Methods and compositions for rapid nucleic acid library preparation

• US 10,450,562 B2
• Filed: 09/09/2015
• Issued: 10/22/2019
• Est. Priority Date: 09/09/2014
• Status: Active Grant

• Current Assignee: iGenomX International Genomics Corporation (Twist Bioscience Corporation)
• Original Assignee: iGenomX International Genomics Corporation (Twist Bioscience Corporation)
• Inventors: Brown, Keith
• Primary Examiner(s): Zhang, Kaijiang
• Application Number:
  US15/509,747
• Publication Number:
  US 20170247689A1
• Time in Patent Office: 1,504 Days
• Field of Search: None
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2537/101 Homogeneous assay format, e.g. one pot reaction
• CPC Class Codes: C12Q 2537/159 Reduction of complexity, e.g. amplification of subsets, removing duplicated genomic regions
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/20 Sequence assembly

Abstract

Rapid nucleic acid libraries, methods of generation, kits, and compositions relating to library synthesis, including reagents, intermediaries and final products are disclosed herein. The disclosure enables rapid synthesis of libraries that allow independent verification of sequence information and rapid identification of sequence information with template of origin.

Claims

1. A method of generating a data set representative of a population of non-identical, tagged nucleic acid molecule sequences each comprising a subset of sequence from a target nucleic acid sample, the method comprising:

a) obtaining a first nucleic acid molecule comprising a first molecular tag sequence and a first target sequence having a first length from said target nucleic acid sample,

wherein said first nucleic acid molecule is obtained through contacting a first primer comprising a first random oligonucleotide sequence to said target nucleic acid sample in the presence of a nucleic acid polymerase and a nucleotide triphosphate, wherein said nucleotide triphosphate is selected by said nucleic acid polymerase from a pool comprising deoxynucleotide triphosphates and dideoxynucleotide triphosphates;

b) annealing an oligonucleotide comprising a second molecular tag sequence to said first nucleic acid molecule;

c) extending said oligonucleotide to obtain a first double-stranded nucleic acid molecule comprising a first molecular tag sequence, a first target sequence having a first length, and a second molecular tag sequence;

d) obtaining a second double-stranded nucleic acid molecule comprising a third molecular tag sequence, a second target sequence having a second length, and a fourth molecular tag sequence;

e) sequencing said first double-stranded nucleic acid molecule and said second double-stranded nucleic acid molecule to generate a data set;

f) observing that;

said third molecular tag sequence is identical to said first molecular tag sequence;

said fourth molecular tag sequence is identical to said second molecular tag sequence;

said second target sequence is identical to said first target sequence; and

said second target sequence length is identical to said first target sequence length; and

g) removing a sequence of said second double-stranded nucleic acid molecule from said data set.

2. The method of claim 1, wherein said contacting said first primer comprises annealing said first primer to a nucleic acid of said target nucleic acid sample.

3. The method of claim 1, wherein said first nucleic acid molecule comprises a molecular ligand.

4. The method of claim 3, wherein said molecular ligand comprises biotin.

5. The method of claim 1, wherein obtaining said second double-stranded nucleic acid molecule in step d) comprises contacting a second primer comprising a second random oligonucleotide sequence to said first nucleic acid molecule obtained in step (a).

6. The method of claim 5, wherein each of said first random oligonucleotide sequence and said second random oligonucleotide sequence is selected to reflect GC content of said target nucleic acid sample.

7. The method of claim 5, wherein said first primer comprises a first universal sequence, wherein said second primer comprises a second universal sequence, and wherein substantially no covalent bonds of said target nucleic acid sample are disrupted in introducing said first universal sequence and said second universal sequence to molecules constituting a data set representative of a population of non-identical, tagged nucleic acid molecule sequences.

8. The method of claim 1, wherein said first random oligonucleotide sequence of said first primer consists of a number of nucleic acid bases selected from the list consisting of 6, 7, 8, 9, and 10 nucleotide bases.

9. The method of claim 8, wherein said first primer comprises an adapter sequence positioned 5′

to said first random oligonucleotide sequence.

10. The method of claim 1, wherein said pool comprises dideoxynucleotide triphosphates in an amount ranging from 0.01% to 5%.

11. The method of claim 1, wherein said pool comprises dideoxynucleotide triphosphates in an amount ranging from 0.05% and 1.0%.

12. The method of claim 1, wherein said nucleic acid polymerase has strand displacement activity.

13. The method of claim 1, wherein said pool comprises at least one of said dideoxynucleotide triphosphates bound to a molecular ligand.

14. The method of claim 13, wherein said molecular ligand comprises biotin.

15. The method of claim 14, comprising contacting said first double-stranded nucleic acid molecule to a ligand binding agent.

16. The method of claim 1, wherein said pool comprises a population of a natural dNTP pair and a population of a semi compatible dNTP pair.

17. The method of claim 1, wherein obtaining said second double-stranded nucleic acid molecule comprises contacting a second primer comprising a locus-specific oligonucleotide sequence and a second molecular tag sequence to said first nucleic acid molecule obtained in step (a).

18. The method of claim 1, wherein steps a)-d) are performed in a single tube.

19. The method of claim 1, wherein no covalent bonds of said target nucleic acid sample are disrupted.

20. A method of generating a data set representative of a population of non-identical, tagged nucleic acid molecule sequences each comprising a subset of sequence from a target nucleic acid sample, the method comprising:

a) obtaining a first nucleic acid molecule comprising a first molecular tag sequence and a first target sequence having a first length from said target nucleic acid sample,

wherein said first nucleic acid molecule is obtained through contacting a first primer comprising a first random oligonucleotide sequence to said target nucleic acid sample in the presence of a nucleic acid polymerase and a nucleotide triphosphate wherein said nucleotide triphosphate is selected by said nucleic acid polymerase from a pool comprising deoxynucleotide triphosphates and dideoxynucleotide triphosphates;

b) annealing an oligonucleotide comprising a second molecular tag sequence to said first nucleic acid molecule;

c) extending said oligonucleotide to obtain a first double-stranded nucleic acid molecule comprising a first molecular tag sequence, a first target sequence having a first length, and a second molecular tag sequence;

d) obtaining a second double-stranded nucleic acid molecule comprising a third molecular tag sequence, a second target sequence having a second length, and a fourth molecular tag sequence;

e) sequencing said first double-stranded nucleic acid molecule and said second double-stranded nucleic acid molecule to generate a data set; and

f) observing that at least one of;

said third molecular tag sequence is different from said first molecular tag sequence;

said fourth molecular tag sequence is different from said second molecular tag sequence;

said second target sequence is different from said first target sequence;

or

said second target sequence length is different from said first target sequence length; and

g) retaining a sequence of said second double-stranded nucleic acid molecule in said data set

thereby generating a population of non-identical, tagged nucleic acid molecules each comprising a subset of sequence from said target nucleic acid sample.