Abstract: Aspects of the disclosure provide methods and compositions for hyper- and hypo-methylation analysis of nucleic acid molecules. In specific aspects, the nucleic acid molecules comprise cell-free DNA. The methods may comprise ligation of a first set of adapters, methylation-sensitive or methylation-dependent restriction enzyme digestion, ligation of a second set of adapters, amplification and sample partitioning, enrichment of nucleic acid molecules tagged with specific adapters, sequencing and analysis of the sequencing data, and detection of diseases such as cancer.

Abstract: The present disclosure provides methods and compositions for preparation of a nucleic acid library. In some embodiments, the nucleic acids comprise cell-free DNA, including cfDNA that is in need of analysis, such as by sequencing. The methods may comprise restriction enzyme digestion, adapter ligation, and subsequent amplification, and may provide improved approaches for reducing the number adapter dimers produced during the process. In an aspect, a method for preparing a library of nucleic acids may comprise: digesting DNA molecules with restriction enzymes to produce DNA fragments; ligating adapters to the DNA fragments by incubating with ligase to produce a mixture of adapter-ligated DNA fragments and adapter dimers; amplifying the adapter-ligated DNA fragments to produce amplified adapter-ligated DNA fragments; and reducing the quantity of the adapter dimers by differentiating between the junction between an adapter and a DNA fragment, and the junction between an adapter and another adapter.