Methods of preparing ligation product and sequencing library, identifying biomarkers, predicting or detecting a disease or condition

A method of identifying one or more biomarkers associated with a disease or condition, comprising the steps of:

• a. obtaining a plurality of samples comprising a plurality of single-strand nucleic acid fragments from a case group of subjects having the disease or condition and from a control group;
• b. for individual sample, ligating a first universal oligonucleotide adaptor to at least one single-strand nucleic acid fragment, wherein the first universal oligonucleotide adaptor is configured for ligating to a 3′ end of individual single-strand nucleic acid fragment;
• c. ligating a second universal oligonucleotide adaptor to the at least one single-strand nucleic acid fragment, wherein the second universal oligonucleotide adaptor is configured for ligating to a 5′ end of individual single-strand nucleic acid fragment, thereby at least one ligation product is formed;
• d. amplifying the at least one ligation product with a pair of sequencing specific adaptor primers to form individual sequencing library, wherein the pair of sequencing specific adaptor primers is at least partially complementary to the first universal oligonucleotide adaptor and the second universal oligonucleotide adaptor respectively,
• e. quantifying and reading the sequencing library to obtain individual sequencing result;
• and
• f. comparing the sequencing results between the case group and the control group, such that one or more biomarkers associated with the disease or condition are identified.