# US 12,180,535 B2 · Tagging nucleic acids for sequence assembly > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 12,180,535 B2 - Document ID: US12180535B2 - Status: Granted - Kind: B2 - Application number: 16/685,855 - Filed: 2019-11-15 - Published: 2020-09-10 - Granted: 2024-12-31 - Earliest claimed: 2014-08-01 - Assignees: Dovetail Genomics, LLC - Applicants: Dovetail Genomics, LLC - Inventors: Andrew Fields; Paul Hartley; Nicholas Putnam; Brandon Rice; Jonathan Stites - Web page: https://patentgrep.com/patent/US12180535B2 ## CPC - C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) - C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation - 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 ## Abstract Various approaches for generating long-distance contiguity information to facilitate contig assembly and phase determination are disclosed. Nucleic acids are assembled into complexes using binding moieties such that, when the nucleic acid backbones are cleaved, the ensuing fragments remain bound. Exposed ends are tagged and ligated either to one another or to tagging moieties such as oligo labels. Ligated junctions are sequenced, and the sequence information is used to assemble contigs into common scaffolds or to assign phase information. Various approaches to tagging the exposed ends are presented. ## Claims ### Claim 1 A method of mapping a sequence to a nucleic acid molecule, comprising the steps of: obtaining a nucleic acid sample comprising a first nucleic acid molecule comprising a first region and a second region; crosslinking said nucleic acid sample outside of a cell such that said first region and said second region of said first nucleic acid molecule are bound independently of a phosphodiester backbone of said first nucleic acid molecule; fragmenting said nucleic acid sample to produce at least one double strand break between said first region and said second region of said first nucleic acid molecule; contacting said nucleic acid sample to a population of oligonucleotides comprising a first plurality of oligonucleotides, wherein each of said first plurality of oligonucleotides comprises a) a 3′ annealing region capable of annealing to said double strand break, and b) a first molecular tag sequence 5′ of said 3′ annealing region, and wherein at least one of said first plurality of oligonucleotides anneals to one or more nucleotides on either side of said double strand break of said first nucleic acid molecule; ligating each side of said double strand break to at least one of said first plurality of oligonucleotides to form a ligation product; recovering said first nucleic acid molecule; and sequencing said ligation product; wherein a first sequence read comprising said first molecular tag sequence corresponds to a sequence of said first nucleic acid molecule. ### Claim 2 (depends on 1) The method of claim 1, wherein a second sequence comprising said first molecular tag corresponds to a sequence of said first nucleic acid molecule. ### Claim 3 (depends on 1) The method of claim 1, wherein said nucleic acid sample is subjected to fragmentation prior to said crosslinking. ### Claim 4 (depends on 1) The method claim 1, wherein said population of oligonucleotides comprises a second plurality of oligonucleotides, wherein each of said second plurality of oligonucleotides comprises a) a 3′ annealing region capable of annealing to said double strand break, and b) a second molecular tag sequence 5′ of said 3′ annealing region, having a sequence different from that of said first molecular tag. ### Claim 5 (depends on 4) The method of claim 4, wherein said second plurality of oligonucleotides is spatially separate from said first plurality of oligonucleotides. ### Claim 6 (depends on 1) The method claim 1, wherein said population of oligonucleotides is attached to a solid surface. ### Claim 7 (depends on 6) The method of claim 6, wherein said solid surface is a nucleic acid array. ### Claim 8 (depends on 1) The method of claim 1, wherein said fragmenting is conducted enzymatically. ### Claim 9 (depends on 8) The method of claim 8, wherein said fragmenting is conducted enzymatically in vitro. ### Claim 10 (depends on 8) The method of claim 8, wherein said fragmenting is conducted enzymatically with one or more restriction enzymes. ### Claim 11 (depends on 8) The method of claim 8, wherein said fragmenting is conducted enzymatically with a DNase. ### Claim 12 (depends on 8) The method of claim 8, wherein said fragmenting is conducted enzymatically with a transposase. ### Claim 13 (depends on 8) The method of claim 8, wherein said fragmenting is conducted enzymatically with a nicking enzyme. ### Claim 14 (depends on 1) The method of claim 1, wherein said fragmenting is conducted mechanically. ### Claim 15 (depends on 14) The method of claim 14, wherein said fragmenting is conducted mechanically with hydro-shear. ### Claim 16 (depends on 14) The method of claim 14, wherein said fragmenting is conducted mechanically with sonication. ### Claim 17 (depends on 14) The method of claim 14, wherein said fragmenting is conducted mechanically with nebulization. ### Claim 18 (depends on 1) The method of claim 1, further comprising, prior to said recovering said first nucleic acid molecule, ligating said ligation product to a second double strand break of said first nucleic acid molecule. ### Claim 19 (depends on 1) The method of claim 1, wherein said recovering comprises protein removal. ### Claim 20 (depends on 19) The method of claim 19, wherein said protein removal comprises protease treatment. ### Claim 21 (depends on 1) The method of claim 1, wherein said recovering comprises crosslink reversal. ### Claim 22 (depends on 1) The method of claim 1, wherein said recovering comprises capturing. ### Claim 23 (depends on 22) The method of claim 22, wherein said capturing comprises capturing via affinity label.