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Andrew S. Wiecek

Methylation analysis methods have always had a trade-off: either they have required significant amount of starting DNA or have provided incomprehensive results. Andrew S. Wiecek takes a look at a new method that promises a comprehensive methylome with less sample than before.

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If you want to study DNA methylation at the genomic level, you have your choice between several methods, but each has a trade-off.

For example, while whole genome bisulfite sequencing provides the highest resolution and most comprehensive detection of methylation patterns across the entire genome, the method requires significant starting material. Over 5 micrograms of genomic DNA is necessary to construct the library through ligation chemistry in this method. As a result, whole genome bisulfite sequencing cannot be used for DNA methylation analysis when the starting material is limited.

(A) Tagmentation-based DNA-seq library construction: Genomic DNA is attacked by transposase homodimers loaded with synthetic, discontinuous oligos (yellow, purple) that allow for fragmentation and adaptor incorporation in a single step. Subsequent PCR appends outer flowcell-compatible primers (pink, green). (B) Tn5mC-seq library construction: Loaded transposase attacks genomic DNA with a single methylated adaptor (yellow). An oligo-replacement approach anneals a second methylated adaptor (purple) which is then subject to gap-repair. Bisulfite treatment then converts unmethylated cytosine to uracil (orange) followed by PCR to append outer flowcellcompatible primers (pink, green). Methylation is represented as black lollipops. Source: Genome Research

In contrast, reduced representation bisulfite sequencing requires less DNA input but sacrifices comprehensiveness. Instead of analyzing the whole genome, the method focuses only on a defined subset of the genome. So, in the end, regardless of the method chosen, there was always a sacrifice.

“When you are looking at cancer, for instance, you have limited amounts of starting material, and there’s evidence that there’s things going on with methylation in cancer tissues. Input is also often an issue in early development, which also has interesting methylation patterns,” says graduate student Andrew Adey who studies genomics in the lab of Jay Shendure at the University of Washington in Seattle.

But now, Adey and Shendure have developed a new bisulfite sequencing method that combines the best of both previous approaches. Their method requires as little as 1 nanogram of starting DNA while still providing high resolution and comprehensive analysis of DNA methylation patterns in the whole genome. Their trick is a library construction method that is more efficient than ligation chemistry, namely tagmentation.

In a paper published in Genome Research last week (1), Adey and Shendure describe their tagmentation-based whole genome bisulfite sequencing method. Instead of using ligation chemistry to construct a sequencing library in a multi-step process, the new approach uses the Tn5 transposase to fragment in vitro DNA and incorporate an adaptor at the same time. In comparison to ligation chemistry, the transposase approach is much more efficient, reducing the amount of starting DNA required.

“The goal is to have as much of our original DNA converted into strands flanked by methylated adaptors prior to the bisulfite treatment, so the yield going into it will as high as possible because the biggest hit is still the bisulfite treatment. But the upfront process is now much more efficient with this transposase process, so we get a lot more out of it,” says Adey.

This transposase approach is not new to Adey and Shedure. In a paper published last year in Genome Biology (2), the two scientists and colleagues described a similar tagmentation library construction approach for genomic sequencing. All in all, they found that even though their approach used less starting DNA than previously described protocols, it still provided comparable coverage. At that time, they developed several protocols, including ones for sub-nanogram library construction and exome capture from 50 ng of DNA.

“It’s really a natural extension of the standard transposase-based DNA-seq library prep,” says Adey. “But you can’t just methylate the adapters and do the standard library prep because of the bisulfite treatment. It yields single-stranded DNA, so you can’t have a second adaptor.”

To adapt the process, Adey and Shendure first incorporated a single adapter that was methylated, except for the transposase recognition sequence. They then employed an oligo replacement strategy to add on a second methylated adaptor by annealing it and performing gap repair on the resulting nine base-pair gap. This resulted in each strand having both a 5’ and 3’ adapter at its ends.

“There were a lot of questions initially that involved whether or not the transposase could be loaded with methylated adaptors, but we seemed to have no problems with that,” say Adey.

To test their transposase library construction, Adey and Shedure analyzed a lymphoblastoid cell line with both their transposase method and the ligation chemistry method, with various starting sample sizes. As a result, they found the two approaches were relatively in agreement even though their approach used significantly less starting material.

In the long run, this approach will provide a new choice for epigenetic researchers looking to do DNA methylation analysis when their sample size is limited, such as in cancer methylation studies. Furthermore, Adey and Shendure plan to continue optimizing the method, continuously trying to pull more out of less starting material.

“It works pretty well now, but ideally we want to get it to the point where you can use this method for extremely low inputs, even lower than we’ve done in the paper,” says Adey. “We have one nanogram libraries that work OK in the paper, and we’d like to boost that up and go into the sub-nanogram range.”


1. Adey, A., and J. Shendure. 2012. Ultra-low-input, tagmentation-based whole genome bisulfite sequencing. Genome Research (March).

2. Adey, A., H. G. Morrison, Asan, X. Xun, J. O. Kitzman, E. H. Turner, B. Stackhouse, A. P. MacKenzie, N. C. Caruccio, X. Zhang, and J. Shendure. 2010. Rapid, low-input, low-bias construction of shotgun fragment libraries by high-density in vitro transposition. Genome Biology 11(12):R119+.