Method for non-invasive prenatal screening for aneuploidy
The present disclosure provides methods for non-invasive prenatal screening (NIPS) of fetal aneuploidies. The present methods are based on analyzing cell-free fetal DNA (cff DNA) found in a pregnant woman's circulation through the next generation sequencing (NGS) technology. Particularly, the present methods analyze the relative abundance of different fetal genomic fragments present in the maternal sample, where the fragments can be aligned to particular chromosomal locations of the fetal genome. The relative abundance information is indicative as to whether a particular chromosome is overrepresented or underrepresented in a fetal genome as compared to normal individuals, and thus can be used to detect fetal aneuploidy. Additionally, methods for increasing the positive predictive values (PPV) of NIPS by excluding false-positive detections are also provided.
1 . A method for detecting a false-positive diagnosis of chromosomal aneuploidy in a fetus by a non-invasive prenatal screening (NIPS), comprising:
(a) sequencing cell-free DNA from a maternal test sample of a pregnant woman carrying the fetus to provide at least 9 million sequence reads; wherein the fetus has been diagnosed to be aneuploid of a chromosome of interest by the NIPS;
(b) dividing a chromosome of interest diagnosed to be aneuploid into a plurality of bins, each bin having a chromosomal location, wherein each bin has a length of between 5 kb and 500 kb;
(c) aligning the at least 9 million sequence reads to the plurality of bins;
(d) generating a raw bin read count by counting the total number of sequence reads aligned to each of the plurality of bins;
(e) calculating a bin-specific test parameter by scaling the raw bin read count with an autosomal total read count, and performing a GC correction of the scaled bin read count to produce a GC corrected scaled bin read count;
(f) plotting the bin-specific test parameters versus the chromosomal locations of corresponding bins to produce an ideogram of the chromosome of interest; and
(g) filtering confounding signals to detect the false-positive diagnosis when the ideogram exhibits an increase of a bin-specific test parameter in at least one bin compared to the mean or median of the bin-specific test parameters for the plurality of bins of the chromosome of interest, and wherein the increase is at least 1.2-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 6.5-fold or at least 7-fold, wherein the diagnosed chromosomal aneuploidy is confirmed by performing an amniocentesis on the pregnant woman when the filtering does not detect the false-positive diagnosis.
2 . The method of claim 1 , further comprising repeating steps (a) to (d) for a confirming chromosome other than the chromosome of interest.
3 . The method of claim 1 , wherein the bin-specific test parameter is reflective of relative abundance of genetic material corresponding to the bin in a maternal test sample.
4 . The method of claim 1 , wherein obtaining the bin-specific test parameter comprises aligning the sequence reads to a plurality of bins of a reference genome comprising the chromosome of interest.
5 . The method of claim 1 , wherein obtaining the bin-specific test parameter comprises calculating the bin-specific test parameter based on a total number of sequence reads aligned to each bin.
6 . The method of claim 1 , wherein the bin-specific test parameter is a normalized bin read count.
7 . The method of claim 1 , wherein the bin-specific test parameter is produced by the NIPS.
8 . The method of claim 1 , wherein the method improves a positive predictive value (PPV) of the NIPS to at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% for human trisomy 21, human trisomy 18 and/or human trisomy 13, as compared to previously available NIPS methods.
9 . The method of claim 8 , wherein the PPV is improved to at least 93% for human trisomy 21, at least 72% for human trisomy 18, and/or at least 39% for human trisomy 13, as compared to previously available NIPS methods.
10 . The method of claim 8 , wherein the PPV for trisomy 21 is improved to 98% for human trisomy 21, 92% for human trisomy 18, and/or 69% for human trisomy 13, as compared to previously available NIPS methods.
11 . The method of claim 1 , wherein the method improves a positive predictive value (PPV) of the NIPS by at least 4%, 10%, 20%, 30%, 40% and 50% for human trisomy 21, human trisomy 18 and/or human trisomy 13, as compared to previously available NIPS methods.
12 . The method of claim 11 , wherein the PPV is improved by at least 4% for human trisomy 21, at least 20% for human trisomy 18, and/or at least 30% for human trisomy 13, as compared to previously available NIPS methods.
13 . The method of claim 1 , wherein each bin is about 50 kb long.
14 . The method of claim 1 , wherein the chromosome of interest is one or more chromosomes.
15 . The method of claim 2 , wherein the confirming chromosome is one or more chromosomes.
16 . The method of claim 1 , further comprising:
(a) dividing a confirming chromosome into a plurality of bins, each bin having a chromosomal location;
(b) obtaining a bin-specific test parameter for each bin;
(c) calculating a first sum of bin-specific test parameters for corresponding bins residing on the confirming chromosome; wherein the confirming chromosome is different from a chromosome of interest diagnosed to be aneuploid;
(d) calculating a second sum of bin-specific test parameters for corresponding bins residing on one or more autosomes;
(e) calculating a chromosome representation value for the confirming chromosome by dividing the first sum by the second sum;
(f) comparing the chromosome representation value to a set of references to generate a chromosome-specific comparison result; and
(g) detecting the false-positive diagnosis when the chromosome-specific comparison result achieves a pre-determined threshold.
17 . The method of claim 16 , wherein obtaining the bin-specific test parameter comprises sequencing cell-free DNA from a maternal test sample of a pregnant woman carrying the fetus to provide sequence reads.
18 . The method of claim 16 , wherein obtaining the bin-specific test parameter further comprises aligning the sequence reads to the plurality of bins of a reference genome.
19 . The method of claim 16 , wherein obtaining the bin-specific test parameter further comprises calculating the bin-specific test parameter based on a total number of sequence reads aligned to each bin.
20 . The method of claim 19 , wherein the bin-specific test parameter is a normalized bin read count.
21 . The method of claim 16 , wherein the confirming chromosome is one or more chromosomes in a reference genome.
22 . The method of claim 16 , wherein the set of references comprises a plurality of chromosome representation values for the confirming chromosome obtained from a random sample of unaffected pregnancies.
23 . The method of claim 16 , wherein step (f) is performed by calculating a Z-score of said chromosome representation value with respect to the set of references.
24 . The method of claim 23 , wherein the threshold is achieved when the Z-score is greater than 4.
25 . The method of claim 16 , wherein the method further comprises assessing a fetal fraction of the cell-free DNA in the maternal test sample before performing step (a).
26 . The method of claim 25 , further comprising excluding the maternal test sample when the fetal fraction is less than 4%.
27 . The method of claim 16 , wherein the chromosomal aneuploidy is a complete or partial chromosomal duplication or a chromosomal trisomy.
28 . The method of claim 16 , wherein the chromosomal aneuploidy is human trisomy 13, human trisomy 18 or human trisomy 21.
29 . The method of claim 16 , wherein the fetus is aneuploid mosaic.
30 . The method of claim 1 , wherein GC correction is performed using local polynomial regression (loess).
31 . The method of claim 1 , wherein performing GC correction includes obtaining the GC content for regions corresponding to the genomic locations of sequenced bins from a reference genome.