Abstract
DNA methylation changes are dynamic processes which occur at cytosines of CpG dinucleotides and contribute to normal development but also to diseases. DNA methylation changes are most effective in promoters and enhancers, the former frequently being CpG-rich and the latter, in contrast, CpG-poor. Many genome-wide methods for DNA methylation analysis interrogate predominantly CpG-rich regions and, hence, spare enhancers and other potentially important genomic regions. Whole genome bisulfite sequencing (WGBS), in contrast, analyzes the DNA methylome in its entirety. Standard tagmentation-based whole genome bisulfite sequencing (TWGBS) is a Tn5 transposon-based method which requires only 30 ng of human input DNA and, hence, is particularly suited for precious biological samples like cells sorted by flow cytometry or laser capture microdissected tissue specimens. In the standard version, tagmentation generates DNA fragments flanked by uniform sequencing adapters. In a subsequent step, the non-covalently bound adapter oligonucleotide needs to be replaced by a novel oligonucleotide to provide the proper adapter sequence for the reverse strand in paired-end sequencing. The presented protocol describes an improved, simplified version of TWGBS where the inefficient oligo-replacement is circumvented by usage of a sequencing-compatible transposase-adapter complex. Consequently, genomic DNA of only a few hundred human cells is required to interrogate the complete human DNA methylome.
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Acknowledgments
We gratefully acknowledge excellent technical support by Marion Bähr. We also acknowledge the excellent support from the sequencing core facility at the DKFZ. Work in the Plass laboratory was supported by the Helmholtz Foundation and the German Federal Ministry of Education and Science (BMBF) in the program for medical genome research (FKZ: 01KU1001A). B.B. and C.P. also receive funding within the DFG Research Consortium FOR 2674. B.B. has been further supported by a grant from BMBF in the framework of the German Epigenome Project DEEP (FKZ: 01KU1216B) and by a grant for the de.NBI-epi project in the context of the German National Network for Bioinformatics Infrastructures (FKZ: 031L0101A). Parts of the bioinformatics workflow are based on methylCtools, which has been originally developed by Volker Hovestadt.
Author Contributions
D.W., C.D.I. and Q.W. conceived the study. B.B. and C.P. contributed materials. D.W. did the experiments and analyzed data. D.W., C.D.I. and C.P. wrote the manuscript.
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Weichenhan, D., Imbusch, C.D., Wang, Q., Brors, B., Plass, C. (2019). Generation of Whole Genome Bisulfite Sequencing Libraries from Very Low DNA Input. In: Küppers, R. (eds) Lymphoma. Methods in Molecular Biology, vol 1956. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9151-8_10
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DOI: https://doi.org/10.1007/978-1-4939-9151-8_10
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