Post-bisulfite Adaptor Tagging Based on an ssDNA Ligation Technique (tPBAT).

Post-bisulfite adaptor tagging (PBAT) is a concept that enables the preparation of an efficient sequencing library from bisulfite-treated DNA, and it also means the protocol implemented the concept. Although the previous PBAT or rPBAT was sensitive enough for single-cell methylome analysis, the protocol had several drawbacks owing to the repeated random priming reactions. To resolve these problems, we developed a unique single-strand DNA ligation technique, termed TACS ligation, and established a new protocol called tPBAT. With tPBAT, the data quality improved, with a longer insert and higher mapping rate than that obtained with rPBAT. In addition, paired-end sequencing and indexing were supported by the default. In this chapter, the tPBAT protocol is introduced, and a thorough description of its application to small samples is provided.

Post-bisulfite Adaptor Tagging with a Highly Efficient Single-Stranded DNA Ligation Technique.

Post-bisulfite adaptor tagging (PBAT) is a procedure for efficiently preparing a sequencing library for whole-genome bisulfite sequencing (WGBS). The original version of the PBAT protocol was highly efficient, such that it helped realize library preparation from samples of limited amounts. However, two rounds of random priming reactions employed in the original protocol limited further improvement of the PBAT protocol in terms of read length and mapping rate. In this chapter, an improved version of the PBAT protocol called tPBAT is described.

Low Input Genome-Wide DNA Methylation Analysis with Minimal Library Amplification.

Whole genome bisulfite sequencing (WGBS) is a high-throughput DNA sequencing-based technique that is used to determine genome-wide DNA methylation patterns at base resolution. Library construction by post-bisulfite adaptor tagging (PBAT ) extends the application of WGBS to several hundred cells and minimizes the required number of library amplification cycles. We herein describe a PBAT protocol to prepare WGBS libraries from 200 cells and introduce the outline of a downstream bioinformatic analysis. The prepared library can typically generate 800 million sequencing reads, which is sufficient to cover the human and mouse genomes approximately 15 times, using the Illumina NovaSeq 6000 sequencing system.

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 (<100 cells), 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.