# US 11,078,532 B2 · Synthetic nucleic acid spike-ins > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,078,532 B2 - Document ID: US11078532B2 - Status: Granted - Kind: B2 - Application number: 15/953,822 - Filed: 2018-04-16 - Published: 2018-08-23 - Granted: 2021-08-03 - Earliest claimed: 2016-03-25 - Assignees: Karius, Inc. - Applicants: Karius, Inc. - Inventors: Fred C. Christians; Igor D. Vilfan; Michael Kertesz; Timothy A. Blauwkamp; Shivkumar Venkatasubrahmanyam; Michael Rosen; Rene Sit - Web page: https://patentgrep.com/patent/US11078532B2 ## CPC - C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation - C12N 15/1065: {Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags} - C12Q 1/6869: Methods for sequencing - C12Q 1/689: for bacteria - G16B 30/00: ICT specially adapted for sequence analysis involving nucleotides or amino acids - G16B 30/10: Sequence alignment; Homology search - C12Q 2600/166: Oligonucleotides used as internal standards, controls or normalisation probes - 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 This disclosure provides methods for determining relative abundance of one or more non-host species in a sample from a host. Also provided are methods involving addition of known concentrations of synthetic nucleic acids to a sample and performing sequencing assays to identify non-host species such as pathogens. Also provided are methods of tracking samples, tracking reagents, and tracking diversity loss in sequencing assays. ## Claims ### Claim 1 A composition comprising at least 1,000 synthetic spike-in nucleic acids comprising nucleic acid sequences that are not naturally occurring wherein at least 1,000 of said at least 1,000 synthetic spike-in nucleic acids comprising nucleic acid sequences that are not naturally occurring comprise one or more identifying tag regions positioned downstream or upstream of a unique variable region, wherein said unique variable region comprises at least five degenerate bases, and wherein said at least 1,000 of said at least 1,000 synthetic spike-in nucleic acids comprising nucleic acid sequences that are not naturally occurring have sequences that are unique to each other. ### Claim 2 (depends on 1) The composition of claim 1, wherein said unique variable region comprises from at least five degenerate bases to about 25 degenerate bases. ### Claim 3 (depends on 2) The composition of claim 2, wherein said unique variable region comprises at least 16 degenerate bases. ### Claim 4 (depends on 1) The composition of claim 1, wherein said at least five degenerate bases are separated into two or more groups. ### Claim 5 (depends on 4) The composition of claim 4, wherein said two or more groups are separated by at least four nucleotides. ### Claim 6 (depends on 4) The composition of claim 4, wherein said two or more groups comprises an equal number of degenerate bases. ### Claim 7 (depends on 6) The composition of claim 6, wherein said equal number of degenerate bases is at least eight degenerate bases. ### Claim 8 (depends on 4) The composition of claim 4, wherein said two or more groups comprises an unequal number of degenerate bases. ### Claim 9 (depends on 1) The composition of claim 1, wherein each of said at least 1,000 of said at least 1,000 unique synthetic spike-in nucleic acids comprising nucleic acid sequences that are not naturally occurring is from about 20 nucleotides up to about 1000 nucleotides in length. ### Claim 10 (depends on 1) The composition of claim 1, wherein said identifying tag region comprises a diversity code, a length identifier domain, a synthetic spike-in nucleic acid-identifier, a feature domain, a process code, or any combination thereof. ### Claim 11 (depends on 1) The composition of claim 1, wherein said at least 1,000 of said at least 1,000 unique synthetic spike-in nucleic acids comprising nucleic acid sequences that are not naturally occurring further comprise an adapter a poly-(A) tail. ### Claim 12 (depends on 1) The composition of claim 1, wherein each of said at least 1,000 of said at least 1,000 unique synthetic spike-in nucleic acids comprising nucleic acid sequences that are not naturally occurring further comprises a modified base or an artificial base. ### Claim 13 (depends on 12) The composition of claim 12, wherein each of said at least 1,000 of said at least 1,000 unique synthetic spike-in nucleic acids comprising nucleic acid sequences that are not naturally occurring comprises a modified base. ### Claim 14 (depends on 13) The composition of claim 13, wherein said modified base comprises 5-methylcytosine (m5C), pseudouridine (T), dihydrouridine (D), inosine (I), or 7-methylguanosine (m7G). ### Claim 15 (depends on 1) The composition of claim 1, wherein each of said at least 1,000 of said at least 1,000 unique synthetic spike-in nucleic acids comprising nucleic acid sequences that are not naturally occurring is attached, ligated or conjugated to a bead, a fluorophore, a polymer, or any combination thereof. ### Claim 16 A composition comprising at least 1,000 unique synthetic nucleic acid sequences wherein each of said at least 1,000 unique synthetic nucleic acids comprise sequences identified in SEQ ID NO: 119 or SEQ ID NO: 120. ### Claim 17 (depends on 1) The composition of claim 1, wherein each of said at least 1,000 of said at least 1,000 unique synthetic spike-in nucleic acids comprising nucleic acid sequences that are not naturally occurring further comprises a second identifying tag region positioned upstream of said unique variable region. ### Claim 18 (depends on 1) The composition of claim 1, wherein said composition further comprises human genomic nucleic acids. ### Claim 19 (depends on 18) The composition of claim 18, wherein said human genomic nucleic acids are not attached to said at least 1,000 of said 1,000 synthetic spike-in nucleic acids comprising nucleic acid sequences that are not naturally occurring. ### Claim 20 (depends on 1) The composition of claim 1, wherein said at least 1,000 synthetic spike-in nucleic acids comprising nucleic acid sequences that are not naturally occurring comprise at least 10,000 synthetic spike-in nucleic acids that are unique to each other. ### Claim 21 (depends on 1) The composition of claim 1, wherein said at least 1,000 synthetic spike-in nucleic acids comprising nucleic acid sequences that are not naturally occurring comprise between 1,000 and 109 or between 1010 and 1.15×1011 synthetic spike-in nucleic acids that are unique to each other. ### Claim 22 (depends on 1) The composition of claim 1, wherein said at least 1,000 of said at least 1,000 synthetic spike-in nucleic acids comprising nucleic acid sequences that are not naturally occurring do not comprise a genomic DNA sequence of at least 20 nucleotides in length. ### Claim 23 (depends on 1) The composition of claim 1, wherein said at least 1,000 of said at least 1,000 synthetic spike-in nucleic acids comprising nucleic acid sequences that are not naturally occurring do not comprise a genomic DNA sequence of at least 100 nucleotides in length. ### Claim 24 A composition comprising: (a) a biological sample from a tissue or body fluid; and (b) at least 1,000 synthetic nucleic acids comprising nucleic acid sequences that are not naturally occurring wherein at least 1,000 of said at least 1,000 synthetic nucleic acids comprising nucleic acid sequences that are not naturally occurring comprise one or more identifying tag regions positioned downstream or upstream of a unique variable region, wherein said unique variable region comprises at least five degenerate bases, and wherein said at least 1,000 of said at least 1,000 synthetic nucleic acids comprising nucleic acid sequences that are not naturally occurring have sequences that are unique to each other. ### Claim 25 (depends on 24) The composition of claim 24, wherein said biological sample from said tissue or body fluid comprises (i) said at least 1,000 synthetic nucleic acids comprising nucleic acid sequences that are not naturally occurring and (ii) a tissue, a body fluid, or a combination thereof. ### Claim 26 (depends on 24) The composition of claim 24, wherein said biological sample from a tissue or body fluid sample is a body fluid sample. ### Claim 27 (depends on 26) The composition of claim 26, wherein said body fluid sample comprises cell-free nucleic acids. ### Claim 28 (depends on 26) The composition of claim 26, wherein said body fluid sample is selected from the group consisting of plasma, blood, serum, synovial fluid, bronchial-alveolar lavage, cerebrospinal fluid, saliva, stool, urine, nasal sample, or any combination thereof. ### Claim 29 (depends on 24) The composition of claim 24, wherein said biological sample is a plasma sample. ### Claim 30 (depends on 24) The composition of claim 24, wherein said biological sample comprises human genomic nucleic acids or microbial nucleic acids, or a combination thereof. ### Claim 31 (depends on 30) The composition of claim 30, wherein said at least 1,000 of said at least 1,000 synthetic nucleic acids comprising nucleic acid sequences that are not naturally occurring are not capable of hybridizing to said human genomic nucleic acids or said microbial nucleic acids, or a combination thereof. ### Claim 32 (depends on 30) The composition of claim 30, wherein said at least 1,000 of said at least 1,000 synthetic nucleic acids comprising nucleic acid sequences that are not naturally occurring do not comprise said human genomic nucleic acids or microbial nucleic acids, or a combination thereof. ### Claim 33 (depends on 26) The composition of claim 26, wherein said at least 1,000 of said at least 1,000 synthetic nucleic acids comprising nucleic acid sequences that are not naturally occurring are up to 200 bases in length. ### Claim 34 (depends on 26) The composition of claim 26, wherein said at least 1,000 of said at least 1,000 synthetic nucleic acids comprise DNA. ### Claim 35 A composition comprising: (a) genomic nucleic acids that are not attached to an adapter; and (b) at least 1,000 synthetic nucleic acids comprising nucleic acid sequences that are not naturally occurring wherein at least 1,000 of said at least 1,000 synthetic nucleic acids comprising nucleic acid sequences that are not naturally occurring comprise one or more identifying tag regions positioned downstream or upstream of a unique variable region, wherein said unique variable region comprises at least five degenerate bases, and wherein said at least 1,000 of said at least 1,000 synthetic nucleic acid sequences have sequences that are unique to each other. ### Claim 36 A composition comprising at least 1,000 synthetic spike-in nucleic acids comprising non-natural nucleic acid sequences that are not naturally occurring wherein at least 1,000 of said at least 1,000 unique synthetic nucleic acids comprising non-natural nucleic acid sequences that are not naturally occurring comprises one or more identifying tag regions positioned downstream or upstream of a unique variable region, wherein said unique variable region comprises at least five degenerate bases and wherein said degenerate bases are split into at least two groups separated by at least four contiguous nucleotides wherein each of said non-natural nucleic acid sequences that are not naturally occurring is unique to each other. ### Claim 37 (depends on 1) The composition of claim 1, wherein said nucleic acid sequences that are not naturally occurring are unique to each other. ### Claim 38 (depends on 24) The composition of claim 24, wherein said nucleic acid sequences that are not naturally occurring are unique to each other. ### Claim 39 (depends on 35) The composition of claim 35, wherein said nucleic acid sequences that are not naturally occurring are unique to each other. ### Claim 40 (depends on 26) The composition of claim 26, wherein said at least 1000 synthetic nucleic acids comprising nucleic acid sequences that are not naturally occuring comprise at least 100,000 synthetic nucleic acids that are unique to each other.