The biological characteristics of long cell-free DNA in spent embryos culture medium as noninvasive biomarker in in-vitro embryo selection

•A Tn5 transposase integrated dual sequencing strategy significantly improved the detection ratio of long cfDNA over 500bp.•A multiparametric index (TQI) displayed potential in differentiating top with normal quality in-vitro embryos (AUC=0.927).•TDual-Seq strategy has opened up analysis and potential clinical utilities of long cfDNA in liquid biopsy. An improved understanding of the cfDNA fragmentomics has proved it as a promising biomarker in clinical applications. However, biological characteristics of cfDNA in spent embryos culture medium (SECM) remain unsolved obstacles before the application in non-invasive in-vitro embryo selection. In this study, we developed a Tn5 transposase and ligase integrated dual-library construction sequencing strategy (TDual-Seq) and revealed the fragmentomic profile of cfDNA of all sizes in early embryonic development. The detected ratio of long cfDNA (>500 bp) was improved from 4.23 % by traditional NGS to 12.80 % by TDual-Seq. End motif analysis showed long cfDNA molecules have a more dominance of fragmentation intracellularly in apoptotic cells with higher predominance of G-end, while shorter cfDNA undergo fragmentation process both intracellularly and extracellularly. Moreover, the mutational pattern of cfDNA and the correlated GO biological process were well differentiated in cleavage and blastocyst embryos. Finally, we developed a multiparametric index (TQI) that employs the fragmentomic profiles of cfDNA, and achieved an area under the ROC curve of 0.927 in screening top quality embryos. TDual-Seq strategy has facilitated characterizing the fragmentomic profile of cfDNA of all sizes in SECM, which are served as a class of non-invasive biomarkers in the evaluation of embryo quality in in-vitro fertilization. And this improved strategy has opened up potential clinical utilities of long cfDNA analysis.