# US 12,180,468 B2 · Transposase compositions for reduction of insertion bias > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 12,180,468 B2 - Document ID: US12180468B2 - Status: Granted - Kind: B2 - Application number: 17/010,349 - Filed: 2020-09-02 - Published: 2021-02-18 - Granted: 2024-12-31 - Earliest claimed: 2014-11-05 - Assignees: Illumina, Inc. - Applicants: Illumina, Inc. - Inventors: Christian Gloeckner; Amirali Kia; Molly He; Trina Faye Osothprarop; Frank J. Steemers; Kevin L. Gunderson; Sasan Amini; Jerome Jendrisak - Web page: https://patentgrep.com/patent/US12180468B2 ## CPC - C12N 15/1065: {Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags} - C12N 9/1241: {Nucleotidyltransferases (2.7.7)} - C12N 15/1093: {General methods of preparing gene libraries, not provided for in other subgroups} - C12Y 207/07: Nucleotidyltransferases (2.7.7) - C12Q 2563/185: Nucleic acid dedicated to use as a hidden marker/bar code, e.g. inclusion of nucleic acids to mark art objects or animals - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12Y: ENZYMES - 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 Presented herein are methods and compositions for tagmentation of nucleic acids. The methods are useful for generating tagged DNA fragments that are qualitatively and quantitatively representative of the target nucleic acids in the sample from which they are generated. ## Claims ### Claim 1 A method for preparing a nucleic acid library, comprising: (a) contacting a target nucleic acid with a first transposome in a reaction buffer comprising manganese cations, thereby generating a first plurality of tagmented nucleic acids, wherein the first transposome comprises a Tn5 transposase; (b) contacting the first plurality of tagmented nucleic acids with a second transposome to generate a second plurality of tagmented nucleic acids, wherein the second transposome comprises a Mos-1 transposase; and (c) amplifying the second plurality of tagmented nucleic acids to generate a nucleic acid library having an increased representation of different sequences of the target nucleic acid compared to a nucleic acid library prepared by a single tagmentation of the target nucleic acid. ### Claim 2 (depends on 1) The method of claim 1, wherein the nucleic acid library comprises an increased representation of A/T-rich genomic sequences or G/C-rich genomic sequences compared to a nucleic acid library prepared by a single tagmentation of the target nucleic acid. ### Claim 3 (depends on 1) The method of claim 1, wherein the first transposome and the second transposome each comprise a different insertion bias into a nucleic acid. ### Claim 4 (depends on 1) The method of claim 1, wherein steps (a) and (b) are performed in the presence of manganese cations. ### Claim 5 (depends on 1) The method of claim 1, wherein the first transposome comprises a transposon comprising a first barcode and a second barcode indicative of a continuity of tagmented nucleic acids in the first plurality of tagmented nucleic acids. ### Claim 6 (depends on 1) The method of claim 1, further comprising amplifying the first plurality of tagmented nucleic acids prior to step (b). ### Claim 7 (depends on 1) The method of claim 1, wherein the first transposome is immobilized on a first solid support. ### Claim 8 (depends on 7) The method of claim 7, wherein the first transposome is removed from the reaction buffer prior to step (b). ### Claim 9 (depends on 8) The method of claim 8, wherein the first plurality of tagmented nucleic acids is attached to the first transposome. ### Claim 10 (depends on 1) The method of claim 1, wherein the second transposome is immobilized on a second solid support. ### Claim 11 (depends on 1) The method of claim 1, wherein the first plurality of tagmented nucleic acids is removed from the reaction buffer prior to step (b). ### Claim 12 (depends on 1) The method of claim 1, wherein a salt concentration of the reaction buffer is adjusted by addition of NaCl to the reaction buffer, prior to step (b). ### Claim 13 (depends on 1) The method of claim 1, wherein the target nucleic acid comprises genomic DNA. ### Claim 14 (depends on 1) The method of claim 1, wherein an amount of first transposome and an amount of the second transposome have a ratio in a range from 1:0.4 to 1:1.7. ### Claim 15 A method for preparing a nucleic acid library, comprising: (a) generating a first plurality of tagmented nucleic acids by contacting a target nucleic acid with a first transposome in the presence of manganese cations, wherein the first transposome comprises a Tn5 transposase; (b) generating a second plurality of tagmented nucleic acids by contacting the first plurality of tagmented nucleic acids with a second transposome, wherein the second transposome comprises a Mos-1 transposase; and (c) amplifying the second plurality of tagmented nucleic acids to generate a nucleic acid library. ### Claim 16 (depends on 15) The method of claim 15, wherein steps (a) and (b) are performed in the presence of manganese cations. ### Claim 17 A method for preparing a nucleic acid library, comprising: (a) contacting a target nucleic acid with a first transposome in a reaction buffer to generate a first plurality of tagmented nucleic acids, wherein the first transposome comprises a Tn5 transposase; (b) adjusting a salt concentration of the reaction buffer by addition of NaCl; (c) contacting the first plurality of tagmented nucleic acids with a second transposome to generate a second plurality of tagmented nucleic acids, wherein the second transposome comprises a Mos-1 transposase; and (d) amplifying the second plurality of tagmented nucleic acids to generate a nucleic acid library having an increased representation of different sequences of the target nucleic acid compared to a nucleic acid library prepared by a single tagmentation of the target nucleic acid.