Sequencing the 5th Base of DNA, 5-Methylcytosine

Whole genome bisulfite converted next generation sequencing (NGS) enables comprehensive per base methylation status detection of the entire genome; however, current library preparation methods introduce significant bias or loss, reducing the representation of the sample’s methylome. For conventional library prep which requires dsDNA, bisulfite conversion must be performed post library prep.  Bisulfite treatment fragments and denatures DNA, which causes greater than 95% of the library to be lost. Random primer-based methods are capable of adapting single stranded DNA molecules, but are still inefficient and generate significant base composition bias. 

Here we present a novel method to prepare NGS libraries, starting with as little as 500 ng (PCR-free) or 100 pg (with PCR) input. This single stranded technology uses denatured, bisulfite converted DNA as the initial input.  Efficient enzyme-mediated chemistry is used to attach the first NGS adapter to the 3’ termini of the single stranded fragments. The second NGS adapter is added in a sequential reaction following second strand synthesis. Additionally, second strand synthesis enables PCR-free compatibility with flow cell cluster formation.

Our data was compared to two market-leading methods: a conventional dsDNA protocol that requires library construction prior to bisulfite conversion, and a single stranded, random primer-based method.  We observed substantially higher yield when compared to both of these methodologies (100-fold and 30-fold, respectively).  Using aligned base composition as a metric, we observed that bias was low, comparable to the conventional protocol and significantly better than the random primed approach.