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Fumihito Miura, Methylome analysis based on enhanced single strand DNA ligation, FRANCE JAPAN EPIGENETICS WORKSHOP 2017, 2017.11, Whole-genome bisulfite sequencing (WGBS) has become the method of choice for analyzing the methylome. If an adaptor-tagged library is treated with bisulfite, vital loss of its structure as a sequencing template would occur, since bisulfite treatment intrinsically cuts DNA. Therefore, tagging adapters to sample DNA should be performed after bisulfite treatment in library preparation for WGBS. Accordingly, we previously proposed a strategy termed post-bisulfite adaptor tagging (PBAT), a highly efficient protocol for constructing sequencing libraries for WGBS. The original protocol for PBAT was implemented with two rounds of random priming reactions. This protocol realized so efficient library preparation for WGBS that enabled PCR-free methylome analysis. The PCR-free protocol greatly contributed to the improvement of evenness for genomic coverage with mapped reads compared with conventional protocols, which depend on intensive PCR amplification and cause a heavily biased representation of template DNA. Despite the improved coverage, because it is based on random priming, GC-rich genomic regions are still more deeply covered by the current PBAT protocol. So, to further improve the quality of libraries for WGBS, novel procedures should be developed. Since DNA is single-stranded after bisulfite treatment, a method for connecting artificial adaptor sequences to single-stranded DNA (ssDNA) is essential. Here, I present a method termed TdT-assisted chimeric ssDNA (TACS) ligation as a highly efficient procedure for connecting adaptor sequences to ssDNA. TACS ligation achieves more sensitive preparation of libraries for WGBS with higher quality than conventional protocols.. |
