SINGLE POINT VARIANT DETECTION

Abstract

The invention relates to a genetic probe, wherein the genetic probe comprises: an oligonucleotide, or an oligonucleotide analogue, with a Raman-active moiety incorporated therein, wherein the Raman-active moiety is incorporated into a base of the oligonucleotide or oligonucleotide thereof; and associated methods, uses, kits and compositions for determining a single point variant nucleotide in a target nucleic acid.

Claims

1. A genetic probe, wherein the genetic probe comprises: an oligonucleotide, or an oligonucleotide analogue, with a Raman-active moiety incorporated therein, wherein the Raman-active moiety is incorporated into a base of the oligonucleotide or oligonucleotide thereof.

2. The genetic probe according to claim 1, wherein the genetic probe is suitable for determining the identity of a single targeted nucleotide in a target nucleic acid.

3. The genetic probe according to claim 1 or 2, wherein the Raman-active moiety comprises or consists of a moiety that resonates at a frequency within the cell-silent range of between 1800 and 2800 cm.sup.1.

4. The genetic probe according to any preceding claim, wherein the Raman-active moiety comprises or consists of one or more of the functional groups selected from a diyne group, an alkyne group, an azide group, a nitrile/cyano group, a metal-carbonyl complex, a carbon-13 label, and a deuterium group.

5. The genetic probe according to any preceding claim, wherein the Raman-active moiety comprises or consists of an alkyne group.

6. The genetic probe according to any preceding claim, wherein the Raman-active moiety is incorporated into a base to form a Raman-active molecule.

7. The genetic probe according to any preceding claim, comprising a Raman-active molecule in the oligonucleotide, wherein the Raman-active molecule comprises or consists of any one of Formulas II to IX, optionally wherein the alkyne group is substituted with an alternative Raman-active moiety.

8. The genetic probe according to any preceding claim, wherein the oligonucleotide comprises 10 or more nucleotides.

9. The genetic probe according to any preceding claim, wherein the oligonucleotide is a 20-mer strand.

10. The genetic probe according to any preceding claim, wherein the Raman-active moiety, or a Raman-active molecule comprising the Raman-active moiety, is located at a position that will be opposing a nucleotide to be interrogated when the oligonucleotide is hybridised/duplexed with the target nucleic acid.

11. The genetic probe according to any preceding claim, wherein the oligonucleotide comprises or consists of: the P21 oligonucleotide: 5-AGTCGCGXCTCAGCT-3, or a complementary sequence thereof; the BRAF V600E oligonucleotide: 5-AGATTTCXCTGTAGC-3, or a complementary sequence thereof; the KRAS oligonucleotide: 5-TACGCCAXCAGCTCC-3, or a complementary sequence thereof; wherein X is the site of a nucleotide or nucleotide analogue comprising the Raman-active moiety.

12. An array of genetic probes, wherein the array of genetic probes comprises two or more genetic probes according to any preceding claim provided in separate wells or on separate surfaces or spatially separated areas of the same surface.

13. A method of determining a single point variant nucleotide in a target nucleic acid in a pool of the target nucleic acid, the method comprising: providing a genetic probe in accordance with any one of claims 1-11, wherein the genetic probe is capable of detecting a single point variant nucleotide, wherein the genetic probe comprises an oligonucleotide that is substantially complimentary to the target nucleic acid, and wherein the Raman-active moiety of the genetic probe is in a base position that is arranged to be paired with a nucleotide of the target nucleic acid to be interrogated; contacting the genetic probe with the pool of target nucleic acid such that the genetic probe hybridises to the target nucleic acid; measuring the Raman frequency shift generated by the duplex formation by Raman spectroscopy.

14. The method according to claim 13, wherein the Raman frequency shift for the target nucleic acid is compared to a known reference standard, and/or a known control molecule.

15. The method according to claim 14, wherein the reference standard is based on nitrogen gas to provide an external calibration band.

16. The method according to any of claims 13-15, wherein the pool of target nucleic acid is in a sample comprising a cell, a cell lysate, a bodily fluid sample, or a nucleic acid sample.

17. The method according to any of claims 13-16, wherein the target nucleic acid is associated with a disease or condition or a known single nucleotide variant.

18. Use of a genetic probe in accordance with any one of claims 1-11, for determining the single nucleotide identity of target nucleic acid in a pool of the target nucleic acid.

19. Use of a genetic probe in accordance with any one of claims 1-11, for diagnosis and/or prognosis of a condition associated with a single point variant in a subject.

20. A kit for the detection of, and/or analysis of the ratio of, a single point variant of a target nucleic acid in a pool of the target nucleic acid, wherein the kit comprises: the genetic probe according to any one of claims 1-11, wherein the Raman-active moiety is in a position that is arranged to be paired with a nucleotide of the target nucleic acid to be interrogated; and an internal reference standard to determine the probe concentration wherein the internal reference standard comprises a molecule of known concentration in the liquid.

21. A composition comprising a plurality of genetic probes according to any one of claims 1-11.

22. A method of determining the status of a condition associated with a known single point variant in a subject, the method comprising: providing a sample from the subject comprising a target nucleic acid, wherein the target nucleic acid may comprise the single point variant; determining the presence or percentage of the single point variant in the sample relative to target nucleic acid not having the single point variant in accordance with the method of any of claims 12-17, wherein the presence or percentage of the single point variant is indicative of the status of the condition associated with the single point variant in the subject.

23. Use of N.sub.2 gas to provide an external calibration band in a Raman spectroscopy assay.

24. A method of Raman spectroscopy, wherein a Raman spectroscopy assay is carried out on a sample together with the use of N.sub.2 gas to provide an external calibration band, optionally wherein the N.sub.2 gas is ambient N.sub.2 gas in air.

25. A method of detecting a target nucleic acid in a pool of nucleic acid, the method comprising: providing a genetic probe in accordance with any one of claims 1-11, wherein the genetic probe is capable of detecting the target nucleic acid, wherein the genetic probe comprises an oligonucleotide that is substantially complimentary to the target nucleic acid, and wherein the Raman-active moiety of the genetic probe is in a base position that is arranged to be paired with a nucleotide of the target nucleic acid to be interrogated; contacting the genetic probe with the pool of nucleic acid such that the genetic probe hybridises to the target nucleic acid; measuring the Raman frequency shift generated by the duplex formation by Raman spectroscopy.

26. Use of a genetic probe in accordance with any one of claims 1-11, for detection of a target nucleic acid, such as microRNA, in a sample.

27. Use of a genetic probe in accordance with any one of claims 1-11, for diagnosis and/or prognosis of concussion in a subject that is associated with the presence of small non-coding RNA as described herein, for example in the saliva of the subject.

28. A kit for the detection of, and/or analysis of target nucleic acid in a sample, wherein the kit comprises: the genetic probe in accordance with any one of claims 1-11, wherein the Raman-active moiety is in a position that is arranged to be paired with a nucleotide of the target nucleic acid to be interrogated; and optionally an internal reference standard to determine the probe concentration wherein the internal reference standard comprises a molecule of known concentration in the liquid.

29. A method of determining the status of a condition associated with a target nucleic acid in a subject, the method comprising: providing a sample from the subject comprising, or potentially comprising, a target nucleic acid, wherein the target nucleic acid; determining the presence or level of the target nucleic acid in accordance with the method of any preceding claim, wherein the presence or level of the target nucleic acid is indicative of the status of the condition associated with the target nucleic acid in the subject.

30. A nucleic acid duplex, the nucleic acid duplex comprising the genetic probe in accordance with any one of claims 1-11, duplexed with a target nucleic acid, wherein the Raman-active moiety incorporated within the genetic probe is base paired with a base of the target nucleic acid.

Description

[0147] FIG. 1: Structure of nucleobase complexes (left) and simulated Raman spectra of ethynyl-labelled nucleobase molecules and their complexes in the region of CC vibration (right). (A, G, U and C denote adenine, guanine, uracil and cytosine molecules, respectively; Elethynyl-labelled molecules. Calculated (B3LYP/6-311++G(df,pd)) Raman spectra of complexes were scaled by 0.9578. Lorenz-type function with full width at half maximum (FWHM) of 3 cm.sup.1 was used in order to simulate the spectra.

[0148] FIG. 1B: Structure of ethynyl uracil and nucleobase complexes (left) and simulated Raman spectra of different complexes in the region of CC vibration (right). (A, G, U, mU and C denote adenine, guanine, uracil, methyl-uracil and cytosine molecules, respectively; Elethynyl-labelled molecule. Calculated (B3LYP/6-311++G(d,p)) Raman spectra of complexes were scaled by 0.95845. Lorenz-type function with full width at half maximum (FWHM) of 1 cm.sup.1 was used in order to simulate the spectra.

[0149] FIG. 2: (A) Baseline corrected and curve fitted Raman spectra of DNA sequences in the region of alkyne and N2 stretching vibrations, (B) variatmoions of alkyne Raman mode frequency in the Raman spectra of different samples and (C) the Raman mode of N2 gas used as external calibration band (X denotes EdU molecule).

[0150] FIG. 3:(A) Baseline corrected and curve fitted Raman spectra of BRAF DNA sequences in the region of alkyne and N2 stretching vibrations, (B) variations of alkyne Raman mode frequency in the Raman spectra of different samples and (C) the Raman mode of N2 gas used as external calibration band (X denotes EdU molecule).

EXAMPLES

[0151] Computer simulations were performed to calculate the Raman shift of the Watson-Crick base pairing compared with free Raman active molecules. The calculation was performed on the Hydrogen bonding of the nitrogenous part of the matching Watson-Crick base pairing molecules. The data (FIG. 1) suggested that the purine pairing C:G and G:C complexes were the most promising, demonstrating the larger frequency shifts (+6.5 and 4.5 cm.sup.1, respectively). The pyrimidine pairing El-U:A and A:U combination gave a predicted frequency shift of (1.7 and 0.5 cm.sup.1). Due to the commercial availability of 5-Ethynyl-dU-CE Phosphoramidite, the El-U:A combination was chosen for our study. In this case the predicted frequency shift is small (1.7 cm.sup.1) but it can still be resolved in the high resolution experimental Raman spectra.

[0152] Whilst the computed C:G and G:C complexes are most promising with larger frequency shifts (+6.5 and 4.5 cm.sup.1, respectively) between alkyne Raman modes of base pairs the clinical relevance of detecting T to A mutations meant the detection of A (by EI-U) was prioritized. In this case, the predicted frequency shift is small (1.7 cm.sup.1) but it still can be resolved in the high-resolution experimental Raman spectra.

[0153] Inclusion of commercially available 5-ethynyl-dU-CE phosphoramidite in a probe sequence builds in the alkyne for Raman and is the natural base compliment for base A.

##STR00005##

[0154] The material was purchased from Glen Research and used directly as instructed by the manufacturer on a solid support DNA synthesiser to make 15-mer strands. The probe and target strands were all made with the modification in the middle as highlighted in table 1. The probe strand had CXC where X is EdU and the target strands would be GXG, with X here being the four nucleobases (A, T, C, G). This would enable the measurement of the match and mis-match of all combinations and maybe reveal the identity of the base opposite the alkyne probe.

[0155] The reason for making a 15-mer strand is that, it offers the best chance to see a difference between match and single mis-match upon hybridisation of the probe and target. This is because there is a thermodynamic/detectable difference upon hybridisation between a match and mis-match strand with the probe if the modification is in the middle of a 15-mer strand. The test strand chosen has been studied by Tucker and co-workers in development of alternate signalling modalities for the proof of principle (POP) testing.

[0156] Sequences Made

TABLE-US-00001 TABLE1 Oligostrandssynthesisedonautomated DNAsynthesiser. Oligonucleotide Strands names sequences S0 Naturalprobe 5-TGGACTCTCTCAATG-3 5-CTC S1 Probe5-CXC 5-TGGACTCXCTCAATG-3 S2 Target3-GAG 3-ACCTGAGAGAGTTAC-5 S3 Target3-GTG 3-ACCTGAGTGAGTTAC-5 S4 Target3-GCG 3-ACCTGAGCGAGTTAC-5 S5 Target3-GGG 3-ACCTGAGGGAGTTAC-5 S6 Probe5-CXC 5-AGATTTCXCTGTAGC-3 (BRAF) S7 Target3-GAG 3-TCTAAAGAGACATCG-5 (cancerous) S8 Target3-GTG 3-TCTAAAGTGACATCG-5 (wildtype)

[0157] Sequence Synthetic Procedure:

[0158] Synthesis (Ultramild Reagents):

[0159] All nucleobases, reagents and solvents were purchased and used from suppliers without any further purification. Oligonucleotides were synthesised on an Applied Biosystems ABI 394 (Foster City, CA, 30 U.S.A). Standard phosphoramidites of Pac-dA, iPr-Pac-dG, Ac-dC, dT were purchased from LGC Genomics. 5-Ethynyl-dU-CE Phosphoramidite was purchase from Glen Research and used directly. The phosphoramidites were dissolved in anhydrous acetonitrile to a concentration of 0.1 M. Strands were synthesised at a 1 mol scale on SynBase CPG 1000/110 solid supports from LGC Genomics.

[0160] The resins with the completed strands were placed in 1 ml solutions of potassium carbonate (0.05 M) in methanol and left overnight, before neutralisation with acetic acid (6 l). Solvent was removed on a Thermo Scientific speed vac. The dried powders were re-dissolved in 1 ml ultrapure water and passed through a NAP-10 desalting column from GE Healthcare to remove any residual resin and potassium carbonate. The collected solutions were stored in the freezer before purification.

[0161] Purification and Characterisation:

[0162] Semi preparative HPLC purification was performed on an Agilent Technologies 1260 Infinity system using a Phenomenex Clarity 5 m Oligo-RP LC 25010 mm column. Collected fractions were evaporated to dryness, redissolved in Milli-Q water (1 ml) and desalted using a NAP-10 column (GE Healthcare), whilst being eluted from the column to 1.5 ml. Purity was determined by analytical HPLC using a Phenomenex Clarity 5 m Oligo RP LC 2504.6 mm column on an Agilent Technologies 1260 Infinity system. The UV/vis absorbance was monitored at 260 nm.

[0163] A solvent gradient system of HPLC grade acetonitrile (Fisher Scientific) and 0.1 M triethylamine acetate (TEAA) in HPLC grade water (Fisher Scientific) was employed for the purification of the oligonucleotides.

[0164] Pure oligonucleotides were characterised by negative mode electrospray mass spectrometry on a Waters Xevo G2-XS mass spectrometer. Sample concentrations were determined by optical density at 260 nm using a BioSpecNano micro-volume UV-Vis spectrophotometer from Shimadzu and the Beer Lambert law, with extinction coefficients obtained from Integrated DNA Technologies' OligoAnalyzer.

[0165] TM Result of Match and Mis-Match (POP Probe)

[0166] Having obtained the sequences, the stability/destabilisation effect of the modification upon duplex formation was determined by measuring the thermal melting (Tm) temperature. The result indicated that the modification did not seem to affect the stability of the strands upon hybridisation (comparing entries 1 and 3 in table 2). Although there was a slightly bigger destabilizing effect in the mis-match strand (comparing entries 2 and 4 in table 2). The numerical value was an approximately 10 C. difference between the match and mis-match in both the probe with modification and ones without. Attention was turned to a biologically relevant sequence that has also been prepared. The BRAF 600E is a single point variation for prostate cancer (T is wild-type. A denotes the cancerous mutant). Therefore, the ability to rapidly detect this SNIPs would be highly desired. The result in entry 5 and 6 indicate that there is a difference in the wild type (mis-match) and the cancerous (match) of around 10 C. This is in line with DNA stability of 15 mer strands observed previously. This indicates that the alkyne probe does not destabilise the parent structure and adds value to it being a non-intrusive probe.

TABLE-US-00002 TABLE2 entry Duplexformations Tmvalues 1 Naturalstrand-S0&S2 Match= (Amatch) 64.0C. 5-TGGACTCTCTCAATG-3 3-ACCTGAGAGAGTTAC-5 2 Naturalstrand-S0&S3 Mis-match= (Tmis-match) 54.5C. 5-TGGACTCTCTCAATG-3 3-ACCTGAGTGAGTTAC-5 3 Modifiedstrand-S1&S2 Match= (Amatch) 64.7C. 5-TGGACTCXCTCAATG-3 3-ACCTGAGAGAGTTAC-5 4 Modifiedstrand-S1&S3 Mis-match= (Tmis-match) 53.7C. 5-TGGACTCXCTCAATG-3 3-ACCTGAGTGAGTTAC-5 5 Modifiedstrand-S6&S7 Match= (cancerous) 62.3C. 5-AGATTTCXCTGTAGC-3 3-TCTAAAGAGACATCG-5 6 Modifiedstrand-S6&S8 Mis-match= (wildtype) 52.5C. 5-AGATTTCXCTGTAGC-3 3-TCTAAAGTGACATCG-5

[0167] Raman Spectroscopy

[0168] The accuracy required to reliably detect small changes in signal of the alkyne between free chain, chain bond to wild-type (healthy modelone mismatch) and mutated-type (disease modelno mismatches) required the development of a new spectroscopy technique.

[0169] Herein the first use of the Raman signal of ambient nitrogen gas in the air as a reference standard to achieve high resolution is disclosed.

[0170] Modelling has been performed to predict the EdU alkyne Raman peak shifts during perfectly matching (El-U:A) and mis-match (El-U:mU.sub.1, El-U:mU.sub.2, El-U:C, El-U:G) hybridizations. The modelling prediction (FIG. 1B.) was supported when Raman measurements were conducted (see table 3). The single strand probe (X=EdU) gave stretch at 2118.4 cm.sup.1. Upon hybridisation with the perfectly matching strand (EdU with A), there was a small shift to 2119.2 cm.sup.1. The result of the other strands containing one base mis-match (EdU with T, C or G) gave larger shifts (table 3). The mis-match of EdU with T gave the largest shift from 2118.4 cm.sup.1 to 2120.5 cm.sup.1. The extent of the shift is different for each mis-match base, suggesting that the identity of the mis-match base opposite the modification could be identified. While the value of the shift does not match the predicted value, the direction of the shifts is in good agreement with the calculated/predicted ones (except El-U:C) FIG. 1B. However, such calculations, and especially the geometry optimisation therein always have some limitations in case of systems consisting of a high number of atoms, whilst the experiments record the Raman response of the whole system. Therefore, the differences between predicted and measured should not be over interpreted. The fundamental result is that Raman Spectroscopy can be used to detect nucleoside hybridisation and single base mis-match.

TABLE-US-00003 TABLE3 TheshiftinRamansignalfrommatchedand mis-matchedstrands. V Av Entries DNAstrands (cm.sup.1) (cm.sup.1) 1 Singlestrand-S1 2118.4 0.8 5-TGGACTCXCTCAATG-3 2 Duplexstrand-S1&S2 2119.2 0.0 (Amatch) (ref.) 5-TGGACTCXCTCAATG-3 3-ACCTGAGAGAGTTAC-5 3 Duplexstrand-S1&S3 2120.5 +1.3 (Tmis-match) 5-TGGACTCXCTCAATG-3 3-ACCTGAGTGAGTTAC-5 4 Duplexstrand-S1&S4 2119.4 +0.2 (Cmis-match) 5-TGGACTCXCTCAATG-3 3-ACCTGAGCGAGTTAC-5 5 Duplexstrand-S1&S5 2119.9 +0.7 (Gmis-match) 5-TGGACTCXCTCAATG-3 3-ACCTGAGGGAGTTAC-5

[0171] (BRAF)

[0172] Attention was turned to a biologically relevant BRAF 600E sequence that has also been prepared. The single strand probe (S6, entry 1, table 4) has a peak at 2118.7 cm.sup.1; upon hybridisation with the matching strand (S7, A, cancerous mutant, entry 2, table 4), there was a small shift to 2119.4 cm.sup.1. With the mis-matching strand (T, wildtype, entry 3, table 4) the shift was larger, moving to 2120.3 cm.sup.1. The result here indicated that potentially Raman spectroscopy could be used to detect the BRAF 600E mutation SNIPs.

TABLE-US-00004 TABLE4 RamanshiftofBRAFsequencewithmatchedand mis-matchedstrands. v v Entries BRAF600Emarker (cm.sup.-1) (cm.sup.-1) 1 Singlestrand-S6 2118.7 -0.7 5-AGATTTCXCTGTAGC-3 2 Duplexstrand-S6& 2119.4 0.0 S7(Amatch) (ref.) 5-AGATTTCXCTGTAGC-3 3-TCTAAAGAGACATCG-5 3 Duplexstrand-S6& 2120.3 +0.9 S8(Tmis-match) 5-AGATTTCXCTGTAGC-3 3-TCTAAAGTGACATCG-5

[0173] Concussion Associated RNAs:

TABLE-US-00005 microRNAs Gene Number hsa-let-7f-5p MIMAT0000067 hsa-miR-1246 MIMAT0005898 hsa-miR-135b-5p MIMAT0000758 hsa-miR-21-5p MIMAT0000076 hsa-miR-425-5p MIMAT0003393 hsa-miR-497-5p MIMAT0002820 hsa-miR-148a-3p MIMAT0000243 hsa-let-7a-5p MIMAT0000062 hsa-let-7i-5p MIMAT0000415 hsa-miR-143-3p MIMAT0000435 hsa-miR-34b-3p MIMAT0004676 hsa-miR-144-3p MIMAT0000436 hsa-miR-16-1-3p MIMAT0004489 hsa-miR-103a-3p MIMAT0000101 hsa-miR-92a-3p MIMAT0000092 hsa-let-7b-5p MIMAT0000063 hsa-miR-142-5p MIMAT0000433 hsa-miR-29c-3p MIMAT0000433 has-miR-339-5p MIMAT0000764 has-miR-107 MIMAT0000104 hsa-miR-126-3p MIMAT0000445 hsa-miR-1271-5p MIMAT0005796 hsa-miR-143-3p MIMAT0000435

TABLE-US-00006 small non coding RNAS Chr Strand Start Stop Sequence RNU4-6p X 16893269 16893390 TATCGTAGCCA ATGAGGTTTAT CCGAGGCGTGA TTATTGCTAAT TGAAAA tRNA18 17 + 73030001 73030073 GCACTGGCCTC ArgCCT CTAAGCCAGGG ATTGTGGGTTC GAGTCCCACCT GGGGTA U6.428 1 + 180727858 180727953 AAGATTAGCAT GAGGATGACAC GCAAATTCGTG AAGCGTTCCAT TTCTTT RNU6-45 11 + 63737942 63738048 GGCCCTTGTGC AAGGATGACAC GCAAATTCGTG AAGCGTTCCAT ATTTTT RNU6-4 1 31970419 31970525 GGCCCCTGCAC AGGGATGACAC GCAAATTCGTG AAGCGTTCCAT ATTTTT RNU6-6 2 + 201694732 201694839 GGCCCCTGTGC AAGGATGACAC GCAAATTCGTG AAGCGTTCCAT ATTTTT RNU6-7 3 + 194935516 194935622 GGCCCCTGCGC AAGGATGACAT GCAAATTCGTG AAGCGTTCCAT ATTTTT RNU6-73 13 + 28402900 28403006 GGCCCCTGTGC AAGGATGACAT GCAAATTCGTG AAGCGTTCCAT ATTTTT SNORD3B 17 + 18965225 18965440 CTTCTCTCCGT TATTGGGGAGT GAGAGGGAGAG AACGCGGTCTG AGTGGT tRNA120- 6 28626014 28625085 GCGCATGCTTA AlaAGC GCATGCATGAG GTCCCGGGTTC GATCCCCAGCA TCTCCA tRNA73- 6 + 28849165 28849237 GCGTCTGATTC ArgCCG CGGATCAGAAG ATTGAGGGTTC GAGTCCCTTCG TGGTCG U6.168 6 18307204 18307310 ATGGCCCCTGC GCAAGGATGAC ACGCAAATTTG TGAAGGATTCC ATATTT U6.375 4 + 109573306 109573412 GGCCCCTGTGC AAGAATGACTC GCAAATTCGTG AAGCGTTCCAT ATTTTT YRNA-684 18 + 20604559 20604666 GCTTCTTTTAC TCTTTCCCTTC ATTCTCACTAC TGTACCTGATT CGTCTT U6.601 19 39287642 39287749 GGCCCCTGCGC AAGGATGACAT GCAAATTTGTG AAGTGTTCCAT ATTTTT YRNA-255 17 + 80375102 80375197 GUGUCACCAAC GUUGGUAUACA ACCCCCCACAA CUAAAUUUGAC UGGCUU tRNA9- 7 + 149255133 149255205 CTTTTTGACTG TyrGTA TAGAGCAAGAG GTCCCTGGTTC AAATCCAGGTT CTCCCT U2.3 1 + 150209315 150209504 TCACTTCACGC ATCGATCTGGT ATTGCAGTACC TCCAGGAACAG TGCACC U4.64 9 + 36267780 36267919 GTATCGTAGCC AATGAGGTTTA TCCAAGGTGCG ATTATTGCTAA TTGAAA SNORA57 10 + 27077946 27078086 TGCTGGCGGCT TCCCATCCGCT GGTTCTATCCT CAAACGCCGGG ACACCG UC022CJG1 Y + 10037846 10037870 CATTGATCATC GACACTTCGAA CGCACTTG tRNA27- 6 + 26766444 26765516 GCGTCAGTCTC MetCAT ATAATCTGAAG GTCCTGAGTTC GAGCCTCAGAG AGGGCA tRNA8- 17 + 8090478 8090551 GCGCCTGTCTA ThrAGT GTAAACAGGAG ATCCTGGGTTC GAATCCCAGCG GTGCCT tRNA2- 4 156384978 156385052 GCATAAAACTT LeuTAA AAAATTTTATA ATCAGAGGTTC AACTCCTCTTC TTAACA YRNA-245 2 25919945 25920057 GTCTTTGTTGA ACTCTTTCCCT CCTTCTCATTA CTGTACTTGAC CAGTCT snoU13.120 4 + 17530560 17530663 GCTACCCTGGA ACCTTGTTATG ACATCTGCACA TTACCCATCTG ACCTGA U6.1249 1 + 67661823 67661926 GATGGCATGAC CCCTGATCAAG GACGGCATGCA AATTTGTGAAG TATTTC tRNA84- 1 1.61E+08 1.61E+08 TTTCACCGCCG GluTCC CGGCCCGGGTT CGATTCCCGGT CAGGGAA tRNA8- 12 + 1.25E+08 1.25E+08 TGCACGTATGA AlaTGC GGCCCCGGGTT CAATCCCCGGC ATCTCCA