# US 11,306,348 B2 · Complex surface-bound transposome complexes

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- Number: US 11,306,348 B2
- Document ID: US11306348B2
- Status: Granted
- Kind: B2
- Application number: 17/306,592
- Filed: 2021-05-03
- Published: 2021-09-02
- Granted: 2022-04-19
- Earliest claimed: 2019-01-11
- Assignees: Illumina Cambridge Limited
- Applicants: Illumina Cambridge Limited
- Inventors: Andrew Slatter; Esther Musgrave-Brown; Susan C. Verity; Niall Anthony Gormley
- Web page: https://patentgrep.com/patent/US11306348B2

## CPC

- C12N 15/1065: {Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags}
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6834: Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q 1/6869: Methods for sequencing
- C12Y 207/07: Nucleotidyltransferases (2.7.7)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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
- C12Y: ENZYMES

## Abstract

The present disclosure relates to methods, compositions, and kits for generating a library of tagged nucleic acid fragments without using PCR amplification, including methods and compositions for fragmenting and tagging nucleic acids (e.g., DNA) using transposome complexes immobilized on solid support.

## Claims

### Claim 1

A transposome complex comprising:
a. a transposase;
b. a first transposon comprising a 3′ transposon end sequence and a 5′ adaptor sequence;
c. a second transposon comprising a 5′ transposon end sequence and a 3′ adaptor sequence;
wherein the 5′ transposon end sequence is complementary to the 3′ transposon end sequence;
wherein the 5′ adaptor sequence is non-complementary to the 3′ adaptor sequence; and
wherein the first transposon and the second transposon form a Y-shape; and
d. an attachment polynucleotide comprising:
i. an attachment adaptor sequence, wherein the attachment adaptor sequence is complementary to and hybridized to one of the two adaptor sequences, and
ii. a binding element, optionally wherein the binding element comprises biotin, optionally wherein the binding element in a 3′ biotin linker; and
wherein the transposome complex comprises at least one uracil.

### Claim 2 (depends on 1)

The transposome complex of claim 1, further comprising a cleavable sequence comprising at least one cleavable nucleotide or nucleobase that is cleavable with an RNase.

### Claim 3 (depends on 2)

The transposome complex of claim 2, wherein the at least one cleavable nucleotide or nucleobase is uracil.

### Claim 4 (depends on 2)

The transposome complex of claim 2, wherein the at least one cleavable nucleotide or nucleobase is 8-oxo-guanine.

### Claim 5 (depends on 2)

The transposome complex of claim 2, wherein the at least one cleavable nucleotide or nucleobase comprises three uracil residues.

### Claim 6 (depends on 2)

The transposome complex of claim 2, wherein the at least one cleavable nucleotide or nucleobase is cleaved in an enzyme mixture comprising uracil DNA glycosylase and endonuclease VIII.

### Claim 7 (depends on 1)

The transposome complex of claim 1, wherein the binding element immobilizes the transposome complex to a solid support.

### Claim 8 (depends on 1)

The transposome complex of claim 1, wherein the binding element is attached to the 5′ adaptor sequence or the 3′ adaptor sequence through a cleavable linker.

### Claim 9 (depends on 1)

The transposome complex of claim 1, wherein the uracil is located between the 3′ transposon end sequence and the 5′ adaptor sequence, or (ii) between the 5′ transposon end sequence and the 3′ adaptor sequence.

### Claim 10 (depends on 1)

A kit comprising:
a. the transposome complex of claim 1; and
b. an index mix comprising index sequences.

### Claim 11 (depends on 7)

A method of generating a library of tagged nucleic acid fragments comprising contacting the immobilized transposome complex of claim 7 with a target nucleic acid under conditions sufficient to fragment the target nucleic acid into a plurality of target fragments, and to join the 3′ end of the first transposon to the 5′ ends of the target fragments to produce a plurality of 5′ tagged target fragments.

### Claim 12 (depends on 11)

The method of claim 11, after the contacting step, further comprising:
a. treating the solid support to remove unbound nucleic acids; or treating the solid support to remove the transposase from the complex, optionally by
i. heating the solid support and/or
ii. washing the solid support with an enzyme denaturing agent, wherein the enzyme denaturing agent optionally comprises sodium dodecyl sulfate (SDS), guanidine hydrochloride, urea, or proteinase;
b. treating the plurality of 5′ tagged target fragments with a polymerase and a ligase to extend and ligate the 5′ tagged target fragments to produce fully double-stranded tagged fragments, optionally wherein the treating with a polymerase and a ligase is done in the presence of a DNA secondary structure disruptor, wherein the disruptor is optionally DMSO;
c. removing the fully double-stranded tagged fragments from the solid support, optionally wherein the removing comprises applying heat and/or a denaturant sufficient to cleave the fully double-stranded tagged fragments from the solid support, optionally wherein the denaturant is NaOH; and
d. selecting the fully double-stranded tagged fragments using capture beads, optionally wherein the capture beads are magnetic beads, further optionally wherein two separate selecting steps are performed.

### Claim 13 (depends on 7)

A method of generating a library of tagged nucleic acid fragments comprising:
a. contacting a first immobilized transposome complex of claim 7 and a second immobilized transposome complex of claim 7 with a target nucleic acid under conditions sufficient to fragment the target nucleic acid into a plurality of target fragments, and to join the 3′ end of each first transposon to the 5′ ends of the target fragments to produce a plurality of first 5′ tagged target fragments generated from the first immobilized transposome complex and a plurality of second 5′ tagged target fragments generated from the second immobilized transposome complex, wherein
i. the first immobilized transposome complex comprises a solid support and a first transposome complex immobilized to the solid support, and wherein the first attachment polynucleotide of the first transposome complex further comprises: an anchor sequence complement, an A14′ sequence, a spacer, and a P5′ sequence, and a binding element comprising biotin, wherein the biotin is immobilized to the solid support; and wherein
ii. the second immobilized transposome complex further comprises a solid support and a second transposome complex immobilized to the solid support, wherein the second transposome complex comprises: an anchor sequence complement, a B15′ sequence, a spacer, and a P7′ sequence, and a binding element comprising biotin, wherein the biotin is immobilized to the solid support;
b. treating the plurality of 5′ tagged target fragments with a ligase to ligate each 5′ tagged target fragment to either a first indexing oligonucleotide or second indexing oligonucleotide by contacting the 5′ tagged target fragments with a pool of first and second indexing oligonucleotides, wherein each first indexing oligonucleotides comprises a A14 sequence, i5 sequence, and P5 sequence and can associate with a first 5′ tagged target fragment; and wherein each second indexing oligonucleotide comprises a B15 sequence, i7 sequence, and P7 sequence and can associate with a second 5′ tagged target fragment, to produce a plurality of 5′ tagged target fragments ligated to indexing oligonucleotides;
c. treating the solid support to remove the transposases from the complex, optionally by
i. heating the solid support and/or
ii. washing the solid support with an enzyme denaturing agent, wherein the enzyme denaturing agent optionally comprises sodium dodecyl sulfate (SDS), guanidine hydrochloride, urea, or proteinase; and
d. treating the plurality of 5′ tagged target fragments ligated to indexing oligonucleotides with a polymerase to extend and produce fully double-stranded tagged fragments.

### Claim 14 (depends on 1)

The transposome complex of claim 1, wherein the adaptor sequence is a sequence for hybridization.

### Claim 15 (depends on 1)

The transposome complex of claim 1, wherein the adaptor sequence is a sequence chosen from A14 sequence, B15 sequence, A14′ sequence, B15′ sequence, and combinations thereof.

### Claim 16 (depends on 1)

The transposome complex of claim 1, wherein the adaptor sequence is chosen from primer sequences, anchor sequences, universal sequences, spacer regions, index sequences, capture sequences, barcode sequences, cleavage sequences, sequencing-related sequences, and combinations thereof.

### Claim 17 (depends on 1)

The transposome complex of claim 1, wherein the attachment polynucleotide further comprises a spacer region, an anchor region, a primer sequence, or an index tag sequence, or a combination thereof, and optionally further comprises a capture sequence, a barcode sequence, a cleavage sequence, or a sequencing-related sequence, or a combination thereof.

### Claim 18 (depends on 17)

The transposome complex of claim 17, wherein:
a. the first transposon further comprises a first primer sequence 5′ of the 5′ adaptor sequence, and the attachment polynucleotide comprises (i) a portion complementary with and hybridized to the 5′ adaptor sequence and (ii) a sequence that is complementary to the first primer sequence, or
b. the second transposon further comprises a second primer sequence 3′ of the 3′ adaptor sequence, and the attachment polynucleotide comprises (i) a portion complementary with and hybridized to the 3′ adaptor sequence and (ii) a sequence that is complementary to the second primer sequence.

### Claim 19 (depends on 17)

The transposome complex of claim 17, wherein the first transposon further comprises a first primer sequence 5′ of the 5′ adaptor sequence, and the attachment polynucleotide comprises an index tag sequence and a primer sequence.

### Claim 20 (depends on 17)

The transposome complex of claim 17, wherein the second transposon comprises the 3′ adaptor sequence and the attachment polynucleotide comprises (i) a portion complementary to and hybridized with the 3′ adaptor sequence, (ii) an index tag sequence, and (iii) a primer sequence.

### Claim 21 (depends on 17)

The transposome complex of claim 17, wherein the attachment polynucleotide comprises a spacer region or a spacer region and an anchor region.

### Claim 22 (depends on 21)

The transposome complex of claim 21, wherein the first transposon comprises the 5′ adaptor sequence, and the attachment polynucleotide comprises (i) a portion complementary with and hybridized to the 5′ adaptor sequence, (ii) a spacer region, and (iii) a primer sequence.

### Claim 23 (depends on 1)

The transposome complex of claim 1, wherein the attachment polynucleotide comprises an A14′ sequence.

### Claim 24 (depends on 21)

The transposome complex of claim 21, wherein the spacer region is a non-DNA spacer.

### Claim 25 (depends on 21)

The transposome complex of claim 21, wherein the spacer region is a length equivalent to 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, or 30 base pairs, optionally wherein the spacer region is a length equivalent to 8 or 10 base pairs or nucleotides.

### Claim 26 (depends on 21)

The transposome complex of claim 21, wherein the spacer region comprises universal bases, optionally wherein the universal bases comprise an inosine or a nitroindole sequence.

### Claim 27 (depends on 21)

The transposome complex of claim 21, wherein the spacer region comprises one or more C18 spacers, one or more C9 spacers, or a combination thereof.

### Claim 28 (depends on 21)

The transposome complex of claim 21, wherein the spacer region comprises a 2 x sp18 synthetic linker, two C18 spacers, a C9 spacer, or two C18 spacers and one C9 spacer.

### Claim 29 (depends on 1)

The transposome complex of claim 1, wherein the attachment polynucleotide is hybridized to the 5′ adaptor sequence and the 3′ adaptor sequence is single-stranded.

### Claim 30 (depends on 1)

The transposome complex of claim 1, wherein the attachment polynucleotide is hybridized to the 3′ adaptor sequence and the 5′ adaptor sequence is single-stranded.