Methods and processes for non-invasive assessment of genetic variations

1. A computer-implemented method for sequencing a test sample nucleic acid from a pregnant female, generating a classification of a presence of a chromosome aneuploidy for the test sample, and performing a medical procedure comprising amniocentesis or chorionic villus sampling, the method comprising:

(a) sequencing test sample nucleic acid by a genome-wide massively parallel sequencer that generates sequence reads, wherein the test sample nucleic acid comprises circulating cell-free nucleic acid from blood of a pregnant female bearing a fetus;

(b) mapping, using a microprocessor, the sequence reads to portions of a reference genome;

(c) counting, using a microprocessor, the sequence reads mapped to the portions, wherein counting generates counts of sequence reads mapped to the portions;

(d) calculating, using a microprocessor, genomic section levels for the portions of the reference genome, wherein calculating generates calculated genomic section levels;

(e) determining a fraction of fetal nucleic acid in the test sample nucleic acid;

(f) calculating, using a microprocessor, a fetal ploidy determination based on (i) the calculated genomic section levels of (d) for a subset of portions of the reference genome, (ii) the fraction of fetal nucleic acid determined in (e), and (iii) an estimated maternal ploidy;

(g) generating a classification of a presence of a chromosome aneuploidy in the fetus according to the fetal ploidy determination calculated in (f); and

(h) performing a medical procedure comprising amniocentesis or chorionic villus sampling for the pregnant female after step (g) based on the classification of the presence of a chromosome aneuploidy in the fetus.

2. A computer-implemented method for sequencing a test sample nucleic acid from a pregnant female and generating a classification of a presence or absence of a chromosome aneuploidy for the test sample, the method comprising:

(a) sequencing test sample nucleic acid by a genome-wide massively parallel sequencer that generates sequence reads, wherein the test sample nucleic acid comprises circulating cell-free nucleic acid from blood of a pregnant female bearing a fetus;

(b) mapping, using a microprocessor, the sequence reads to portions of a reference genome;

(c) counting, using a microprocessor, the sequence reads mapped to the portions, wherein counting generates counts of sequence reads mapped to the portions;

(d) calculating, using a microprocessor, genomic section levels for the portions of the reference genome, wherein calculating generates calculated genomic section levels;

(e) determining a fraction of fetal nucleic acid in the test sample nucleic acid;

(f) calculating, using a microprocessor, a fetal ploidy determination based on (i) the calculated genomic section levels of (d) for a subset of portions of the reference genome, (ii) the fraction of fetal nucleic acid determined in (e), and (iii) an estimated maternal ploidy; and

(g) generating, using a microprocessor, a classification of a presence or absence of a chromosome aneuploidy in the fetus according to the fetal ploidy determination calculated in (f).

3. The method of claim 2 , wherein the fetal ploidy determination is calculated in (f) according to Equation 8 below, or a rearranged format thereof:

y i (1− F ) M i f i +FXf i   (8)

where y i represents the calculated genomic section level for portion i of a reference genome, F represents the fraction of fetal nucleic acid determined in (d), f i represents a reference count for i, X represents the fetal ploidy, and M i represents the maternal ploidy of portion i.

4. The method of claim 2 , wherein the sequencing is at about 1-fold coverage or less.

5. The method of claim 2 , wherein the nucleic acid is from blood plasma or blood serum.

6. The method of claim 2 , wherein calculating the genomic section level for the portions of the reference genome comprises normalizing counts of reads mapped to the reference genome according to guanine and cytosine (GC) content for each of the portions.

7. The method of claim 2 , wherein the classification of the presence or absence of a chromosome aneuploidy is generated for one or more of chromosome 13, chromosome 18 and chromosome 21.

8. The method of claim 2 , wherein the subset of portions of the reference genome in (f) is portions of a chromosome or segment thereof.

9. The method of claim 8 , wherein the chromosome is chosen from chromosome 13, chromosome 18 and chromosome 21.

10. The method of claim 2 , comprising determining a reference count and determining an uncertainty value according to the reference count, wherein:

the reference count is determined according to calculated genomic section levels for a subset of portions of the reference genome for one or more pregnant females bearing a fetus;

the subset of portions of the reference genome for one or more pregnant females are known to be euploid for the fetus and/or the mother;

the reference count is not determined from the sample; and

the reference count is determined from the same subset of portions of the reference genome as in (f).

11. The method of claim 10 , wherein the fetal ploidy determination is calculated in (f) according to (i) the calculated genomic section levels for a subset of portions of the reference genome determined in (d), (ii) the fraction of fetal nucleic acid determined in (e), (iii) the estimated maternal ploidy, and (iv) the reference count.

12. The method of claim 11 , wherein the fetal ploidy determination is calculated in (f) by linear regression.

13. The method of claim 12 , wherein the fetal ploidy determination is calculated in (f) according to Equation 20 below:

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wherein yi represents the calculated genomic section level for portion i of a reference genome, F represents the fraction of fetal nucleic acid determined in (e), fi represents a reference count for i, σ represents the uncertainty value for fi, X represents the fetal ploidy determination, and Mi represents the estimated maternal ploidy of portion i.

14. The method of claim 12 , wherein a classification of an absence of a fetal chromosome trisomy is determined when the fetal ploidy determination is about 1 and a classification of a presence of a fetal chromosome trisomy is determined when the fetal ploidy determination is not about 1.

15. The method of claim 2 , wherein determining the fraction of fetal nucleic acid comprises analyzing one or more loci in sample nucleic acid, wherein at least one of the one or more loci vary between fetal nucleic acid and maternal nucleic acid.

16. The method of claim 2 , wherein determining the fraction of fetal nucleic acid in (e) comprises:

(1) generating an experimental Y chromosome representation, which experimental Y chromosome representation is a ratio of (i) counts of sequence reads mapped to the genomic sections of the reference genome in the Y chromosome, and (ii) counts of sequence reads mapped to genomic sections of the reference genome in the genome or segment thereof;

(2) determining the fraction of the fetal nucleic acid in the blood of the pregnant female according to the experimental Y chromosome representation generated in (1) and a fitted relationship, wherein:

the fitted relationship is between (i) an experimental Y chromosome representation determined from a set of pregnant females bearing a male fetus and (ii) an X chromosome representation determined from a set of pregnant females; and

the fitted relationship is fitted to a median chromosome X representation and a median chromosome Y representation for a set of pregnant females bearing a female fetus.

17. The method of claim 2 , further comprising generating and transmitting a laboratory prenatal test report comprising a classification of a presence of a chromosome aneuploidy in the fetus.

18. The method of claim 2 , further comprising monitoring health of the fetus and pregnant female from whom the test sample was obtained based on the classification of a chromosome aneuploidy in the fetus.

19. The method of claim 2 , further comprising performing a medical procedure comprising amniocentesis or chorionic villus sampling when the classification is a presence of three copies of a chromosome for the test sample.