Systems and methods to detect rare mutations and copy number variation

• US 9,598,731 B2
• Filed: 05/14/2015
• Issued: 03/21/2017
• 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:
  US14/712,754
• Publication Number:
  US 20150299812A1
• Time in Patent Office: 677 Days
• Field of Search: None
• US Class Current: 1/1
• 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 quantifying single nucleotide variant tumor markers in cell-free DNA from a subject, comprising:

(a) providing at least 10 ng of cell-free DNA obtained from a bodily sample of the subject;

(b) attaching tags comprising barcodes having from 5 to 1000 distinct barcode sequences to said cell-free DNA obtained from said bodily sample of the subject, to generate non-uniquely tagged parent polynucleotides, wherein each barcode sequence is at least 5 nucleotides in length;

(c) amplifying the non-uniquely tagged parent polynucleotides to produce amplified non-uniquely tagged progeny polynucleotides;

(d) sequencing the amplified non-uniquely tagged progeny polynucleotides to produce a plurality of sequence reads from each parent polynucleotide, wherein each sequence read comprises a barcode sequence and a sequence derived from cell-free DNA;

(e) grouping the plurality of sequence reads produced from each non-uniquely tagged parent polynucleotide into families based on i) the barcode sequence and ii) at least one of;

sequence information at a beginning of the sequence derived from cell-free DNA, sequence information at an end of the sequence derived from cell-free DNA, and length of the sequence read, whereby each family comprises sequence reads of non-uniquely tagged progeny polynucleotides amplified from a unique polynucleotide among the non-uniquely tagged parent polynucleotides;

(f) comparing the sequence reads grouped within each family to each other to determine consensus sequences for each family, wherein each of the consensus sequences corresponds to a unique polynucleotide among the non-uniquely tagged parent polynucleotides;

(g) providing one or more reference sequences from a human genome, said one or more reference sequences comprising one or more loci of reported tumor markers, wherein each of the reported tumor markers is a single nucleotide variant;

(h) identifying consensus sequences that map to a given locus of said one or more loci of reported tumor markers; and

(i) calculating a number of consensus sequences that map to the given locus that include the single nucleotide variant thereby quantifying single nucleotide variant tumor markers in said cell-free DNA from said subject.

2. The method of claim 1, further comprising detecting an indel, copy number variation, transversion, translocation, inversion, deletion, aneuploidy, partial aneuploidy, polyploidy, chromosomal instability, chromosomal structure alteration, gene fusion, chromosome fusion, gene truncation, gene amplification, gene duplication, chromosomal lesion, DNA lesion, abnormal change in nucleic acid chemical modification, abnormal changes in epigenetic pattern, abnormal change in nucleic acid methylation infection or cancer.

3. The method of claim 1, further comprising determining copy number variation by mapping the consensus sequences to a plurality of reference sequences, counting consensus sequences corresponding to each reference sequence, and normalizing the number of consensus sequences between different reference sequences.

4. The method of claim 1, wherein grouping uses information about the length of each of the sequence reads.

5. The method of claim 1, further comprising filtering out sequence reads that fail to meet a set quality control threshold.

6. The method of claim 1, wherein the cell-free DNA is between about 10 ng and 10 μ

g.

7. The method of claim 1, wherein the cell-free DNA comprises no more than 100 ng of polynucleotides.

8. The method of claim 1, wherein the attaching comprises blunt-end ligation or sticky end ligation.

9. The method of claim 1, wherein the cell-free DNA is tagged with a double-stranded DNA barcode.

10. The method of claim 1, further comprising selectively enriching regions from the subject'"'"'s genome or transcriptome prior to sequencing.

11. The method of claim 1, wherein the grouping uses the barcode sequence and more than one of:

sequence information at a beginning of the sequence derived from cell-free DNA, sequence information at an end of the sequence derived from cell-free DNA, and the length of the sequence read.

12. The method of claim 1, wherein the barcode sequence comprises sequence information from barcodes attached at both ends of the cell-free DNA.

13. The method of claim 1, wherein the one or more reference sequences is from a healthy control.

14. The method of claim 1, wherein the one or more reference sequences is a human genome assembly.

15. The method of claim 1, further comprising attaching said tags to said cell-free DNA in a single container.

16. The method of claim 1, wherein the tags are attached to the cell-free DNA by ligation.

17. The method of claim 1, further comprising calculating a ratio of (1) a number of consensus sequences that map to the given locus that include the single nucleotide variant to (2) a total number of consensus sequences that map to the given locus, thereby quantifying single nucleotide variant tumor markers in said cell-free DNA from said subject.