Methods of fetal abnormality detection

• US 10,718,020 B2
• Filed: 09/28/2016
• Issued: 07/21/2020
• Est. Priority Date: 01/23/2010
• Status: Active Grant

• Current Assignee: Verinata Health Incorporated (Illumina Incorporated)
• Original Assignee: Verinata Health Incorporated (Illumina Incorporated)
• Inventors: Chuu, Yue-Jen, Rava, Richard P.
• Primary Examiner(s): Mahatan, Channing S
• Application Number:
  US15/279,066
• Publication Number:
  US 20170073757A1
• Time in Patent Office: 1,392 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2600/112 Disease subtyping, staging or classification
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

Abstract

Methods and kits for selectively enriching non-random polynucleotide sequences are provided. Methods and kits for generating libraries of sequences are provided. Methods of using selectively enriched non-random polynucleotide sequences for detection of fetal aneuploidy are provided.

Claims

1. A method for determining the presence or absence of fetal aneuploidy in a maternal blood sample, the method comprising:

(a) obtaining a maternal blood sample comprising fetal and maternal cell-free DNA;

(b) selectively enriching a plurality of non-random polynucleotide sequences of genomic DNA from cell-free DNA in a maternal blood sample to generate a library of enriched non-random polynucleotide sequences found in said fetal and maternal cell-free DNA, wherein said plurality of non-random polynucleotide sequences comprises at least 100 different non-random polynucleotide sequences selected from a chromosome tested for being aneuploid, said enriching comprising;

(i) a first amplification step to generate a plurality of first reaction products, said amplification comprising at least 100 first primers configured to amplify at least 100 different non-random polynucleotide sequences;

(ii) a second amplification step to generate a second reaction product, said amplification comprising a second set of primers comprising sequences contained in the first reaction products; and

(iii) a third amplification step to generate a third reaction product comprising said library of enriched non-random polynucleotide sequences, said amplification comprising a third set of primers comprising sequences contained in the second reaction products;

wherein at least one primer of at least one of the second and third sets of primers includes a sequence configured to be added to the different non-random polynucleotide sequences to permit the enriched non-random polynucleotide sequences of the library to anneal to a same sequencing primer for the enriched non-random polynucleotide sequences of the library; and

wherein at least one primer of at least one of the second and third sets of primers includes a sequence configured to be added to the different non-random polynucleotide sequences to add an index to the enriched non-random polynucleotide sequences of the library, the index being indicative of the maternal blood sample from which the library was generated;

(c) sequencing said enriched non-random polynucleotide sequences;

(d) enumerating sequence reads from said sequencing step; and

(e) determining the presence or absence of fetal aneuploidy based on said enumerating.

2. The method of claim 1, wherein at least one primer of the third set of primers includes a sequence configured to be added to the different non-random polynucleotide sequences to add an index to the enriched non-random polynucleotide sequences of the library, the index being indicative of the maternal blood sample from which the library was generated.

3. The method of claim 1, wherein primers of the second set of primers contain universal sequences such that nucleic acids of the second reaction product contain common universal sequences added to the different non-random polynucleotide sequences, and wherein the third set of primers are configured to hybridize at least in part to the common universal sequences.

4. The method of claim 3, wherein at least one primer of the third set of primers includes a sequence configured to be added to the different non-random polynucleotide sequences to add an index to the enriched non-random polynucleotide sequences of the library, the index being indicative of the maternal blood sample from which the library was generated.

5. The method of claim 1, wherein said plurality of non-random polynucleotide sequences comprises at least 300 different non-random polynucleotide sequences selected from the chromosome tested for being aneuploid.

6. The method of claim 1, wherein said plurality of non-random polynucleotide sequences comprises at least 500 different non-random polynucleotide sequences selected from the chromosome tested for being aneuploid.

7. The method of claim 1, wherein each of said plurality of non-random polynucleotide sequences is from 10 to 500 nucleotide bases in length.

8. The method of claim 1, wherein each of said plurality of non-random polynucleotide sequences is from 50 to 150 nucleotide bases in length.

9. The method of claim 1, wherein said selectively enriching comprises performing PCR.

10. The method of claim 1, wherein said selectively enriching comprises linear amplification.

11. The method of claim 1, wherein said non-random polynucleotide sequences comprise sequences that are sequenced at a rate of greater than 5-fold more than other sequences on the chromosome.

12. The method of claim 1, wherein said chromosome tested is selected from the group consisting of chromosome 13, chromosome 18, chromosome 21, chromosome X, and chromosome Y.

13. A method for determining the presence or absence of fetal aneuploidy in a maternal blood sample, the method comprising:

(a) obtaining a maternal blood sample comprising fetal and maternal cell-free DNA;

(b) selectively enriching a plurality of non-random polynucleotide sequences of genomic DNA from cell-free DNA in a maternal blood sample to generate a library of enriched non-random polynucleotide sequences found in said fetal and maternal cell-free DNA, wherein said plurality of non-random polynucleotide sequences comprises at least 100 different non-random polynucleotide sequences selected from a chromosome tested for being aneuploid, wherein said selectively enriching comprises;

(i) amplifying said plurality of non-random polynucleotide sequences from said maternal and fetal genomic DNA using a first pair of primers to form a first product, wherein said plurality of non-random polynucleotide sequences comprises at least 100 different non-random polynucleotide sequences selected from a chromosome tested for being aneuploid;

(ii) amplifying the first product of (i) with a second set of primers comprising sequences contained in the first product of (i) to form a second product; and

(iii) amplifying the second product of (ii) with a third set of primers comprising sequences contained in the second product of (ii);

wherein one of said second or third sets of primers includes an indexing sequence;

(c) sequencing said enriched non-random polynucleotide sequences;

(d) enumerating sequence reads from said sequencing step; and

(e) determining the presence or absence of fetal aneuploidy based on said enumerating.

14. The method of claim 13, wherein said third set of primers includes the indexing sequence.

15. The method of claim 13, wherein said indexing sequence distinguishes polynucleotides in the maternal blood sample from polynucleotides in a different maternal blood sample.

16. The method of claim 13, wherein said cell-free DNA is from a plurality of maternal blood samples from a plurality of different individuals, and wherein said indexing sequence identifies a maternal blood sample from each of said plurality of different individuals.

17. The method of claim 13, wherein said plurality of non-random polynucleotide sequences comprises at least 300 different non-random polynucleotide sequences selected from the chromosome tested for being aneuploid.

18. The method of claim 13, wherein said plurality of non-random polynucleotide sequences comprises at least 500 different non-random polynucleotide sequences selected from the chromosome tested for being aneuploid.

19. The method of claim 13, wherein each of said plurality of non-random polynucleotide sequences is from 10 to 500 nucleotide bases in length.

20. The method of claim 13, wherein each of said plurality of non-random polynucleotide sequences is from 50 to 150 nucleotide bases in length.

21. The method of claim 13, wherein said selectively enriching comprises performing a polymerase chain reaction (PCR).

22. The method of claim 13, wherein said selectively enriching comprises linear amplification.

23. The method of claim 13, wherein said chromosome tested is selected from the group consisting of chromosome 13, chromosome 18, chromosome 21, chromosome X, and chromosome Y.