# US 10,501,808 B2 · Systems and methods to detect rare mutations and copy number variation

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 10,501,808 B2
- Document ID: US10501808B2
- Status: Granted
- Kind: B2
- Application number: 15/978,848
- Filed: 2018-05-14
- Published: 2018-11-15
- Granted: 2019-12-10
- Earliest claimed: 2012-09-04
- Assignees: Guardant Health, Inc.
- Applicants: Guardant Health, Inc.
- Inventors: AmirAli Talasaz
- Web page: https://patentgrep.com/patent/US10501808B2

## CPC

- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- C12N 15/1065: {Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags}
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6869: Methods for sequencing
- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- G16B 30/00: ICT specially adapted for sequence analysis involving nucleotides or amino acids
- G16B 30/10: Sequence alignment; Homology search
- C12Q 2600/118: Prognosis of disease development
- C12Q 2600/156: Polymorphic or mutational markers
- C12Q 2600/158: Expression markers
- C12Q 2600/16: Primer sets for multiplex assays
- 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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 system comprising:
a nucleic acid sequencer configured to sequence a nucleic acid library comprising non-uniquely tagged parent polynucleotides and non-uniquely tagged progeny polynucleotides derived therefrom, to generate sequencing reads corresponding to the non-uniquely tagged parent polynucleotides, wherein the non-uniquely tagged parent polynucleotides comprise nucleic acid molecules ligated to non-unique molecular barcodes, wherein the non-unique molecular barcodes have from 2 to 1000 different sequences and lengths of at least 5 nucleotides, wherein the sequencing reads comprise (1) nucleic acid sequences of the nucleic acid molecules, and (2) nucleic acid sequences of the non-unique molecular barcodes attached to the nucleic acid molecules;
a communication interface that receives, over a communication network, the sequencing reads generated by the nucleic acid sequencer; and
a computer in communication with the communication interface, wherein the computer comprises one or more computer processors and a computer-readable medium comprising machine-executable code that, upon execution by the one or more computer processors, implements a method comprising:
(i) receiving, over the communication network, the sequencing reads generated by the nucleic acid sequencer;
(ii) mapping the sequencing reads to one or more reference sequences from a human genome;
(iii) grouping the sequencing reads into a plurality of families, wherein a family of the plurality of families comprises sequencing reads comprising identical nucleic acid sequences of the non-unique molecular barcodes and having the same start or stop positions, wherein the family of the plurality of families comprises sequencing reads corresponding to a same original cell-free nucleic acid molecule, wherein the family of the plurality of families comprises sequencing reads of non-uniquely tagged progeny polynucleotides amplified from a unique polynucleotide among the non-uniquely tagged parent polynucleotides; and
(iv) generating a base call for at least the family of the plurality of families at a genetic locus of a plurality of genetic loci in the one or more reference sequences.

### Claim 2 (depends on 1)

The system of claim 1, wherein the nucleic acid sequencer performs sequencing-by-synthesis on the sequencing library to generate the sequencing reads.

### Claim 3 (depends on 1)

The system of claim 1, wherein the nucleic acid sequencer performs pyrosequencing, single-molecule sequencing, nanopore sequencing, semiconductor sequencing, sequencing-by-ligation, or sequencing-by-hybridization on the sequencing library to generate the sequencing reads.

### Claim 4 (depends on 1)

The system of claim 1, wherein the nucleic acid sequencer uses a clonal single molecule array derived from the sequencing library to generate the sequencing reads.

### Claim 5 (depends on 1)

The system of claim 1, wherein the nucleic acid sequencer comprises a chip having an array of microwells for sequencing the sequencing library to generate the sequencing reads.

### Claim 6 (depends on 1)

The system of claim 1, wherein the computer-readable medium comprises a memory, a hard drive, or a computer server.

### Claim 7 (depends on 1)

The system of claim 1, wherein the communication network comprises a telecommunication network, an internet, an extranet, or an intranet.

### Claim 8 (depends on 1)

The system of claim 1, wherein the communication network includes one or more computer servers capable of distributed computing.

### Claim 9 (depends on 8)

The system of claim 8, wherein the distributed computing is cloud computing.

### Claim 10 (depends on 1)

The system of claim 1, wherein the computer is located on a computer server that is remotely located from the nucleic acid sequencer.

### Claim 11 (depends on 1)

The system of claim 1, wherein the non-unique molecular barcodes are located on both ends of the nucleic acid molecules.

### Claim 12 (depends on 1)

The system of claim 1, wherein the sequences of the non-unique molecular barcodes are randomly ligated to the nucleic acid molecules.

### Claim 13 (depends on 1)

The system of claim 1, further comprising: an electronic display in communication with the computer over a network, wherein the electronic display comprises a user interface for displaying results upon implementing (i)-(iv).

### Claim 14 (depends on 13)

The system of claim 13, wherein the user interface is a graphical user interface (GUI) or a web-based user interface.

### Claim 15 (depends on 13)

The system of claim 13, wherein the electronic display is in a personal computer.

### Claim 16 (depends on 13)

The system of claim 13, wherein the electronic display is in an internet-enabled computer.

### Claim 17 (depends on 16)

The system of claim 16, wherein the internet-enabled computer is located at a location remote from the computer.

### Claim 18 (depends on 1)

The system of claim 1, wherein the method further comprises:
(iv) counting a number of families mapping to each of a plurality of predefined regions of the one or more reference sequences.

### Claim 19 (depends on 1)

The system of claim 1, wherein the nucleic acid sequences comprise sequences of circulating tumor deoxyribonucleic acid (DNA) and circulating non-tumor DNA.

### Claim 20 (depends on 1)

The system of claim 1, wherein the method further comprises:
(v) measuring a frequency of one or more bases called at the genetic locus from among the plurality of families.

### Claim 21 (depends on 1)

The system of claim 1, wherein (iv) of the method comprises:
collapsing sequencing reads in the family to yield a base call for the family at the genetic locus.

### Claim 22 (depends on 1)

The system of claim 1, wherein the nucleic acid sequencer performs next-generation sequencing to generate the sequencing reads.

### Claim 23 (depends on 1)

The system of claim 1, wherein the non-unique molecular barcodes have from 5 to 100 different sequences.

### Claim 24 (depends on 1)

The system of claim 1, wherein the non-unique molecular barcodes have from 10 to 100 different sequences.

### Claim 25 (depends on 1)

The system of claim 1, wherein the non-unique molecular barcodes have from 50 to 100 different sequences.

### Claim 26 (depends on 1)

The system of claim 1, wherein the nucleic acid molecules are derived from a sample comprising between 100 and 100,000 haploid genome equivalents of cell-free nucleic acid molecules.

### Claim 27 (depends on 1)

The system of claim 1, wherein the nucleic acid sequences of the nucleic acid molecules correspond to sequences of cell-free nucleic acid molecules from which the nucleic acid molecules are derived.

### Claim 28 (depends on 1)

The system of claim 1, wherein the family of the plurality of families comprises sequencing reads comprising identical nucleic acid sequences of the non-unique molecular barcodes and having the same start and stop positions.