Abstract
Epigenetic modifications such as carbon 5 methylation of the cytosine base in a CpG dinucleotide context are involved in the onset and progression of human diseases. A comprehensive understanding of the role of genome-wide DNA methylation patterns, the methylome, requires quantitative determination of the methylation states of all CpG sites in a genome. So far, analyses of the complete methylome by whole-genome bisulfite sequencing (WGBS) are rare because of the required large DNA quantities, substantial bioinformatic resources and high sequencing costs. Here we describe a detailed protocol for tagmentation-based WGBS (T-WGBS) and demonstrate its reliability in comparison with conventional WGBS. In T-WGBS, a hyperactive Tn5 transposase fragments the DNA and appends sequencing adapters in a single step. T-WGBS requires not more than 20 ng of input DNA; hence, the protocol allows the comprehensive methylome analysis of limited amounts of DNA isolated from precious biological specimens. The T-WGBS library preparation takes 2 d.
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Acknowledgements
We gratefully acknowledge excellent technical support by M. Helf and helpful discussions with D. Lipka. We also acknowledge the excellent support by the sequencing core facility at DKFZ. Work in the Plass laboratory was supported by the Helmholtz Foundation and the German Federal Ministry of Education and Science in the program for medical genome research (FKZ; no. 01KU1001A). Q.W. obtained support by the Humboldt Research Fellowship for Postdoctoral Researchers. A.A. is funded by a National Science Foundation Graduate Research Fellowship.
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Contributions
A.A., J.S. and D.W. conceived the study. B.R., W.W. and V.H. contributed data. B.R., R.E. and C.P. contributed materials. W.W., M.B., S.W. and D.W. performed the experiments and analyzed data. Q.W., L.G. and V.H. performed the bioinformatic analysis. Q.W., L.G. and D.W. wrote the manuscript.
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A provisional US patent application has been deposited for aspects of these methods (A.A., J.S.).
Integrated supplementary information
Supplementary Figure 1 Control of size distribution of three DNA samples after tagmentation.
(a) Prior to oligo replacement/gap repair, the DNA has a size range of about 700–10,000 bp. The non-tagmented DNA (not shown) was ≥ 20 kb in size. (b) Three sequencing libraries prepared from the DNA shown in a without bisulfite treatment. FU, fluorescence units.
Supplementary Figure 2 PCR amplification curves and size distributions of T-WGBS libraries prepared from six different human blood samples.
Input DNA amount was 20 ng each. (a) PCR amplification curves indicate that all 6 samples are close to the plateau phase at cycle 12. (b) Size distributions of the six T-WGBS libraries ranging from about 200 bp to 600 bp with size peaks around 300 bp. The high spikes flanking the curves represent size markers. FU, fluorescence units.
Supplementary Figure 3 Base composition.
(a,b) Consistency in base composition of sequencing reads between T-WGBS (a) and conventional WGBS (b). The base composition bias in the first bases of the T-WGBS reads is caused by the Tn5 transposase which has a slight base preference at certain positions of the integration target sequence (please see the paragraph entitled ‘Limitations’ in the INTRODUCTION for further information).
Supplementary Figure 4 Scatter plot versions of the comparative methylation levels shown in Figure 4a,b.
(a) High consistency of methylation level estimates between T-WGBS and conventional WGBS at single CpGs covered at least 30-fold (r = 0.95). (b) High reproducibility of T-WGBS indicated by the similarity of the methylation levels (r = 0.92) in windows of 5 CpGs (read numbers too low for single CpG analysis) in libraries from two independent tagmentations analyzed on a single HiSeq2000 lane each.
Supplementary Figure 5 High consistency, 97.8% and 98.3%, between T-WGBS and conventional WGBS methylation data from two human blood samples, M and K, respectively.
For each sample, two lanes of T-WGBS data and three lanes of conventional WGBS were compared. (a,b) Methylation levels were calculated based on scanning windows of 5 CpGs with at least 30-fold coverage and displayed on density plots (a) and scatter plots (b). The dotted lines mark the 0.2 difference between the x and y axes. Consistency values were calculated by subtracting from 100% the respective numbers in the corners of the density plots displayed in (a); these numbers indicate the percentage of data points that fall above/below the envelope marked with dotted lines. The r values in (b) indicate Pearson correlation.
Supplementary Figure 6 High consistency of methylation levels between T-WGBS and conventional WGBS.
(a) 98.5% consistency between T-WGBS and conventional WGBS. (b) 98.1% consistency is in an analogous analysis as in (a) between the two sequencing strands, designated Watson and Crick, from the conventional WGBS. The lower figures show scatter plot versions of the figures at the top. Consistency defined as less than 0.2 (20%) difference in methylation level in windows of 5 CpGs. Consistency values were calculated by subtracting from 100% the respective numbers in the corners of the upper two density plots; these numbers indicate the percentage of data points that fall above/below the envelope marked with dotted lines (marking the 0.2 difference between the x and y axes). The r-values indicate Pearson correlation.
Supplementary Figure 7 Comparison of sequencing coverage of cytosines in CpG, CHG and CHH context (H can be A, C or T) between T-WGBS and conventional WGBS.
Coverage versus cytosine density in 1-kb windows for WGBS (red) and T-WGBS (blue). The coverage appears stable in genomic regions with 100–400 cytosines, likely because the majority of 1-kb regions has a cytosine density between 10% and 40%. Variation becomes larger in the two extremes of the cytosine density for both T-WGBS and conventional WGBS due to low counts in each category.
Supplementary Figure 8 Comparison of sequencing coverage in relation to the GC content in 200-bp windows between T-WGBS and conventional WGBS.
Coverage versus local GC content in 200-bp windows, roughly the median length of genomic DNA in the library fragments, for WGBS (red) and T-WGBS (blue). Overall, T-WGBS has a higher coverage along a wide range of GC content.
Supplementary information
Supplementary Methods
Sequencing data alignment and methylation calling methods (PDF 84 kb)
Supplementary Table 1
Comparison of read numbers, duplications, coverage, methylation level and conversion between T-WGBS and conventional WGBS. (XLS 29 kb)
Supplementary Table 2
Comparison of CpG coverage between conventional WGBS and T-WGBS. (XLS 22 kb)
Supplementary Figure 1
Control of size distribution of three DNA samples after tagmentation. (PDF 176 kb)
Supplementary Figure 2
PCR amplification curves and size distributions of T-WGBS libraries prepared from six different human blood samples. (PDF 74 kb)
Supplementary Figure 3
Base composition. (PDF 117 kb)
Supplementary Figure 4
Scatter plot versions of the comparative methylation levels shown in Figure 4a,b. (PDF 321 kb)
Supplementary Figure 5
High consistency, 97.8% and 98.3%, between T-WGBS and conventional WGBS methylation data from two human blood samples, M and K, respectively. (PDF 255 kb)
Supplementary Figure 6
High consistency of methylation levels between T-WGBS and conventional WGBS. (PDF 456 kb)
Supplementary Figure 7
Comparison of sequencing coverage of cytosines in CpG, CHG and CHH context (H can be A, C or T) between T-WGBS and conventional WGBS. (PDF 79 kb)
Supplementary Figure 8
Comparison of sequencing coverage in relation to the GC content in 200-bp windows between T-WGBS and conventional WGBS. (PDF 47 kb)
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Wang, Q., Gu, L., Adey, A. et al. Tagmentation-based whole-genome bisulfite sequencing. Nat Protoc 8, 2022–2032 (2013). https://doi.org/10.1038/nprot.2013.118
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DOI: https://doi.org/10.1038/nprot.2013.118
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