Systems and methods to detect rare mutations and copy number variation

• US 10,457,995 B2
• Filed: 01/16/2018
• Issued: 10/29/2019
• Est. Priority Date: 09/04/2012
• Status: Active Grant

• Current Assignee: Guardant Health Inc.
• Original Assignee: Guardant Health Inc.
• Inventors: Talasaz, AmirAli
• Primary Examiner(s): Horlick, Kenneth R
• Application Number:
  US15/872,831
• Publication Number:
  US 20180223374A1
• Time in Patent Office: 651 Days
• Field of Search: None
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• 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 2537/165 Mathematical modelling, e.g. logarithm, ratio
• CPC Class Codes: C12Q 2545/114 involving a quantitation step
• CPC Class Codes: C12Q 2600/118 Prognosis of disease development
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• 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

The present disclosure provides a system and method for the detection of rare mutations and copy number variations in cell free polynucleotides. Generally, the systems and methods comprise sample preparation, or the extraction and isolation of cell free polynucleotide sequences from a bodily fluid; subsequent sequencing of cell free polynucleotides by techniques known in the art; and application of bioinformatics tools to detect rare mutations and copy number variations as compared to a reference. The systems and methods also may contain a database or collection of different rare mutations or copy number variation profiles of different diseases, to be used as additional references in aiding detection of rare mutations, copy number variation profiling or general genetic profiling of a disease.

Claims

1. A method for detecting copy number variation, comprising:

a) attaching a set of molecular barcodes to cell-free nucleic acid molecules obtained or derived from a sample of a subject to produce tagged parent polynucleotides, wherein a plurality of the tagged parent polynucleotides has identical molecular barcodes;

b) amplifying the plurality of the tagged parent polynucleotides, thereby producing progeny polynucleotides;

c) sequencing at least a portion of the progeny polynucleotides to generate sequence reads;

d) mapping the sequence reads derived from the sequencing to a reference sequence to produce mapped reads;

e) grouping the mapped sequence reads into families, wherein, of the families, a family corresponds to sequence reads of one or more progeny polynucleotides amplified from a same tagged parent polynucleotide of the plurality of the tagged parent polynucleotides;

f) quantifying numbers of families mapping to predefined regions of the reference sequence; and

g) determining copy number variation in one or more of the predefined regions from the numbers of families mapping to the predefined regions of the reference sequence.

2. The method of claim 1, wherein the sample is a bodily fluid selected from the group consisting of blood, plasma, and serum.

3. The method of claim 1, wherein the subject has cancer.

4. The method of claim 1, wherein the cell-free nucleic acid molecules comprise circulating tumor DNA.

5. The method of claim 1, further comprising selectively enriching regions from a genome or transcriptome of the subject prior to sequencing.

6. The method of claim 1, wherein, in a) attaching comprises ligating the set of molecular barcodes to the cell-free nucleic acid molecules.

7. The method of claim 1, wherein the set of molecular barcodes comprises between 2 to 1,000,000 different molecular barcodes.

8. The method of claim 7, wherein the set of molecular barcodes comprises between 5 to 100 different molecular barcodes.

9. The method of claim 1, wherein, in a), a molecular barcode is attached to each end of a cell-free nucleic acid molecule.

10. The method of claim 1, wherein a predefined region of the predefined regions is at least a single base.

11. The method of claim 1, wherein a predefined region of the predefined regions comprises at least a portion of a gene.

12. The method of claim 1, wherein the reference sequence is a sequence of a human genome.

13. The method of claim 1, wherein the sample comprises no more than 1% of cell-free nucleic acids from a cancer genome.

14. The method of claim 1, wherein the sample comprises no more than 0.5% of cell-free nucleic acids from a cancer genome.

15. The method of claim 1, wherein the sample comprises no more than 0.1% of cell-free nucleic acids from a cancer genome.

16. The method of claim 1, wherein, in e), grouping mapped sequence reads into families is based on a combination of sequence information from the molecular barcodes and sequence information from a cell-free nucleic acid molecule portion of the progeny polynucleotides.

17. The method of claim 1, wherein the amount of cell-free nucleic acid molecules in the sample is between 10 nanograms (ng) and 100 ng.

18. The method of claim 1, wherein the cell-free nucleic acid molecules are double-stranded deoxyribonucleic acid (DNA).

19. The method of claim 8, wherein the length of the molecular barcodes is at least 5 nucleotides in length.

20. The method of claim 1, further comprising determining a quantity of sequence reads in one or more of the families.

21. The method of claim 1, wherein the predefined regions are overlapping.

22. The method of claim 1, wherein the predefined regions are non-overlapping.

23. The method of claim 1, wherein the numbers of families mapping to the predefined regions are normalized for representational bias during the sequencing process.

24. The method of claim 1, wherein g) comprises normalizing the numbers of families mapping to the predefined regions to each other to determine copy number variation.

25. The method of claim 24, further comprising adjusting the normalized numbers of the families mapping to the predefined regions with a normalized number of families mapping to the predefined regions obtained from at least one control sample.

26. The method of claim 1, wherein g) comprises normalizing the numbers of families mapping to the predefined regions against numbers of families mapping to the predefined regions in at least one control sample.