population isolated from animal models or human samples.

Estimating Global Methylation and Erasure Using Low-Coverage Whole-Genome Bisulfite Sequencing (WGBS ).

Whole-genome bisulfite sequencing (WGBS) is a popular method for characterizing cytosine methylation because it is fully quantitative and has base-pair resolution. While WGBS is prohibitively expensive for experiments involving many samples, low-coverage WGBS can accurately determine global methylation and erasure at similar cost to high-performance liquid chromatography (HPLC) or enzyme-linked immunosorbent assays (ELISA). Moreover, low-coverage WGBS has the capacity to distinguish between methylation in different cytosine contexts (e.g., CG, CHH, and CHG), can tolerate low-input material (<100cells), and can detect the presence of overrepresented DNA originating from mitochondria or amplified ribosomal DNA. In addition to describing a WGBS library construction and quantitation approach, here we detail computational methods to predict the accuracy of low-coverage WGBS using empirical bootstrap samplers and theoretical estimators similar to those used in election polling. Using examples, we further demonstrate how non-independent sampling of cytosines can alter the precision of error calculation and provide methods to improve this.

Systematic evaluation of library preparation methods and sequencing platforms for high-throughput whole genome bisulfite sequencing

Whole genome bisulfite sequencing (WGBS), with its ability to interrogate methylation status at single CpG site resolution epigenome-wide, is a powerful technique for use in molecular experiments. Here, we aim to advance strategies for accurate and efficient WGBS for application in future large-scale epidemiological studies. We systematically compared the performance of three WGBS library preparation methods with low DNA input requirement (Swift Biosciences Accel-NGS, Illumina TruSeq and QIAGEN QIAseq) on two state-of-the-art sequencing platforms (Illumina NovaSeq and HiSeq X), and also assessed concordance between data generated by WGBS and methylation arrays. Swift achieved the highest proportion of CpG sites assayed and effective coverage at 26x (P < 0.001). TruSeq suffered from the highest proportion of PCR duplicates, while QIAseq failed to deliver across all quality metrics. There was little difference in performance between NovaSeq and HiSeq X, with the exception of higher read duplication rate on the NovaSeq (P < 0.05), likely attributable to the higher cluster densities on its flow cells. Systematic biases exist between WGBS and methylation arrays, with lower precision observed for WGBS across the range of depths investigated. To achieve a level of precision broadly comparable to the methylation array, a minimum coverage of 100x is recommended.

1 Whole-genome bisulfite sequencing of bovine gametes and invivo-produced pre-implantation embryos

Dynamic changes in DNA methylation are crucial in the epigenetic regulation of mammalian embryogenesis. Global DNA methylation studies in the bovine, however, remain mostly at the immunostaining level. We adopted the single-cell whole-genome bisulfite sequencing method to characterise stage-specific genome-wide DNA methylation in bovine sperm, individual oocytes derived invivo and invitro, and invivo-developed embryos at the 2-, 4-, 8-, and 16-cell stages. This method allowed us to theoretically cover all CpG sites in the genome using a limited number of cells from single embryos. Pools of 20 sperm were selected from a bull with proven fertility. Single oocytes (n=6) and embryos (n=4 per stage) were collected from Holstein cows (n=10). Single-cell whole-genome bisulfite sequencing libraries were prepared and sequenced using the Illumina HiSEqn 4000 platform (Illumina, San Diego, CA, USA). Sequencing reads were filtered and aligned to the bovine reference genome (UMD 3.1.1) using Bismark (Krueger and Andrews 2011Bioinformatics27, 1571-1572, DOI: 10.1093/bioinformatics/btr167).A 300-bp tile-based method was applied to bin the genome into consecutive windows to facilitate comparison across samples. The DNA methylation level was calculated as the fraction of read counts of the total number of cytosines (methylated) in the total read counts of reported cytosines and thymines (methylated and unmethylated), only if more than 3 CpG sites were covered in this tile. Gamete-specific differentially methylated regions were identified when DNA methylation levels were greater than 75% in one type of gamete and less than 25% in the other with false discovery rate-corrected Fisher’s exact test P-values of less than 0.05. The major wave of genome-wide DNA demethylation was complete at the 8-cell stage when de novo methylation became prominent. Sperm and oocytes had numerous differentially methylated regions that were enriched in intergenic regions. Differentially methylated regions were also identified between invivo- and invitro-matured oocytes. Moreover, X chromosome methylation followed the global dynamic patterns. Virtually no (less than 1.5%) DNA methylation was found in mitochondrial DNA. Finally, using our RNA sequencing data generated from the same developmental stages (Jiang et al. 2014 BMC Genomics 15, 756; DOI: 10.1186/1471-2164-15-756), we revealed an inverse correlation between gene expression and promoter methylation. Our study provides the first fully comprehensive analysis of the global dynamics of DNA methylation in bovine gametes and single early embryos using single-cell whole-genome bisulfite sequencing. These data provide insights into the critical features of the methylome of bovine embryos and serve as an important reference for embryos produced by assisted reproduction, such as IVF and cloning, and a model for human early embryo epigenetic regulation.

Comparison of whole-genome bisulfite sequencing library preparation strategies identifies sources of biases affecting DNA methylation data

BackgroundWhole-genome bisulfite sequencing (WGBS) is becoming an increasingly accessible technique, used widely for both fundamental and disease-oriented research. Library preparation methods benefit from a variety of available kits, polymerases and bisulfite conversion protocols. Although some steps in the procedure, such as PCR amplification, are known to introduce biases, a systematic evaluation of biases in WGBS strategies is missing.ResultsWe perform a comparative analysis of several commonly used pre- and post-bisulfite WGBS library preparation protocols for their performance and quality of sequencing outputs. Our results show that bisulfite conversion per se is the main trigger of pronounced sequencing biases, and PCR amplification builds on these underlying artefacts. The majority of standard library preparation methods yield a significantly biased sequence output and overestimate global methylation. Importantly, both absolute and relative methylation levels at specific genomic regions vary substantially between methods, with clear implications for DNA methylation studies.ConclusionsWe show that amplification-free library preparation is the least biased approach for WGBS. In protocols with amplification, the choice of bisulfite conversion protocol or polymerase can significantly minimize artefacts. To aid with the quality assessment of existing WGBS datasets, we have integrated a bias diagnostic tool in the Bismark package and offer several approaches for consideration during the preparation and analysis of WGBS datasets.

Large-scale comparative epigenomics reveals hierarchical regulation of non-CG methylation in Arabidopsis

Genome-wide characterization by next-generation sequencing has greatly improved our understanding of the landscape of epigenetic modifications. Since 2008, whole-genome bisulfite sequencing (WGBS) has become the gold standard for DNA methylation analysis, and a tremendous amount of WGBS data has been generated by the research community. However, the systematic comparison of DNA methylation profiles to identify regulatory mechanisms has yet to be fully explored. Here we reprocessed the raw data of over 500 publicly available Arabidopsis WGBS libraries from various mutant backgrounds, tissue types, and stress treatments and also filtered them based on sequencing depth and efficiency of bisulfite conversion. This enabled us to identify high-confidence differentially methylated regions (hcDMRs) by comparing each test library to over 50 high-quality wild-type controls. We developed statistical and quantitative measurements to analyze the overlapping of DMRs and to cluster libraries based on their effect on DNA methylation. In addition to confirming existing relationships, we revealed unanticipated connections between well-known genes. For instance, MET1 and CMT3 were found to be required for the maintenance of asymmetric CHH methylation at nonoverlapping regions of CMT2 targeted heterochromatin. Our comparative methylome approach has established a framework for extracting biological insights via large-scale comparison of methylomes and can also be adopted for other genomics datasets.

Generation of Whole Genome Bisulfite Sequencing Libraries for Comprehensive DNA Methylome Analysis.

Whole genome bisulfite sequencing (WGBS) enables the detection of DNA methylation at single base-pair resolution. The treatment of DNA with sodium bisulfite allows the discrimination of methylated and unmethylated cytosines, but the power of this technology can be limited by the input amounts of DNA and the length of DNA fragments due to DNA damage caused by the desulfonation process. Here, we describe a WGBS library preparation protocol that minimizes the loss and damage of DNA, generating high quality libraries amplified with fewer PCR cycles, and hence data with fewer PCR duplicates, from lower amounts of input material. Briefly, genomic DNA is sheared, end-repaired, 3'-adenylated, and ligated to adaptors with fewer cleanup steps in between, minimizing DNA loss. The adapter-ligated DNA is then treated with sodium bisulfite and amplified with few PCR cycles to reach the yield needed for sequencing.

Tagmentation-Based Library Preparation for Low DNA Input Whole Genome Bisulfite Sequencing.

Aberrations of the DNA methylome contribute to onset and progression of diseases. Whole genome bisulfite sequencing (WGBS) is the only analytical method covering the complete methylome. Alternative methods requiring less DNA than WGBS analyze only a minor portion of the methylome and do not cover important regulatory features like enhancers and noncoding RNAs. In tagmentation-based WGBS (TWGBS), several DNA and time-consuming steps of the conventional WGBS library preparation are circumvented by the use of a hyperactive transposase, which simultaneously fragments DNA and appends sequencing adapters. TWGBS requires only nanogram amounts of DNA and, thus, is well suited to study precious biological specimens such as sorted cells or micro-dissected tissue samples.

Software updates in the Illumina HiSeq platform affect whole-genome bisulfite sequencing

BackgroundMethylation 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.ResultsWe 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.ConclusionsSoftware 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.

Identifying Methylation Changes Driving Evolution of Relapse in MLL-Rearranged Acute Lymphoblastic Leukemias

IntroductionThe MLL gene is rearranged (MLL-r) in 80% of infants with B-lymphoblastic leukemia (B-ALL). MLL-r infant B-ALL has a poor prognosis, with 4-year event-free survival less than 45%. The typical pattern of failure in MLL-r infant ALL is successful remission induction followed by early relapse, suggesting rapid emergence and/or selection of one or more chemoresistant subclones. The markedly lower observed remission induction rates at relapse (<40%) vs. diagnosis (>90%) are consistent with this hypothesis. Genomic studies of MLL-r B-ALL have revealed a striking paucity of cooperating genomic abnormalities compared to other subsets of B-ALL, which suggests heritable epigenetic changes may drive leukemogenesis, chemoresistance and evolution of relapse in MLL-r B-ALL. MLL, its fusion partners and various components of its large complexes have functional domains with known or suspected epigenetic activity. Thus, MLL rearrangement in B-ALL may trigger chromatin modifications and DNA CpG methylation changes that interplay to disrupt normal gene transcription and expression. We hypothesized that infant MLL-r B-ALL cells dynamically acquire heritable DNA CpG methylation changes, some of which may contribute to chemoresistance and evolution of relapse. To test this hypothesis we performed whole-genome bisulfite sequencing (WGBS) using paired diagnosis-relapse (DX-RL) samples to identify methylation changes that may drive relapse.MethodsWe evaluated paired DX-RL specimens with >95% leukemic blasts from two infants with B-ALL harboring MLL-ENL fusions: A 4 month-old female who relapsed after 1 year (case a), and an 8 month-old male who relapsed after 5 months (case b). We prepared WGBS libraries and ran paired-end sequencing (2x100bp) using Illumina HiSeq at average 25X coverage. We used Bismark for aligning reads and calling CpG methylation states. We developed an analysis pipeline to convert CpG states into CpG haplotypes, detect methylation changes at the haplotype level between DX and RL, annotate methylation changes to genes and promoters, and perform gene set enrichment analysis (GSEA) on these genes.ResultsFirst, we filtered reads falling into repeated DNA. We then obtained methylation calls at 15.6/14.3 (DX/RL) million CpGs in case (a) and 15.5/15.6 (DX/RL) million CpGs in case (b). We extracted uniquely aligned reads covering 4 contiguous CpGs, which defined a “4 CpG-site”, or “site”. We required that each site in both DX and RL is covered by at least 4 reads, which yielded 26,747 and 85,174 sites for (a) and (b). We compared the methylation level at each site in DX versus RL (e.g. a “1-0-1-1” 4 CpG-site has a methylation level of 0.75). For (a), 165 sites showed a 50% increase in methylation level at relapse (e.g., from 0.2 to 0.7), and 468 showed a 50% decrease in methylation level at relapse. For (b) there were 605 increased and 57 decreased.The 633 methylation changes in (a) mapped to 175 genes (2kb upstream to transcription end site) and the 662 changes in (b) mapped to 135 genes. Some of these genes are known to be involved in MLL-r ALL [e.g., IKZF1, in (a) and TLX2 in (b)]. GSEA was performed independently using the 175 and 135 genes in (a) and (b), respectively. Interestingly, there was substantial overlap: The top 3 gene sets for (b) (FDR q-value ≤ 2.9E-8) were also within the top 9 sets for (a) (FDR ≤ 1.1E-5). Strikingly, all 3 of these sets involved components of the polycomb repressive complex 2 (PRC2) (1. EED target genes, 2. H3K27me3 bound genes and 3. Genes increased after EZH2 knockdown). Also in common was the KEGG gene set for antigen processing and presentation [FDR 3.4E-6 in (a) and 1.7E-4 in (b)].ConclusionWGBS allows an in-depth look at the epigenetic state of leukemic cell populations. While >98% of the methylome remained relatively stable between diagnosis and relapse (< 50% change), the hundreds of differentially methylated sites may be sufficient to influence expression of key genes and drive selection during the evolution of relapse. In addition, the ~15 million CpG methylation states are stably inherited yet variable, and comprise a rich source of information that can be used to identify evolving subclones, particularily in MLL-r ALL given its silent genomic landscape. Validation of the functional significance of the methylation changes using RNA-seq and identification of functionally important epigenetically-defined subclones is underway. DisclosuresNo relevant conflicts of interest to declare.

Coverage recommendations for methylation analysis by whole-genome bisulfite sequencing.

Whole genome bisulfite sequencing (WGBS) allows genome-wide DNA methylation profiling but the associated high sequencing costs continue to limit its widespread application. We utilized several high coverage reference data sets to experimentally determine minimal sequencing requirements. Here, we present data derived recommendations for minimum sequencing depth for WGBS libraries, highlight what is gained with increasing coverage and discuss the trade off between sequencing depth and number of assayed replicates.

EpiGnome™ Methyl-Seq Kit: a novel post–bisulfite conversion library prep method for methylation analysis

Epigenomics is increasingly becoming an important field of research, and the ability to detect and quantify DNA methylation accurately is now critical for numerous fields of study, including disease biology and gene expression. The differential reactivities of methylated and nonmethylated cytosines in DNA with sodium bisulfite forms the basis for their identification in the genome by sequencing. Current whole-genome bisulfite sequencing methods require substantial amounts of starting DNA to compensate for the loss due to bisulfite-mediated DNA degradation. To address issues with sample preparation, we have developed the EpiGnome™ Methyl-Seq Kit, which utilizes a novel pre-library bisulfite conversion scheme to prepare whole-genome bisulfite sequencing libraries with no sample loss and with only 50 ng of input genomic DNA.