Polynucleotide barcodes for long read sequencing

Patent metadata

Field	Value
Publication number	US-12139754-B2
Application number	US-202117486656-A
Country	US
Kind code	B2
Filing date	Sep 27, 2021
Priority date	Dec 31, 2019
Publication date	Nov 12, 2024
Grant date	Nov 12, 2024

Abstract

Provided herein are methods of making, amplifying, and sequencing tagged nucleic acid complements, compositions including interposing oligonucleotide barcodes, and kits useful in obtaining long-range sequence data.

First claim

What is claimed:

1. A method of amplifying a tagged complement of a sample polynucleotide, the method comprising:
a) hybridizing a first hybridization sequence of a first terminal adapter to the 3′ end of said sample polynucleotide, wherein said first terminal adapter comprises from 5′ to 3′, a primer binding sequence, a barcode sequence, and said first hybridization sequence;
b) hybridizing a second hybridization sequence of a second terminal adapter to the 5′ end of said sample polynucleotide, wherein said second terminal adapter comprises from 5′ to 3′, the second hybridization sequence, a barcode sequence, and a primer binding sequence;
c) hybridizing one or more interposing oligonucleotide barcode probe(s) to the sample polynucleotide, wherein each of the interposing oligonucleotide barcode probes comprise from 5′ to 3′, a first target hybridization sequence, a first stem sequence, a loop region comprising a barcode sequence; a second stem sequence; and a second target hybridization sequence;
d) extending the 3′ end of the first terminal adapter and extending the 3′ end(s) of the second target hybridization sequences with one or more polymerases to create extension products; and
e) ligating adjacent ends of the extension products hybridized to the sample polynucleotide, and amplifying an integrated strand comprising a tagged complement of the sample polynucleotide.

2. The method of claim 1 , wherein step c) comprises hybridizing one interposing oligonucleotide barcode probe to the sample polynucleotide.

3. The method of claim 1 , wherein step c) comprises hybridizing a plurality of interposing oligonucleotide barcode probes to the sample polynucleotide.

4. The method of claim 1 , wherein the first terminal adapter, the second terminal adapter, or both the first terminal adapter and the second terminal adapter comprise one or more phosphorothioate-containing nucleotides or one or more LNAs.

5. The method of claim 1 , wherein the first terminal adapter, the second terminal adapter, or both the first terminal adapter and the second terminal adapter comprise a modified nucleotide comprising an affinity tag.

6. The method of claim 1 , wherein the first target hybridization sequence and the second target hybridization sequence each comprise about 9 to about 15 nucleotides and each terminal adapter hybridization sequence comprises about 10 to about 30 nucleotides.

7. The method of claim 1 , wherein each of the one or more interposing oligonucleotide barcode probe(s) comprises a phosphorylated 5′ end.

8. The method of claim 1 , wherein the barcode of the first terminal adapter and the barcode of the second terminal adapter each comprise about 5 to 15 nucleotides.

9. The method of claim 1 , wherein amplifying comprises hybridizing an amplification primer to the integrated strand and extending the amplification primer with a strand-displacing polymerase and nucleotides.

10. The method of claim 1 , further comprising sequencing the complement of the integrated strand.

11. The method of claim 10 , wherein the sequencing comprises: (A) fragmenting the complement of the integrated strand to produce fragments, (B) ligating adapters to the fragments, (C) amplifying the resultant products from step (B) to generate a polynucleotide, and (D) performing a sequencing reaction on the polynucleotide from step (C).

12. The method of claim 10 , wherein the sequencing further comprises producing a plurality of sequencing reads; grouping sequencing reads based on co-occurrence of barcode sequences; and within each group, aligning the reads that belong to the same strand of the sample polynucleotide based on the sequences of the barcode sequences.

13. The method of claim 12 , wherein each of the sequencing reads comprise at least a portion of two or more barcode sequences, or complements thereof.

14. The method of claim 12 , further comprising computationally reconstructing sequences of a plurality of individual strands of the sample polynucleotide by removing interposing oligonucleotide barcode probe-derived sequences and joining sequences for adjacent portions of the sample polynucleotide.

15. The method of claim 12 , wherein the sequencing further comprises forming a consensus sequence for reads having the same barcode sequence, or a portion thereof.

16. The method of claim 15 , wherein the consensus sequence is obtained by comparing all sequencing reads aligning at a given nucleotide position, and identifying the nucleotide at that position as the one shared by a majority of the aligned reads.

17. The method of claim 1 , wherein the complement of the integrated strand is greater than 500 bases in length.

18. A method of amplifying a nucleic acid molecule, said method comprising
binding a first hybridization sequence of a first oligonucleotide probe to the nucleic acid molecule, wherein the first oligonucleotide probe comprises a first barcode sequence;
binding a second oligonucleotide probe to the nucleic acid molecule, wherein the second oligonucleotide probe comprises a first target hybridization sequence, a second barcode sequence, and a second target hybridization sequence;
binding a second hybridization sequence of a third oligonucleotide probe to the nucleic acid molecule, wherein the third oligonucleotide probe comprises a third barcode sequence;
extending the first hybridization sequence and ligating the extended first hybridization sequence to the first target hybridization sequence, and extending the second target hybridization sequence and ligating the extended second target hybridization sequence to the second hybridization sequence to form an integrated strand comprising the first, second and third barcode sequences; and
amplifying the integrated strand.

Assignees

• Singular Genomics Systems Inc

Inventors

• Glezer Eli N
• Fabani Martin Maria
• Shultzaberger Ryan
• Sridhar Bharat
• Stengel Gudrun
• Wei Christopher Jen-Yue

Classifications

• C12Q1/6869

  Methods for sequencing · CPC title

• C12Q2525/301

  Hairpin oligonucleotides · CPC title

• C12Q2563/179

  the label being a nucleic acid · CPC title

• C12Q2525/161

  incorporating target specific and non-target specific sites · CPC title

• C12Q2535/122

  Massive parallel sequencing · CPC title