Methods and compositions for detecting genomic methylation

A method of preparing a polynucleotide library, comprising:

• (a) obtaining a plurality of double-stranded template nucleic acids, and a first adapter comprising a hairpin and a double-stranded region comprising a nick or a nickable site, wherein the nickable site comprises a uracil or a ribonucleotide;
• (b) ligating the first adapter to each end of the double-stranded template nucleic acids by double-stranded ligation;
• (c) denaturing the double-stranded template nucleic acids to obtain single-stranded template nucleic acids comprising a hairpin;
• (d) extending the hairpin in the presence of a conversion-resistant cytosine analog to obtain extended hairpins comprising a template strand and a complementary strand;
• and
• (e) ligating a Y-adapter to an end of the extended hairpins by double-stranded ligation to obtain a polynucleotide library.

A method of preparing a polynucleotide library, comprising:

• (a) obtaining a plurality of double-stranded template nucleic acids, and a first adapter comprising a first adapter strand and second adapter strand, wherein the first adapter strand comprises a hairpin and a double-stranded region formed between a 5′ end of the first adapter strand and a 3′ end of the second adapter strand, wherein a 5′ end of the second adapter strand is single-stranded;
• (b) ligating the first adapter to each end of the double-stranded template nucleic acids by double-stranded ligation;
• (c) denaturing the double-stranded template nucleic acids to obtain single-stranded template nucleic acids;
• and
• (d) extending the hairpin in the presence of a conversion-resistant cytosine analog to obtain a polynucleotide library comprising a template strand and a complementary strand.

A method of preparing a polynucleotide library, comprising:

• (a) obtaining a plurality of double-stranded template nucleic acids by (i) contacting double stranded DNA with a plurality of transposomes comprising a first adapter to obtain DNA fragments, and (ii) end-filling each DNA fragment, wherein each end of the double-stranded template nucleic acids comprises the first adapter;
• (b) denaturing the double-stranded template nucleic acids to obtain single-stranded template nucleic acids;
• (c) hybridizing a first tailed primer to a region at an end of the single-stranded template nucleic acids, and extending the hybridized first tailed primer in the presence of a conversion-resistant cytosine analog to obtain extended polynucleotides comprising a template strand, a complementary strand and a double-stranded end;
• and
• (d) ligating a second adapter comprising a hairpin to the double-stranded end of the extended polynucleotides by double-stranded ligation to obtain a polynucleotide library.

A method of preparing a polynucleotide library, comprising:

• (a) obtaining a plurality of double-stranded template nucleic acids by (i) contacting double-stranded DNA with a plurality of transposomes comprising a first adapter to obtain DNA fragments, wherein the first adapter comprises a hairpin and cleavable site, and (ii) end-filling each DNA fragment, wherein each end of the double-stranded template nucleic acids comprises the first adapter;
• (b) denaturing the double-stranded template nucleic acids to obtain single-stranded template nucleic acids comprising the cleavable sites;
• (c) cleaving the cleavable sites to remove a portion of the first adapter from the single-stranded template nucleic acids such that an end of the cleaved single-stranded template nucleic acids comprises a hairpin;
• and
• (d) extending the hairpins of the cleaved single-stranded template nucleic acids in the presence of a conversion-resistant cytosine analog to obtain a polynucleotide library comprising extended hairpins comprising a template strand and a complementary strand.