Sequencing methods for prenatal diagnoses

1. A method for determining a fetal aneuploidy of chromosome 21 in a maternal test sample comprising a mixture of fetal and maternal nucleic acid molecules, said method comprising the steps:

(a) preparing a sequencing library from said mixture of fetal and maternal nucleic acid molecules in said maternal test sample, wherein preparing said library comprises the consecutive steps of end-repairing, dA-tailing and adaptor ligating said nucleic acids, wherein said consecutive steps exclude purifying the end-repaired products prior to said dA-tailing and exclude purifying the dA-tailing products prior to said adaptor-ligating;

(b) sequencing at least a portion of said nucleic acid molecules from said sequencing library thereby obtaining sequence information for a plurality of fetal and maternal nucleic acid molecules of a maternal test sample;

(c) using the sequence information obtained in step (b), identifying a number of sequence tags for at least one normalizing chromosome and for chromosome 21, wherein said at least one normalizing chromosome is a chromosome or a group of chromosomes selected from the group consisting of chromosome 9, chromosome 1, chromosome 2, chromosome 11, chromosome 12, and chromosome 14;

(d) using the number of sequence tags identified for chromosome 21 and the number of sequence tags identified for the at least one normalizing chromosome in step (c) to calculate a chromosome dose for chromosome 21, wherein said chromosome dose is a ratio of the number of sequence tags identified for chromosome 21 and the number of sequence tags identified for said at least one normalizing chromosome; and

(e) comparing said chromosome dose to at least one threshold value, and thereby identifying the presence or of chromosome 21.

2. The method of claim 1 , wherein step (d) comprises:

(i) calculating a sequence tag density ratio for chromosome 21, by relating the number of sequence tags identified for chromosome 21 in step (c) to the length of chromosome 21;

(ii) calculating a sequence tag density ratio for said at least one normalizing chromosome, by relating the number of sequence tags identified for said at least one normalizing chromosome in step (c) to the length of said at least one normalizing chromosome; and

(iii) using the sequence tag density ratios calculated in steps (i) and (ii) to calculate a chromosome dose for chromosome 21, wherein the chromosome dose is calculated as the ratio of the sequence tag density ratio for chromosome 21 and the sequence tag density ratio for said at least one normalizing chromosome.

3. The method of claim 1 , wherein said maternal sample is a biological fluid selected from blood, plasma, serum, urine and saliva.

4. The method of claim 1 , wherein said fetal and maternal nucleic acid molecules are cell-free DNA (cfDNA) molecules.

5. The method of claim 1 , wherein said sequencing is next generation sequencing (NGS).

6. The method of claim 1 , wherein said sequencing is massively parallel sequencing using sequencing-by synthesis with reversible dye terminators.

7. The method of claim 1 , wherein said sequencing is massively parallel sequencing using sequencing-by-ligation.

8. The method of claim 1 , wherein said sequencing comprises an amplification.

9. The method of claim 1 , wherein said sequencing is single molecule sequencing.

10. A method for determining a fetal aneuploidy of chromosome 18 in a maternal test sample comprising a mixture of fetal and maternal nucleic acids molecules, said method comprising the steps:

(a) preparing a sequencing library from said mixture of fetal and maternal nucleic acid molecules in said maternal test sample, wherein preparing said library comprises the consecutive steps of end-repairing, dA-tailing and adaptor ligating said nucleic acids, wherein said consecutive steps exclude purifying the end-repaired products prior to said dA-tailing and exclude purifying the dA-tailing products prior to said adaptor-ligating;

(b) sequencing at least a portion of said nucleic acid molecules from said sequencing library thereby obtaining sequence information for a plurality of fetal and maternal nucleic acid molecules of a maternal test sample;

(c) using the sequence information obtained in step (b), identifying a number of sequence tags for at least one normalizing chromosome and for chromosome 18, wherein said at least one normalizing chromosome is a chromosome or a group of chromosomes selected from the group consisting of selected from chromosome 8, chromosome 2, chromosome 3, chromosome 5, chromosome 6, chromosome 12, and chromosome 14;

(d) using the number of sequence tags identified for chromosome 18 and the number of sequence tags identified for the at least one normalizing chromosome in step (c) to calculate a chromosome dose for chromosome 18, wherein said chromosome dose is a ratio of the number of sequence tags identified for chromosome 18 and the number of sequence tags identified for said at least one normalizing chromosome; and

(e) comparing said chromosome dose to at least one threshold value, and thereby identifying the presence or absence of fetal aneuploidy of chromosome 18.

11. The method of claim 10 , wherein step (d) comprises:

(i) calculating a sequence tag density ratio for chromosome 18, by relating the number of sequence tags identified for chromosome 18 in step (c) to the length of chromosome 18;

(ii) calculating a sequence tag density ratio for said at least one normalizing chromosome, by relating the number of sequence tags identified for said at least one normalizing chromosome in step (c) to the length of said at least one normalizing chromosome; and

(iii) using the sequence tag density ratios calculated in steps (i) and (ii) to calculate a chromosome dose for chromosome 18, wherein the chromosome dose is calculated as the ratio of the sequence tag density ratio for chromosome 18 and the sequence tag density ratio for said at least one normalizing chromosome.

12. The method of claim 10 , wherein said maternal sample is a biological fluid selected from blood, plasma, serum, urine and saliva.

13. The method of claim 10 , wherein said fetal and maternal nucleic acid molecules are cell-free DNA (cfDNA) molecules.

14. The method of claim 10 , wherein said sequencing is next generation sequencing (NGS).

15. The method of claim 10 , wherein said sequencing is massively parallel sequencing using sequencing-by synthesis with reversible dye terminators.

16. The method of claim 10 , wherein said sequencing is massively parallel sequencing using sequencing-by-ligation.

17. The method of claim 10 , wherein said sequencing comprises an amplification.

18. The method of claim 10 , wherein said sequencing is single molecule sequencing.

19. A method for determining a fetal aneuploidy of chromosome X in a maternal test sample comprising a mixture of fetal and maternal nucleic acids molecules, said method comprising the steps:

(a) preparing a sequencing library from said mixture of fetal and maternal nucleic acid molecules in said maternal test sample, wherein preparing said library comprises the consecutive steps of end-repairing, dA-tailing and adaptor ligating said nucleic acids, wherein said consecutive steps exclude purifying the end-repaired products prior to said dA-tailing and exclude purifying the dA-tailing products prior to said adaptor-ligating;

(b) sequencing at least a portion of said nucleic acid molecules from said sequencing thereby obtaining sequence information for a plurality of fetal and maternal nucleic acid molecules of a maternal test sample;

(c) using the sequence information obtained in step (b), identifying a number of sequence tags for at least one normalizing chromosome and for chromosome X, wherein said at least one normalizing chromosome is selected from the group consisting of chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, and chromosome 8;

(d) using the number of sequence tags identified for chromosome X and the number of sequence tags identified for the at least one normalizing chromosome in step (c) to calculate a chromosome dose for chromosome X, wherein said chromosome dose is a ratio of the number of sequence tags identified for chromosome X and the number of sequence tags identified for said at least one normalizing chromosome; and

(e) comparing said chromosome dose to at least one threshold value, and thereby identifying the presence or absence of fetal aneuploidy of chromosome X.

20. The method of claim 19 , wherein step (d) comprises:

(i) calculating a sequence tag density ratio for chromosome X, by relating the number of sequence tags identified for chromosome X in step (c) to the length of chromosome X;

(ii) calculating a sequence tag density ratio for said at least one normalizing chromosome, by relating the number of sequence tags identified for said at least one normalizing chromosome in step (c) to the length of said at least one normalizing chromosome; and

(iii) using the sequence tag density ratios calculated in steps (i) and (ii) to calculate a chromosome dose for chromosome X, wherein the chromosome dose is calculated as the ratio of the sequence tag density ratio for chromosome X and the sequence tag density ratio for said at least one normalizing chromosome.

21. The method of claim 19 , wherein said maternal sample is a biological fluid selected from blood, plasma, serum, urine and saliva.

22. The method of claim 19 , wherein said fetal and maternal nucleic acid molecules are cell-free DNA (cfDNA) molecules.

23. The method of claim 19 , wherein said sequencing is next generation sequencing (NGS).

24. The method of claim 19 , wherein said sequencing is massively parallel sequencing using sequencing-by synthesis with reversible dye terminators.

25. The method of claim 19 , wherein said sequencing is massively parallel sequencing using sequencing-by-ligation.

26. The method of claim 19 , wherein said sequencing comprises an amplification.

27. The method of claim 19 , wherein said sequencing is single molecule sequencing.