# US 10,870,880 B2 · Systems and methods to detect rare mutations and copy number variation

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- Number: US 10,870,880 B2
- Document ID: US10870880B2
- Status: Granted
- Kind: B2
- Application number: 16/870,476
- Filed: 2020-05-08
- Published: 2020-09-24
- Granted: 2020-12-22
- Earliest claimed: 2014-03-05
- Assignees: Guardant Health, Inc.
- Applicants: Guardant Health, Inc.
- Inventors: AmirAli Talasaz; Stefanie Ann Ward Mortimer
- Web page: https://patentgrep.com/patent/US10870880B2

## CPC

- C12Q 1/6827: for detection of mutation or polymorphism
- C12Q 1/6837: using probe arrays or probe chips (C12Q1/6874 takes precedence)
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6855: Ligating adaptors
- G16B 20/10: Ploidy or copy number detection
- G16B 20/20: Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
- G16B 30/00: ICT specially adapted for sequence analysis involving nucleotides or amino acids
- G16B 30/10: Sequence alignment; Homology search
- G16B 30/20: Sequence assembly
- C12Q 1/6869: Methods for sequencing
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY

## 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

### Claim 1

A method for identifying one or more somatic genetic variants in a population of cell-free deoxyribonucleic acid (cfDNA) molecules, comprising:
(a) tagging the population of cfDNA molecules with tags comprising molecular barcodes from a set of molecular barcodes to tag at least 20% of the cfDNA molecules in the population, wherein the tagging comprises attaching the tags to both ends of cfDNA molecules from the population of cfDNA molecules using more than a 10× molar excess of tags relative to the cfDNA molecules in the population, thereby generating tagged parent polynucleotides;
(b) determining, based at least on sequence information from the molecular barcodes, sequences of a plurality of the tagged parent polynucleotides to provide sequenced cfDNA molecules; and
(c) identifying, from among a plurality of the sequenced cfDNA molecules, the one or more somatic genetic variants as compared to a reference sequence.

### Claim 2 (depends on 1)

The method of claim 1, further comprising amplifying a plurality of the tagged parent polynucleotides prior to (b) to produce amplified progeny polynucleotides.

### Claim 3 (depends on 2)

The method of claim 2, wherein (b) comprises determining sequences of a plurality of the amplified progeny polynucleotides.

### Claim 4 (depends on 3)

The method of claim 3, wherein (b) further comprises grouping a plurality of the sequences of the plurality of the amplified progeny polynucleotides into families, the members of a family having the same molecular barcode sequences and the same start and stop base positions that map to the reference sequence.

### Claim 5 (depends on 4)

The method of claim 4, wherein (b) further comprises determining consensus sequences for the families from the sequences of their respective members.

### Claim 6 (depends on 5)

The method of claim 5, wherein (b) further comprises determining a frequency of bases called at one or more genetic loci from among the plurality of consensus sequences.

### Claim 7 (depends on 1)

The method of claim 1, further comprising (d) calculating a ratio of a number of the plurality of the sequenced cfDNA molecules having a somatic genetic variant that map to a genetic locus of the reference sequence relative to a total number of the plurality of the sequenced cfDNA molecules that map to the genetic locus.

### Claim 8 (depends on 1)

The method of claim 1, wherein the population of cfDNA molecules is obtained from a sample of a subject having cancer or suspected of having cancer.

### Claim 9 (depends on 8)

The method of claim 8, wherein the sample is blood, plasma, or serum.

### Claim 10 (depends on 1)

The method of claim 1, wherein the set of molecular barcodes comprises molecular barcodes having 2 to 1,000,000 different molecular barcode sequences.

### Claim 11 (depends on 1)

The method of claim 1, wherein the set of molecular barcodes comprises molecular barcodes having 5 to 10,000 different molecular barcode sequences.

### Claim 12 (depends on 1)

The method of claim 1, wherein the molecular barcodes have pre-determined, fixed nucleic acid sequences.

### Claim 13 (depends on 1)

The method of claim 1, wherein the tagging comprises using more than an 80× molar excess of tags relative to the cfDNA molecules in the population.

### Claim 14 (depends on 13)

The method of claim 13, wherein at least 40% of the cfDNA molecules in the population are tagged.

### Claim 15 (depends on 1)

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

### Claim 16 (depends on 1)

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

### Claim 17 (depends on 2)

The method of claim 2, further comprising, prior to (b), enriching a plurality of the amplified progeny polynucleotides for target sequences associated with cancer.

### Claim 18 (depends on 4)

The method of claim 4, further comprising quantifying numbers of families mapping to pre-defined regions of the reference sequence.

### Claim 19 (depends on 18)

The method of claim 18, further comprising normalizing the numbers of families mapping to the pre-defined regions to each other.

### Claim 20 (depends on 1)

The method of claim 1, wherein the one or more somatic genetic variants comprise single nucleotide variants (SNVs), insertions or deletions (indels), copy number variations (CNVs), or gene fusions.