Systems and methods to detect rare mutations and copy number variation

• US 10,704,085 B2
• Filed: 09/18/2019
• Issued: 07/07/2020
• Est. Priority Date: 03/05/2014
• Status: Active Grant

• Current Assignee: Guardant Health Inc.
• Original Assignee: Guardant Health Inc.
• Inventors: Talasaz, AmirAli, Mortimer, Stefanie Ann Ward
• Primary Examiner(s): Wilder, Cynthia B
• Application Number:
  US16/575,079
• Publication Number:
  US 20200032323A1
• Time in Patent Office: 293 Days
• 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 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2565/513 characterised by the pattern of the arrayed oligonucleotides
• 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
• CPC Class Codes: G16B 30/20 Sequence assembly

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 generating a genetic profile of a tumor from a blood sample of double-stranded cell-free deoxyribonucleic acids (cfDNA) molecules from a subject having cancer or suspected of having a cancer, the method comprising:

(a) obtaining a population comprising the double-stranded cfDNA molecules from the blood sample from the subject;

(b) ligating a set of molecular barcodes to both ends of a plurality of the double-stranded cfDNA molecules using more than a 30×

molar excess of molecular barcodes relative to the double-stranded cfDNA molecules to produce tagged parent polynucleotides, wherein a given molecular barcode is a member of a set of molecular barcodes comprising 2 to 1,000,000 different molecular barcode sequences,

wherein at least 20% of the double-stranded cfDNA molecules from the population of cfDNA molecules are attached to molecular barcodes;

(c) amplifying a plurality of the tagged parent polynucleotides to produce progeny polynucleotides with associated molecular barcodes;

(d) selectively enriching a subset of the progeny polynucleotides for target regions associated with cancer, whereby enriched progeny polynucleotides are generated;

(e) sequencing a portion of the enriched progeny polynucleotides to produce sequencing reads of the progeny polynucleotides with associated molecular barcodes;

(f) aligning a plurality of the sequencing reads to a reference sequence;

(g) grouping a plurality of the sequencing reads into a plurality of families based at least on sequence information of the molecular barcodes, a start base position of a given sequencing read from among the sequencing reads at which the given sequencing read is determined to start aligning to the reference sequence, and a stop base position of the given sequencing read at which the given sequencing read is determined to stop aligning to the reference sequence, wherein a family of the plurality of families is representative of a cell-free nucleic acid molecule in the sample;

(h) detecting, from among the families, the presence or absence of somatic genetic variants; and

(i) quantifying a plurality of somatic genetic variants detected as present to generate the genetic profile of the tumor.

2. The method of claim 1, wherein the population comprises 1 nanogram (ng) to 100 ng of double-stranded cfDNA molecules.

3. The method of claim 1, wherein the ligating is blunt-end ligation or sticky-end ligation.

4. The method of claim 1, wherein a given molecular barcode is a member of the set of molecular barcodes having 5 to 1,000 different molecular barcode sequences.

5. The method of claim 1, wherein a given molecular barcode is a member of a set of molecular barcodes having 5 to 100 different molecular barcode sequences.

6. The method of claim 5, wherein the molecular barcode sequences are from 5 to 20 nucleotides in length.

7. The method of claim 1, wherein the ligating comprises using more than a 80×

molar excess of molecular barcodes relative to the double-stranded cfDNA molecules.

8. The method of claim 1, wherein at least 40% of the double-stranded cfDNA molecules from the population of double-stranded cfDNA molecules are attached to molecular barcodes.

9. The method of claim 1, wherein the molecular barcodes are part of sequencing adapters.

10. The method of claim 1, further comprising filtering out sequencing reads that fail to meet a quality threshold.

11. The method of claim 1, further comprising generating a consensus sequence for a family from among the plurality of families.

12. The method of claim 1, further comprising generating a base call for at least the family at a genetic locus of a plurality of genetic loci of the reference sequence.

13. The method of claim 12, further comprising determining a frequency of one or more bases called at the genetic locus from among a plurality of families.

14. The method of claim 11, wherein detecting in (h) comprises identifying a subset of consensus sequences that include a somatic genetic variant as compared to the reference sequence.

15. The method of claim 1, further comprising determining a treatment regimen for the subject based on the genetic profile of the tumor.

30. The method of claim 1, wherein the plurality of somatic genetic variants comprise a single nucleotide variant (SNV) or an insertion or deletion (indel).

16. A method for quantifying somatic genetic variants from a bodily fluid sample of cell-free nucleic acid molecules from a subject, the method comprising:

(a) ligating a set of molecular barcodes to both ends of a plurality of cell-free nucleic acid molecules from a population of the cell-free nucleic acid molecules obtained from the bodily fluid sample using more than a 30×

molar excess of molecular barcodes relative to the cell-free nucleic acid molecules to produce tagged parent polynucleotides,

wherein at least 20% of the cell-free nucleic acid molecules from the population of cell-free nucleic acid molecules are attached to molecular barcodes;

(b) amplifying a plurality of the tagged parent polynucleotides to produce progeny polynucleotides with associated molecular barcodes;

(c) sequencing a portion of the progeny polynucleotides to produce sequencing reads of the progeny polynucleotides with associated molecular barcodes;

(d) detecting, from among a plurality of the sequencing reads, the presence or absence of somatic genetic variants; and

(e) quantifying a plurality of the somatic genetic variants detected as present.

17. The method of claim 16, wherein the bodily fluid sample is blood, plasma, or serum.

18. The method of claim 16, wherein the population comprises 1 nanogram (ng) to 100 ng of double-stranded DNA molecules.

19. The method of claim 16, wherein the ligating is performed using blunt-end ligation or sticky-end ligation.

20. The method of claim 16, wherein the ligating comprises using more than an 80×

molar excess of molecular barcodes relative to the cell-free nucleic acid molecules.

21. The method of claim 16, wherein at least 40% of the cell-free nucleic acid molecules from the population of cell-free nucleic acid molecules are attached to molecular barcodes.

22. The method of claim 16, wherein the detecting in (d) comprises aligning a plurality of sequencing reads to a reference sequence and grouping a subset of the sequencing reads from the alignment into families based at least on sequence information from the molecular barcodes and the start and/or stop base positions of the sequencing reads aligning to the reference sequence.

23. The method of claim 22, further comprising generating a consensus sequence from a family from among the plurality of families.

24. The method of claim 23, wherein the detecting in (d) is based on a frequency of consensus sequences that include one or more somatic genetic variants as compared to the reference sequence.

25. The method of claim 16, wherein the plurality of somatic genetic variants comprise a single nucleotide variant (SNV) or an insertion or deletion (indel).

26. The method of claim 16, wherein a given molecular barcode is a member of the set of molecular barcodes having 5 to 1,000 different molecular barcode sequences that are from 5 to 20 nucleotides in length.

27. The method of claim 16, further comprising generating a genetic profile of the tumor based at least on the plurality of somatic genetic variants quantified.

28. The method of claim 27, further comprising determining a treatment regimen for the subject based on the genetic profile of the tumor.

29. The method of claim 16, further comprising selectively enriching a subset of the progeny polynucleotides for target regions associated with cancer.