IDENTIFICATION OF POLYMORPHIC SEQUENCES IN MIXTURES OF GENOMIC DNA BY WHOLE GENOME SEQUENCING

• US 20110230358A1
• Filed: 01/19/2011
• Published: 09/22/2011
• Est. Priority Date: 01/19/2010
• Status: Active Grant

• Current Assignee: Verinata Health Incorporated (Illumina Incorporated)
• Original Assignee: Artemis Health, Inc. (Nomi Health, Inc.)
• Inventors: Rava, Richard P.
• Granted Patent: US 10,662,474 B2
• Time in Patent Office: Days
• US Class Current: 506/7
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2565/125 Electrophoretic separation
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• CPC Class Codes: C12Q 2600/172 Haplotypes

Abstract

The present invention relates to methods comprising whole genome sequencing for identifying polymorphisms in samples comprising mixtures of genomes, and for determining and/or monitoring the presence or absence of disorders associated with the identified polymorphisms.

Claims

1. A method for identifying multiple polymorphisms in a first genome of a blood sample comprising a mixture of cfDNA of a first and a second genome, said method comprising:

whole genome sequencing at least a portion of said mixture of cfDNA, thereby obtaining a plurality of sequence tags, wherein said mixture is unenriched for said multiple polymorphisms;

comparing the sequence of said plurality of tags to the sequence of multiple reference polymorphisms;

identifying said multiple polymorphisms in said first and second genome of said mixture; and

associating the multiple polymorphisms identified in step (c) with said first and second genome, thereby identifying said multiple polymorphisms in said first genome of said mixture.

2. The method of claim 1, wherein step (c) comprises genotyping said second genome in a sample that is substantially free of said first genome

3. The method of claim 1, wherein step (c) comprises counting sequence tags mapped to said multiple reference polymorphisms.

4. The method of claim 1, wherein said multiple polymorphisms in said first genome are associated with at least one disorder.

5. The method of claim 1, wherein said sample is unenriched for polymorphic target sequences in said first and second genomes.

6. The method of claim 1, wherein said sample is a blood sample obtained from a pregnant woman.

7. The method of claim 1, wherein said first genome is a fetal genome and said second genome is a maternal genome.

8. The method of claim 7, further comprising identifying said multiple polymorphisms in a paternal genome.

9. The method of claim 1, wherein said sample is a blood sample obtained from a subject that is known or suspected of having cancer.

27. The method of claim 1, 10 or 20, wherein said sequencing is massively parallel sequencing of clonally amplified cfDNA molecules or of single cfDNA molecules.

28. The method of claim 1, 10 or 20, wherein said sequencing is performed using massively parallel sequencing-by-synthesis with reversible dye terminators.

29. The method of claim 1, 10 or 20, wherein said sequencing is performed using massively parallel sequencing-by-ligation.

30. The method of claim 1, 10 or 20, wherein said sample is a plasma sample.

10. A method for determining the presence or absence of multiple disorders in a blood sample comprising a mixture of cfDNA of a first and a second genome, said method comprising:

whole genome sequencing at least a portion of said mixture of cfDNA, thereby providing a plurality of sequence tags;

identifying multiple polymorphisms in said plurality of sequence tags, wherein said multiple polymorphisms are associated with said number of disorders; and

associating said multiple polymorphisms with said first and/or second genome in said mixture, wherein said mixture is unenriched for said multiple polymorphisms.

11. The method of claim 10, further comprising comparing the sequence of said plurality of tags to the sequence of multiple reference polymorphisms, thereby identifying said multiple polymorphisms in said mixture of cfDNA.

12. The method of claim 10, wherein step (b) comprises counting sequence tags mapped to said multiple polymorphisms.

13. The method of claim 10, wherein step (c) comprises genotyping said second genome in a sample that is substantially free of said first genome.

14. The method of claim 10, wherein said first genome is a fetal genome and said second genome is a maternal genome.

15. The method of claim 10, wherein said first genome is a genome of an unaffected cell and said second genome is a genome from an affected cell.

16. The method of claim 10, wherein said at least one disorder is a monogenic disease.

17. The method of claim 10, wherein said at least one disorder is a polygenic disease.

18. The method of claim 10, wherein said at least one disorder is cancer.

19. The method of claim 14, further comprising determining the presence or absence of said multiple polymorphisms in a cellular paternal genome.

20. A method for determining the presence or absence of multiple fetal disorders in a maternal blood sample comprising a mixture of fetal and maternal cfDNA, said method comprising;

whole genome sequencing at least a portion of said mixture of fetal and maternal cfDNA, thereby obtaining a plurality of sequence tags;

identifying multiple fetal polymorphisms in said plurality of sequence tags, thereby determining multiple fetal haplotypes associated with said multiple fetal disorders; and

determining the presence or absence of said at least one fetal disorder.

21. The method of claim 20, wherein step (b) comprises genotyping maternal cellular DNA thereby identifying multiple polymorphisms in said maternal cellular DNA.

22. The method of claim 20, wherein step (b) comprises counting tags mapped to said fetal and maternal polymorphisms in said mixture.

23. The method of claim 20, further comprising sequencing paternal cellular DNA.

24. The method of claim 20, wherein each of said multiple fetal polymorphisms comprise at least one SNP, a tandem SNP, or an STR.

25. The method of claim 24, wherein said at least one SNP is a tag SNP.

26. The method of claim 24, wherein said at least one STR is a tag STR.