[2] This technique can be employed when there is a lack of precious sample. Evaluation of single cell XRBS profiles, Fig. Gaiti, F. et al. The Transition from Cancer "omics" to "epi-omics" through Next- and Third-Generation Sequencing. This analysis shows that RRBS of MspI fragments 40120 bases in length covers only 0.9% of the genome, but enriches for 5.6% of genomic CpGs. })},1000); Finally, Anchor-Based Bisulfite Sequencing (ABBS) was developed by Ben Delatte's group at Active Motif. Cell 167, 233247 (2016). . Reduced representation bisulfite sequencing (RRBS), which combines restriction enzymes and bisulfite sequencing to enrich for areas of the genome with a high CpG content, is an efficient and high-throughput technique for analyzing the genome-wide methylation profiles on a single nucleotide level. e52246 (2015). These results suggest that one source of single cell DNA methylation heterogeneity is decreased fidelity of maintenance DNA methylation in late replicating domains. Careers. developed this technique in 2005 to reduce the amount of nucleotides required to sequence to 1% of the genome. Cell line-specific homozygous alleles are indicated by white boxes boxes and were used for single cell SNP analysis in Fig. At Illumina, our goal is to apply innovative technologies to the analysis of genetic variation and function, making studies possible that were not even imaginable just a few years ago. Our protocols typically require nine cycles of polymerase chain reaction (PCR) amplification to obtain a sufficient amount of library for sequencing when 100 ng of genomic DNA is used as a starting material; moreover, it takes 3 days to complete library preparation and quality control procedures for up to eight samples. Here we present an extended-representation bisulfite sequencing (XRBS) method for targeted profiling of DNA methylation. Enable insights and variant interpretation for diverse genomic testing applications at scale, Our instrument performance service helps reduce unplanned downtime and minimize instrument requalification, New configurations will bring longer read capabilities with more output for immune repertoire, shotgun metagenomics and more, Understanding cardiovascular diseases through genomic sequencing, Our mission is to improve human health by unlocking the power of the genome, Get instructions for using DRAGEN Secondary Analysis v4.0, Linking the causes and consequences of complex phenotypes through multiomics, Save on the Ribo-Zero Plus Microbiome rRNA Depletion Kit, restrictions apply, More than just a sweet treat, sugarcane can also be a source of greener energy, The NovaSeq 6000Dx is our first IVD-compliant high-throughput sequencing instrument for the clinical lab. 7 . Reduced-representation bisulfite sequencing (RRBS-Seq ) or single-cell reduced-representation bisulfite sequencing (scRRBS ) is a protocol that uses one or multiple restriction enzymes on the genomic DNA to produce sequence-specific fragmentation. Shared peaks are indicated in gray. Nature 569, 576580 (2019). A simple algorithm for choosing a suitable method for DNA methylation analysis is illustrated in Figure 4 (Kurdyukov and Bullock, 2016). Google Scholar. if ($(this).prop("checked") == true) { It is mission critical for us to deliver innovative, flexible, and scalable solutions to meet the needs of our customers. 2009. Science 356, eaaj2239 (2017). F) Heatmap depicts copy number variations inferred for single GM12878 (n=32, top) and K562 cells (n=27, bottom). Abstract. Upstream and downstream regions (200bp) are divided into 25 equally sized windows. [2] This technique is cost-effective especially when focusing on common CpG regions. While hypomethylation of compartment B is most pronounced in K562 cells, a similar trend is also observed in other cultured cell lines and in primary mammary epithelial cells. MspI digestion covers the majority, but not all the CG regions in the genome. Shareef, S.J., Bevill, S.M., Raman, A.T. et al. 2 . HHS Vulnerability Disclosure, Help The protocol by Guo et al. Genes Dev. CAS mRRBS achieves similar CpG coverage to the original RRBS protocol, while the higher throughput and . We describe a large-scale random approach termed reduced representation bisulfite sequencing (RRBS) for analyzing and comparing genomic methylation patterns. Dynamic enhancer DNA methylation as basis for transcriptional and cellular heterogeneity of ESCs. Only differentially methylated elements between monocytes and T cells are shown. Random hexamer-primed second strand synthesis recovers fragmented DNA. Meissner, A. et al. Cancer 11, 726734 (2011). Cell Biol. Filter by batch size, system, software, and more. Cooper, D.N. National Library of Medicine "Preparation of reduced representation bisulfite sequencing libraries for genome-scale DNA methylation profiling. Unauthorized use of these marks is strictly prohibited. Wu, T.P., Wang, T., Seetin, M.G., Lai, Y., Zhu, S., Lin, K., Liu, Y., Byrum, S.D., Mackintosh, S.G., Zhong, M. Reduced Representation Bisulfite Sequencing (RRBS) detects millions of novel CpG sites based on the improved bisulfite sequencing method, providing coverage to approximately all gene promoters, CpG islands, gene bodies, repetitive DNA sequences, and regulatory elements. Reach out to us and we will get back to you shortly. The .gov means its official. Biomolecules. To construct a methylation library, RNA fragments of various lengths are created using a restriction enzyme treatment, followed by sodium bisulfite conversion of unmethylated cytosines into uracils. Robust single-cell DNA methylome profiling with snmC-seq2. Extended Data Fig. Single cells from Hou et al. Differential nuclear protein binding to 5-azacytosine-containing DNA as a potential mechanism for 5-aza-2-deoxycytidine resistance. We describe a large-scale random approach termed reduced representation bisulfite sequencing (RRBS) for analyzing and comparing genomic methylation patterns. }, function() { Tax Reg: 105-87-87282 | Sequence alignment and analysis: Due to the unique properties of RRBS, special software is needed for alignment and analysis. Differential methylation analysis of reduced representation bisulfite sequencing experiments using edgeR F1000Res. With RRBS, scientists enrich for GC-rich parts of the genome by digesting the DNA with an enzyme that generates fragments with CpG dinucleotides at . 1 . J) Scatterplots compare single cell average DNA methylation within various early and late replicating regions (y-axis) against genome-wide average DNA methylation levels (x-axis). Liu, X. S. et al. McGahon, A. J. et al. The fragmented genomic DNA is then treated with bisulfite and sequenced. [7] This is unfortunate in regards to methylation studies, as these repeated sequences often contain methylated cytosines. DNA methylation and cancer. This is the method of choice to study specific regions of interest. Ghandi, M. et al. was supported by a Human Frontier Science Program long-term fellowship (LT000596/2016-L). Mukherjee, K., Twyman, R.M., and Vilcinskas, A. Nature 517, 321326 (2015). Epub 2015 Apr 2. Panels from left to right show: methylation calls from 450k methylation array, RRBS, XRBS, and WGBS. Each bar plot represents a single cell XRBS library. Only CpGs within 75 bases of an MspI cut site were considered for scRRBS libraries to adjust for differences in read lengths. DNA contains four kinds of nitrogenous bases, namely cytosine, guanine, thymine and adenine (Figure 1A). Insects as models to study the epigenetic basis of disease. Several cost-effective reduced representation bisulfite sequencing approaches (RRBS) have been recently developed and applied to different organisms that lack a well annotated reference genome. Sci. The site is secure. The heatmap for methylation density in CHH context 4. is the Bernard and Mildred Kayden Endowed MGH Research Institute Chair and an American Cancer Society Research Professor. Nature 569, 503508 (2019). Reduced representation bisulfite sequencing (RRBS) enriches CpG-rich genomic regions using the MspI restriction enzymewhich cuts DNA at all CCGG sites, regardless of their DNA methylation status at the CG siteand enables the measurement of DNA methylation levels at 5% ~ 10% of all CpG sites in the mammalian genome. Song, Y. et al. Epub 2012 Nov 28. A.T.R. Of these peaks, 7.5% and 2.1% are specifically hypomethylated in K562 (methylation difference 0.5) and OCI-AML3 (0.5) cells, respectively. The Novogene RRBS service comprises four steps. Enhanced reduced representation bisulfite sequencing for assessment of DNA methylation at base pair resolution. Both strands of a CpG dinucleotide are assessed individually. Luo, C., Hajkova, P. & Ecker, J. R. Dynamic DNA methylation: in the right place at the right time. Ben-Hattar, J. S.J.S. 2023. FOIA wrote the manuscript with assistance from other authors, all of whom approved the final submission. Libraries were generated from 100 sorted cells. Rev. Individual promoters (dots) are colored if specifically hypermethylated (red) and hypermethylated (blue) in K562 cells. 14, R21 (2013). Epigenetics Chromatin 11, 21 (2018). and S.M.B. A) Heatmap depicts 8kb regions (rows, n=15,202 peaks) centered on H3K27ac peaks, grouped into regions that are hypomethylated specifically in K562 or OCI-AML3 cells (as in Fig. Article CAS Google Scholar What is RRBS? Wang J, Xia Y, Li L, Gong D, Yao Y, Luo H, Lu H, Yi N, Wu H, Zhang X, Tao Q, Gao F. BMC Genomics. and transmitted securely. Li, G. et al. However, whole-genome bisulfite sequencing (WGBS) can be very expensive when the genome size is large. Adusumalli S, Mohd Omar MF, Soong R, Benoukraf T. Brief Bioinform. 4a) and divided into 100 equally sized windows. Libraries from Gaiti et al. Cloney, R. (2016). B) Plot shows unique reads as a function of aligned reads in libraries from unsorted human bone marrow, HSPCs, monocytes, and T cells. Extended-representation bisulfite sequencing of gene regulatory elements in multiplexed samples and single cells Nat Biotechnol. Researchers have found that cytosine and adenine can be methylated. The https:// ensures that you are connecting to the We describe epiGBS, a reduced representation bisulfite sequencing method for cost-effective exploration and comparative analysis of DNA methylation and genetic variation in hundreds of . Nature Biotechnology Illumina innovative sequencing and array technologies are fueling groundbreaking advancements in life science research, translational and consumer genomics, and molecular diagnostics. A) Plot shows unique reads as a function of aligned reads in 1,000 cell high-coverage libraries of four cell lines. Please enable it to take advantage of the complete set of features! An extended representation DNA, Fig. Percentages indicate the fraction of CpGs that differed between conditions (difference in beta-values >0.5). The onlylimitations are the cost and difficulties in the analysis of NGS data. S.J.S. One of the other most commonly used techniques for profiling methylation is MeDiP-Seq. C) Plot shows expression levels for genes that were associated with cell line-specific promoter hyper- and hypo-methylation. eCollection 2023. Guo H, Zhu P, Guo F, Li X, Wu X, Fan X, Wen L, Tang F. Nat Protoc. a, Plots show results from an in silico MspI restriction digest analysis of the human genome. This analysis shows that RRBS of MspI fragments 40-120 bases in length covers only 0.9% of the genome, but enriches for 5.6% of genomic CpGs. 1000 cells were used for the unsorted bone marrow library. [10] If samples from the patient's tumor and normal cells can be obtained, a comparison between these two cell types can be observed. Decitabine reduces methylation in both compartments to a similar extent. Individual peaks (dots) are colored if specifically hypermethylated in K562 or HL-60 cells (as in Fig. ERRBS initiates with restriction enzyme digestion of DNA to generate low molecular weight fragments for use in library preparation. E) Genome plot shows average DNA methylation and the Hi-C-derived first eigenvector for K562 cells for a 80Mb genomic segment of chromosome 2. Methodological aspects of whole-genome bisulfite sequencing analysis. PMID: 34179291 PMCID: PMC8203956 DOI: 10.21769/BioProtoc.2778 Baylin, S. B. Figure 1. official website and that any information you provide is encrypted Article G) Genome plot for the same region as in panel E shows the Hi-C-derived first eigenvector illustrating compartments A (green) and B (orange) for HL-60 cells , and average DNA methylation for HL-60 or OCI-AML3 treated with decitabine or control. With greater sequencing depth the fraction of unique reads decreases, as the chance of sampling a non-unique read (that is PCR duplicate) increases. Gel-free multiplexed reduced representation bisulfite sequencing for large-scale DNA methylation profiling. https://doi.org/10.1038/s41587-021-00910-x. But there are also some limitations in the specific protocol steps. TSG: tumor suppressor gene. Stage-specific changes can be observed in all living organisms. Life (Basel). c, Barplot shows the fraction of unique reads (that is reads not representing PCR duplicates) per single cell library. Med. Different sizes of the fragments are separated using, Bisulfite conversion: The DNA fragments are then bisulfite converted, which is a process that deaminates unmethylated cytosine into a, PCR amplification: The bisulfite converted DNA is then amplified using. Yin, Y. et al. Cells are sorted into separate barcoding reactions, and then pooled for subsequent bisulfite conversion, hexamer extension, and library amplification. Genet. Reduced Representation Bisulfite Sequencing (RRBS) and sperm quality analysis were assessed in bull sperm collected from the caput, corpus and cauda of the epididymis, to investigate the variation in sperm methylation and chromatin condensation during sperm epididymal maturation. The cumulative number of MspI fragments (total of 2.3 million, left), of basepairs (total of 3.1 billion, middle), and of CpGs (total of 29.4 million, right) is shown relative to increasing MspI fragment length. Cell Res. Shared peaks are indicated in gray. (Robertson, 2005). BglII restriction fragments were size-selected to 500-600 bp, equipped with adapters, treated with bisulfite, PCR amplified, cloned and sequenced. Some CpGs are missed and some regions thus have lower coverage. 3 . d, Heatmap compares alternate allele frequencies from SNP array data for K562 and HL-60 cell lines. f, Scatterplot shows merged CTCF peaks from K562 and HL-60 ChIP-seq datasets. A) Plot shows unique reads as a function of aligned reads in single cell XRBS profiles (n=96 cells). Reduced Representation Bisulfite Sequencing An alternative approach to WGBS is reduced representation bisulfite sequencing (RRBS). Another positive aspect is that fresh or live samples are not required. Adapted from (Kurdyukov and Bullock, 2016). Single-cell methylomes identify neuronal subtypes and regulatory elements in mammalian cortex. Nat. However, these differences likely represent true copy number variations between K562 cells used for these experiments. performed decitabine experiments. The heatmap for methylation density in CHG context 3. Profiling DNA methylome landscapes of mammalian cells with single-cell reduced-representation bisulfite sequencing. Dendrogram derived from unsupervised clustering is indicated to the left. Periodicity of DNA methylation around CTCF binding sites is consistent with positioned nucleosomes, but more pronounced for CTCF-only sites. 2015 May;16(3):369-79. doi: 10.1093/bib/bbu016. 2015 Jul-Aug;49(4):689-99. doi: 10.7868/S0026898415040187. Unauthorized use of these marks is strictly prohibited. J Vis Exp 96:e52246, Guo H, Zhu P, Guo F et al (2015) Profiling DNA methylome landscapes of mammalian cells with single-cell reduced-representation bisulfite sequencing. 29, 750761 (2019). Twenty four barcoded cells were in each of 4 independent libraries. High-throughput bisulfite sequencing in mammalian genomes. and transmitted securely. Left plot shows the number of paired-end reads sequenced for each cell. J. Vis. Disclaimer. Sequencing: The fragments are then sequenced. Chem. 20, 590607 (2019). (2015) Quantitative and multiplexed DNA methylation analysis using long-read single-molecule real-time bisulfite sequencing (SMRT-BS). Nat. Reduced representation bisulfite sequencing ( RRBS) is an efficient and high-throughput technique for analyzing the genome-wide methylation profiles on a single nucleotide level. The bisulfite treatment of DNA mediates the deamination of cytosineinto uracil, and these converted residues will be read as thymine, as determined by PCR-amplificationand subsequent sequencing analysis (Figure 5). was supported by a Medical Scientist Training Award from the National Institute of General Medical Sciences (T32GM007753). While hypomethylation of compartment B is most pronounced in K562 cells, a similar trend is also observed in other cultured cell lines and in primary mammary epithelial cells. An official website of the United States government. V.H. Barcoding samples in a single well through sequential lysis, digestion, and ligation minimizes DNA loss. The data obtained on RRBS and the Illumina Infinium methylation are highly comparable, with a Pearson correlation of 0.92. Methylation levels of the unsorted bone marrow are shown for comparison (left). doi: 10.1002/ctm2.1165. Panels show average methylation from 1,000-cell XRBS profiles for the indicated cell types. CAS 9, 3824 (2018). "Technical considerations for reduced representation bisulfite sequencing with multiplexed libraries. Gu, H. et al. a, Plot shows unique reads as a function of aligned reads in low-coverage XRBS libraries from K562, HL-60, OCI-AML3, and Kasumi-1 cells. d, Scatterplot compares gene expression level and H3K4me3 ChIP-seq signal for gene promoters that were identified as differentially methylated between all four cell lines. $("div[id='pelase_specify_main']").show(); A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Promoters (rows, 25-fold combined coverage in every cell line) are grouped by the cell line in which they are specifically hypermethylated (top) or hypomethylated (bottom). Varley, K. E. et al. Application of XRBS to single leukemia cells enabled us to evaluate genetic copy number variations and methylation variability across individual cells. This is especially limiting for studies involving profiling cancer genomes, as a loss of methylation in this repeated sequences is observed in many cancer types. Expanded encyclopaedias of DNA elements in the human and mouse genomes. [2] This technique can rapidly determine the overall methylation status of cancer genomes which is cost and time effective. We have used Circos plot to represent the methylation density on a chromosome (Silin Zhong , James J Giovannoni et al. Correlation of DNA methylation with histone marks and compartment calls, Extended Data Fig. Authors Epub 2011 Mar 18. Enrichment for functional elements at a uniform sequencing depth of 10 billion base pairs is indicated. The detection of DNA methylation patterns is a rapidly advancing area of research and several methods have been available for the assessment of DNA methylation, with bisulfite treatment being a central procedure to a majority. Single GM12878 cells are largely copy number neutral, while single K562 cells exhibit multiple chromosomal and sub-chromosomal abnormalities. Nat Protoc 10: 645-659, Guo H., Zhu P., Guo F., Li X., Wu X., et al. Barcoded DNA fragments are pooled before bisulfite conversion, allowing multiplex processing and technical consistency in low-input samples. 2a). Adenine methylation happens at the 6-nitrogen position of adenine (N6-methyladenine or N6mA). Retailer Reg: 2019--2018 | Thus, a step for PCR purification is required. if($(".R_boxmap").height()<$("#pic").height()+e.pageY){picy=e.pageY-$("#pic").height()-150;console.log($(".R_boxmap").height())} Average gene expression levels are indicated by horizontal lines. DNA methylation refers to the addition of a methyl group (CH3) to the DNA strand which is catalyzed by DNA methyltransferases (DNMTs). government site. Mulqueen, R. M. et al. A) Plots show the gating strategy for fluorescence assisted cell sorting (FACS) of human bone marrow of CD34+ HSPCs, CD3+ T cells, and CD14+ monocytes.