| BACKGROUND AND OBJECTIVECurrently,low content of cffDNA(approximately 10%only)among cell free DNA in the maternal circulation remains one of the main technical obstacles towards precise noninvasive test.An ideal noninvasive screening approach of monogenetic disorders requires the direct determination of the fetal genotype without the prior knowledge of parent pathogenic mutations or linked SNPs.Moreover,mutiple gene mutations should be detected simultaneously.Here,we have established an innovative technology termed cfDNA Barcode-Enabled Single-molecule Test(cfBEST).It can meet the requirements above.MATERIALS AND METHODS1)The principle of cfBEST and proof-of-concept experimentsThe DNA templates were tagged by specific barcodes and prelibrary was prepared.The prelibrary was separated to two equal parts,then,two pairs of primer located near the targeted sites and two universal primers were used to perform two PCR amplications.The products were put together and performed the third PCR.Consequently.massively parrel sequencing was done and the ratio ofalleic mutation of the targeted sites could be decided by cfBEST.Acording to the Mendel’s laws and maximum likelihood estimate,the combination of maternal and fetal genotype can be deduced.We evaluated the performance of cfBEST by detecting known low-abundance mutations in commercial reference standards.Then,we compared cfBEST with the gold standard method for fetal DNA fraction determination.2)Quality indicators were determined by standard samples from gDNA and cfDNA of β-thalassemia carriers,including minimal amount of starting DNA,low limit of fetal fraction and low limit of unique reads(1000).3)157 maternal plasma samples with known maternal and fetal genotypes were tested blindly to determine the accuracy of cfBEST.RESULTS1.Experiments for proof-of-concept of cfBEST1)Comparison of accuracy for cfBEST and ddPCR in detecting low-abundance mutationsThe results of detecting of known low-abundance mutations in commercial reference standards showed that cfBEST had comparable performance with ddPCR when detecting samples containing 0%,0.05%,0.1%,and 0.5%mutation2)Comparison of fetal fraction calculated by SNPs-assay and Y-assay in 26 samples The fetal fraction calculated by SNPs and by Y-assay was highly consistent in the maternal plasma of 26 samples from pregnant women with male fetuses.(R2=0.97).2.Establishment and optimization of cfBESTStandard samples from gDNA and cfDNA were used to acquire various indicators for quality control of cfBEST,including minimal amount of starting DNA(10ng),low limit of fetal fraction(5%)and low limit of unique reads(1000).3.Blind validation experiments in 157 samples from at-risk families1)The fetal genotypes determined by invasive prenatal diagnosisSubjects:age:20-38 years old,gestation:11-24+6 weeks.The fetal samples were tested by RDB or Sanger sequencing to determine the fetal genotypes.2)Noninvasive prenatal testing of 157 samples by cfBESTThe fetal fraction varied from 2.27%to 22.85%among 157 maternal plasma samples tested by cfBEST.Fourteen samples were excluded because they failed to pass quality control.Cohen’s kappa coefficient was used to analyse the accuracy of cfBEST(κ=0.98).The sensitivity,specifity,positive predictive value and negative value of cfBEST for 16 mutations foβ-thalassemia is 99.19%(95%Cl,97.62-100%),99.92%(95%Cl,99.82-100%),97.62%(95%CI,94.96-100%)and 99.97%(95%Cl,99.92-100%)respectively.CONCLUSION1.Mutiple gene mutations have been tested simultaneously by cfBEST using cell free DNA in the maternal plasma.2.cfBESt has high sensitivity and specifity for NIPT of point mutation and indel mutation,which meet the requirements in clinic practice.3.Without the prior knowledge of parental genotype or linked SNPs,cfBEST could be applied to noninvasive prenatal diagnosis for couples at-risk of β-thalassemia and large-scale noninvasive prenatal screening for β-thalassemia and other high prevalence monogenic diseases in certain population. |
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