An oligonucleotide-based strategy for capture, detection, adaptation, and sequencing of trna using nanopore technology

Abstract

Disclosed are methods of preparing a sequencing library to quantify tRNA abundance and tRNA modifications in an RNA sample that comprises contacting RNA with oligonucleotides in the presence of a ligating agent and performing nanopore direct sequencing. It also discloses a kit to perform said method.

Description

[0001] AN OLIGONUCLEOTIDE-BASED STRATEGY FOR CAPTURE, DETECTION, ADAPTATION, AND SEQUENCING OF TRNA USING NANOPORE TECHNOLOGY

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit of priority to U.S. Provisional Patent Application serial number 63 / 546,632, filed October 31, 2023.

[0004] GOVERNMENT SUPPORT

[0005] This invention was made with government support under Grant No. 5R01HG010053-02 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] BACKGROUND

[0007] Transfer RNAs (tRNAs) are small, non-coding RNA molecules that play an essential role in protein translation. They are highly modified, and these modifications are crucial for their stability and function. Specifically, modifications act as identity markers that enable accurate aminoacylation, ensuring the fidelity of the genetic code. Notably, tRNA modifications are reversible and can change dynamically in response to environmental conditions, cell cycle stages, and tumor development. In addition to modifications, the abundance of tRNAs is also altered by environmental factors and is associated with various human diseases. Thus, accurately quantifying tRNA levels and monitoring modification dynamics are essential for understanding their regulatory roles and their implications in disease. However, current methods for tRNA sequencing have limitations, highlighting the need for new, advanced techniques to sequence tRNAs effectively.

[0008] SUMMARY

[0009] Disclosed are methods of preparing a sequencing library. The method may comprise a) contacting a sample comprising tRNA and a RNA ligase, with i) a population of 3' splinted oligonucleotides, each 3' splinted oligonucleotide comprising in 5' to 3' order: 1) a first hybridization region, 2) a first extended oligonucleotide region, and 3) an adapter DNA oligonucleotide hybridization region, and ii) a population of 5' splinted oligonucleotides, each 5' splinted oligonucleotide comprising in 5' to 3' order: 1) a second extended oligonucleotide region, 2) a second hybridization region complementary to the first hybridization region of the 3' splinted oligonucleotide, and 3) a tRNA hybridization region. At the end of step a), the 5' splinted oligonucleotide is adjacent to the 5' end of the tRNA and annealed to both the 3' end of the tRNA by the tRNA hybridization region and to the 3' splinted oligonucleotide by the second hybridization region thereby forming a first product. The method may further comprise b) contacting the first product with a DNA ligase with: i) a first adapter DNA oligonucleotide comprising a third hybridization region, and ii) a second adapter DNA oligonucleotide comprising in 5' to 3' order: 1) a fourth hybridization region complementary to the third hybridization region of the first adapter DNA oligonucleotide, and 2) a DNA hybridization region complementary to the adapter DNA oligonucleotide hybridization region of the 3' splinted oligonucleotide. At the end of step b), the first adapter DNA oligonucleotide is adjacent to the 3' end of the terminal region of the 3' splinted oligonucleotide, and the second adapter DNA oligonucleotide is annealed to both the 3' splinted oligonucleotide and the first adapter DNA oligonucleotide thereby forming a second product.

[0010] In some embodiments, the method further comprising c) performing reverse transcription to linearize the second product thereby forming a third product. In some embodiments, the method further comprising d) carrying out nanopore direct sequencing to obtain the abundance of tRNA and its modifications in the sample.

[0011] In some embodiments, the RNA ligase is T4 RNA ligase 2. In some embodiments, the DNA ligase is T4 DNA ligase. In some embodiments, the first adapter DNA oligonucleotide and the second adapter DNA oligonucleotide are a motor associated sequencing adapter (such as an Oxford Nanopore Technologies (ONT)-DNA RT Adapter (RTA) motor associated sequencing adapter).

[0012] In some embodiments, the 5' splinted oligonucleotide is 30 to 200 nucleotides in size. In some embodiments, the 5' splinted oligonucleotide is 30 to 50 nucleotides in size, 50 to 100 nucleotides in size, 100 to 150 nucleotides in size, or 150 to 200 nucleotides in size. In some embodiments, the 5' splinted oligonucleotide is RNA. In some embodiments, the second hybridization region is 15 to 50 nucleotides in size. In some embodiments, the second hybridization region is 15 to 20 nucleotides in size, 20 to 30 nucleotides in size, 30 to 40 nucleotides in size, or 40 to 50 nucleotides in size.

[0013] In some embodiments, the tRNA hybridization region comprises the nucleotide sequence of NGGU, and N is A, C, G, or U. In some embodiments, the first hybridization region and the first extended oligonucleotide region of the 3' splinted oligonucleotide is 10 to 200 nucleotides in size. In some embodiments, the first hybridization region and the first extended oligonucleotide region of the 3' splinted oligonucleotide is 10 to 50 nucleotides in size, 50 to 100 nucleotides in size, 100 to 150 nucleotides in size, or 150 to 200 nucleotides in size. In some embodiments, the first hybridization region and the first extended oligonucleotide region of the 3' splinted oligonucleotide is RNA. In some embodiments, the first hybridization region is 15 to 50 nucleotides in size. In some embodiments, the first hybridization region is 15 to 20 nucleotides in size, 20 to 30 nucleotides in size, 30 to 40 nucleotides in size, or 40 to 50 nucleotides in size. In some embodiments, the adapter DNA oligonucleotide hybridization region is 15 to 50 nucleotides in size. In some embodiments, the adapter DNA oligonucleotide hybridization region is 15 to 20 nucleotides in size, 20 to 30 nucleotides in size, 30 to 40 nucleotides in size, or 40 to 50 nucleotides in size.

[0014] In some embodiments, the 3' splinted oligonucleotide is 5' phosphorylated. In some embodiments, the 3' splinted oligonucleotide comprises the nucleotide sequence of SEQ ID NO: 1, 5, or 7. In some embodiments, the 5' splinted oligonucleotide comprises the nucleotide sequence of SEQ ID NO: 3, 4, or 6, and N is A, C, G, or U. In some embodiments, the 3' splinted oligonucleotide and the 5' splinted oligonucleotide are annealed prior to step a). In some embodiments, the first adapter DNA oligonucleotide binds to a helicase motor.

[0015] In some embodiments, the nanopore direct sequencing comprises: loading the second product or the third product to a flow cell which comprises a membrane in which is present a nanopore that provides a channel through the membrane, coupled to a current intensity, wherein the second product or the third passes through the nanopore, causes disruptions in the current intensity, and analyzing said sequences to obtain the abundance of tRNA and its modifications in the sample. In some embodiments, the duration of step a) is between 1 hr and 3hr at a temperature comprised between 20°C and 26°C. In some embodiments, the method sequenced tRNA, identifies tRNA associated disorders, or identifies tRNA modifications.

[0016] In another aspect, disclosed are kits comprising: i) a population of 3' splinted oligonucleotides, each 3' splinted oligonucleotide comprising in 5' to 3' order: 1) a first hybridization region, 2) a first extended oligonucleotide region, and 3) an adapter DNA oligonucleotide hybridization region, and ii) a population of 5' splinted oligonucleotides, each 5' splinted oligonucleotide comprising in 5' to 3' order: 1) a second extended oligonucleotide region, 2) a second hybridization region complementary to the first hybridization region of the 3' splinted oligonucleotide, and 3) a tRNA hybridization region.

[0017] In some embodiments, the kit further comprises i) a first adapter DNA oligonucleotide comprising a third hybridization region, and ii) a second adapter DNA oligonucleotide comprising in 5' to 3' order: 1) a fourth hybridization region complementary to the third hybridization region of the first adapter DNA oligonucleotide, and 2) a DNA hybridization region complementary to the adapter DNA oligonucleotide hybridization region of the 3' splinted oligonucleotide.

[0018] In some embodiments, the first adapter DNA oligonucleotide and the second adapter DNA oligonucleotide are a motor associated sequencing adapter (such as an Oxford Nanopore Technologies (ONT)-DNA RT Adapter (RTA) motor associated sequencing adapter).

[0019] In some embodiments, the 5' splinted oligonucleotide is 30 to 200 nucleotides in size. In some embodiments, the 5' splinted oligonucleotide is 30 to 50 nucleotides in size, 50 to 100 nucleotides in size, 100 to 150 nucleotides in size, or 150 to 200 nucleotides in size. In some embodiments, the 5' splinted oligonucleotide is RNA. In some embodiments, the second hybridization region is 15 to 50 nucleotides in size. In some embodiments, the second hybridization region is 15 to 20 nucleotides in size, 20 to 30 nucleotides in size, 30 to 40 nucleotides in size, or 40 to 50 nucleotides in size. In some embodiments, the tRNA hybridization region comprises the nucleotide sequence of NGGU, and N is A, C, G, or U. In some embodiments, the first hybridization region and the first extended oligonucleotide region of the 3' splinted oligonucleotide is 10 to 200 nucleotides in size. In some embodiments, the first hybridization region and the first extended oligonucleotide region of the 3' splinted oligonucleotide is 10 to 50 nucleotides in size, 50 to 100 nucleotides in size, 100 to 150 nucleotides in size, or 150 to 200 nucleotides in size. In some embodiments, the first hybridization region and the first extended oligonucleotide region of the 3' splinted oligonucleotide is RNA. In some embodiments, the first hybridization region is 15 to 50 nucleotides in size. In some embodiments, the first hybridization region is 15 to 20 nucleotides in size, 20 to 30 nucleotides in size, 30 to 40 nucleotides in size, or 40 to 50 nucleotides in size. In some embodiments, the adapter DNA oligonucleotide hybridization region is 15 to 50 nucleotides in size. In some embodiments, the adapter DNA oligonucleotide hybridization region is 15 to 20 nucleotides in size, 20 to 30 nucleotides in size, 30 to 40 nucleotides in size, or 40 to 50 nucleotides in size. In some embodiments, the 3' splinted oligonucleotide is 5' phosphorylated. In some embodiments, the 3' splinted oligonucleotide comprises the nucleotide sequence of SEQ ID NO: 1, 5, or 7. In some embodiments, the 5' splinted oligonucleotide comprises the nucleotide sequence of SEQ ID NO: 3, 4, or 6, and N is A, C, G, or U. In some embodiments, the 3' splinted oligonucleotide and the 5' splinted oligonucleotide are annealed prior to step a). In some embodiments, the first adapter DNA oligonucleotide binds to a helicase motor.

[0020] BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Fig- 1 shows a diagram of the components used for tRNA nanopore sequencing reaction (not to scale). * Splint adapter may also be designed with short oligonucleotides that allow ligation to the Oxford Nanopore Technologies (ONT)-DNA RT Adapter (RTA) adapter and used with a reverse transcriptase step (Lucas M.C., et al. Nat Biotechnol. 2023 Apr 6;42(l):72-86), but the overall design is generally the same.

[0022] Fig- 2 shows an overview of ligation scheme to make the sequencing ready product.

[0023] Fig- 3 shows the difference between the older design and the disclosed design using a long adapter for tRNA nanopore sequencing reaction.

[0024] Fig. 4A-4C show that longer adapters facilitate successful ionic current alignments with Nanopolish. Fig. 4A shows an illustration of the adaptation method from Fig. 1 using longer splints. It shows the 5' adapter, the 3' adapter in yellow, and the ONT adapters. Fig. 4B shows ionic current trace from a long adapted tRNA. Fig. 4C shows Nanopolish generated segmentation of the ionic current from Fig. 4B. Region between each vertical dashed line represents an ionic current segment associated with a 5mer assigned by Nanopolish. The mean current (current; pA), standard deviation (current; pA), and dwell time (seconds; s) can be calculated for machine learning.

[0025] DETAILED DESCRIPTION tRNA is an important genetic molecule composed of standard and modified nucleotides. Nanopore tRNA sequencing requires adding additional nucleotides (adaptors) to each tRNA molecule, and assessing modifications has only been done using inference-based methods. In order to directly detect and characterize tRNA modifications in Nanopore data, one must be able to analyze the ionic current (raw nucleotide measurements). In the past, adaptors were made of both deoxyribonucleotide and ribonucleotide subunits and were relatively short (20-30 nucleotides long). While this short adaptor design allowed for successful tRNA sequencing, it still prohibited direct tRNA modification detection because ionic current based modification analysis requires a minimum data input. In this rendition, our adaptor designs contain mostly ribonucleotide components and are much longer (60-120 nucleotides long) which permits the use of modification detection software.

[0026] This disclosure provides 120 ribonucleotide long 5 prime adaptor strand and 46 ribonucleotide and 14 deoxyribonucleotide hybrid 3 prime adaptor strand. The length of the strands permits addition of unique strand markers for RNA multiplexing. The length of the strands permits ionic current to ionic current alignments and permits direct modification detection and characterization. The length of the strands may permit higher quality sequence alignments.

[0027] This disclosure provides medicals diagnostics of tRNA associated disorders, discovery of tRNA modifications, characterization of tRNA modifications, understanding regulation of tRNA modifications, determining the abundance of tRNA modifications, and functionality of tRNA modifications. This allows direct RNA sequencing and ionic current analysis.

[0028] Disclosed are methods of preparing a sequencing library. The method may comprise a) contacting a sample comprising tRNA and a RNA ligase, with i) a population of 3' splinted oligonucleotides, each 3' splinted oligonucleotide comprising in 5' to 3' order: 1) a first hybridization region, 2) a first extended oligonucleotide region, and 3) an adapter DNA oligonucleotide hybridization region, and ii) a population of 5' splinted oligonucleotides, each 5' splinted oligonucleotide comprising in 5' to 3' order: 1) a second extended oligonucleotide region, 2) a second hybridization region complementary to the first hybridization region of the 3' splinted oligonucleotide, and 3) a tRNA hybridization region. At the end of step a), the 5' splinted oligonucleotide is adjacent to the 5' end of the tRNA and annealed to both the 3' end of the tRNA by the tRNA hybridization region and to the 3' splinted oligonucleotide by the second hybridization region thereby forming a first product. The method may further comprise b) contacting the first product with a DNA ligase with: i) a first adapter DNA oligonucleotide comprising a third hybridization region, and ii) a second adapter DNA oligonucleotide comprising in 5' to 3' order: 1) a fourth hybridization region complementary to the third hybridization region of the first adapter DNA oligonucleotide, and 2) a DNA hybridization region complementary to the adapter DNA oligonucleotide hybridization region of the 3' splinted oligonucleotide. At the end of step b), the first adapter DNA oligonucleotide is adjacent to the 3' end of the terminal region of the 3' splinted oligonucleotide, and the second adapter DNA oligonucleotide is annealed to both the 3' splinted oligonucleotide and the first adapter DNA oligonucleotide thereby forming a second product.

[0029] Definitions

[0030] Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclature used in connection with, and techniques of, chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics and protein and nucleic acid chemistry, described herein, are those well-known and commonly used in the art.

[0031] The methods and techniques of the present disclosure are generally performed, unless otherwise indicated, according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout this specification. See, e.g. “Principles of Neural Science”, McGraw-Hill Medical, New York, N.Y. (2000); Motulsky, “Intuitive Biostatistics”, Oxford University Press, Inc. (1995); Lodish et al., “Molecular Cell Biology, 4th ed.”, W. H. Freeman & Co., New York (2000); Griffiths et al., “Introduction to Genetic Analysis, 7th ed.”, W. H. Freeman & Co., N.Y. (1999); and Gilbert et al., “Developmental Biology, 6th ed.”, Sinauer Associates, Inc., Sunderland, MA (2000).

[0032] Chemistry terms used herein, unless otherwise defined herein, are used according to conventional usage in the art, as exemplified by “The McGraw-Hill Dictionary of Chemical Terms”, Parker S., Ed., McGraw-Hill, San Francisco, C.A. (1985).

[0033] All of the above, and any other publications, patents and published patent applications referred to in this application are specifically incorporated by reference herein. In case of conflict, the present specification, including its specific definitions, will control.

[0034] In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean " includes," "including," and the like; "consisting essentially of1or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

[0035] Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural.

[0036] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0037] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0038] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0039] As used herein, the terms “optional” or “optionally” mean that the subsequently described event or circumstance may occur or may not occur, and that the description includes instances where the event or circumstance occurs as well as instances in which it does not. For example, “optionally substituted alkyl” refers to the alkyl may be substituted as well as where the alkyl is not substituted.

[0040] The term "complementary" and "complementarity" are interchangeable and refer to the ability of polynucleotides to form base pairs with one another. Base pairs are typically formed by hydrogen bonds between nucleotide units in antiparallel polynucleotide strands or regions. Complementary polynucleotide strands or regions can base pair in the Watson-Crick manner (e.g., A to T, A to U, C to G). 100% (or total) complementary refers to the situation in which each nucleotide unit of one polynucleotide strand or region can hydrogen bond with each nucleotide unit of a second polynucleotide strand or region. Less than perfect (or partial) complementarity refers to the situation in which some, but not all, nucleotide units of two strands or two regions can hydrogen bond with each other and can be expressed as a percentage.

[0041] The term “hybridization” is used to refer to the structure formed by 2 independent strands of RNA that form a double stranded structure via base pairings from one strand to the other. These base pairs are considered to be G-C, A-U, and G-U. (A - Adenine, C - Cytosine, G - Guanine, U - Uracil). As in the case of the complementarity, the hybridization can be total or partial.

[0042] The term “oligonucleotide” refers to RNA, DNA, or RNA: DNA oligonucleotides.

[0043] The term “nucleotide” can refer to both, ribonucleotide or deoxyribonucleotide, unless otherwise explained. Methods

[0044] To quantify tRNA modifications, researchers typically use mass spectrometry-based methods such as liquid chromatography -tandem mass spectrometry (LC-MS / MS). These methods are highly sensitive and effective at detecting various modifications but lack the ability to identify the exact positions of these modifications within tRNA molecules. For quantifying tRNA abundance, next-generation sequencing (NGS) is commonly used. This approach requires converting tRNA molecules into complementary DNA (cDNA) prior to sequencing, which unfortunately makes it unable to detect most tRNA modifications.

[0045] A promising alternative to NGS-based techniques is direct RNA nanopore sequencing (DRS), developed by Oxford Nanopore Technologies (ONT). DRS enables sequencing of native tRNA molecules directly, potentially allowing simultaneous detection and quantification of both tRNA modifications and abundances without needing reverse transcription or PCR. Recent studies have demonstrated the feasibility of sequencing native tRNAs using nanopore technology, including both solid-state and ONT’s biological nanopores, highlighting its potential for comprehensive tRNAome analysis.

[0046] The disclosed method implements longer splint adapters with more RNA nucleotides for nanopore tRNA direct RNA sequencing. The purpose is to enable the use of nanopolish for ionic current analysis for tRNA and potentially other short classes of RNA that will help in modification detection.

[0047] We ran nanopore based tRNA sequencing runs of total yeast tRNA using the short versions of 5 double stranded splint adapters. These splint adapters had 18 RNA nt (nucleotides) on the strand that abuts the 5’ end of the tRNA and 6 RNA nt that abut the 3’ end of the tRNA. When analyzed, 0% of the reads could be segmented into kmers by nanopolish. K-mers are short DNA sequences of a fixed length that are used in bioinformatics to analyze genome sequences.

[0048] We then ran tRNA sequencing runs of total yeast tRNA using the long splint adapters described in this disclosure. The adapted molecules had longer RNA sequence, and hence an increased RNA translocation times. An example of this is shown in Figs. 4A-4C. In this experiment, we were then able to perform nanopolish for 10,901 reads out of 47,612 aligned reads, or 23%. Previously, none of the reads were amenable to nanopolish analysis. This approach improves ionic current analysis. An improved adaptation strategy for tRNA DRS ionic current analysis enhances quantitative capabilities of DRS: 60mer RNA / DNA strand

[0049] 5Phos rGrGrC rUrUrC rUrUrC rUrUrG rCrUrC rCrArU rCrArU rCrArU rCr ArU rCrArU rCrArU rCrArU rCrArU rCrArU rCrArU rUTA GGT AGT AGG TTC 3’ (SEQ ID NO: 5)

[0050] UrGrGrUCTTGGATGATGGATrArCrTrArCrTrArCrTrArCrTrArCrTrArCrTrArCrTrA rCrCrTrCrGrTrTrCrTrTrCrUrUrCrGrG-P5’ (SEQ ID NO: 6)

[0051] 3’rGrArUrGrArUrGrArUrGrArUrGrArUrGrArUrGrGrArGrCrArArGrArArGrArArGrCr CrUrGrGrU (SEQ ID NO: 7)

[0052] Extending adapter length for tRNA ionic current analysis

[0053] The analyses presented thus far allowed us to associate base miscalls with modifications in the T-loop. These associations were confirmed using genetic controls and LC-MS / MS. However, the correlation between miscalls and modifications that we observed could be viewed as a serendipitous accident. A more principled modification detection approach requires examination of the raw Nanopore ionic current signal. Presently, most DRS-based modification analysis tools use the Nanopolish algorithm to correlate ionic current data (mean current amplitude (pA), standard deviation (pA), and dwell time (s)) with 5nt kmers for individual aligned reads. This has been challenging for tRNA principally because the signal corresponding to tRNA translocation is brief (typically under 1 second long) relative to the sequencing adapter (typically 2-4 seconds). As a result, these molecules fail to be processed by ionic current analysis tools such as Nanopolish. Extending the tRNA splint adapter in length using RNA nucleotides should aid this process. To that end, we increased the number of ribonucleotides from 18 to 120 for the 5'-tRNA splint adapter, and from 6 to 46 for the 3'-tRNA splint adapter (Fig. 4A). We then sequenced wild-type yeast tRNA using these longer splint adapters. The adapted molecules had longer RNA sequence, and hence increased RNA translocation times. An example of this is shown in Fig. 4B. In this experiment, we were then able to perform Nanopolish for 10,901 reads out of 47,612 aligned reads, or 23%. Previously, none of the reads were amenable to Nanopolish analysis. While this approach needs further optimization, we anticipate that it will help improve ionic current analysis of tRNAs. The RNA sample comprising tRNA used in the methods described herein may be from any source including, human beings, animals, plants, bacteria and fungi or yeast. For example, a body fluid (blood or plasma), tissue sample, organ, organelle, or single cells obtained using methods known in the art. Commercial kits are available for isolation of RNA including, for example, RNeasy Kits (Qiagen). Approaches, reagents and kits for isolating, purifying and / or concentrating RNA from sources of interest are known in the art and commercially available. For example, kits for isolating RNA from a source of interest include the nucleic acid isolation / purification kits by Qiagen, Inc. (Germantown, Me); the ChargeSwitch®, Purelink®, nucleic acid isolation / purification kits by Life Technologies, Inc. (Carlsbad, CA). In certain aspects, the nucleic acid is isolated from a fixed biological sample, e.g., formalin- fixed, paraffin- embedded (FFPE) tissue. RNE from FFPE tissue may be isolated using commercially available kits - such as the AllPrep® DNA / RNA FFPE kit by Qiagen, Inc. (Germantown, Me), and the Recover All® Total Nucleic Acid Isolation kit for FFPE by Life Technologies, Inc. (Carlsbad, CA). In a particular embodiment the RNA composition comprising tRNA is treated with a DNAse before the method of the invention.

[0054] For the pre-annealing of the splinted oligonucleotides, both the 3’ splinted oligonucleotide and the 5 ’ splinted oligonucleotide are mixed in a molar ratio about 3 : 1 , 2.5 : 1 , 2: 1, 1.5: 1, or about 1: 1 molar ratio 3’ splinted oligonucleotide: 5’ splinted oligonucleotide. In some embodiments, said 3’ and 5’ splinted oligonucleotides are mixed in a molar ratio about 1 :1, 1: 1.5, 1:2, 1 :2.5, or about 1:3 3’ splinted oligonucleotide: 5’ splinted oligonucleotide, preferably a 1 : 1 molar ratio.

[0055] For the pre-annealing of the adapter oligonucleotides, both the first adapter DNA oligonucleotide and the second DNA oligonucleotide are mixed in a molar ratio about 3:1, 2.5: 1, 2: 1, 1.5: 1, 1.2: 1 or about 1: 1 first adapter DNA oligonucleotide: DNA oligonucleotide. In some embodiments, said first and second adapter DNA oligonucleotides are mixed in a molar ratio about 1: 1, 1: 1.2, 1 :1.5, 1:2, 1 :2.5, or aboutl :3 first adapter DNA oligonucleotide: DNA oligonucleotide, preferably a 1 : 1 molar ratio.

[0056] The conditions for the annealing can be the general conditions used in the field following regular protocols known by a skilled person. In a particular embodiment the above-mentioned splinted or adapter oligonucleotides are pre-annealed in a solution of about 8-12 mM Tris-HCI (pH 7.5), 45-55 mM NaCI and RNasin® Ribonuclease Inhibitor (Promega,N251 A), with a final concentration of about 50 ng / pL, and heated to 65°C to 85C for about 15 s and cooled to 18 to 27°C at a rate of about 0.1 °C / s to hybridize said adapters.

[0057] In some embodiments, the tRNA hybridization region of the 5’ splinted oligonucleotide has at least 3 nucleotides (preferably RNA nucleotides) matching the CCA overhang of the tRNA, this is UGG plus a random nucleotide (N) in the 3' end. In some embodiments, the 3' end sequence of the 5’ splinted oligonucleotide is 5'-rUrGrG(rN)- 3’, being N any ribonucleotide selected form rA, rU, rG or rC.

[0058] In some embodiments, the second adapter DNA oligonucleotide hybridization region of the 3 ’ splinted oligonucleotide can be any sequence of at least 10 nucleotides, such as a poly-A tail of at least 10 A nucleotides. This overhang of at least 10 nucleotides ensures that the RNA sample comprising tRNA with the 3’ and 5’ splinted oligonucleotide can be coupled with default ONT oligonucleotides known in the state of the art for direct RNA library preparation (RTA ligation step).

[0059] The size of at least 10 nucleotides, preferably 20 nucleotides in the adapter design improves the mappability of the reads. The size has to be as long as possible to improve the mapping of the tRNAs, and it has to be as small as possible so that it can be removed by the clean-up; any commercial RNA cleanup kit can be used, preferably one that retains any size larger than 17nt, such as the Zymo cleanup kit. For this reason, bead clean up can be used as alternative to these clean up kits, which gives more flexibility in terms of which RNAs are lost during the clean-up step.

[0060] In some embodiments, the terminal DNA nucleotide(s) is part of the second adapter DNA oligonucleotide hybridization region of the second splinted RNA: DNA oligonucleotide. For instance, it can be one, or two, or three “A” of the poly-A tail of 10 A nucleotides.

[0061] The conditions for the reverse transcription are known by anyone skilled in the art; for instance, using a temperature comprised between 55°C and 65°C for a period of time between 45 min and 75 min. Any commercial reverse transcriptase can be used. For instance, Maxima Reverse Transcriptase from ThermoFisher, Superscript™ II reverse transcriptase or Superscript™ IV reverse transcriptase.

[0062] The helicase protein locates in one strand of the double-stranded sequencing adapter RNA oligonucleotide possibilities such particular RNA strand to be translocated by the nanopore at a constant speed and thus sequenced. The sequencing of the linearized product of step b) or c) can be performed by any direct sequencing method that comprises a nanopore, for instance Oxford Nanopore technologies. The nanopore direct sequencing and the materials and protocols to perform it are known in the art. For instance, in US Patent Number 6,015,714. In some embodiments, the oligonucleotide adapter configured to perform nanopore direct sequencing is a double-stranded sequencing adapter DNA oligonucleotide with a helicase protein bound to one of the strands and having the complementary strand, a first DNA adapter oligonucleotide hybridization region. In some embodiments, the nanopore direct sequencing comprises a membrane, said membrane can be either solid-state or biological membranes.

[0063] Any known nanopore direct sequencing method or product can be used, for instance the one disclosed in US Patent Number 6,015,714 or US6,362,002.

[0064] The analysis or performing algorithm used can be any commercial one known by a skilled of many performing algorithms known in the art suitable for nanopore direct RNA sequencing. The first step is extracting the reads. This step can be done by a commercial software, for instance MinKNOW or any software configured to analyze the sequencing results of the nanopore direct sequencing. Next step of the analysis is the base calling, which can be done by a skilled person using any of several known performing algorithms in the field, such as Guppy or Bonito. Last step of the analysis is mapping, which can be done by several known performing algorithms. For example, Minimap2 or BWA which is a versatile sequence alignment program that aligns nucleic acid sequences against a large reference database. In some embodiments, the performing algorithm is configured to capture (and sequence) more tRNAs in a quantitative way compared to the default or adjusted parameters of MinKnow.

[0065] Kits

[0066] In one aspect, disclosed are kits comprising: i) a population of 3' splinted oligonucleotides, each 3' splinted oligonucleotide comprising in 5' to 3' order: 1) a first hybridization region, 2) a first extended oligonucleotide region, and 3) an adapter DNA oligonucleotide hybridization region, and ii) a population of 5' splinted oligonucleotides, each 5' splinted oligonucleotide comprising in 5' to 3' order: 1) a second extended oligonucleotide region, 2) a second hybridization region complementary to the first hybridization region of the 3' splinted oligonucleotide, and 3) a tRNA hybridization region.

[0067] In some embodiments, the kit further comprises i) a first adapter DNA oligonucleotide comprising a third hybridization region, and ii) a second adapter DNA oligonucleotide comprising in 5' to 3' order: 1) a fourth hybridization region complementary to the third hybridization region of the first adapter DNA oligonucleotide, and 2) a DNA hybridization region complementary to the adapter DNA oligonucleotide hybridization region of the 3' splinted oligonucleotide.

[0068] In some embodiments, the first adapter DNA oligonucleotide and the second adapter DNA oligonucleotide are a motor associated sequencing adapter (such as an Oxford Nanopore Technologies (ONT)-DNA RT Adapter (RTA) motor associated sequencing adapter).

[0069] In some embodiments, the 5' splinted oligonucleotide is 30 to 200 nucleotides in size. In some embodiments, the 5' splinted oligonucleotide is 30 to 50 nucleotides in size, 50 to 100 nucleotides in size, 100 to 150 nucleotides in size, or 150 to 200 nucleotides in size. In some embodiments, the 5' splinted oligonucleotide is RNA. In some embodiments, the second hybridization region is 15 to 50 nucleotides in size. In some embodiments, the second hybridization region is 15 to 20 nucleotides in size, 20 to 30 nucleotides in size, 30 to 40 nucleotides in size, or 40 to 50 nucleotides in size. In some embodiments, the tRNA hybridization region comprises the nucleotide sequence of NGGU, and N is A, C, G, or U. In some embodiments, the first hybridization region and the first extended oligonucleotide region of the 3' splinted oligonucleotide is 10 to 200 nucleotides in size. In some embodiments, the first hybridization region and the first extended oligonucleotide region of the 3' splinted oligonucleotide is 10 to 50 nucleotides in size, 50 to 100 nucleotides in size, 100 to 150 nucleotides in size, or 150 to 200 nucleotides in size. In some embodiments, the first hybridization region and the first extended oligonucleotide region of the 3' splinted oligonucleotide is RNA. In some embodiments, the first hybridization region is 15 to 50 nucleotides in size. In some embodiments, the first hybridization region is 15 to 20 nucleotides in size, 20 to 30 nucleotides in size, 30 to 40 nucleotides in size, or 40 to 50 nucleotides in size. In some embodiments, the adapter DNA oligonucleotide hybridization region is 15 to 50 nucleotides in size. In some embodiments, the adapter DNA oligonucleotide hybridization region is 15 to 20 nucleotides in size, 20 to 30 nucleotides in size, 30 to 40 nucleotides in size, or 40 to 50 nucleotides in size. In some embodiments, the 3' splinted oligonucleotide is 5' phosphorylated. In some embodiments, the 3' splinted oligonucleotide comprises the nucleotide sequence of SEQ ID NO: 1, 5, or 7. In some embodiments, the 5' splinted oligonucleotide comprises the nucleotide sequence of SEQ ID NO: 3, 4, or 6, and N is A, C, G, or U. In some embodiments, the 3' splinted oligonucleotide and the 5' splinted oligonucleotide are annealed prior to step a). In some embodiments, the first adapter DNA oligonucleotide binds to a helicase motor.

[0070] EXAMPLES

[0071] The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.

[0072] Example 1 : tRNA nanopore sequencing

[0073] This method implements longer splint adapters with more RNA nucleotides for nanopore tRNA direct RNA sequencing. The purpose of lengthening the adapter is to increase the overall length of the samples for nanopore sequencing. This enables the use of nanopolish, a program used for ionic current analysis, for tRNA and potentially other short classes of RNA. Ionic current based analyses allows for improved detection of RNA modification compared to the current method of using base calling errors to infer their presence in nanopore direct RNA sequencing data.

[0074] Fig- 1 shows a diagram of the components used for tRNA nanopore sequencing reaction (not to scale). Fig. 2 shows an overview of ligation scheme to make the sequencing ready product.

[0075] Use of the longer adapter design modifies existing methods for preparing tRNA for sequencing. The major steps are ligation of tRNA to the splint adapter with T4 RNA Ligase 2, cleanup of the product and ligation of the splint-adapted tRNA to the ONT motor associated sequencing adapter as per manufacturer protocol.

[0076] This method concerns design of longer splint adapters diagrammed in Fig. 3. The 5' strand of the old adapter design was composed of 6 DNA and 18 RNA nucleotides and the 3' portion of the splint adapter was composed of 6 RNA nucleotides followed by 24 DNA nucleotides. The new long adapter splint is designed to have 120 RNA nucleotides on the 5' strand and 46 RNA nucleotides plus 14 DNA nucleotides strand for the 3' splint strand. The strands that make up the long splint adapter are ordered from a supplier such as IDT.

[0077] Below are some examples of sequences of the long adapter strands. RNA nucleotides are indicated with a lowercase r preceding the base below.

[0078] 3' splint adapter strand (60mer):RNA / DNA strand: common strand for all splints

[0079] / 5Phos / rGrGrCrUrUrCrUrUrCrUrUrGrCrUrCrCrArUrCrArUrCrArUrCrArUrCrArUrCr ArUrCrArUrCrArUrCrArUrCrArUrUTAGGTAGTAGGTTC3' (SEQ ID NO: 1)

[0080] The 60mer could be extended to a up to 120mer. The example above contains 46 RNA nucleotides and 14 DNA nucleotides.

[0081] What is critical is that this strand be 5' phosphorylated, that at least the first 15 5 'nucleotides (underlined above) be complementary to the 5' strand of the splint (following 4 nt region that anneals to the tRNA overhang, e.g., NGGU) and that the 3' 14 nucleotides be complementary to the RNA sequencing adapter, RMX (as in sequence above TAGGTAGTAGGTTC (SEQ ID NO: 2)) or RTA adapter. Best practices are to order the 3' splint adapter strand to be HPLC purified under RNAse free conditions.

[0082] 5' splint adapter strands- 120mers

[0083] Below are examples of sequences for 5' splint adapter strands specific to different types of tRNA 3 'ends.

[0084] 120mer 5' splint strands, a total of five versions designed to anneal and hybridize to the 3' overhang to mature tRNA with ACCA, GCCA, CCCA and CCA ends.

[0085] Four as follows:

[0086] 5- 'rArArArArArArArArArArArArArUrUrCrUrArCrCrGrUrUrUrGrGrUrArArUrCrGrU rArArGrGrUrArCrGrUrArCrArGrUrArCrArGrUrArArGrCrUrArCrArUrArCrGrCrArArUrCrAr ArGrUrGrCrUrArUrGrArArUrGrArUrGrArUrGrArUrGrArUrGrArUrGrArUrGrGrArGrCrArAr GrArArGrArArGrCrCrNrGrGrU -3' (SEQ ID NO: 3)

[0087] Where N=A, C, G, or U, the underlined section is designed to anneal to the 3' overhang of mature tRNA. Version of adapter that terminates in UGGU-3', for example is a perfect complement to tRNA that have an ACCA 3' overhang. And one more designed for tRNA terminating with CCA overhang:

[0088] 5'rArAArArArArArArArArArArArArUrUrCrUrArCrCrGrUrUrUrGrGrUrArArUrCrGr UrArArGrGrUrArCrGrUrArCrArGrUrArCrArGrUrArArGrCrUrArCrArUrArCrGrCrArArUrCr ArArGrUrGrCrUrArUrGrArArUrGrArUrGrArUrGrArUrGrArUrGrArUrGrArUrGrGrArGrCrAr ArGrAr ArGrArArGrCrCrGr GrU-3 ' (SEQ ID NO: 4)

[0089] This version of the long adapter is designed to anneal with tRNAHlsthat post- transcriptionally includes an additional 5' rG typically and thus has a CCA rather than an NCCA 3' overhang.

[0090] These as oligonucleotides may be ordered from IDT. The oligonucleotides may be purified using PAGE, bead or column-based method.

[0091] If a single type of tRNA with known NCCA end is to be sequenced, a single splint adapter specific for the known NCCA end may be used.

[0092] If total tRNA is to be sequenced, 5 sets of adapters should be used.

[0093] The adapters are prepared as a combined stock or prepared individually, essentially as described in Thomas N.K., et. al (ACS Nano. Vol 15, Issue 10, 2021). Make an annealing reaction containing 10 pM of 3' adapter strand (common adapter) and 10 pM of 5' adapter strand specific adapter in IX TNE and nuclease free water. The amount of 3' adapter strand should be equimolar to the amount of 5' adapter strand used. So, if one uses 10 pM each of the five versions of the 5' splint adapter, a total of 50 pM of the 3' splint must be used. It also works to anneal the separate splints in separate annealing reactions. In either case, once the annealing reaction is assembled, anneal by first heating to 75°C for 1 min, then slowly cool to room temperature, optimally at a rate of approximated 2 degree / min in a PCR machine.

[0094] The 120mer is at the current limit for ordering an RNA oligonucleotide from a vendor such as IDT, but other methods could be employed to lengthen it if needed. The 5' splint could be redesigned to include a short sequence allowing for multiplexing for different samples in the same experiment. This specific sequence could be, for example, a 20nt RNA stretch positioned near the middle replacing 20nt of the 120mer. If placed too close to the 5' end it may not be sequenced well. Likewise, it must not interfere with the 3' terminus required to ligate to the tRNA.

[0095] Library prep using long oligonucleotide follows essentially the same procedure found in the methods of Thomas et al. 2021. The steps are: 1. Ligation of the tRNA to the long splint adapters with RNA ligase2 (New England Biolabs)

[0096] 2. Bead clean-up of the ligation reaction.

[0097] 3. Ligation to the sequencing adapter (RMX tube that comes with ONT kit)

[0098] 4. Standard run procedures for Direct RNA sequencing using mini ON or promeTHION platform (currently version SQK-002-RNA)

[0099] Presently, most direct RNA sequencing (DRS)-based modification analysis tools use the Nanopolish algorithm to correlate ionic current data (mean current amplitude (pA), standard deviation (pA), and dwell time (s)) with 5nt kmers for individual aligned reads. This has been challenging for tRNA principally because the signal corresponding to tRNA translocation is brief (typically under 1 second long) relative to the sequencing adapter (typically 2-4 seconds). As a result, these molecules fail to be processed by ionic current analysis tools such as Nanopolish. Extending the tRNA splint adapter in length using RNA nucleotides aids this process. To that end, we increased the number of ribonucleotides from 18 to 120 for the 5'-tRNA splint adapter, and from 6 to 46 for the 3 '-tRNA splint adapter (Fig. 4A). We then sequenced wild-type yeast tRNA using these longer splint adapters. The adapted molecules had longer RNA sequence, and hence increased RNA translocation times. An example of this is shown in Figs. 4B-4C. In this experiment, we were then able to perform Nanopolish for 10,901 reads out of 47,612 aligned reads, or 23%. Previously, none of the reads were amenable to Nanopolish analysis. This method improves ionic current analysis of tRNAs.

[0100] Example 2: Methods

[0101] Enzymes: T4 RNA ligase 2 (10,000 units / mL), T4 DNA ligase (2,000,000 units / mL), T4 polynucleotide kinase (10,000 units / mL), and corresponding buffer solutions were purchased from New England Biolabs. Nuclease Pl (Sigma Aldrich), antarctic phosphatase (5,000 units / mL) (New England Biolabs), and phosphodiesterase 1 (Thermo Fisher Scientific) were used to prepare samples for liquid chromatography / mass spectrometry.

[0102] Annealing Splint Adapters. Ten pM stocks of each double-stranded splint adapters were prepared in 10 mM Tris (pH 8.0), 50 mM NaCl, and 1 mM EDTA by adding 100 pmol of each strand in a total volume of 10 pL. The solution was heated to 75 °C for 15 s and slowly cooled to 25 °C to hybridize the adapter strands.

[0103] Library Preparation. tRNA libraries were prepared using the SQKRNA002 (Oxford Nanopore Technologies) kit as described below.

[0104] First Ligation: tRNA to Splint Adapter. In the first ligation reaction, the splint adapter to tRNA molar ratio was 1 : 1.25. The tRNA sample is first heated to 95 °C for 2 min, allowed to cool for 2 min, then placed on ice for 2 min. The reaction was carried out at room temperature in a DNA LoBind tube for 45 min. Its constituents were 1 xRNA ligase 2 buffer (NEB) supplemented with 5% PEG 8000, 2 mM ATP, 6.25 mM DTT, 6.25 mM MgC12, and 0.5 umts / pL T4 RNA ligase 2 (10,000 units / mL). For single tRNA isotype libraries (for tRNAfMet, tRNALys , or tRNAPhe ), 16 pmol of splint adapter and 20 pmol of tRNA (~500 ng), in a total reaction volume of 20 pL, were used. The tRNAs for these libraries have ACCA 3' termini. The splint adapter used was the form with a UGGU overhang. Total tRNA reactions using all four splint adapters were performed using 32 pmol of adapter (8 pmol of each of the four adapters) and 40 pmol (~1 pg) of total tRNA in a reaction volume of 40 pL. For total tRNA runs using only one of the four adapters, 16 pmol of adapter and 20 pmol (~500 ng) of total tRNA were used in a reaction volume of 20 pL.

[0105] Gel Purification of Ligation I Product. Gel excision and purification was performed for all samples in this study. It is recommended for this procedure. Unligated and partially ligated tRNA carried forward to subsequent reactions may decrease the throughput and coverage.

[0106] PAGE Gel Separation and Excision of the tRNA / Splint Ligation Product. The ligation reaction was diluted to 1 * with 2 RNA loading dye (NEB). Standards (low range ssRNA ladder, NEB) and 10 pmol of unligated tRNA sample were prepared in an equivalent buffer to the first ligation reaction (1 x RNA ligase 2 buffer (NEB), 5% PEG 8000, 2 mM ATP, 6.25 mM DTT, 6.25 mM MgC12) and diluted with an equal volume of 2 RNA loading dye.

[0107] The size standard, unligated tRNA, and ligation reaction samples were run on a denaturing 7 M urea / TBE PAGE gel (8%) in 1 x TBE buffer for ~ 1.5 h at 28 W. The gel was poststained in the dark, in a 1 x TBE solution containing 2x (diluted 1 to 5000) SybrGoldTM (Life Technologies) for 20 min. The gel was transferred to a piece of saran wrap, and using UV shadowing, the gel region corresponding to the fully ligated product (~130 nt for tRNAfXIct, tRNALys, and tRNApheor from ~ 120-170 nt for total tRNA) was excised. Gel Purification of tRNA / Splint Ligation Product by Electroelution. The excised gel fragment was electroeluted in l x TAE buffer using 3.5 kDa MWCO D-tube dialyzers (Novagen) according to the manufacturer’s protocol with minor modifications. For the ethanol precipitation step, the solution was precipitated overnight at -20 °C with 0.1 x sodium acetate (pH 5.2), 1 pL of glycogen (20 mg / ml, RNA grade), and 2.5-3x ethanol. Following centrifugation at 4 °C for 30 min at 12,000g, the solution was removed. 200 pL of 80% ethanol was added to wash each pellet. After centrifugation at 4 °C for 15 min, the ethanol was removed, and the pellets were air-dried briefly. The pellets were resuspended and pooled using NF H2O. For single isotype tRNA libraries or total tRNA libraries where one of the four adapters was used, a total of 12 pL of nuclease-free (NF) H2O was used to resuspend the pellets. For total tRNA libraries where all four adapters were ligated, the resuspension volume was 24 pL of NF H2O. The concentration of the sample at this point may be quantified by nanodrop or the Qubit fluorometer RNA HS assay. The amount of material recovered and carried forward to the second ligation varied between ~ 60 and 200 ng for single isotype tRNA libraries and total tRNA libraries using a single version of double stranded splint adapter. Approximately 500 ng was recovered in total tRNA libraries ligated to all four adapter versions. For this library, the amount of input tRNA (1 pg) was twice as much as other libraries (0.5 pg). On the order of 25% of the material by weight of the input tRNA is recovered following purification of the full-length product, but this can vary substantially.

[0108] Second Ligation: Splint-Ligated tRNA and RMX Adapter. For single isotype tRNA libraries or total tRNA libraries where one version of the splint adapters was used, the second ligation reaction was composed of 11 pL of the gel purified splint ligation product, 5 pLof5x quick ligation reaction buffer (NEB: B6058S), 6 pL of the RMX adapter, and 3 pL of T4 DNA ligase (2,000,000 units / mL). RMX adapter is included in ONT’s RNA sequencing kits.

[0109] For total tRNA libraries previously ligated to all four types of double-stranded splint adapters, the second ligation reaction included 23 pL of the purified splint ligation products, 8 pL of 5x quick ligation reaction buffer (NEB: B6058S), 6 pL of the RMX adapter, and 3 pL of T4 DNA ligase (2,000,000 units / mL).

[0110] Ligation reactions were carried out at room temperature for 30 min. A 1.5x volume of Ampure RNAClean XP beads (Beckman-Coulter) was then added and mixed into the reaction by pipetting up and down. The tube was incubated for 15 min at room temperature with occasional light tapping and pelleted on the magnet, and the supernatant was removed. Two 150 pL washes with WSB (wash buffer in the ONT kit) were conducted, during which the pellet was vigorously resuspended by flicking and returned to the magnet to pellet, and the wash solution was removed. Following the second wash, the pellet was resuspended in 12.5 pL elution buffer (EB) and incubated for 20 min at room temperature with light tapping. Following pelleting of the beads on the magnet, the eluate was recovered to a fresh tube.

[0111] Flow Cell Quality Control, Priming the Flow Cell, and Loading the Sample on the MinlON. The ONT SQK-RNA002 protocol was followed for mini ON flow cell (FLO-MIN-106) quality control, priming, and preparation of the sample in RNA running buffer and for loading the library onto the flow cell.

[0112] RNA Handling Practices. Care was taken to avoid introducing RNases into the samples or into stock solutions by wearing gloves at all times, using RNase-free filter tips and NF water. Pipettes, benches, and equipment were cleaned with RNase AWAY.

[0113] MinlON Running Parameters. Sequencing runs were done with live base-calling off. The experiments were set for the standard 48 h period, but were typically run for <24 h due to a deterioration in functional channels over time seen using our samples. For sequencing runs where the nanopores in the flow cell became clogged (indicated by reduced functional pores on the MinKnow GUI), the experiment was restarted up to five times.

[0114] INCORPORATION BY REFERENCE

[0115] All publications, US patents, and US and PCT published patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0116] EQUIVALENTS

[0117] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

We claim:

1. A method of preparing a sequencing library, comprising: a) contacting a sample comprising tRNA and a RNA ligase, with i) a population of 3' splinted oligonucleotides, each 3' splinted oligonucleotide comprising in 5' to 3' order:1) a first hybridization region,2) a first extended oligonucleotide region, and3) an adapter DNA oligonucleotide hybridization region, and ii) a population of 5' splinted oligonucleotides, each 5' splinted oligonucleotide comprising in 5' to 3' order:1) a second extended oligonucleotide region,2) a second hybridization region complementary to the first hybridization region of the 3' splinted oligonucleotide, and3) a tRNA hybridization region; wherein at the end of step a), the 5' splinted oligonucleotide is adjacent to the 5' end of the tRNA and annealed to both the 3' end of the tRNA by the tRNA hybridization region and to the 3' splinted oligonucleotide by the second hybridization region thereby forming a first product, and b) contacting the first product with a DNA ligase: i) a first adapter DNA oligonucleotide comprising a third hybridization region, and ii) a second adapter DNA oligonucleotide comprising in 5' to 3' order:1) a fourth hybridization region complementary to the third hybridization region of the first adapter DNA oligonucleotide, and2) a DNA hybridization region complementary to the adapter DNA oligonucleotide hybridization region of the 3' splinted oligonucleotide, wherein at the end of step b), the first adapter DNA oligonucleotide is adjacent to the 3' end of the terminal region of the 3' splinted oligonucleotide, and the second adapter DNA oligonucleotide is annealed to both the 3' splinted oligonucleotide and the first adapter DNA oligonucleotide thereby forming a second product.

2. The method of claim 1 , further comprising c) performing reverse transcription to linearize the second product thereby forming a third product.

3. The method of claim 1 or 2, further comprising d) carrying out nanopore direct sequencing to obtain the abundance of tRNA and its modifications in the sample.

4. The method of any one of claims 1-3, wherein the RNA ligase is T4 RNA ligase 2.

5. The method of any one of claims 1-4, wherein the DNA ligase is T4 DNA ligase.

6. The method of any one of claims 1-5, wherein the first adapter DNA oligonucleotide and the second adapter DNA oligonucleotide are a motor associated sequencing adapter.

7. The method of any one of claims 1-6, wherein the 5' splinted oligonucleotide is 30 to 200 nucleotides in size.

8. The method of any one of claims 1-7, wherein the 5' splinted oligonucleotide is 30 to 50 nucleotides in size, 50 to 100 nucleotides in size, 100 to 150 nucleotides in size, or 150 to 200 nucleotides in size.

9. The method of any one of claims 1-8, wherein the 5' splinted oligonucleotide is RNA.

10. The method of any one of claims 1-9, wherein the second hybridization region is 15 to 50 nucleotides in size.

11. The method of any one of claims 1-10, wherein the second hybridization region is 15 to 20 nucleotides in size, 20 to 30 nucleotides in size, 30 to 40 nucleotides in size, or 40 to 50 nucleotides in size.

12. The method of any one of claims 1-11, wherein the tRNA hybridization region comprises the nucleotide sequence of NGGU, and N is A, C, G, or U.

13. The method of any one of claims 1-12, wherein the first hybridization region and the first extended oligonucleotide region of the 3' splinted oligonucleotide is 10 to 200 nucleotides in size.

14. The method of any one of claims 1-13, wherein the first hybridization region and the first extended oligonucleotide region of the 3' splinted oligonucleotide is 10 to 50 nucleotides in size, 50 to 100 nucleotides in size, 100 to 150 nucleotides in size, or 150 to 200 nucleotides in size.

15. The method of any one of claims 1-14, wherein the first hybridization region and the first extended oligonucleotide region of the 3' splinted oligonucleotide is RNA.

16. The method of any one of claims 1-15, wherein the first hybridization region is 15 to 50 nucleotides in size.

17. The method of any one of claims 1-16, wherein the first hybridization region is 15 to 20 nucleotides in size, 20 to 30 nucleotides in size, 30 to 40 nucleotides in size, or 40 to 50 nucleotides in size.

18. The method of any one of claims 1-17, wherein the adapter DNA oligonucleotide hybridization region is 15 to 50 nucleotides in size.

19. The method of any one of claims 1-18, wherein the adapter DNA oligonucleotide hybridization region is 15 to 20 nucleotides in size, 20 to 30 nucleotides in size, 30 to 40 nucleotides in size, or 40 to 50 nucleotides in size.

20. The method of any one of claims 1-19, wherein the 3' splinted oligonucleotide is 5' phosphorylated.

21. The method of any one of claims 1-20, wherein the 3' splinted oligonucleotide comprises the nucleotide sequence of SEQ ID NO: 1, 5, or 7.

22. The method of any one of claims 1-21, wherein the 5' splinted oligonucleotide comprises the nucleotide sequence of SEQ ID NO: 3, 4, or 6, and N is A, C, G, or U.

23. The method of any one of claims 1-22, wherein the 3' splinted oligonucleotide and the 5' splinted oligonucleotide are annealed prior to step a).

24. The method of any one of claims 1-23, wherein the first adapter DNA oligonucleotide binds to a helicase motor.

25. The method of any one of claims 3-24, wherein the nanopore direct sequencing comprises: loading the second product or the third product to a flow cell which comprises a membrane in which is present a nanopore that provides a channel through the membrane, coupled to a current intensity, wherein the second product or the third passes through the nanopore, causes disruptions in the current intensity, and analyzing said sequences to obtain the abundance of tRNA and its modifications in the sample.

26. The method of any one of claims 1-25, wherein the duration of step a) is between 1 hr and 3hr at a temperature comprised between 20°C and 26°C.

27. The method of any one of claims 1-26, wherein the method sequenced tRNA, identifies tRNA associated disorders, or identifies tRNA modifications.

28. A kit comprises: i) a population of 3' splinted oligonucleotides, each 3' splinted oligonucleotide comprising in 5' to 3' order:1) a first hybridization region,2) a first extended oligonucleotide region, and3) an adapter DNA oligonucleotide hybridization region, and ii) a population of 5' splinted oligonucleotides, each 5' splinted oligonucleotide comprising in 5' to 3' order:1) a second extended oligonucleotide region,2) a second hybridization region complementary to the first hybridization region of the 3' splinted oligonucleotide, and3) a tRNA hybridization region.

29. The kit of claim 28, further comprises i) a first adapter DNA oligonucleotide comprising a third hybridization region, and ii) a second adapter DNA oligonucleotide comprising in 5' to 3' order:1) a fourth hybridization region complementary to the third hybridization region of the first adapter DNA oligonucleotide, and2) a DNA hybridization region complementary to the adapter DNA oligonucleotide hybridization region of the 3' splinted oligonucleotide.

30. The kit of any one of claims 28-29, wherein the first adapter DNA oligonucleotide and the second adapter DNA oligonucleotide are a motor associated sequencing adapter.

31. The kit of any one of claims 28-30, wherein the 5' splinted oligonucleotide is 30 to 200 nucleotides in size.

32. The kit of any one of claims 28-31, wherein the 5' splinted oligonucleotide is 30 to 50 nucleotides in size, 50 to 100 nucleotides in size, 100 to 150 nucleotides in size, or 150 to 200 nucleotides in size.

33. The kit of any one of claims 28-32, wherein the 5' splinted oligonucleotide is RNA.

34. The kit of any one of claims 28-33, wherein the second hybridization region is 15 to 50 nucleotides in size.

35. The kit of any one of claims 28-34, wherein the second hybridization region is 15 to 20 nucleotides in size, 20 to 30 nucleotides in size, 30 to 40 nucleotides in size, or 40 to 50 nucleotides in size.

36. The kit of any one of claims 28-35, wherein the tRNA hybridization region comprises the nucleotide sequence of NGGU, and N is A, C, G, or U.

37. The kit of any one of claims 28-36, wherein the first hybridization region and the first extended oligonucleotide region of the 3' splinted oligonucleotide is 10 to 200 nucleotides in size.

38. The kit of any one of claims 28-37, wherein the first hybridization region and the first extended oligonucleotide region of the 3' splinted oligonucleotide is 10 to 50 nucleotides in size, 50 to 100 nucleotides in size, 100 to 150 nucleotides in size, or 150 to 200 nucleotides in size.

39. The kit of any one of claims 28-38, wherein the first hybridization region and the first extended oligonucleotide region of the 3' splinted oligonucleotide is RNA.

40. The kit of any one of claims 28-39, wherein the first hybridization region is 15 to 50 nucleotides in size.

41. The kit of any one of claims 28-40, wherein the first hybridization region is 15 to 20 nucleotides in size, 20 to 30 nucleotides in size, 30 to 40 nucleotides in size, or 40 to 50 nucleotides in size.

42. The kit of any one of claims 28-41 , wherein the adapter DNA oligonucleotide hybridization region is 15 to 50 nucleotides in size.

43. The kit of any one of claims 28-42, wherein the adapter DNA oligonucleotide hybridization region is 15 to 20 nucleotides in size, 20 to 30 nucleotides in size, 30 to 40 nucleotides in size, or 40 to 50 nucleotides in size.

44. The kit of any one of claims 28-43, wherein the 3' splinted oligonucleotide is 5' phosphorylated.

45. The kit of any one of claims 28-44, wherein the 3' splinted oligonucleotide comprises the nucleotide sequence of SEQ ID NO: 1, 5, or 7.

46. The kit of any one of claims 28-45, wherein the 5' splinted oligonucleotide comprises the nucleotide sequence of SEQ ID NO: 3, 4, or 6, and N is A, C, G, or U.

47. The kit of any one of claims 28-46, wherein the 3' splinted oligonucleotide and the 5' splinted oligonucleotide are annealed prior to step a).

48. The kit of any one of claims 28-47, wherein the first adapter DNA oligonucleotide binds to a helicase motor.

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