Abstract
Whole-genome bisulfite sequencing (WGBS) is the current gold standard of methylome analysis. Post-bisulfite adaptor tagging (PBAT) is an increasingly popular WGBS protocol because of high sensitivity and low bias. 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. To overcome this limitation, we developed terminal deoxyribonucleotidyl transferase (TdT)-assisted adenylate connector-mediated ssDNA (TACS) ligation as an alternative to random priming. 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. A protocol that uses TACS ligation instead of the second random priming step substantially increased the lengths of PBAT library fragments. 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. 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. 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.
Highlights
The methylome is a genome-wide distribution of 5methylcytosine, and its patterns are often specific for cells and tissues but dynamically modulated in response to various stimuli and environmental changes
We focused on terminal deoxyribonucleotidyl transferase (TdT) because it can add a few ribonucleotides to the 3 -end of single-stranded DNA (ssDNA), notably, in a self-limiting manner [19]
These findings indicated the potential of a combinatorial use of TdT and RNA ligase as a method for adaptor-tagging of ssDNA
Summary
The methylome is a genome-wide distribution of 5methylcytosine, and its patterns are often specific for cells and tissues but dynamically modulated in response to various stimuli and environmental changes. Whole-genome bisulfite sequencing (WGBS) has made it possible to draw an almost complete picture of the methylome at singlenucleotide resolution [1,2], which has led to various novel findings of biomedical importance. The initial protocols for WGBS library preparation required microgram quantities of input DNA [1,2] and were not readily applicable to samples whose quantities were limited. Tn5mC-seq used Tn5 transposasemediated tagmentation, an extremely efficient adaptor tagging method for dsDNA, to successfully reduce the amount of input DNA to tens of nanograms [3]. 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]
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