Sequencing adaptors and methods are provided for preparation of polynucleotides for sequencing. The sequencing adaptors contain a portion of a recognition sequence for a methyl-dependent endonuclease. Unwanted adaptor dimers that form during ligation of adaptors to target polynucleotides produce a complete restriction sequence and are cleaved by the endonuclease, followed by exonuclease digestion, thereby removing the dimers.

Disclosed herein are processes for enriching low-copy elements in DNA samples. More particularly, the present disclosure relates to the use of a methylation-dependent restriction endonuclease that recognizes methylated DNA and that cleaves upstream and downstream of the methylated DNA in processes for depleting repetitive methylated DNA elements and for enriching low-copy elements. The disclosed processes have particular utility in methods for analyzing features of low-copy elements with increased sensitivity.

Disclosed herein are processes for enriching low-copy elements in DNA samples. More particularly, the present disclosure relates to the use of a methylation-dependent restriction endonuclease that recognizes methylated DNA and that cleaves upstream and downstream of the methylated DNA in processes for depleting repetitive methylated DNA elements and for enriching low-copy elements. The disclosed processes have particular utility in methods for analyzing features of low-copy elements with increased sensitivity.

The present disclosure relates to methods of enzymatic treatment of double-stranded PCR amplified products for eliminating or minimizing primer-dimers in multiplex PCR reactions and for the efficient ligation of adapters. The present disclosure relates to methods and compositions that allow more efficient highly multiplex target amplification compared to conventional methods, compositions and kits by minimizing laboratory steps, eliminating primer-dimers and increasing the efficiency of adapter ligation. The disclosed methods use multiple target-specific primers for specific and selective amplification of targets in a subject's genome. The disclosed methods can be used for numerous downstream procedures and analysis, including DNA sequencing.

The present disclosure relates to methods of enzymatic treatment of double-stranded PCR amplified products for eliminating or minimizing primer-dimers in multiplex PCR reactions and for the efficient ligation of adapters. The present disclosure relates to methods and compositions that allow more efficient highly multiplex target amplification compared to conventional methods, compositions and kits by minimizing laboratory steps, eliminating primer-dimers and increasing the efficiency of adapter ligation. The disclosed methods use multiple target-specific primers for specific and selective amplification of targets in a subject's genome. The disclosed methods can be used for numerous downstream procedures and analysis, including DNA sequencing.

Providing herein, among other things, is a method for preparing a nucleic acid for sequencing. In some embodiments, the method comprises a) amplifying a nucleic acid template using a dNTP mix that contains 5-methyl dCTP, thereby producing product nucleic acid molecules that contains methylcytosines; b) digesting the product nucleic acid molecules with a methylation-dependent restriction endonuclease, thereby cleaving the product nucleic acid molecules at sites that are adjacent to at least some of the methylcytosine and producing fragments of the product nucleic acid molecules; and c) ligating double-stranded adaptors onto the ends of the fragments to produce adaptor-ligated products.

Providing herein, among other things, is a method for preparing a nucleic acid for sequencing. In some embodiments, the method comprises a) amplifying a nucleic acid template using a dNTP mix that contains 5-methyl dCTP, thereby producing product nucleic acid molecules that contains methylcytosines; b) digesting the product nucleic acid molecules with a methylation-dependent restriction endonuclease, thereby cleaving the product nucleic acid molecules at sites that are adjacent to at least some of the methylcytosine and producing fragments of the product nucleic acid molecules; and c) ligating double-stranded adaptors onto the ends of the fragments to produce adaptor-ligated products.

Provided herein, among other things, are various compositions and methods for analyzing chromatin. In some embodiments, the composition may comprise a mixture of a nicking enzyme, four dNTPs, at least one labeled dNTP and, optionally, a polymerase. In some embodiments, this method may comprise: obtaining a sample comprising chromatin, reacting the sample with the composition to selectively label the open chromatin in the sample, and analyzing the labeled sample.

Provided herein, among other things, are various compositions and methods for analyzing chromatin. In some embodiments, the composition may comprise a mixture of a nicking enzyme, four dNTPs, at least one labeled dNTP and, optionally, a polymerase. In some embodiments, this method may comprise: obtaining a sample comprising chromatin, reacting the sample with the composition to selectively label the open chromatin in the sample, and analyzing the labeled sample.

The present disclosure relates to methodology for fast and cost-effective identification of the source of DNA samples. DNA samples obtained from unknown or unrecognized tissues or cell types are analyzed according to the methodology described herein, yielding an identification of the tissue and/or cell type source. Identification is based on sequential biochemical procedures including methylation sensitive/dependent restriction and polymerase chain reaction, followed by analysis of the data. All biochemical steps are performed in a single test tube. The disclosure has immediate applications in forensic science for identification of the tissue source of DNA obtained from biological stains. The disclosure also has immediate applications in cancer diagnosis for identification.