Figure 1: Brief flow chart of the present study.

In “Preparation of DNA fragments in different length” section: DNA fragments in a series of length were obtained by PCR and phosphorylated at the 5′-ends. DNA fragments with blunt ends were marked as T-1~T-8 from small to large, respectively. DNA fragments with 3′-A overhanging ends were marked as T-1A~T-8A from small to large, respectively. In “Preparation of double stranded unidirectional linkers” section: four kinds of double stranded unidirectional linkers were made. L-T-P had 3′-T overhanging ends and 5′-end phosphorylation; L-P had blunt ends and 5′-end phosphorylation; L-T had 3′-T overhanging ends without 5′-end phosphorylation; L had blunt ends without 5′-end phosphorylation. In “Pretreatment of cfDNA” section: cfDNA was pre-treated by producing blunt ends and adding adenine to 3′-ends. Then, T-1-P was ligated with L or L-P while T-1A-P was ligated with L-T or L-T-P to assess the ligation effect and select the most suitable combination. Following this step, an equal amount of T-1A-P~T-8A-P were mixed (marked as T-Mix) and ligated with L-T-P (marked as T-Mix-L), followed by overall amplified (marked as T-Mix-OA) to optimize the ligation and overall amplification reaction. Afterwards, the optimized ligation reaction conditions were utilized for ligating pretreated cfDNA and double stranded unidirectional linkers (product marked as cfDNA-BAL), and the optimized amplification reaction conditions were utilized for overall amplifying cfDNA (product marked as cfDNA-BAL-PCR). T-Mix-OA was overall amplified at a series of denaturation temperatures to optimize the optimum temperature for selective overall amplification of shorter fragments in cfDNA. The amplification features of DNA fragments of different length were determined and cffDNA was verified in the selectively overall amplified small fragment cfDNA.