# US 2021/0172012 A1 · Preparation of dna sequencing libraries for detection of dna pathogens in plasma > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2021/0172012 A1 - Document ID: US20210172012A1 - Status: Published application - Kind: A1 - Application number: 17/109,348 - Filed: 2020-12-02 - Published: 2021-06-10 - Earliest claimed: 2019-12-04 - Assignees: Illumina, Inc. - Applicants: Illumina, Inc. - Inventors: Tong Liu; Fiona Kaper; Clifford Wang - Web page: https://patentgrep.com/patent/US20210172012A1 ## CPC - 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 - C12N 15/1006: {by means of a solid support carrier, e.g. particles, polymers} - C12Q 1/6888: for detection or identification of organisms - C12Q 1/689: for bacteria - C12Q 1/6893: for protozoa - C12Q 1/6895: for plants, fungi or algae - C12Q 1/70: involving virus or bacteriophage {(immunoassay for viruses G01N33/56983)} - C12Q 2523/32: Centrifugation - C12Q 2563/149: Particles, e.g. beads - 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) ## Abstract The application provides an agnostic, shotgun nucleic acid sequencing-based method for the detection of pathogens in samples from human patients, animals, or plants. The method includes dehosting the sample of the nucleic acid molecules of host origin and provides for the detection of pathogens without prior knowledge of their genome sequences. ## Claims ### Claim 1 A sample preparation method comprising: obtaining a host organism sample; removing intact cells from the host organism sample; removing nucleic acid molecules of less than 1000 basepairs (bp) from the host organism sample to obtain a dehosted sample. ### Claim 2 A method of dehosting a sample obtained from a host organism, the method comprising: removing intact cells from the host organism sample; removing nucleotide acid molecules of less than 1000 basepairs (bp) from the host organism sample to obtain a dehosted sample. ### Claim 3 (depends on 1) The method of claim 1 further comprising sequencing the nucleic acid molecules remaining in the dehosted sample. ### Claim 4 (depends on 1) The method of claim 1 further comprising preparing a sequencing library from the nucleic acid molecules remaining in the dehosted sample. ### Claim 5 (depends on 4) The method of claim 4 further comprising sequencing the nucleotide sequences of the sequencing library. ### Claim 6 (depends on 3) The method of claim 3 further comprising identifying pathogen sequences within the sequenced sequences. ### Claim 7 A method of identifying pathogen nucleotide sequences in a sample obtained from a host organism, the method comprising: removing intact cells from the host organism sample; removing nucleotide acid molecules of less than 1000 basepairs (bp) from the host organism sample to obtain a dehosted sample; preparing a sequencing library from the nucleic acid molecules remaining in the dehosted sample; sequencing the nucleotide sequences of the sequencing library; and identifying pathogen sequences within the sequenced sequences. ### Claim 8 (depends on 7) The method of claim 7, wherein the sequencing library is prepared by a transposon-based library preparation method. ### Claim 9 (depends on 8) The method of claim 8, wherein the transposon-based library preparation method comprises NEXTERA transposons or NEXTERA bead-based transposons. ### Claim 10 (depends on 3) The method of claim 3, wherein sequencing is by high throughput sequencing. ### Claim 11 (depends on 1) The method of e of claim 1, comprising removing nucleic acid molecules of less than 600 bp from the host organism sample to obtain the dehosted sample. ### Claim 12 (depends on 1) The method of claim 1, wherein removing intact cells from the host organism sample comprises centrifugation. ### Claim 13 (depends on 1) The method of claim 1, wherein removing intact cells from the host organism sample comprises binding cell free nucleic acids to functionalized controlled pore glass (CPG) beads. ### Claim 14 (depends on 13) The method of claim 13, wherein the functionalized controlled pore glass (CPG) beads are functionalized with a copolymer of N-vinyl pyrrolidone (70%) and N-methyl-N-vinyl imidazolium chloride (30%). ### Claim 15 The method of 1, wherein removing nucleotide acid molecules of less than 1000 bp from the host organism sample comprises solid phase reversible immobilization (SPRI) beads under conditions favoring capture of nucleotide molecules of 1000 bp or greater. ### Claim 16 (depends on 6) The method of claim 6, wherein the pathogen sequences comprise viral, bacterial, fungal, and/or parasitic sequence. ### Claim 17 (depends on 6) The method of claim 6, wherein the pathogen sequences comprise a pathogen with a DNA genome. ### Claim 18 (depends on 1) The method of claim 1, wherein the host organism sample comprises blood. ### Claim 19 (depends on 1) The method of claim 1, wherein the host organism sample comprises plasma. ### Claim 20 (depends on 1) The method of claim 1, wherein the host comprises a eukaryotic organism. ### Claim 21 (depends on 20) The method of claim 20, wherein the host comprises an animal or plant. ### Claim 22 (depends on 20) The method of claim 20, wherein the host comprises a mammal. ### Claim 23 (depends on 22) The method of claim 22, wherein the host comprises a human.