# US 10,501,810 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 10,501,810 B2 - Document ID: US10501810B2 - Status: Granted - Kind: B2 - Application number: 16/283,629 - Filed: 2019-02-22 - Published: 2019-06-20 - 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/US10501810B2 ## 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 method for detecting somatic genetic variants of cell-free deoxyribonucleic acid (DNA) in a human subject, the method comprising: a) obtaining 10 to 100 nanograms (ng) of double-stranded cell-free DNA from a blood sample from the human subject; b) ligating adapters comprising molecular barcodes to ends of a plurality of molecules of the double-stranded cell-free DNA to produce tagged parent polynucleotides, wherein the molecular barcodes together constitute a set of 5-100 molecular barcodes from 5-20 nucleotides in length; c) amplifying a plurality of the tagged parent polynucleotides to produce progeny polynucleotides with associated molecular barcodes; d) selectively enriching 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 mappable portions of the sequencing reads to a human reference genome; g) grouping a plurality of the sequencing reads into families based on the sequence information of the molecular barcodes and the beginning and end base positions of the mapped portion of the progeny polynucleotides; and h) detecting, from among a plurality of the families, the presence or absence of one or more somatic genetic variants comprising a single nucleotide variant (SNV), a copy number variation (CNV), an insertion or deletion (indel), a gene fusion, or any combination thereof. ### Claim 2 (depends on 1) The method of claim 1, wherein the human subject has a cancer. ### Claim 3 (depends on 1) The method of claim 1, wherein ligating comprises blunt-end ligation. ### Claim 4 (depends on 1) The method of claim 1, wherein ligating comprises sticky-end ligation. ### Claim 5 (depends on 1) The method of claim 1, wherein at least 20% of the double-stranded cell-free DNA are ligated with adapters. ### Claim 6 (depends on 1) The method of claim 1, wherein at least 40% of the double-stranded cell-free DNA are ligated with adapters. ### Claim 7 (depends on 1) The method of claim 1, wherein the molecular barcodes have a predetermined sequence. ### Claim 8 (depends on 1) The method of claim 1, wherein the target regions comprise genomic sequences of actionable cancer-related genes. ### Claim 9 (depends on 1) The method of claim 1, wherein the target regions comprise a genomic sequence from one of TP53, KRAS, PIK3CA, APC, EGFR, BRAF, MET, and MYC. ### Claim 10 (depends on 1) The method of claim 1, wherein sequencing comprises next generation sequencing. ### Claim 11 (depends on 1) The method of claim 1, further comprising filtering out sequencing reads that fail to meet a quality control threshold. ### Claim 12 (depends on 1) The method of claim 1, wherein the number of sequencing reads in a family is between 2 and 10. ### Claim 13 (depends on 1) The method of claim 1, wherein detecting comprises generating a set of consensus sequences from among the sequence reads in the families. ### Claim 14 (depends on 13) The method of claim 13, wherein a consensus sequence within the set of consensus sequences corresponds to a mapped portion of the progeny polynucleotides. ### Claim 15 (depends on 13) The method of claim 13, further comprising determining the presence or absence of the one or more somatic genetic variants in the set of consensus sequences compared with the human reference genome. ### Claim 16 (depends on 1) The method of claim 1, wherein detecting comprises generating a base call at a genetic locus of the human reference genome for a family from among the plurality of families. ### Claim 17 (depends on 1) The method of claim 1, further comprising quantifying the number of families. ### Claim 18 (depends on 1) The method of claim 1, wherein the presence of at least one or more somatic genetic variants detected is an SNV. ### Claim 19 (depends on 1) The method of claim 1, wherein the presence of at least one or more somatic genetic variants detected is an indel. ### Claim 20 (depends on 1) The method of claim 1, wherein the presence of at least one or more somatic genetic variants detected is a CNV. ### Claim 21 (depends on 1) The method of claim 1, wherein the presence of at least one or more somatic genetic variants detected is a gene fusion. ### Claim 22 (depends on 1) The method of claim 1, wherein the presence of at least one or more somatic genetic variants detected is a SNV and CNV. ### Claim 23 (depends on 1) The method of claim 1, wherein the double-stranded cell-free DNA comprises circulating tumor DNA (ctDNA). ### Claim 24 (depends on 1) The method of claim 1, wherein less than 5% of the double-stranded cell-free DNA is from one or more cancer genomes. ### Claim 25 (depends on 1) The method of claim 1, wherein less than 1% of the double-stranded cell-free DNA is from one or more cancer genomes. ### Claim 26 (depends on 1) The method of claim 1, wherein less than 0.1% of the double-stranded cell-free DNA is from one or more cancer genomes. ### Claim 27 (depends on 1) The method of claim 1, further comprising generating a report that includes a cell-free tumor mutation profile of the human subject based on the detection of the presence or absence of the one or more somatic genetic variants. ### Claim 28 (depends on 27) The method of claim 27, wherein the report further includes a treatment recommendation for the human subject based on the cell-free tumor mutation profile.