RU2787079C1 - Depletion of nuclease-based rna - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: present invention relates to the field of biotechnology, more specifically to new methods and materials for removal of undesired types of nucleic acids (in particular, RNA) from a sample of target nucleic acid.

EFFECT: invention discloses a sequence of actions for effective removal from a nucleic acid preparation of undesired rRNA, tRNA, mRNA, or other types, which can prevent analysis, manipulation, and study of target RNA molecules in a sample.

49 cl, 8 ex, 3 tbl, 9 dwg

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of U.S. Provisional Application Nos. 62/783,869, filed December 21, 2018, and 62/847,797, filed May 14, 2019, each of which is incorporated herein by reference in its entirety for all purposes. .

SEQUENCE LIST

[0002] This application is filed with a sequence listing in electronic format. The sequence listing is provided as a file named "2019-12-09_01243-0012-00PCT_Sequence_Listing_ST25.txt", created on December 9, 2019, which is 94,208 bytes in size. The information contained in the electronic sequence listing file is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

[0003] Unwanted RNA molecules in a nucleic acid sample, for example, taken from human cells or tissues, can complicate its analysis, for example, analysis of gene expression, microarray analysis, and deciphering the genetic sequence of the sample. Since ribosomal RNA (rRNA) makes up approximately 95% of the RNA in a cell, its presence is one example of those types of RNA that can interfere with and obscure the results of the analysis of the target nucleic acid in the sample, or those nucleic acids that the researcher or diagnostician intends to study. . For example, unwanted rRNA species can make it difficult to analyze the RNA molecules of interest in a sample, such as tRNA or mRNA. This is a persistent problem, especially for tissues that have been fixed, such as Formalin Fixed Paraffin Embedded (FFPE) specimens from biopsy specimens. If unwanted rRNAs are not removed from FFPE tissues, they can interfere with the measurement and characterization of the target RNA in the tissue, thereby making it extremely difficult to obtain medical information from the target RNA, such as disease and cancer identification, treatment options, and diagnosis and prognosis. disease or cancer. Although FFPE tissue is given as an example, the same problems associated with rRNA remain for samples of all kinds, such as blood, cells, and other samples containing nucleic acids.

[0004] Methods currently on the market for removing unwanted RNA molecules from a nucleic acid sample include the RiboZero® (Epicentre) and NEBNext® (NEB) RNA depletion kits, as well as the methods described in US Patent Nos. 9,745,570 and 9,005 891. However, these methods, while useful in RNA depletion, have their drawbacks, including complexity of application, high requirements for input samples, time spent by a technician on their use, cost and/or removal efficiency. What is needed are materials and methods that can more easily or economically remove unwanted RNA species from a sample, thereby unlocking possibly hidden information in the target RNA, such as rare or difficult to identify sequence variants. The simple and reliable methods of the present invention can greatly increase the availability of target RNA molecules for research purposes by making it possible to discover the information they store about the sample and the organism from which they are derived.

SUMMARY OF THE INVENTION

[0005] Samples, for example, obtained from eukaryotes or prokaryotes, contain many nucleic acids, many of which are not of interest to researchers. Quite often, there is a need to examine only a specific type of nucleic acid, such as DNA or RNA. When studying RNA, the sample of interest may contain many different types of RNA that can suppress and obscure the target RNA under study. Thus, RNA depletion refers to the removal of unwanted RNA and/or DNA species from a nucleic acid sample, whereby the nucleic acid sample for analysis remains enriched in the target RNA.

[0006] The present invention provides a solution for depleting an excess amount of unwanted RNA molecules in a nucleic acid sample prior to further investigation. For example, an RNA sample of interest includes not only the target RNA of interest, but also repetitive transcripts such as rRNA, globin mRNA, viral contamination, or any other undesirable nucleic acids that may dominate the sample and silence the target of interest, thereby greatly reducing the opportunity for the researcher to accurately analyze the part of the transcriptome of interest.

[0007] Thus, removing unwanted RNA molecules from a nucleic acid sample prior to analysis, such as expression microarrays or sequencing, increases the specificity and precision of the assay for the target RNAs of interest. In the present invention, the depletion of non-target RNA through the degradation of specific DNA:RNA hybrids allows for the efficient removal of unwanted RNA species from a sample prior to library preparation and analysis. Once the level of unwanted RNA molecules in the sample is reduced, the remaining target RNA can be converted to cDNA. A reliable sample will provide acceptable data, as well as improve potential treatment options, prognosis and diagnosis of cancer, understanding of the microbiome, its significance in our and other eukaryotic systems, as well as improve the analysis of the expression of genes of interest, etc.

[0008] In one embodiment, the present invention describes a method for depleting non-target RNA from a nucleic acid sample, which includes:

a) bringing a nucleic acid sample containing at least one target sequence in the RNA or DNA structure and at least one sequence of non-target RNA molecules into contact with a set of probes containing at least two DNA probes complementary to non-contiguous sequences along the entire length of at least one sequence of a non-target RNA molecule, thereby hybridizing DNA probes with non-target RNA molecules to form DNA:RNA hybrids, with each DNA:RNA hybrid separated by at least 5 bases or at least 10 bases along a given sequence of a non-target RNA molecule from another DNA:RNA hybrid; and

b) bringing the DNA:RNA hybrids into contact with a ribonuclease that cleaves RNA from the DNA:RNA hybrids, thereby destroying the non-target RNA molecules in the nucleic acid sample to form a digested mixture.

[0009] In one embodiment, the present invention relates to a composition with a set of probes that includes at least two DNA probes that are complementary to non-contiguous sequences along the entire length of at least one sequence of non-target RNA molecules (for example, by at least 5 or 10 bases apart along their entire length) in the nucleic acid sample. In some embodiments of the present invention, the composition also contains a ribonuclease capable of degrading RNA in a DNA:RNA hybrid. In another embodiment, the present invention relates to a composition with a set of probes that includes at least two DNA probes hybridized with at least one sequence of non-target RNA molecules, with each DNA probe hybridizing at a distance of at least 5 or 10 bases along the entire length of the non-target RNA molecule sequence from another DNA probe in the kit.

[0010] In one embodiment, the present invention describes a probe set kit that includes at least two DNA probes that are complementary to non-contiguous sequences along the entire length of at least one sequence of non-target rRNA molecules (e.g., at a distance at least 5 bases apart or at least 10 bases along its entire length) in a nucleic acid sample and a ribonuclease capable of cleaving RNA in a DNA:RNA hybrid.

[0011] In one embodiment, the present invention describes a method for adding a set of probes to remove non-target RNA nucleic acid molecules from a nucleic acid sample. The method includes the following: a) bringing a nucleic acid sample containing at least one target sequence in the RNA or DNA structure and at least one sequence of non-target RNA molecules of the first species into contact with a set of probes containing at least two DNA probes that are complementary to non-contiguous sequences along the entire length of at least one sequence of a non-target RNA molecule of the second species, thereby hybridizing DNA probes with non-target RNA molecules to form DNA:RNA hybrids, with each hybrid DNA:RNA is separated by at least 5 bases or at least 10 bases along a given sequence of the non-target RNA molecule from another DNA:RNA hybrid; b) bringing the DNA:RNA hybrids into contact with a ribonuclease that cleaves RNA from the DNA:RNA hybrids, thereby destroying the non-target RNA molecules in the nucleic acid sample to form a digested mixture; c) separating the digested rRNA from the digested mixture; d) sequencing the remaining RNA from the sample; e) evaluating the remaining RNA sequences for the presence of non-target RNA molecules of the first kind, thus determining gap sequence regions; and f) adding to the set additional DNA probes that are complementary to non-contiguous sequences in one or more gap regions.

BRIEF DESCRIPTION OF GRAPHICS

[0012] In FIG. 1 shows an example workflow for depleting RNA species from a sample. Step 1 involves the denaturation of nucleic acids followed by the addition of DNA probes to deplete and hybridize the probes with unwanted RNA species and thus create DNA:RNA hybrids. Step 2 involves digesting RNA from DNA:RNA hybrids using a ribonuclease such as RNase H. Step 3 involves digesting residual DNA probes from the digested mixture by adding DNase. Step 4 involves capturing the remaining target RNA in the sample, with optional subsequent additional manipulations that ultimately result in depletion of the RNA species in the sample, which can be sequenced, microarrayed, qPCR, or other assay techniques.

[0013] In FIG. 2A-2C are exemplary rRNA depletion data in sample b. subtilis by adding formamide to the rRNA depletion process (2A) 0% formamide, (2B) 25% formamide, (2C) 45% formamide). In each panel, the x-axis shows the detected rRNA species, and the y-axis shows rRNA depletion data according to the results of reading the sequences in percentage terms.

[0014] In FIG. Figure 3 shows an example of next-generation sequencing (NGS) sequence data for rRNA-reduced Human Brain RNA (HBR) and Universal Human RNA (UHR) samples comparing different amounts of sequenced sample. (100 ng, 10 ng or 1 ng).

[0015] In FIG. 4 shows an example of NGS sequence data for rRNA-reduced mouse and rat RNA samples using different concentrations of formamide (0%, 25%, 45%) added to the rRNA-reduced workflow.

[0016] In FIG. Figure 5 provides exemplary data on rRNA removal from various microbial species using low entry samples comparing methodologies for reducing the level of enzymatic removal of rRNA by RiboZero® and RNase H. Sample read depths were normalized. The x-axis represents the method for reducing the level of unwanted rRNA molecules (RZ - RiboZero or ED - the RNase H enzyme depletion method), and the y-axis represents the percentages of rRNA read.

[0017] In FIG. 6 shows examples of transcript detection data at different reading depths for B. subtilis and E. coli after rRNA depletion by RNase H (ED) enzymes on the left side of the graph compared to no rRNA depletion (None) on the right side of the graph. The x-axis shows the sequencing read values (M) and the y-axis shows the number of detected transcripts.

[0018] In FIG. 7A-7B are exemplary plots of pairwise linear regression data on gene expression demonstrating the reproducibility of the described rRNA depletion methods. Panel 7A shows two levels of E. coli gene expression and panel 7B shows two levels of b gene expression. subtilis . Both types of bacteria show a high correlation between gene expression levels after RNA depletion by RNase H enzymes.

[0019] In FIG. 8 shows examples of triplicate rRNA read data for a mixed sample of strain 20 (MSA-2002, left side) and strain 12 (MSA-2006, right side). In a mixed triplicate of samples, rRNA was removed by the RiboZero (RZ) method or by RNase H (ED) enzymes as described herein. The input RNA for MSA2002 samples was 10 ng, while for MSA2006 it was 80 ng. The x-axis shows the mode of rRNA depletion, and the y-axis represents the percentage of rRNA reads.

[0020] In FIG. 9 shows the read coverage of mouse 12S rRNA sequencing (mt-Rnr1 and 16S (mt-Rnr2) loci (bottom of figure) and the effect of a set of 333 DNA probes (SEQ ID NOs: 1-333) on mouse 16S rRNA depletion from universal samples. mouse reference RNA (UMR - universal mouse reference RNA) Squares show location of 90% match along base length 50 or 70% match along base pair length 30 with a set of 333 DNA probes In the absence of additional mouse and rat probes, gaps without probe coating correspond to the peaks at the residual or non-depleted rRNA level for the two replicas (Rep 1 and Rep 2) shown at the top of the figure.

DETAILED DESCRIPTION

[0021] The creation of nucleic acid libraries from RNA for sequencing is often difficult due to the large number of unwanted transcripts, such as ribosomal RNA, globin mRNA, viral contamination, etc., which can dominate the sample and overwhelm the RNA sequences of interest. If undesired transcripts are not removed, transcriptome analysis with possible prognostic, diagnostic, or investigative effect may be compromised. Thus, removing unwanted RNA molecules from a nucleic acid sample prior to analysis, such as sequencing or other 3'-side applications, can increase the specificity and precision of the desired assay.

[0022] The present invention provides methods and materials used to remove unwanted RNA species from a nucleic acid sample in order to explore and not lose important RNA data among a significant number of unwanted RNA transcripts.

[0023] In the described RNA depletion method, compared to the existing ones, smaller volumes of input total RNA sample can be used, while maintaining similar performance indicators. Thus, the described method can be used when the researcher has a small amount of source material that is not suitable for other methods. In addition, the described method can be implemented with a single pool of probes simultaneously targeting many different undesirable RNA species in the body without compromising the efficiency of depletion. For example, the present invention can simultaneously remove unwanted eukaryotic and prokaryotic RNA species from an RNA sample, including, without limitation, human, bacterial, viral, and/or Archaea sources of unwanted RNA.

[0024] The term "sample or mixture of nucleic acids" means a sample containing RNA or DNA, or both, including unwanted (non-target) and desired (target) nucleic acids. The DNA or RNA in a sample may be either unmodified or modified and includes, without limitation, single or double stranded DNA or RNA or derivatives thereof (e.g., some regions of DNA or RNA are double stranded while other regions of DNA or RNA are single-stranded), etc. Typically, a nucleic acid sample includes all chemically, enzymatically and/or metabolically modified forms of nucleic acids, as well as all unmodified forms of nucleic acids, or combinations thereof. The nucleic acid sample may contain both desired and undesired nucleic acids, such as genomic DNA, or total cellular RNA, or a combination thereof. Undesired nucleic acids include nucleic acids from eukaryotes that are not targets for research, as well as contamination of nucleic acids from bacteria, viruses, Archaea species, etc. Desirable or necessary nucleic acids are nucleic acids that are the basis or focus of research - nucleic acids -targets. For example, the researcher may want to analyze the expression of mRNA, where rRNA, tRNA and DNA can be considered unwanted nucleic acids, and mRNA as a target nucleic acid. In addition, total RNA may need to be studied, while rRNA, mRNA, and DNA would be considered unwanted or unnecessary nucleic acids, and total RNA would be the target nucleic acid. Unwanted RNA includes, without limitation, ribosomal RNA (rRNA), mitochondrial rRNA, nuclear rRNA, mRNA such as globin RNA or transfer tRNA, or a mixture thereof. In some embodiments, the non-target RNA is rRNA. In some embodiments, the non-target RNA is globin mRNA.

[0025] For example, a nucleic acid sample may contain the desired messenger RNA (mRNA) or total RNA, while including unwanted ribosomal RNA (rRNA), transfer RNA (tRNA), and possibly unwanted DNA. General methods for extracting RNA from a macroscopic sample such as blood, tissue, cells, fixed tissues, etc. are well known in the art, as described in Current Protocols for Molecular Biology, John Wiley & Sons) and in numerous manuals on techniques in molecular biology. RNA isolation can be performed using commercially available purification kits such as Qiagen RNeasy mini columns, MasterPure Complete DNA and RNA purification kits (Epicenter), Parrafin Block RNA RNA isolation kit (Ambion), RNA-Stat-60 ( Tel-Test) or cesium chloride density gradient centrifugation. The present methods are not limited to isolating RNA from a sample prior to RNA depletion.

[0026] There is natural skepticism that mixing probes that target bacterial and human rRNA in the same pool will result in significant off-target removal of the desired transcripts (Mauro et al., Proc. Natl. Acad. Sci. USA 1997, 94:422-427; Mignone and Pesole, Appl Bioinformatics 2002, 1:145-54). Surprisingly, studies carried out in the development of the inventive methods demonstrate that this is not the case, since the specificity of hybridization of the DNA probe to unwanted RNA transcripts results in the unwanted RNA species being effectively removed from the sample. It has also been found that the addition of a destabilizer such as formamide helps to remove some unwanted RNA which has been shown to be more problematic for depletion in the absence of formamide. While there is no need to understand how formamide assists in the removal of these RNAs, it is believed that formamide may serve to weaken structural barriers in unwanted RNA so that DNA probes can bind more efficiently. In addition, the addition of formamide has shown the additional benefit of improving the detection of certain non-target transcripts, possibly by denaturing/relaxing regions of the mRNA, for example those having very stable secondary or tertiary structures and generally poorly represented in other library preparation methods.

Samples or mixtures of nucleic acids

[0027] The present invention is not limited to the source of the nucleic acid sample, for example, the source may be eukaryotes or prokaryotes, including, without limitation, human, non-human primates, mammals, birds, reptiles, plants, bacteria, viruses, nucleic acids present in soils, water or other liquids, and other environmental samples. The sample may be obtained from cells, tissues, organs, the environment, lysates, and the like. It may be freshly prepared, frozen, lyophilized, and reconstituted, or fixed, such as a formalin-fixed, paraffin-embedded biopsy (FFPE) tissue specimen, or subjected to other cytological or histological manipulations.

[0028] The kind of nucleic acid sample for which RNA depletion techniques may be useful can be eukaryotic or prokaryotic (eg, non-human, mouse, rat, microorganism, etc.). One sample may contain one or more species. In addition, the present depletion methods can be used on freshly prepared or fixed biopsies or tissue, including formalin-treated and paraffin-embedded (FFPE) samples. In some embodiments, implementation of the present invention provides nucleic acid samples of human or non-human origin, such as non-human eukaryotes, bacteria, viruses, plants, soil, or a mixture thereof. Once unwanted RNA species have been removed from the sample, the remaining desired targets can be converted to cDNA for further processing at the discretion of those skilled in the art.

[0029] In some embodiments, the invention provides nucleic acid samples of human or non-human primate origin. In some embodiments, the implementation of the present invention, the nucleic acid sample is obtained from a rat or mouse. In some embodiments of the present invention, the nucleic acid sample contains nucleic acids of non-human origin. In some embodiments, the non-human nucleic acids are derived from non-human eukaryotes, bacteria, viruses, plants, soil, or mixtures thereof.

Depletion methods

[0030] Unwanted RNA in a nucleic acid sample is removed by the methods described. The unwanted RNA is converted into a DNA:RNA hybrid by hybridization of partially or fully complementary DNA probes with unwanted RNA molecules. Methods for hybridizing nucleic acid probes to nucleic acids are well known in the art. Regardless of whether the probe is partially or completely complementary to the partner sequence, the fact that the DNA probe hybridizes to unwanted RNA species after washes and other sample manipulations demonstrates the ability to use the DNA probe in the methods of the present invention. Samples from any eukaryotic species, eg, human, mice, rats, and the like, are suitable for use with the RNA depletion kit. Sample RNA depletion has a beneficial effect on subsequent 3'-end studies such as sequencing (eg, improved results reduced by the presence of undesired nucleic acid species). DNA can also be considered as an unwanted nucleic acid if the target for investigation is RNA, in which the DNA can be removed by depletion.

[0031] In one embodiment, the present invention describes a method for depleting non-target RNA from a nucleic acid sample, which includes:

a) bringing a nucleic acid sample containing at least one target sequence in the RNA or DNA structure and at least one sequence of non-target RNA molecules into contact with a set of probes containing at least two DNA probes complementary to non-contiguous sequences along the entire length of at least one sequence of a non-target RNA molecule, thereby hybridizing DNA probes with non-target RNA molecules to form DNA:RNA hybrids, with each DNA:RNA hybrid separated by at least 5 bases or at least 10 bases along a given sequence of a non-target RNA molecule from another DNA:RNA hybrid; and

b) bringing the DNA:RNA hybrids into contact with a ribonuclease that cleaves RNA from the DNA:RNA hybrids, thereby destroying the non-target RNA molecules in the nucleic acid sample to form a digested mixture.

[0032] In one embodiment of the present invention, the RNA sample is denatured in the presence of DNA probes. An example workflow is shown in Fig. 1. In the example of FIG. 1 DNA probes are added to the denatured RNA sample (at 95°C for 2 min), after which the reaction mixture is cooled to 37°C for 15-30 min. This results in the hybridization of the DNA probes with the corresponding RNA target sequences, thus creating DNA:RNA hybrid molecules.

[0033] In some embodiments of the present invention, contacting the probe array comprises treating the nucleic acid sample with a destabilizer. In some embodiments of the present invention, the destabilizer is a thermal stabilizer or a chemical to destabilize the nucleic acid. In some embodiments, the nucleic acid destabilizing chemical is betaine, DMSO, formamide, glycerol, or a derivative or mixture thereof. In some embodiments, the nucleic acid destabilizing chemical is formamide or a derivative thereof, where the formamide or derivative thereof will optionally be present at a concentration of approx. 10 to 45% of the total volume of the hybridization reaction. In some embodiments of the present invention, the heat treatment of the sample uses a temperature above the melting point of at least one DNA:RNA hybrid.

[0034] In some embodiments of the present invention, formamide is added to the hybridization reaction regardless of the source of the RNA sample (eg, human, mouse, rat, etc.). For example, in some embodiments of the present invention, hybridization with DNA probes is performed with at least 3%, 5%, 10%, 20%, 25%, 30%, 35%, 40%, or 45% by volume of formamide. In one embodiment of the present invention, the hybridization reaction for RNA depletion includes from about 25 to 45 vol.% formamide.

[0035] After the hybridization reaction, a ribonuclease that cleaves RNA from the DNA:RNA hybrid is added to the reaction. In some embodiments of the present invention, the ribonuclease is an RNase H or a hybridase. RNase H (NEB) or hybridase (Lucigen) are examples of enzymes that cleave RNA from a DNA:RNA hybrid. Digestion with a ribonuclease, such as RNase H or hybridase, breaks the RNA into small molecules that can then be removed. For example, RNase H is reported to cleave RNA from a DNA:RNA hybrid approximately every 7-21 bases (Schultz et al., J. Biol. Chem. 2006, 281:1943-1955; Champoux and Schultz, FEBS J. 2009, 276 :1506-1516). In some embodiments of the present invention, RNA cleavage of the DNA:RNA hybrid may occur at 37° C. for approximately 30 minutes, as shown in FIG. 1, step 2 and example 1.

[0036] In some embodiments, after the DNA:RNA hybrid molecule is cleaved, the remaining DNA probes and any non-target DNA in the nucleic acid sample are cleaved. Thus, in some embodiments of the present invention, the methods comprise contacting the RNase-cleaved mixture with a DNA-cleaving enzyme, thereby cleaving the DNA in the mixture. In some embodiments of the present invention, the digested sample is exposed to a DNA-cleaving enzyme, such as DNase I, which cleaves DNA probes. The DNA cleavage reaction with DNase is incubated, for example, at 37°C for 30 minutes, after which the DNase enzyme can be denatured at 75°C for the time required to denature the DNase, for example, up to 20 minutes.

[0037] In some embodiments, the depletion method comprises separating the digested RNA from the digested mixture. In some embodiments of the present invention, separation comprises purifying the target RNA from digested RNA (and digested DNA, if present), for example, using a nucleic acid purification medium such as RNA capture beads such as RNAClean XP (Beckman Coulter). Thus, in some embodiments of the present invention, after enzymatic(s) cleavage(s), the target RNA can be enriched by removing the cleaved products, leaving behind the desired and longer target RNA. Suitable enrichment methods include treating the digested mixture with magnetic microcarriers binding to a fragment of the enriched target RNA, spin columns, and the like. In some embodiments, magnetic beads such as AMPure XP, SPRISelect, RNAClean XP beads (Beckman Coulter) may be used, provided they are RNase-free (eg, quality controlled without RNases). These beads provide various size options for nucleic acid binding, for example, RNAClean XP beads target nucleic acid fragments of 100 or more nucleotides, while SPRISelect beads target nucleic acid fragments from 150 to 800 nucleotides and do not target shorter nucleotide sequences, such as digested RNA and DNA resulting from the enzymatic digestion of RNase H and DNase. If the mRNA is the target RNA of interest, then the mRNA can be further enriched by capture using, for example, beads that contain oligodT sequences to capture adenylated mRNA tails. Methods for capturing mRNA are well known to those skilled in the art.

[0038] Once the target RNA has been purified of reaction components, including unwanted cleaved nucleic acids, further sample manipulation can be performed. Examples 2 and 3 of the present invention describe workflows for synthesizing cDNA from an enriched total target RNA and subsequent library production workflow typical of further sequencing, for example using an Illumina sequencer. However, it should be understood that these workflows are exemplary only and one skilled in the art will appreciate that enriched RNA can be used for a variety of additional applications such as PCR, qPCR, microarray analysis, and the like, either directly or after additional manipulations such as converting RNA to cDNA using established and well-understood protocols.

[0039] The RNA depletion methods described herein result in a sample enriched in target RNA molecules. For example, the methods described herein produce a purified RNA sample containing less than 15%, 13%, 11%, 9%, 7%, 5%, 3%, 2%, or 1% (or any range in between) unwanted RNA species. The enriched RNA sample will then contain at least 99%, 98%, 97%, 95%, 93%, 91%, 89%, or 87% (or any range in between) of the total target RNA. Once enriched, the sample can be used for library preparation or other 3' manipulations.

DNA Probe Kits/DNA Probes

[0040] The term "DNA probe" refers to a single-stranded DNA oligonucleotide whose sequence is complementary to unwanted RNA species. The DNA probe sequence may be partially or fully complementary to the unwanted RNA for depleting the nucleic acid sample. Unwanted RNA for depletion includes, without limitation, rRNA, tRNA and mRNA, as well as mixtures thereof. In some embodiments, each DNA probe is about 10 to 100 nucleotides long, or about 20 to 80 nucleotides long, or about 40 to 60 nucleotides long, or about 50 nucleotides long. DNA probes are able to hybridize with unwanted RNA species, thus creating DNA:RNA hybrid molecules. Although in some embodiments of the present invention, at least two DNA probes hybridize to a particular non-target RNA molecule, the DNA probes do not span the entire sequence length of the unwanted RNA molecule. For example, in some embodiments of the present invention, the probe set exits gaps or regions of unwanted RNA without a complementary DNA probe in the probe set. DNA probes fully or partially hybridize to unwanted RNA without overlap, leaving gaps 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 of the present invention, each DNA probe hybridizes at least 5 or 10 bases along the entire length of at least one target RNA molecule from any other DNA probe in the probe set. Thus, unwanted RNA generally does not fully hybridize with DNA probes. In addition, the present invention provides a plurality of DNA probes that hybridize to a single RNA for depletion. As such, there is no "one DNA probe for one RNA" concept, instead a plurality of discontinuous DNA probes in a set of probes targeting a given unwanted RNA is used. For example, in some embodiments of the present invention, a given set of depletion RNAs uses a set of DNA probes, where each probe is approximately 20-80 nucleotides in length and hybridizes to the unwanted RNA anywhere within 5-15 nucleotides of another DNA probe in the set. . The DNA probe may be fully or partially complementary to a specific location on the RNA to be depleted, for example, the sequence of the DNA probe may be at least 80%, 85%, 90%, 95%, or 100% complementary to the target location on the RNA transcript to be depleted. The only limitation on complementarity is that the DNA probe must hybridize to the depletion target RNA such that the DNA:RNA hybrid results in enzymatic cleavage as described herein. In some cases, the mRNA is the target of interest and does not target depletion, then the DNA probes will not contain the poly-T sequence and, as a result, will not hybridize with the mRNA species. In some embodiments, DNA probes are not labeled with a capture moiety such as biotin, avidin, streptavidin, or a magnetic bead to allow physical depletion of the hybrid, while in other embodiments, DNA probes are labeled with a capture moiety. for capture, such as biotin, avidin, streptavidin, or a magnetic bead that depletes the hybrid by physical means.

[0041] In some embodiments, the probe set comprises at least DNA probes that hybridize to non-target RNA molecules from human and microbial sources. In some embodiments, the probe set comprises at least DNA probes that hybridize to non-target RNA molecules from human, microorganism, and Archaea . In some embodiments, the probe set comprises at least DNA probes that hybridize to non-target RNA molecules from human, microbial, mouse, and rat sources. In some embodiments, the probe set comprises at least DNA probes that hybridize to non-target RNA molecules from human, microorganism, mouse, rat, and Archaea . In some embodiments of the present invention, the source of non-target RNA molecules are microorganisms, Gram-positive or Gram-negative bacteria, or mixtures thereof. In some embodiments of the present invention, the probe set comprises at least two DNA probes that hybridize to one or more non-target RNA molecules sourced from Archaea species. In some embodiments, the probe set comprises at least two DNA probes that are complementary to two or more rRNA sequences of Archaea species.

[0042] In some embodiments, the probe set comprises at least two DNA probes that hybridize 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 that hybridize to one or more or two or more non-target RNA molecules selected from human 28S, 18S, 5.8S, 5S, 16S, and 12S fragments. origin. In some embodiments of the present invention, the probe set comprises at least two DNA probes that hybridize to one or more or two or more non-target rat and/or mouse RNA molecules, optionally selected from rat 16S, rat 28S fragments. , 16S mouse, 28S mouse and combinations thereof. In some embodiments, the probe set comprises at least two DNA probes that hybridize to one or more non-target RNA molecules selected from HBA-A1, HBA-A2, HBB, HBB-B1, HBB-B2, HBG1 and HBG2 from hemoglobin. In some embodiments, the probe set comprises at least two DNA probes that hybridize to one or more non-target RNA molecules selected from 23S, 16S, and 5S from Gram-positive and/or Gram-negative bacteria. Depleted globin mRNAs may include, but are not limited to, mRNAs in mice or rats including HBA-A1, HBA-A2, HBB, HBB-B1, HBB-B2, and in humans including HBA-A1, HBA-A2 , HBB, HGB1 and HGB2. Mitochondrial rRNAs suitable for depletion include 18S and 12S (human and rodent). Nuclear rRNAs suitable for depletion include 28S, 18S, 5.8S and 5S (human and rodent) and prokaryotic rRNAs including 5S, 16S and 23S. Depletion of Archaea species rRNA, such as 23S, 16S or 5S rRNA, may also be desirable in some samples. In additional embodiments of the present invention, 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 bacterial 5S, 16S, and 23S rRNA. In some embodiments, the probe set comprises at least two (or five, or 10, or 20) DNA probes complementary to each of 28S, 18S, 5.8S, 5S, 16S, and 12S globin mRNA HBA-A1, HBA-A2 , HBB, human HBG1 and HBG2 and gram-positive or gram-negative bacterial 5S, 16S and 23S rRNA. In some embodiments, probes for a particular non-target RNA molecule are about 80-85% complementary to its sequence, with gaps between each probe hybridization site of at least 5 or 10 bases.

[0043] In some embodiments of the present invention, the set of probes contains two or more, or five or more, or 10 or more, or 25 or more, or 50 or more, or 100 or more, or 150 or more, or 200 or more , or 250 or more, or 300 or more, or 333 sequences from SEQ ID NOs: 1-333 (human, Gram-positive and Gram-negative bacteria). In some embodiments of the present invention, the set of probes contains two or more, or five or more, or 10 or more, or 25 or more, or 50 or more, or 100 or more, or 150 or more, or 200 or more, or 250 or more, or 300 or more, or 350 or more, or 400 or more, or 428 sequences from SEQ ID NOs: 1-428 (human, Gram-positive and Gram-negative bacteria, Archaea , mice and rats). In some embodiments of the present invention, the set of probes contains two or more, or five or more, or 10 or more, or 25 or more, or 50 or more, or 100 or more, or 150 or more, or 200 or more, or 250 or more, or 300 or more, or 350 or more, or 377 sequences from SEQ ID NOs: 1-377 (human, Gram-positive and Gram-negative bacteria and Archaea ). In some embodiments of the present invention, the set of probes contains two or more, or five or more, or 10 or more, or 25 or more, or 50 or more, or 100 or more, or 150 or more, or 200 or more, or 250 or more, or 300 or more, or 350 or more, or 384 sequences from SEQ ID NOs: 1-333 (human, Gram-positive and Gram-negative bacteria) and SEQ ID NOs: 378-428 (mice and rats). In some embodiments, the probe set contains two or more, or five or more, or 10 or more, or 25 or more, or 44 sequences from SEQ ID NOs: 334-377 ( Archaea ). In some embodiments, the probe set contains two or more, or five or more, or 10 or more, or 25 or more, or 50 or more, or 51 sequences from SEQ ID NOs: 378-428 (mice and rats) .

[0044] In some embodiments, the DNA probes are partially or fully complementary and contain sequences that hybridize to human 28S, 18S, 5.8S, and/or 5S rRNA, such as DNA probe sequences according to Table 1, from SEQ ID NO : 40 to SEQ ID NO: 150. In a second embodiment of the present invention, DNA probes include sequences that hybridize to mitochondrial 16S and/or 12S rRNA, such as DNA probe sequences as shown in Table 1 from SEQ ID NO: 1 to SEQ ID NO: 39. In other embodiments, the DNA probes include sequences that hybridize to hemoglobin mRNA, including HBA-A1, HBA-A2, HBB, HBB-B1, HBB-B2, HBG1 and/or HBG2, such as the DNA probe sequences shown in Table 1, from SEQ ID NO: 151 to SEQ ID NO: 194. In some embodiments, implementation of the present invention, DNA probes include sequences that hybridize with bacterial and rRNA, such as Gram-positive and/or Gram-negative bacterial rRNA 23S, 16S, and/or 5S, such as DNA probe sequences as shown in Table 1, from SEQ ID NO: 195 to SEQ ID NO: 262 (gram-negative bacterial representative E. coli ) and SEQ ID NO: 263 to SEQ ID NO: 333 (gram-positive bacterial representative of Bacillus subtilis ). In other embodiments of the present invention, the DNA probes include sequences that hybridize to Archaea rRNA, such as 23S, 16S, and/or 5S rRNA, such as the DNA probe sequences shown in Table 1, from SEQ ID NO: 334 to SEQ ID NO : 384 that hybridize with rRNA from Archaea species Methanobrevibacter smithii . In some embodiments of the present invention, DNA probes include sequences that hybridize to mouse rRNA, such as mouse 16S and/or 28S, such as the DNA probe sequences shown in Table 1, from SEQ ID NO: 385 to SEQ ID NO: 393 and SEQ ID NO: 400 to SEQ ID NO: 419. In some embodiments, the DNA probes include sequences that hybridize to rat rRNA, such as rat 16S and/or 28S, such as the DNA probe sequences shown in Table 1, SEQ ID NO: 394 to SEQ ID NO: 399 and SEQ ID NO: 420 to SEQ ID NO: 428.

Table 1. DNA probe sequences for removing unwanted RNA molecules

SEQID NO Probe name Probe sequence 5'-3' 1 12S_P1 GTTCGTCCAAGTGCACTTTCCAGTACACTTACCATGTTACGACTTGTCTC 2 12S_P2 TAGGGGTTTTAGTTAAATGTCCTTTGAAGTATACTTGAGGAGGGTGACGG 3 12S_P3 TTCAGGGCCCTGTTCAACTAAGCACTCTACTCTCAGTTTACTGCTAAATC four 12S_P4 AGTTTCATAAGGGCTATCGTAGTTTTCTGGGGTAGAAAATGTAGCCCATT five 12S_P5 GGCTACACCTTGACCTAACGTCTTTACGTGGGTACTTGCGCTTACTTTGT 6 12S_P6 TTGCTGAAGATGGCGGTATATAGGCTGAGCAAGAGGTGGTGAGGTTGATC 7 12S_P7 CAGAACAGGCTCCTCTAGAGGGATATGAAGCACCGCCAGGTCCTTTGAGT 8 12S_P8 GTAGTGTTCTGGCGAGCAGTTTTGTTGATTTAACTGTTGAGGTTTAGGGC nine 12S_P9 ATCTAATCCCAGTTTGGGTCTTAGCTATTGTGTGTTCAGATATGTTAAAG 10 12S_P10 ATTTTGTGTCAACTGGAGTTTTTTTACAACTCAGGTGAGTTTTAGCTTTAT eleven 12S_P11 CTAAAACACTCTTTACGCCGGCTTCTATTGACTTGGGTTAATCGTGTGAC 12 12S_P12 GAAATTGACCAACCCTGGGGTTAGTATAGCTTAGTTAAACTTTCGTTTAT 13 12S_P13 ACTGCTGTTTCCCGTGGGGGTGTGGCTAGGCTAAGCGTTTTGAGCTGCAT fourteen 12S_P14 GCTTGTCCCTTTTGATCGTGGTGATTTAGAGGGTGAACTCACTGGAACGG 15 12S_P15 TAATCTTACTAAGAGCTAATAGAAAGGCTAGGACCAAACCTATTTGTTTA sixteen 16S_P1 AAACCCTGTTCTTGGGTGGGTGTGGGTATAATACTAAGTTGAGATGATAT 17 16S_P2 GCGCTTTGTGAAGTAGGCCTTATTTCTCTTGTCCTTTCGTACAGGGAGGA eighteen 16S_P3 AAACCGACCTGGATTACTCCGGTCTGAACTCAGATCACGTAGGACTTTAA nineteen 16S_P4 ACCTTTAATAGCGGCTGCACCATCGGGATGTCCTGATCCAACATCGAGGT 20 16S_P5 TGATATGGACTCTAGAATAGGATTGCGCTGTTATCCCTAGGGTAACTTGT 21 16S_P6 ATTGGATTCAATTGAGTATAGTAGTTCGCTTTGACTGGTGAAGTCTTAGCA 22 16S_P7 TTGGGTTCTGCTCCGAGGTCGCCCCAACCGAAATTTTTAATGCAGGTTTG 23 16S_P8 TGGGTTTGTTAGGTACTGTTTGCATTAATAAATTAAAGCTCCATAGGGTC 24 16S_P9 GTCATGCCCGCCTCTTCACGGGCAGGTCAATTTCACTGGTTAAAAGTAAG 25 16S_P10 CGTGGAGCCATTCATACAGGTCCCTATTTAAGGAACAAGTGATTTATGCTA 26 16S_P11 GGTACCGCGGCCGTTAAACATGTGTCACTGGGCAGGCGGTGCCTCTAATA 27 16S_P12 GTGATGTTTTTGGTAAACAGGCGGGGTAAGGTTTGCCGAGTTCCTTTTAC 28 16S_P13 CTTATGAGCATGCCTGTGTTGGGTTGACAGTGAGGGTAATAATGACTTGT 29 16S_P14 ATTGGGCTGTTAATTGTCAGTTCAGTGTTTTGATCTGACGCAGGCTTATG thirty 16S_P15 TCATGTTACTTATACTAACATTAGTTCTTCTATAGGGTGATAGATTGGTC 31 16S_P16 AGTTCAGTTATATGTTTGGGATTTTTTAGGTAGTGGGTGTTGAGCTTGAA 32 16S_P17 TGGCTGCTTTAGGCCTACTATGGGTGTTAAATTTTTTTACTCTCTCTACA 33 16S_P18 GTCCAAAGAGCTGTTCCTCTTTGGACTAACAGTTAAATTTACAAGGGGAT 34 16S_P19 GGCAAATTTAAAGTTGAACTAAGATTCTATCTTGGACAACCAGCTATCAC 35 16S_P20 TGTCGCCTCTACCTATAAATCTTCCCACTATTTTGCTACATAGACGGGTG 36 16S_P21 TCTTAGGTAGCTCGTCTGGTTTCGGGGGTCTTAGCTTTGGCCTCTCCTTGC 37 16S_P22 TAATTCATTATGCAGAAGGTATAGGGGTTAGTCCTTGCTATATTATGCTT 38 16S_P23 TCTTCCCTTGCGGTACTATATCTATTGCGCCAGGTTTCAATTTCTATCG 39 16S_P24 GGTAAATGGTTTGGCTAAGGTTGTCTGGTAGTAAGGTGGAGTGGGTTTGG 40 18S_P1 TAATGATCCTTCCGCAGGTTCACCTACGGAAACCTTGTTACGACTTTTAC 41 18S_P2 AAGTTCGACCGTCTTCTCAGCGCTCCGCCAGGGCCGTGGGCCGACCCCGG 42 18S_P3 GGCCTCACTAAACCATCCAATCGGTAGTAGCGACGGGCGGTGTGTACAAA 43 18S_P4 CAACGCAAGCTTATGACCCGCACTTACTCGGGAATTCCCTCGTTCATGGG 44 18S_P5 CCGATCCCCATCACGAATGGGGTTCAACGGGTTACCCGCGCCTGCCGGCG 45 18S_P6 CTGAGCCAGTCAGTGTAGCGCGCGTGCAGCCCCGGACATCTAAGGGCATC 46 18S_P7 CTCAATCTCGGGTGGCTGAACGCCACTTGTCCCTCTAAGAAGTTGGGGGA 47 18S_P8 GGTCGCGTAACTAGTTAGCATGCCAGAGTCTCGTTCGTTATCGGAATTAA 48 18S_P9 CACCAACTAAGAACGGCCATGCACCACCACCCACGGAATCGAGAAAGAGC 49 18S_P10 CCTGTCCGTGTCCGGGCCGGGTGAGGTTTCCCGTGTTGAGTCAAATTAAG 50 18S_P11 CTGGTGGTGCCCTTCCGTCAATTCCTTTAAGTTTCAGCTTTGCAACCATA 51 18S_P12 AAAGACTTTGGTTTCCCGGAAGCTGCCCGGCGGGTCATGGGAATAACGCC 52 18S_P13 GGCATCGTTTATGGTCGGAACTACGACGGTATCTGATCGTCTTCGAACCT 53 18S_P14 GATTAATGAAAAACATTCTTGGCAAATGCTTTCGCTCTGGTCCGTCTTGCG 54 18S_P15 CACCTCTAGCGGCGCAATACGAATGCCCCCGGCCGTCCCCTTAATCATG 55 18S_P16 ACCAACAAAATAGAACCGCGGTCCTATTCCATTATTCCTAGCTGCGGTAT 56 18S_P17 CTGCTTTGAACACTCTAATTTTTTCAAAGTAAACGCTTCGGGCCCCGCGG 57 18S_P18 GCATCGAGGGGGCGCCGAGAGGCAAGGGGCGGGGACGGGCGGTGGCTCGC 58 18S_P19 CCGCCCGCTCCCAAGATCCAACTACGAGCTTTTTAACTGCAGCAACTTTA 59 18S_P20 GCTGGAATTACCGCGGCTGCTGGCACCAGACTTGCCCTCCAATGGATCCT 60 18S_P21 AGTGGACTCATTCCAATTACAGGGCCTCGAAAGAGTCCTGTATTGTTATT 61 18S_P22 CCCGGGTCGGGAGTGGGTAATTTGCGCGCCTGCTGCCTTCCTTGGATGTG 62 18S_P23 GCTCCCTCTCCGGAATCGAACCCTGATTCCCCGTCACCCGTGGTCACCAT 63 18S_P24 TACCATCGAAAGTTGATAGGGCAGACGTTCGAATGGGTCGTCGCCGCCAC 64 18S_P25 GGCCCGAGGTTATCTAGAGTCACCAAAGCCGCCGGCGCCCGCCCCCCGGC 65 18S_P26 GCTGACCGGGTTGGTTTTGATCTGATAAATGCACGCATCCCCCCCGCGAA 66 18S_P27 TCGGCATGTATTAGCTCTAGAATTACCACAGTTATCCAAGTAGGAGAGGA 67 18S_P28 AACCATAACTGATTTAATGAGCCATTCGCAGTTTCACTGTACCGGCCGTG 68 18S_P29 ATGGCTTAATCTTTGAGACAAGCATATGCTACTGGCAGGATCAACCAGGT 69 28S_P1 GACAAACCCTTGTGTCGAGGGCTGACTTTCAATAGATCGCAGCGAGGGAG 70 28S_P2 CGAAACCCCGACCCAGAAGCAGGTCGTCTACGAATGGTTTAGCGCCAGGT 71 28S_P3 GGTGCGTGACGGGCGAGGGGGCGGCCGCCTTTCCGGCCGCGCCCCGTTTC 72 28S_P4 CTCCGCACCGGACCCCGGTCCCGGCGCGCGGCGGGGCACGCGCCCTCCCG 73 28S_P5 AGGGGGGGGCGGCCCGCCGGCGGGACAGGCGGGGGACCGGCTATCCGAG 74 28S_P6 GCGGCGCTGCCGTATCGTTCGCCTGGGCGGGATTCTGACTTAGAGGCGTT 75 28S_P7 AGATGGTAGCTTCGCCCCATTGGCTCCTCAGCCAAGCACATACACCAAAT 76 28S_P8 TCCTCTCGTACTGAGCAGGATTACCATGGCAACAACACATCATCAGTAGG 77 28S_P9 CTCACGACGGTCTAAACCCAGCTCACGTTCCCTATTAGTGGGTGAACAAT 78 28S_P10 TTCTGCTTCACAATGATAGGAAGAGCCGACATCGAAGGATCAAAAAGCGA 79 28S_P11 TTGGCCGCCACAAGCCAGTTATCCCTGTGGTAACTTTTCTGACACCTCCT 80 28S_P12 GGTCAGAAGGATCGTGAGGCCCCGCTTTCACGGTCTGTATTCGTACTGAA 81 28S_P13 AGCTTTTGCCCTTCTGCTCCACGGGAGGTTTCTGTCCTCCCTGAGCTCGC 82 28S_P14 TTACCGTTTGACAGGTGTACCGCCCCAGTCAAACTCCCCACCTGGCACTG 83 28S_P15 GCGCCCGGCCGGGCGGGCGCTTGGCGCCAGAAGCGAGAGCCCCTCGGGCT 84 28S_P16 CCGGGTCAGTGAAAAAACGATCAGAGTAGTGGTATTTCACCGGCGGCCCG 85 28S_P17 CGCCCCGGGCCCCTCGCGGGGACACCGGGGGGGCGCCGGGGCCTCCCAC 86 28S_P18 CATGTCTCTTCACCGTGCCAGACTAGAGTCAAGCTCAACAGGGTCTTCTT 87 28S_P19 CCAAGCCCGTTCCCTTGGCTGTGGTTTCGCTGGATAGTAGGTAGGGACAG 88 28S_P20 TCCATTCATGCGCGTCACTAATTAGATGACGAGGCATTTGGCTACCTTAA 89 28S_P21 TCCCGCCGTTTACCGCGCGCTTCATTGAATTTCTTCACTTTGACATTCAGA 90 28S_P22 CACATCGCGTCAACACCCGCCGCGGGCCTTCGCGATGCTTTGTTTTAATT 91 28S_P23 CCTGGTCCGCACCAGTTCTAAGTCGGCTGCTAGGCGCCGGCCGAGGCGAG 92 28S_P24 CGGCCCCGGGGGCGGACCCGGCGGGGGGACCGGCCCGCGGCCCCTCCGC 93 28S_P25 CCGCCGCGCGCCGAGGAGGAGGGGGGAACGGGGGGCGGACGGGGCCGGGG 94 28S_P26 ACGAACCGCCCCGCCCCGCCGCCCGCCGACCGCCGCCGCCCGACCGCTCC 95 28S_P27 CGCGCGCGACCGAGACGTGGGGTGGGGGTGGGGGGCGCGCCGCGCCGCCG 96 28S_P28 GCGGCCGCGACGCCCGCCGCAGCTGGGGCGATCCACGGGAAGGGCCCGGC 97 28S_P29 GCGCCGCCGCCGGCCCCCCGGGTCCCCGGGGCCCCCCTCGCGGGGACCTG 98 28S_P30 CCGGCGGCCGCCGCGCGGCCCCTGCCGCCCCGACCCTTCTCCCCCCGCCG 99 28S_P31 CTCCCCCGGGGAGGGGGGAGGACGGGGAGCGGGGGAGAGAGAGAGAGA one hundred 28S_P32 AGGGAGCGAGCGGCGCGCGCGGGTGGGGCGGGGGAGGGCCGCGAGGGGGG 101 28S_P33 GGGGGCGCGCGCCTCGTCCAGCCGCGGCGCGCGCCCAGCCCCGCTTCGCG 102 28S_P34 CCCAGCCCTTAGAGCCAATCCTTATCCCGAAGTTACGGATCCGGCTTGCC 103 28S_P35 CATTGTTCCAACATGCCAGAGGCTGTTCACCTTGGAGACCTGCTGCGGAT 104 28S_P36 CGCGAGATTTACACCCTCTCCCCCGGATTTTCAAGGGCCAGCGAGAGCTC 105 28S_P37 AACCGCGACGCTTTCCAAGGCACGGGCCCCTCTCTCGGGGCGAACCCATT 106 28S_P38 CTTCACAAAGAAAAGAGAACTCTCCCCGGGGCTCCCGCCGGCTTTCTCCGG 107 28S_P39 CGCACTGGACGCCTCGCGGCGCCCATCTCCGCCACTCCGGATTCGGGGAT 108 28S_P40 TTTCGATCGGCCGAGGGCAACGGAGGCCATCGCCCGTCCCTTCGGAACGG 109 28S_P41 CAGGACCGACTGACCCATGTTCAACTGCTGTTCACATGGAACCCTTCTCC 110 28S_P42 GTTCTCGTTTTGAATATTTGCTACTACCACCAAGATCTGCACCTGCGGCGG 111 28S_P43 CGCCCTAGGCTTCAAGGCTCACCCGCAGCGGCCCTCCTACTCGTCGCGGCG 112 28S_P44 TCCGGGGGCGGGGAGCGGGGCGTGGGCGGGAGGAGGGGAGGAGGCGTGGG 113 28S_P45 AGGACCCCACACCCCCGCCGCCGCCGCCGCCGCCGCCCTCCGACGCACAC 114 28S_P46 GCGCGCCGCCCCCGCCGCTCCCGTCCACTCTCGACTGCCGGCGACGGCCG 115 28S_P47 CTCCAGCGCCATCCATTTTCAGGGCTAGTTGATTCGGCAGGTGAGTTGTT 116 28S_P48 GATTCCGACTTCCATGGCCACCGTCCTGCTGTCTATATCAACCAACACCT 117 28S_P49 GAGCGTCGGCATCGGGCGCCTTAACCCGGCGTTCGGTTCATCCCGCAGCG 118 28S_P50 AAAAGTGGCCCACTAGGCACTCGCATTCCACGCCCGGCTCCACGCCAGCG 119 28S_P51 CCATTTAAAGTTTGAGAATAGGTTGAGATCGTTTCGGCCCCAAGACCTCT 120 28S_P52 CGGATAAAACTGCGTGGCGGGGGTGCGTCGGGTCTGCGAGAGCGCCAGCT 121 28S_P53 TCGGAGGGAACCAGCTACTAGATGGTTCGATTAGTCTTTCGCCCCTATAC 122 28S_P54 GATTTGCACGTCAGGACCGCTACGGACCTCCACCAGAGTTTCCTCTGGCT 123 28S_P55 ATAGTTCACCATCTTTCGGGTCCTAACACGTGCGCTCGTGCTCCACCTCC 124 28S_P56 AGACGGGCCGGTGGTGCGCCCTCGGCGGACTGGAGAGGCCTCGGGATCCC 125 28S_P57 CGCGCCGGCCTTCACCTTCATTGCGCCACGGCGGCTTTCGTGCGAGCCCC 126 28S_P58 TTAGACTCCTTGGTCCGTGTTTCAAGACGGGTCGGGTGGGTAGCCGACGT 127 28S_P59 GCGCTCGCTCCGCCGTCCCCCTCTTCGGGGGACGCGCGCGTGGCCCCGAG 128 28S_P60 CCCGACGGCGCGACCCGCCCGGGGCGCACTGGGGACAGTCCGCCCCGCCC 129 28S_P61 GCACCCCCCCCGTCGCCGGGGCGGGGGCGCGGGGAGGAGGGGTGGGAGAG 130 28S_P62 AGGGGTGGCCCCGGCCCCCCCACGAGGAGACGCCGGCGCGCCCCCGCGGGG 131 28S_P63 GGGGATTCCCCGCGGGGGTGGGCGCCGGGAGGGGGGAGAGCGCGGCGACG 132 28S_P64 GCCCCGGGATTCGGCGAGTGCTGCTGCCGGGGGGGCTGTAACACTCGGGG 133 28S_P65 CCGCCCCCGCCGCCGCCGCCACCGCCGCCGCCGCCGCCGCCCCGACCCGC 134 28S_P66 AGGACGCGGGGCCGGGGGGCGGAGACGGGGGAGGAGGAGGACGGACGGAC 135 28S_P67 AGCCACCTTCCCCGCCGGGCCTTCCCAGCCGTCCCGGAGCCGGTCGCGGC 136 28S_P68 AAATGCGCCCGGCGGCGGCCGGTCGCCGGTCGGGGGACGGTCCCCCGCCG 137 28S_P69 CCGCCCGCCCACCCCCGCACCCGCCGGAGCCCGCCCCCTCCGGGGAGGAG 138 28S_P70 GGGAAGGGAGGGCGGGTGGAGGGGTCGGGAGGAACGGGGGGCGGGAAAGA 139 28S_P71 ACACGGCCGGACCCGCCGCCGGGTTGAATCCTCCGGGCGGACTGCGCGGA 140 28S_P72 TCTTAACGGTTTCACGCCCTCTTGAACTCTCTCTTCAAAGTTCTTTTCAA 141 28S_P73 CTTGTTGACTATCGGTCTCGTGCCGGTATTTAGCCTTAGATGGAGTTTAC 142 28S_P74 GCATTCCCAAGCAACCCGACTCCGGGAAGACCCGGGCGCGCGCCGGCCGC 143 28S_P75 GTCCACGGGCTGGGCCTCGATCAGAAGGACTTGGGCCCCCCACGAGCGGC 144 28S_P76 TTCCGTACGCCACATGTCCCGCGCCCCGCGGGGCGGGGATTCGGCGCTGG 145 28S_P77 CTCGCCGTTACTGAGGGAATCCTGGTTAGTTTCTTTTCCTCCGCTGACTA 146 28S_P78 GCGGGTCGCCACGTCTGATCTGAGGTCGCGTCTCGGAGGGGGACGGGCCG 147 5.8S_P1 AAGCGACGCTCAGACAGGCGTAGCCCCGGGAGGAACCCGGGGCCGCAAGT 148 5.8S_P3 GCAGCTAGCTGCGTTTCTTCATCGACGCACGAGCCGAGTGATCCACCGCTA 149 5S_P1 AAAGCCTACAGCACCCGGTATTCCCAGGCGGTCTCCCATCCAAGTACTAA 150 5S_P3 TTCCGAGATCAGACGAGATCGGGCGCGTTCAGGGTGGTATGGCCGTAGAC 151 HBA1_P1 GCCGCCCACTCAGACTTTATTCAAAGACCACGGGGGTACGGGTGCAGGAA 152 HBA1_P2 GGGGGAGGCCCAAGGGGCAAGAAGCATGGCCACCGAGGCTCCAGCTTAAC 153 HBA1_P3 GCACGGTGCTCACAGAAGCCAGGAACTTGTCCAGGGAGGCGTGCACCGCA 154 HBA1_P4 GGGAGGTGGGCGGCCAGGGTCACCAGCAGGCAGTGGCTTAGGAGCTTGAA 155 HBA1_P5 CCGAAGCTTGTGCGCGTGCAGGTCGCTCAGGGCGGACAGCGCGTTGGGCA 156 HBA1_P6 CCACGGCGTTGGTCAGCGCGTCGGCCACCTTTCTTGCCGTGGCCCTTAACC 157 HBA1_P7 CTCAGGTCGAAGTGCGGGAAGTAGGTCTTGGTGGTGGGGAAGGACAGGAA 158 HBA1_P8 CTCCGCACCATACTCGCCAGCGTGCGCGCCGACCTTTACCCCAGGCGGCCT 159 HBA1_P9 CGGCAGGAGACAGCACCATGGTGGGTTCTCTCTGAGTCTGTGGGGACCAG 160 HBA2_P1 GAGGGGAGGAGGGCCCGTTGGGAGGCCCAGCGGGCAGGAGGAACGGCTAC 161 HBA2_P2 ACGGTATTTGGAGGTCAGCACGGTGCTCACAGAAGCCAGGAACTTGTCCA 162 HBA2_P3 CAGGGGTGAACTCGGCGGGGAGGTGGGCGGCCAGGGTCACCAGCAGGCAG 163 HBA2_P4 AAGTTGACCGGGTCCACCCGAAGCTTGTGCGCGTGTCAGGTCGCTCAGGGC 164 HBA2_P5 CATGTCGTCCACGTGCGCCACGGCGTTGGTCAGCGCGTCGGCCACCTTCT 165 HBA2_P6 CCTGGGCAGAGCCGTGGCTCAGGTCGAAGTGCGGGAAGTAGGTCTTGGTG 166 HBA2_P7 AACATCCTCTCAGGGCCTCCGCACCATACTCGCCAGCGTGCGCGCCGAC 167 HBA2_P8 CTTGACGTTGGTCTTGTCGGCAGGAGACAGCACCATGGTGGGTTCTCTCT 168 HBB_P1 GCAATGAAAATAAATGTTTTTTATTAGGCAGAATCCAGATGCTCAAGGCC 169 HBB_P2 CAGTTTAGTAGTTGGACTTAGGGAACAAAGGAACCCTTTAATAGAAATTGG 170 HBB_P3 GCTTAGTGATACTTGTGGGCCAGGGCATTAGCCACACCAGCCACCACTTT 171 HBB_P4 CACTGGTGGGGTGAATTCTTTGCCAAAGTGATGGGCCAGCACACAGACCA 172 HBB_P5 GCCTGAAGTTCTCAGGATCCACGTGCAGCTTGTCACAGTGCAGCTCACTC 173 HBB_P6 CCCTTGAGGTTGTCCAGGTGAGCCAGGCCATCACTAAAGGCACCGAGCAC 174 HBB_P7 CTTCACCCTTAGGGTTGCCCATAACAGCATCAGGAGTGGACAGATCCCCAA 175 HBB_P8 TCTGGGTCCAAGGGTAGACCACCAAGCAGCCTGCCCAGGGCCTCACCACCA 176 HBB_P9 ACCTTGCCCCACAGGGCAGTAACGGCAGACTTCTCTCAGGAGTCAGATG 177 HBG1_P1 GTGATCTCTCAGCAGAATAGATTTATTATTTGTATTGCTTGCAGAATAAA 178 HBG1_P2 CTCTGAATCATGGGCAGTGAGCTCAGTGGTATCTGGAGGACAGGGCACTG 179 HBG1_P3 ATCTTCTGCCAGGAAGCCTGCACCTCAGGGGTGAATTCTTTGCCGAAATG 180 HBG1_P4 CACCAGCACATTTCCCAGGAGCTTGAAGTTCTCAGGATCCACATGCAGCT 181 HBG1_P5 CACTCAGCTGGGCAAAGGTGCCCTTGAGATCATCCAGGTGCTTTGTGGCA 182 HBG1_P6 AGCACCTTCTTGCCATGTGCCTTGACTTTGGGGTTGCCCATGATGGCAGA 183 HBG1_P7 GCCAAAGCTGTCAAAGAACCTCTGGGTCCATGGGTAGACAACCAGGAGCC 184 HBG1_P8 CTCCAGCATCTTCCACATTCACCTTGCCCCACAGGCTTGTGTGATAGTAGCC 185 HBG1_P9 AAATGACCCATGGCGTCTGGACTAGGAGCTTATTGATAACCTCAGACGTT 186 HBG2_P1 GTGATCTCTTAGCAGAATAGATTTATTATTTGATTGCTTGCAGAATAAAG 187 HBG2_P2 TCTGCATCATGGGCAGTGAGCTCAGTGGTATCTGGAGGACAGGGCACTGG 188 HBG2_P3 TCTTCTGCCAGGAAGCCTGCACCTCAGGGGTGAATTCTTTGCCGAAATGG 189 HBG2_P4 ACCAGCACATTTCCCAGGAGCTTGAAGTTCTCAGGATCCACATGCAGCTT 190 HBG2_P5 ACTCAGCTGGGCAAAGGTGCCCTTGAGATCATCCAGGTGCTTTATGGCAT 191 HBG2_P6 GCACCTTCTTGCCATGTGCCTTGACTTTGGGGTTGCCCATGATGGCAGAG 192 HBG2_P7 CCAAAGCTGTCAAAGAACCTCTGGGTCCATGGGTAGACAACCAGGAGCCT 193 HBG2_P8 TCCAGCATCTTCCACATTCACCTTGCCCCACAGGCTTGTGATAGTAGCCT 194 HBG2_P9 AATGACCCATGGCGTCTGGACTAGGAGCTTATTGATAACCTCAGACGTTC 195 5S_GNbac_P1 ATGCCTGGCAGTTCCCTACTCTCGCATGGGGAGACCCCACACTACCATCG 196 5S_GNbac_P2 ACTTCTGAGTTCGGCATGGGGTCAGGTGGGACCACCGCGCTACGGCCGCC 197 16S_GNbac_P1 GGTTACCTTGTTACGACTTCACCCCAGTCATGAATCACAAAGTGGTAAGT 198 16S_GNbac_P2 AAGCTACCTACTTCTTTTGCAACCCACTCCCATGGTGTGACGGGCGGTGT 199 16S_GNbac_P3 ACGTATTCACCGTGGCATTCTGATCCACGATTACTAGCGATTCCGACTTC 200 16S_GNbac_P4 AGACTCCAATCCGGACTACGACGCACTTTATGAGGTCCCGCTTGCTCTCGC 201 16S_GNbac_P5 TGTATGGCGCCATTGTAGCACGTGTGTAGCCCTGGTCGTAAGGGCCATGAT 202 16S_GNbac_P6 CCACCTTCCTCCAGTTTATCACTGGCAGTCTCCTTTGAGTTCCCGGCCGG 203 16S_GNbac_P7 GGATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATTTCACAACACG 204 16S_GNbac_P8 TGCAGCACCTGTCTCACGGTTCCCCGAAGGCACATTCTCATCTCTGAAAAC 205 16S_GNbac_P9 GACCAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACC 206 16S_GNbac_P10 CGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCG 207 16S_GNbac_P11 TCCGGAAGCCACGCCTCAAGGGCACAACCTCCAAGTCGACATCGTTTACG 208 16S_GNbac_P12 GTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACTGAGCGTCAGTCTTC 209 16S_GNbac_P13 TTCGCCACCGGTATTCCTCCAGATCTCTACGCATTTCACCGCTACACCTG 210 16S_GNbac_P14 CTACGAGACTCAAGCTTGCCAGTATCAGATGCAGTTCCCAGGTTGAGCCC 211 16S_GNbac_P15 GACTTAACAAACCGCCTGCGTGCGCTTTACGCCCAGTAATTCCGATTAAC 212 16S_GNbac_P16 ATTACCGCGGCCTGCTGGCACGGAGTTAGCCGGTGCTTCTTCTGCGGGTAA 213 16S_GNbac_P17 GTATTAACTTTACTCCCTTCCTCCCCGCTGAAAGTACTTTACAACCCGAA 214 16S_GNbac_P18 CGCGGCATGGCTGCATCAGGCTTGCGCCCATTGTGCAGTATTCCCCACTG 215 16S_GNbac_P19 GTCTGGACCGTGTCTCAGTTCCAGTGTGTGGCTGGTCATCCTCTCAGACAG 216 16S_GNbac_P20 TAGGTGAGCCGTTACCCCCACCTACTAGCTAATCCCATCTGGGCACATCCG 217 16S_GNbac_P21 AAGGTCCCCCTCTTTGGTCTTGCGACGTTATGCGGTATTAGCTACCGTTT 218 16S_GNbac_P22 CTCCATCAGGCAGTTTCCCAGACATTACTCACCCGTCCGCCACTCGTCAG 219 23S_GNbac_P1 AAGGTTAAGCCTCACGGTTCATTAGTACCGGTTAGCTCAACGCATCGCTG 220 23S_GNbac_P2 CCTATCAACGTCGTCGTCTTCAACGTTTCCTTCAGGACCCTTAAAGGGTCA 221 23S_GNbac_P3 GGGGCAAGTTTCGTGCTTAGATGCTTTCAGCACTTATCTCTTCCGCATTT 222 23S_GNbac_P4 CCATTGGCATGACAACCCGAACACCAGTGATGCGTCCACTCCGGTCCTCT 223 23S_GNbac_P5 CCCCCTCAGTTCTCCAGCGCCCACGGCAGATAGGGACCGAACTGTCTCAC 224 23S_GNbac_P6 GCTCCGCGTACCACTTTAAATGGCGAACAGCCATACCCTTGGGACCTACTT 225 23S_GNbac_P7 ATGAGCCGACATCGAGGTGCCAAACACCGCCGTCGATATGAACTCTTGGG 226 23S_GNbac_P8 ATCCCCGGAGTACCTTTTATCCGTTGAGCGATGGCCCTTCCATTCAGAAC 227 23S_GNbac_P9 ACCTGCTTTCGCACCTGCTCGCGCCGTCACGCTCGCAGTCAAGCTGGCTT 228 23S_GNbac_P10 CCTCCTGATGTCCGACCAGGATTAGCCAACCTTCGTGCTCCTCCGTTACT 229 23S_GNbac_P11 GCCCCAGTCAAACTACCCACCAGAACACTGTCCGCAACCCGGATTACGGGT 230 23S_GNbac_P12 AAACATTAAAGGGTGGTATTTTCAAGGTCGGCTCCATGCAGACTGGCGTCC 231 23S_GNbac_P13 CCACCTATCCTACACATCAAGGCTCAATGTTCAGTGTCAAGCTATAGTAA 232 23S_GNbac_P14 TTCCGTCTTGCCGCGGGTACACTGCATCTTCACAGCGAGTTCAATTTCAC 233 23S_GNbac_P15 GACAGCCTGGCCATCATTACGCCATTCGTGTCAGGTCGGAACTTACCCGAC 234 23S_GNbac_P16 CTTAGGACCGTTATAGTTACGGCCCGCCGTTTACCGGGGCTTTCGATCAAGA 235 23S_GNbac_P17 ACCCCATCAATTAACCTTCCGGCACCGGGCAGGCGTCACACCGTATACGT 236 23S_GNbac_P18 CACAGTGCTGTGTTTTTAATAAACAGTTGCAGCCAGCTGGTATCTTCGAC 237 23S_GNbac_P19 CCGCGAGGGACCTCACCTACATATCAGCGTGCCTTTCTCCCGAAGTTACGG 238 23S_GNbac_P20 TTCCTTCACCCGAGTTCTCTCAAGCGCCTTGGTATTCTCTACCTGACCAC 239 23S_GNbac_P21 GTACGATTTGATGTTACCTGATTGCTTAGAGGCTTTTCCTGGAAGCAGGGC 240 23S_GNbac_P22 ACCGTAGTGCCTCGTCATCACGCCTCAGCCTTGATTTTCCGGATTTTGCCT 241 23S_GNbac_P23 ACGCTTAAACCGGGACAACCGTCGCCCGGCCAACATAGCCTTCTCCGTCC 242 23S_GNbac_P24 ACCAAGTACAGGAATATTAACCTGTTTCCCATCGACTACGCCTTTCGGCC 243 23S_GNbac_P25 ACTCACCCTGCCCCGATTAACGTTGGACAGGAACCCTTGGTCTTCCGGCG 244 23S_GNbac_P26 CGCTTTATCGTTACTTATGTCAGCATTCGCACTTCTGATACCTCAGCAT 245 23S_GNbac_P27 TTCGCAGGCTTACAGACGCTCCCCTACCCAACAACGCATAAGCGTCGCT 246 23S_GNbac_P28 CATGGTTTAGCCCCGTTACATCTTCCCGCGCAGGCCGACTCGACCAGTGAG 247 23S_GNbac_P29 TAAATGATGGCTGCTTCTAAGCCAACATCCTGGCTGTCTGGGCCTTCCCA 248 23S_GNbac_P30 AACCATGACTTTGGGACCTTAGCTGGCGGTCTGGGTTGTTTCCCTCTTCA 249 23S_GNbac_P31 CCCGCCGTGTGTCTCCCGTGATAACATTCTCCGGTATTCGCAGTTTGCAT 250 23S_GNbac_P32 GGATGACCCCCTTGCCGAAACAGTGCTCTACCCCCGGAGATGAATTCACG 251 23S_GNbac_P33 AGCTTTCGGGGAGAACCAGCTATCTCCCGGTTTGATTGGCCTTTCACCCC 252 23S_GNbac_P34 CGCTAATTTTTCAACATTAGTCGGTTCGGTCCTCCAGTTAGTGTTACCCA 253 23S_GNbac_P35 ATGGCTAGATCACCGGGTTTCGGGTCTATACCCTGCAACTTAACGCCCAG 254 23S_GNbac_P36 CCTTCGGCTCCCCTATTCGGTTAACCTTGCTACAGAATATAAGTCGCTGA 255 23S_GNbac_P37 GTACGCAGTCACACGCCTAAGCGTGCTCCCACTGCTTGTACGTACACGGT 256 23S_GNbac_P38 ACTCCCCTCGCCGGGGTTCTTTTCGCCTTTCCCTCACGGTACTGGTTCAC 257 23S_GNbac_P39 AGTATTTAGCCTTGGAGGATGGTCCCCCCATATTCAGACAGGATACCACG 258 23S_GNbac_P40 ATCGAGCTCACAGCATGTGCATTTTTGTGTGTACGGGGCTGTCACCCTGTAT 259 23S_GNbac_P41 ACGCTTCCACTAACACACACACTGATTCAGGCTCTGGGCTGCTCCCCGTT 260 23S_GNbac_P42 GGGGAATCTCGGTTGATTTCTTTTCCTCGGGGTACTTAGATGTTTCAGTT 261 23S_GNbac_P43 ATTAACCTATGGATTCAGTTAATGATAGTGTGTCGAAACACACTGGGTTT 262 23S_GNbac_P44 GCCGGTTATAACGGTTCATATCACCTTACCGACGCTTATCGCAGATTAGC 263 5S_GPbac_P1 GCTTGGCGGCGTCCTACTCTCACAGGGGGAAACCCCCGACTACCATCGGC 264 5S_GPbac_P2 TTCCGTGTTCGGTATGGGAACGGGTGTGACCTCTTCGCTATCGCCACCAA 265 16S_GPbac_P1 TAGAAAGGAGGTGATCCAGCCGCACCTTCCGATACGGCTACCTTGTTACG 266 16S_GPbac_P2 TCTGTCCCACCTTCGGCGGCTGGCCTCCTAAAAGGTTACCTCACCGACTTC 267 16S_GPbac_P3 TCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCG 268 16S_GPbac_P4 ATTACTAGCGATTCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGA 269 16S_GPbac_P5 GTGGGATTGGCTTAACCTCGCGGTTTCGCCTGCCCTTTGTTCTGTCCATTG 270 16S_GPbac_P6 CCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGG 271 16S_GPbac_P7 CACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGC 272 16S_GPbac_P8 ACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCAC 273 16S_GPbac_P9 GACGTCCTATCTCTAGGATTGTCAGAGGATGTCAAGACCTGGTAAGGTTC 274 16S_GPbac_P10 ATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGA 275 16S_GPbac_P11 CCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGG 276 16S_GPbac_P12 ACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGT 277 16S_GPbac_P13 TCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTG 278 16S_GPbac_P14 ACGCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAA 279 16S_GPbac_P15 ATGACCCTCCCCGGTTGAGCCGGGGGCTTTTCACATCAGACTTAAGAAACC 280 16S_GPbac_P16 ACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTG 281 16S_GPbac_P17 CCGTGGCTTTCTGGTTAGGTACCGTCAAGGTACCGCCCTATTCGAACGGT 282 16S_GPbac_P18 ACAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGCGGCGTTGCT 283 16S_GPbac_P19 CCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTG 284 16S_GPbac_P20 GGCCGATCACCCTCTCAGGTCGGCTACGCATCGTCGCCTTGGTGAGCCGT 285 16S_GPbac_P21 CTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGCCGAAGCCACCTTTTAT 286 16S_GPbac_P22 TTCAAACAACCATCCGGTATTAGCCCCGGTTTCCCGGAGTTATCCCAGTC 287 16S_GPbac_P23 CCACGTGTTACTCACCCGTCCGCCCGCTAACATCAGGGAGCAAGCTCCCAT 288 16S_GPbac_P24 GCATGTATTAGGCACGCCGCCAGCGTTCGTCCTGAGCCAGGATCAAACTC 289 23S_GPbac_P1 TGGTTAAGTCTCGATCGATTAGTATCTGTCAGCTCCATGTGTCGCCACA 290 23S_GPbac_P2 TATCAACCTGATCATCTTTTCAGGGATCTTACTTCCTTGCGGAATGGGAAA 291 23S_GPbac_P3 GGCTTCATGCTTAGATGCTTTCAGCACTTATCCCGTCCGCACATAGCTAC 292 23S_GPbac_P4 GCAGAACAACTGGTACACCAGCGGTGCGTCCATCCCGGTCCTCTCGTACT 293 23S_GPbac_P5 CAAATTTCCTGCGCCCGCGACGGATAGGGACCGAACTGTCTCACGACGTT 294 23S_GPbac_P6 GTACCGCTTTAATGGGCGAACAGCCCAACCCTTGGGACTGACTACAGCCC 295 23S_GPbac_P7 CGACATCGAGGTGCCAAACCTCCCCGTCGATGTGGACTCTTGGGGGAGAT 296 23S_GPbac_P8 GGGGTAGCTTTTATCCGTTGAGCGATGGCCCTTCCATGCGGAACCACCGG 297 23S_GPbac_P9 TTTCGTCCCTGCTCGACTTGTAGGTCTCGCAGTCAAGCTCCCTTGTGCCT 298 23S_GPbac_P10 GATTTCCAACCATTCTGAGGGAACCTTTGGGCGCCTCCGTTACCTTTAG 299 23S_GPbac_P11 GTCAAACTGCCCACCTGACACTGTCTCCCCGCCCGATAAGGGCGGCGGGT 300 23S_GPbac_P12 GCCAGGGTAGTATCCCACCGATGCCTCCACCGAAGCTGGCGCTCCGGTTT 301 23S_GPbac_P13 ATCCTGTACAAGCTGTACCAACATTCAATATCAGGCTGCAGTAAAGCTCC 302 23S_GPbac_P14 CCTGTCGCGGGTAACCTGCATCTTCACAGGTACTATAATTTCACCGAGTC 303 23S_GPbac_P15 GCCCAGATCGTTGCGCCTTTCGTGCGGGTCGGAACTTACCCGACAAGGAA 304 23S_GPbac_P16 ACCGTTATAGTTACGGCCGCCGTTTACTGGGGCTTCAATTCGCACCTTCG 305 23S_GPbac_P17 CCTCTTAACCTTCCAGCACCGGGCAGGCGTCAGCCCCCTATACTTCGCCTT 306 23S_GPbac_P18 CCTGTGTTTTTGCTAAACAGTCGCCTGGGCCTATTCACTGCGGCTCTCTC 307 23S_GPbac_P19 CAGAGCACCCCTTCTCCCGAAGTTACGGGGTCATTTTGCCGAGTTCCTTA 308 23S_GPbac_P20 ATCACCTTAGGATTTCTCCTCGCCTACCTGTGTCGGTTTGCGGTACGGG 309 23S_GPbac_P21 TAGAGGCTTTTCTTGGCAGTGTGGAATCAGGAACTTCGCTACTATATTTC 310 23S_GPbac_P22 TCAGCCTTATGGGAAACGGATTTGCCTATTTCCCAGCCTAACTGCTTGGA 311 23S_GPbac_P23 CCGCGCTTACCCTATCCTCCTGCGTCCCCCCATTGCTCAAATGGTGAGGA 312 23S_GPbac_P24 TCAACCTGTTGTCCATCGCCTACGCCTTTCGGCCTCGGCTTAGGTCCCGA 313 23S_GPbac_P25 CGAGCCTTCCTCAGGAAACCTTAGGCATTCGGTGGAGGGGATTCTCACCC 314 23S_GPbac_P26 TACCGGCATTCTCACTTCTAAGCGTCTCCACCAGTCCTTCCGGTCTGGCTT 315 23S_GPbac_P27 GCTCTCCTACCACTGTTCGAAGAACAGTCCGCAGCTTCGGTGATACGTTT 316 23S_GPbac_P28 TCGGCGCAGAGTCACTCGACCAGTGAGCTATTACGCACTCTTTAAATGGT 317 23S_GPbac_P29 AACATCCTGGTTGTCTAAGCAACTCCACATCCTTTTCCACTTAACGTATA 318 23S_GPbac_P30 TGGCGGTCTGGGCTGTTTCCCTTTCGACTACGGATCCTTATCACTCGCAGT 319 23S_GPbac_P31 AAGTCATTGGCATTCGGAGTTTGACTGAATTCGGTAACCCGGTAGGGGCC 320 23S_GPbac_P32 GCTCTACCTCCAAGACTCTTACCTTGAGGCTAGCCCTAAAGCTATTTCGG 321 23S_GPbac_P33 TCCAGGTTCGATTGGCATTTCACCCCTACCCAACACCTCATCCCCGCACTT 322 23S_GPbac_P34 TTCGGGCCTCCATTCAGTGTTACCTGAACTTCACCCTGGACATGGGTAGA 323 23S_GPbac_P35 TCTACGACCACGTACTCATGCGCCCCTATTCAGACTCGCTTTCGCTGCGGC 324 23S_GPbac_P36 TAACCTTGCACGGGATCGTAACTCGCCGGTTCATTCTACAAAAGGCACGC 325 23S_GPbac_P37 GGCTCTGACTACTTGTAGGCACACGGTTTCAGGATCTCTTTCACTCCCCCT 326 23S_GPbac_P38 ACCTTTCCCTCACGGTACTGGTTCACTATCGGTCACTAGGGAGTATTAG 327 23S_GPbac_P39 CTCCCGGATTCCGACGGAATTTCACGTGTTCCGCCGTACTCAGGATCCAC 328 23S_GPbac_P40 GTTTTGACTACAGGGCTGTTACCTCCTATGGCGGGCCTTTCCAGACCTCT 329 23S_GPbac_P41 CTTTGTAACTCCGTACAGAAGTGTCCTACTACAACCCCAAGAGGCAAGCCTCTT 330 23S_GPbac_P42 CGTTTCGCTCGCCGCTACTCAGGGAATCGCATTTGCTTTCTCTTCCTCCG 331 23S_GPbac_P43 CAGTTCCCCGGGTCTGCCCTTCTCATATCCTATGAATTCAGATATGGATAC 332 23S_GPbac_P44 GGTGGGTTTCCCCATTCGGAAATCTCCGGATCAAAGCTTGCTTACAGCTC 333 23S_GPbac_P45 TGTTCGTCCCGTCCTTCATCGGCTCCTAGTGCCAAGGCATCCACCGTGCG 334 16S:A1 AAACTAGATTCGAATATAACAAAACATTACATCCTCATCCAATCCCTTTT 335 16S:A2 GCGGTGTGTGCAAGGAGCAGGGACGTATTCACCGCGCGATTGTGACACGC 336 16S:A3 GCCTTTTCGGCGTCGGAACCCATTGTCTCAGCCATTGTAGCCCGCGTGTGTTG 337 16S:A4 GCATACGGACCTACCGTCGTCCACTCCTTCCTCCTATTTATCATAGGCGG 338 16S:A5 CGGCATCCAAAAAAGGATCCGCTGGTAACTAAGAGCGTGGGTCTCGCTCG 339 16S: A6 CAACCTGGCTATCATACAGCTGTCGCCCTCTGGTGAGATGTCCGGCGTTGA 340 16S:A7 AGGCTCCACGCGTTGTGGTGCTCCCCCGCCAATTCCTTTAAGTTTCAGTC 341 16S:A8 CCAGGCGGCGGACTTAACAGCTTCCCTTCGGCACTGGGACAGCTCAAAGC 342 16S:A9 TCCGCATCGTTTACAGCTAGGACTACCCGGGTATCTAATCCGGTTCGCGC 343 16S:A10 TTCCCACAGTTAAGCTGCAGGATTTCACCAGAGACTTATTAAACCGGCTA 344 16S:A12 CTCTTATTCCAAAAGCTCTTTACACTAATGAAAAGCCATCCCGTTAAGAA 345 16S:A13 CCCCCGTCGCGATTCTCACATTGCGGAGGTTTCGCGCCTGCTGCACCCC 346 16S:A14 TTGTCTCAGGTTCCATCTCCGGGCTCTTGCTCTCACAACCCGTACCGATC 347 16S:A16 CATTACCTAACCAACTACCTAATCGGCCGCAGACCCATCCTTAGGCGAAA 348 16S:A17 AAACCATTACAGGAATAATTGCCTATCCAGTATTATCCCCAGTTTCCCAG 349 16S:A18 AAGGGTAGGTTATCCACGTGTTACTGAGCCGTACGCCACGAGCCTAAACT 350 23S:A1 ACCTAGCGCGTAGCTGCCCGGCACTGCCTTATCAGACAACCGGTCGACCA 351 23S:A2 CGTTCCTCTCGTACTGGAGCCACCTTCCCCTCAGACTACTAACACATCCA 352 23S:A3 CCTGTCTCACGACGGTCTAAACCCAGCTCACGTTCCCCTTTAATGGGCGA 353 23S:A4 GGTGCTGCTGCACCCAGGATGGAAAGAACCGACATCGAAGTAGCAAGC 354 23S:A5 GGCTCTTGCCTGCGACCACCAAGTTATCCCCGAGGTAGTTTTTCTGTCAT 355 23S:A6 AGGAGGACTCTGAGGTTCGCTAGGCCCGGCTTTCGCCTCTGGATTTCTTG 356 23S:A7 CAAAGTAAGTTAGAAACACAGTCATAAGAAAGTGGTGTCTCAAGAACGAA 357 23S:A8 GACTTATAATCGAATTCTCCCACTTACACTGCATACCTATAACCAAGCTT 358 23S:A9 GTAAAACTCTACGGGGTCTTCGCTTCCCAATGGAAGACTCTGGCTTGTGC 359 23S:A10 TCACTAAGTTCTAGCTAGGGACAGTGGGGACCTCGTTCTACCATTCATGC 360 23S:A11 CGACAAGGCATTTCGCTACCTTAAGAGGGTTATAGTTACCCCCGCCGTTT 361 23S:A12 AACTGAACTCCAGCTTCACGTGCCAGCACTGGGCAGGTGTCGCCCTCTGT 362 23S:A13 CTAGCAGAGAGCTATGTTTTTATTAAACAGTCGGGCCCCCCTAGTCACTG 363 23S:A14 TTAAAACGCCTTAGCCTACTCAGCTAGGGGCACCTGTGACGGATCTCGGT 364 23S:A15 ACAAAACTAACTCCCTTTTCAAGGACTCCATGAATCAGTTAAACCAGTAC 365 23S:A16 ATAATGCCTACACCTGGTTCTCGCTATTACACCTCTCCCAGGCTTAAAC 366 23S:A17 CAATCCTACAAAACATATCTCGAAGTGTCAGAAATTAGCCCTCAACGTCA 367 23S:A18 CTTTGCTGCTACTACTACCAGGATCCACATACCTGCAAGGTCCAAAGGAA 368 23S:A19 CAACCCACACAGGTCGCCACTCTACACAATCACCAAAAAAAAGGTGTTCC 369 23S:A20 GGATTAATTCCCGTCCATTTTAGGTGCCCTCTGACCTCGATGGGTGATCTG 370 23S:A21 AGGGTGGCTGCTTCTAAGCCCACCTTCCCATTGTCTTGGGCCAAAAGACTC 371 23S:A22 GTATTTAGGGGCCTTAACCATAGTCTGAGTTGTTTCTCTTTCGGGACACA 372 23S:A23 CCTCACTCCAACCTTCTACGACGGTGACGAGTTCGGAGTTTTACAGTACG 373 23S:A24 CCCTAAACGTCCAATTAGTGCTCTACCCCGCCACCAACCTCCAGTCAGGC 374 23S:A25 AATAGATCGACCGGCTTCGGGTTTCAATGCTGTGATTCCAGGCCCTATTA 375 23S:A26 ACAACGCTGCGGGCATATCGGTTTCCCTACGACTACAAGGATAAAAAACCT 376 23S:A27 ACAAAGAACTCCCTGGCCCGTGTTTCAAGACGGACGATGCAACACTAGTC 377 23S:A28 ACAATGTTACCACTGATTCTTTCGGAAGAATTCATTCCTTACGCGCCACA 378 23S:A29 CTGGTTTCAGGTACTTTTCACCCCCCTATAGGGGTACTTTTCAGCATTCC 379 23S:A30 CTCTATCGGTCTTGAGACGTATTTAGAATTGGAAGTTGATTGCCTCCCACA 380 23S:A31 ATCACCCTCTACGGTTCTAAAATTCCAAATAAAATTCGATTTATCCCACG 381 23S:A32 TCTATACACCACATCTCCCTAATATTACTAAAAGGGATTCAGTTTGTTCT 382 23S:A33 GCCGTTACTAACGACATCGCATATTGCTTTCTTTTCCTCCGCCTACTAAG 383 23S:A34 GGGTTCCCAATCCTACACGGATCAACACAAAAAAAATGTGCTAGGAAGTC 384 5S:A1 ACTACTGGGATCGAAACGAGACCAGGTATAACCCCCCATGCTATGACCGCA 385 MM_16S_P10 GCGTATGCCTGGAGAATTGGAATTCTTGTTACTCATACTAACAGTGTTGC 386 MM_16S_P11 GATTAACCCAATTTTAAGTTTAGGAAGTTGGTGTAAATTATGGAATTAAT 387 MM_16S_P12 AGCTTGAACGCTTTCTTTATTGGTGGCCTGCTTTTAGGCCTACAATGGTTA 388 MM_16S_P13 ATTATTCACTATTAAAGGTTTTTTCCGTTCCAGAAGAGCTGTCCCTCTTTT 389 MM_16S_P14 CTTACTTTTTGATTTTGTTGTTTTTTTAGCAAGTTTAAAATTGAACTTAA 390 MM_16S_P15 AACCAGCTATCACCAAGCTCGTTAGGCTTTTCACCTCTACCTAAAAAATCT 391 MM_16S_P7 AATACTTGTAATGCTAGAGGTGATGTTTTTGGTAAACAGGCGGGGTTCTT 392 MM_16S_P8 TTTATCTTTTTGGATCTTTCCTTTAGGCATTCCGGTGTTGGGTTAACAGA 393 MM_16S_P9 TTATTTATAGTGTGATTATTGCCTATAGTCTGATTAACTAACAATGGTTA 394 RN_16S_P4 AGTGATTGTAGTTGTTTTATTCACTATTTAAGGTTTTTTCCTTTTCCTAAA 395 RN_16S_P5 TGGCTATATTTTAAGTTTACATTTTGATTTGTTGTTCTGATGGTAAGCTT 396 RN_16S_P6 TTTTTTTAATCTTTCCTTAAAGCACGCCTGTGTTGGGCTAACGAGTTAGG 397 RN_16S_P7 TGTTGGGTTAGTACCTATGATTCGATAATTGACAATGGTTATCCGGGTTG 398 RN_16S_P8 AGGAGAATTGGTTCTTGTTACTCATATTAACAGTATTTTCATCTATGGATC 399 RN_16S_P9 TTTGTGATATAGGAATTTATTGAGGTTTGTGGAATTAGTGTGTGTGTAAGTA 400 MM_28S_P1 GCCGGGGAGTGGGTCTTCCGTACGCCACATTTCCCACGCCGCGACGCGCG 401 MM_28S_P10 ACCTCGGGCCCCCGGGCGGGGCCCTTCACCTTCATTGCGCCACGGCGGCT 402 MM_28S_P14 TCGCGTCCAGAGTCGCCGCCGCCCGCCGGCCCCCCGAGTGTCCGGGCCCCC 403 MM_28S_P15 CGCTGGTTCCTCCCGCTCCGGAACCCCCGCGGGGTTGGACCCGCCGCCCC 404 MM_28S_P16 CGCCGACCCCCGACCCGCCCCCCGACGGGAAGAAGGAGGGGGGAAGAGAG 405 MM_28S_P17 GGGACGACGGGGCCCCGCGGGGAAGAGGGGAGGGCGGGCCCGGGCGGAAA 406 MM_28S_P18 GGCGCCGCGCGGAAAACCGCGGCCCGGGGGGCGGACCCGGCGGGGGAACA 407 MM_28S_P19 CCCCCACACGCGCGGGACACGCCCGCCCGCCCCCGCCACGCACCTCGGGA 408 MM_28S_P2 CACCCGCTTTGGGCTGCATTCCCAAGCAACCCGACTCCGGGAAGACCCGA 409 MM_28S_P20 TGGAGCGAGGCCCCGCGGGGAGGGGACCCGCGCCGGCACCCGCCGGGCTC 410 MM_28S_P21 CGAGGCCGGCGTGCCCCGACCCCGACGCGAGGACGGGGCCGGGCGCCGGG 411 MM_28S_P22 TCCCCGGAGCGGTCGCGCCCGCCCGCACGCGCGGGACGGACGCTTGGCG 412 MM_28S_P23 TCCACACGAACGTGCGTTCAACGTGACGGGCGAGAGGGCGGCCCCCTTTC 413 MM_28S_P24 TCCCAAGACGAACGGCTCTCCGCACCGGACCCCGGTCCCGACGCCCGGCG 414 MM_28S_P25 CCGCCGCGGGGACGACGCGGGGACCCCGCCGAGCGGGGACGGACGGGGAC 415 MM_28S_P3 GCACCGCCACGGTGGAAGTGCGCCCGGCGGCGGCCGGTCGCCGGCCGGGG 416 MM_28S_P6 CCCACCGGGCCCCGAGAGAGGCGACGGAGGGGGGTGGGAGAGCGGTCGCG 417 MM_28S_P7 CCCGGCCCCCACCCCCACGCCCGCCCGGGAGGCGGACGGGGGGAGAGGGA 418 MM_28S_P8 TATCTGGCTTCCTCGGCCCCGGGATTCGGCGAAAAGCGCGGCCGGAGGGCT 419 MM_28S_P9 CGCCGCCGACCCCGTGCGCTCGGCTTCGTCGGGAGACGCGTGACCGACGG 420 RN_28S_P12 GCGCCCCCCCGCACCCGCCCCGTCCCCCCCGCGGACGGGGAAGAAGGGAG 421 RN_28S_P14 CGAACCCCGGGAACCCCCGACCCCGCGGAGGGGGAAGGGGGAGGACGAGG 422 RN_28S_P16 CACCCGGGGGGGCGACGAGGCGGGGACCCGCCGGACGGGGACGGACGGGG 423 RN_28S_P17 GCCAACCGAGGCTCCTTCGGCGCTGCCGTATCGTTCCGCTTGGGCGGATT 424 RN_28S_P4 CCCGGGCCCCCGGACCCCCGAGGGACGACGGAGGCGACGGGGGGTGGG 425 RN_28S_P5 TGGGAGGGGCGGCCCGGCCCCCGCGACCGCCCCCCTTTCCGCCACCCCAC 426 RN_28S_P6 GGGAGAGGCCGGGGGGAGAGCGCGGCGACGGGTATCCGGCTCCCTCGGCC 427 RN_28S_P7 CGCTGCTGCCGGGGGGCTGTAACACTCGGGGCGGGGTGGTCCGGCGCCCA 428 RN_28S_P8 CGCCGCCGACCCCGTGCGCTCGGCTTCGCTCCCCCCCACCCCGAGAAGGG

[0045] In one embodiment of the present invention, the RNA sample is from a human and the DNA probe set includes probes specific for unwanted human RNAs, such as rRNA and mitochondrial RNA transcripts, as described herein. In another embodiment of the present invention, a set of DNA probes for removing unwanted RNA molecules from a human RNA sample includes probes specific for human rRNA and mitochondrial mRNA transcripts, as well as probes specific for Gram-positive and Gram-negative unwanted RNA transcripts, as described herein. document. In a further embodiment of the present invention, a set of DNA probes for removing unwanted RNA molecules from a human RNA sample includes probes specific for Archaea bacterial species, an example of which is M. smithii , as described herein. Thus, in some embodiments of the present invention, a set of DNA probes for depleting rRNA from a human RNA sample contains only probes that target unwanted human RNA species, or a mixed set of DNA probes that also target unwanted non-human RNA transcripts. . The skilled artisan will appreciate that the set of probes to be used for RNA depletion will depend on the research objectives, the medium from which the sample was taken, and other factors of the RNA sample depletion experimental design.

[0046] In one embodiment of the present invention, the RNA sample is derived from a non-human eukaryote, and the set of DNA probes includes probes specific for unwanted RNA in the resulting eukaryotic sample. For example, if the RNA sample is from a mouse or rat, the DNA probe set will include probes specific for unwanted mouse or rat RNA, which may also include DNA probes specific for unwanted Gram-positive and Gram-negative bacterial RNA, or other bacterial species. , such as Archaea species.

[0047] In some embodiments of the present invention, DNA probes do not hybridize to the entire contiguous length of the RNA molecules to be removed. During the experiments, it was unexpectedly found that for a full-length sequence of target RNA species for depletion, it is not necessary to use a full-length DNA probe or probe set that will continuously cover the entire RNA sequence; indeed, the DNA probes described herein leave gaps such that the DNA:RNA hybrids formed are not contiguous. Surprisingly, gaps of at least 5 nt, 10 nt, 15 nt, or 20 nt between DNA:RNA hybrids were found to provide efficient RNA depletion. In addition, gapping probe sets can more efficiently hybridize to unwanted RNA because DNA probes do not interfere with hybridization of adjacent probes, which can potentially occur with probes that span the entire sequence of the depletion target RNA or with probes that overlap. with friend.

[0048] In addition, probe sets can be supplemented to improve RNA depletion methods for a given species. A method for adding a set of probes to remove non-target RNA nucleic acid molecules from a nucleic acid sample may include the following: a) bringing a nucleic acid sample containing at least one target sequence in the RNA or DNA structure and at least one molecular sequence A first non-target RNA species in contact with a probe set comprising at least two DNA probes complementary to non-contiguous sequences along the entire length of at least one non-target second species RNA molecule sequence, thereby hybridizing the DNA probes with non-target RNA molecules to form DNA:RNA hybrids, with each DNA:RNA hybrid separated by at least 5 bases or at least 10 bases along a predetermined sequence of a non-target RNA molecule from another DNA hybrid : RNA; b) bringing the DNA:RNA hybrids into contact with a ribonuclease that cleaves RNA from the DNA:RNA hybrids, thereby destroying the non-target RNA molecules in the nucleic acid sample to form a digested mixture; c) separating the digested RNA from the sample; d) sequencing the remaining RNA from the sample; e) evaluating the remaining RNA sequences for the presence of non-target RNA molecules of the first kind, thus determining gap sequence regions; and f) adding to the set at least one DNA probe that is complementary to non-contiguous sequences in one or more gap regions. In some embodiments, the gap sequence regions are at least 50, 60, or 70 bp long. In some embodiments, the implementation of the present invention, the first species does not apply, and the second species refers to the person. In some embodiments of the present invention, the first species is rats or mice. Exemplary methods for adding a set of probes to improve the removal of unwanted rRNA nucleic acid molecules in mouse samples are presented in Example 8 and in FIG. nine.

[0049] In some embodiments, the implementation of the present invention, the first species does not apply, and the second species refers to a person. In some embodiments of the present invention, the first species is rats or mice. In some embodiments of the present invention, the second species is a human, gram-positive bacteria, gram-negative bacteria, or a mixture thereof.

Compositions and sets

[0050] In one embodiment, the present invention relates to compositions containing a set of probes, as described herein. In some embodiments, implementation of the present invention, the composition contains a set of probes and a ribonuclease capable of cleaving RNA in a DNA:RNA hybrid, such as RNase H or hybridase. In some embodiments, the probe set comprises at least two DNA probes that are complementary to at least one non-target rRNA molecule in the nucleic acid sample, and the probes do not overlap or are contiguous in length (e.g., are at least 5 or at least 10 bases apart along their entire length). In some embodiments of the present invention, the composition comprises a set of probes containing at least two DNA probes hybridizable with at least one non-target RNA molecule, with each DNA probe hybridizing at a distance of at least 5 or at least 10 bases apart along the length of the non-target RNA molecule of any other DNA probe in the set. In some embodiments, the implementation of the present invention, the composition contains a chemical substance that destabilizes the nucleic acid, such as formamide, betaine, DMSO, glycerol, derivatives or mixtures thereof. In one embodiment of the present invention, the destabilizing chemical is formamide or its derivative, the concentration of which in the total volume of the hybridization reaction is 10-45%.

[0051] In one embodiment, the present invention describes a kit with a set of probes that includes at least two DNA probes that are complementary to non-contiguous sequences along the entire length of at least one sequence of non-target rRNA molecules (for example, at a distance at least 5 bases apart or at least 10 bases along its entire length) in a nucleic acid sample and a ribonuclease capable of cleaving RNA in a DNA:RNA hybrid. In some embodiments of the present invention, the set of probes contains any of the DNA probes described herein, or any combination thereof.

[0052] In some embodiments, the implementation of the present invention, the kit contains a buffer and environment for purification of nucleic acids. In some embodiments, the kit includes one or more buffers, nucleic acid purification media, and a set of DNA probes as described herein. In some embodiments, implementation of the present invention, the set of probes contains two or more sequences of SEQ ID NO: 1-333. In some embodiments, the implementation of the present invention, the set of probes contains two or more sequences of SEQ ID NO: 1-428. In some embodiments of the present invention, the set of probes contains two or more, or five or more, or 10 or more, or 25 or more, or 50 or more, or 100 or more, or 150 or more, or 200 or more, or 250 or more, or 300 or more, or 350 or more, or 377 sequences from SEQ ID NOs: 1-377 (human, Gram-positive and Gram-negative bacteria and Archaea ). In some embodiments of the present invention, the set of probes contains two or more, or five or more, or 10 or more, or 25 or more, or 50 or more, or 100 or more, or 150 or more, or 200 or more, or 250 or more, or 300 or more, or 350 or more, or 384 sequences from SEQ ID NOs: 1-333 and SEQ ID NOs: 378-428 (human, Gram-positive bacteria, Gram-negative bacteria, mice and rats). In some embodiments, the probe set contains two or more, or five or more, or 10 or more, or 25 or more, or 44 sequences from SEQ ID NOs: 334-377 ( Archaea ). In some embodiments, the probe set contains two or more, or five or more, or 10 or more, or 25 or more, or 50 or more, or 51 sequences from SEQ ID NOs: 378-428 (mice and rats) .

[0053] In some embodiments, the implementation of the present invention, the kit includes: 1) a set of probes, as described herein; 2) ribonuclease; 3) DNase; and 4) RNA purification beads. In some embodiments, the kit includes an RNA depletion buffer, a depletion probe buffer, and a probe removal buffer.

Analysis of depleted samples

[0054] The described methods also find application in the analysis of transcriptomes from single or mixed samples. Transcriptomic analysis may be hindered by a high relative content of ribosomal RNA, for example, a sample may contain ≥ 85% rRNA molecules in total RNA from bacterial cells. With so many rRNAs competing for sequencing or other analysis reagents, it can be difficult to focus on the more informative parts of the transcriptome that can be lost in the analysis of unwanted rRNA. The described methods can facilitate enhanced transcriptomic analysis of microbial or eukaryotic isolates, for example, with a small DNA sample volume, resulting in fewer rRNA sequencing reads, lower sequencing costs, and allowing metatranscriptomic analysis of low biomass samples. This is shown in Example 4, in which small volume (<80 ng) in mixed samples was evaluated using the RNase H rRNA depletion methods described herein. The methods described herein can be used in conjunction with various 3'-side applications such as the creation of libraries for nucleic acid sequencing techniques, the use of enriched samples for RT-PCR followed by microarray analysis, PCR, qPCR, etc. However, it should be understood that the enriched RNA samples produced by the RNA depletion methods described herein are not limited to any particular downstream application such as sequencing.

[0055] For example, depleted RNA samples can be used to create sequencing libraries such that the generated libraries can be attached at fixed locations on the chip such that their relative positions do not change and the chip is repeatedly imaged. In particular, embodiments are applicable in which images are obtained in different color channels, for example, matching different labels used to distinguish one type of nucleotide base from another. In some embodiments, the process of determining the nucleotide sequence of the target nucleic acid may be an automated process. Preferred embodiments include sequencing-by-synthesis (SBS) techniques.

[0056] SBS techniques essentially involve enzymatic extension of the nascent nucleic acid strand by iterative addition of nucleotides to the template strand. In traditional SBS methods, a single nucleotide monomer can be introduced into the target nucleotide in the presence of a polymerase with each delivery. Nucleotide monomers with or without terminator functionality can be used in SBS. Methods for using nucleotide monomers without terminators include, for example, pyrosequencing and sequencing using γ-phosphate-labeled nucleotides. In methods for using nucleotide monomers that do not contain terminators, the number of nucleotides added in each cycle essentially varies and depends on the template sequence and the method of delivery of the nucleotides. For SBS techniques that use terminally functionalized nucleotide monomers, the terminator may be effectively irreversible under sequencing conditions, as is the case for traditional Sanger sequencing using dideoxynucleotides, or the terminator may be reversible, as is the case for sequencing methods developed by Solexa. (currently Illumina, Inc.).

[0057] Sequencing methodologies that can utilize RNA depletion processes and RNA-enriched samples include, but are not limited to, run sequencing, which is performed by stepwise addition of reversible terminator nucleotides containing, for example, a cleavable or photoleachable dye. Examples of Illumina instruments that may use the methods described herein include commercially available HiSeq ^TM , MiSeq TM , NextSeq ^TM , NovaSeq ^TM , NextSeq ^TM , and iSeq ^TM instruments.

[0058] Additional sequencing techniques include a ligation method. Such methods use DNA ligase to incorporate oligonucleotides and identify their inclusion.

[0059] In addition, when sequencing nanopores, the described RNA samples after depletion can also be used to obtain a library. Nanopore sequencing methods determine the sequence of a strand of nucleic acids that pass through a pore, with the change flowing through the pore being indicative of which nucleotide passes through it.

[0060] In addition, when sequencing using real-time monitoring of DNA polymerase activity, post-depleted RNA samples can be used.

[0061] Additional SBS techniques for generating sequencing libraries using depleted RNA samples described herein include detecting a proton released when a nucleotide is inserted into the extension product. For example, sequencing based on the detection of released protons can use an electrical detector and related solutions that are commercially available from Ion Torrent (a subsidiary of Life Technologies, Guilford, CT).

[0062] Additional 3'-end applications in which depleted enriched RNA samples as described herein include PCR, qPCR, microarray analysis, and the like. For example, microarray analysis is an efficient way to study gene expression. Enriched samples can be used in microarray analysis by converting the enriched RNA into cDNA by methods known to those skilled in the art (eg RT-PCR reverse transcription polymerase chain reaction). The cDNA can then be immobilized on substrates, microarray probes applied, and expression analysis determined following any of a variety of microarray analysis methods (eg, Agilent, Affymetrix, and Illumina, and many others sell commercial microarray analysis systems). Polymerase chain reaction (PCR) or quantitative PCR (qPCR) can also use an enriched sample as a substrate according to established procedures (current molecular biology protocols).

[0063] Thus, RNA-reduced samples obtained by the methods described herein can be used to generate sequencing libraries, amplification products, and the like, which can be used for 3'-side analysis methodologies. The methods described are not limited to any use at the 3' ends.

EXAMPLES

[0064] The following examples are illustrative only and are not intended to limit the scope of the application. Modifications will be obvious and understandable to those skilled in the art and are included within the spirit and scope of the invention described in this application.

EXAMPLE 1 Depletion of unwanted RNA species from a sample

[0065] In this example, total RNA is the target nucleic acid in the sample, and RNA depletion involves four main steps: 1) hybridization, 2) RNase H treatment, 3) DNase treatment, and 4) target RNA purification.

[0066] Hybridization is carried out by annealing a specific DNA probe set to denatured RNA in the sample. An RNA sample, 10-100 ng, is incubated in a tube with 1 µl of a set of 1 µM/oligo-DNA oligonucleotide probes (probes corresponding to SEQ ID NOs: 1-333 as indicated in Table 1), 3 µl of 5X hybridization buffer ( 500 mM Tris-HCl pH 7.5 and 1000 mM KCl), 2.5 µl 100% formamide and enough 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, slowly cooled to 37°C by lowering the temperature by 0.1°C/sec and maintained at 37°C. After the reaction mixture reaches a temperature of 37°C, no time is required for incubation. The total time required for the denaturation to reach 37°C is approximately 15 minutes.

[0067] After hybridization, the following components are added to the reaction tube to remove unwanted RNA species from the DNA duplex: RNA; 4 µl 5X RNase H buffer (100 mM Tris pH 7.5, 5 mM DTT, 40 mM MgCl ₂ ) and 1 µl RNase H enzyme. The enzyme reaction mixture is incubated at 37° C. for 30 minutes. The reaction tube may be kept on ice.

[0068] Upon removal of the RNA from the DNA:RNA hybrid, the DNA probes are cleaved. The following components are added to a 20 µl reaction tube: 3 µl 10X Turbo DNase buffer (200 mM Tris pH 7.5, 50 mM CaCl ₂ , 20 mM MgCl ₂ ), 1.5 µl Turbo DNase (Thermo Fisher Scientific) and 5, 5 µl H ₂ O for a total volume of 30 µl. The enzymatic reaction mixture is incubated at 37°C for 30 minutes and then at 75°C for 15 minutes. An incubation at 75° C. can be used to fragment the total target RNA to the required insert sizes for further processing at the 3' end. In this example, the size of the target insert is approximately 200 nt of total RNA. The timing of this incubation step can be adjusted depending on the size of the insert required for subsequent reactions, at the discretion of the skilled artisan. After incubation, the reaction tube can be stored on ice.

[0069] After hybridization of probes with unwanted RNA, removal of RNA, and removal of DNA, the total target RNA in the sample can be isolated from the reaction conditions. The reaction tube is removed from the storage medium at 4° C. and allowed to warm to room temperature, 60 µl of RNAClean XP beads (Beckman Coulter) are added and incubated for 5 min. After incubation, the tube is placed on a magnet for 5 min, after which the supernatant is carefully removed and discarded. Without removing the magnet, the beads with attached total RNA are washed twice in 175 µl of freshly prepared 80% ethanol. After the second wash, the pellets are centrifuged in a microcentrifuge to settle the pellets at the bottom of the tube. The tube is then placed back on the magnet and as much of the residual ethanol as possible is removed, being especially careful to preserve the integrity of the pellets. The beads are air-dried for a few minutes, resuspended in 9.5 µl of ELB buffer (Illumina), left for a few more minutes at room temp, and placed back on the magnet to collect the beads. 8.5 µl of the supernatant is transferred to an unused tube and placed on ice for further processing at the 3' end, such as generating cDNA from a total target RNA.

[0070] In another example, 100 ng of total RNA is diluted in 11 μl of nuclease-free ultrapure water in each well of a 96-well PCR plate. 4 μl of DNA probes (SEQ ID NOs: 1-333) are added to each well with hybridization buffer. The contents of the wells are mixed and centrifuged if necessary. The plate is heated at 95°C for 2 min and then the temperature is lowered at a rate of 0.1°C/sec until the temperature reaches 37°C and then held at 37°C to hybridize the probes. The plate is centrifuged at 280xg for 10 seconds. To decompose DNA : RNA hybrids, 5 μl of RNase in buffer is added to each well and the contents of the wells are mixed. The tablet is heated at 37°C for 15 min and then kept at 4°C. 10 µl of DNase in buffer is added to each well and the contents of the well are mixed. The tablet is heated at 37°C for 15 min and then kept at 4°C. The sample plate is centrifuged at 280xg for 10 seconds. 60 µl RNAClean XP beads are added to each well and the contents of the wells are mixed. The plate is incubated at room temperature for 5 minutes. The plate is placed on a magnetic stand until the supernatant is clear (approximately 5 minutes). The supernatant is removed from each well and discarded. The granules are washed twice with 80% ethanol. Residual ethanol is removed from each well. The plate is air dried on a magnetic stand for 1 min. 10.5 µl of elution buffer is added to each well, the contents of the well are mixed. The plate is incubated at room temperature for 2 minutes. It is then sealed and centrifuged at 280xg for 10 seconds. The plate is placed on a magnetic stand until the supernatant is clear (approximately 2 min). From each well, transfer 8.5 µl of the supernatant to the appropriate well of a new plate.

EXAMPLE 2 Synthesis of cDNA

[0071] Upon further processing of the RNA of Example 1, a library of target RNA nucleic acid preparations can be obtained that can be sequenced, for example, by next generation sequencing (NGS) methods. To 8.5 µl of the final reaction from example 1, add 8.5 µl of eluate to a high concentration random hexamer mix primary buffer (EPH Buffer, TruSeq Stranded Total RNA Kit, Illumina) to a total volume of 17 µl. The sample is incubated at 65°C for 2 min to denature the nucleic acids. After denaturation, the reaction tube can be stored on ice. First strand synthesis is performed by adding 8 µl reverse transcription enzyme mix (9 µl first strand synthesis mix (FSA, TruSeq Straned Total RNA Kit, Illumina) and 1 µl Reverse Transcription Mix II RT (NEB)) to the denatured sample until total volume 25 µl. The reaction mixture is incubated in a thermal cycler with a heated lid under the following conditions: 25°C for 5 min, 42°C for 25 min, 70°C for 15 min. After completion of the first chain synthesis reaction, the reaction tube can be stored on ice.

[0072] Second strand cDNA synthesis can be performed by adding 5 µl of resuspension buffer (RSB, TruSeq Straned Total RNA Kit, Illumina) and 20 µl of second strand labeling mix (SSM buffer, TruSeq Straned Total RNA Kit, Illumina) to the sample, kept on ice. The reaction tube is incubated at 16°C for 60 minutes and then the sample can be stored on ice.

[0073] After the synthesis steps, cDNA can be purified and separated from the reaction components, for example, by adding 90 μl of SPB (Illumina) to the reaction tube and incubating for 5 minutes at room temperature. After incubation, the tube is placed on a magnet for about 8 minutes to collect the paramagnetic microcarriers. The supernatant is carefully removed and discarded. Without removing the magnet, the beads are washed twice with 175 µl of freshly prepared 80% ethanol. After washing, the pellets are centrifuged towards the bottom of the tube. The tube is placed back on the magnet and the ethanol is carefully removed and discarded. The pellets are dried for a few minutes and resuspended in 18.5 µl of resuspension buffer (RSB - Resuspension Buffer), mixed well and incubated at room temperature for approximately 5 minutes, after which they are placed back on the magnet. Depending on the 3' end applications, the required amount of purified cDNA can be transferred to an unused tube. In this example, the library is being prepared for sequencing from the 3' end, so 17.5 µl of the supernatant is transferred to an unused tube that can be stored on ice.

EXAMPLE 3 Library preparation for next generation sequencing

[0074] One method of obtaining a library for sequencing includes A-tails of cDNA fragments, ligation adapters, amplification of target fragments, and quantification of the resulting fragments prior to sequencing.

[0075] A tube containing 17.5 μl of the purified cDNA from Example 2 was used for processing. 12.5 μl of ATL buffer (Illumina) was added to the purified cDNA to add adenine to the 3' end of the fragmented DNA. The reaction tube is incubated at 37°C for 30 min followed by incubation at 70°C for 5 min and return the tube to ice. The adapters are ligated to the sample with adenine added to the 3' end of the fragmented DNA by adding, in order: 2.5 µl of resuspension buffer, 2.5 µl of index adapters (TruSeq Straned Total RNA Kit, Illumina) and 2.5 µl of ligation buffer (Illumina). The reaction tube is incubated at 30°C for 10 min, after which 5 μl of stop ligation buffer (Illumina) is added. The reaction mixture was kept on ice.

[0076] Upon completion of the adapter ligation reaction, the ligated fragments are separated from the reaction components. To purify adapter-ligated fragments, add 34 µl of SPB to the reaction tube and incubate at room temperature for approximately 5 minutes. The tube is then placed on a magnet to trap the paramagnetic beads and washed twice with 175 µl of 80% EtOH. The EtOH is carefully removed after the second wash. After 3 minutes of air drying, the beads are resuspended in 52 µl of RSB. The suspension is stirred, left at room temperature for another 5 minutes and placed back on the magnet. The supernatant (50 µl) is transferred to an unused tube for the second bead purification cycle.

[0077] In the second cycle, 40 µl of SPB is added to 50 µl of sample and the above method is repeated, except that the final purified fragments are resuspended in 21 µl of RSB and 20 µl of the final purified sample and transferred to a new reaction tube for subsequent amplification, which increases the amount of target sequence to optimize sequencing results.

[0078] To 20 µl of the purified sample ligated to the adapter, add 5 µl of PCR primer mix (PPC, TruSeq Straned Total RNA Kit, Illumina) and 25 µl of primer and probe mix (TruSeq Straned Total RNA Kit, Illumina) and perform the following amplification program in thermal cycler with heated lid: At 98°C for 30 sec followed by a cycling program of 98°C for 10 sec, 60°C for 30 sec, 72°C for 30 sec. The number of amplification cycles depends on the amount of RNA at the beginning of the whole process. For example, 100 ng of RNA may require approximately 12-13 cycles, 10 ng 15-16 cycles, and 1 ng 17-18 cycles. The number of amplification cycles is generally optimized for any formulation, at the discretion of the skilled artisan.

[0079] Amplicons can be purified from reaction conditions by adding 50 μl of SPB to the reaction tube, incubating at room temperature, centrifuging the tube to pellet the beads, and capturing the beads with a magnetic field. The supernatant can be discarded and the beads washed as above, followed by resuspension of the washed beads in 26 μl of RSB, capture of the magnetic beads, and transfer of the supernatant containing the DNA library to an unused tube for sequencing. The library is typically quantified and analyzed prior to sequencing, for example, by measuring an aliquot using a QubitTM High Sensitivity kit (Thermo Fisher Scientific) and/or running an aliquot on a bioanalyzer (Agilent). One of skill in the art will appreciate the many ways to quantify nucleic acids in a sample.

[0080] The resulting library can then be used for next generation sequencing, microarray analysis, or other 3' end applications. In applications such as sequencing, the methodology for obtaining a library is determined by the sequencing instrument used and the method of obtaining an auxiliary library for that sequencing instrument. In this example, the library acquisition method is a characteristic of library generation when sequencing on Illumina instruments. One of skill in the art will appreciate that methods for obtaining libraries may vary depending on the sequencing instrument, so the present examples are illustrative and RNA depletion methods are not limited to any particular workflow for obtaining a library. With the present methods, the use of an RNA sample after removal of unwanted RNA species will have a positive impact on any applications downstream of transcription.

EXAMPLE 4 Transcriptome Analysis of Microorganisms

[0081] In this example, microbial isolates, a mixed sample of bacterial species, and a standard cell mixture were obtained from ATCC for testing.

sample type Types of microorganisms tested Isolates of microorganisms E. coli, B. subtilis, S. epidermidis, E. cloacae and B. cereus ATCC-MSA2002 Mix 20 strains A. Baumannii, A. odontolyhticus, B. cereus, B. vulgatus, B. adolescentis, C. beijerinckii, C. acnes, D. radiodurans, E. faecalis, E. coli, H. pylori, L. gasseri., N meningitidis, P. ginvalis, P. aeruginosa, R. sphaeroides, S. aureus, S. epidermidis, S. agalactiae, S. mutans ATCC MSA2006 Mixture of human intestinal samples B. tragilis, B. vulgatus, B. adolescentis, C. difficile, E. faecalis, L. plantarum, E. cloacae, E. coli, H. pylori, S. enterica, y. enterococolitica, F. ucleatum

[0082] Total RNA can be extracted using the RNeasy Power Microbiome Kit (Qiagen) according to the manufacturer's protocol and assessed for integrity and quantified by Bioanalyzer RNA Electrophoresis (Agilent). To reduce the concentration of rRNA in each sample, 10-250 ng of total RNA can be used according to the RiboZero method (Illumina, following the manufacturer's protocol) or the methods described herein using RNase H enzymatic digestion of unwanted rRNA. Samples with deleted rRNA and other unwanted RNA (controls) can be prepared for sequencing using the TruSeq Straned Total RNA Sample Preparation Kit (Illumina) according to the manufacturer's instructions. Libraries can be pooled and sequenced, for example, on MiSeq or NextSeq (Illumina) sequencing analyzers for 2×76 paired end reads.

[0083] Filtering, alignment and coverage of transcripts can be performed using the BaseSpace Sequence Hub (BSSH - BaseSpace Sequencing Hub) and the following processing examples, for example: 1) Partition rRNA Sequences , 2) the RNA Custom Genome Builder RNA Genome Builder application (creating a STAR-compatible microbial transcriptome) and 3) the RNA-Seq Alignment application (STAR alignment and quantification of the Salmon transcript). To quantify rRNA in multiple strains of microbial samples, rRNA sequences can be extracted from National Center for Biotechnological Information (NCBI) annotated genomes and used as input to the BSSH workflow.

[0084] Transcriptomes of microbial isolates, microbial mixtures and controls were sequenced and compared to % rRNA reads. The RNase H enzyme method described herein is highly effective in removing unwanted rRNA in the species tested (<5% rRNA reads). Removal of ribosomal RNA is most significant for the low volume (10 ng) E. coli sample using the RNase H method compared to the established RiboZero method; <0.5% vs. 13% mean rRNA reads, respectively (FIG. 5).

[0085] The data was used to gain access to the enrichment of biologically important RNA reads using RNase H enzyme rRNA depletion methods, and compared to sample data without rRNA removal. On FIG. 6 shows the results of an evaluation where a 20- to 50-fold reduction in reading depth was generally observed for sample b. subtilis or E. coli if rRNA was removed prior to library preparations and sequencing using RNase H methods compared to a sample without rRNA removal.

[0086] The data collected was evaluated to determine the reproducibility of experimental microbial transcriptome sequencing efforts. Pairwise linear regression of gene expression levels was determined between rRNA-deleted replicates by RNase H enzymes for E. coli and B. subtilis as exemplary systems. A high correlation (R ² >0.99) indicated the ability of the rRNA depletion method with RNase H enzymes to reproducibly remove rRNA from samples (FIG. 7).

[0087] To evaluate whether the RNase H enzyme rRNA depletion method is suitable for removing rRNA from mixed samples, FIG. 8 shows sample data for mixed samples of 20 MSA2002 strains and MSA2006 human intestine in triplicate. Low levels of 10 mg total RNA from MSA2002 or 80 ng total RNA from MSA2006 were used for rRNA removal methods. For 20 MSA2002 strain samples, the RNase H rRNA removal method reduced the number of rRNA reads by 83% or <2% of sequence reads, while the RiboZero rRNA removal method resulted in more variable and higher rRNA content compared to non-depleted samples. For 12 strain MSA2006 samples, the same result was observed where the RNase H method reduced the number of rRNA reads by about 95% to <13% sequencing reads compared to non-depleted samples, the RiboZero method gave more variable results.

[0088] In summary, in sample evaluation experiments (mixed or otherwise), the RNase H enzyme rRNA depletion method has been found to provide a reliable and efficient workflow for removing unwanted rRNA in samples for high quality microbial whole transcriptome analysis. The method of removing rRNA with RNase H enzymes was also very efficient and compatible with small sample volumes.

EXAMPLE 5 Effect of Formamide on RNA Depletion

[0089] In FIG. 2 shows examples of data from an RNA sample in which unwanted RNA species have been removed. Unwanted RNA was removed from the sample using the methods described here, and the effect of formamide concentration on RNA depletion was evaluated. In this example, DNA probes target the removal of unwanted rRNA species from gram-positive bacteria (23S, 16S, 5S), gram-negative bacteria (23S, 16S, 5S incl.), human mitochondria (16S, 12S), human rRNA (28S, 18S, 5.8S, 5S), human hemoglobin mRNA (HBA-A1, HBA-A2, HBB, HBG1, HBG2), while the target RNA species are the total RNA from b. subtilis . As the concentration of formamide increases, the percentage of unwanted RNA species decreases significantly. For example, formamide, upon RNA depletion, did not read non-target RNA of Gram-positive bacteria 23S and 16S and Gram-negative bacteria (including E. coli ) 23S and 16S, including specific E. coli sequences. The addition of 25% formamide to the hybridization reaction resulted in undetectable off-target reads for Gram-negative 23S and 16S (with a significant decrease in off-target reads consistent with E. coli ) and a significant decrease in off-target reads for Gram-positive 23S and 16S. The addition of formamide to the 45% hybridization reaction resulted in an additional significant reduction in off-target reads for Gram-positive unwanted 23S and 16S rRNAs, as well as a further reduction in E. coli off-target reads. Thus, the addition of formamide to the RNA hybridization reaction has been shown to increase the amount of gram-positive and gram-negative unwanted RNAs, as indicated by a decrease in the number of off-target reads for these species. In general, the addition of formamide was found to improve the removal of unwanted rRNA transcripts. When using B. subtilis RNA as the target RNA for analysis, for example, analysis for the presence of E. coli and human rRNA sequences can serve as an indicator of potential contamination.

EXAMPLE 6 Various starting material options

[0090] Experiments were carried out to determine the effect of the introduced starting material on RNA depletion and subsequent analysis from the 3'-ends, as shown in FIG. 3, where RNA was used to create sequencing libraries on the Illumina NextSeq ^TM 500 or 550 instrument after removal of unwanted RNA species and enriched human brain RNA (HBR - human brain RNA) and universal human RNA (UHR - universal human RNA). After reducing the concentration of RNA using 100 ng, 10 ng or 1 ng of the original samples, sequencing libraries were prepared as shown in examples 1-3. Sequencing was performed as recommended in the NextSeq ^TM User Guide, followed by data analysis using two BaseSpace applications (Illumina), the RNASeq Alignment application, and the RNAExpress application. Data analysis for the presence of B. subtilis and E. coli was also performed using a modified Fastqscreen tool (https://www.bioinformatics.babraham.ac.uk/projects/fastq_screen/). Data showing that RNA depletion remains constant for both HBR and UHR, regardless of the amount of input RNA sample and % total alignment for target RNA, although there is a reduction with decreasing volume, still demonstrates that valid and useful data o sequences can be collected even when using 1 ng of the input sample. In addition, in a comparative experiment, the current RNA depletion method results in a lower percentage of off-target reads at all input levels (100ng ~ 3%, 25ng ~ 4%, 10ng ~ 3%, and 1ng ~ 3%) compared to data when using the RiboZero rRNA depletion kit (Epicentre) (100 ng ~ 3%; 25 ng ~ 5%, 10 ng ~ 8% and 1 ng ~ 35%) or NEBext rRNA depletion methods (NEB) (100 ng ~ 8%, 25ng ~ 8%, 10ng ~ 9% and 1ng ~ 30%).

EXAMPLE 7 RNA Depletion in Mouse and Rat RNA Samples

[0091] To demonstrate that RNA depletion methods can be used on non-human RNA samples, mouse and rat RNA samples were used for RNA depletion methods. On FIG. 4, unwanted RNA was removed from mouse or rat RNA samples using equivalent methods and DNA probes as in human RNA depletion. The value of formamide again varied in each rodent species, including its absence in the hybridization reaction (25% or 45% formamide). Although the increase in formamide does not affect the overall % matched reads, there may be a trend towards an increase in off-target reads as the amount of formamide increases. Thus, the addition of formamide to the hybridization reaction may be beneficial for some sample types as it may improve the detection of some transcripts. The addition of formamide must be optimized.

EXAMPLE 8 Obtaining Additional Mouse Probes

[0092] In the pool of 333 DNA probes described above for the enzymatic removal of unwanted sequences (SEQ ID NOs: 1-333), DNA oligonucleotides for eukaryotic rRNA removal 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 on total human RNA, this pool of 333 DNA probes was very effective in removing rRNA reads. However, when testing mouse ( Mus musculus ) or rat ( Rattus norvegicus ) total RNA samples, the depletion was less consistent, suggesting that the probes do not hybridize and remove certain regions of rodent rRNA sequences effectively, since these regions in mice and rats differ from the sequences person.

[0093] FASTRQ files containing total sequencing reads from the 333 DNA probe experiment were matched to mouse and rat ribosomal RNA sequences and to 333 DNA probe sequences. The alignment results indicated that the coverage of the probes across all ribosomal RNA sequences was essentially good, but there are some areas where the probe sequences did not align as well as with rodent RNA. More specifically, most mouse and rat rRNA reads that do not match the probe pool map belong to rodent 28S or 16S rRNA transcripts (Table 2). Alignment was performed using the Bowtie2 program ( see Langmead and Salzberg, Nature Methods 2012, 9:357-359), version 2.1.0, with default settings. Most of the ribosomal RNA that was not enzymatically removed with the 333 DNA probes was from the same regions lacking probe alignment (FIG. 9).

Table 2. Genetic sequence data of mice and rats from the GenBank repository used for the study

Genome 16S 28S Mus muscle NC_005089.1:1094-2675 NR_003279.1 Rattus norvegicus NC_001665.2:1094-2664 NR_046246.1

[0094] To more effectively deplete these regions, additional probes have been developed covering the regions described above for mouse and rat ribosomal RNA sequences. To minimize the number of additional probes and their redundancy, additional probes have been designed to cover gaps in mouse rRNA sequences. These data were then informatively pooled together with a set of 333 DNA probes to identify remaining gaps in rat rRNA by matching the pooled pool to rat rRNA transcripts. This sequential process resulted in a total of 44 additional oligonucleotide probes to provide an additional pool of 377 probes. Sequencing experiments as described above were repeated with a set of 377 DNA probes. The addition of the new 44 probes in both mouse and rat samples resulted in a decrease in the percentage of rRNA reads from libraries compared to a set of 333 DNA probes, indicating increased depletion efficiency (Table 3).

Table 3. Percentage of ribosomal RNA in sequencing reads using sets of 333 and 377 probes

RNase H probe set mouse pattern rat pattern Set of 333 DNA probes 9.5% 5.3% Set of 377 DNA probes 7.0% 3.7%

[0095] Adding additional probes to a pool of 333 DNA probes to certain rodent sequences increased the level of rRNA removal in the rodent samples tested. Examples of mouse 16S probes include SEQ ID NOs: 385-393. Examples of mouse 28S probes include SEQ ID NOs: 400-419. Examples of anti-16S rat probes include SEQ ID NOs: 394-399. Examples of anti-28S rat probes include SEQ ID NOs: 420-428.

--->

SEQUENCE LIST

<110> EPICENTRE TECHNOLOGIES CORPORATION

<120> RNA DEPLETION BASED ON NUCLEASE

<130> 01243-0012-00PCT

<150> US 62/783,869

<151> 2018-12-21

<150> US 62/847,797

<151> 2019-05-14

<160> 428

<170> Patent Version - 3.5

<210> 1

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 1

gttcgtccaa gtgcactttc cagtacactt accatgttac gacttgtctc 50

<210> 2

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 2

taggggtttt agttaaatgt cctttgaagt atacttgagg agggtgacgg 50

<210> 3

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 3

ttcagggcc tgttcaacta agcactctac tctcagttta ctgctaaatc 50

<210> 4

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 4

agtttcataa gggctatcgt agttttctgg ggtagaaaat gtagcccatt 50

<210> 5

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 5

ggctacacct tgacctaacg tctttacgtg ggtacttgcg cttactttgt 50

<210> 6

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 6

ttgctgaaga tggcggtata taggctgagc aagaggtggt gaggttgatc 50

<210> 7

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 7

cagaacaggc tcctctagag ggatatgaag caccgccagg tcctttgagt 50

<210> 8

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 8

gtagtgttct ggcgagcagt tttgttgatt taactgttga ggtttagggc 50

<210> 9

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 9

atctaatccc agtttgggtc ttagctattg tgtgttcaga tatgttaaag 50

<210> 10

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 10

attttgtgtc aactggagtt ttttacaact caggtgagtt ttagctttat 50

<210> 11

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 11

ctaaaacact ctttacgccg gcttctattg acttgggtta atcgtgtgac 50

<210> 12

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 12

gaaattgacc aaccctgggg ttagtatagc ttagttaaac tttcgtttat 50

<210> 13

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 13

actgctgttt cccgtggggg tgtggctagg ctaagcgttt tgagctgcat 50

<210> 14

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 14

gcttgtccct tttgatcgtg gtgatttaga gggtgaactc actggaacgg 50

<210> 15

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 15

taatcttact aagagctaat agaaaggcta ggaccaaacc tatttgttta 50

<210> 16

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 16

aaaccctgtt cttgggtggg tgtgggtata atactaagtt gagatgatat 50

<210> 17

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 17

gcgctttgtg aagtaggcct tatttctctt gtcctttcgt acagggagga 50

<210> 18

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 18

aaaccgacct ggattactcc ggtctgaact cagatcacgt aggactttaa 50

<210> 19

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 19

acctttaata gcggctgcac catcgggatg tcctgatcca acatcgaggt 50

<210> 20

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 20

tgatatggac tctagaatag gattgcgctg ttatccctag ggtaacttgt 50

<210> 21

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 21

attggatcaa ttgagtatag tagttcgctt tgactggtga agtcttagca 50

<210> 22

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 22

ttgggttctg ctccgaggtc gccccaaccg aaattttttaa tgcaggtttg 50

<210> 23

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 23

tgggtttgtt aggtactgtt tgcattaata aattaaagct ccatagggtc 50

<210> 24

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 24

gtcatgcccg ccctcttcacg ggcaggtcaa tttcactggt taaaagtaag 50

<210> 25

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 25

cgtggagcca ttcatacagg tccctattta aggaacaagt gattatgcta 50

<210> 26

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 26

ggtaccgcgg ccgttaaaca tgtgtcactg ggcaggcggt gcctctaata 50

<210> 27

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 27

gtgatgtttt tggtaaacag gcggggtaag gtttgccgag ttccttttac 50

<210> 28

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 28

cttatgagca tgcctgtgtt gggttgacag tgagggtaat aatgacttgt 50

<210> 29

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 29

attgggctgt taattgtcag ttcagtgttt tgatctgacg caggcttatg 50

<210> 30

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 30

tcatgttact tatactaaca ttagttcttc tatagggtga tagattggtc 50

<210> 31

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 31

50

<210> 32

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 32

tggctgcttt taggcctact atgggtgtta aatttttttac tctctctaca 50

<210> 33

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 33

gtccaaagag ctgttcctct ttggactaac agttaaattt acaaggggat 50

<210> 34

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 34

ggcaaattta aagttgaact aagattctat cttggacaac cagctatcac 50

<210> 35

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 35

tgtcgcctct acctataaat cttcccacta ttttgctaca tagacgggtg 50

<210> 36

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 36

tcttaggtag ctcgtctggt ttcgggggtc ttagctttgg ctctccttgc 50

<210> 37

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 37

taattcatta tgcagaaggt ataggggtta gtccttgcta tattatgctt 50

<210> 38

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 38

tctttccctt gcggtactat atctattgcg ccaggtttca atttctatcg 50

<210> 39

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 39

ggtaaatggt ttggctaagg ttgtctggta gtaaggtgga gtgggtttgg 50

<210> 40

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 40

taatgatcct tccgcaggtt cacctacggga aaccttgtta cgacttttac 50

<210> 41

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 41

aagttcgacc gtcttctcag cgctccgcca gggccgtggg ccgaccccgg 50

<210> 42

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 42

ggcctcacta aaccatccaa tcggtagtag cgacgggcgg tgtgtacaaa 50

<210> 43

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 43

caacgcaagc ttatgacccg cacttactcg ggaattccct cgttcatggg 50

<210> 44

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 44

ccgatcccca tcacgaatgg ggttcaacgg gttacccgcg cctgccggcg 50

<210> 45

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 45

ctgagccagt cagtgtagcg cgcgtgcagc cccggacatc taagggcatc 50

<210> 46

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 46

ctcaatctcg ggtggctgaa cgccacttgt ccctctaaga agttggggga 50

<210> 47

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 47

ggtcgcgtaa ctagttagca tgccagagtc tcgttcgtta tcggaattaa 50

<210> 48

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 48

caccaactaa gaacggccat gcaccaccac ccacggaatc gagaaagagc 50

<210> 49

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 49

cctgtccgtg tccgggccgg gtgaggtttc ccgtgttgag tcaaattaag 50

<210> 50

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 50

ctggtggtgc ccttccgtca attcctttaa gtttcagctt tgcaaccata 50

<210> 51

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 51

aaagactttg gtttcccggga agctgcccgg cgggtcatgg gaataacgcc 50

<210> 52

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 52

ggcatcgttt atggtcggaa ctacgacggt atctgatcgt cttcgaacct 50

<210> 53

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 53

gattaatgaa aacattcttg gcaaatgctt tcgctctggt ccgtcttgcg 50

<210> 54

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 54

cacctctagc ggcgcaatac gaatgccccc ggccgtccct cttaatcatg 50

<210> 55

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 55

accaacaaaa tagaaccgcg gtcctattcc attattccta gctgcggtat 50

<210> 56

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 56

ctgctttgaa cactctaatt ttttcaaagt aaacgcttcg ggccccgcgg 50

<210> 57

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 57

gcatcgaggg ggcgccgaga ggcaaggggc ggggacgggc ggtggctcgc 50

<210> 58

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 58

ccgcccgctc ccaagatcca actacgagct ttttaactgc agcaacttta 50

<210> 59

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 59

gctggaatta ccgcggctgc tggcaccaga cttgccctcc aatggatcct 50

<210> 60

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 60

agtggactca ttccaattac agggcctcga aagagtcctg tattgtttatt 50

<210> 61

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 61

cccgggtcgg gagtgggtaa tttgcgcgcc tgctgccttc cttggatgtg 50

<210> 62

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 62

gctccctctc cggaatcgaa ccctgattcc ccgtcacccg tggtcaccat 50

<210> 63

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 63

taccatcgaa agttgatagg gcagacgttc gaatgggtcg tcgccgccac 50

<210> 64

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 64

ggcccgaggt tatctagagt caccaaagcc gccggcgccc gccccccggc 50

<210> 65

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 65

gctgaccggg ttggttttga tctgataaat gcacgcatcc cccccgcgaa 50

<210> 66

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 66

tcggcatgta ttagctctag aattaccaca gttatccaag taggagagga 50

<210> 67

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 67

aaccataact gatttaatga gccattcgca gtttcactgt accggccgtg 50

<210> 68

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 68

atggcttaat ctttgagaca agcatatgct actggcagga tcaaccaggt 50

<210> 69

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 69

gacaaaccct tgtgtcgagg gctgactttc aatagatcgc agcgagggag 50

<210> 70

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 70

cgaaaccccg acccagaagc aggtcgtcta cgaatggttt agcgccaggt 50

<210> 71

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 71

ggtgcgtgac gggcgagggg gcggccgcct ttccggccgc gccccgtttc 50

<210> 72

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 72

ctccgcaccg gaccccggtc ccggcgcgcg gcggggcacg cgccctcccg 50

<210> 73

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 73

aggggggggc ggcccgccgg cggggacagg cgggggaccg gctatccgag 50

<210> 74

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 74

gcggcgctgc cgtatcgttc gcctgggcgg gattctgact tagaggcgtt 50

<210> 75

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 75

agatggtagc ttcgccccat tggctcctca gccaagcaca tacaccaaat 50

<210> 76

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 76

tcctctcgta ctgagcagga ttaccatggc aacaacacat catcagtagg 50

<210> 77

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 77

ctcacgacgg tctaaaccca gctcacgttc cctattagtg ggtgaacaat 50

<210> 78

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 78

ttctgcttca caatgatagg aagagccgac atcgaaggat caaaaagcga 50

<210> 79

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 79

ttggccgcca caagccagtt atccctgtgg taacttttct gacacctcct 50

<210> 80

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 80

ggtcagaagg atcgtgaggc cccgctttca cggtctgtat tcgtactgaa 50

<210> 81

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 81

agcttttgcc cttctgctcc acgggaggtt tctgtcctcc ctgagctcgc 50

<210> 82

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 82

ttaccgtttg acaggtgtac cgccccagtc aaactcccca cctggcactg 50

<210> 83

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 83

gcgcccggcc gggcgggcgc ttggcgccag aagcgagagc ccctcgggct 50

<210> 84

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 84

ccgggtcagt gaaaaaacga tcagagtagt ggtatttcac cggcggcccg 50

<210> 85

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 85

cgccccgggc ccctcgcggg gacaccgggg gggcgccggg ggcctcccac 50

<210> 86

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 86

catgtctctt caccgtgcca gactagagtc aagctcaaca gggtcttctt 50

<210> 87

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 87

ccaagcccgt tcccttggct gtggtttcgc tggatagtag gtagggacag 50

<210> 88

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 88

tccattcatg cgcgtcacta attagatgac gaggcatttg gctaccttaa 50

<210> 89

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 89

tcccgccgtt tacccgcgct tcattgaatt tcttcacttt gacattcaga 50

<210> 90

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 90

cacatcgcgt caacacccgc cgcgggcctt cgcgatgctt tgttttaatt 50

<210> 91

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 91

cctggtccgc accagttcta agtcggctgc taggcgccgg ccgaggcgag 50

<210> 92

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 92

cggccccggg ggcggacccg gcggggggga ccggcccgcg gcccctccgc 50

<210> 93

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 93

ccgccgcgcg ccgaggagga ggggggaacg gggggcggac ggggccgggg 50

<210> 94

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 94

acgaaccgcc ccgccccgcc gcccgccgac cgccgccgcc cgaccgctcc 50

<210> 95

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 95

cgcgcgcgac cgagacgtgg ggtgggggtg gggggcgcgc cgcgccgccg 50

<210> 96

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 96

gcggccgcga cgcccgccgc agctggggcg atcacggga agggcccggc 50

<210> 97

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 97

gcgccgccgc cggccccccg ggtccccggg gcccccctcg cggggacctg 50

<210> 98

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 98

ccggcggccg ccgcgcggcc cctgccgccc cgacccttct ccccccgccg 50

<210> 99

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 99

ctcccccgggg gaggggggag gacggggagc gggggagaga gagagagaga 50

<210> 100

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 100

agggagcgag cggcgcgcgc gggtggggcg ggggagggcc gcgagggggg 50

<210> 101

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 101

gggggcgcgc gcctcgtcca gccgcggcgc gcgcccagcc ccgcttcgcg 50

<210> 102

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 102

cccagccctt agagccaatc cttatcccga agttacggat ccggcttgcc 50

<210> 103

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 103

cattgttcca acatgccaga ggctgttcac cttggagacc tgctgcggat 50

<210> 104

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 104

cgcgagattt acaccctctc ccccggattt tcaagggcca gcgagagctc 50

<210> 105

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 105

aaccgcgacg ctttccaagg cacgggcccc tctctcgggg cgaacccatt 50

<210> 106

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 106

cttcacaaag aaaagagaac tctccccggg gctcccgccg gcttctccgg 50

<210> 107

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 107

cgcactggac gcctcgcggc gcccatctcc gccactccgg attcggggat 50

<210> 108

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 108

tttcgatcgg ccgagggcaa cggaggccat cgcccgtccc ttcggaacgg 50

<210> 109

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 109

caggaccgac tgacccatgt tcaactgctg ttcacatgga acccttctcc 50

<210> 110

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 110

gttctcgttt gaatatttgc tactaccacc aagatctgca cctgcggcgg 50

<210> 111

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 111

cgccctaggc ttcaaggctc accgcagcgg ccctcctact cgtcgcggcg 50

<210> 112

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 112

tccggggggcg gggagcgggg cgtgggcggg aggagggag gaggcgtggg 50

<210> 113

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 113

aggacccac acccccgccg ccgccgccgc cgccgccctc cgacgcacac 50

<210> 114

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 114

gcgcgccgcc cccgccgctc ccgtccactc tcgactgccg gcgacggccg 50

<210> 115

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 115

ctccagcgcc atccattttc agggctagtt gattcggcag gtgagttgtt 50

<210> 116

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 116

gattccgact tccatggcca ccgtcctgct gtctatatca accaacacct 50

<210> 117

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 117

gagcgtcggc atcgggcgcc ttaacccggc gttcggttca tcccgcagcg 50

<210> 118

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 118

aaaagtggcc cactaggcac tcgcattcca cgcccggctc cacgccagcg 50

<210> 119

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 119

ccatttaaag tttgagaata ggttgagatc gtttcggccc caagacctct 50

<210> 120

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 120

cggataaaac tgcgtggcgg gggtgcgtcg ggtctgcgag agcgccagct 50

<210> 121

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 121

tcggagggaa ccagctacta gatggttcga ttagtctttc gcccctatac 50

<210> 122

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 122

gatttgcacg tcaggaccgc tacggacctc caccagagtt tcctctggct 50

<210> 123

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 123

atagttcacc atctttcggg tcctaacacg tgcgctcgtg ctccacctcc 50

<210> 124

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 124

agacgggccg gtggtgcgcc ctcggcggac tggagaggcc tcgggatccc 50

<210> 125

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 125

cgcgccggcc ttcaccttca ttgcgccacg gcggctttcg tgcgagcccc 50

<210> 126

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 126

ttagactcct tggtccgtgt ttcaagacgg gtcgggtggg tagccgacgt 50

<210> 127

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 127

gcgctcgctc cgccgtcccc ctcttcgggg gacgcgcgcg tggccccgag 50

<210> 128

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 128

cccgacggcg cgacccgccc ggggcgcact ggggacagtc cgccccgccc 50

<210> 129

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 129

gcaccccccc cgtcgccggg gcgggggcgc ggggaggagg ggtgggagag 50

<210> 130

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 130

aggggtggcc cggccccccc acgaggagac gccggcgcgc ccccgcgggg 50

<210> 131

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 131

ggggattccc cgcgggggtg ggcgccggga ggggggagag cgcggcgacg 50

<210> 132

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 132

gccccgggat tcggcgagtg ctgctgccgg gggggctgta acactcgggg 50

<210> 133

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 133

ccgcccccgc cgccgccgcc accgccgccg ccgccgccgc cccgacccgc 50

<210> 134

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 134

aggacgcggg gccggggggc ggagacgggg gaggaggagg acggacggac 50

<210> 135

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 135

agccaccttc cccgccgggc cttcccagcc gtcccggagc cggtcgcggc 50

<210> 136

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 136

aaatgcgccc ggcggcggcc ggtcgccggt cggggggacgg tcccccgccg 50

<210> 137

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 137

ccgcccgccc acccccgcac ccgccggagc ccgccccctc cggggaggag 50

<210> 138

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 138

gggaagggag ggcgggtgga ggggtcggga ggaacgggggg gcgggaaaga 50

<210> 139

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 139

acacggccgg acccgccgcc gggttgaatc ctccgggcgg actgcgcgga 50

<210> 140

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 140

tcttaacggt ttcacgccct cttgaactct ctcttcaaag ttcttttcaa 50

<210> 141

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 141

cttgttgact atcggtctcg tgccggtatt tagccttaga tggagtttac 50

<210> 142

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 142

gcattcccaa gcaacccgac tccgggaaga cccgggcgcg cgccggccgc 50

<210> 143

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 143

gtccacgggc tgggcctcga tcagaaggac ttgggccccc cacgagcggc 50

<210> 144

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 144

ttccgtacgc cacatgtccc gcgccccgcg gggcggggat tcggcgctgg 50

<210> 145

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 145

ctcgccgtta ctgagggaat cctggttagt ttcttttcct ccgctgacta 50

<210> 146

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 146

gcgggtcgcc acgtctgatc tgaggtcgcg tctcggaggg ggacgggccg 50

<210> 147

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 147

aagcgacgct cagacaggcg tagccccggg aggaacccgg ggccgcaagt 50

<210> 148

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 148

gcagctagct gcgttcttca tcgacgcacg agccgagtga tccaccgcta 50

<210> 149

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 149

aaagcctaca gcacccggta ttcccaggcg gtctcccatc caagtactaa 50

<210> 150

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 150

ttccgagatc agacgagatc gggcgcgttc agggtggtat ggccgtagac 50

<210> 151

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 151

gccgcccact cagactttat tcaaagacca cggggggtacg ggtgcaggaa 50

<210> 152

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 152

gggggaggcc caaggggcaa gaagcatggc caccgaggct ccagcttaac 50

<210> 153

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 153

gcacggtgct cacagaagcc aggaacttgt ccagggaggc gtgcaccgca 50

<210> 154

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 154

gggaggtggg cggccagggt caccagcagg cagtggctta ggagcttgaa 50

<210> 155

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 155

ccgaagcttg tgcgcgtgca ggtcgctcag ggcggacagc gcgttggggca 50

<210> 156

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 156

ccacggcgtt ggtcagcgcg tcggccacct tcttgccgtg gcccttaacc 50

<210> 157

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 157

ctcaggtcga agtgcgggaa gtaggtcttg gtggtgggga aggacaggaa 50

<210> 158

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 158

ctccgcacca tactcgccag cgtgcgcgcc gaccttaccc caggcggcct 50

<210> 159

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 159

cggcaggaga cagcaccatg gtgggttctc tctgagtctg tggggaccag 50

<210> 160

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 160

gaggggagga gggcccgttg ggaggcccag cgggcaggag gaacggctac 50

<210> 161

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 161

acggtatttg gaggtcagca cggtgctcac agaagccagg aacttgtcca 50

<210> 162

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 162

caggggtgaa ctcggcgggg aggtggggcgg ccagggtcac cagcaggcag 50

<210> 163

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 163

aagttgaccg ggtccacccg aagcttgtgc gcgtgcaggt cgctcagggc 50

<210> 164

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 164

catgtcgtcc acgtgcgcca cggcgttggt cagcgcgtcg gccaccttct 50

<210> 165

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 165

cctgggcaga gccgtggctc aggtcgaagt gcgggaagta ggtcttggtg 50

<210> 166

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 166

aacatcctct cggggcctc cgcaccatac tcgccagcgt gcgcgccgac 50

<210> 167

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 167

cttgacgttg gtcttgtcgg caggagacag caccatggtg ggttctctct 50

<210> 168

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 168

gcaatgaaaa taaatgtttt ttattaggca gaatccagat gctcaaggcc 50

<210> 169

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 169

cagtttagta gttggactta gggaacaaag gaacctttaa tagaaattgg 50

<210> 170

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 170

gcttagtgat acttgtgggc cagggcatta gccacaccag ccaccacttt 50

<210> 171

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 171

cactggtggg gtgaattctt tgccaaagtg atgggccagc acacagacca 50

<210> 172

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 172

gcctgaagtt ctcaggatcc acgtgcagct tgtcacagtg cagctcactc 50

<210> 173

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 173

cccttgaggt tgtccaggtg agccaggcca tcactaaagg caccgagcac 50

<210> 174

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 174

cttcacctta gggttgccca taacagcatc aggagtggac agatccccaa 50

<210> 175

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 175

tctgggtcca agggtagacc accagcagcc tgcccagggc ctcaccacca 50

<210> 176

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 176

accttgcccc aacagggcagt aacggcagac ttctcctcag gagtcagatg 50

<210> 177

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 177

gtgatctctc agcagaatag atttattatt tgtattgctt gcagaataaa 50

<210> 178

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 178

ctctgaatca tgggcagtga gctcagtggt atctggagga cagggcactg 50

<210> 179

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 179

atcttctgcc aggaagcctg cacctcaggg gtgaattctt tgccgaaatg 50

<210> 180

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 180

caccagcaca tttccgga gcttgaagtt ctcaggatcc acatgcagct 50

<210> 181

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 181

cactcagctg ggcaaaggtg cccttgagat catccaggtg ctttgtggca 50

<210> 182

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 182

agcaccttct tgccatgtgc cttgactttg gggttgccca tgatggcaga 50

<210> 183

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 183

gccaaagctg tcaaagaacc tctgggtcca tgggtagaca accaggagcc 50

<210> 184

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 184

ctccagcatc ttccacattc accttgcccc acaggcttgt gatagtagcc 50

<210> 185

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 185

aaatgaccca tggcgtctgg actaggagct tattgataac ctcagacgtt 50

<210> 186

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 186

gtgatctctt agcagaatag atttattatt tgattgcttg cagaataaag 50

<210> 187

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 187

tctgcatcat gggcagtgag ctcagtggta tctggaggac agggcactgg 50

<210> 188

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 188

tcttctgcca ggaagcctgc acctcagggg tgaattcttt gccgaaatgg 50

<210> 189

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 189

accagcacat ttcccaggag cttgaagttc tcaggatcca catgcagctt 50

<210> 190

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 190

actcagctgg gcaaaggtgc ccttgagatc atccaggtgc tttatggcat 50

<210> 191

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 191

gcaccttctt gccatgtgcc ttgactttgg ggttgcccat gatggcagag 50

<210> 192

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 192

ccaaagctgt caaagaacct ctgggtccat gggtagacaa ccaggagcct 50

<210> 193

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 193

tccagcatct tccacattca ccttgcccca caggcttgtg atagtagcct 50

<210> 194

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 194

aatgacccat ggcgtctgga ctaggagctt attgataacc tcagacgttc 50

<210> 195

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 195

atgcctggca gttccctact ctcgcatggg gagaccccac actaccatcg 50

<210> 196

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 196

acttctgagt tcggcatggg gtcaggtggg accaccgcgc tacggccgcc 50

<210> 197

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 197

ggttaccttg ttacgacttc accccagtca tgaatcacaa agtggtaagt 50

<210> 198

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 198

aagctaccta cttcttttgc aacccactcc catggtgtga cgggcggtgt 50

<210> 199

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 199

acgtattcac cgtggcattc tgatccacga ttactagcga ttccgacttc 50

<210> 200

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 200

agactccaat ccggactacg acgcacttta tgaggtccgc ttgctctcgc 50

<210> 201

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 201

tgtatgcgcc attgtagcac gtgtgtagcc ctggtcgtaa gggccatgat 50

<210> 202

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 202

ccaccttcct ccagtttatc actggcagtc tcctttgagt tcccggccgg 50

<210> 203

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 203

ggataagggt tgcgctcgtt gcgggactta acccaacatt tcacaacacg 50

<210> 204

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 204

tgcagcacct gtctcacggt tcccgaaggc acattctcat ctctgaaaac 50

<210> 205

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 205

gaccaggtaa ggttcttcgc gttgcatcga attaaaccac atgctccacc 50

<210> 206

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 206

cgtcaattca tttgagtttt aaccttgcgg ccgtactccc caggcggtcg 50

<210> 207

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 207

tcgggaagcc acgcctcaag ggcacaacct ccaagtcgac atcgtttacg 50

<210> 208

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 208

gtatctaatc ctgtttgctc cccacgcttt cgcactgagc gtcagtcttc 50

<210> 209

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 209

ttcgccaccg gtattcctcc agatctctac gcatttcacc gctacacctg 50

<210> 210

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 210

ctacgagact caagcttgcc agtatcagat gcagttccca ggttgagccc 50

<210> 211

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 211

gacttaacaa accgcctgcg tgcgctttac gcccagtaat tccgattaac 50

<210> 212

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 212

attaccgcgg ctgctggcac ggagttagcc ggtgcttctt ctgcgggtaa 50

<210> 213

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 213

gtattaactt tactcccttc ctccccgctg aaagtacttt acaacccgaa 50

<210> 214

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 214

cgcggcatgg ctgcatcagg cttgcgccca ttgtgcagta ttccccactg 50

<210> 215

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 215

gtctggaccg tgtctcagtt ccagtgtggc tggtcatcct ctcagaccag 50

<210> 216

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 216

taggtgagcc gttaccccac ctactagcta atcccatctg ggcacatccg 50

<210> 217

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 217

aaggtccccc tctttggtct tgcgacgtta tgcggtatta gctaccgttt 50

<210> 218

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 218

ctccatcagg cagtttccca gacattactc acccgtccgc cactcgtcag 50

<210> 219

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 219

aaggttaagc ctcacggttc attagtaccg gttagctcaa cgcatcgctg 50

<210> 220

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 220

cctatcaacg tcgtcgtctt caacgttcct tcaggaccct taaagggtca 50

<210> 221

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 221

ggggcaagtt tcgtgcttag atgctttcag cacttatctc ttccgcattt 50

<210> 222

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 222

ccattggcat gacaacccga acaccagtga tgcgtccact ccggtcctct 50

<210> 223

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 223

ccccctcagt tctccagcgc ccacggcaga tagggaccga actgtctcac 50

<210> 224

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 224

gctcgcgtac cactttaaat ggcgaacagc catacccttg ggacctactt 50

<210> 225

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 225

atgagccgac atcgaggtgc caaacaccgc cgtcgatatg aactcttggg 50

<210> 226

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 226

atccccggag taccttttat ccgttgagcg atggcccttc cattcagaac 50

<210> 227

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 227

acctgctttc gcacctgctc gcgccgtcac gctcgcagtc aagctggctt 50

<210> 228

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 228

ccctcctgatg tccgaccagg attagccaac cttcgtgctc ctccgttact 50

<210> 229

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 229

gccccagtca aactacccac cagacactgt ccgcaacccg gattacgggt 50

<210> 230

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 230

aaacattaaa gggtggtatt tcaaggtcgg ctccatgcag actggcgtcc 50

<210> 231

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 231

ccacctatcc tacacatcaa ggctcaatgt tcagtgtcaa gctatagtaa 50

<210> 232

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 232

ttccgtcttg ccgcgggtac actgcatctt cacagcgagt tcaatttcac 50

<210> 233

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 233

gacagcctgg ccatcattac gccattcgtg caggtcggaa cttacccgac 50

<210> 234

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 234

cttaggaccg ttatagttac ggccgccgtt taccggggct tcgatcaaga 50

<210> 235

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 235

accccatcaa ttaaccttcc ggcaccggggc aggcgtcaca ccgtatacgt 50

<210> 236

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 236

cacagtgctg tgtttttaat aaacagttgc agccagctgg tatcttcgac 50

<210> 237

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 237

ccgcgaggga cctcacctac atatcagcgt gccttctccc gaagttacgg 50

<210> 238

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 238

ttccttcacc cgagttctct caagcgcctt ggtattctct acctgaccac 50

<210> 239

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 239

gtacgatttg atgttacctg atgcttagag gcttttcctg gaagcaggggc 50

<210> 240

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 240

accgtagtgc ctcgtcatca cgcctcagcc ttgattttcc ggatttgcct 50

<210> 241

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 241

acgcttaaac cgggacaacc gtcgcccggc caacatagcc ttctccgtcc 50

<210> 242

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 242

accaagtaca ggaatattaa cctgtttccc atcgactacg cctttcggcc 50

<210> 243

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 243

actcaccctg ccccgattaa cgttggacag gaacccttgg tcttccggcg 50

<210> 244

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 244

cgctttatcg ttacttatgt cagcattcgc acttctgata cctccagcat 50

<210> 245

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 245

ttcgcaggct tacagaacgc tcccctaccc aacaacgcat aagcgtcgct 50

<210> 246

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 246

catggtttag ccccgttaca tcttccgcgc aggccgactc gaccagtgag 50

<210> 247

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 247

taaatgatgg ctgcttctaa gccaacatcc tggctgtctg ggccttccca 50

<210> 248

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 248

aaccatgact ttgggacctt agctggcggt ctgggttgtt tccctcttca 50

<210> 249

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 249

cccgccgtgt gtctcccgtg ataacattct ccggtattcg cagtttgcat 50

<210> 250

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 250

ggatgacccc cttgccgaaa cagtgctcta cccccggaga tgaattcacg 50

<210> 251

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 251

agctttcggg gagaaccagc tatctcccgg tttgattggc ctttcacccc 50

<210> 252

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 252

cgctaatttt tcaacattag tcggttcggt cctccagtta gtgttaccca 50

<210> 253

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 253

atggctagat caccgggttt cgggtctata ccctgcaact taacgcccag 50

<210> 254

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 254

ccttcggctc ccctattcgg ttaaccttgc tacagaatat aagtcgctga 50

<210> 255

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 255

gtacgcagtc acacgcctaa gcgtgctccc actgcttgta cgtacacggt 50

<210> 256

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 256

actcccctcg ccggggttct tttcgccttt ccctcacggt actggttcac 50

<210> 257

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 257

agtatttagc cttggaggat ggtcccccca tattcagaca ggataccacg 50

<210> 258

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 258

atcgagctca cagcatgtgc atttttgtgt acggggctgt caccctgtat 50

<210> 259

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 259

acgcttccac taacacacac actgattcag gctctgggct gctccccgtt 50

<210> 260

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 260

ggggaatctc ggttgatttc ttttcctcgg ggtacttaga tgtttcagtt 50

<210> 261

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 261

attaacctat ggattcagtt aatgatagtg tgtcgaaaca cactgggttt 50

<210> 262

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 262

gccggttata acggttcata tcaccttacc gacgcttatc gcagattagc 50

<210> 263

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 263

gcttggcggc gtcctactct cacaggggga aacccccgac taccatcggc 50

<210> 264

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 264

ttccgtgttc ggtatgggaa cgggtgtgac ctcttcgcta tcgccaccaa 50

<210> 265

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 265

tagaaaggag gtgatccagc cgcaccttcc gatacggcta ccttgttacg 50

<210> 266

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 266

tctgtcccac cttcggcggc tggctcctaa aaggttacct caccgacttc 50

<210> 267

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 267

tcgtggtgtg acgggcggtg tgtacaaggc ccgggaacgt attcaccgcg 50

<210> 268

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 268

attactagcg attccagctt cacgcagtcg agttgcagac tgcgatccga 50

<210> 269

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 269

gtgggattgg cttaacctcg cggtttcgct gccctttgtt ctgtccattg 50

<210> 270

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 270

ccaggtcata aggggcatga tgatttgacg tcatccccac cttcctccgg 50

<210> 271

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 271

caccttagag tgcccaactg aatgctggca actaagatca agggttgcgc 50

<210> 272

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 272

acccaacatc tcacgacacg agctgacgac aaccatgcac cacctgtcac 50

<210> 273

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 273

gacgtcctat ctctaggatt gtcagaggat gtcaagacct ggtaaggttc 50

<210> 274

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 274

attaaaccac atgctccacc gcttgtgcgg gcccccgtca attcctttga 50

<210> 275

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 275

ccgtactccc caggcggagt gcttaatgcg ttagctgcag cactaagggg 50

<210> 276

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 276

acttagcact catcgtttac ggcgtggact accagggtat ctaatcctgt 50

<210> 277

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 277

tcgctcctca gcgtcagtta cagaccagag agtcgccttc gccactggtg 50

<210> 278

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 278

acgcatttca ccgctacacg tggaattcca ctctcctctt ctgcactcaa 50

<210> 279

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 279

atgaccctcc ccggttgagc cggggggcttt cacatcagac ttaagaaacc 50

<210> 280

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 280

acgcccaata attccggaca acgcttgcca cctacgtatt accgcggctg 50

<210> 281

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 281

ccgtggcttt ctggttaggt accgtcaagg taccgcccta ttcgaacggt 50

<210> 282

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 282

acaacagagc tttacgatcc gaaaaccttc atcactcacg cggcgttgct 50

<210> 283

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 283

ccattgcgga agattcccta ctgctgcctc ccgtaggagt ctgggccgtg 50

<210> 284

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 284

ggccgatcac cctctcaggt cggctacgca tcgtcgcctt ggtgagccgt 50

<210> 285

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 285

ctaatgcgcc gcgggtccat ctgtaagtgg tagccgaagc caccttttat 50

<210> 286

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 286

ttcaaacaac catccggtat tagccccggt ttcccggagt tatcccagtc 50

<210> 287

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 287

ccacgtgtta ctcacccgtc cgccgctaac atcagggagc aagctcccat 50

<210> 288

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 288

gcatgtatta ggcacgccgc cagcgttcgt cctgagccag gatcaaactc 50

<210> 289

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 289

tggttaagtc ctcgatcgat tagtatctgt cagctccatg tgtcgccaca 50

<210> 290

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 290

tatcaacctg atcatctttc agggatctta cttccttgcg gaatgggaaa 50

<210> 291

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 291

ggcttcatgc ttagatgctt tcagcactta tcccgtccgc acatagctac 50

<210> 292

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 292

gcagaacaac tggtacacca gcggtgcgtc catcccggtc ctctcgtact 50

<210> 293

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 293

caaatttcct gcgcccgcga cggataggga ccgaactgtc tcacgacgtt 50

<210> 294

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 294

gtaccgcttt aatgggcgaa cagcccaacc cttgggactg actacagccc 50

<210> 295

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 295

cgacatcgag gtgccaaacc tccccgtcga tgtggactct tgggggagat 50

<210> 296

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 296

ggggtagctt ttatccgttg agcgatggcc cttccatgcg gaaccaccgg 50

<210> 297

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 297

tttcgtccct gctcgacttg taggtctcgc agtcaagctc ccttgtgcct 50

<210> 298

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 298

gatttccaac cattctgagg gaacctttgg gcgcctccgt taccttttag 50

<210> 299

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 299

gtcaaactgc ccacctgaca ctgtctcccc gcccgataag ggcggcgggt 50

<210> 300

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 300

gccagggtag tatcccaccg atgcctccac cgaagctggc gctccggttt 50

<210> 301

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 301

atcctgtaca agctgtacca acattcaata tcaggctgca gtaaagctcc 50

<210> 302

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 302

cctgtcgcgg gtaacctgca tcttcacagg tactataatt tcaccgagtc 50

<210> 303

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 303

gccgatcg ttgcgccttt cgtgcgggtc ggaacttacc cgacaaggaa 50

<210> 304

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 304

accgttatag ttacggccgc cgtttactgg ggcttcaatt cgcaccttcg 50

<210> 305

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 305

cctcttaacc ttccagcacc gggcaggcgt cagcccctat acttcgcctt 50

<210> 306

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 306

cctgtgtttt tgctaaacag tcgcctgggc ctattcactg cggctctctc 50

<210> 307

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 307

cagagcaccc cttctcccga agttacgggg tcattttgcc gagttcctta 50

<210> 308

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 308

atcaccttag gattctctcc tcgcctacct gtgtcggttt gcggtacggg 50

<210> 309

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 309

tagaggcttt tcttggcagt gtggaatcag gaacttcgct actatatttc 50

<210> 310

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 310

tcagccttat gggaaacgga tttgcctatt tcccagccta actgcttgga 50

<210> 311

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 311

ccgcgcttac cctatcctcc tgcgtccccc cattgctcaa atggtgagga 50

<210> 312

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 312

tcaacctgtt gtccatcgcc tacgcctttc ggcctcggct taggtcccga 50

<210> 313

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 313

cgagccttcc tcaggaaacc ttaggcattc ggtggagggg attctcaccc 50

<210> 314

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 314

taccggcatt ctcacttcta agcgctccac cagtccttcc ggtctggctt 50

<210> 315

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 315

gctctcctac cactgttcga agaacagtcc gcagcttcgg tgatacgttt 50

<210> 316

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 316

tcggcgcaga gtcactcgac cagtgagcta ttacgcactc tttaaatggt 50

<210> 317

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 317

aacatcctgg ttgtctaagc aactccacat ccttttccac ttaacgtata 50

<210> 318

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 318

tggcggtctg ggctgtttcc ctttcgacta cggatcttat cactcgcagt 50

<210> 319

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 319

aagtcattgg cattcggagt ttgactgaat tcggtaaccc ggtaggggcc 50

<210> 320

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 320

gctctacctc caagactctt accttgaggc tagccctaaa gctatttcgg 50

<210> 321

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 321

tccaggttcg attggcattt cacccctacc cacacctcat ccccgcactt 50

<210> 322

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 322

ttcgggcctc cattcagtgt tacctgaact tcaccctgga catgggtaga 50

<210> 323

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 323

tctacgacca cgtactcatg cgccctattc agactcgctt tcgctgcggc 50

<210> 324

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 324

taaccttgca cgggatcgta actcgccggt tcattctaca aaaggcacgc 50

<210> 325

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 325

ggctctgact acttgtaggc acacggtttc aggatctctt tcactcccct 50

<210> 326

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 326

acctttccct cacggtactg gttcactatc ggtcactagg gagtatttag 50

<210> 327

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 327

ctcccggatt ccgacggaat ttcacgtgtt ccgccgtact caggatccac 50

<210> 328

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 328

gttttgacta cagggctgtt acctcctatg gcgggccttt ccagacctct 50

<210> 329

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 329

ctttgtaact ccgtacagag tgtcctacaa ccccaagagg caagcctctt 50

<210> 330

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 330

cgtttcgctc gccgctactc agggaatcgc atttgctttc tcttcctccg 50

<210> 331

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 331

cagttccccg ggtctgcctt ctcatatcct atgaattcag atatggatac 50

<210> 332

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 332

ggtgggtttc cccattcgga aatctccgga tcaaagcttg cttacagctc 50

<210> 333

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 333

tgttcgtccc gtccttcatc ggctcctagt gccaaggcat ccaccgtgcg 50

<210> 334

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 334

aaactagatt cgaatataac aaaacattac atcctcatcc aatccctttt 50

<210> 335

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 335

gcggtgtgtg caaggagcag ggacgtattc accgcgcgat tgtgacacgc 50

<210> 336

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 336

gcctttcggc gtcggaaccc attgtctcag ccattgtagc ccgcgtgttg 50

<210> 337

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 337

gcatacggac ctaccgtcgt ccactccttc ctcctattta tcataggcgg 50

<210> 338

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 338

cggcatccaa aaaaggatcc gctggtaact aagagcgtgg gtctcgctcg 50

<210> 339

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 339

caacctggct atcatacagc tgtcgcctct ggtgagatgt ccggcgttga 50

<210> 340

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 340

aggctccacg cgttgtggtg ctcccccgcc aattccttta agtttcagtc 50

<210> 341

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 341

ccaggcggcg gacttaacag cttcccttcg gcactgggac agctcaaagc 50

<210> 342

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 342

tccgcatcgt ttacagctag gactacccgg gtatctaatc cggttcgcgc 50

<210> 343

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 343

ttcccacagt taagctgcag gatttcacca gagacttatt aaaccggcta 50

<210> 344

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 344

ctcttattcc aaaagctctt tacactaatg aaaagccatc ccgttaagaa 50

<210> 345

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 345

cccccgtcgc gatttctcac attgcgggagg tttcgcgcct gctgcacccc 50

<210> 346

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 346

ttgtctcagg ttccatctcc gggctcttgc tctcacaacc cgtaccgatc 50

<210> 347

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 347

cattacctaa ccaactacct aatcggccgc agacccatcc ttaggcgaaa 50

<210> 348

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 348

aaaccattac aggaataatt gcctatccag tattatcccc agtttcccag 50

<210> 349

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 349

aagggtaggt tatccacgtg ttactgagcc gtacgccacg agcctaaact 50

<210> 350

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 350

acctagcgcg tagctgcccg gcactgcctt atcagacaac cggtcgacca 50

<210> 351

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 351

cgttcctctc gtactggagc caccttcccc tcagactact aacacatcca 50

<210> 352

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 352

cctgtctcac gacggtctaa acccagctca cgttcccctt taatgggcga 50

<210> 353

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 353

ggtgctgctg cacacccagg atggaaagaa ccgacatcga agtagcaagc 50

<210> 354

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 354

ggctcttgcc tgcgaccacc cagttatccc cgaggtagtt tttctgtcat 50

<210> 355

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 355

aggaggactc tgaggttcgc taggcccggc tttcgcctct ggatttcttg 50

<210> 356

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 356

caaagtaagt tagaaacaca gtcataagaa agtggtgtct caagaacgaa 50

<210> 357

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 357

gacttataat cgaattctcc cacttacact gcatacctat aaccaagctt 50

<210> 358

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 358

gtaaaactct acggggtctt cgcttcccaa tggaagactc tggcttgtgc 50

<210> 359

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 359

tcactaagtt ctagctaggg acagtgggga cctcgttcta ccattcatgc 50

<210> 360

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 360

50

<210> 361

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 361

aactgaactc cagcttcacg tgccagcact gggcaggtgt cgccctctgt 50

<210> 362

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 362

ctagcagaga gctatgtttt tattaaacag tcgggccccc ctagtcactg 50

<210> 363

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 363

ttaaaacgcc ttagcctact cagctaggggg cacctgtgac ggatctcggt 50

<210> 364

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 364

acaaaactaa ctcccttttc aaggactcca tgaatcagtt aaaccagtac 50

<210> 365

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 365

ataatgccta cacctggttc tcgctattac acctctcccc aggcttaaac 50

<210> 366

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 366

caatcctaca aaacatatct cgaagtgtca gaaattagcc ctcaacgtca 50

<210> 367

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 367

ctttgctgct actactacca ggatccacat acctgcaagg tccaaaggaa 50

<210> 368

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 368

caacccacac aggtcgccac tctacacaat caccaaaaaa aaggtgttcc 50

<210> 369

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 369

ggattaattc ccgtccattt taggtgcctc tgacctcgat gggtgatctg 50

<210> 370

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 370

agggtggctg cttctaagcc caccttccca ttgtcttgggg ccaaagactc 50

<210> 371

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 371

gtatttaggg gccttaacca tagtctgagt tgtttctctt tcgggacaca 50

<210> 372

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 372

cctcactcca accttctacg acggtgacga gttcggagtt ttacagtacg 50

<210> 373

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 373

ccctaaacgt ccaattagtg ctctaccccg ccaccaacct ccagtcaggc 50

<210> 374

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 374

aatagatcga ccggcttcgg gtttcaatgc tgtgattcca ggccctatta 50

<210> 375

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 375

acaacgctgc gggcatatcg gtttccctac gactacaagg ataaaaacct 50

<210> 376

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 376

acaaagaact ccctggcccg tgtttcaaga cggacgatgc aacactagtc 50

<210> 377

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 377

acaatgttac cactgattct ttcggaagaa ttcattcctt acgcgccaca 50

<210> 378

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 378

ctggtttcag gtacttttca cccccctata ggggtacttt tcagcattcc 50

<210> 379

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 379

ctctatcggt cttgagacgt atttagaatt ggaagttgat gcctcccaca 50

<210> 380

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 380

atcaccctct acggttctaa aattccaaat aaaattcgat ttatccccg 50

<210> 381

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 381

tctatacacc acatctccct aatattacta aaagggattc agtttgttct 50

<210> 382

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 382

gccgttacta acgacatcgc atattgcttt cttttcctcc gcctactaag 50

<210> 383

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 383

gggttcccaa tcctacacgg atcaacacaa aaaaaatgtg ctaggaagtc 50

<210> 384

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> DNA probe

<400> 384

actactggga tcgaaacgag accaggtata acccccatgc tatgaccgca 50

<210> 385

<211> 50

<212> PROTEIN

<213> Mus musculus

<400> 385

Gly Cys Gly Thr Ala Thr Gly Cys Cys Thr Gly Gly Ala Gly Ala Ala

1 5 10 15

Thr Thr Gly Gly Ala Ala Thr Thr Cys Thr Thr Gly Thr Thr Ala Cys

20 25 30

Thr Cys Ala Thr Ala Cys Thr Ala Ala Cys Ala Gly Thr Gly Thr Thr

35 40 45

Gly Cys

50

<210> 386

<211> 50

<212> PROTEIN

<213> Mus musculus

<400> 386

Gly Ala Thr Thr Ala Ala Cys Cys Cys Ala Ala Thr Thr Thr Thr Thr Ala

1 5 10 15

Ala Gly Thr Thr Thr Ala Gly Gly Ala Ala Gly Thr Thr Gly Gly Thr

20 25 30

Gly Thr Ala Ala Ala Thr Thr Ala Thr Gly Gly Ala Ala Thr Thr Ala

35 40 45

Ala Thr

50

<210> 387

<211> 50

<212> PROTEIN

<213> Mus musculus

<400> 387

Ala Gly Cys Thr Thr Gly Ala Ala Cys Gly Cys Thr Thr Thr Cys Thr

1 5 10 15

Thr Thr Ala Thr Thr Gly Gly Thr Gly Gly Cys Thr Gly Cys Thr Thr

20 25 30

Thr Thr Ala Gly Gly Cys Cys Thr Ala Cys Ala Ala Thr Gly Gly Thr

35 40 45

Thr Ala

50

<210> 388

<211> 50

<212> PROTEIN

<213> Mus musculus

<400> 388

Ala Thr Thr Ala Thr Thr Cys Ala Cys Thr Ala Thr Thr Ala Ala Ala

1 5 10 15

Gly Gly Thr Thr Thr Thr Thr Thr Cys Cys Gly Thr Thr Cys Cys Ala

20 25 30

Gly Ala Ala Gly Ala Gly Cys Thr Gly Thr Cys Cys Cys Thr Cys Thr

35 40 45

Thr Thr

50

<210> 389

<211> 50

<212> PROTEIN

<213> Mus musculus

<400> 389

Cys Thr Thr Ala Cys Thr Thr Thr Thr Thr Gly Ala Thr Thr Thr Thr

1 5 10 15

Gly Thr Thr Gly Thr Thr Thr Thr Thr Thr Thr Ala Gly Cys Ala Ala

20 25 30

Gly Thr Thr Thr Ala Ala Ala Ala Thr Thr Gly Ala Ala Cys Thr Thr

35 40 45

Ala Ala

50

<210> 390

<211> 50

<212> PROTEIN

<213> Mus musculus

<400> 390

Ala Ala Cys Cys Ala Gly Cys Thr Ala Thr Cys Ala Cys Cys Ala Ala

1 5 10 15

Gly Cys Thr Cys Gly Thr Thr Ala Gly Gly Cys Thr Thr Thr Thr Cys

20 25 30

Ala Cys Cys Thr Cys Thr Ala Cys Cys Thr Ala Ala Ala Ala Ala Thr

35 40 45

Cys Thr

50

<210> 391

<211> 50

<212> PROTEIN

<213> Mus musculus

<400> 391

Ala Ala Thr Ala Cys Thr Thr Gly Thr Ala Ala Thr Gly Cys Thr Ala

1 5 10 15

Gly Ala Gly Gly Thr Gly Ala Thr Gly Thr Thr Thr Thr Thr Gly Gly

20 25 30

Thr Ala Ala Ala Cys Ala Gly Gly Cys Gly Gly Gly Gly Thr Thr Cys

35 40 45

Thr Thr

50

<210> 392

<211> 50

<212> PROTEIN

<213> Mus musculus

<400> 392

Thr Thr Thr Ala Thr Cys Thr Thr Thr Thr Thr Gly Gly Ala Thr Cys

1 5 10 15

Thr Thr Thr Cys Cys Thr Thr Thr Ala Gly Gly Cys Ala Thr Thr Cys

20 25 30

Cys Gly Gly Thr Gly Thr Thr Gly Gly Gly Thr Thr Ala Ala Cys Ala

35 40 45

Gly Ala

50

<210> 393

<211> 50

<212> PROTEIN

<213> Mus musculus

<400> 393

Thr Thr Ala Thr Thr Thr Ala Thr Ala Gly Thr Gly Thr Gly Ala Thr

1 5 10 15

Thr Ala Thr Thr Gly Cys Cys Thr Ala Thr Ala Gly Thr Cys Thr Gly

20 25 30

Ala Thr Thr Ala Ala Cys Thr Ala Ala Cys Ala Ala Thr Gly Gly Thr

35 40 45

Thr Ala

50

<210> 394

<211> 50

<212> PROTEIN

<213> Rattus norvegicus

<400> 394

Ala Gly Thr Gly Ala Thr Thr Gly Thr Ala Gly Thr Thr Gly Thr Thr

1 5 10 15

Thr Ala Thr Thr Cys Ala Cys Thr Ala Thr Thr Thr Ala Ala Gly Gly

20 25 30

Thr Thr Thr Thr Thr Thr Cys Cys Thr Thr Thr Thr Cys Cys Thr Ala

35 40 45

Ala Ala

50

<210> 395

<211> 50

<212> PROTEIN

<213> Rattus norvegicus

<400> 395

Thr Gly Gly Cys Thr Ala Thr Ala Thr Thr Thr Thr Ala Ala Gly Thr

1 5 10 15

Thr Thr Ala Cys Ala Thr Thr Thr Thr Gly Ala Thr Thr Thr Gly Thr

20 25 30

Thr Gly Thr Thr Cys Thr Gly Ala Thr Gly Gly Thr Ala Ala Gly Cys

35 40 45

Thr Thr

50

<210> 396

<211> 50

<212> PROTEIN

<213> Rattus norvegicus

<400> 396

Thr Thr Thr Thr Thr Thr Thr Thr Ala Ala Thr Cys Thr Thr Thr Cys Cys

1 5 10 15

Thr Thr Ala Ala Ala Gly Cys Ala Cys Gly Cys Cys Thr Gly Thr Gly

20 25 30

Thr Thr Gly Gly Gly Cys Thr Ala Ala Cys Gly Ala Gly Thr Thr Ala

35 40 45

Gly Gly

50

<210> 397

<211> 50

<212> PROTEIN

<213> Rattus norvegicus

<400> 397

Thr Gly Thr Thr Gly Gly Gly Thr Thr Ala Gly Thr Ala Cys Cys Thr

1 5 10 15

Ala Thr Gly Ala Thr Thr Cys Gly Ala Thr Ala Ala Thr Thr Gly Ala

20 25 30

Cys Ala Ala Thr Gly Gly Thr Thr Ala Thr Cys Cys Gly Gly Gly Thr

35 40 45

Thr Gly

50

<210> 398

<211> 50

<212> PROTEIN

<213> Rattus norvegicus

<400> 398

Ala Gly Gly Ala Gly Ala Ala Thr Thr Gly Gly Thr Thr Cys Thr Thr

1 5 10 15

Gly Thr Thr Ala Cys Thr Cys Ala Thr Ala Thr Thr Ala Ala Cys Ala

20 25 30

Gly Thr Ala Thr Thr Thr Thr Cys Ala Thr Cys Thr Ala Thr Gly Gly Ala

35 40 45

Thr Cys

50

<210> 399

<211> 50

<212> PROTEIN

<213> Rattus norvegicus

<400> 399

Thr Thr Thr Gly Thr Gly Ala Thr Ala Thr Ala Gly Gly Ala Ala Thr

1 5 10 15

Thr Thr Ala Thr Thr Gly Ala Gly Gly Thr Thr Thr Gly Thr Gly Gly

20 25 30

Ala Ala Thr Thr Ala Gly Thr Gly Thr Gly Thr Gly Thr Ala Ala Gly

35 40 45

Thr Ala

50

<210> 400

<211> 50

<212> PROTEIN

<213> Mus musculus

<400> 400

Gly Cys Cys Gly Gly Gly Gly Ala Gly Thr Gly Gly Gly Thr Cys Thr

1 5 10 15

Thr Cys Cys Gly Thr Ala Cys Gly Cys Cys Ala Cys Ala Thr Thr Thr

20 25 30

Cys Cys Cys Ala Cys Gly Cys Cys Gly Cys Gly Ala Cys Gly Cys Gly

35 40 45

Cys Gly

50

<210> 401

<211> 50

<212> PROTEIN

<213> Mus musculus

<400> 401

Ala Cys Cys Thr Cys Gly Gly Gly Cys Cys Cys Cys Cys Gly Gly Gly

1 5 10 15

Cys Gly Gly Gly Gly Cys Cys Cys Thr Thr Cys Ala Cys Cys Thr Thr

20 25 30

Cys Ala Thr Thr Gly Cys Gly Cys Cys Ala Cys Gly Gly Cys Gly Gly

35 40 45

Cys Thr

50

<210> 402

<211> 50

<212> PROTEIN

<213> Mus musculus

<400> 402

Thr Cys Gly Cys Gly Thr Cys Cys Ala Gly Ala Gly Thr Cys Gly Cys

1 5 10 15

Cys Gly Cys Cys Gly Cys Cys Gly Cys Cys Gly Gly Cys Cys Cys Cys

20 25 30

Cys Cys Gly Ala Gly Thr Gly Thr Cys Cys Gly Gly Gly Cys Cys Cys

35 40 45

Cys Cys

50

<210> 403

<211> 50

<212> PROTEIN

<213> Mus musculus

<400> 403

Cys Gly Cys Thr Gly Gly Thr Thr Cys Cys Thr Cys Cys Cys Gly Cys

1 5 10 15

Thr Cys Cys Gly Gly Ala Ala Cys Cys Cys Cys Cys Gly Cys Gly Gly

20 25 30

Gly Gly Thr Thr Gly Gly Ala Cys Cys Cys Gly Cys Cys Gly Cys Cys

35 40 45

Cys Cys

50

<210> 404

<211> 50

<212> PROTEIN

<213> Mus musculus

<400> 404

Cys Gly Cys Cys Gly Ala Cys Cys Cys Cys Cys Gly Ala Cys Cys Cys

1 5 10 15

Gly Cys Cys Cys Cys Cys Cys Gly Ala Cys Gly Gly Gly Ala Ala Gly

20 25 30

Ala Ala Gly Gly Ala Gly Gly Gly Gly Gly Gly Ala Ala Gly Ala Gly

35 40 45

Ala Gly

50

<210> 405

<211> 50

<212> PROTEIN

<213> Mus musculus

<400> 405

Gly Gly Gly Ala Cys Gly Ala Cys Gly Gly Gly Gly Cys Cys Cys Cys

1 5 10 15

Gly Cys Gly Gly Gly Gly Ala Ala Gly Ala Gly Gly Gly Gly Ala Gly

20 25 30

Gly Gly Cys Gly Gly Gly Cys Cys Cys Gly Gly Gly Cys Gly Gly Ala

35 40 45

Ala Ala

50

<210> 406

<211> 50

<212> PROTEIN

<213> Mus musculus

<400> 406

Gly Gly Cys Gly Cys Cys Gly Cys Gly Cys Gly Gly Ala Ala Ala Ala

1 5 10 15

Cys Cys Gly Cys Gly Gly Cys Cys Cys Gly Gly Gly Gly Gly Gly Cys

20 25 30

Gly Gly Ala Cys Cys Cys Gly Gly Cys Gly Gly Gly Gly Gly Ala Ala

35 40 45

Cys Ala

50

<210> 407

<211> 50

<212> PROTEIN

<213> Mus musculus

<400> 407

Cys Cys Cys Cys Cys Ala Cys Ala Cys Gly Cys Gly Cys Gly Gly Gly

1 5 10 15

Ala Cys Ala Cys Gly Cys Cys Cys Gly Cys Cys Cys Gly Cys Cys Cys

20 25 30

Cys Cys Gly Cys Cys Ala Cys Gly Cys Ala Cys Cys Thr Cys Gly Gly

35 40 45

Gly Ala

50

<210> 408

<211> 50

<212> PROTEIN

<213> Mus musculus

<400> 408

Cys Ala Cys Cys Cys Gly Cys Thr Thr Thr Gly Gly Gly Cys Thr Gly

1 5 10 15

Cys Ala Thr Thr Cys Cys Cys Ala Ala Gly Cys Ala Ala Cys Cys Cys

20 25 30

Gly Ala Cys Thr Cys Cys Gly Gly Gly Ala Ala Gly Ala Cys Cys Cys

35 40 45

Gly Ala

50

<210> 409

<211> 50

<212> PROTEIN

<213> Mus musculus

<400> 409

Thr Gly Gly Ala Gly Cys Gly Ala Gly Gly Cys Cys Cys Cys Gly Cys

1 5 10 15

Gly Gly Gly Gly Ala Gly Gly Gly Gly Ala Cys Cys Cys Gly Cys Gly

20 25 30

Cys Cys Gly Gly Cys Ala Cys Cys Cys Gly Cys Cys Gly Gly Gly Cys

35 40 45

Thr Cys

50

<210> 410

<211> 50

<212> PROTEIN

<213> Mus musculus

<400> 410

Cys Gly Ala Gly Gly Cys Cys Gly Gly Cys Gly Thr Gly Cys Cys Cys

1 5 10 15

Cys Gly Ala Cys Cys Cys Cys Gly Ala Cys Gly Cys Gly Ala Gly Gly

20 25 30

Ala Cys Gly Gly Gly Gly Cys Cys Gly Gly Gly Cys Gly Cys Cys Gly

35 40 45

Gly Gly

50

<210> 411

<211> 50

<212> PROTEIN

<213> Mus musculus

<400> 411

Thr Cys Cys Cys Cys Gly Gly Ala Gly Cys Gly Gly Gly Thr Cys Gly

1 5 10 15

Cys Gly Cys Cys Cys Gly Cys Cys Cys Gly Cys Ala Cys Gly Cys Gly

20 25 30

Cys Gly Gly Gly Ala Cys Gly Gly Ala Cys Gly Cys Thr Thr Gly Gly

35 40 45

Cys Gly

50

<210> 412

<211> 50

<212> PROTEIN

<213> Mus musculus

<400> 412

Thr Cys Cys Ala Cys Ala Cys Gly Ala Ala Cys Gly Thr Gly Cys Gly

1 5 10 15

Thr Thr Cys Ala Ala Cys Gly Thr Gly Ala Cys Gly Gly Gly Cys Gly

20 25 30

Ala Gly Ala Gly Gly Gly Cys Gly Gly Cys Cys Cys Cys Cys Thr Thr

35 40 45

Thr Cys

50

<210> 413

<211> 50

<212> PROTEIN

<213> Mus musculus

<400> 413

Thr Cys Cys Cys Ala Ala Gly Ala Cys Gly Ala Ala Cys Gly Gly Cys

1 5 10 15

Thr Cys Thr Cys Cys Gly Cys Ala Cys Cys Gly Gly Ala Cys Cys Cys

20 25 30

Cys Gly Gly Thr Cys Cys Cys Gly Ala Cys Gly Cys Cys Cys Gly Gly

35 40 45

Cys Gly

50

<210> 414

<211> 50

<212> PROTEIN

<213> Mus musculus

<400> 414

Cys Cys Gly Cys Cys Gly Cys Gly Gly Gly Gly Ala Cys Gly Ala Cys

1 5 10 15

Gly Cys Gly Gly Gly Gly Ala Cys Cys Cys Cys Gly Cys Cys Gly Ala

20 25 30

Gly Cys Gly Gly Gly Gly Ala Cys Gly Gly Ala Cys Gly Gly Gly Gly

35 40 45

Ala Cys

50

<210> 415

<211> 50

<212> PROTEIN

<213> Mus musculus

<400> 415

Gly Cys Ala Cys Cys Gly Cys Cys Ala Cys Gly Gly Thr Gly Gly Ala

1 5 10 15

Ala Gly Thr Gly Cys Gly Cys Cys Cys Gly Gly Cys Gly Gly Cys Gly

20 25 30

Gly Cys Cys Gly Gly Thr Cys Gly Cys Cys Gly Gly Cys Cys Gly Gly

35 40 45

Gly Gly

50

<210> 416

<211> 50

<212> PROTEIN

<213> Mus musculus

<400> 416

Cys Cys Cys Ala Cys Cys Gly Gly Gly Cys Cys Cys Cys Gly Ala Gly

1 5 10 15

Ala Gly Ala Gly Gly Cys Gly Ala Cys Gly Gly Ala Gly Gly Gly Gly

20 25 30

Gly Gly Thr Gly Gly Gly Ala Gly Ala Gly Cys Gly Gly Thr Cys Gly

35 40 45

Cys Gly

50

<210> 417

<211> 50

<212> PROTEIN

<213> Mus musculus

<400> 417

Cys Cys Cys Gly Gly Cys Cys Cys Cys Cys Ala Cys Cys Cys Cys Cys

1 5 10 15

Ala Cys Gly Cys Cys Cys Gly Cys Cys Cys Gly Gly Gly Ala Gly Gly

20 25 30

Cys Gly Gly Ala Cys Gly Gly Gly Gly Gly Gly Ala Gly Ala Gly Gly

35 40 45

Gly Ala

50

<210> 418

<211> 50

<212> PROTEIN

<213> Mus musculus

<400> 418

Thr Ala Thr Cys Thr Gly Gly Cys Thr Thr Cys Cys Thr Cys Gly Gly

1 5 10 15

Cys Cys Cys Cys Gly Gly Gly Ala Thr Thr Cys Gly Gly Cys Gly Ala

20 25 30

Ala Ala Gly Cys Gly Cys Gly Gly Cys Cys Gly Gly Ala Gly Gly Gly

35 40 45

Cys Thr

50

<210> 419

<211> 50

<212> PROTEIN

<213> Mus musculus

<400> 419

Cys Gly Cys Cys Gly Cys Cys Gly Ala Cys Cys Cys Cys Gly Thr Gly

1 5 10 15

Cys Gly Cys Thr Cys Gly Gly Cys Thr Thr Cys Gly Thr Cys Gly Gly

20 25 30

Gly Ala Gly Ala Cys Gly Cys Gly Thr Gly Ala Cys Cys Gly Ala Cys

35 40 45

Gly Gly

50

<210> 420

<211> 50

<212> PROTEIN

<213> Mus musculus

<400> 420

Gly Cys Gly Cys Cys Cys Cys Cys Cys Cys Gly Cys Ala Cys Cys Cys

1 5 10 15

Gly Cys Cys Cys Cys Gly Thr Cys Cys Cys Cys Cys Cys Cys Gly Cys

20 25 30

Gly Gly Ala Cys Gly Gly Gly Gly Ala Ala Gly Ala Ala Gly Gly Gly

35 40 45

Ala Gly

50

<210> 421

<211> 50

<212> PROTEIN

<213> Mus musculus

<400> 421

Cys Gly Ala Ala Cys Cys Cys Cys Gly Gly Gly Ala Ala Cys Cys Cys

1 5 10 15

Cys Cys Gly Ala Cys Cys Cys Cys Gly Cys Gly Gly Ala Gly Gly Gly

20 25 30

Gly Gly Ala Ala Gly Gly Gly Gly Gly Ala Gly Gly Ala Cys Gly Ala

35 40 45

Gly Gly

50

<210> 422

<211> 50

<212> PROTEIN

<213> Mus musculus

<400> 422

Cys Ala Cys Cys Cys Gly Gly Gly Gly Gly Gly Gly Cys Gly Ala Cys

1 5 10 15

Gly Ala Gly Gly Cys Gly Gly Gly Gly Ala Cys Cys Cys Gly Cys Cys

20 25 30

Gly Gly Ala Cys Gly Gly Gly Gly Ala Cys Gly Gly Ala Cys Gly Gly

35 40 45

Gly Gly

50

<210> 423

<211> 50

<212> PROTEIN

<213> Mus musculus

<400> 423

Gly Cys Cys Ala Ala Cys Cys Gly Ala Gly Gly Cys Thr Cys Cys Thr

1 5 10 15

Thr Cys Gly Gly Cys Gly Cys Thr Gly Cys Cys Gly Thr Ala Thr Cys

20 25 30

Gly Thr Thr Cys Cys Gly Cys Thr Thr Gly Gly Gly Cys Gly Gly Ala

35 40 45

Thr Thr

50

<210> 424

<211> 50

<212> PROTEIN

<213> Mus musculus

<400> 424

Cys Cys Cys Gly Gly Gly Cys Cys Cys Cys Cys Gly Gly Ala Cys Cys

1 5 10 15

Cys Cys Cys Gly Ala Gly Ala Gly Gly Gly Ala Cys Gly Ala Cys Gly

20 25 30

Gly Ala Gly Gly Cys Gly Ala Cys Gly Gly Gly Gly Gly Gly Thr Gly

35 40 45

Gly Gly

50

<210> 425

<211> 50

<212> PROTEIN

<213> Mus musculus

<400> 425

Thr Gly Gly Gly Ala Gly Gly Gly Gly Cys Gly Gly Cys Cys Cys Gly

1 5 10 15

Gly Cys Cys Cys Cys Cys Gly Cys Gly Ala Cys Cys Gly Cys Cys Cys

20 25 30

Cys Cys Cys Thr Thr Thr Thr Cys Cys Gly Cys Cys Ala Cys Cys Cys Cys

35 40 45

Ala Cys

50

<210> 426

<211> 50

<212> PROTEIN

<213> Mus musculus

<400> 426

Gly Gly Gly Ala Gly Ala Gly Gly Cys Cys Gly Gly Gly Gly Gly Gly

1 5 10 15

Ala Gly Ala Gly Cys Gly Cys Gly Gly Cys Gly Ala Cys Gly Gly Gly

20 25 30

Thr Ala Thr Cys Cys Gly Gly Cys Thr Cys Cys Cys Thr Cys Gly Gly

35 40 45

Cys Cys

50

<210> 427

<211> 50

<212> PROTEIN

<213> Mus musculus

<400> 427

Cys Gly Cys Thr Gly Cys Thr Gly Cys Cys Gly Gly Gly Gly Gly Gly

1 5 10 15

Cys Thr Gly Thr Ala Ala Cys Ala Cys Thr Cys Gly Gly Gly Gly Cys

20 25 30

Gly Gly Gly Gly Thr Gly Gly Thr Cys Cys Gly Gly Cys Gly Cys Cys

35 40 45

Cys Ala

50

<210> 428

<211> 50

<212> PROTEIN

<213> Mus musculus

<400> 428

Cys Gly Cys Cys Gly Cys Cys Gly Ala Cys Cys Cys Cys Gly Thr Gly

1 5 10 15

Cys Gly Cys Thr Cys Gly Gly Cys Thr Thr Cys Gly Cys Thr Cys Cys

20 25 30

Cys Cys Cys Cys Cys Ala Cys Cys Cys Cys Gly Ala Gly Ala Ala Gly

35 40 45

Gly Gly

50

<---

Claims (94)

1. A method for removing non-target RNA molecules from a nucleic acid sample, comprising: (a) bringing a nucleic acid sample containing at least one target RNA or DNA sequence and at least one non-target RNA molecule into contact with a probe set containing 50 or more DNA probes complementary to non-contiguous sequences along the entire length of at least one sequence of a non-target RNA molecule, thereby hybridizing 50 or more DNA probes with non-target RNA molecules to form DNA:RNA hybrids, characterized in that i) each of the 50 or more DNA probes is 40 to 60 bases in length, and ii) each DNA:RNA hybrid is separated by at least 5 to 40 bases along a predetermined sequence of a non-target RNA molecule from any other DNA:RNA hybrid; and (b) contacting the DNA:RNA hybrids with a ribonuclease that cleaves RNA from the DNA:RNA hybrids, thereby destroying non-target RNA molecules in the nucleic acid sample to form a digested mixture. 2. The method according to p. 1, including: (c) optionally cleaving the remaining DNA probes by contacting the cleaved mixture with a DNA cleaving enzyme to form a cleaved DNA mixture, wherein the DNA cleaving enzyme may optionally be DNase I; and (d) separating the digested RNA from the digested mixture or digested DNA mixture. 3. The method of claim 1 or 2, wherein contacting the probe set comprises treating the nucleic acid sample with a destabilizer. 4. The method of claim 3, wherein the destabilizer is heat or a nucleic acid destabilizing chemical, wherein the nucleic acid destabilizing chemical is optionally betaine, DMSO, formamide, glycerol, derivatives thereof, or a mixture thereof. 5. The method of claim 4, wherein the nucleic acid destabilizing chemical is formamide, optionally the formamide is present at the time of contact with the probe set at a concentration of from about 10% to 45% by volume. 6. The method of claim 4, wherein treating the sample with heat comprises applying heat above the melting point of at least one DNA:RNA hybrid. 7. The method according to any one of paragraphs. 1-6, wherein the ribonuclease is an RNase H or a hybridase. 8. The method according to any one of paragraphs. 1-7, wherein the nucleic acid sample is from a human or non-human primate. 9. The method according to any one of paragraphs. 1-7, wherein the nucleic acid sample is from a rat or mouse. 10. The method according to any one of paragraphs. 1-8, wherein the nucleic acid sample contains nucleic acids of non-human origin. 11. The method of claim 10, wherein the non-human nucleic acids are derived from non-human eukaryotes, bacteria, viruses, plants, soil, or mixtures thereof. 12. The method according to any one of paragraphs. 1-11, wherein the non-target RNA is rRNA, mRNA, tRNA, or a mixture thereof. 13. The method of claim 12 wherein the non-target RNA is rRNA and globin mRNA. 14. The method according to any one of paragraphs. 1-12, wherein the probe set comprises at least two DNA probes that hybridize 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. 15. The method of claim 14, wherein the probe set comprises at least two DNA probes that hybridize to two or more non-target RNA molecules selected from human 28S, 18S, 5.8S, 5S, 16S, and 12S. 16. The method according to any one of paragraphs. 1-15, in which the set of probes contains at least two DNA probes that hybridize with one or more non-target RNA molecules selected from HBA-A1, HBA-A2, HBB, HBG1 and HBG2 hemoglobin and 23S, 16S and 5S Gram-positive or Gram-negative bacteria. 17. The method according to any one of paragraphs. 1-16, wherein the probe set comprises at least two DNA probes that hybridize to one or more non-target RNA molecules derived from Archaea species. 18. The method according to any one of paragraphs. 1-17, wherein the probe set comprises at least two DNA probes that hybridize to one or more non-target rat and/or mouse RNA molecules, which can be selected from 16S rat, 28S rat, 16S mouse, 28S mouse and combinations thereof. 19. The method according to any one of paragraphs. 1-18, wherein the probes for a particular non-target RNA molecule are about 80 to 85% complementary to the sequence of the non-target RNA molecule. 20. The method according to any one of paragraphs. 1-11, in which DNA probes contain: (a) two or more, or five or more, or 10 or more, or 25 or more, or 50 or more, or 100 or more, or 150 or more, or 200 or more, or 250 or more, or 300 or more, or 333 sequences selected from SEQ ID NO: 1-333; or (b) two or more, or five or more, or 10 or more, or 25 or more, or 50 or more, or 100 or more, or 150 or more, or 200 or more, or 250 or more, or 300 or more, or 350 or more, or 400 or more, or 428 sequences selected from SEQ ID NO: 1-428; or (c) two or more, or five or more, or 10 or more, or 25 or more, or 50 or more, or 100 or more, or 150 or more, or 200 or more, or 250 or more, or 300 or more, or 350 or more, or 377 sequences selected from SEQ ID NO: 1-377; or (d) two or more, or five or more, or 10 or more, or 25 or more, or 50 or more, or 100 or more, or 150 or more, or 200 or more, or 250 or more, or 300 or more, or 350 or more, or 384 sequences selected from SEQ ID NO: 1-333 and SEQ ID NO: 378-428; or (e) two or more, or five or more, or 10 or more, or 25 or more, or 44 sequences selected from SEQ ID NO: 334-377; or (f) two or more, or five or more, or 10 or more, or 25 or more, or 50 or more, or 51 sequences selected from SEQ ID NO: 378-428; or their combinations. 21. Composition for removing non-target RNA molecules from a nucleic acid sample, comprising a set of probes containing (a) 50 or more DNA probes complementary to non-contiguous sequences that are 5 to 40 bases apart along the entire length of at least one non-target RNA molecule in the nucleic acid sample; where each of the 50 or more DNA probes is 40 to 60 bases in length; and (b) a ribonuclease capable of cleaving RNA in a DNA:RNA hybrid. 22. The composition of claim 21 wherein the ribonuclease is RNase H. 23. The composition according to any one of paragraphs. 21-22 containing a destabilizing chemical. 24. The composition of claim 23 wherein the destabilizing chemical is formamide. 25. The composition according to any one of paragraphs. 21-24, wherein the non-target RNA is rRNA, mRNA, tRNA, or a mixture thereof. 26. The composition of claim 25 wherein the non-target RNA is rRNA and globin mRNA. 27. The composition according to any one of paragraphs. 21-26, wherein the probe set comprises at least two DNA probes that hybridize 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. 28. The composition of claim 27, wherein the probe set comprises at least two DNA probes that hybridize to two or more non-target RNA molecules selected from human 28S, 18S, 5.8S, 5S, 16S, and 12S. 29. The composition according to any one of paragraphs. 21-28, wherein the probe set comprises at least two DNA probes that hybridize to one or more non-target RNA molecules selected from HBA-A1, HBA-A2, HBB, HBG1 and HBG2 hemoglobin and 23S, 16S and 5S Gram-positive or Gram-negative bacteria. 30. The composition according to any one of paragraphs. 21-29, wherein the probe set contains at least two DNA probes complementary to one or more Archaea species rRNA molecules. 31. The composition according to any one of paragraphs. 21-30, wherein the probe set comprises DNA probes that hybridize to one or more non-target rat and/or mouse RNA molecules that are optionally selected from rat 16S, rat 28S, mouse 16S, mouse 28S, and their combinations. 32. The composition according to any one of paragraphs. 21-31, in which DNA probes contain: (a) two or more, or five or more, or 10 or more, or 25 or more, or 50 or more, or 100 or more, or 150 or more, or 200 or more, or 250 or more, or 300 or more, or 333 sequences selected from SEQ ID NO: 1-333; or (b) two or more, or five or more, or 10 or more, or 25 or more, or 50 or more, or 100 or more, or 150 or more, or 200 or more, or 250 or more, or 300 or more, or 350 or more, or 400 or more, or 428 sequences selected from SEQ ID NO: 1-428; or (c) two or more, or five or more, or 10 or more, or 25 or more, or 50 or more, or 100 or more, or 150 or more, or 200 or more, or 250 or more, or 300 or more, or 350 or more, or 377 sequences selected from SEQ ID NO: 1-377; or (d) two or more, or five or more, or 10 or more, or 25 or more, or 50 or more, or 100 or more, or 150 or more, or 200 or more, or 250 or more, or 300 or more, or 350 or more, or 384 sequences selected from SEQ ID NO: 1-333 and SEQ ID NO: 378-428; or (e) two or more, or five or more, or 10 or more, or 25 or more, or 44 sequences selected from -SEQ ID NOs: 334-377; or (f) two or more, or five or more, or 10 or more, or 25 or more, or 50 or more, or 51 sequences selected from -SEQ ID NO: 378-428; or their combinations. 33. Kit for removing non-target RNA molecules from a nucleic acid sample, containing a set of probes with 50 or more DNA probes complementary to non-contiguous sequences that are at a distance of 5 to 40 bases from each other along the entire length of at least at least one non-target RNA molecule in a nucleic acid sample, where each of the 50 or more DNA probes is 40 to 60 bases in length; (b) a ribonuclease capable of cleaving RNA in a DNA:RNA hybrid; (c) buffer; (d) a nucleic acid purification medium; and (e) a destabilizing chemical. 34. The kit of claim 33, wherein the non-target RNA is rRNA, mRNA, tRNA, or a mixture thereof. 35. The kit of claim 34 wherein the non-target RNA is rRNA and globin mRNA. 36. Set according to any one of paragraphs. 33-35, wherein the probe set comprises at least two DNA probes hybridizing 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. 37. The kit of claim 36, wherein the probe kit contains at least two DNA probes that hybridize to two or more non-target RNA molecules selected from human 28S, 18S, 5.8S, 5S, 16S, and 12S. 38. Set according to any one of paragraphs. 33-37, in which the set of probes contains at least two DNA probes that hybridize with one or more non-target RNA molecules selected from HBA-A1, HBA-A2, HBB, HBG1 and HBG2 hemoglobin and 23S, 16S and 5S Gram-positive or Gram-negative bacteria. 39. Set according to any one of paragraphs. 33-38, wherein the probe set contains at least two DNA probes complementary to one or more Archaea species rRNA molecules. 40. Set according to any one of paragraphs. 33-39, wherein the probe set comprises DNA probes that hybridize to one or more non-target rat and/or mouse RNA molecules, which can be selected from rat 16S, rat 28S, mouse 16S, mouse 28S and their combinations. 41. Set according to any one of paragraphs. 33-40, in which DNA probes contain: (a) two or more, or five or more, or 10 or more, or 25 or more, or 50 or more, or 100 or more, or 150 or more, or 200 or more, or 250 or more, or 300 or more, or 333 sequences selected from -SEQ ID NO: 1-333; or (b) two or more, or five or more, or 10 or more, or 25 or more, or 50 or more, or 100 or more, or 150 or more, or 200 or more, or 250 or more, or 300 or more, or 350 or more, or 400 or more, or 428 sequences selected from SEQ ID NO: 1-428; or (c) two or more, or five or more, or 10 or more, or 25 or more, or 50 or more, or 100 or more, or 150 or more, or 200 or more, or 250 or more, or 300 or more, or 350 or more, or 377 sequences selected from SEQ ID NO: 1-377; or (d) two or more, or five or more, or 10 or more, or 25 or more, or 50 or more, or 100 or more, or 150 or more, or 200 or more, or 250 or more, or 300 or more, or 350 or more, or 384 sequences selected from SEQ ID NOs: 1-333 and SEQ ID NOs: 378-428; or (e) two or more, or five or more, or 10 or more, or 25 or more, or 44 sequences selected from SEQ ID NO: 334-377; or (f) two or more, or five or more, or 10 or more, or 25 or more, or 50 or more, or 51 sequences selected from SEQ ID NO: 378-428; or their combinations. 42. The set according to claim 33, containing: (a) a set of probes containing SEQ ID NO: 1-333; (b) ribonuclease, which may be RNase H; (c) DNase; and (d) RNA purification beads; additional RNA depletion buffer, probe depletion buffer, and probe removal buffer may be present. 43. A method for replenishing a set of probes for use in removing non-target RNA nucleic acid molecules from a nucleic acid sample, comprising: (a) bringing a nucleic acid sample comprising at least one target RNA or DNA sequence and at least one non-target RNA molecule from the first species into contact with a probe set containing 50 or more complementary DNA probes to non-contiguous sequences along the entire length of at least one non-target RNA molecule sequence from the second species, thereby hybridizing 50 or more DNA probes with non-target RNA molecules to form DNA:RNA hybrids, while i) each of the 50 or more DNA probes is 40 to 60 bases in length, and ii) each DNA:RNA hybrid is separated from each other by a distance of 5 to 40 bases along a given sequence of a non-target RNA molecule from any other DNA:RNA hybrid; (b) contacting the DNA:RNA hybrids with a ribonuclease that cleaves RNA from the DNA:RNA hybrids, thereby destroying non-target RNA molecules in the nucleic acid sample to form a digested mixture; (c) separating the digested RNA from the digested mixture; (d) sequencing the remaining RNA from the sample; (e) evaluating the remaining RNA sequences for the presence of non-target RNA molecules of the first species, thereby determining gap sequence regions; and (f) replenishing the probe set with additional DNA probes complementary to non-contiguous sequences in one or more gap sequence regions. 44. The method of claim 43, wherein the gap regions in the sequence are 50 or more base pairs. 45. The method of claim 43 or 44, wherein the first species is non-human and the second is human. 46. The method of claim 45 wherein the first species is a rat or mouse. 47. The method according to p. 45 or 46, in which the composition according to any one of paragraphs. 21-33 are used to supply ribonuclease and the probe set contains DNA probes that are complementary to non-contiguous sequences along the entire length of at least one non-target human RNA molecule. 48. The method according to claim 46 or 47, which uses a method to identify DNA probes that hybridize with one or more rat and/or mouse non-target RNA molecules, which are optionally selected from rat 16S, rat 28S, 16S mice, 28S mice and their combinations. 49. The method according to any one of paragraphs. 43-48, where ribonuclease is RNase H.

Images