Detecting genetic aberrations associated with cancer using genomic sequencing

• US 10,619,214 B2
• Filed: 03/30/2017
• Issued: 04/14/2020
• Est. Priority Date: 07/23/2007
• Status: Active Grant

• Current Assignee: Chinese University of Hong Kong
• Original Assignee: Chinese University of Hong Kong
• Inventors: Lo, Yuk-Ming Dennis, Chiu, Rossa Wai Kwun, Chan, Kwan Chee
• Primary Examiner(s): Lin, Jerry
• Application Number:
  US15/474,995
• Publication Number:
  US 20170218450A1
• Time in Patent Office: 1,111 Days
• Field of Search: 702 19
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• 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 2537/16 Assays for determining copy number or wherein the copy number is of special importance
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: G16B 20/00 ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
• CPC Class Codes: G16B 20/10 Ploidy or copy number detection
• CPC Class Codes: G16B 20/20 Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/10 Sequence alignment; Homology search

Abstract

Methods, systems, and apparatus determine whether a first chromosomal region exhibits a deletion or an amplification associated with cancer in a sample from a subject (e.g., where the sample includes a mixture of cell-free DNA from tumor cells and non-malignant cells. Nucleic acid molecules of the biological sample are sequenced. Respective amounts of a clinically-relevant chromosomal region and of background chromosomal region(s) are determined from results of the sequencing. A parameter derived from these amounts (e.g. a ratio) is compared to one or more cutoff values, thereby determining a classification of whether first chromosomal region exhibits a deletion or an amplification associated with cancer.

Claims

1. A method of analyzing a biological sample obtained from a subject being screened for a genetic aberration in a chromosomal region associated with cancer, said biological sample comprising cell-free nucleic acid molecules, said method comprising:

(a) performing sequencing of said cell-free nucleic acid molecules from said biological sample of said subject to generate sequence reads;

(b) receiving, at a computer system, said sequence reads obtained from said sequencing of said cell-free nucleic acid molecules from said biological sample;

(c) aligning at least a portion of said sequence reads to a reference genome;

(d) determining a parameter of a relative amount between (i) a first amount of said sequence reads that align to a first chromosomal region that is part of a first chromosome in the reference genome, and (ii) a second amount of said sequence reads that align to one or more second chromosomal regions in the reference genome; and

(e) determining, using said parameter, whether said first chromosomal region comprises a genetic aberration associated with cancer in cell-free nucleic acid molecules of said biological sample that are derived from a tumor.

2. The method of claim 1, wherein said first chromosomal region comprises said first chromosome.

3. The method of claim 1, wherein said genetic aberration comprises a copy number variation.

4. The method of claim 1, wherein said sequencing comprises massively parallel sequencing.

5. The method of claim 4, wherein said massively parallel sequencing comprises sequencing clonally-expanded single DNA molecules captured on a solid surface and generating said sequence reads.

6. The method of claim 1, wherein said sequencing comprises paired-end sequencing.

7. The method of claim 1, wherein determining said parameter based on said first amount of said sequence reads that align to said first chromosomal region that is part of said first chromosome comprises counting said sequence reads that align to said first chromosomal region.

8. The method of claim 1, wherein sequencing of said cell-free nucleic acid molecules from said biological sample of said subject results in at least 120,000 sequence reads.

9. The method of claim 1, wherein said genetic aberration comprises an amplification.

10. The method of claim 1, wherein said sequencing comprises random sequencing.

11. The method of claim 1, further comprising ligating adaptors to said cell-free nucleic acid molecules from said biological sample.

12. The method of claim 1, wherein said biological sample is blood or plasma.

13. The method of claim 1, wherein said cell-free nucleic acid molecules comprise DNA fragments.

14. The method of claim 1, wherein said first amount is determined based on a pool of sequence reads that align to a plurality of positions of said first chromosomal region.

15. The method of claim 1, wherein sequence reads identified as aligning to said first chromosomal region uniquely align to said first chromosomal region.

16. The method of claim 1, wherein said biological sample is blood or plasma, wherein said cell-free nucleic acid molecules comprise DNA fragments, wherein said sequencing comprises massively parallel sequencing and results in at least 120,000 sequence reads, wherein said genetic aberration comprises a copy number variation, and wherein said determining comprises counting said sequence reads that align to said first chromosomal region.

17. The method of claim 1, wherein said cell-free nucleic acid molecules include at least 30% of said reference genome.

18. The method of claim 17, wherein said cell-free nucleic acid molecules provides at least one-fold coverage of said reference genome.

19. The method of claim 1, wherein the parameter includes a ratio of the first amount and the second amount.

20. A system for analyzing a biological sample obtained from a subject being screened for a genetic aberration in a chromosomal region associated with cancer, said biological sample comprising cell-free nucleic acid molecules, said system comprising:

a sequencing device configured for sequencing a plurality of cell-free nucleic acid molecules from the biological sample of the subject, thereby generating sequence reads; and

one or more computer processors communicably coupled with the sequencing device and individually or collectively programmed for;

receiving said sequence reads from sequencing of said cell-free nucleic acid molecules from said biological sample;

aligning at least a portion of said sequence reads to a reference genome;

determining a parameter based on (i) a first amount of said sequence reads that align to a first chromosomal region that is part of a first chromosome in the reference genome, and (ii) a second amount of said sequence reads that align to one or more second chromosomal regions in the reference genome; and

determining, using said parameter, whether said first chromosomal region comprises a genetic aberration associated with cancer in cell-free nucleic acid molecules of said biological sample that are derived from a tumor.

21. The system of claim 20, wherein said genetic aberration comprises a copy number variation.

22. The system of claim 20, wherein said sequencing comprises massively parallel sequencing.

23. The system of claim 20, wherein said sequencing comprises paired-end sequencing.

24. The system of claim 20, wherein determining said parameter based on said first amount of said sequence reads that align to said first chromosomal region that is part of said first chromosome comprises counting said sequence reads that align to said first chromosomal region.

25. The system of claim 20, wherein sequencing of said cell-free nucleic acid molecules from said biological sample of said subject results in at least 120,000 sequence reads.

26. The system of claim 20, wherein said genetic aberration comprises an amplification.

27. The system of claim 20, wherein said sequencing comprises random sequencing.

28. The system of claim 20, wherein said biological sample is blood or plasma.

29. The system of claim 20, wherein said first amount is determined based on a pool of sequence reads that align to a plurality of positions of said first chromosomal region.

30. The system of claim 20, wherein said sequence reads identified as aligning to said first chromosomal region uniquely align to said first chromosomal region.

31. The system of claim 20, wherein said biological sample is blood or plasma, wherein said cell-free nucleic acid molecules comprise DNA fragments, wherein said sequencing comprises massively parallel sequencing and results in at least 120,000 sequence reads, wherein said genetic aberration comprises a copy number variation, and wherein said determining comprises counting said sequence reads that align to said first chromosomal region.