TECHNICAL FIELD

The invention generally relates to negative selection of nucleic acids.

BACKGROUND

The advent of high-throughput DNA sequencing has the potential to revolutionize modern biology and transform diagnostic medicine. Instruments for next-generation sequencing (NGS) continue to generate more data and become more inexpensive at a rate far outpacing Moore's Law. However, the most popular sequencers have an extremely short read length, limiting their ability to characterize any gene containing paralogous sequence or repetitive elements. As nearly two thirds of the genome is highly repetitive and over 20,000 pseudogenic regions exist, much of the genome is very difficult to characterize in a modern whole-genome sequencing experiment. Unfortunately, for many genes of clinical interest, characterizing those genes is made difficult by the presence of paralogs, pseudogenic homologs, and other segments of the genome that may be similar to the gene of interest and thus stymie attempts to detect, sequence, or isolate the gene of interest. As a result, despite the power of NGS instruments, some disease-related genes and mutations, even where known, are difficult to detect.

SUMMARY

The invention provides methods and systems that remove unwanted segments of nucleic acid in a sample so that a target gene or region of interest may be analyzed without interference from the unwanted segments. A sample is obtained that includes single-stranded nucleic acid with one or more unwanted segments. Primers that are specific or preferentially bind to the unwanted segment are hybridized to the single-stranded nucleic acid within the unwanted region or in a non-repetitive section upstream of the unwanted region and extended by a polymerase to create a double-stranded region that includes the unwanted segment. The double-stranded region is then digested, leaving single-stranded nucleic acid that includes the target gene or region of interest. This allows paralogs, pseudogenes, repetitive elements, and other segments of the genome that may be similar to the target gene or region of interest to be removed from the sample. The target gene or region of interest may thus be detected or characterized by analysis without interference from the unwanted segments. This may provide an improved ability to detect features such as disease-related genes and mutations, thus improving the clinical value of NGS technologies.

Systems and methods of the invention may be used to remove unwanted regions from genomic DNA (such as homologous genes, pseudogenes, or repetitive elements) prior to any DNA-based experimental procedure, including but not limited to microarray hybridization, quantitative or standard polymerase chain reaction, multiplex target capture, or DNA sequencing (either targeted or shotgun). Systems and methods of the invention provide for the identification of mutations in previously difficult-to-characterize genes, and therefore allow practitioners to expand the number of genes included in a targeted or whole-genome sequencing assay.

In certain aspects, the invention provides a method of removing unwanted segments of a nucleic acid from a sample. The method includes annealing a nucleic acid primer to a portion of a single-stranded nucleic acid that flanks an unwanted segment of the nucleic acid, extending the annealed primer in order to create a double-stranded region that includes the unwanted segment; and digesting the double-stranded region, thereby removing the unwanted segment from the nucleic acid.

The nucleic acid in the sample may include DNA, RNA, modified nucleic acids, or combinations thereof. The method may include obtaining a sample from a subject and denaturing double-stranded DNA in the sample. Denaturing can include the use of methods such as exposing the sample to heat, a detergent, or an acidic or basic solution.

The primer may be annealed within the unwanted segment or within an area upstream of the unwanted segment and extended. A pair or a number of primers may be used and primers that flank the unwanted segment may be used. In certain embodiments, a plurality of primers are annealed to a plurality of portions of that nucleic acid that flank an unwanted segment. The primer or primers are preferably extended using a polymerase enzyme under conditions sufficient to cause extension of the primer in a template-dependent manner. In some embodiments, a primer or oligonucleotide is hybridized to the unwanted segment to create the double-stranded region containing the unwanted segment without need for an extension step.

The double-stranded region is digested. This can include exposing the sample to an enzyme that preferentially digests double-stranded nucleic acid such as certain double-stranded endonucleases, restriction endonucleases, or nicking enzymes. After digestion, the enzyme may be de-activated (e.g., by heat, chemicals, etc.). Digestion preferably results in intact genomic DNA lacking one or more unwanted segment and that is compatible with a nucleic acid analysis assay. Nucleic acid that is not digested may be analyzed by a nucleic acid analysis assay.

Assays suitable for analysis of the remaining un-digested nucleic acid may make use of molecular inversion probe capture, hybrid capture, Haloplex, sequencing (e.g., Sanger sequencing, NGS, or both), other methodologies, or combinations thereof. Where the unwanted segment is a paralog, a pseudogene, or non-paralogous repetitive element, such elements may be removed from the sample by methods of the invention.

In certain aspects, the invention provides a method of removing nucleic acid from a sample. The method includes annealing at least one oligonucleotide to single-stranded DNA in a sample, wherein the single-stranded DNA comprises target and non-target sequence. The oligonucleotide may be annealed to the non-target sequence to create double-stranded DNA that includes the non-target sequence or the oligonucleotide may be annealed elsewhere and extended to create double-stranded DNA that includes the non-target sequence. The non-target sequence is removed from the sample by digesting the double-stranded DNA. The target sequence may be analyzed using, e.g., molecular inversion probes, microarray hybridization, multiplex ligation-dependent probe amplification (MLPA), sequencing, fingerprinting techniques such as RFLP/AFLP, chromatography, others, or combinations thereof. In some embodiments, the method includes first obtaining the sample from a subject and denaturing double-stranded subject DNA to produce the single-stranded DNA. Preferably, that single-stranded DNA consists essentially of genomic DNA from the subject prior to the annealing of the oligo. The annealing may include annealing a pair of oligonucleotides to the single-stranded DNA at sites that flank the non-target sequence (i.e., to remove both strands of the unwanted segment or non-target sequence. In certain embodiments, the target and non-target sequence are both located on at least one single strand of the single-stranded DNA, and extending the at least one oligonucleotide and digesting the double-stranded DNA results in removing the non-target sequence from the at least one single strand of the single-stranded DNA.

