Enhancer oligonucleotides for nucleic acid hybridization

Abstract

To provide methods for enriching target nucleic acids and methods for sequencing nucleic acids.SOLUTION: The invention is a method of enriching target nucleic acids, the method comprising: contacting a mixture of target and non-target nucleic acids with a composition comprising two or more probe oligonucleotides, where each probe oligonucleotide comprises a target-binding region, a first and a second primer-binding region, and one or more enhancer oligonucleotides capable of hybridizing to at least one of the primer binding regions; incubating the mixture under hybridization conditions; and separating probe-bound target nucleic acids from unbound nucleic acids. The invention is also a method of sequencing nucleic acids comprising: contacting a mixture of target and non-target nucleic acids with a composition comprising two or more probe oligonucleotides where each probe oligonucleotide comprises a target-binding region, a first and a second primer-binding region, and one or more enhancer oligonucleotides hybridizing to at least one of the primer binding regions; incubating the mixture under hybridization conditions; capturing hybrids formed between the probes and the target nucleic acids to obtain enriched nucleic acids; and sequencing the enriched nucleic acids.SELECTED DRAWING: None

Description

[Background technology]

[0001] Background of the Invention Target enrichment (TE) techniques are widely used in genomic research, including human disease research and clinical applications. These techniques offer a focused and cost-effective solution compared to whole-genome analyses such as whole-genome sequencing. By focusing analysis on only the regions of interest in the genome, disease- or phenotype-associated genetic variants and other relevant genomic features can be identified, and cost-effective clinical diagnostic assays can be designed for such features.

[0002] Initially, target enrichment utilized single-stranded DNA (ssDNA) probes and probe pools to capture regions of interest in high-complexity samples, such as genomic samples. More recently, double-stranded DNA (dsDNA) probes have become popular in TE workflows. dsDNA probes are preferred for their ability to capture both the plus (+) and minus (-) strands of the target region, thereby improving data quality by minimizing DNA strand capture bias. Unfortunately, the double-stranded nature of these probes can cause self-annealing, cross-annealing, and other artifacts, resulting in poor assay performance and ultimately a loss of assay sensitivity.

[0003] Given the importance of target enrichment in bringing cost-effective genomic analysis to the clinic, there is a need to improve the performance of probes in target enrichment assays. Summary of the Invention

[0004] In one embodiment, the present invention provides a composition for nucleic acid hybridization comprising two or more probe oligonucleotides, each probe oligonucleotide comprising a target binding region and a first and second primer binding region, and one or more enhancer oligonucleotides capable of hybridizing to at least one of the primer binding regions. In some embodiments, the two or more probe oligonucleotides comprise multiple probe oligonucleotides capable of specifically hybridizing to multiple nucleic acid targets under hybridization conditions. In some embodiments, the hybridization conditions are stringent hybridization conditions. In some embodiments, the probe oligonucleotides are double-stranded. In some embodiments, the probe oligonucleotides are single-stranded. In some embodiments, all probe oligonucleotides have the same first and second primer binding regions. In some embodiments, the enhancer oligonucleotide comprises a mixture of oligonucleotides capable of hybridizing to the first and second primer binding regions. In some embodiments, the enhancer oligonucleotide comprises a mixture of oligonucleotides capable of hybridizing to each strand of the first and second primer binding regions. In some embodiments, the enhancer oligonucleotide comprises a mixture of four oligonucleotides, each capable of hybridizing to one of the Watson or Crick strands of the first or second primer binding region. In some embodiments, the enhancer oligonucleotide comprises a mixture of more than four oligonucleotides grouped into four groups, each group of oligonucleotides capable of hybridizing to one of the Watson or Crick strands of the first or second primer binding region.

[0005] In one embodiment, the present invention is a composition for enriching nucleic acid targets, comprising two or more probe oligonucleotides, each probe oligonucleotide comprising a target binding region, a first primer binding region, and a second primer binding region, and one or more enhancer oligonucleotides capable of hybridizing to at least one of the primer binding regions. In some embodiments, the two or more probe oligonucleotides comprise multiple probe oligonucleotides capable of specifically hybridizing to multiple nucleic acid targets present in a mixture with non-target nucleic acids under hybridization conditions. In some embodiments, the composition further comprises a mixture of target nucleic acids and non-target nucleic acids. In some embodiments,

[0006] In one embodiment, the present invention is a method for concentrating a target nucleic acid, the method comprising: contacting a mixture of a target nucleic acid and a non-target nucleic acid with a composition comprising two or more probe oligonucleotides, each probe oligonucleotide comprising a target binding region and a first and a second primer binding region, and one or more enhancer oligonucleotides hybridizing to at least one of the primer binding regions; incubating the mixture under hybridization conditions; and separating the probe-bound target nucleic acid from unbound nucleic acid. In some embodiments, each of the target nucleic acid, non-target nucleic acid, two or more probe oligonucleotides, and one or more enhancer oligonucleotides is single-stranded. In some embodiments, the method further comprises incubating the mixture under conditions that result in denaturation of the nucleic acid prior to hybridization.

[0007] In some embodiments, the mixture of target nucleic acids and non-target nucleic acids comprises the genomic DNA of the organism. In some embodiments, the mixture of target nucleic acids and non-target nucleic acids comprises a library formed from the genomic DNA of the organism. In some embodiments, the library comprises nucleic acids isolated from the organism, each nucleic acid being conjugated to at least one adaptor nucleic acid, for example, two adaptor nucleic acids. In some embodiments, the adaptor nucleic acid comprises a nucleic acid barcode and a universal primer binding site.

[0008] In some embodiments, the method further comprises removing any single-stranded nucleic acids from the mixture, for example, by capturing the hybridized nucleic acids via a capture moiety present on the probe oligonucleotide.

[0009] In one embodiment, the present invention is a method for sequencing nucleic acids, comprising contacting a mixture of target nucleic acids and non-target nucleic acids with a composition comprising two or more probe oligonucleotides, each probe oligonucleotide comprising a target binding region and a first and second primer binding region, and one or more enhancer oligonucleotides hybridizing to at least one of the primer binding regions; incubating the mixture under hybridization conditions; capturing hybrids formed between the probe and target nucleic acid to obtain enriched nucleic acids; and sequencing the enriched nucleic acids. In some embodiments, each of the target nucleic acid, non-target nucleic acid, two or more probe oligonucleotides, and one or more enhancer oligonucleotides is single-stranded. In some embodiments, denaturation is required prior to hybridization. In some embodiments, the method further comprises amplifying the enriched nucleic acids, for example, using a universal primer that binds to the universal primer binding site of the enriched nucleic acids. In some embodiments, the present invention is an enriched library of nucleic acids formed by the methods described herein.

[0010] In one embodiment, the present invention provides a reaction mixture comprising a plurality of nucleic acids, including target nucleic acids and non-target nucleic acids; two or more probe oligonucleotides, each probe oligonucleotide comprising a target binding region, a first primer binding region, and a second primer binding region; and one or more enhancer oligonucleotides capable of hybridizing to at least one of the primer binding regions. In some embodiments, the two or more probe oligonucleotides comprise a plurality of probe oligonucleotides capable of specifically hybridizing to a plurality of nucleic acid targets present in a mixture with non-target nucleic acids under hybridization conditions. In some embodiments, the plurality of nucleic acids, including target nucleic acids and non-target nucleic acids, constitutes a library formed from the genomic DNA of an organism, the library comprising nucleic acids isolated from the organism, each nucleic acid being conjugated to at least one adaptor nucleic acid.

[0011] In one embodiment, the invention is a method of assessing a disease or condition in a patient, the method comprising providing a nucleic acid-containing sample from the patient, enriching target nucleic acids in the sample by a method described herein, and detecting the disease or condition in the patient by determining the mutation status of one or more genetic loci in the enriched target nucleic acids that are known to be biomarkers for the disease or condition.

[0012] In one embodiment, the invention is a method of selecting a treatment for a disease or condition in a patient, the method comprising providing a nucleic acid-containing sample from a patient having the disease or condition, enriching target nucleic acids in the sample by a method described herein, determining the mutation status of one or more genetic loci in the enriched target nucleic acids that are known to be biomarkers for the disease or condition, and selecting an appropriate treatment for mutations detected in the enriched nucleic acids.

[0013] In one embodiment, the invention is a method of diagnosing or screening for the presence of a cancerous tumor in a patient, the method comprising providing a nucleic acid-containing sample from the patient, enriching a target nucleic acid in the sample by a method described herein, and detecting the presence of a cancerous tumor in the patient by determining the mutation status of one or more genetic loci in the enriched nucleic acid that are known to indicate the presence of a cancerous tumor.

[0014] In one embodiment, the invention is a method of selecting a treatment that targets a cancerous tumor in a patient based on the mutational status of the tumor, the method comprising: providing a nucleic acid-containing sample from the patient; enriching target nucleic acids in the sample by a method described herein; determining the mutational status of one or more genetic loci in the enriched nucleic acids that are known to be mutated in cancerous tumors; and selecting a treatment that targets the mutational status found.

[0015] In one embodiment, the invention is a method of monitoring tumor growth or shrinkage, the method comprising periodically sampling circulating cell-free DNA (cfDNA) from a patient, enriching one or more target sequences in the cfDNA by methods described herein, and detecting changes in the amount of mutated cfDNA comprising one or more mutations in the target sequences known to be mutated in cancerous tumors, wherein an increase in the level of such mutated cfDNA indicates tumor growth, while a decrease in the level of such mutated cfDNA indicates tumor shrinkage.

[0016] In one embodiment, the invention is a method of monitoring the effectiveness of a treatment for cancer in a patient, the method comprising periodically sampling circulating cell-free DNA (cfDNA) from the patient, enriching for one or more target sequences in the cfDNA by a method described herein, and detecting changes in the amount of cfDNA containing one or more mutations in the target sequences known to be mutated in cancerous tumors, wherein an increase in the level of such mutant cfDNA indicates tumor growth and ineffectiveness of the treatment, while a decrease in the level of such mutant cfDNA indicates tumor shrinkage and effective treatment, and a stable level of such mutant cfDNA indicates stable disease and effective treatment.

[0017] In one embodiment, the invention provides a method for diagnosing minimal residual disease (MRD) in a cancer patient, the method comprising obtaining circulating cell-free DNA (cfDNA) from the patient, enriching one or more target sequences in the cfDNA by a method described herein, and detecting the mutational status of one or more genetic loci known to be mutated in cancerous tumors in the enriched cfDNA, wherein the presence of mutated cfDNA indicates the presence of MRD in the patient.

[0018] In one embodiment, the present invention is a kit for improved hybridization of nucleic acids, comprising one or more probe oligonucleotides, each probe oligonucleotide comprising a target binding region and a first and second primer binding region, and one or more enhancer oligonucleotides capable of hybridizing to at least one of the probe oligonucleotides, the primer binding region. In some embodiments, the one or more probe oligonucleotides are double-stranded, and the kit comprises four enhancer oligonucleotides capable of hybridizing to four primer binding regions. In some embodiments, the kit comprises one or more of reagents for purifying and isolating nucleic acids, reagents for forming a library of nucleic acids, reagents for amplifying nucleic acids, and reagents for sequencing nucleic acids.

[0019] In one embodiment, the invention is a method for concentrating target nucleic acids, the method comprising: contacting a mixture of target nucleic acids and non-target nucleic acids with a composition comprising two or more probe oligonucleotides, each probe oligonucleotide comprising a target binding region and a first primer binding region and a second primer binding region, wherein the first primer binding region hybridizes to a capture oligonucleotide attached to a solid support, and one or more enhancer oligonucleotides that hybridize to the second primer binding region; incubating the mixture under hybridization conditions; and separating the probe-bound target nucleic acids from unbound nucleic acids by contacting the mixture with one or more enhancer oligonucleotides that hybridize to the first primer binding region under conditions suitable for dissociating the first primer binding region from the capture oligonucleotide. [Brief explanation of the drawings]

[0020] [Figure 1A] FIG. 1 shows the design and engineering of enhancer oligonucleotides. [Figure 1B] FIG. 1 shows the design and engineering of enhancer oligonucleotides. [Figure 1C] FIG. 1 shows the design and engineering of enhancer oligonucleotides.

[0021] [Figure 2A] 1 shows the results of sequencing performed on nucleic acids enriched by hybridization in the presence of enhancer oligonucleotides. [Figure 2B] 1 shows the results of sequencing performed on nucleic acids enriched by hybridization in the presence of enhancer oligonucleotides. DETAILED DESCRIPTION OF THE INVENTION

[0022] Detailed Description of the Invention definition

[0023] The following definitions will aid in the understanding of this disclosure: All technical terms not specifically defined in this section have their ordinary and accustomed meanings.

[0024] The term "probe" refers to a nucleic acid (either single-stranded or double-stranded) comprising an oligonucleotide capable of specifically binding to a target nucleic acid under stringent hybridization conditions.

[0025] The term "oligonucleotide" refers to a nucleic acid that is typically shorter than a naturally occurring nucleic acid. The terms oligonucleotide and nucleic acid can be used interchangeably. Unless otherwise specified, an oligonucleotide is single-stranded.

[0026] The term "enhancer oligonucleotide" refers to a type of oligonucleotide described and claimed herein that has the specific property of hybridizing to certain elements present in a hybridization probe, improving the performance of the hybridization probe.

[0027] The term "blocker oligonucleotide" refers to an oligonucleotide that is added to a hybridization reaction, for example, containing a nucleic acid library prepared for sequencing. Blocker oligonucleotides have the specific property of hybridizing to and blocking a specific element present in all library molecules. Some commercially available blocker oligonucleotides are sold under the name "universal enhancer oligonucleotides." For the avoidance of doubt, the term "enhancer oligonucleotide" as defined herein is different from "universal enhancer oligonucleotide." The term "universal enhancer oligonucleotide" is not used in this disclosure.

[0028] The term "primer binding region" includes a primer binding site, which is a sequence within a nucleic acid to which an amplification primer binds to initiate strand synthesis. In the context of the present disclosure, the term "primer binding region" also includes the reverse complement of the primer binding site. For example, a double-stranded nucleic acid resulting from amplification using a primer will contain four primer binding regions, one at each of the two ends of each of the two strands, where two primer binding regions are primer binding sites and the other two primer binding regions are reverse complements of the primer binding sites.

[0029] Target enrichment (TE) technology is widely utilized in genomic research, both in life sciences and human disease research, as well as in clinical applications. Target enrichment offers a focused and cost-effective solution compared to whole-genome sequencing for identifying disease- and phenotype-associated gene variants and genomic regions. Double-stranded DNA (dsDNA) probes have become a common probe type in TE workflows in recent years due to their ability to capture both the plus (+) and minus (-) strands of the enriched target region. dsDNA probes improve data quality by minimizing DNA strand capture bias. To control production costs, major dsDNA probe providers manufacture these probes in large quantities via polymerase chain reaction (PCR) amplification. To enable PCR, each dsDNA probe produced must contain a primer binding site (PBS) at its end. The PBS is typically identical for all probes synthesized by the manufacturer as part of a probe lot or pool. While reducing production costs, the production of these primer binding sites results in the formation of artifacts that impair probe performance. The reduced performance is due to the tendency of the plus (+) and minus (-) strands of the probe molecules to concatenate through their complementary PBSs (Figure 1A, top left) or to self-anneal or cross-anneal (Figure 1A, bottom left). These artifacts adversely affect hybridization efficiency, thus resulting in suboptimal target enrichment and reduced quality of downstream analyses, such as nucleic acid sequencing.

[0030] Hybridization blockers are known in the art. However, hybridization blocker oligonucleotides are conventionally used to block adapter sequences in nucleic acid libraries (see US20200102611). During target enrichment hybridization, such blocker oligonucleotides bind to library molecules and do not bind to hybridization probes. Existing blocker oligonucleotides prevent adapter-adapter hybridization of library molecules and do not address any of the problems or artifacts associated with hybridization probes. For example, existing blockers do not address the problems of concatenation, cross-annealing or self-annealing of hybridization probes.

[0031] The present disclosure provides solutions to problems associated with hybridization probes, such as target enrichment hybridization probes. The present invention includes probe enhancer oligonucleotides (dPEOs) that improve capture efficiency and target enrichment performance. The enhancer oligonucleotides are designed to bind to common sequences shared among a pool of hybridization probes. In some embodiments, the enhancer oligonucleotides are designed to bind to primer binding sites present in dsDNA probes. PCR is commonly used in the production of hybridization probes. In such cases, each probe contains forward and reverse universal primer binding sites. The enhancer oligonucleotides of the present invention are designed to bind to these universal sites and prevent undesired interactions between probes in the hybridization mixture. As a result, the enhancer oligonucleotides minimize probe concatenation (shown in Figure 1B, upper left) and reduce the spreading of re-annealed or cross-annealed double-stranded probes (Figure 1B, lower left), thereby increasing the number of effective probe molecules in the hybridization reaction. As shown in Figures 2A and 2B, which summarize the sequencing data generated using different doses of enhancer oligonucleotides, the use of enhancer oligonucleotides dose-dependently improves capture uniformity (Figure 2A) and reduces read overlap levels (Figure 2B).

[0032] Various aspects of the invention are described in further detail below.

[0033] The present invention includes methods for manipulating nucleic acids from a sample. In some embodiments, the sample is derived from a subject or patient. In some embodiments, the sample may include solid tissue or a fragment of a solid tumor derived from a subject or patient, for example, by biopsy. The sample may also include bodily fluids that may contain nucleic acids (e.g., urine, sputum, serum, blood or blood fractions, i.e., plasma, lymph, saliva, sputum, sweat, tears, cerebrospinal fluid, amniotic fluid, synovial fluid, pericardial fluid, ascites, pleural fluid, cyst fluid, bile, gastric juice, intestinal fluid, or fecal samples). In some embodiments, the sample is a plasma or urine sample containing cell-free DNA (cfDNA), including circulating tumor DNA (ctDNA). In other embodiments, the sample is a cultured sample, e.g., a tissue culture containing cells and fluids from which nucleic acids can be isolated. In some embodiments, the nucleic acid of interest in the sample is derived from an infectious pathogen, such as a virus, bacteria, protozoan, or fungus.

[0034] The present invention involves manipulating isolated nucleic acids isolated or extracted from a sample. Methods for nucleic acid extraction are well known in the art. See J. Sambrook et al., "Molecular Cloning: A Laboratory Manual," 1989, 2nd ed., Cold Spring Harbor Laboratory Press: New York, NY). Various kits for extracting nucleic acids (DNA or RNA) from biological samples are commercially available, such as KAPA Express Extract (Roche Sequencing Solutions, Pleasanton, Calif.), and other similar products from BD Biosciences Clontech (Palo Alto, Calif.), Epicentre Technologies (Madison, Wis.); Gentra Systems (Minneapolis, Minn.); and Qiagen (Valencia, Calif.), Ambion (Austin, Tex.); BioRad Laboratories (Hercules, Calif.).

[0035] In some embodiments, nucleic acids are extracted, separated by size, and optionally concentrated by epitachophoresis, for example, as described in publications WO2019092269 and WO2020074742.

[0036] Target enrichment is a method of capturing one or more target nucleic acids or separating one or more target nucleic acids from any non-target nucleic acids in a sample or reaction mixture. In some embodiments, target enrichment is a method of increasing the concentration of one or more target nucleic acids relative to the concentration of any non-target nucleic acids present in the sample or reaction mixture.

[0037] A target nucleic acid is a nucleic acid of interest that may be present in a sample. Each target is characterized by its nucleic acid sequence. In some embodiments, the target nucleic acid is a gene or gene fragment (including exons and introns). In some embodiments, the target is a gene, gene fragment, or intergenic region involved in a fusion event, e.g., a region where a fusion breakpoint is located. In some embodiments, the target is present as RNA and is a gene transcript or a portion thereof. In some embodiments, the target nucleic acid comprises a biomarker, i.e., a gene whose variant, such as a single nucleotide variation (SNV), copy number variation (CNV), or gene fusion, is associated with a disease or condition. For example, the target nucleic acid can be selected from a panel of disease-associated markers described in U.S. Patent Application No. 14 / 774,518, filed September 10, 2015. Such a panel is available as the AVENIO ctDNA Analysis Kit (Roche Sequencing Solutions, Pleasanton, Calif.). In some embodiments, the target nucleic acid is one or more of the genes listed in Table 1 or Table 2.

[0038] [Table 1]

[0039] [Table 2]

[0040] In some embodiments, the target nucleic acid is one or more genes involved in clinically relevant gene fusions. In some embodiments, the target nucleic acid is one or more genes known to undergo fusions in tumors. In some embodiments, the target nucleic acid is one or more fusion sites associated with the following genes: ALK, RET, ROS, FGFR2, FGFR3, NTRK1, ALK, PPARG, BRAF, EGFR, FGFR1, FGFR2, FGFR3, MET, NRG1, NTRK1, NTRK2, NTRK3, RET, ROS1, AXL, PDGFRA, PDGFB, ABL1, ABL2, AKT1, AKT2, AKT3, ARHGAP26, BRD3 , BRD4, CRLF2, CSF1R, EPOR, ERBB2, ERBB4, ERG, ESR1, ESRRA, ETV1, ETV4, ETV5, ETV6, EWSR1, FGR, IL2RB, INSR, JAK1, JAK2, JA K3, KIT, MAML2, MAST1, MAST2, MSMB, MUSK, MYB, MYC, NOTCH1, NOTCH2, NUMBL, NUT, PDGFRB, PIK3CA, PKN1, PRKCA, PRKCB, PTK2B, RAF1, RARA, RELA, RSPO2, RSPO3, SYK, TERT, TFE3, TFEB, THADA, TMPRSS2, TSLP, TY, BCL2, BCL6, BCR, CAMTA1, CBFB, CCNB3, CCN D1, CIC, CRFL2, DUSP22, EPC1, FOXO1, FUS, GLI1, GLIS2, HMGA2, JAZF1, KMT2A, MALT1, MEAF6, MECOM, MKL1, MKL2, MTB, NCOA2, NU P214, NUP98, PAX5, PDGFB, PICALM, PLAG1, RBM15, RUNX1, RUNX1T1, SS18, STAT6, TAF15, TAL1, TCF12, TCF3, TFG, TYK2, USP6, Y WHAE, AR, BRCA1, BRCA2, CDKN2A, ERB84, FLT3, KRAS, MDM4, MYBL1, NF1, NOTCH4, NUTM1, PRKACA, PRKACB, PTEN, RAD51B, and RB1.

[0041] In some embodiments, the target nucleic acid is one or more genes or genomic regions involved in epigenetic modifications such as DNA methylation. In some embodiments, the target nucleic acid is one or more genes involved in genome maintenance or mismatch repair. In some embodiments, the target nucleic acid comprises a microsatellite locus that exhibits microsatellite instability (MSI). In some embodiments, the target nucleic acid comprises one or more genes involved in mismatch repair that are known to confer the microsatellite instability (MSI) phenotype when mutated.

[0042] In some embodiments, the target nucleic acid is RNA (including mRNA). In some embodiments, the target nucleic acid is cDNA derived from RNA, for example, via reverse transcription. In some embodiments, the target nucleic acid is DNA, including cellular DNA, or cell-free DNA (cfDNA), including circulating tumor DNA (ctDNA) and cell-free fetal DNA. The target nucleic acid can exist in short or long forms. In some embodiments, longer target nucleic acids are fragmented by enzymatic or physical treatment, as described below. In some embodiments, the target nucleic acid is naturally fragmented DNA, including, for example, circulating cell-free DNA (cfDNA), or chemically degraded DNA, such as that found in chemically preserved or old samples.

[0043] The present invention involves the use of hybridization probes that target nucleic acids of interest (target nucleic acids) in a sample. Hybridization probes can be either single-stranded or double-stranded. In some embodiments, the probes are pools of more than one probe, for example, up to 10, or 10-100 probes, or 100-500 probes, or 500-1,000 probes, or 1,000-10,000 probes. In some embodiments, there is one probe for each target locus, i.e., gene or region of interest. In other embodiments, there are multiple probes, for example, 2-10 probes, or 10-100 probes, or 100-500 probes, covering the same gene or region of interest. Many organism-specific hybridization probes and probe pools are available, including custom probes and probe pools. Hybridization probes are typically manufactured by a workflow that includes amplification, for example, by PCR or non-exponential amplification methods. For this reason, the probes contain an amplification primer binding site, such as a universal primer binding site.

[0044] The present invention involves the use of enhancer oligonucleotides specific to the amplification primer binding site, e.g., the universal primer binding site, of a probe. These enhancer oligonucleotides are different from the "universal enhancer oligonucleotides" currently available (e.g., as part of the KAPA HyperCap workflow). Existing universal enhancer oligonucleotides bind to adapter sequences of library molecules. In contrast, the enhancer oligonucleotides of the present invention are designed to bind to the primer binding site of a hybridization probe. (Figure 1B) In some embodiments, four enhancer oligonucleotides are added, each complementary to the forward and reverse primer binding sites of a double-stranded probe oligonucleotide and reverse-complementary to the forward and reverse primer binding sites, as shown in Figure 1B. In other embodiments, fewer than four enhancer oligonucleotides are added, for example, when the probe is single-stranded.

[0045] In some embodiments, enhancer oligonucleotide has the same length as the primer binding site.In other embodiments, enhancer oligonucleotide is shorter or longer than the primer binding site.Those skilled in the art can determine the optimal length of enhancer oligonucleotide so that under given hybridization conditions (for example, the conditions used for target enrichment), enhancer oligonucleotide forms a stable hybrid with the primer binding site of hybridization probe, thereby achieving the desired hybridization enhancement described herein.

[0046] Those skilled in the art can further calculate the desired ratio between enhancer oligonucleotides and hybridization probes, taking into account the fact that between one and four enhancer oligonucleotides are required to bind to each double-stranded hybridization probe, depending on the number of enhancer oligonucleotides used. In some embodiments, the molar ratio of probe to enhancer oligonucleotide is 1:4. In other embodiments, a molar excess of enhancer oligonucleotides is added so that the molar ratio of probe to enhancer oligonucleotide is 1:6, 1:8, 1:10, or greater. In some embodiments, the final concentration of enhancer oligonucleotides is about 0.2 mM, 0.02 mM, 0.002 mM, or 0.0002 mM. In principle, it can be beneficial to have a molar excess of enhancer oligonucleotides relative to the probes.

[0047] Under the hybridization conditions used in the target enrichment process, the temperature (T m It may be beneficial to optimize the design of the enhancer oligonucleotide to have a desired melting point according to the predicted T mis determined experimentally, or using either manual calculations or in silico tools available for this purpose. In some embodiments, the desired T of the enhancer oligonucleotide m is higher than the incubation temperature used in the hybridization conditions used in target enrichment. In some embodiments, the desired T m is the T of a hypothetical probe-probe hybrid m or higher than the T of the double-stranded probe m Higher than that. Such a high T m To achieve this, in some embodiments, the enhancer oligonucleotide comprises one or more modified nucleotides or nucleotide variants selected from, for example, 5-methylcytosine, 2,6-diaminopurine, 5-hydroxybutynyl-2'-deoxyuridine, 8-aza-7-deazaguanosine, ribonucleotides, 2'O-methylribonucleotides, or locked nucleic acids.

[0048] The length of the enhancer oligonucleotide also affects the melting temperature. The primer binding site is most often about 10-20 nucleotides long, but can be between about 10 and about 40 nucleotides long. The length of the enhancer oligonucleotide does not have to exactly match the length of the primer binding site it blocks. For example, the enhancer oligonucleotide can be one or more nucleotides shorter than the primer binding site it blocks on one or both sides of the enhancer oligonucleotide.

[0049] Also, the enhancer oligonucleotide need not be perfectly complementary to the blocked primer binding site. In some embodiments, the enhancer oligonucleotide is less than 100% complementary to the blocked primer binding site, such as greater than 90%, 80-90%, or 70-80% complementary.

[0050] In some embodiments, the nucleic acids in the sample are present in the form of a library. In some embodiments, the library is formed from the genomic DNA of an organism. In some embodiments, the library is a genomic library. The library consists of multiple nucleic acids that have been modified to enable downstream applications such as sequencing, amplification, or another type of detection method. The library is formed from multiple nucleic acids in the sample, for example, by adding one or more common elements to the multiple nucleic acids in the sample.

[0051] In some embodiments, libraries are formed by adding a common adapter molecule to one or both ends of nucleic acids in a sample. Adapters of various shapes and functions are known in the art (see, for example, PCT / EP2019 / 05515, filed February 28, 2019, US8822150, and US8455193). In some embodiments, the adapter comprises certain elements, such as a nucleic acid barcode, a primer binding site, and a ligatable site. The adapter comprises at least one element selected from a barcode, a primer binding site, and a ligatable site. The adapter can be double-stranded, partially single-stranded, or single-stranded. In some embodiments, a Y-shaped, hairpin, or stem-loop adapter is used, and the double-stranded portion of the adapter is ligated to the formed double-stranded nucleic acid as described herein. In some embodiments, the adapter is an artificial sequence synthesized in vitro. In other embodiments, the adapter is a naturally occurring sequence synthesized in vitro. In yet other embodiments, the adaptor is an isolated naturally occurring molecule or an isolated non-naturally occurring molecule.

[0052] In some embodiments, adapters are added by extending adapter sequence-containing primers annealed to multiple nucleic acids in a sample. Such primers are referred to as "tailed primers." A tailed primer comprises a 3' portion that hybridizes to the target and a non-hybridizing 5' tail that includes the adapter sequence. In some embodiments, the target hybridizing sequence is specific to one nucleic acid in the library, e.g., gene-specific. In some embodiments, the target hybridizing sequence is specific to one type of nucleic acid, e.g., a poly-T sequence. In some embodiments, the target hybridizing sequence is random, e.g., a random hexamer nucleotide sequence. Upon extension of the tailed primers hybridized to the nucleic acids in the sample, the nucleic acids form a library of adapted nucleic acids.

[0053] In some embodiments, adapters are added to each end of a plurality of nucleic acids in a sample by ligation. In some embodiments, the adapters are double-stranded or partially double-stranded adapter oligonucleotides with overhangs or blunt ends. In some embodiments, the double-stranded DNA can contain blunt ends to which blunt-ended adapters can be ligated using blunt-end ligation. In other embodiments, the blunt-ended DNA is A-tailed, in which a single A nucleotide is added to the 3' end of the blunt end. The corresponding adapter is designed to have a single T nucleotide extending from the 3' end of the blunt end to facilitate ligation between the DNA and the adapter. Commercially available kits for adapter ligation include the AVENIO ctDNA Library Prep Kit or the KAPA HyperPrep and HyperPlus Kit (Roche Sequencing Solutions, Pleasanton, CA). In some embodiments, the adapter-ligated (matched) library nucleic acids can be separated from excess adapters and unligated nucleic acids in the sample.

[0054] In some embodiments, the adapters present in the library nucleic acids are used in nucleic acid sequencing. Analyzing individual molecules by massively parallel sequencing typically requires different levels of barcoding for sample identification and error correction. The use of molecular barcodes, as described in U.S. Patent Nos. 7,393,665, 8,168,385, 8,481,292, 8,685,678, and 8,722,368, is used. A unique molecular barcode is added to each molecule to be sequenced to mark the molecule and its progeny (e.g., the original molecule and its amplicon generated by PCR). Unique molecular identifier barcodes (UIDs) (also known as unique molecular identifiers (UMIs)) have multiple uses, including counting the number of original target molecules in a sample and error correction (Newman, A. et al., (2014) An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage, Nature Medicine doi:10.1038 / nm.3519).

[0055] In some embodiments, unique molecular barcodes (UIDs) are used for error correction in sequencing. All descendants of a single target molecule are marked with the same barcode, forming a barcoded family. Sequence variations that are not shared by all members of the barcoded family are discarded as artifacts. Because the entire family represents a single molecule in the original sample, barcodes can also be used for positional deduplication and target quantification (Newman, A. et al., (2016) Integrated digital error suppression for improved detection of circulating tumor DNA, Nature Biotechnology 34:547).

[0056] In some embodiments of the present invention, the adapters ligated to one or both ends of the barcoded target nucleic acid contain one or more barcodes used in sequencing. The barcodes can be UIDs or multiplexed sample IDs (MIDs or SIDs) used to identify the source of the sample when samples are mixed (multiplexed). The barcodes can also be a combination of UIDs and MIDs. In some embodiments, a single barcode is used as both a UID and MID. In some embodiments, each barcode contains a predefined sequence. In other embodiments, the barcodes contain random sequences. In some embodiments of the present invention, the barcodes are between about 4 and 20 bases long, resulting in 96 to 384 different adapters (each with a different pair of identical barcodes) being added to a human genomic sample. In some embodiments, the number of UIDs in a reaction can exceed the number of molecules being labeled. One of skill in the art will recognize that the number of barcodes depends on the complexity of the sample (i.e., the expected number of unique target molecules) and can generate a suitable number of barcodes for each experiment.

[0057] In some embodiments, the present invention provides an improved method for concentrating one or more target nucleic acids present in a sample or reaction mixture that also contains non-target nucleic acids. The method involves contacting the sample or reaction mixture with one or more probes that specifically hybridize to the target nucleic acids. More specifically, the present invention involves the use of improved probe mixtures. The improved probe mixtures include two or more probe oligonucleotides, e.g., a plurality of probe oligonucleotides. In some embodiments, the plurality of probes includes 2, 3, 4, 5, 6, 7, 8, 9, less than 10, or between 100 probes, or between 100 and 500 probes, or between 500 and 1,000 probes, or between 1,000 and 10,000 probes. One or more probes in the probe mixture include an amplification primer binding region. The improved probe mixtures further include a hybridization enhancer oligonucleotide capable of hybridizing to the primer binding region of the probe. In some embodiments, the probe mixture includes one or more enhancer oligonucleotides that hybridize to at least one of the primer binding regions. In some embodiments, the probe mixture comprises an enhancer oligonucleotide that can hybridize to the first primer binding region and the second primer binding region of the probe. In some embodiments, the molar ratio of the probe to the enhancer oligonucleotide in the probe mixture is optimized to achieve blocking without cross-reaction of the probe with additional hybridization sites, such as partially complementary sites. In some embodiments, the molar ratio of the probe oligonucleotide to the enhancer oligonucleotide is 1:2, 1:4, 1:8, or more.

[0058] The method further includes incubating a reaction mixture containing the target nucleic acid, the non-target nucleic acid, the probe, and the enhancer oligonucleotide under hybridization conditions, and separating the target nucleic acid hybridized to the probe from the non-hybridized nucleic acid.

[0059] In some embodiments, the nucleic acid in the mixture comprising target nucleic acid, non-target nucleic acid, two or more probe oligonucleotides, and one or more enhancer oligonucleotides is single-stranded.In some embodiments, at least one of the nucleic acids in the mixture comprising target nucleic acid, non-target nucleic acid, two or more probe oligonucleotides, and one or more enhancer oligonucleotides is double-stranded, and the method comprises a preliminary step of incubating sample or reaction mixture under conditions that cause nucleic acid denaturation.Nucleic acid denaturation can be carried out by high temperature, alkali, or a combination thereof.

[0060] In some embodiments, the target enrichment procedures described herein are performed on the genomic DNA of an organism. In some embodiments, the genomic DNA of the organism is converted into a genomic library before the target enrichment procedures described herein. In some embodiments, the genomic DNA or genomic DNA library is depleted of repetitive sequences before the target enrichment procedures described herein.

[0061] In some embodiments, removal of repetitive sequences from genomic DNA or genomic DNA libraries is achieved by the target enrichment methods described herein, i.e., hybridization procedures utilizing the improved probe mixtures described herein are applied to probes capable of hybridizing to repetitive sequences in the genome of an organism.

[0062] In some embodiments, the method further comprises removing any unhybridized or single-stranded nucleic acids from the reaction mixture after hybridization. In some embodiments, the unhybridized or single-stranded nucleic acids are removed by capture. In some embodiments, the hybridization probe comprises a capture moiety (e.g., biotin) that allows for the capture of the sample nucleic acid hybridized to the probe.

[0063] In some embodiments, the present invention provides an economical method for sequencing nucleic acids, comprising: contacting a mixture of target nucleic acids and non-target nucleic acids with a composition comprising two or more probe oligonucleotides, each probe oligonucleotide comprising a target binding region, a first primer binding region, and a second primer binding region; and one or more enhancer oligonucleotides hybridizing to at least one of the primer binding regions; incubating the mixture under hybridization conditions; capturing the hybridized target nucleic acids; and sequencing only the captured nucleic acids. In some embodiments, the economical sequencing method is applied to the genomic DNA of an organism. In some embodiments, the genomic DNA of the organism is converted into a genomic library before the sequencing procedure.

[0064] In some embodiments, the method further comprises amplifying the enriched nucleic acids prior to sequencing, hi some embodiments, the amplification utilizes universal primer binding sites present in the adapters of the library nucleic acids prior to sequencing.

[0065] In some embodiments, the present invention includes a step of amplifying nucleic acids. In some embodiments, amplification occurs before the sequencing step. In some embodiments, amplification occurs before the target enrichment step. In some embodiments, amplification occurs after the target enrichment step but before the sequencing step. Amplification utilizes an upstream primer and a downstream primer. In some embodiments, both primers are target-specific primers, i.e., primers that contain a sequence complementary to the target sequence of a methylation biomarker. In other embodiments, one or both primers are universal primers. In some embodiments, the universal primer binding site is present in an adapter ligated to the target to be sequenced, as described herein. In some embodiments, the universal primer binding site is present in the 5' region (tail) of the target-specific primer. Thus, after one or more rounds of primer extension with the tailed target-specific primer, the universal primer can be used for subsequent rounds of amplification. In some embodiments, the universal primer is paired with another universal primer (of the same or different sequence). In other embodiments, the universal primer is paired with a target-specific primer.

[0066] In some embodiments, the nucleic acids enriched by the methods described herein are sequenced. Any of several sequencing techniques or sequencing assays can be utilized. As used herein, the term "next-generation sequencing (NGS)" refers to a sequencing method that allows for massively parallel sequencing of clonally amplified molecules and single nucleic acid molecules.

[0067] Non-limiting examples of sequencing assays suitable for use with the methods disclosed herein include nanopore sequencing (U.S. Patent Application Publication Nos. 2013 / 0244340, 2013 / 0264207, 2014 / 0134616, 2015 / 0119259, and 2015 / 0337366), Sanger sequencing, capillary array sequencing, thermal cycle sequencing (Sears et al., Biotechniques, 13:626-633 (1992)), solid-phase sequencing (Zimmerman et al., Methods Mol. Cell Biol., 3:39-42 (1992)), and sequencing using mass spectrometry, e.g., matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF / MS; Fu et al., Nature 106:101-102 (1992)). Biotech., 16:381-384 (1998)), sequencing by hybridization (Drmanac et al., Nature Biotech., 16:54-58 (1998)), and NGS methods including, but not limited to, sequencing by synthesis (e.g., HiSeq™, MiSeq™, or Genome Analyzer, each available from Illumina), sequencing by ligation (e.g., SOLiD™, Life Technologies), ion semiconductor sequencing (e.g., Ion Torrent™, Life Technologies), and SMRT™ sequencing (e.g., Pacific Biosciences).

[0068] Commercially available sequencing technologies include the sequencing-by-hybridization platform from Affymetrix Inc. (Sunnyvale, Calif.), the sequencing-by-synthesis platforms from Illumina / Solexa (San Diego, Calif.) and Helicos Biosciences (Cambridge, Mass.), and the sequencing-by-ligation platform from Applied Biosystems (Foster City, Calif.). Other sequencing technologies include, but are not limited to, Ion Torrent technology (ThermoFisher Scientific), and nanopore sequencing (Genia Technology, part of Roche Sequencing Solutions, Santa Clara, Calif.), and Oxford Nanopore Technologies (Oxford, UK).

[0069] In some embodiments, the sequencing step includes sequence alignment. In some embodiments, alignment is used to determine a consensus sequence from multiple sequences, for example, multiple sequences with the same unique molecular ID (UID). The molecular ID is a barcode that can be added to each molecule prior to sequencing or, if an amplification step is included, prior to the amplification step. In some embodiments, the UID is present in the 5' portion of the RT primer. Similarly, the UID can be present at the 5' end of the last barcode subunit that is added to the compound barcode. In other embodiments, the UID is present in an adapter and is added to one or both ends of the target nucleic acid by ligation.

[0070] In some embodiments, a consensus sequence is determined from multiple sequences that all share the same UID. Sequences with the same UID are presumed to be derived from the same original molecule through amplification. In other embodiments, UIDs are used to eliminate artifacts, i.e., variations present in the progeny of a single molecule (characterized by a specific UID). Such artifacts resulting from PCR or sequencing errors can be eliminated using UIDs.

[0071] In some embodiments, the number of each sequence in a sample can be quantified by quantifying the relative number of sequences with each UID in a population with the same multiple sample ID (MID). Because each UID represents a single molecule in the original sample, counting the distinct UIDs associated with each sequence variant can determine the fraction of each sequence variant in the original sample where all molecules share the same MID. One skilled in the art can determine the number of sequence reads required to determine a consensus sequence. In some embodiments, a reasonable number is the number of reads per UID ("sequence depth") required for accurate quantitative results. In some embodiments, the desired depth is 5-50 reads per UID.

[0072] In some embodiments, the present invention provides a composition for nucleic acid hybridization, comprising two or more probe oligonucleotides, each probe oligonucleotide comprising a target binding region and a first and second primer binding region, and one or more enhancer oligonucleotides capable of hybridizing to at least one of the primer binding regions. In some embodiments, the composition is obtained by contacting a sample with a probe mixture containing a plurality of probe oligonucleotides capable of specifically hybridizing to a plurality of nucleic acid targets under hybridization conditions. The probe mixture further comprises an enhancer oligonucleotide comprising a mixture of oligonucleotides capable of hybridizing to the first and second primer binding regions. Various mixtures of enhancer oligonucleotides are contemplated within the scope of the present invention. For example, an oligonucleotide capable of hybridizing to each strand of the first and second primer binding regions. The enhancer oligonucleotide can be a mixture of four oligonucleotides, each capable of hybridizing to one of the Watson or Crick strands of the first or second primer binding region. The enhancer oligonucleotide can also be a mixture of more than four oligonucleotides that can be grouped into four groups, each group of oligonucleotides capable of hybridizing to either the Watson strand or the Crick strand of the first primer binding region or the second primer binding region.

[0073] In some embodiments, at least some of the nucleic acids in the composition are double-stranded. In some embodiments, all of the nucleic acids in the composition, including target and non-target nucleic acids, probes, and enhancer oligonucleotides, are single-stranded.

[0074] In some embodiments, the present invention provides a composition for enriching nucleic acid targets, comprising two or more probe oligonucleotides, each probe oligonucleotide comprising a target binding region capable of hybridizing to a nucleic acid to be enriched, as well as a first primer binding region and a second primer binding region, and one or more enhancer oligonucleotides capable of hybridizing to at least one of the primer binding regions. The probe oligonucleotides in the composition are capable of specifically hybridizing to multiple nucleic acid targets to be enriched in a mixture with non-target nucleic acids under hybridization conditions. In some embodiments, the composition further comprises a mixture of target nucleic acids and non-target nucleic acids. In some embodiments, the mixture of target nucleic acids and non-target nucleic acids present in the composition is genomic DNA of an organism. In some embodiments, the mixture of target nucleic acids and non-target nucleic acids present in the composition is a genomic DNA library derived from the genome of an organism.

[0075] In some embodiments, hybridization between the sample nucleic acid and the capture probe occurs in solution. In other embodiments, hybridization occurs on a solid support, such as a surface or a particle such as a slide or bead. In this embodiment, the hybridization probe is covalently or non-covalently tethered to the solid support. In some embodiments, the probe is attached to the solid support via a capture moiety (e.g., biotin) present on the probe. In some embodiments, the probe is attached to the solid support via hybridization of a sequence in the probe to a capture oligonucleotide covalently or non-covalently attached to the solid support. The sample nucleic acid is present in solution in contact with the solid support. In some embodiments, the probe is attached to the solid support via a primer binding site. In such cases, the enhancer oligonucleotide of the present invention can be used to elute the probe or probe-target complex from the solid support.

[0076] In other embodiments, the sample nucleic acids (i.e., the library nucleic acids) are covalently or non-covalently tethered to a solid support (e.g., via a capture moiety present on an adapter or another portion of the library molecule), and the probes are in solution in contact with the solid support.

[0077] In some embodiments, the present invention provides a reaction mixture comprising a plurality of nucleic acids, including target and non-target nucleic acids; two or more probe oligonucleotides, each probe oligonucleotide comprising a target binding region and a first and second primer binding region; and one or more enhancer oligonucleotides capable of hybridizing to at least one of the primer binding regions. In some embodiments, the reaction mixture comprises a plurality of probe oligonucleotides capable of specifically hybridizing to a plurality of nucleic acid targets present in a mixture with non-target nucleic acids under hybridization conditions. In some embodiments, the reaction mixture comprises genomic DNA of an organism or a genomic library derived from an organism. In some embodiments, all nucleic acids in the reaction mixture are single-stranded. In some embodiments, all nucleic acids in the reaction mixture are double-stranded. In some embodiments, each probe has four primer binding regions, and the enhancer oligonucleotide binds to all four primer binding regions. In some embodiments, the enhancer oligonucleotides comprise a mixture of four oligonucleotides, each capable of hybridizing to one of the Watson or Crick strands of the first or second primer binding region, or the enhancer oligonucleotides comprise a mixture of more than four oligonucleotides, which can be grouped into four groups, each capable of hybridizing to one of the Watson or Crick strands of the first or second primer binding region. In some embodiments, the reaction mixture comprises genomic DNA of an organism. In some embodiments, the reaction mixture comprises a genomic library formed from genomic DNA of an organism.

[0078] In some embodiments, the present invention is a kit containing components and tools for target capture by hybridization in the presence of enhancer oligonucleotides. In some embodiments, the kit contains one or more aliquots of hybridization probes (each in a separate vial or as one or more probe pools) and one or more aliquots of enhancer oligonucleotides (each in a separate vial or as a mixture of two or more enhancer oligonucleotides). In some embodiments, the kit further contains solutions and buffers for hybridization and one or more post-hybridization washes. In some embodiments, the kit further contains reagents for intermediate purification of nucleic acids, including capture particles (e.g., magnetic or paramagnetic particles), wash buffers, and magnets.

[0079] In some embodiments, the kit further comprises reagents and tools for performing steps upstream of target capture by hybridization. In some embodiments, the kit comprises reagents for preparing a library from nucleic acids in a sample. The library preparation reagents include one or more of DNA ligase, DNA polymerase, adapters, and buffers required for A-tailing and ligation of adapters to sample nucleic acids.

[0080] In some embodiments, the kit further comprises reagents and tools for performing steps downstream of target capture by hybridization, hi some embodiments, the kit comprises reagents for isolation, amplification and sequencing of captured nucleic acids.

[0081] In some embodiments, the method further comprises assessing a disease or condition in a subject (e.g., a patient) based on the mutation status of one or more loci in the patient's genome.

[0082] The mutation status is selected from no mutation (wild-type sequence) and one or more mutations selected from mutations including at least one single nucleotide variation (SNV), at least one copy number variation (CNV), (including sequence deletion, duplication or higher order amplification), translocation or fusion.

[0083] In some embodiments, the invention is a method comprising enriching nucleic acids from a patient by the methods described herein and detecting or diagnosing a disease or condition in the patient by determining the mutation status of one or more genetic loci in the enriched nucleic acids that are known to be biomarkers for the disease or condition. In some embodiments, the method further comprises selecting or altering treatment based on the mutation status of the one or more genetic loci enriched from the patient sample.

[0084] In some embodiments, the present invention is a method of diagnosing or screening for the presence of a cancerous tumor in a patient or subject. In some embodiments, the present invention comprises enriching the patient's nucleic acid by a method described herein and detecting the presence of a cancerous tumor in the patient by determining, in the enriched nucleic acid, the mutational status of one or more genetic loci known to indicate the presence of a cancerous tumor. In some embodiments, the method further comprises selecting or altering a treatment targeted to the cancerous tumor based on the mutational status of one or more genetic loci enriched from the patient's sample by a method described herein.

[0085] In some embodiments, the invention is a method of monitoring tumor growth or regression, the method comprising periodically sampling circulating cell-free DNA (cfDNA) from a patient, enriching for one or more target sequences in the cfDNA, and measuring changes in the amount of cfDNA containing one or more mutations in the target sequences, wherein an increase in the level of such mutated cell-free DNA indicates tumor growth, while a decrease in the level of such mutated cell-free DNA indicates tumor regression.

[0086] In some embodiments, the invention is a method of monitoring the effectiveness of a cancer treatment in a patient or subject, the method comprising periodically sampling circulating cell-free DNA (cfDNA) from the patient, enriching for one or more target sequences in the cfDNA, and measuring changes in the amount of cfDNA containing one or more mutations in the target sequences, wherein an increase in the level of such mutant cell-free DNA indicates tumor growth and ineffectiveness of the treatment, while a decrease in the level of such mutant cell-free DNA indicates tumor shrinkage and effective treatment, and a stable level of such mutant cell-free DNA indicates stable disease and effective treatment.

[0087] In some embodiments, the present invention includes a method for diagnosing minimal residual disease (MRD) in cancer patients after treatment.The National Cancer Institute defines MRD as a very small number of cancer cells that remain in the body during or after treatment when the patient has no signs or symptoms of disease.In some embodiments, the present invention is a method for diagnosing MRD, the method includes obtaining circulating cell-free DNA (cfDNA) from a patient, enriching one or more target sequences in cfDNA, and detecting one or more mutations characteristic of tumors in the enriched cfDNA, and the presence of such mutant cell-free DNA indicates the presence of MRD in the patient.

[0088] Example

[0089] Example 1. Enhancer Oligonucleotides in Target Capture

[0090] In this experiment, the probe hybridization step of the KAPA HyperCap Workflow (v3.0, available from Roche Sequencing Solutions, Inc. Pleasanton, Calif.) was performed in the presence of hybridization enhancer oligonucleotides.

[0091] To prepare for hybridization, 130 μL of KAPA HyperPure Beads were added to each tube containing the DNA sample library (composed of sheared human genomic DNA ligated to adapters) and the COT human DNA mixture. The mixture was thoroughly mixed by vortexing for 10 seconds and centrifuged. The mixture was incubated at room temperature for 10 minutes to ensure that the DNA sample library and COT human DNA bound to the beads. The sample was placed on a magnet, and the beads were collected until the liquid was clear. The supernatant was removed and discarded. With the sample still on the magnet, 200 μL of freshly prepared 80% ethanol was added, and the sample was incubated at room temperature for 30 seconds or more. The ethanol was removed and discarded without disturbing the beads. The residual ethanol was allowed to evaporate at room temperature. A hybridization master mix was prepared as follows:

[0092] [Table 3]

[0093] Next, 43 μL of hybridization master mix was added to the bead-bound DNA mixture, which was suspended in a solution containing blocker oligonucleotides designed to bind to the adapters attached to the library molecules. The reaction mixture was mixed thoroughly, centrifuged, and incubated at room temperature for 2 minutes. The sample was placed on a magnet to collect the beads and incubated until the liquid was clear. 56.4 μL of the eluate (total volume) was then transferred to a new tube containing 4 μL of KAPA target enrichment probes (a pool of biotinylated 120-nt probes) and an enhancer oligonucleotide of the present invention. The enhancer oligonucleotide was added at four different concentrations (0.234 mM, 0.0234 mM, 0.00234 mM, and 0.000234 mM) relative to the final volume of the hybridization mixture. Control reactions did not contain the enhancer oligonucleotide (Figures 2A and 2B).

[0094] The reaction mixture was mixed thoroughly by vortexing for 10 seconds and centrifuged. Hybridization was performed in a thermocycler using a program set at 105°C lid temperature, 95°C for 5 minutes, and 55°C overnight.

[0095] Hybridized DNA was washed, recovered, and amplified according to the manufacturer's recommendations in KAPA HyperCap Workflow v3.0. Amplified DNA was sequenced on an Illumina instrument.

[0096] The sequencing results are shown in Figures 2A and 2B. Figure 2A: The dsDNA probe enhancer oligonucleotide of the present invention improved capture uniformity in a dose-dependent manner. The fold 80 base penalty is defined as the additional sequencing times required to achieve an average coverage depth of 80%; therefore, a lower fold 80 base penalty indicates better capture uniformity. Figure 2B: The inclusion of the dsDNA probe enhancer oligo resulted in a dose-dependent decrease in the overall duplication rate of the sequencing data.

Claims

1. A composition for nucleic acid hybridization, comprising: a. two or more probe oligonucleotides, each probe oligonucleotide comprising a target binding region, and a first primer binding region and a second primer binding region; b. one or more enhancer oligonucleotides capable of hybridizing to at least one of said primer binding regions; A composition comprising:

2. 2. The composition of claim 1, wherein all of the probe oligonucleotides have the same first primer binding region and the same second primer binding region.

3. The composition of claim 1 , wherein the enhancer oligonucleotide comprises a mixture of oligonucleotides capable of hybridizing to the first primer binding region and the second primer binding region.

4. 1. A composition for enrichment of a nucleic acid target, comprising: a. two or more probe oligonucleotides, each probe oligonucleotide comprising a target binding region, and a first primer binding region and a second primer binding region; b. one or more enhancer oligonucleotides capable of hybridizing to at least one of said primer binding regions; A composition comprising:

5. The composition of claim 4 , wherein all of the probe oligonucleotides have the same first primer binding region and the same second primer binding region.

6. The composition of claim 4 , wherein the enhancer oligonucleotide comprises a mixture of oligonucleotides capable of hybridizing to the first primer binding region and the second primer binding region.

7. 1. A method for concentrating a target nucleic acid, comprising: a. a mixture of target and non-target nucleic acids; two or more probe oligonucleotides, each probe oligonucleotide comprising a target binding region and a first primer binding region and a second primer binding region; one or more enhancer oligonucleotides that hybridize to at least one of the primer binding regions; contacting the composition comprising: b. incubating the mixture under hybridization conditions; c. Separating probe-bound target nucleic acids from unbound nucleic acids; A method comprising:

8. the mixture of target and non-target nucleic acids constitutes a library formed from the genomic DNA of an organism; 8. The method of claim 7, wherein the library comprises nucleic acids isolated from the organism, each nucleic acid being conjugated to at least one adaptor nucleic acid.

9. 9. The method of claim 8, wherein each nucleic acid in the library is conjugated to two adaptor nucleic acids.

10. 1. A method for sequencing a nucleic acid, comprising: a. a mixture of target and non-target nucleic acids; two or more probe oligonucleotides, each probe oligonucleotide comprising a target binding region and a first primer binding region and a second primer binding region; one or more enhancer oligonucleotides that hybridize to at least one of the primer binding regions; contacting the composition comprising: b. incubating the mixture under hybridization conditions; c. capturing hybrids formed between the probe and the target nucleic acid to obtain enriched nucleic acids; d. sequencing the enriched nucleic acids; A method comprising:

11. A reaction mixture comprising: a. a plurality of nucleic acids, including target nucleic acids and non-target nucleic acids; b. two or more probe oligonucleotides, each probe oligonucleotide comprising a target binding region, and a first primer binding region and a second primer binding region; c. one or more enhancer oligonucleotides capable of hybridizing to at least one of said primer binding regions; Including, A reaction mixture wherein all of said probe oligonucleotides have the same first primer binding region and the same second primer binding region.

12. 1. A method for assessing a disease or condition in a patient, comprising: a. providing a nucleic acid-containing sample from a patient; b. concentrating the target nucleic acid in the sample by the method of claim 7; c. detecting said disease or condition in said patient by determining the mutation status of one or more genetic loci in said enriched target nucleic acids that are known to be biomarkers for said disease or condition; A method comprising:

13. 1. A method of diagnosing or screening for the presence of a cancerous tumor in a patient, comprising: a. providing a nucleic acid-containing sample from a patient; b. concentrating the target nucleic acid in the sample by the method of claim 7; c. detecting the presence of a cancerous tumor in the patient by determining the mutation status of one or more genetic loci in the enriched nucleic acid that are known to indicate the presence of a cancerous tumor; A method comprising:

14. 1. A method for monitoring tumor growth or regression, comprising: a. periodically sampling circulating cell-free DNA (cfDNA) from a patient; b. Enriching one or more target sequences in the cfDNA by the method of claim 7; c. detecting changes in the amount of mutated cfDNA containing one or more mutations in said target sequence known to be mutated in cancerous tumors, wherein an increase in the level of such mutated cfDNA indicates tumor growth, while a decrease in the level of such mutated cfDNA indicates tumor shrinkage; A method comprising:

15. 1. A method for diagnosing minimal residual disease (MRD) in a cancer patient, comprising: a. obtaining circulating cell-free DNA (cfDNA) from a patient; b. Enriching one or more target sequences in the cfDNA by the method of claim 7; c. detecting the mutational status of one or more genetic loci in the enriched cfDNA known to be mutated in cancerous tumors, wherein the presence of mutated cfDNA indicates the presence of MRD in the patient; A method comprising:

16. 1. A kit for improved hybridization of nucleic acids, comprising: a. one or more probe oligonucleotides, each probe oligonucleotide comprising a target binding region, and a first primer binding region and a second primer binding region; b. one or more enhancer oligonucleotides capable of hybridizing to at least one of said primer binding regions; Includes a kit.

17. 1. A method for concentrating a target nucleic acid, comprising: a. a mixture of target and non-target nucleic acids; i. two or more probe oligonucleotides, each probe oligonucleotide comprising a target binding region and a first primer binding region and a second primer binding region, wherein the first primer binding region hybridizes to a capture oligonucleotide attached to a solid support; ii. one or more enhancer oligonucleotides that hybridize to the second primer binding region; and contacting the composition comprising: b. incubating the mixture under hybridization conditions; c. separating probe-bound target nucleic acid from unbound nucleic acid by contacting the mixture with one or more enhancer oligonucleotides that hybridize to the first primer binding region under conditions suitable for dissociating the first primer binding region from the capture oligonucleotide; A method comprising:

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