Methods to mitigate methylation bias

Abstract

Methods for mitigating methylation bias during the preparation of sequencing libraries are described. The methods may include, for example, extracting a DNA fragment from a sample and performing at least one of the following: (i) a first end repair reaction, wherein the first end repair reaction comprises the use of a non-stranded substitution DNA polymerase, or (ii) a second end repair reaction, wherein the second end repair reaction comprises the use of a chain arrest mechanism, and (iii) a tailing reaction for adding an overhanging polynucleotide chain to the repaired end DNA fragment, wherein the tailing reaction comprises the use of a single deoxynucleotide triphosphate (dNTP), or (iv) a nick / gap repair reaction, wherein a DNA ligase is used to fill single-stranded nicks or gaps in the repaired end DNA fragment to produce a plurality of modified DNA fragments, and performing methylation analysis of nucleic acid molecules derived from the plurality of modified DNA fragments.

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