Abstract: Disclosed herein are compositions and methods for the detection of DNAs in a sample, including single-stranded DNA, denatured double-stranded DNAs and DNA fragments for research and clinical diagnostic purposes. The methods and compositions disclosed herein may be used for preparing next generation sequencing libraries of highly fragmented DNA molecules isolated from biofluids (such as plasma, serum, saliva and urine), FFPE samples and ancient organisms. The methods involve the ligation of hairpin adaptors to the 3?-end Sand the 5?-end of the ssDNA.