# US 2021/0340632 A1 · 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 2021/0340632 A1 - Document ID: US20210340632A1 - Status: Published application - Kind: A1 - Application number: 17/370,941 - Filed: 2021-07-08 - Published: 2021-11-04 - Earliest claimed: 2015-03-02 - Assignees: Guardant Health, Inc. - Applicants: Guardant Health, Inc. - Inventors: AmirAli Talasaz - Web page: https://patentgrep.com/patent/US20210340632A1 ## 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 tagging cell-free deoxyribonucleic acid (cfDNA) molecules from a sample of a subject, the method comprising: attaching tags comprising molecular barcodes to a plurality of polynucleotides derived from the cfDNA molecules to generate tagged polynucleotides, wherein n is at least 2 and no more than 100,000*z, wherein z is a mean of an expected number of duplicate molecules in the sample of cfDNA molecules that map to identical start and stop positions on a reference sequence. ### Claim 2 (depends on 1) The method of claim 1, wherein the sample comprises a blood sample, plasma sample, or serum sample. ### Claim 3 (depends on 1) The method of claim 1, wherein the sample is from a subject having cancer. ### Claim 4 (depends on 1) The method of claim 1, wherein the sample comprises between 5 nanograms (ng) and 100 ng of nucleic acid molecules. ### Claim 5 (depends on 1) The method of claim 1, wherein the sample of cfDNA molecules comprises 100 to 100,000 haploid human genome equivalents. ### Claim 6 (depends on 1) The method of claim 1, comprising amplifying a plurality of the cfDNA molecules from the sample of the subject prior to the attaching of the tags. ### Claim 7 (depends on 1) The method of claim 1, wherein the attaching is performed by blunt-end ligation. ### Claim 8 (depends on 1) The method of claim 1, wherein the attaching is performed by sticky-end ligation. ### Claim 9 (depends on 1) The method of claim 1, wherein the attaching is performed by single-strand ligation. ### Claim 10 (depends on 1) The method of claim 1, wherein n different molecular barcodes is no more than 10,000*z. ### Claim 11 (depends on 1) The method of claim 1, wherein n different molecular barcodes is no more than 1,000*z. ### Claim 12 (depends on 1) The method of claim 1, wherein n different molecular barcodes is no more than 100*z. ### Claim 13 (depends on 1) The method of claim 1, wherein the molecular barcodes have a length of between 5 and 20 nucleotides. ### Claim 14 (depends on 1) The method of claim 1, wherein the tags are part of sequencing adapters. ### Claim 15 (depends on 1) The method of claim 1, wherein the tags are attached to both ends of a molecule of the polynucleotides derived from the cfDNA molecules. ### Claim 16 (depends on 6) The method of claim 6, further comprising amplifying a plurality of the tagged polynucleotides to generate amplified tagged polynucleotides. ### Claim 17 (depends on 16) The method of claim 16, wherein amplification of the tagged polynucleotides is performed by selective amplification. ### Claim 18 (depends on 17) The method of claim 17, wherein the tagged polynucleotides are selectively amplified for regions of interest that comprise genomic sequences of genes, oncogenes, tumor suppressor genes, promoters, or regulatory sequence elements. ### Claim 19 (depends on 17) The method of claim 17, wherein the tagged polynucleotides are selectively amplified for regions of interest that comprise exon sequences of one or more actionable cancer-related genes. ### Claim 20 (depends on 16) The method of claim 16, further comprising sequencing a plurality of the amplified tagged polynucleotides. ### Claim 21 A method for preparing cell-free deoxyribonucleic acid (cfDNA) molecules for sequencing, the method comprising: (a) amplifying a plurality of the cfDNA molecules from a sample of a subject to generate amplified polynucleotides; (b) attaching tags comprising molecular barcodes to a plurality of the amplified polynucleotides to generate tagged polynucleotides, wherein n is at least 2 and no more than 100,000*z, wherein z is a mean of an expected number of duplicate molecules in the plurality of cfDNA molecules from the sample that map to identical start and stop positions on a reference sequence, wherein the tags are attached to at least one end of a molecule of the plurality of amplified polynucleotides; (c) amplifying a plurality of the tagged polynucleotides to generate amplified tagged polynucleotides; and (d) sequencing a plurality of the amplified tagged polynucleotides. ### Claim 22 (depends on 21) The method of claim 21, wherein the sample is from a subject having cancer. ### Claim 23 (depends on 21) The method of claim 21, wherein the sample comprises a blood sample, plasma sample, or serum sample. ### Claim 24 (depends on 21) The method of claim 21, wherein the sample of cfDNA molecules comprises 100 to 100,000 haploid human genome equivalents. ### Claim 25 (depends on 21) The method of claim 21, wherein the attaching is performed by single-strand ligation. ### Claim 26 (depends on 21) The method of claim 21, wherein n different molecular barcodes is no more than 10,000*z. ### Claim 27 (depends on 21) The method of claim 21, wherein amplification of the tagged polynucleotides is performed by selective amplification.