METHODS FOR TARGETED NUCLEIC ACID SEQUENCE ENRICHMENT WITH APPLICATIONS TO ERROR CORRECTED NUCLEIC ACID SEQUENCING

• US 20200131561A1
• Filed: 03/23/2018
• Published: 04/30/2020
• Est. Priority Date: 03/23/2017
• Status: Active Grant

• Current Assignee: University of Washington
• Original Assignee: University of Washington
• Inventors: Kennedy, Scott R., Salk, Jesse J., Hipp, Michael, Schmidt, Elizabeth, Risques, Rosa Ana, Nachmanson, Daniela
• Granted Patent: US 11,479,807 B2
• Time in Patent Office: Days
• 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 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2535/119 Double strand sequencing
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid

Abstract

The present technology relates generally to methods and compositions for targeted nucleic acid sequence enrichment, as well as uses of such enrichment for error-corrected nucleic acid sequencing applications. In some embodiments, highly accurate, error corrected and massively parallel sequencing of nucleic acid material is possible using a combination of uniquely labeled strands in a double-stranded nucleic acid complex in such a way that each strand can be informatically related to its complementary strand, but also distinguished from it following sequencing of each strand or an amplified product derived therefrom. In various embodiments, this information can be used for the purpose of error correction of the determined sequence.

Claims

1. A method comprising:

providing double-stranded nucleic acid material comprising one or more double-stranded nucleic acid molecules, wherein each double-stranded nucleic acid molecule comprises a single molecule identifier sequence on each strand and an adapter on at least one of the 5′ and

/or 3′

ends of the nucleic acid molecule, and wherein, for each nucleic acid molecule, a first adapter sequence is associated with a first strand and a second adapter sequence is associated with a second strand of the nucleic acid molecule;

amplifying the nucleic acid material;

separating the amplified nucleic acid material into a first sample and a second sample;

amplifying the first strand in the first sample through use of a primer specific to the first adapter sequence to provide a first nucleic acid product;

amplifying the second strand in the second sample through use of a primer specific to the second adapter sequence to provide a second nucleic acid product;

sequencing each of the first nucleic acid product and second nucleic acid product; and

comparing the sequence of the first nucleic acid product to the sequence of the second nucleic acid product.

2. The method of claim 1, wherein the nucleic acid material is or comprises at least one of double-stranded DNA and double-stranded RNA.

3. The method of claim 1, wherein the providing step comprises

ligating a double-stranded nucleic acid material to at least one degenerate or semi-degenerate barcode sequence to form a double-stranded nucleic acid molecule barcode complex, wherein the barcode sequence comprises the single molecule identifier sequence.

4. The method of claim 1, wherein the single molecule identifier sequence is at least of one of a degenerate or semi-degenerate barcode sequence, one or more nucleic acid fragment ends of the nucleic acid material, or a combination thereof that uniquely labels the double-stranded nucleic acid molecule.

5. The method of claim 1, wherein the single molecule identifier sequence comprises an endogenous shear point or an endogenous sequence that can be positionally related to the shear point.

6. The method of claim 1, wherein amplifying the nucleic acid material includes generating a plurality of amplicons derived from the first strand and a plurality of amplicons derived from the second strand.

7. The method of claim 1, wherein amplifying the nucleic acid material in the first sample comprises

amplifying nucleic acid material derived from a single nucleic acid strand from an original double-stranded nucleic acid molecule using at least one single-stranded oligonucleotide at least partially complementary to a sequence present in the first adapter sequence and at least one single-stranded oligonucleotide at least partially complementary to a target sequence of interest such that the single molecule identifier sequence is at least partially maintained.

8. The method of claim 1, wherein the amplifying the nucleic acid material in the second sample comprises

amplifying nucleic acid material derived from a single nucleic acid strand from an original double-stranded nucleic acid molecule using at least one single-stranded oligonucleotide at least partially complementary to a sequence present in the second adapter sequence and at least one single-stranded oligonucleotide at least partially complementary to a target sequence of interest such that the single molecule identifier sequence is at least partially maintained.

9. The method of claim 1, wherein at least some of the nucleic acid material is damaged.

10. The method of claim 9, wherein the damage is or comprises at least one of oxidation, alkylation, deamination, methylation, hydrolysis, hydroxylation, nicking, intra-strand crosslinks, inter-strand cross links, blunt end strand breakage, staggered end double strand breakage, phosphorylation, dephosphorylation, sumoylation, glycosylation, deglycosylation, putrescinylation, carboxylation, halogenation, formylation, single-stranded gaps, damage from heat, damage from desiccation, damage from UV exposure, damage from gamma radiation damage from X-radiation, damage from ionizing radiation, damage from non-ionizing radiation, damage from heavy particle radiation, damage from nuclear decay, damage from beta-radiation, damage from alpha radiation, damage from neutron radiation, damage from proton radiation, damage from cosmic radiation, damage from high pH, damage from low pH, damage from reactive oxidative species, damage from free radicals, damage from peroxide, damage from hypochlorite, damage from tissue fixation such formalin or formaldehyde, damage from reactive iron, damage from low ionic conditions, damage from high ionic conditions, damage from unbuffered conditions, damage from nucleases, damage from environmental exposure, damage from fire, damage from mechanical stress, damage from enzymatic degradation, damage from microorganisms, damage from preparative mechanical shearing, damage from preparative enzymatic fragmentation, damage having naturally occurred in vivo, damage having occurred during nucleic acid extraction, damage having occurred during sequencing library preparation, damage having been introduced by a polymerase, damage having been introduced during nucleic acid repair, damage having occurred during nucleic acid end-tailing, damage having occurred during nucleic acid ligation, damage having occurred during sequencing, damage having occurred from mechanical handling of DNA, damage having occurred during passage through a nanopore, damage having occurred as part of aging in an organism, damage having occurred as a result if chemical exposure of an individual, damage having occurred by a mutagen, damage having occurred by a carcinogen, damage having occurred by a clastogen, damage having occurred from in vivo inflammation damage due to oxygen exposure, damage due to one or more strand breaks, and any combination thereof.

11. The method of claim 1, wherein the nucleic acid material is provided from a sample comprising one or more double stranded nucleic acid molecules originating from a subject or an organism.

12. The method of claim 11, wherein the sample is or comprises a body tissue, a biopsy, a skin sample, blood, serum, plasma, sweat, saliva, cerebrospinal fluid, mucus, uterine lavage fluid, a vaginal swab, a pap smear, a nasal swab, an oral swab, a tissue scraping, hair, a finger print, urine, stool, vitreous humor, peritoneal wash, sputum, bronchial lavage, oral lavage, pleural lavage, gastric lavage, gastric juice, bile, pancreatic duct lavage, bile duct lavage, common bile duct lavage, gall bladder fluid, synovial fluid, an infected wound, a non-infected wound, an archaeological sample, a forensic sample, a water sample, a tissue sample, a food sample, a bioreactor sample, a plant sample, a bacterial sample, a protozoan sample, a fungal sample, an animal sample, a viral sample, a multi-organism sample, a fingernail scraping, semen, prostatic fluid, vaginal fluid, a vaginal swab, a fallopian tube lavage, a cell free nucleic acid, a nucleic acid within a cell, a metagenomics sample, a lavage or a swab of an implanted foreign body, a nasal lavage, intestinal fluid, epithelial brushing, epithelial lavage, tissue biopsy, an autopsy sample, a necropsy sample, an organ sample, a human identification sample, a non-human identification sample, an artificially produced nucleic acid sample, a synthetic gene sample, a banked or stored sample, tumor tissue, a fetal sample, an organ transplant sample, a microbial culture sample, a nuclear DNA sample, a mitochondrial DNA sample, a chloroplast DNA sample, an apicoplast DNA sample, an organelle sample, and any combination thereof.

13. The method of claim 1, wherein the nucleic acid material comprises nucleic acid molecules of a substantially or near uniform length.

18. The method of claim 1, wherein before the providing step, the method comprises:

cutting the nucleic acid material with one or more targeted endonucleases such that a target nucleic acid fragment of a substantially known length is formed; and

isolating the target nucleic acid fragment based on the substantially known length.

19. The method of claim 18, wherein the one or more targeted endonucleases is selected from the group consisting of a ribonucleoprotein, a Cas enzyme, a Cas9-like enzyme, a meganuclease, a transcription activator-like effector-based nuclease (TALEN), a zinc-finger nuclease, an argonaute nuclease or a combination thereof.

20. The method of claim 18, wherein the one or more targeted endonucleases comprises Cas9 or CPF1 or a derivative thereof.

32. The method of claim 1, wherein sequencing each of the first nucleic acid product and second nucleic acid product comprises

comparing the sequence of a plurality of strands in the first nucleic acid product to determine a first strand consensus sequence;

and

comparing the sequence of a plurality of strands in the second nucleic acid product to determine a second strand consensus sequence.

33. The method of claim 32, wherein comparing the sequence of the first nucleic acid product to the sequence of the second nucleic acid product comprises comparing the first strand consensus sequence and the second strand consensus sequence to provide an error-corrected consensus sequence.

34. The method of claim 1, wherein sequencing each of the first nucleic acid product and second nucleic acid product comprises

sequencing at least one of the first strand to determine a first strand sequence read;

sequencing at least one of the second strand to determine a second strand sequence read; and

comparing the first strand sequence read and the second strand sequence read to generate an error-corrected sequence read.

35. The method of claim 34, wherein the error-corrected sequence read comprises nucleotide bases that agree between the first strand sequence read and the second strand sequence read.

36. The method of claim 34, wherein a variation occurring at a particular position in the error-corrected sequence read is identified as a true variant.

37. The method of claim 34, wherein a variation that occurs at a particular position in only one of the first strand sequence read or the second strand sequence read is identified as a potential artifact.

14-17. -17. (canceled)

21-31. -31. (canceled)

38-55. -55. (canceled)