Nuclease-based RNA depletion

Abstract

The present invention relates to a method for depleting unwanted RNA species from a nucleic acid sample and to a material thereof. In particular, the present invention describes a method for removing unwanted rRNA, tRNA, mRNA, or other RNA species that may interfere with the analysis, manipulation, and study of target RNA molecules in a sample.

Description

Technology Field

[0001] Cross-reference of related applications

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 783,869 filed December 21, 2018 and U.S. Provisional Application No. 62 / 847,797 filed May 14, 2019, each of which is incorporated herein by reference in its entirety for the purposes thereof.

[0003] Sequence list

[0004] The present application is submitted together with a sequence listing in electronic format. The sequence listing is 94,208 bytes in size and is provided as a file named "2019-12-09_01243-0012-00PCT_Sequence_Listing_ST25.txt" created on December 9, 2019. The information in electronic format of the sequence listing is incorporated herein by reference in its entirety. Background Technology

[0005] Unwanted RNA in nucleic acid samples, such as those taken from human cells or tissues, can complicate the analysis of those samples, including gene expression analysis, microarray analysis, and sequencing. Since ribosomal RNA (rRNA) accounts for approximately 95% of RNA within cells, its presence is an example of an RNA species that can interfere with and obfuscate results regarding target nucleic acids in a sample, or an example of a nucleic acid that researchers or diagnosticians wish to understand more deeply. For instance, unwanted rRNA species can make it particularly difficult to analyze RNA molecules of interest in a sample, such as tRNA or mRNA. This is a problem that is always present, especially in the case of fixed tissues, such as those fixed in formalin and subsequently embedded in wax, like formalin-fixed paraffin-embedded (FFPE) tissue from biopsies. If rRNA species are not removed from FFPE tissues, they can interfere with the measurement and characterization of target RNA within the tissue, thereby making it very difficult to derive medically actionable information from target RNA, such as disease and cancer identification, potential treatment options, and disease or cancer diagnosis and prognosis. While FFPE tissues are one example, the same problem with rRNA applies to all kinds of samples, such as blood, cells, and other types of nucleic acid-containing samples.

[0006] Current commercially available methods for depleting unwanted RNA from nucleic acid samples include RiboZero® (Epicentre) and NEBNext® rRNA depletion kits (NEB) and RNA depletion methods as described in U.S. Patents No. 9,745,570 and No. 9,005,891. However, while these methods are useful for depleting RNA, they have their own disadvantages, including ease of use, high sample input requirements, and the time, cost, and / or efficiency of the technician in depleting unwanted RNA from samples. There is a need for materials and methods that can deplete unwanted RNA species from samples more easily or cost-effectively to reveal information about target RNA that may be hidden, such as rare or hard-to-identify sequence changes. Simple and reliable methods as described herein can significantly increase the availability of target RNA molecules for testing purposes, thereby enabling the discovery of information held about the sample and the organism from which it originated.

[0007] Nucleic acid samples, such as those from eukaryotes or prokaryotes, contain a multitude of nucleic acids, many of which do not interest researchers. Researchers often wish to study specific types of nucleic acids, such as DNA or RNA. When studying RNA, the sample of interest may contain various types of RNA species that can overwhelm and obscure the target RNA, which is the focus of the study. As such, RNA depletion refers to the removal of unwanted RNA and / or DNA species from a nucleic acid sample, leaving a nucleic acid sample rich in the desired RNA for research.

[0008] Before proceeding to further study, the present invention provides a solution for depleting nucleic acid samples with an excess of unwanted RNA species. For example, an RNA sample of interest contains not only the target RNA to be studied but also an abundance of transcripts, such as rRNA, globin mRNA, viral contaminants, or any other unwanted nucleic acids, that are dominant in the sample and can neutralize the target of interest, thereby significantly reducing the researcher's ability to accurately analyze the desired portion of the transcriptome.

[0009] Therefore, depleting unwanted RNA from nucleic acid samples prior to analysis, such as expression microarrays or sequencing, increases the specificity and accuracy of the analysis for desired RNA targets. In the present invention, the depletion of off-target RNA through the degradation of specific DNA:RNA hybrids enables the efficient removal of unwanted RNA species from samples prior to library preparation and analysis. Once unwanted RNA species are depleted from the sample, residual target RNA can be converted into cDNA. Obtaining actionable data from stable samples can lead to a better understanding of potential therapeutic options for cancer prognosis and diagnosis, a better understanding of the human microbiome and its importance in humans and other eukaryotic cell systems, and a more thorough understanding of the expression analysis of genes of interest.

[0010] In one embodiment, the present invention,

[0011] a) a nucleic acid sample comprising at least one RNA or DNA target sequence and at least one non-target RNA molecule, in contact with a probe set comprising at least two DNA probes complementary to a discontinuous sequence along the full length of at least one non-target RNA molecule, thereby hybridizing the DNA probes to the non-target RNA molecule to form a DNA:RNA hybrid—wherein each DNA:RNA hybrid is separated from any other DNA:RNA hybrid by at least 5 bases or at least 10 bases along a given non-target RNA molecule sequence—; and

[0012] b) A method for depleting non-target RNA molecules from a nucleic acid sample is described, comprising the step of contacting a DNA:RNA hybrid with a ribonuclease that degrades RNA from the DNA:RNA hybrid to degrade non-target RNA molecules of the nucleic acid sample and form a degradation mixture.

[0013] In one embodiment, the present invention relates to a composition comprising a probe set comprising at least two DNA probes complementary to a discontinuous sequence along the full length of at least one non-target rRNA molecule in a nucleic acid sample (e.g., separated by at least 5 or at least 10 bases along the full length). In some embodiments, the composition also comprises a ribonuclease capable of degrading RNA in a DNA:RNA hybrid. In another embodiment, the present invention relates to a composition comprising a probe set comprising at least two DNA probes hybridized to at least one non-target RNA molecule—wherein each DNA probe is hybridized from any other DNA probe in the probe set separated by at least 5 or at least 10 bases along the length of the non-target RNA molecule.

[0014] In one embodiment, the present invention describes a kit comprising a set of probes including at least two DNA probes complementary to a discontinuous sequence along the full length of at least one non-target rRNA molecule in a nucleic acid sample (e.g., separated by at least 5 or at least 10 bases along the full length) and a ribonuclease capable of degrading RNA in a DNA:RNA hybrid.

[0015] In one embodiment, the present invention comprises: a) contacting a nucleic acid sample comprising at least one RNA or DNA target sequence and at least one non-target RNA molecule of a first species with a probe set comprising at least two DNA probes complementary to a discontinuous sequence along the entire length of at least one non-target RNA molecule of a second species to hybridize the DNA probes to the non-target RNA molecule to form a DNA:RNA hybrid—wherein each DNA:RNA hybrid is separated from any other DNA:RNA hybrid by at least 5 bases or by at least 10 bases along a given non-target RNA molecule sequence—; b) contacting the DNA:RNA hybrid with a ribonuclease that degrades RNA from the DNA:RNA hybrid to degrade the non-target RNA molecule of the nucleic acid sample to form a degradation mixture; c) separating the degraded rRNA from the degradation mixture; d) sequencing residual RNA from the sample; e) evaluating the residual RNA sequence for the presence of a non-target RNA molecule of a first species to determine a gap sequence region; and f) a method for replenishing a probe set used to deplete non-target RNA nucleic acid molecules from a nucleic acid sample, comprising the step of supplementing the probe set with additional DNA probes complementary to discontinuous sequences in one or more gap sequence regions. Brief explanation of the drawing

[0016] Figure 1 illustrates an exemplary workflow for performing RNA species depletion from a sample. Step 1 involves adding a depletion DNA probe following nucleic acid denaturation and hybridizing the probe with the unwanted RNA species to create a DNA:RNA hybrid. Step 2 involves degrading the RNA from the DNA:RNA hybrid using a ribonuclease such as RNase H. Step 3 involves adding DNase to degrade the residual DNA probe from the degradation mixture. Step 4 involves capturing residual target RNA in the sample, which is optionally followed by further manipulation, and ultimately produces a sample depleted of the unwanted RNA species, which can be sequenced or exposed to microarray expression analysis, qPCR, or other analytical techniques. FIGS. 2a to 2c show Bacillus subtilis (when formamide is added to an rRNA depletion workflow) B. subtilis ) Exemplary data on rRNA depletion from samples are shown: (2a) 0% formamide, (2b) 25% formamide, (2c) 45% formamide. In each panel, the X-axis enumerates the detected rRNA species, and the Y-axis represents % depletion through % ​​sequence reads. Figure 3 shows exemplary next-generation sequencing (NGS) sequence data for rRNA-depleted samples of human brain RNA (HBR) and universal human RNA (UHR) comparing various amounts of sequenced samples (100 ng, 10 ng, or 1 ng). Figure 4 shows exemplary NGS sequence data for rRNA depletion samples from mouse RNA and rat RNA using various concentrations of formamide (0%, 25%, 45%) added to the rRNA depletion workflow. Figure 5 shows exemplary data on the removal of rRNA from various microbial species using low sample inputs, comparing the Ribozero® and RNase H enzymatic removal rRNA depletion methods. All sample read depths were normalized. The X-axis represents the rRNA depletion method (RZ = Ribozero or ED = RNase H enzymatic depletion method), and the Y-axis represents % rRNA reads. Figure 6 shows Bacillus subtilis and Escherichia coli (after RNase H rRNA depletion (ED) on the left side of the graph, compared to the absence of rRNA depletion (no) on the right side of the graph) E.coli This shows exemplary transcriptome detection data at various read depths for ). The X-axis represents sequencing reads (M), and the Y-axis represents the number of detected transcriptomes. Figures 7a and 7b show exemplary graphs of pairwise linear regression data for gene expression demonstrating the reproducibility of the disclosed method for rRNA depletion. Panel 7a illustrates gene expression levels for two E. coli clones, and Panel 7b illustrates gene expression levels for two Bacillus subtilis clones. The two bacterial types show a high correlation between gene expression levels after RNase H rRNA depletion. FIG. 8 shows exemplary trimeric rRNA read data for a mixed sample of 20 strains (MSA-2002, left) and 12 strains (MSA-2006, right). The mixed sample trimeric was rRNA depleted by the ribozero method (RZ) or the RNase H (ED) depletion method described herein. The RNA input for the MSA2002 sample was 10 ng, whereas for MSA2006 it was 80 ng. The X-axis represents the rRNA depletion method, and the Y-axis represents the % rRNA read. Figure 9 shows the sequencing read coverage (bottom of the figure) of mouse mitochondrial 12S (mt-Rnr1) and 16S (mt-Rnr2) rRNA loci and the effect of the 333 DNA probe set (sequence numbers 1 to 333) on depleting mouse 16S rRNA from a universal mouse reference RNA (UMR) sample. Squares indicate the 333 DNA probe set and the locations of a 90% match over a 50-base-pair length or a 70% match over a 30-base-pair length. In the absence of additional mouse and rat probes, gaps with no probe coverage correspond to peaks of residual or undepleted rRNA for the two replicates (Rep 1 and Rep2) shown at the top of the figure. Specific details for implementing the invention

[0017] Constructing nucleic acid libraries from RNA for sequencing is often difficult because samples are rich in unwanted transcripts, such as ribosomal RNA, globin mRNA, and viral contaminants, which are often dominant and can neutralize RNA sequences of interest. If these unwanted transcripts are not removed, they can impede the analysis of transcriptomes for prognostic, diagnostic, or research purposes. Therefore, depleting unwanted RNA from nucleic acid samples prior to analysis, such as sequencing or other downstream applications, can increase the specificity and accuracy of the desired analysis.

[0018] The present invention describes a method and material useful for depleting unwanted RNA species from nucleic acid samples so that important RNA can be studied and is not lost from numerous unwanted RNA transcripts.

[0019] Compared to existing methods for RNA depletion, the disclosed method can utilize a smaller amount of total input RNA while still maintaining equivalent performance indicators. Therefore, the disclosed method can be used when researchers have small amounts of starting material that cannot be accommodated by other methods. Additionally, the disclosed method can be performed with a single probe pool that simultaneously targets unwanted RNA species of various different organisms without compromising depletion efficiency. For example, the present invention can simultaneously deplete unwanted eukaryotic and prokaryotic RNA species from RNA samples, including but not limited to human, bacterial, viral, and / or archaea sources of unwanted RNA.

[0020] A nucleic acid sample or mixture refers to a sample containing RNA or DNA, or both, that includes both undesirable (non-target or unwanted) nucleic acids and desired (target) nucleic acids. The DNA or RNA in the sample may be unmodified or modified, and may include, but is not limited to, single-stranded or double-stranded DNA or RNA or derivatives thereof (e.g., where some regions of DNA or RNA are double-stranded while other regions of DNA or RNA are single-stranded). Generally, a nucleic acid sample includes all unmodified forms of nucleic acids as well as all chemically, enzymatically, and / or metabolically modified forms of nucleic acids, or combinations thereof. A nucleic acid sample may include desired or unwanted nucleic acids, for example, genomic DNA or whole-cell RNA, or a combination of both. Undesirable nucleic acids include nucleic acids from eukaryotes that are not the subject of study and contaminating nucleic acids from bacterial, viral, archaea species, etc. Desired or desirable nucleic acids are the nucleic acids that serve as the basis or focus of the study, which are the target nucleic acids. For example, a researcher may wish to study mRNA expression analysis, where rRNA, tRNA, and DNA will be considered unwanted nucleic acids, and mRNA will be the target nucleic acid. Additionally, while a researcher may wish to study total RNA, rRNA, mRNA, and DNA will be considered unwanted or undesirable nucleic acids, and total RNA will be considered the target nucleic acid. Undesirable RNA includes, but is not limited to, ribosomal RNA (rRNA), mitochondrial rRNA, nuclear rRNA, mRNA, such as globin RNA, or transfer or tRNA, or a mixture thereof. In some embodiments, the untarget RNA is rRNA. In some embodiments, the untarget RNA is globin mRNA.

[0021] For example, nucleic acid samples may contain the desired messenger RNA (mRNA) or total RNA, while also containing undesirable ribosomal RNA (rRNA), transfer RNA (tRNA), and possibly undesirable DNA. General methods for extracting RNA from total samples, such as blood, tissue, cells, and fixed tissue, are well known in the art, as found in the literature [Current Protocols for Molecular Biology (John Wiley & Sons)] and numerous molecular biology method manuals. RNA isolation can be performed by commercially available purification kits, such as the Qiagen RNeasy mini-column, MasterPure Complete DNA and RNA Purification Kit (Epicenter), Paraffin Block RNA Isolation Kit (Ambion), RNA-Stat-60 (Tel-Test), or by cesium chloride density gradient centrifugation. Current methods are not limited by how RNA is isolated from the sample prior to RNA depletion.

[0022] There is an inherent distrust that mixing probes targeting bacterial rRNA and human rRNA in the same pool will lead to widespread non-target depletion of desirable transcripts (Mauro et al., Proc. Natl. Acad. Sci. USA 1997, 94:422-427; Mignone and Pesole, Appl. Bioinformatics 2002, 1:145-54). Surprisingly, research conducted during the development of the disclosed method demonstrates that this is not the case, as the specificity of DNA probe hybridization with unwanted RNA transcripts produces samples in which the unwanted RNA species are efficiently depleted. Furthermore, it was found that the addition of destabilizers, such as formamide, helps remove some unwanted RNAs that appear to be much more problematic to deplete in the absence of formamide. Although it is not necessary to understand how formamide helps remove these RNAs, it is believed that formamide relaxes the structural barriers of unwanted RNA, allowing DNA probes to bind more efficiently. Additionally, the addition of formamide has demonstrated an additional benefit of improving the detection of some non-target transcripts by denaturing / relaxing regions of mRNA that, for example, have very stable secondary or tertiary structures and are not typically visible in other library preparation methods.

[0023] nucleic acid samples or mixtures

[0024] The present invention is not limited to sources of nucleic acid samples, and, for example, said sources may be derived from nucleic acids found in soil, water or other liquids and other environmental samples, including but not limited to humans, non-human primates, mammals, birds, reptiles, plants, bacteria, and viruses, eukaryotes or prokaryotes. Samples may be obtained from cells, tissues, organs, environments, lysates, etc., and may be derived from any state of the sample such as fresh, frozen, freeze-dried, and reconstituted, or from fixed samples from tissue or biopsy specimens that were formalin-fixed paraffin-embedded (FFPE) or other cytological or histological sample manipulations.

[0025] Nucleic acid samples that can benefit from the RNA depletion method may be derived from any species, eukaryotes or prokaryotes, such as humans, non-humans, mice, rats, bacteria, etc., and may contain a single or multiple species in a single sample. Furthermore, the depletion method of the present invention may be used on fresh or preserved samples, such as biopsy or tissue samples, comprising samples treated with formalin and embedded in paraffin (e.g., formalin-fixed paraffin embedded, FFPE, samples). In some embodiments, the nucleic acid samples are derived from human or non-human sources, e.g., non-human eukaryotes, bacteria, viruses, plants, soil, or mixtures thereof. Once the unwanted RNA species are depleted from the sample, the remaining desired targets may be converted into cDNA for further processing known to those skilled in the art.

[0026] In some embodiments, the nucleic acid sample is derived from a human or non-human primate. In some embodiments, the nucleic acid sample is derived from a rat or mouse. In some embodiments, the nucleic acid sample comprises nucleic acid of non-human origin. In some embodiments, the nucleic acid of non-human origin is derived from a non-human eukaryote, bacteria, viruses, plants, soil, or a mixture thereof.

[0027] depletion method

[0028] Thus, unwanted or undesirable RNA in nucleic acid samples is depleted by the described method. The unwanted RNA is converted into a DNA:RNA hybrid by hybridizing a DNA probe that is partially or fully complementary to the unwanted RNA molecule. Methods for hybridizing nucleic acid probes to nucleic acids are well established in the scientific field, and the fact that a DNA probe is hybridized to an unwanted RNA species after washing and other manipulations of the sample, regardless of whether the probe is partially or fully complementary to the partner sequence, demonstrates the DNA probes that can be used in the method of the present invention. The set of unwanted RNA for depletion may be derived from any eukaryote, e.g., humans, mice, rats, etc., where depletion of RNA from the sample may lead to more advantageous downstream research, e.g., sequencing (e.g., fewer results from unwanted nucleic acid species). Additionally, if the subject of study is RNA, DNA may be considered an unwanted nucleic acid, in which case the DNA may also be removed by depletion.

[0029] In one embodiment, the present invention,

[0030] a) a nucleic acid sample comprising at least one RNA or DNA target sequence and at least one non-target RNA molecule, in contact with a probe set comprising at least two DNA probes complementary to a discontinuous sequence along the entire length of at least one non-target RNA molecule, thereby hybridizing the DNA probes to the non-target RNA molecule to form a DNA:RNA hybrid—wherein each DNA:RNA hybrid is separated from any other DNA:RNA hybrid by at least 5 bases or by at least 10 bases along a given non-target RNA molecule sequence—; and

[0031] b) A method for depleting non-target RNA molecules from a nucleic acid sample is described, comprising the step of contacting a DNA:RNA hybrid with a ribonuclease that degrades RNA from the DNA:RNA hybrid to degrade non-target RNA molecules of the nucleic acid sample and form a degradation mixture.

[0032] In one embodiment, an RNA sample is denatured in the presence of a DNA probe. An exemplary workflow is illustrated in FIG. 1. In the example of FIG. 1, a DNA probe is added to a denatured RNA sample (denatured at 95°C for 2 minutes) and the reaction mixture is cooled to 37°C for 15 to 30 minutes, so that the DNA probe hybridizes to each of these target RNA sequences to produce a DNA:RNA hybrid molecule.

[0033] In some embodiments, the step of contacting the probe set includes the step of treating the nucleic acid sample with a destabilizing agent. In some embodiments, the destabilizing agent is heat or a nucleic acid destabilizing chemical. In some embodiments, the nucleic acid destabilizing chemical is betaine, DMSO, formamide, glycerol or a derivative thereof, or a mixture thereof. In some embodiments, the nucleic acid destabilizing chemical is formamide or a derivative thereof, optionally the formamide or its derivative is present at a concentration of about 10 to 45% of the total hybridization reaction volume. In some embodiments, the step of treating the sample with heat includes the step of applying heat higher than the dissolution temperature of at least one DNA:RNA hybrid.

[0034] In some embodiments, formamide is added to the hybridization reaction regardless of the RNA sample source (e.g., human, mouse, rat, etc.). For example, in some embodiments, hybridization to a DNA probe is performed in the presence of at least 3 volume%, 5 volume%, 10 volume%, 20 volume%, 25 volume%, 30 volume%, 35 volume%, 40 volume%, or 45 volume% of formamide. In one embodiment, the hybridization reaction for RNA depletion comprises about 25 volume% to 45 volume% of formamide.

[0035] After the hybridization reaction, a ribonuclease that degrades RNA from the DNA:RNA hybrid is added to the reactants. In some embodiments, the ribonuclease is RNase H or hybridase. RNase H (NEB) or hybridase (Lucigen) are examples of enzymes that degrade RNA from the DNA:RNA hybrid. Ribonucles such as RNase H or hybridase can degrade RNA into small molecules and remove them. For example, RNase H has been reported to degrade RNA from the DNA:RNA hybrid at intervals of about 7 to 21 bases (see [Schultz et al., J. Biol. Chem. 2006, 281:1943-1955; Champoux and Schultz, FEBS J. 2009, 276:1506-1516]). In some embodiments, RNA degradation of the DNA:RNA hybrid may occur at 37°C for about 30 minutes as described in FIG. 1, Step 2 and Example 1.

[0036] In some embodiments, after DNA:RNA hybrid molecular degradation, all non-target DNA of the remaining DNA probe and nucleic acid sample is degraded. Accordingly, in some embodiments, the method comprises the step of contacting the ribonuclease-degradation mixture with a DNA degrading enzyme to degrade the DNA in the mixture. In some embodiments, the degraded sample is exposed to a DNA degrading enzyme, e.g., DNase I, which degrades the DNA probe. The DNase DNA degradation reaction mixture is incubated, for example, at 37°C for 30 minutes, and then the DNase enzyme may be denatured at 75°C for the time required to denature the DNase, e.g., up to 20 minutes.

[0037] In some embodiments, the depletion method includes the step of separating the degraded RNA from the degradation mixture. In some embodiments, the separation step includes purifying the target RNA from the degraded RNA (and, if present, degraded DNA) using, for example, a nucleic acid purification medium, such as RNA capture beads like RNAClean XP beads (Beckman Coulter). Thus, in some embodiments, after enzymatic degradation(s), the target RNA can be enriched by removing degradation products while leaving the desired longer RNA target. A suitable enrichment method includes the step of treating the degradation mixture with magnetic beads that bind to the enriched RNA target, such as a spin column, to the desired fragment size. In some embodiments, magnetic beads such as AMPure XP beads, SPRISelect beads, and RNAClean XP beads (Beckman Coulter) may be used if the beads are RNase-free (e.g., of RNase-free quality). These beads provide various size selection options for nucleic acid binding; for example, RNAClean XP beads target nucleic acid fragments of 100 nt or larger, and SPRISelect beads target nucleic acid fragments of 150 to 800 nt, and do not target shorter nucleic acid sequences such as degraded RNA and DNA resulting from enzymatic degradation by RNase H and DNase. If mRNA is the target RNA to be studied, the mRNA can be further enriched by capture using beads containing an oligo dT sequence to capture, for example, the mRNA adenylated tail. mRNA capture methods are well known to those skilled in the art.

[0038] If the target RNA is purified from reaction components containing unwanted degraded nucleic acids, additional sample manipulation may occur. In the present invention, Examples 2 and 3 provide an exemplary library preparation workflow typical for subsequent sequencing, for example on an Illumina sequencer, following an exemplary workflow for cDNA synthesis from concentrated target total RNA. However, this workflow should be understood as merely exemplary, and those skilled in the art will understand that the concentrated RNA may be used directly in a number of additional applications, such as PCR, qPCR, microarray analysis, etc., or after additional manipulation, such as converting RNA to cDNA using established and fully understood protocols.

[0039] The method described herein for RNA depletion will produce a sample enriched with target RNA molecules. For example, the method described herein produces a depleted RNA sample containing less than 15%, 13%, 11%, 9%, 7%, 5%, 3%, 2%, or 1% of unwanted RNA species, or any range in between. The enriched RNA sample contains at least 99%, 98%, 97%, 95%, 93%, 91%, 89%, or 87% of the total target RNA, or any range in between. Once the sample is enriched, it may be used for library preparation or other downstream operations.

[0040] DNA probe set / DNA probe

[0041] A DNA probe refers to a single-stranded DNA oligonucleotide having sequence complementarity to an unwanted RNA species. The DNA probe sequence may be partially or fully complementary to the unwanted RNA for depletion in a nucleic acid sample. The unwanted RNA for depletion includes, but is not limited to, rRNA, tRNA, and mRNA, and mixtures thereof. In some embodiments, each DNA probe is about 10 to 100 nucleotides, about 20 to 80 nucleotides, about 40 to 60 nucleotides, or about 50 nucleotides in length. The DNA probe may be hybridized to the unwanted RNA species to produce a DNA:RNA hybrid molecule. In some embodiments, at least two DNA probes are hybridized to a specific non-target RNA molecule, but the DNA probes do not cover the entire length of the unwanted RNA molecule sequence. For example, in some embodiments, the probe set leaves a gap or region of the unwanted RNA without the DNA probe complementary to the probe set. The DNA probe is completely or partially hybridized to the unwanted RNA in a non-overlapping manner, leaving a gap of at least 5, 10, 15, 20, 30, 40, 50, 60, 70, 80 or more nucleotides between the resulting DNA:RNA hybrids. Thus, in some embodiments, each DNA probe is hybridized from any other DNA probe in the probe set by at least 5 or at least 10 bases along the entire length of at least one non-target RNA molecule. In this way, the unwanted RNA is not completely hybridized to the DNA probe in its entirety.Additionally, the present invention provides a plurality of DNA probes hybridized to a single RNA for depletion, so that instead of "one DNA probe for one RNA," there are instead a plurality of discontinuous DNA probes in a set of probes targeting a given unwanted RNA. For example, in some embodiments, for a given set of RNA for depletion, each probe is of length about 20 to 80 nucleotides, and a set of DNA probes is used in which each probe hybridizes to an unwanted RNA somewhere separated by 5 to 15 nucleotides from other DNA probes in the set. The DNA probes may be completely or partially complementary to a specific location on the RNA to be depleted, for example, the DNA probe sequence may be at least 80%, 85%, 90%, 95%, or 100% complementary to a target location on the RNA transcript to be depleted, or complementary to any range in between. The only limitation regarding complementarity is that the DNA probe must be hybridized to the target RNA such that it is depleted in a manner that generates an enzymatically degradable DNA:RNA hybrid as described herein. In some cases, mRNA is the target of interest and not the target of depletion, and in this case, the DNA probe does not contain a polyT sequence so that the probe is not hybridized to the mRNA species. In some embodiments, the DNA probe does not include a tag having a capture portion such as biotin, avidin, streptavidin, or magnetic beads capable of depleting the hybrid by physical means, whereas in other embodiments, the DNA probe includes a tag having a capture portion such as biotin, avidin, streptavidin, or magnetic beads capable of depleting the hybrid by physical means.

[0042] In some embodiments, the probe set comprises at least a DNA probe that hybridizes to non-target RNA molecules of human and bacterial origin. In some embodiments, the probe set comprises at least a DNA probe that hybridizes to non-target RNA molecules of human, bacterial, and archaea origin. In some embodiments, the probe set comprises at least a DNA probe that hybridizes to non-target RNA molecules of human, bacterial, mouse, and rat origin. In some embodiments, the probe set comprises at least a DNA probe that hybridizes to non-target RNA molecules of human, bacterial, mouse, rat, and archaea origin. In some embodiments, the non-target RNA molecules of bacterial origin are derived from Gram-positive bacteria or Gram-negative bacteria, or a mixture thereof. In some embodiments, the probe set comprises at least two DNA probes that hybridize to one or more non-target RNA molecules of archaea origin. In some embodiments, the probe set comprises at least two DNA probes that are complementary to two or more rRNA sequences of archaea origin.

[0043] In some embodiments, the probe set comprises at least two DNA probes hybridized to at least one or at least two non-target RNA molecules selected from 28S, 23S, 18S, 5.8S, 5S, 16S, 12S, HBA-A1, HBA-A2, HBB, HBB-B1, HBB-B2, HBG1, and HBG2. In some embodiments, the probe set comprises at least two DNA probes complementary to two or more rRNA sequences selected from the group consisting of 28S, 23S, 18S, 5.8S, 5S, 16S, 12S, HBA-A1, HBA-A2, HBB, HBB-B1, HBB-B2, HBG1, and HBG2. In some embodiments, the probe set comprises at least two DNA probes hybridized to one or more or two or more non-target RNA molecules selected from human-derived 28S, 18S, 5.8S, 5S, 16S, and 12S. In some embodiments, the probe set comprises at least two DNA probes hybridized to one or more or two or more non-target RNA molecules of rat and / or mouse-derived selected from rat 16S, rat 28S, mouse 16S, and mouse 28S, and combinations thereof. In some embodiments, the probe set comprises at least two DNA probes hybridized to one or more non-target RNA molecules selected from hemoglobin-derived HBA-A1, HBA-A2, HBB, HBB-B1, HBB-B2, HBG1, and HBG2. In some embodiments, the probe set comprises at least two DNA probes that are hybridized to one or more non-target RNA molecules selected from 23S, 16S, and 5S of Gram-positive and / or Gram-negative bacteria.Globin mRNA for depletion may include, but is not limited to, those found in rodents such as mice or rats, including HBA-A1, HBA-A2, HBB, HBB-B1, and HBB-B2, and those found in humans, including HBA-A1, HBA-A2, HBB, HGB1, and HGB2. Mitochondrial rRNA suitable for depletion includes 18S and 12S (humans and rodents). Nuclear rRNA suitable for depletion includes 28S, 18S, 5.8S, and 5S (humans and rodents), and prokaryotic rRNA including 5S, 16S, and 23S. In some samples, depletion of rRNA derived from archaea, such as rRNA 23S, 16S, or 5S, may also be required. In additional embodiments, the probe set comprises at least two DNA probes complementary to two or more rRNA sequences selected from the group consisting of Gram-positive or Gram-negative rRNA 5S, 16S, and 23S. In some embodiments, the probe set comprises at least two (or at least five, at least ten, or at least twenty) DNA probes complementary to each of human 28S, 18S, 5.8S, 5S, 16S, and 12S, globin mRNA HBA-A1, HBA-A2, HBB, HBG1, and HBG2, and Gram-positive or Gram-negative rRNA 5S, 16S, and 23S. In some embodiments, the probe for a specific non-target RNA molecule is complementary to about 80 to 85% of the sequence of the non-target RNA molecule, and there is a gap of at least 5 or at least 10 bases between each probe hybridization site.

[0044] In some embodiments, the probe set comprises two or more, five or more, ten or more, twenty or more, fifty or more, ten or more, fifty or more, ten or more, ten or more, 100 or more, ten or more, 150 or more, twenty or more, twenty or more, 300 or more, or 333 sequences from SEQ NOs 1 to 333 (human, Gram-positive, Gram-negative, archaea, mouse, and rat). In some embodiments, the probe set comprises two or more, five or more, ten or more, twenty or more, twenty or more, fifty or more, ten or more, ten or more, fifty or more, ten or more, ten or more, 100 or more, ten or more, ten or more, twenty or more, twenty or more, twenty or more, twenty or more, twenty or more, 350 or more, four or more, or 428 sequences from SEQ NOs 1 to 428 (human, Gram-positive, Gram-negative, archaea, mouse, and rat). In some embodiments, the probe set comprises two or more, five or more, ten or more, twenty or more, fifty or more, ten or more, fifty or more, ten or more, ten or more, ten or more, ten or more, 150 or more, twenty or more, twenty or more, twenty or more, twenty or more, three or more, three or more, or three or more, or 377 sequences from SEQ NOs 1 to 377 (human, Gram-positive, Gram-negative bacteria, and archaea). In some embodiments, the probe set comprises two or more, five or more, ten or more, twenty or more, twenty or more, fifty or more, ten or more, ten or more, ten or more, ten or more, ten or more, ten or more, ten or more, twenty or more, twenty or more, three or more, three or more, or three or more, or 384 sequences from SEQ NOs 1 to 333 (human, Gram-positive, and Gram-negative bacteria) and SEQ NOs 378 to 428 (mouse and rat). In some embodiments, the probe set comprises two or more, five or more, ten or more, twenty or more, or forty-four sequences from sequence numbers 334 to 377 (archaea). In some embodiments, the probe set comprises two or more, five or more, ten or more, twenty or more, fifty or more, or fifty-one sequences from sequence numbers 378 to 428 (mice and rats).

[0045] In some embodiments, the DNA probe comprises a sequence that is partially or completely complementary and hybridizes to human 28S, 18S, 5.8S, and / or 5S rRNA, for example, DNA probe sequences shown in SEQ ID NOs 40 to 150 of Table 1. In another embodiment, the DNA probe comprises a sequence that hybridizes to mitochondrial rRNA 16S and / or 12S, for example, DNA probe sequences shown in SEQ ID NOs 1 to 39 of Table 1. In another embodiment, the DNA probe comprises a sequence that hybridizes to hemoglobin mRNA including HBA-A1, HBA-A2, HBB, HBB-B1, HBB-B2, HBG1, and / or HBG2, for example, DNA probe sequences shown in SEQ ID NOs 151 to 194 of Table 1. In some embodiments, the DNA probe comprises a sequence that hybridizes to bacterial rRNA such as Gram-positive and / or Gram-negative rRNA 23S, 16S, and / or 5S, for example, the DNA probe sequences shown in SEQ ID NOs 195 to 262 of Table 1 (E. coli representing Gram-negative bacteria) and SEQ ID NOs 263 to 333 (Bacillus subtilis representing Gram-positive bacteria). In other embodiments, the DNA probe comprises a sequence that hybridizes to archaea rRNA such as rRNA 23S, 16S, and / or 5S, for example, archaea species, Methanobrevibacter smitii ( Methanobrevibacter smithiiIt comprises DNA probe sequences shown in SEQ NOs 334 to 384 of Table 1 that hybridize to rRNA derived from ). In some embodiments, the DNA probe comprises sequences that hybridize to mouse rRNA, such as mouse 16S and / or 28S, for example, DNA probe sequences shown in SEQ NOs 385 to 393 and SEQ NOs 400 to 419 of Table 1. In some embodiments, the DNA probe comprises sequences that hybridize to rat rRNA, such as rat 16S and / or 28S, for example, DNA probe sequences shown in SEQ NOs 394 to 399 and SEQ NOs 420 to 428 of Table 1.

[0046] [Table 1]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058] In one embodiment, the RNA sample is derived from a human, and the DNA probe set comprises probes specific to human unwanted RNA species as described herein, such as rRNA and mitochondrial mRNA transcripts. In another embodiment, the DNA probe set for depleting unwanted RNA from a human RNA sample comprises probes specific to human rRNA and mitochondrial mRNA transcripts as described herein, and probes specific to unwanted RNA transcripts of Gram-positive and Gram-negative bacteria. In a further embodiment, the DNA probe set for depleting unwanted RNA from a human RNA sample comprises probes specific to archaea species, examples of which are Methanobrevibacter smitii as described herein. Thus, in some embodiments, the DNA probe set for depleting rRNA from a human RNA sample comprises only probes relating to human unwanted RNA species, or comprises a mixed DNA probe set targeting non-human unwanted RNA transcripts. Those skilled in the art will understand that the set of probes used for RNA depletion depends on the research intent regarding the sample, the environment in which the sample was collected, and any other factors leading to the experimental design for RNA depletion of the RNA sample.

[0059] In one embodiment, the RNA sample is derived from a non-human eukaryote, and the DNA probe set includes probes specific to unwanted RNA in the eukaryotic source sample. For example, if the RNA sample is derived from a mouse or rat, the DNA probe set will include probes specific to unwanted RNA species of the mouse or rat, and may also include DNA probes specific to unwanted Gram-positive and Gram-negative RNA species, or other bacterial species, such as archaea.

[0060] In some embodiments, the DNA probe is not hybridized over the entire continuous length of the RNA species to be depleted. Surprisingly, it was found during experiments that the full-length sequence of the RNA species targeted for depletion does not need to be targeted by a full-length DNA probe or a set of probes tiling continuously across the entire RNA sequence; indeed, the DNA probes described herein leave gaps so that the formed DNA:RNA hybrids are not continuous. Surprisingly, gaps of at least 5 nt, 10 nt, 15 nt, or 20 nt between DNA:RNA hybrids provided efficient RNA depletion. Furthermore, a set of probes containing gaps can be hybridized more efficiently to the unwanted RNA because the DNA probe does not interfere with the hybridization of adjacent probes that could potentially occur by probes covering the entire RNA sequence targeted for depletion or by probes overlapping each other.

[0061] Additionally, the probe set may be supplemented to improve the RNA depletion method for a given species. A method for supplementing a probe set used to deplete non-target RNA nucleic acid molecules from a nucleic acid sample comprises: a) contacting a nucleic acid sample comprising at least one RNA or DNA target sequence and at least one non-target RNA molecule of a first species with a probe set comprising at least two DNA probes complementary to a discontinuous sequence along the entire length of at least one non-target RNA molecule of a second species to hybridize the DNA probes to the non-target RNA molecule to form a DNA:RNA hybrid—wherein each DNA:RNA hybrid is separated from any other DNA:RNA hybrid by at least 5 bases or by at least 10 bases along a given non-target RNA molecule sequence—; b) contacting the DNA:RNA hybrid with a ribonuclease that degrades RNA from the DNA:RNA hybrid to degrade the non-target RNA molecule of the nucleic acid sample to form a degradation mixture; c) separating the degraded RNA from the sample; and d) sequencing the residual RNA from the sample. e) evaluating the residual RNA sequence for the presence of non-target RNA molecules of the first species to determine a gap sequence region; and f) supplementing the probe set with at least one DNA probe complementary to the discontinuous sequence in one or more gap sequence regions. In some embodiments, the gap sequence region comprises 50 or more, 60 or more, or 70 or more base pairs. In some embodiments, the first species is a non-human species and the second species is human. In some embodiments, the first species is a rat or a mouse. An exemplary method for supplementing the probe set to improve the depletion of non-target rRNA nucleic acid molecules in mouse samples is summarized in Example 8 and FIG. 9.

[0062] In some embodiments, the first species is a non-human species, and the second species is human. In some embodiments, the first species is a rat or a mouse. In some embodiments, the second species is human, Gram-positive bacteria, Gram-negative bacteria, or a mixture thereof.

[0063] Composition and kit

[0064] In one embodiment, the present invention relates to a composition comprising a probe set as described herein. In some embodiments, the composition comprises a probe set and a ribonuclease capable of degrading RNA in a DNA:RNA hybrid, such as RNase H or hybridase. In some embodiments, the probe set comprises at least two DNA probes complementary to at least one non-target rRNA molecule in a nucleic acid sample, said probes not overlapping and discontinuous with respect to the length of said non-target rRNA molecule (e.g., separated by at least 5 or at least 10 bases along the entire length). In some embodiments, the present composition comprises a probe set comprising at least two DNA probes that hybridize to at least one non-target RNA molecule, each DNA probe being hybridized from any other DNA probe in the probe set by at least 5 or at least 10 bases along the length of the non-target RNA molecule. In some embodiments, the composition comprises a nucleic acid destabilizing chemical, such as formamide, betaine, DMSO, glycerol, or a derivative thereof, or a mixture thereof. In one embodiment, the nucleic acid destabilizing chemical is formamide or a derivative thereof present at a concentration of 10 to 45% of the total hybridization reaction volume.

[0065] In one embodiment, the present invention describes a kit comprising a probe set including at least two DNA probes complementary to a discontinuous sequence along the full length of at least one non-target rRNA molecule in a nucleic acid sample (e.g., separated by at least 5 or at least 10 bases along the full length) and a ribonuclease capable of degrading RNA in a DNA:RNA hybrid. In some embodiments, the probe set includes any of the DNA probes described herein or any combination thereof.

[0066] In some embodiments, the kit comprises a buffer and a nucleic acid purification medium. In some embodiments, the kit comprises a buffer, a nucleic acid purification medium, and one or more of a DNA probe set as described herein. In some embodiments, the probe set comprises two or more sequences from SEQ NOs 1 to 333. In some embodiments, the probe set comprises two or more sequences from SEQ NOs 1 to 428. In some embodiments, the probe set comprises two or more, five or more, ten or more, twenty or more, fifty or more, one hundred or more, one hundred or more, one hundred or more, two hundred or more, two hundred or more, three hundred or more, three hundred or more, or 377 sequences from SEQ NOs 1 to 377 (human, Gram-positive, Gram-negative, and archaea). In some embodiments, the probe set comprises two or more, five or more, ten or more, twenty or more, fifty or more, ten or more, twenty or more, 100 or more, 150 or more, twenty or more, twenty or more, twenty or more, thirty or more, thirty or more, or 384 sequences from SEQ NOs 1 to 333 and SEQ NOs 378 to 428 (human, Gram-positive bacteria, Gram-negative bacteria, mouse and rat). In some embodiments, the probe set comprises two or more, five or more, ten or more, twenty or more, twenty or more, or 44 sequences from SEQ NOs 334 to 377 (archaea). In some embodiments, the probe set comprises two or more, five or more, ten or more, twenty or more, fifty or more, or 51 sequences from SEQ NOs 378 to 428 (mouse and rat).

[0067] In some embodiments, the kit comprises 1) a set of probes as described herein; 2) a ribonuclease; 3) a DNase; and 4) RNA purification beads. In some embodiments, the kit comprises an RNA depletion buffer, a probe depletion buffer, and a probe removal buffer.

[0068] Analysis of depleted samples

[0069] The disclosed method also finds utility in analyzing transcriptomes from single or mixed samples. Transcriptome analysis can be hindered by high relative abundance of ribosomal RNA; for example, a sample may contain more than 85% of rRNA molecules in the total RNA from bacterial cells. Due to such large amounts of rRNA competing with sequencing or other analytical reagents, it can be difficult to focus on more beneficial portions of the transcriptome that may be lost in the background of unwanted rRNA analysis. The disclosed method can facilitate the analysis of transcriptomes rich in microbial or eukaryotic isolates, for example, with low DNA input, thereby reducing rRNA sequencing reads, enabling lower sequencing costs, and allowing metatranscriptomic analysis of low biomass samples. This is exemplified in Example 4, where low input (<80 ng) from a mixed sample was evaluated using the RNase H rRNA depletion method described in the present invention. The method described herein may be used with various downstream applications, such as the production of libraries for nucleic acid sequencing technology, by using the enriched sample in RT-PCR, followed by microarray analysis, PCR, qPCR, etc. However, it should be understood that the enriched RNA sample produced by the RNA depletion method described herein is not limited to any specific downstream application, such as sequencing.

[0070] For example, a sequencing library can be constructed using an RNA-depleted sample so that the constructed library can be attached to a fixed position within the array, and thus the array can be repeatedly imaged without their relative positions changing. For example, an embodiment in which images are acquired in different color channels that match different labels used to identify one nucleotide base type from another is particularly applicable. In some embodiments, the method for determining the nucleotide sequence of the target nucleic acid may be an automated method. A preferred embodiment includes sequencing-by-synthesis ("SBS") technology.

[0071] SBS technology generally involves the enzymatic elongation of an initial nucleic acid strand through the repetitive addition of nucleotides to a template strand. In conventional SBS methods, a single nucleotide monomer may be provided to a target nucleotide in the presence of polymerase in each delivery. SBS may utilize nucleotide monomers having a terminator moiety or nucleotide monomers lacking any terminator moiety. Methods using terminator-free nucleotide monomers include, for example, pyrosequencing and sequencing using γ-phosphate-labeled nucleotides. In methods using terminator-free nucleotide monomers, the number of nucleotides added in each cycle is generally variable and depends on the template sequence and the nucleotide delivery method. In SBS technology using a nucleotide monomer having a terminator portion, the terminator may be substantially irreversible under the sequencing conditions used, as in the case of conventional Sanger sequencing using dideoxynucleotides, or the terminator may be reversible, as in the case of sequencing methods developed by Solexa (currently Illumina Incorporates).

[0072] Sequencing methods capable of utilizing RNA depletion workflows and RNA enrichment samples include, for example, cycle sequencing achieved by the stepwise addition of reversible terminator nucleotides containing cleavable or photobleachable dyes, but are not limited thereto. Examples of Illumina instruments capable of utilizing the methods described herein include commercially available HiSeq™, MiSeq™, NextSe™, NovaSeq™, NextSeq™, and iSeq™ instruments.

[0073] Additional sequencing techniques include sequencing by ligation. These techniques use DNA ligase to incorporate oligonucleotides and confirm the incorporation of these oligonucleotides.

[0074] In addition, nanopore sequencing can also be used for the preparation of libraries using RNA-depleted samples. The nanopore sequencing method sequences nucleic acid strands passing through pores, where a change in current through the pores indicates that nucleotides are passing through the pores.

[0075] In addition, sequencing using real-time monitoring of DNA polymerase activity can be performed on RNA-depleted samples.

[0076] Additional SBS techniques for constructing a library for sequencing using RNA-depleted samples described herein include the detection of protons released when nucleotides are incorporated into an extension product. For example, sequencing based on the detection of released protons may use electrical detectors and related technologies commercially available from Ion Torrent (a subsidiary of Life Technologies, Guildford, Connecticut, USA).

[0077] Further downstream applications that may utilize the enriched samples after RNA depletion described herein include PCR, qPCR, microarray analysis, etc. For example, microarray analysis is a powerful technique for studying gene expression. The enriched samples may be used in microarray analysis by converting the enriched RNA into cDNA according to a method known to those skilled in the art (e.g., reverse transcriptase polymerase chain reaction (RT-PCR)). Subsequently, the cDNA may be immobilized on a substrate and a microarray probe applied to determine expression analysis according to a number of microarray analysis methods (e.g., commercially available microarray analysis systems sold by Agilent, Affymetrix, Illumina, etc.). Polymerase chain reaction (PCR) or quantification PCR (qPCR) may also utilize the enriched samples as substrates according to established techniques (Current Protocols for Molecular Biology).

[0078] As such, RNA-depleted samples produced by the method described herein may be used to produce sequencing libraries, amplification products, etc., which can be used in downstream analysis methods. The disclosed method is not limited to any downstream application.

[0079] Examples

[0080] The following examples are merely illustrative and are not intended to limit the scope of this application. Variations will be obvious and understood by those skilled in the art and are incorporated into the spirit and disclosure of this application.

[0081] Example 1 - Depletion of unwanted RNA species from samples

[0082] In this embodiment, total RNA is the target nucleic acid of the sample, and RNA depletion comprises four main steps: 1) hybridization, 2) RNase H treatment, 3) DNase treatment, and 4) target RNA cleanup.

[0083] Hybridization is performed by annealing a prescribed set of DNA probes onto the denatured RNA of the sample. 10 to 100 ng of RNA samples are incubated in a tube containing 1 μL of a 1 μM / oligo DNA oligo probe set (probes corresponding to sequence numbers 1 to 333 as listed in Table 1), 3 μL of 5X hybridization buffer (500 mM Tris HCl pH 7.5 and 1000 mM KCl), 2.5 μL of 100% formamide, and sufficient water for a total reaction volume of 15 μL. The hybridization reaction mixture is incubated at 95°C for 2 minutes to denature the nucleic acids, and then slowly cooled to 37°C by reducing the temperature at a rate of 0.1°C / sec and maintained at 37°C. Once the reaction mixture reached 37°C, no further incubation time was required. The total time required to reach 37°C for denaturation is about 15 minutes.

[0084] After hybridization, add the following ingredients to the reaction tube to remove unwanted RNA species with RNase H from the DNA:RNA duplex: 4 μL of 5X RNase H buffer (100 mM Tris pH 7.5, 5 mM DTT, 4 μL of 0 mM MgCl2) and 1 μL of RNase H enzyme. Incubate the enzyme reaction mixture at 37°C for 30 minutes. The reaction tube may be kept on ice.

[0085] After removing RNA from the DNA:RNA hybrid, the DNA probe is degraded. In a 20 μL reaction tube, add the following ingredients to make a total volume of 30 μL: 3 μL 10X Turbo DNase buffer (200 mM Tris pH 7.5, 50 mM CaCl2, 20 mM MgCl2), 1.5 μL Turbo DNase (Thermo Fisher Scientific), and 5.5 μL H2O. Incubate the enzyme mixture at 37°C for 30 minutes, followed by at 75°C for 15 minutes. The 75°C incubation serves to fragment the target total RNA to a desired insertion size for use in downstream processing; in this example, the target insertion size is approximately 200 nt of total RNA. The timing of this incubation step can be adjusted according to the insertion size required for the subsequent reaction, as is known to those skilled in the art. After incubation, the reaction tube can be kept on ice.

[0086] After hybridizing the probe to unwanted RNA to remove the RNA and DNA, the target total RNA of the sample can be isolated under reaction conditions. The reaction tube was taken at 4°C, allowed to reach room temperature, and 60 μL of RNAClean XP beads (Beckman Coulter) was added, and the reaction tube was incubated for 5 minutes. After incubation, the tube was placed on a magnet for 5 minutes, and the supernatant was carefully removed and discarded. While still on the magnet, the beads with attached total RNA were washed twice with 175 μL of fresh 80% EtOH. After the second wash, the beads were spun down in a microcentrifuge to pellet the beads at the bottom of the tube, and the tube was placed back on the magnet to remove the EtOH, taking care to remove as much residual EtOH as possible without disturbing the beads. Air-dry the beads for a few minutes, resuspend them in 9.5 μL of ELB buffer (Illumina), let them stand at room temperature for a few more minutes, place them back on a magnet, and collect the beads. Transfer 8.5 μL of the supernatant to a new tube and place it on ice for further downstream processing, such as generating cDNA from target total RNA.

[0087] In another example, 100 ng of total RNA is diluted with 11 μL of nuclease-free ultrapure water in each well of a 96-well PCR plate. 4 μL of DNA probes (SEQS NOs 1 to 333) in hybridization buffer is added to each well, the well contents are mixed, and centrifuged optionally. The plate is heated to 95°C for 2 minutes, then the temperature is reduced at 0.1°C / sec until it reaches 37°C, and then maintained at 37°C to hybridize the probes. The plate is centrifuged at 280 × g for 10 seconds. To degrade the DNA:RNA hybrid, 5 μL of RNase in buffer is added to each well, and the well contents are mixed. The plate is heated to 37°C for 15 minutes, then maintained at 4°C. Add 10 μL of RNase in buffer to each well and mix the well contents. Heat the plate to 37°C for 15 minutes, then maintain at 4°C. Centrifuge the sample plate at 280 × g for 10 seconds. Add 60 μL of RNAClean XP beads to each well and mix the well contents. Incubate the plate at room temperature for 5 minutes. Place the plate on a magnetic stand until the supernatant becomes clear (approximately 5 minutes). Remove and discard the supernatant from each well. Wash the beads twice with 80% ethanol. Remove residual ethanol from each well and air-dry the plate on a magnetic stand for 1 minute. Add 10.5 μL of elution buffer to each well and mix the well contents, then incubate the plate at room temperature for 2 minutes. Seal the plate and centrifuge at 280 × g for 10 seconds. Place the plate on a magnetic stand until the supernatant becomes clear (about 2 minutes). Transfer 8.5 μL of supernatant from each well to the corresponding well of a new plate.

[0088] Example 2 - cDNA Synthesis

[0089] Further processing of the RNA in Example 1 may, for example, involve preparing a library formulation from RNA target nucleic acids that can be sequenced by NGS. To 8.5 μL of the final reaction mixture of Example 1, 8.5 μL of Elute, Prime High Concentration Random Hexamer Mix buffer (EPH buffer, TruSeq Stranded Total RNA Kit, Illumina) is added to make a total volume of 17 μL. The sample is incubated at 65°C for 2 minutes to denature the nucleic acids. After denaturation, the reaction tube may be kept on ice. First strand synthesis is performed by adding 8 μL of reverse transcriptase mixture (9 μL of First Strand Synthesis Mix (FSA, TruSeq Stranded Total RNA Kit, Illumina) and 1 μL of Protoscript II RT (NEB)) to the denatured sample to a total volume of 25 μL. The reaction mix is ​​incubated in a heated lid thermocycler under the following conditions: incubation at 25°C for 5 minutes, at 42°C for 25 minutes, and at 70°C for 15 minutes. Once the first strand synthesis reaction is complete, the reaction tube may be kept on ice.

[0090] Second strand cDNA synthesis can be performed by adding 5 μL of resuspension buffer (RSB, TruSeq Stranded Total RNA Kit, Illumina) and 20 μL of second strand marking mix (SSM buffer, TruSeq Stranded Total RNA Kit, Illumina) to an ice-cooled sample. The reaction tube can be incubated at 16°C for 60 minutes, and then the sample can be kept on ice.

[0091] After the cDNA synthesis step, the cDNA can be cleaned up and separated from the reaction components by adding, for example, 90 μL of SPB (Illumina) to the reaction tube and incubating at room temperature for 5 minutes. After incubation, the tube is placed on a magnet for about 8 minutes to collect the paramagnetic beads, and the supernatant is carefully removed and discarded. While still on the magnet, the beads are washed twice with 175 μL of fresh 80% EtOH. After washing, the beads are centrifuged to the bottom of the tube, the tube is placed back on the magnet, and the EtOH is carefully removed and discarded. The beads are dried for a few minutes, resuspended in 18.5 μL of RSB, mixed well, incubated at room temperature for about 5 minutes, and then placed back on the magnet. Depending on the downstream application, the desired amount of purified cDNA can be removed into a new tube. In this embodiment, a library preparation for downstream sequencing is prepared, and 17.5 μL of the supernatant is transferred to a new tube that can be kept on ice.

[0092] Example 3 - Preparation of a library for next-generation sequencing

[0093] One method for preparing a sequencing library includes, prior to sequencing, A-tailing of the cDNA fragment, ligating of the adapter, amplification of the target fragment, and quantification of the obtained fragment.

[0094] Use the tube containing 17.5 μL of purified cDNA from Example 2 for processing. Add 12.5 μL of ATL (Illumina) to the purified cDNA to A-tail the fragment. Incubate the reaction tube at 37°C for 30 minutes, then incubate at 70°C for 5 minutes, and place the tube back on ice. Ligate the adapter to the A-tailed sample by adding 2.5 μL of RSB, 2.5 μL of Index Adapter (TruSeq Stranded Total RNA Kit, Illumina), and 2.5 μL of Ligation Buffer (Illumina) in that order. Incubate the reaction tube at 30°C for 10 minutes, then add 5 μL of Stop Ligation Buffer (Illumina) and keep the reaction mixture on ice.

[0095] Once the adapter ligation reaction is complete, the ligated fragment is separated from the reaction components. To purify the adapter-ligated fragment, 34 μL of SPB is added to the reaction tube and incubated at room temperature for about 5 minutes. Subsequently, the tube is placed on a magnet to capture the paramagnetic beads, and the beads are washed twice with 175 μL of 80% EtOH, with the EtOH carefully removed after the second wash. After air-drying the beads for 3 minutes, the beads are resuspended in 52 μL of RSB, the slurry is mixed, left to stand at room temperature for an additional 5 minutes, and placed back on the magnet. The supernatant (50 μL) is transferred to a new tube for the second round of bead cleanup.

[0096] In the second round above, the process described above is repeated to increase the amount of the target sequence for optimized sequencing results, except that 40 μL of SPB is added to a 50 μL sample, the final purified fragment is resuspended in 21 μL of RSB, and 20 μL of the final purified sample is transferred to a new reaction tube for subsequent amplification.

[0097] To 20 μL of purified adapter-ligated sample, 5 μL of PCR primer cocktail (PPC, TruSeq Stranded Total RNA Kit, Illumina) and 25 μL of PPM (TruSeq Stranded Total RNA Kit, Illumina) are added, and the following amplification program is performed in a heated lead thermocycler, namely, a cycling program consisting of 30 seconds at 98°C, followed by 10 seconds at 98°C, 30 seconds at 60°C, and 30 seconds at 72°C. The number of amplification cycles depends on the amount of RNA input at the start of the entire process. For example, for 100 ng RNA, about 12 to 13 cycles may be appropriate, for 10 ng, 15 to 16 cycles, and for 1 ng, 17 to 18 cycles may be required. The number of amplification cycles is typically optimized for all preparations known to those skilled in the art.

[0098] 50 μL of SPB is added to the reaction tube and incubated at room temperature. The tube is centrifuged to pellet the beads, and the amplicon can be purified by removing the beads from the reaction conditions through magnetic capture. The supernatant can be discarded, and after washing the beads as described above, the washed beads are resuspended in 26 μL of RSB, the beads are magnetically captured, and the supernatant containing the DNA library for sequencing can be transferred to a new tube. The library is typically quantified and analyzed prior to sequencing by measuring aliquots using, for example, the Qubit™ High Sensitivity Kit (Thermo Fisher Scientific) and / or by running aliquots on a bioanalyzer (Agilent). Those skilled in the art will recognize many methods for quantifying nucleic acids in a sample.

[0099] Subsequently, the obtained library formulation can be used for next-generation sequencing, microarray analysis, or other downstream applications. For applications such as sequencing, the library preparation method is determined by the sequencing instrument used and the companion library preparation method defined for that sequencing instrument. In this embodiment, the library preparation method describes the characteristic of the library being prepared when sequencing on an Illumina sequencing instrument. Those skilled in the art will understand that the library preparation method may vary depending on the sequencing instrument; therefore, the embodiments of the present invention are merely illustrative, and the RNA depletion method of the present invention is not limited to a specific library preparation workflow. In practice, the method of the present invention provides an RNA-depleted sample that can be input into any downstream application that can benefit from the RNA sample from which unwanted RNA species have been depleted.

[0100] Example 4 - Microbial Transcriptome Analysis

[0101] In this embodiment, microbial isolates, mixed samples of bacterial species, and standard cell mixtures were obtained from ATCC for testing.

[0102]

[0103] Total RNA can be extracted using the RNeasy Power Microbiome Kit (Qiagen) according to the manufacturer's protocol, evaluated for integrity, and quantified using BioAnalyzer RNA Electrophoresis (Agilent). 10 to 250 ng of total RNA from each sample can be used for rRNA depletion according to the method disclosed herein using the RiboZero method (Illumina, according to the manufacturer's protocol) or the RNase H enzymatic degradation of unwanted rRNA. Ribo-depleted and non-ribo-depleted RNA (control) samples can be prepared for sequencing using the TruSeq Stranded Total RNA Sample prep kit (Illumina) according to the manufacturer's instructions. The library can be pooled and sequenced on a MiSeq or NextSeq sequencing instrument (Illumina) for 2 × 76 paired terminal reads, for example.

[0104] Sequence filtering, alignment, and transcriptome coverage can be performed using the online BaseSpace Sequencing Hub (BSSH) and the following exemplary workflows, for example: 1) Partition rRNA Sequence App (analyzing rRNA sequences to be marked as enriched sequences in the analysis), 2) RNA Custom Genome Builder App (generating a STAR-compatible microbial transcriptome), and 3) RNA-Seq Alignment App (STAR-alignment and salmon-transcriptome quantification). To quantify rRNA from multiple strains within a microbial sample, rRNA sequences can be retrieved from NCBI-annotated genomes and used as input to the BSSH workflow.

[0105] Transcriptomes of microbial isolates, microbial mixtures, and control samples were sequenced to compare % rRNA reads. The RNase H enzyme method disclosed herein is highly effective in depleting unwanted rRNA in the tested species (<5% rRNA reads). Ribosomal RNA depletion is most pronounced in low-dose samples (10 ng) of E. coli using the RNase H method compared to the established ribozero method; with average rRNA reads of <0.5% versus 13% (Fig. 5).

[0106] Data were used to evaluate the enrichment of biologically important RNA reads compared to the absence of rRNA depletion using the RNase H rRNA depletion method. Figure 6 shows the evaluation results, which generally show a 20 to 50-fold reduction in read depth observed for Bacillus subtilis or E. coli samples when the samples were rRNA depleted prior to library preparation and sequencing using the RNase H method, compared to the absence of rRNA depletion.

[0107] The collected data were evaluated to determine the reproducibility of the experimental microbial transcriptome sequencing effort. Pairwise linear regression of gene expression levels was determined between RNase H rRNA-depleted replicas of Escherichia coli and Bacillus subtilis as example systems. High correlation (R 2 >0.99) demonstrated the ability of the RNase H rRNA depletion method to reproducibly remove rRNA from the sample (Fig. 7).

[0108] To evaluate whether the RNase H enzyme rRNA depletion method could be useful for rRNA depletion in mixed samples, Figure 8 shows exemplary data for mixed samples of 20 strains MSA2002 and human enteric bacteria MSA2006 in triplicate. Low input samples of 10 mg total RNA from MSA2002 or 80 ng total RNA from MSA2006 were used for the rRNA depletion method. For the 20 strain MSA2002 samples, the RNase H rRNA depletion method reduced rRNA reads to 83% or <2% of sequence reads, whereas the ribozero method of rRNA depletion showed higher rRNA abundance and greater variability compared to the non-depleted samples. For the 12 strain MSA2006 samples, the RNase H method showed similar results, reducing rRNA reads to approximately 95% to <13% of sequence reads compared to the non-depleted samples, while the ribozero method yielded more variable results.

[0109] Thus, in experiments to evaluate samples, it was determined that the RNase H rRNA depletion method, whether mixed or otherwise, provides a stable and effective workflow for reducing unwanted rRNA in samples for high-quality microbial whole transcriptome studies. The RNase H rRNA depletion method was also highly effective and suitable for low-input samples.

[0110] Example 5 - Effect of formamide on RNA depletion

[0111] FIG. 2 shows exemplary data on the depletion of unwanted RNA species in RNA samples. Using the method described herein, the effect of formamide concentration on unwanted RNA depletion was evaluated while depleting unwanted RNA in RNA samples. In this embodiment, the DNA probe targets the depletion of unwanted rRNA species derived from Gram-positive bacteria (23S, 16S, 5S), Gram-negative bacteria (including 23S, 16S, 5S), human mitochondria (16S, 12S), human rRNA (28S, 18S, 5.8S, 5S), and human hemoglobin mRNA (HBA-A1, HBA-A2, HBB, HBG1, HBG2), and the target RNA species is total RNA derived from Bacillus subtilis. As the concentration of formamide increases, the proportion of unwanted RNA species leads significantly decreases. For example, in the absence of formamide upon RNA depletion, non-target RNA reads were generated for Gram-positive 23S and 16S, including E. coli-specific sequences, and for Gram-negative 23S and 16S (including E. coli). When 25% formamide was added to the hybridization reaction mixture, undetectable non-target reads were generated for Gram-negative 23S and 16S (E. coli-specific non-target reads were significantly reduced), and non-target reads were significantly reduced for Gram-positive 23S and 16S. When 45% formamide was added to the hybridization reaction mixture, not only were non-target reads for undesirable Gram-positive rRNA 23S and 16S further significantly reduced, but non-target E. coli reads were also further reduced. As such, the addition of formamide to RNA-depleted hybridization reactants appears to increase the amount of undesirable RNA in depleted Gram-positive and Gram-negative bacteria, as evidenced by the reduction of off-target leads for these species. In general, the addition of formamide has been found to improve the depletion of unwanted rRNA transcripts.When Bacillus subtilis RNA is used as the target RNA for analysis, for example, analysis of E. coli and human rRNA sequences can provide a measure of potential contamination.

[0112] Example 6 - Change in Input Amount of Starting Material

[0113] As shown in Figure 3, experiments were conducted to determine the effect of input starting materials on RNA depletion and subsequent downstream analysis. Libraries for sequencing were constructed on Illumina NextSeq™ 500 or 550 sequencing instruments using RNA depleted and enriched RNA samples from human brain (HBR) and universal human RNA (UHR). Sequencing libraries were prepared after RNA depletion using input samples of 100 ng, 10 ng, or 1 ng as exemplified in Examples 1 through 3. Following data analysis using two Basespace (Illumina) applications, the RNASeq alignment application, and the RNAExpress application, sequencing was performed as recommended in the NextSeq™ User Guide. Data analysis for the presence of Bacillus subtilis and Escherichia coli was also performed using the modified tool Fastqscreen (https: / / www.bioinformatics.babraham.ac.uk / projects / fastq_screen / ). The data show that regardless of the RNA sample input amount and the amount of % total alignment to the target RNA, RNA depletion remains constant for both HBR and UHR, although it decreases with decreasing input amount; however, viable and useful sequence data can still be collected even when using a 1 ng input sample. Additionally, in comparative experiments, the current method for RNA depletion shows a lower % abundance of non-target reads at all input levels (100 ng–3%, 25 ng–4%, 10 ng–3%, and 1 ng–35%) when compared to data from using the Ribozero rRNA Depletion Kit (Epicenter) for RNA depletion (100 ng–3%, 25 ng–5%, 10 ng–8%, and 1 ng–35%) or the NEBNext rRNA Depletion Method (NEB) (100 ng–8%, 25 ng–8%, 10 ng–9%, and 1 ng–30%).

[0114] Example 7 - RNA Depletion of Mouse and Rat RNA Samples

[0115] To demonstrate that the RNA depletion method may be useful for non-human RNA samples, both mouse and rat RNA samples were used in the RNA depletion method. In the case of Figure 4, unwanted RNA was depleted from mouse or rat RNA samples using the same method and DNA probes as for human RNA samples. Formamide was then varied for each rodent species, including no formamide, 25% formamide, or 45% formamide in the hybridization reactants. The total % aligned reads were not affected by the increase in formamide, but there may be a tendency for the detection of non-target reads to increase as formamide increases. As such, adding formamide to the hybridization reactants may be useful for some sample types, as it can improve the detection of certain transcripts, and thus its addition should be optimized.

[0116] Example 8 - Preparation of additional mouse probes

[0117] Within the aforementioned pool of 333 DNA probes for the enzymatic removal of unwanted sequences (Sequence Nos. 1 to 333), DNA oligonucleotides for eukaryotic rRNA depletion were designed based on major human rRNA transcripts, namely 5S, 5.8S, 18S, and 28S, as well as two mitochondrial rRNA sequences, 12S and 16S. When tested against human total RNA, this pool of 333 DNA probes was highly effective in removing rRNA reads. However, mice (Mus musculus ( Mus musculus )) or rat(ratus norvezicus( Rattus norvegicus When tested with total RNA samples, depletion was less stable, which suggests that the probe failed to efficiently hybridize and remove some regions of the rodent rRNA sequence, because these mouse and rat regions are inconsistent with the human sequence.

[0118] The fastq file containing the total sequencing reads obtained from the 333-DNA probe experiment was aligned to mouse and rat ribosomal RNA sequences and to 333 DNA probe sequences. The alignment results indicated that while probe coverage across all ribosomal RNA sequences was generally excellent, there were some regions where probe sequences were not well aligned with rodent rRNA. More specifically, most of the mouse and rat rRNA reads not aligned to the probe pool map belonged to either the 28S or 16S rodent rRNA transcripts (Table 2). Bowtie2 (Langmead and Salzberg, Nature Methods [Refer to 2012, 9:357-359]), alignment was performed using its default settings in version 2.1.0. Most of the ribosomal RNA not depleted by the 333 DNA probe enzyme method originated from the same region lacking probe alignment (Fig. 9).

[0119] [Table 2]

[0120]

[0121] To deplete these regions more effectively, additional probes were designed to cover the regions identified above for mouse and rat ribosomal RNA sequences. To minimize the number of additional probes and probe duplications, additional probes were designed for gaps in mouse rRNA sequences, and these data were informatively pooled with a 333-DNA probe set to identify any remaining gaps in rat rRNA coverage by aligning the pool bound to rat rRNA transcripts. This sequential process yielded a total of 44 additional oligonucleotide probes, providing a supplemental pool of 377 probes. The sequencing experiments described above were repeated using the 377-DNA probe set. For both mouse and rat samples, the addition of the 44 new probes resulted in a decrease in the proportion of rRNA reads from the library compared to the 333-DNA probe set, indicating increased depletion efficiency (Table 3).

[0122] [Table 3]

[0123]

[0124] Supplementing the 333 DNA probe pool with additional probes for specific rodent sequences improved rRNA depletion in tested rodent samples. Exemplary probes for mouse 16S include sequence numbers 385 through 393. Exemplary probes for mouse 28S include sequence numbers 400 through 419. Exemplary probes for rat 16S include sequence numbers 394 through 399. Exemplary probes for rat 28S include sequence numbers 420 through 428.

Claims

Claim 1 A method for depleting off-target RNA molecules from a nucleic acid sample, comprising: a) contacting a nucleic acid sample comprising at least one target RNA or DNA sequence and at least one off-target RNA molecule with a probe set comprising at least 40 DNA probes complementary to a discontinuous sequence along the full length of the at least one off-target RNA molecule, thereby hybridizing the at least 40 DNA probes to the off-target RNA molecule to form a DNA and RNA hybrid, wherein (i) each of the at least 40 DNA probes is 40 to 60 bases long and matches at least 70% with the off-target RNA molecule, and (ii) each of the DNA and RNA hybrids is 5 to 40 bases apart from the given off-target RNA molecule sequence from another DNA and RNA hybrid to form the DNA and RNA hybrid; and b) a step of contacting the DNA and RNA hybrid with a ribonuclease that degrades RNA from the DNA and RNA hybrid to degrade non-target RNA molecules of the nucleic acid sample and form a degradation mixture; comprising a method. Claim 2 A method according to claim 1, comprising: c) a step of forming a DNA degradation mixture by degrading any residual DNA probe by contacting the degradation mixture with a DNA degradation enzyme, wherein the DNA degradation enzyme is DNase I; and d) a step of separating the degraded RNA from the degradation mixture or the DNA degradation mixture. Claim 3 A method according to claim 1, wherein the step of contacting the probe set comprises the step of treating the nucleic acid sample with a destabilizer. Claim 4 A method according to paragraph 3, wherein the destabilizing agent is heat or a nucleic acid destabilizing chemical, and the nucleic acid destabilizing chemical is betaine, DMSO, formamide, glycerol, or a mixture thereof. Claim 5 In claim 4, the nucleic acid destabilizing chemical is formamide, and the formamide is present at a concentration of 10 to 45 volume% while in contact with the probe set, method. Claim 6 In claim 4, the step of treating the sample with heat comprises applying heat higher than the dissolution temperature of the at least one DNA and RNA hybrid. Claim 7 A method according to claim 1, wherein the ribonuclease is RNase H or hybridase. Claim 8 In claim 1, the nucleic acid sample is derived from a human or non-human primate. Claim 9 In claim 1, the nucleic acid sample is derived from a rat or mouse. Claim 10 A method according to claim 1, wherein the nucleic acid sample comprises nucleic acid of non-human origin. Claim 11 In paragraph 10, the above-mentioned non-human derived nucleic acid is derived from non-human eukaryotes, bacteria, viruses, plants, soil, or a mixture thereof. Claim 12 The method according to claim 1, wherein the non-target RNA is rRNA, mRNA, tRNA, or a mixture thereof. Claim 13 In paragraph 12, the above non-target RNA is rRNA and globin mRNA, method. Claim 14 The method of claim 1, wherein the probe set comprises at least two DNA probes hybridized to at least one non-target RNA molecule selected from 28S, 23S, 18S, 5.8S, 5S, 16S, 12S, HBA-A1, HBA-A2, HBB, HBB-B1, HBB-B2, HBG1, and HBG2. Claim 15 A method according to claim 14, wherein the probe set comprises at least two DNA probes hybridized to two or more non-target RNA molecules selected from human-derived 28S, 18S, 5.8S, 5S, 16S, and 12S. Claim 16 The method of claim 1, wherein the probe set comprises at least two DNA probes hybridized to one or more non-target RNA molecules selected from HBA-A1, HBA-A2, HBB, HBG1, and HBG2 derived from hemoglobin, and 23S, 16S, and 5S derived from Gram-positive or Gram-negative bacteria. Claim 17 The method of claim 1, wherein the probe set comprises at least two DNA probes that hybridize to one or more non-target RNA molecules derived from Archaea species. Claim 18 The method of claim 1, wherein the probe set comprises at least two DNA probes hybridized to one or more non-target RNA molecules of one or both of rat and mouse, selected from rat 16S, rat 28S, mouse 16S and mouse 28S, and combinations thereof. Claim 19 A method according to claim 1, wherein the probe for a specific non-target RNA molecule is complementary to 80 to 85% of the sequence of the non-target RNA molecule, and there is a gap of at least 5 or at least 10 bases between each probe hybridization site. Claim 20 The method of claim 1, wherein the DNA probe comprises two or more sequences selected from SEQ ID NOs 1 to 428. Claim 21 A composition for depleting a non-target RNA molecule from a nucleic acid sample, comprising a probe set including at least 40 DNA probes complementary to a discontinuous sequence spaced 5 to 40 bases apart along the entire length of at least one non-target RNA molecule in the nucleic acid sample, and a ribonuclease capable of degrading RNA within a DNA and RNA hybrid, wherein each of the at least 40 DNA probes is 40 to 60 bases long and matches at least 70% with the non-target RNA molecule. Claim 22 A composition according to claim 21, wherein the ribonuclease is RNase H. Claim 23 A composition according to claim 21, wherein each DNA probe is hybridized from any other DNA probe in the set of probes by at least 10 bases along the entire length of the at least one non-target RNA molecule. Claim 24 A composition comprising an unstable chemical substance in paragraph 21. Claim 25 In paragraph 24, the above-mentioned destabilizing chemical is formamide, a composition. Claim 26 A composition according to claim 21, wherein the non-target RNA is rRNA, mRNA, tRNA, or a mixture thereof. Claim 27 In claim 26, the composition wherein the above-mentioned non-target RNA is rRNA and globin mRNA. Claim 28 A composition according to claim 21, wherein the probe set comprises at least two DNA probes hybridized to at least one non-target RNA molecule selected from 28S, 23S, 18S, 5.8S, 5S, 16S, 12S, HBA-A1, HBA-A2, HBB, HBG1, and HBG2. Claim 29 A composition according to claim 28, wherein the probe set comprises at least two DNA probes hybridized to two or more non-target RNA molecules selected from human-derived 28S, 18S, 5.8S, 5S, 16S, and 12S. Claim 30 A composition according to claim 21, wherein the probe set comprises at least two DNA probes hybridized to one or more non-target RNA molecules selected from HBA-A1, HBA-A2, HBB, HBG1, and HBG2 derived from hemoglobin, and 23S, 16S, and 5S derived from Gram-positive or Gram-negative bacteria. Claim 31 A composition according to claim 21, wherein the probe set comprises at least two DNA probes complementary to one or more rRNA molecules derived from archaea species. Claim 32 A composition according to claim 21, wherein the probe set comprises a DNA probe hybridized to one or more non-target RNA molecules of one or both of rat and mouse, selected from rat 16S, rat 28S, mouse 16S and mouse 28S, and combinations thereof. Claim 33 In claim 21, the composition wherein the DNA probe comprises two or more sequences selected from SEQ ID NOs 1 to 428. Claim 34 A kit for depleting a non-target RNA molecule from a nucleic acid sample, comprising a probe set including at least 40 DNA probes complementary to a discontinuous sequence spaced 5 to 40 bases apart along the entire length of at least one non-target RNA molecule in the nucleic acid sample, and a ribonuclease capable of degrading RNA within a DNA and RNA hybrid, wherein each of the at least 40 DNA probes is 40 to 60 bases long and matches at least 70% with the non-target RNA molecule. Claim 35 In paragraph 34, a kit comprising a buffer solution and a nucleic acid purification medium, and an destabilizing chemical. Claim 36 In paragraph 34, the above-mentioned non-target RNA is rRNA, mRNA, tRNA, or a mixture thereof, Kit. Claim 37 In paragraph 36, the above-mentioned non-target RNA is rRNA and globin mRNA, Kit. Claim 38 In claim 34, the probe set comprises at least two DNA probes hybridized to at least one non-target RNA molecule selected from 28S, 23S, 18S, 5.8S, 5S, 16S, 12S, HBA-A1, HBA-A2, HBB, HBG1, and HBG2, a kit. Claim 39 In claim 38, the probe set comprises at least two DNA probes hybridized to two or more non-target RNA molecules selected from human-derived 28S, 18S, 5.8S, 5S, 16S, and 12S, a kit. Claim 40 A kit according to claim 34, wherein the probe set comprises at least two DNA probes hybridized to one or more non-target RNA molecules selected from HBA-A1, HBA-A2, HBB, HBG1, and HBG2 derived from hemoglobin, and 23S, 16S, and 5S derived from Gram-positive or Gram-negative bacteria. Claim 41 In claim 34, the probe set comprises at least two DNA probes complementary to one or more rRNA molecules derived from archaea species, in a kit. Claim 42 In claim 34, the probe set comprises a kit comprising a DNA probe hybridized to one or more non-target RNA molecules of one or both of rat and mouse, selected from rat 16S, rat 28S, mouse 16S and mouse 28S, and combinations thereof. Claim 43 In claim 34, the DNA probe comprises two or more sequences selected from SEQ ID NOs 1 to 428, in a kit. Claim 44 A kit according to claim 34, comprising (1) a probe set including sequence numbers 1 to 333; (2) a ribonuclease which is RNase H; (3) a DNase; and (4) RNA purification beads; and further comprising an RNA depletion buffer, a probe depletion buffer, and a probe removal buffer. Claim 45 A method for replenishing a set of probes used to deplete non-target RNA nucleic acid molecules from a nucleic acid sample, comprising: a) contacting a nucleic acid sample comprising at least one RNA or DNA target sequence and at least one non-target RNA molecule of a first species with a set of probes comprising at least 40 DNA probes complementary to a discontinuous sequence along the entire length of at least one non-target RNA molecule of a second species, thereby hybridizing the DNA probes to the non-target RNA molecule to form a DNA and RNA hybrid, wherein each DNA and RNA hybrid is 5 to 40 bases apart from another DNA and RNA hybrid along a given non-target RNA molecule sequence, each of the at least 40 DNA probes is 40 to 60 bases long, and the DNA and RNA hybrid is at least 70% matched with the non-target RNA molecule; b) contacting the DNA and RNA hybrid with a ribonuclease that degrades RNA from the DNA and RNA hybrid to degrade the non-target RNA molecule of the nucleic acid sample to form a degradation mixture; c) the degraded RNA A method comprising: a step of separating from a decomposition mixture; d) a step of sequencing residual RNA from the sample; e) a step of determining a gap sequence region by evaluating the residual RNA sequence for the presence of non-target RNA molecules of the first species; and f) a step of supplementing a set of probes with additional DNA probes complementary to discontinuous sequences in one or more gap sequence regions. Claim 46 In claim 45, the method wherein the gap sequence region comprises 50 or more base pairs. Claim 47 In paragraph 45, the above first species is a non-human species, and the above second species is a human species, a method. Claim 48 In paragraph 47, the above first species is a rat or a mouse. Claim 49 A method according to claim 47, wherein the following composition is used to supply a probe set comprising a ribonuclease and DNA probes complementary to discontinuous sequences along the entire length of at least one human non-target RNA molecule, the composition comprising a probe set comprising at least 40 DNA probes complementary to discontinuous sequences spaced 5 to 40 bases apart along the entire length of at least one non-target RNA molecule in a nucleic acid sample, and a ribonuclease capable of degrading RNA within a DNA-RNA hybrid, wherein each of the at least 40 DNA probes is 40 to 60 bases long and matches at least 70% with the non-target RNA molecule. Claim 50 A method according to claim 48, wherein the method is used to identify a DNA probe hybridized to one or more non-target RNA molecules of one or both of rats and mice selected from rat 16S, rat 28S, mouse 16S and mouse 28S, and combinations thereof.

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