# US 11,078,533 B2 · Single cell nucleic acid detection and analysis > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,078,533 B2 - Document ID: US11078533B2 - Status: Granted - Kind: B2 - Application number: 16/872,571 - Filed: 2020-05-12 - Published: 2020-09-03 - Granted: 2021-08-03 - Earliest claimed: 2011-03-24 - Assignees: President and Fellows of Harvard College - Applicants: President and Fellows of Harvard College - Inventors: Xiaoliang Sunney Xie; Katsuyuki Shiroguchi; Peter A. Sims; Tony Z. Jia - Web page: https://patentgrep.com/patent/US11078533B2 ## 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 1/6853: using modified primers or templates - C12N 2320/10: in screening processes - 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 Methods and compositions for digital profiling of nucleic acid sequences present in a sample are provided. ## Claims ### Claim 1 A method, comprising: (a) tagging a population of double-stranded nucleic acid molecules, each molecule having two ends, with asymmetric sequencing adapters, each adapter having a barcode sequence, to generate a plurality of tagged nucleic acid molecules, wherein the population comprises nucleic acid molecules that differ in sequence from each other, wherein an adapter is joined to both ends of the nucleic acid molecules; (b) amplifying the tagged nucleic acid molecules, whereby amplicons of the tagged nucleic acid molecules are derived from individual double-stranded nucleic acid molecules from the population of double-stranded nucleic acid molecules, wherein the amplicons comprise two complementary single-stranded nucleic acid molecules; (c) sequencing a plurality of the amplicons to generate paired-end sequence reads derived from sequencing the complementary single-stranded nucleic acid molecules, wherein the sequence reads comprise the sequence of the amplicons and the sequence of the associated barcodes, (d) aligning the sequence reads to a reference sequence, wherein the starting position and ending position of the sequence of the amplicons from which the reads are derived can be determined, and wherein the aligning is performed by a computer implemented method, and (e) forming groups of the paired-end sequence reads that can be aligned to the same starting and ending position on the reference sequence and that have the same barcode sequences, wherein paired-end sequence reads obtained from both of the complementary single-strands are identified, and (f) counting the number of groups that comprise paired-end reads that can be aligned to the same starting and ending position and have the different barcode sequences. ### Claim 2 (depends on 1) The method of claim 1, wherein the plurality of nucleic acid molecules comprises nucleic acid molecules with identical sequences, and the number of barcodes is sufficient to tag nucleic acid molecules having identical sequences, wherein the nucleic acids having the identical sequence can be distinguished from each other based on the sequence of the tag, and wherein the number of barcodes is not sufficient to uniquely barcode each nucleic acid molecule in the sample. ### Claim 3 (depends on 1) The method of claim 1, wherein the plurality of nucleic acid molecules is obtained from a biological fluid of a subject. ### Claim 4 (depends on 1) The method of claim 1, wherein the barcodes comprise pseudo-random or random sequences. ### Claim 5 (depends on 1) The method of claim 1, wherein the barcodes are double stranded. ### Claim 6 (depends on 1) The method of claim 1, further comprising comparing the counted number of tagged nucleic acid molecules that align to a first region of the reference sequence to the counted number of tagged nucleic acid molecules that align to a second region of the reference sequence. ### Claim 7 (depends on 1) The method of claim 1, wherein the sequencing comprises performing massively parallel sequencing. ### Claim 8 (depends on 1) The method of claim 1, wherein the sequencing comprises performing paired end sequencing. ### Claim 9 (depends on 1) The method of claim 1, wherein the plurality of nucleic acid molecules comprises nucleic acid molecules with identical sequences, and the number of barcodes is sufficient to differently tag nucleic acid molecules with identical sequences, and the number of barcodes is not sufficient to uniquely barcode each nucleic acid molecule in the sample. ### Claim 10 (depends on 1) The method of claim 1, wherein there is an error-resistant distance between the barcode sequences. ### Claim 11 (depends on 10) The method of claim 10, wherein the error-resistant distance between the barcode sequence is at least 9. ### Claim 12 (depends on 1) The method of claim 1, wherein the asymmetric sequencing adapter is a Y-shaped adapter. ### Claim 13 (depends on 9) The method of claim 9, further comprising amplifying the plurality of tagged nucleic acid molecules to produce amplicons. ### Claim 14 (depends on 9) The method of claim 9, wherein there is an error-resistant distance between the barcode sequences. ### Claim 15 (depends on 1) The method of claim 1, wherein a value is measured for the number of groups formed from the paired-end sequence reads that are aligned to the same starting and ending position on the reference sequence and that have the same barcode sequences.