Method of detecting fetal chromosomal aneuploidy

Abstract

Provided are a method of detecting chromosomal aneuploidy of a targeted fetal chromosome, and a computer-readable medium having recorded thereon a program to be applied to performing the method. According to the present disclosure, fetal chromosomal aneuploidy may be non-invasively and prenatally diagnosed with excellent sensitivity and specificity.

1 . A method of detecting chromosomal aneuploidy of a target fetal chromosome of a fetus using at least one processor, the method comprising:
 isolating a plurality of nucleic acid fragments from a biological sample of a pregnant woman, wherein the biological sample was collected without performing an invasive method selected from chorionic villus sampling, amniocentesis, and sampling from an umbilical cord, and wherein the plurality of nucleic acid fragments includes cell-free fetal nucleic acid fragments;   obtaining sequence information (reads) indicating the plurality of nucleic acid fragments using the at least one processor;   mapping, using the at least one processor, the obtained reads to a reference human genome, to assign corresponding fragments among the plurality of the nucleic acid fragments to the target fetal chromosome as a test sample;   calculating, using the at least one processor, a guanine (G) and cytosine (C) (GC) content of the corresponding fragments on the target fetal chromosome and a fraction-of-reads (Rf) of the number of the corresponding fragments on the target fetal chromosome to the number of the plurality of the nucleic acid fragments, based on the reads indicating the plurality of the nucleic acid fragments mapped to the reference human genome including the corresponding fragments assigned to the target fetal chromosome as the test sample;   selecting, using the at least one processor, from reference samples obtained from biological samples of pregnant women carrying euploid fetuses, adaptive reference samples belonging to a shared range of Rf unit values and GC content unit values shared between the adaptive reference samples and the test sample, based on the calculated Rf content and the calculated GC content;   calculating, using the at least one processor, z scores of the selected adaptive reference samples and a z score of the sequences of the plurality of the nucleic acid fragments assigned to the target fetal chromosome as the test sample;   detecting, using the at least one processor, that the target chromosome has chromosomal aneuploidy without performing the invasive method, by comparing the calculated z scores of the selected adaptive reference samples with the z score of the test sample, when, the z score of the test sample is larger than the z scores of the selected adaptive reference samples.   
     
     
         2 . The method of  claim 1 , further comprising performing an invasive method selected from chorionic villus sampling, amniocentesis, and sampling from an umbilical cord, based on the determining that the target chromosome has chromosomal aneuploidy. 
     
     
         3 . The method of  claim 1 , wherein the sequence information (the reads) is obtained by a massively parallel sequencing system. 
     
     
         4 . The method of  claim 1 , wherein the selected adaptive reference samples have a lower coefficient of variance than reference samples without adaptive selection 
     
     
         5 . The method of  claim 1 , wherein the biological sample is blood, plasma, serum, urine, saliva, mucus, sputum, feces, tears, or a combination thereof. 
     
     
         6 . The method of  claim 1 , further comprising excluding, by the at least one processor, intervals with a low confidence level for the reads by examining depth distribution of the reads assigned to the reference human genome at each interval, after mapping the reads and assigning the corresponding fragments to the target fetal chromosome. 
     
     
         7 . The method of  claim 6 , wherein the interval is an interval set in units of about 5 kb to about 50 kb. 
     
     
         8 . The method of  claim 6 , wherein the excluding of intervals with a low confidence level for the reads comprises removing mismatches, removing multi-mapped reads, removing duplicated reads, or a combination thereof. 
     
     
         9 . The method of  claim 1 , further comprising performing locally weighted scatterplot smoothing (LOWESS or LOESS) regression analysis of the reads according to the following Equation 1 to reduce GC content bias, after assigning the corresponding fragments to the target fetal chromosome:
     Rf   ij′   =R   ij /Σ j=1   22   RC   ij   (Equation 1)
   
       wherein Rfij′ represents a corrected fraction of reads on chromosome j in sample i, and RCij represents a corrected number of unique reads on chromosome j in sample i. 
     
     
         10 . The method of  claim 1 , further comprising performing normalization of the reads of the nucleic acid fragments according to the following Equation 2, after assigning the reads of the nucleic acid fragments to the chromosome:
     Rf   i′j′   =Rf   ij′ /Σ i=1   N   Rf   ij′   (Equation 2)
   wherein Rfi′j′ represents a normalized fraction of reads on chromosome j in sample i, and N represents the total number of samples.   
     
     
         11 . The method of  claim 1 , further comprising establishing a linear regression model from all of the reference samples. 
     
     
         12 . The method of  claim 1 , further comprising extending the unit values of Rf of the reference samples according to Rf values of the test sample, extending the unit values of GC of the reference samples according to GC contents of the test sample, or a combination thereof. 
     
     
         13 . The method of  claim 1 , wherein the calculating of z scores of the selected adaptive reference samples and z score of the test sample comprises performing a linear regression analysis according to the following Equation 3 and calculating a linear predicted value of Rf according to the following Equation 4:
     Rfi′j′=α+β×GCi′j′+e   (Equation 3)
   
       Wherein, in Equation 3, Rfi′j′ represents a normalized fraction of reads on chromosome j in sample i, α represents a constant, β represents a coefficient factor between GC content and Rf, and e represents a residual (R); and
     Rf′i′j′=α+β×GCi′j′   (Equation 4)
 
 in Equation 4, Rfi′j′ represents a fitted predicted value of a fraction of reads on chromosome j in sample i, α represents a constant, and β represents a coefficient factor between GC content and Rf. 
 
     
     
         14 . The method of  claim 13 , wherein the calculating of z scores of the selected adaptive reference samples and z score of the test sample comprises calculating a residual (R) from a calculated value from the linear regression analysis and the calculated linear predicted value according to the following Equation 5, and calculating a Z score from the calculated residual according to the following Equation 6:
     R=Rfi′j′−Rf′i′j′   (Equation 5); and
       z  score=( R−R ′)/σ′  (Equation 6)
   wherein, in Equation 6, R′ represents a mean value of a residual of an adaptive reference sample, R represents a residual value of the test sample, and σ′ represents a standard deviation of the residual of the adaptive reference sample.   
     
     
         15 . The method of  claim 1 , further comprising
 selecting adaptive reference samples belonging to GC content±unit value of the target chromosome or GC content±unit value of the adaptive reference samples, as verification samples;   calculating z scores of the verification samples; and   verifying that the target chromosome has chromosomal aneuploidy by comparing the calculated z scores of the selected adaptive samples with the z score of the test sample.   
     
     
         16 . The method of  claim 1 , wherein the target chromosome is chromosome 13, chromosome 18, chromosome 21, an X chromosome, a Y chromosome, or a combination thereof. 
     
     
         17 . The method of  claim 1 , wherein the chromosomal aneuploidy is trisomy 13, trisomy 18, trisomy 21, XO, XXX, XXY, XYY, or a combination thereof. 
     
     
         18 . A system containing a computer-readable medium having recorded thereon a program that performs the steps of:
 obtaining sequence information (reads) indicating a plurality of nucleic acid fragments using at least one processor, wherein the plurality of nucleic acid fragments is isolated from a biological sample of a pregnant woman, wherein the biological sample was collected without performing an invasive method selected from chorionic villus sampling, amniocentesis, and sampling from an umbilical cord, and wherein the plurality of nucleic acid fragments includes cell-free fetal nucleic acid fragments;   mapping, using the at least one processor, the obtained reads to a reference human genome, to assign corresponding fragments among the plurality of the nucleic acid fragments to a target fetal chromosome as a test sample;   calculating, using the at least one processor, a guanine (G) and cytosine (C) (GC) content of the corresponding fragments on the target fetal chromosome and a fraction-of-reads (Rf) of the number of the corresponding fragments on the target fetal chromosome to the number of the plurality of the nucleic acid fragments, based on the reads indicating the plurality of the nucleic acid fragments mapped to the reference human genome including the corresponding fragments assigned to the target fetal chromosome as the test sample;   selecting, using the at least one processor, from reference samples obtained from biological samples of pregnant women carrying euploid fetuses, adaptive reference samples belonging to a shared range of Rf unit values and GC content unit values shared between the adaptive reference samples and the test sample, based on the calculated Rf content and the calculated GC content;   calculating, using the at least one processor, z scores of the selected adaptive reference samples and a z score of the sequences of the plurality of the nucleic acid fragments assigned to the target fetal chromosome as the test sample;   detecting, using the at least one processor, that the target chromosome has chromosomal aneuploidy without performing the invasive method, by comparing the calculated z scores of the selected adaptive reference samples with the z score of the test sample, when, the z score of the test sample is larger than the z scores of the selected adaptive reference samples.   
     
     
         19 . The system of  claim 18 , further comprising performing an invasive method selected from chorionic villus sampling, amniocentesis, and sampling from an umbilical cord, based on the determining that the target chromosome has chromosomal aneuploidy. 
     
     
         20 . The system of  claim 18 , wherein the sequence information (the reads) is obtained by a massively parallel sequencing system.