As for the study of genome-scale DNA methylation, there are several common methods to be chosen, such as traditional whole-genome bisulfite sequencing. Despite its advantages of high resolution and comprehensive detection for methylation, this method needs relatively great amount of starting material. At least 5μg of genomic DNA is needed for the library construction. So it is inappropriate for the samples with limited starting material.
Another bisulfite sequencing method with low representative needs less starting DNA, but comprehensive analysis can not be achieved. This method focuses on the specific region of genome, which differs from the analysis of the whole genome. For researchers working on study of cancer and development, analysis methods are very limited by the small amount of starting material.
Comparing with traditional methods mentioned above, Andrew Adey and Jay Shendure from University of Washington had developed a new method, Tn5mC-seq, which is published on Genome Research in 2012, for whole-genome bisulfite sequencing. As little as 1 ng of input DNA is enough to obtain comprehensive analysis of whole-genome methylation patterns. They utilized a hyperactive derivative of the Tn5 transposase loaded with discontinuous synthetic oligonucleotides to simultaneously fragment and append adaptors to genomic DNA. The resulting products are subjected to PCR amplification followed by high-throughput sequencing. The increased efficiency of genomic DNA conversion to viable amplicons and the greatly reduced number of steps allow the construction of low-bias, highly complex libraries from <50ng of genomic DNA. [1]
Jay Shendure cooperated with BGI and developed a similar library construction method based on tagmentation for genome sequencing. They found this method can still offer high coverage of cytosine positions with less DNA.
Researchers had improved previous method. Loaded transposase attacks genomic DNA with a single methylated adaptor. An oligo-replacement approach anneals a second methylated adaptor, which is then subject to gap-repair. Bisulfite treatment then converts unmethylated cytosine to uracil followed by PCR to append outer flowcell-compatible primers.
In order to detect the preparation of libraries, researchers compared Tn5mC-seq with ligation chemistry–based library construction to analyze a lymphoblastoid cell line. Finally, they found that, despite the different inputs of starting material, these two methods are relatively consistent.
In a word, this method offers a new selection for researchers working on epigenetics.
Refference:
[1] Andrew Adey, Jay Shendure. Ultra-low-input, tagmentation-based whole-genome bisulfite sequencing. Genome Research 22:1139–1143.
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