genomic DNA sandwiched by two adaptors, Supplementary Figure S1) ( 8). This is because bisulfite-induced DNA degradation leads to a drastic reduction of intact library molecules (i.e. adaptor-tagging followed by bisulfite treatment) shared by all WGBS protocols, including MethylC-seq ( 2) and Tn5mC-seq ( 3), was the primary cause of low efficiency of library preparation. On the contrary, we found that the order of operations (i.e. Therefore, the major cause of the low yields in WGBS library preparation is not the mass yields of bisulfite treatment. However, to our surprise, the mass yields of DNA prepared with commercially available bisulfite treatment kits were not so low, ranging from 30 to 70% ( 8). Since it had been well documented that the mass yield of bisulfite treatment is fairly low ( 4–7), we first thought that the poor yield of library was attributed to the low yields of bisulfite treatment. We independently investigated the cause of low yields in WGBS library preparation. However, even with the ultimate efficiency of adaptor tagging, Tn5mC-seq still required global amplification of the library with more than ten cycles of polymerase chain reaction (PCR) ( 3). For example, Tn5mC-seq used Tn5 transposase-mediated tagmentation, an extremely efficient adaptor tagging method for dsDNA, to successfully reduce the amount of input DNA to tens of nanograms ( 3). Accordingly, various efforts have been made to improve the efficacy of library preparation to expand the WGBS targets. However, the initial protocols for WGBS library preparation required microgram quantities of input DNA ( 1, 2) and were hence not readily applicable to samples whose quantities were limited. Whole-genome bisulfite sequencing (WGBS) has made it possible to draw an almost complete picture of the methylome at single-nucleotide resolution ( 1, 2), which has led to various novel findings of biomedical importance. The methylome is a genome-wide distribution of 5-methylcytosine, and its patterns are often specific for cells and tissues but dynamically modulated in response to various stimuli and environmental changes. Adopting the above strategies to the HiSeq X Ten and NovaSeq 6000 platforms, we established a cost-effective, high-quality WGBS, which should accelerate various methylome analyses. This strategy ensured an ideal base–color balance to eliminate the need for DNA spike-in for color compensation, further improving the throughput and quality of WGBS. Moreover, we devised a dual-library strategy that splits the input DNA to prepare two libraries with reciprocal adaptor polarity, combining them prior to sequencing. A protocol that uses TACS ligation instead of the second random priming step substantially increased the lengths of PBAT library fragments. In this method, TdT attaches adenylates to the 3′-end of input ssDNA, which are then utilized by RNA ligase as an efficient connector to the ssDNA adaptor. To overcome this limitation, we developed terminal deoxyribonucleotidyl transferase (TdT)-assisted adenylate connector-mediated ssDNA (TACS) ligation as an alternative to random priming. ![]() PBAT originally relied on two rounds of random priming for adaptor-tagging of single-stranded DNA (ssDNA) to attain high efficiency but at a cost of library insert length. Post-bisulfite adaptor tagging (PBAT) is an increasingly popular WGBS protocol because of high sensitivity and low bias. NOT FOR USE IN DIAGNOSTIC PROCEDURES (EXCEPT AS SPECIFICALLY NOTED).Whole-genome bisulfite sequencing (WGBS) is the current gold standard of methylome analysis. Our mission is to develop high-quality innovative tools and services to accelerate discovery.įOR RESEARCH USE ONLY. As a member of the Takara Bio Group, Takara Bio USA is part of a company that holds a leadership position in the global market and is committed to improving the human condition through biotechnology. provides kits, reagents, instruments, and services that help researchers explore questions about gene discovery, regulation, and function.
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