Research Of Cell-free DNA Reference Material For Noninvasive Prenatal Tesing Of Fetal Chromosomal Aneuploidy Based On Matched Mother-child Cell Lines And Enzymes Digestion

In 1997,Professor Lo of The Chinese University of Hong Kong found the fetal DNA sequences in maternal plasma,and with the emergence and maturity of next-generation sequencing(NGS),noninvasive prenatal testing(NIPT)based on plasma cell-free DNA(cfDNA)has become possible.The most commonly used NIPT methods based on NGS include random massively parallel sequencing(MPS)and targeted sequencing.NIPT holds no risk of miscarriage and has demonstrated excellent performance in terms of sensitivity and specificity.However,due to the extremely low proportion of fetal DNA in maternal plasma,high accuracy of NIPT method is required.And during the complex NIPT processes,many factors,such as the quality of extracted plasma cfDNA,library preparation,and the methods for GC bias correction,could lead to discordant results between laboratories.Thus,standardization of NIPT is urgently needed,and the preparation of reference material close to real plasma samples is the key to ensure quality assurance and standardization of NIPT in clinical laboratories.Currently,reference materials for NIPT available in clinical practice include natural maternal plasma samples and simulated samples based on fragmentation of human genomic DNA.Understandably,it is not feasible to use natural maternal plasma as reference material because of the limited amount of clinical samples.And the simulated samples can not be applicable to various NIPT methods since they did not meet the biological properties and genetic characteristics of natural plasma samples from pregnant women.Therefore,in order to solve the exsiting problems,DNA fragmentation factor(DFF)and micrococcal nuclease(MNase)digesting techniques involved in apoptosis mechanism were adopted in this study combined with matched mother-child cell lines.And a novel cfDNA reference material for NIPT of fetal chromosomal aneuploidy which can be easily obtained in vitro and own the properties identical to clinical samples was prepared for the urgent requirement in clincal laboratories.In this study,we cultivated matched mother-child cell lines with euploid mother and euploid fetus or fetal aneuploidy of chromosomes 21,18,and 13.Then the nuclei derived from mother cells were prepared and digested with DNA fragmentation factor(DFF),and the nuclei of child cells were digested with micrococcal nuclease(MNase).The DNA products obtained from DFF digestion and MNase digestion were adopted to mimic cell-free maternal DNA(D-cfDNA)and cell-free fetal DNA(M-cfDNA),respectively.The M-cfDNA containing trisomies 21,18,and 13 were serially diluted into matched D-cfDNA at 2.5%,5%,10%,and 20%fetal fraction(FF).The dual enzyme-digested DNA mixtures were added into simulated human plasma to generate a novel cfDNA reference material for NIPT of fetal chromosomal aneuploidy.To evaluate the suitability of the reference material,the enzymes digested samples were validated in this study using random MPS,targeted sequencing and imaging single DNA molecules technique.We also performed genome-wide sequencing analysis of DNA fragmentation patterns of reference material and clinical plasma to confirm that they owned the same biological characteristics.The results showed that when the FF of the novel reference material was higher than the minimum detection limit,the aneuploid samples could be correctly detected by all four routine NIPT assays,and no false positive results were reported,which indicated that the reference material was suitable for NIPT using all the methodologies mentioned above.The good linear fit of Y chromosome-based FF to the expected FF(R2>0.990)indicated that Y chromosome-based assay for FF could reflect the linear FF increase accurately in the prepared samples.Moreover,the X chromosome-based assay and SeqFF-based assay also kept an excellent linear fit to Y chromosome-based assay(R2>0.999;R2=0.995),indicating that the FF of reference material can be determined using multiple methods.In addition,the deep sequencing analysis revealed that the reference material and clinical plasma sample exhibited a high concordance in biological characteristics such as dinucleotide composition and distribution,nucleosome protection peak,and nucleosome occupancy around the transcription start site(TSS).In summary,we have prepared a novel cell-free DNA reference material for NIPT of fetal chromosomal aneuploidy,and compared with other reference materials,it is easily obtained in vitro,can be accurately quantified,and suitable for NIPT using various methodologies.This novel reference material can be used for the quality assurance of NIPT in clinical laboratories and provide the essential material basis for the standardization of NIPT of fetal chromosomal aneuploidy.Additionally,this study also provides a new idea and technology platform for the preparation of reference materials used in other NIPT fields such as single gene disorders.