Systems and methods for detection of genomic copy number changes

• US 9,890,425 B2
• Filed: 03/13/2014
• Issued: 02/13/2018
• Est. Priority Date: 03/15/2013
• Status: Active Grant

• Current Assignee: Abbott Molecular Incorporated (Abbott Laboratories)
• Original Assignee: Abbott Molecular Incorporated (Abbott Laboratories)
• Inventors: Domanus, Marc H., Ho, Shiaolan Y., Kim, Dae Hyun
• Primary Examiner(s): Treptow, Nancy
• Application Number:
  US14/208,874
• Publication Number:
  US 20140296094A1
• Time in Patent Office: 1,433 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• 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 2537/16 Assays for determining copy number or wherein the copy number is of special importance
• CPC Class Codes: C12Q 2600/158 Expression markers

Abstract

The present invention relates to systems and methods for detecting genomic copy number changes. In particular, the present invention relates to next generation sequencing methods for detection of copy number changes.

Claims

1. A method of detecting chromosomal copy number changes or altered expression of a nucleic acid region of interest, comprising:

a) amplifying nucleic acid from said region of interest and at least one control region on the same chromosome as said region of interest and at least one control region on a different chromosome than said region of interest to generate a library of one or more target and one or more control amplicons;

b) sequencing said target and control amplicons to generate sequencing reads wherein said sequencing is selected from the group consisting of pyrosequencing, sequencing-by-ligation, single molecule sequencing, sequence-by-synthesis (SBS), massive parallel clonal sequencing, massive parallel single molecule SBS, massive parallel single molecule real-time sequencing, and massive parallel single molecule real-time nanopore sequencing;

c) using said sequencing reads to compare the level of target amplicon to the level of control amplicon; and

d) detecting chromosomal copy number changes or altered expression of said nucleic acid region of interest when the level of target amplicon is different than the level of at least one said control amplicon generated from said nucleic acid from said at least one control region on the same chromosome as said region of interest.

2. The method of claim 1, wherein using said sequencing reads to compare the level of target amplicon to the level of control amplicon comprises comparing the ratio of the level of a first control amplicon to the level of a second control amplicon to the ratio of the level of said target amplicon to the level of said second control amplicon.

3. The method of claim 1, wherein said detected chromosomal copy number is altered by at least 2-fold.

4. The method of claim 1, wherein said detected chromosomal copy number is altered by at least 10-fold.

5. The method of claim 1, wherein said detected chromosomal copy number is altered by at least 20-fold.

6. The method of claim 1, wherein said amplifying comprises generating at least 2 amplicons from said region of interest.

7. The method of any claim 1, wherein said method further comprises the addition of nucleic acid adaptors to said amplicons during or after amplification.

8. The method of claim 1, wherein said amplifying comprises 45 or fewer cycles of amplification.

9. The method of claim 1, wherein said using said sequencing reads comprises base composition analysis.

10. The method of claim 1, comprising at least one control region comprising a plasmid, mitochondrial DNA, or synthetic DNA.

11. The method of claim 1, wherein said control sequencing reads are averaged.

12. The method of claim 1, comprising simultaneous amplification of a plurality of regions of interest using multiplex PCR.