| Birth defects in China each year by 4%to 6%of the total population,of which trisomy 21(Down syndrome,DS),18 trisomy(Edward’s syndrome)and trisomy 13(Patau syndrome)total birth defects 80%to 90%of the aneupldoidy population.Birth defects are important issues that affect the quality of births,the family and society with a heavy burden.Reducing the rate of birth defects to improve the quality of our population is particularly important,while prenatal screening is one of the effective measures to reduce birth defects.In recent years,with the rapid development of next generation sequencing technology,the noninvasive prenatal DNA testing(NIPT)by next generation sequencing(NGS)method is of increasing people’s attention to detect fetal chromosomal aneuploidy in its non-invasive,high accuracy characteristics,and in the major hospitals have been carried out.This method is the use of cell-free fetal DNA that is present in maternal blood in this fact.Cell-free fetal DNA(cff DNA)was first discovered in 1997 by Lo et al in the maternal circulating blood,and its chimera of fetal and maternal DNA was confirmed.Then scholars have confirmed the maternal fetal DNA circulating in the presence and research on cell-free fetal DNA of maternal plasma became a hot spot in the academic community quickly.Application of these studies focused on detection of chromosomal aneuploidy,single gene disorders,fetal sex-linked disease diagnosis and screening for fetal RhD blood group.These tests are mainly based on the qualitative or quantitative analysis of cell-free fetal DNA,and accurate quantitation of cell-free fetal DNA is very important in chromosome aneuploidy screening.Currently NIPT results analysis is mainly used to screening for chromosomal aneuploidy by calculating the Z score value(normal range:-3 to 3)reflecting the proportion of the chromosome in sequencing data.The distribution of the Z score value shows a Gaussian distribution,and there is a certain intersection area between the Z score distribution of normal pregnant women and pregnant women with a trisomy defect fetus,and this intersection area was the called gray area.There exist many reasons that will cause the sample to fall into the gray area,such as GC content,its structure and cell-free fetal DNA fraction is too low,so only in terms of the absolute value of Z score is greater than 3 to determine the chromosomal the aneuploidy for those samples falling into the gray area prone to false negatives and false positives.In addition,when cff DNA fraction is less than 4%,the result will be reduced reliability and results provided by NIPT are often inaccurate.Therefore,the concentration of cff DNA is a key parameter for result of the determination and analysis of aneuploidy.If the concentration of cff DNA can be added into the analysis of results assisted by some judge or to improve the proportion of cff DNA by sequencing process optimization,it will help to reduce the number of re-sequencing samples that falling into the gray area.What’s more?It can also increase the accuracy of determination for aneuploidy and shortened chromosome aneuploidy screening period.According to current literature on cff DNA quantification,the most used method is to be quantified by real time PCR(QPCR).There are some research through the SNP alleles and methylation loci conducted by Mass Spectrometry.Recently,There also came out method to quantify cff DNA by the droplet digital PCR(ddPCR).However,these methods require additional quantitative experiments and the supporting equipment,etc.,and these methods are not well reflecting the actual proportion of fetal DNA inside the sequencing data.Therefore,to develop a method that can quantify the cff DNA concentration directly from the NIPT sequencing data of screening for fetal aneuploidy is of great significance.Consequenctly,the objective of this study was to establish a method to quantify cell-free fetal DNA fractions directly from NIPT sequencing data using next generation sequencing technology and bioinformatics.We took QPCR fluorescence probe assay and so on to assess the accuracy of the method we had developed.To analyse the clinical NIPT test sample,we use the established methods to obtain cff DNA fraction and optimize the procedure of NIPT next generation sequencing experiments in order to achieve an increase fetal DNA concentration in the NIPT sequencing sample.Method:1.Study participants and sample collection:112 cases of trisomy 21,45 cases of trisomy 18,20 cases of trisomy 13 and 183 cases of aneuploidy were collected by tissue that confirmed by comparative genomic hybridization(CGH)karyotype.2063 cases of whole blood were collected from high-risk pregnant women with fetal defect of chromosome aneuploidy suggested by chemical and ultrasound screening results.50 cases of peripheral blood were collected from healthy nonpregnant women.2.Establishment of cell-free fetal DNA quantification method based on the Y chromosome2.1 Artificial mixture samples preparation:Genomic DNAs were extracted from trisomy 21,trisomy 18,trisomy 13 tissue and normal euploidy tissue,and interrupted break into 140～200bp by Covaris S2 ultrasonic instrument,respectively.The interrupted DNA fragments were purified with XP beads and then DNA were quantified their concentrations using Qubit(?)2.0.50 cases of peripheral blood plasma from healthy nonpregnant women were separated and plasma DNAs extracted using a commercial blood DNA kit following the manufactuer’s instructions,and quantified by Qubit(?)2.0.Cell-free DNA extracted from healthy nonpregnant women were added to the corresponding proportion of said interrupted DNA,formulated into three artificial samples,cell-free fetal DNA content of 3.5%,5%,10%.2.2 Artificial samples and clinical samples undergo NIPT detection:DNA libraries of the artificial mixture samples and clinical samples were constructed,and sequenced using Proton.2.3 To develop a method to determine the cff DNA fraction based on Y chrmosone with 360 cases of artificial mixture DNA samples and 1119 cases of cell-free DNA samples in maternal plasma from clinical pregnant women.2.4 Evaluation of established method based on Y chrmosome for cell-free fetal DNA quantification:To validated our method,among the 2063 pregnancies recruited,cff DNA fractions of those carrying a male fetus were quantify with the established method based on Y chrmosome for cell-free fetal DNA quantification.And the cff DNA concentration in maternal plasma reported in the literature were compared.The Z score value was calculated to determine aneuploidies for chromosome 21,18 and 13.To assess the impact of cff DNA concentration on the NIPT results,we investigated the relationship between Z scores and cff DNA fractions.The relationships between cff DNA fractions and gestational age or maternal age were also analyzed in order to investigate the impact of pregnancies’ characteristics on the cff DNA concentration.The libraries of the artificial mixture samples and clinical maternal plasma samples with the presence of male fetal DNAs were selected to performe QPCR assay,and compared consistency of the cff DNA fractions quantify by the method to determine the cff DNA fraction based on Y chrmosone and QPCR assay.2.5 Application of the cell-free fetal DNA concentration quantitative method based on Y chromosome in non-invasive prenatal chromosomal aneuploidy detection:Through the establishment of paired experiments with paired clinical maternal plasma sample to assess whether the removal of larger DNA fragments effectively increase the concentration of fetal DNA in the library construction process.The cff DNA fractions of these two groups were quantify by the cell-free fetal DNA concentration quantitative method based on Y chromosome,and results were underwent paired t-test statistical analysis.3.To develop a method to determine the cff DNA fraction based on DNA fragment size3.1 Among the 706 pregnancies recruited,cff DNA fractions of those carrying a male fetus were quantify with the established method based on Y chrmosome for cell-free fetal DNA quantification.Samples with different levels of cff DNA fractions were enrolled into investigation of DNA fragments size distributions,and their sequencing read length distributions were observed and compared in order to identify differences between these two DNA fragment size ranges,and draw mainly belonged to cell-free fetal and total DNA fragment size range,respectively.3.2 The found DNA fragment size ranges that mianly belong to fetal and maternal total free DNA were used to interception area that can represent fetal and total free DNA in the NIPT library distribution zone produced by 2100 bioanalyzer.Then use the area representive the part of fetal DNA and the area representive the part of total DNA to calculate a ratio and compared these ratios with the cell-free fetal DNA concentrations quantify by the method based on Y chromosome,and obtain the linear relationship as well as a function of the fitted curve.The cff DNA concentration was deduced from the function with the area ratio.According to the results obtained,size ranges of interception area can be adjusted until the cff DNA concentration determined by this method were as as close as possible to those quantify by the method based on the Y chromosome.And then we can establish a method to quantify cff DNA fraction based on NIPT DNA library fragments length.3.3 To calculate size ratio of fetus sequence fragment by the determined substantially sequence belonging to the fetus and the total maternal cell-free DNA and compared the size ratio with the cff DNA concentration determined by the method based on the Y chromosome,and obtain the linear relationship.And through continuous adjustment of fetal and maternal roughly divided sequence length distribution range,to arrive at the most appropriate fragment length distribution range,launched by a linear relationship between the function of the concentration of fetal DNA.Finally,a cell-free fetal DNA concentration quantitative method based on the sequencing fragments the length was established.3.4 While using the established cell-free fetal DNA concentration quantitative methods based on cell-free DNA fragment size to quantify the cff DNA fraction,we deduced the cff DNA fraction by the cell-free fetal DNA concentration quantitative method based on Y chromosome for clinical NIPT samples,and the results obtained by these two methods were compared to assess the accuracy of the established method.Result:By 360 cases of artificial mixture samples with known cell-free fetal DNA concentration,we successfully established a cell-free fetal DNA concentration quantitative method based on Y chromosome using NIPT sequencing data directly,and realizes the quantitative of NIPT sequencing samples from pregnant women bearing male fetuses.The cff DNA fractions of those carrying a male fetus among 2063 cases of clinical plasma samples were quantified with the established cell-free fetal DNA concentration quantitative method based on Y chromosome.The results showed that the average cell-free fetal DNA concentration was 13.89%,with a range of 4.81%to 31.88%,which was consistent with the results of existing literature reports.In addition,the quantitative results of QPCR assay were also consistent with the results of the cell-free fetal DNA concentration quantitative method based on Y chromosome in the error range.Base on the correlation analysis between Z values of artificial mixture samples and the known cell-free fetal DNA concentration,we found that there was a positive correlation between Z score value of trisomy 21,18 and 13 samples and cell-free fetal DNA concentration.There also exist the same positive correlation between Z score value of trisomy positive pregnant women samples and cell-free fetal DNA concentration(rT21=0.905,PT21,0.00;rT18＝0.887,PT18=0.00;rT13＝0.858,PT13=0.014).Additionally,there was a positive correlation between fetal free DNA concentration and gestational age(r＝ 0.321,P = 0.00)but no significant correlation for maternal age(r=-0.03,P = 0.92).Moreover,cell-free fetal DNA concentration in removal of large DNA fragment group is on average 1.50%higher than those in not removal of large DNA fragment group(t = 13.66,P = 13.66),and the difference is statistically significant.By analyzing the sequencing read length distribution of samples with the high,medium and low cff DNA concentration quantified by the cell-free fetal DNA concentration quantitative method based on Y chromosome,the determined optimal interval belong fetal and maternal total free DNA fragments were 129bp～137bp and 161bp～170bp,respectively.There was a strong positive correlation between the size ratio of fragments mainly belonged to fetus and maternal total free DNA and the cff DNA concentration quantified by the cell-free fetal DNA concentration quantitative method based on Y chromosome,Y = 0.7884X + 0.1296,r = 0.937,P<0.001.Besides,a cell-free fetal DNA concentration quantitative method based on NIPT DNA library fragments length and a cell-free fetal DNA concentration quantitative method based on the sequencing fragments the length were successfully developed,which could used to quantify samples from pregnant women with female fetuses.Compared with the method to quantify cff DNA concentration based on the Y chromosome,the cell-free fetal DNA concentration quantitative method based on NIPT DNA library fragments length shows a relatively higher accuracy.Paired t-test results:t =1.920,P = 0.064>0.05.These two methods showed no significant difference between the results,and the results obtained by the two methods exist significant correlation,r = 0.914,P<0.0001.The cell-free fetal DNA concentration quantitative method based on the sequencing fragments length also show a relatively accuracy compared with the method to quantify cff DNA concentration based on the Y chromosome,and these two methods showed no significant difference between the results(Paired t-test results:t=-0.454,P=0.652>0.05)and the results obtained by the two methods exist significant correlation,r=0.887,P<0.001.Conclusion:The above results show that the study successfully established a cell-free fetal DNA concentration quantitative method based on Y chromosome that is suitable for those NIPT sequencing samples from pregnant women carrying male fetuses,and a cell-free fetal DNA concentration quantitative method based on cell-free DNA fragment size towards both male and female fetus-bearing pregnancies were established,including a method to quantify cff DNA fraction based on NIPT DNA library fragments length and a cell-free fetal DNA concentration quantitative method based on the sequencing fragments the length.The cell-free fetal DNA concentration quantitative methods we have developed are relatively accurate and reliable.Moreover,by optimizing,an increased cell-free fetal DNA concentration of sequencing sample can be abtianed,which may be contribute to improvements in the detection rate and accuracy for NIPT detection. |
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