WO2026151663A1 - Selective rna tagging methods of total nucleic samples for sequencing applications - Google Patents

Abstract

The present disclosure provides methods of tagging RNA molecules in a total nucleic acid sample. Also disclosed herein are methods for preparing sequencing libraries from DNA and RNA simultaneously, i.e., providing genomic and transcriptomic information in a single tube without the need for separate DNA and RNA library preparation workflows.

Description

Attorney Docket No P39431-WO-1

SELECTIVE RNA TAGGING METHODS OF TOTAL NUCLEIC SAMPLES FOR SEQUENCING APPLICATIONS

BACKGROUND OF THE DISCLOSURE

[0001] With advances in sequencing and reduced sequencing costs, the focus has been on new ways of obtaining more information from library preparation. This includes multi-omics or looking at more than one source of information for a given sample.

[0002] Methods which permit genomic and transcriptomic sequencing require that nucleic acid samples to be split into parallel, but physically separated, workflows. More particularly , if information is desired from both DNA and RNA from a single source (e.g., a patient sample), two separate specimens are required - a first to isolate DNA, and a second to isolate RNA. This, it is believed, results in a waste of sample material as well as labor and reagent costs. For precious samples, such as clinical biopsy samples, forensic or historical, there may not be enough material to perform two separate isolations for DNA and RNA.

[0003] The library preparation methods which do offer a one tube solution either do not allow for distinguishing the reads for linking back to the originating nucleic acid molecule; or rely on amplicon-based sequencing by only targeting specific DNA and RNA amplicons.

BRIEF SUMMARY OF THE DISCLOSURE

[0004] The present disclosure is directed to methods of preparing a library suitable for sequencing or other downstream analysis where the library includes nucleic acid molecules that originate from both cellular RNA and cellular DNA. While DNA and RNA targets are processed simultaneously in a single workflow, they remain distinguishable as originating from DNA or RNA respectively after completion of the method.

[0005] Also disclosed are methods of selectively tagging an RNA fraction within a total nucleic acid sample (i.e., a sample which includes both DNA and RNA). Applicant has found that sequencing metrics may be enhanced by optimizing the tagging of RNA molecules in a sample. Additionally, Applicant has discovered the RNA tagging methods of the present disclosure permit higher efficiency tagging and higher RNA library yields.

[0006] A first aspect of the present disclosure is a method of preparing a library comprising one or more cDNA molecules and one or more DNA molecules, comprising: (a) obtaining a sample comprising one or more DNA molecules and one or more RNA molecules; (b) fragmenting the one or more RNA molecules in the sample and optionally fragmenting the one or more DNA molecules in the same to provide a sample including at least one or moreAttorney Docket No P39431-WO-1

fragmented RNA molecules; (c) selectively ligating a tag to the one or more fragmented RNA molecules in the sample to provide one or more tagged RNA molecules; and (d) generating one or more double stranded cDNA molecules from the one or more tagged RNA molecules, wherein the one or more generated double stranded cDNA molecules each include an adapter sequence. In some embodiments, the method further comprises sequencing the library comprising the one or more cDNA molecules and the one or more DNA molecules. In some embodiments, the sequencing comprises next-generation sequencing. In some embodiments, the one or more DNA molecules and the one or more RNA molecules are not physically separated prior to the fragmentation of the one or more RNA molecules in the sample. In some embodiments, the method further comprises amplifying the one or more cDNA molecules and/or the one or more DNA molecules in the library. In some embodiments, the amplification comprises isothermal amplification. In some embodiments, the method further comprises performing an optional target enrichment step.

[0007] In some embodiments, the one or more RNA molecules in the obtained sample are selectively fragmented. In some embodiments, the one or more RNA molecules in the obtained sample are selectively fragmented using an RNA endonuclease. In some embodiments, the RNA endonuclease is RNase III. In some embodiments, the one or more RNA molecules in the obtained sample are selectively fragmented using a RNaseT2 endonuclease. In some embodiments, the tag is ligated to the 5' ends of the one or more fragmented RNA molecules. In some embodiments, the tag comprises at least 5 nucleotides. In some embodiments, the tag comprises at least 10 nucleotides. In some embodiments, the ligation is performed using an RNA ligase. In some embodiments, the RNA ligase is RtcB ligase. In some embodiments, the one or more DNA molecules in the obtained sample are fragmented (but separately from the one or more RNA molecules). In some embodiments, the one or more DNA molecules are fragmented enzymatically. In some embodiments, the enzymatic fragmentation of the one or more DNA molecules comprises contacting the sample with a DNase. In some embodiments, the one or more fragmented DNA molecules are end repaired. In some embodiments, the one or more cDNA molecules are prepared by (i) performing a first-strand synthesis to provide one or more DNA / RNA hybrid molecules; (ii) performing a second-strand synthesis on the provided one or more DNA / RNA hybrid molecules to generate the one or more cDNA molecules; and (iii) ligating adapters to the one or more generated cDNA molecules. In some embodiments, the method further comprises ligating an adapter to the one or more fragmented DNA molecules or the one or more end repaired fragmented DNA molecules.Attorney Docket No P39431-WO-1

[0008] In some embodiments, the one or more RNA molecules and the one or more DNA molecules in the sample are each fragmented (e.g., simultaneously). In some embodiments, the fragmentation is selected from the group consisting of the application of mechanical force, sonication, and restriction endonuclease cleavage. In some embodiments, the one or more fragmented RNA molecules are selectively tagged by polyadenylating the one or more fragmented RNA molecules to provide one or more fragmented RNA molecules each including a polyA tail. In some embodiments, the polyadenylating of the one or more fragmented RNA molecules comprises contacting the sample with a Poly(a) Polymerase. In some embodiments, the one or more cDNA molecules are prepared by (i) performing a first-strand synthesis to provide one or more DNA / RNA hybrid molecules; (ii) performing a second-strand synthesis on the provided one or more DNA / RNA hybrid molecules to generate the one or more cDNA molecules; and (iii) ligating adapters to the one or more generated cDNA molecules. In some embodiments, the method further comprises ligating an adapter to the one or more fragmented DNA molecules or the one or more end repaired fragmented DNA molecules.

[0009] In some embodiments, the tag selectively ligated to the one or more fragmented RNA molecules includes a primer binding site. In some embodiments, the tag selectively ligated to the one or more fragmented RNA molecules includes (i) a 5' sequence index; (ii) an adapter sequence; and (iii) a 3' terminal di-deoxyribonucleotide. In some embodiments, the tag is ligated to the one or more fragmented RNA molecules using an RNA ligase or a T4 RNA ligase II. In some embodiments, the one or more cDNA molecules are generated by (i) performing a first-strand synthesis on the one or more tagged RNA molecules to provide one or more DNA I RNA hybrid molecules; (ii) ligating adapters to the one or more DNA / RNA hybrid molecules; (iii) extending the one or more adapter ligated DNA / RNA hybrid molecules; and (iv) amplifying the one or more adapter ligated DNA / RNA hybrid molecules. In some embodiments, the first-strand synthesis is performed using a reverse transcriptase and a primer specific to at least a portion of the tag selectively ligated to the one or more fragmented RNA molecules. In some embodiments, the method further comprises ligating an adapter to the one or more fragmented DNA molecules. In some embodiments, the method further comprises extending the one or more adapter ligated DNA molecules to provide one or more extended DNA molecules. In some embodiments, the method further comprises amplifying the one or more extended DNA molecules.

[0010] A second aspect of the present disclosure is a method of preparing a library comprising one or more adapter ligated double stranded cDNA molecules and one or moreAttorney Docket No P39431-WO-1

adapter ligated DNA molecules, the method comprising: (a) obtaining a sample comprising one or more RNA molecules and one or more DNA molecules; (b) selectively fragmenting the one or more RNA molecules within the sample; (c) ligating a tag to the 5' ends of the one or more RNA molecules within the sample; (d) fragmenting the DNA molecules within the sample to provide one or more fragmented DNA molecules; (e) performing a first strand synthesis on the one or more fragmented tagged RNA molecules in the sample to provide one or more hybrid DNA/RNA molecules; (f) performing a second strand synthesis on the one or more hybrid DNA/RNA molecules to provide one or more double stranded cDNA molecules; (g) ligating adapters to the one or more double stranded cDNA molecules and the one or more DNA molecules in the sample to provide the library including the one or more adapter ligated double stranded cDNA molecules and the one or more adapter ligated DNA molecules. In some embodiments, the method further comprises sequencing the one or more adapted ligated double stranded cDNA molecules and the one or more adapter ligated DNA molecules in the library.

[0011] In some embodiments, the one or more RNA molecules in the obtained sample are selectively fragmented using an RNA endonuclease. In some embodiments, the RNA endonuclease is RNase III. In some embodiments, the one or more RNA molecules in the obtained sample are selectively fragmented using a RNaseT2 endonuclease. In some embodiments, the tag comprises at least 5 nucleotides. In some embodiments, the ligation is performed using an RNA ligase. In some embodiments, the RNA ligase is RtcB ligase. In some embodiments, the one or more DNA molecules in the obtained sample are separately fragmented. In some embodiments, the one or more DNA molecules are fragmented enzymatically. In some embodiments, the enzymatic fragmentation of the one or more DNA molecules comprises contacting the sample with a DNase. In some embodiments, the one or more fragmented DNA molecules are end repaired. In some embodiments, the sequencing comprises next-generation sequencing.

[0012] A third aspect of the present disclosure is a method of preparing a library comprising one or more adapter ligated double stranded cDNA molecules and one or more adapter ligated DNA molecules, the method comprising: (a) obtaining a sample comprising one or more RNA molecules and one or more DNA molecules; (b) fragmenting both the one or more RNA molecules and the one or more DNA molecules in the obtained sample; (c) selectively polyadenylating the one or more fragmented RNA molecules to provide a sample including one or more polyA tagged fragmented RNA molecules; (d) performing a first strandAttorney Docket No P39431-WO-1

synthesis on the one or more polyA tagged fragmented RNA molecules in the sample using a primer which anneals to the polyA tail to provide one or more DNA/RNA hybrid molecules; (e) performing a second strand synthesis on the one or more DNA / RNA hybrid molecules to provide one or more double stranded cDNA molecules; and (f) ligating adapters to the one or more double stranded cDNA molecules and the one or more DNA molecules in the sample to provide the library including the one or more adapter ligated double stranded cDNA molecules and the one or more adapter ligated DNA molecules. In some embodiments, the method further comprises sequencing the one or more adapted ligated double stranded cDNA molecules and the one or more adapter ligated DNA molecules in the library. In some embodiments, the fragmentation of both the one or more RNA molecules and the one or more DNA molecules in the obtained sample is selected from the group consisting of the application of mechanical force, sonication, and restriction endonuclease cleavage. In some embodiments, the polyadenylating of the one or more fragmented RNA molecules comprises contacting the sample with a Poly(a) Polymerase. In some embodiments, wherein the sequencing comprises next-generation sequencing.

[0013] A fourth aspect of the present disclosure is a method of preparing a library comprising one or more adapter ligated double stranded cDNA molecules and one or more adapter ligated DNA molecules, the method comprising: (a) obtaining a sample comprising one or more RNA molecules and one or more DNA molecules; (b) fragmenting both the one or more RNA molecules and the one or more DNA molecules in the obtained sample; (c) selectively ligating a tag to the 3' ends of the one or more fragmented RNA molecules in the sample; (d) performing a first strand synthesis of the one or more tagged RNA molecules in the sample, wherein the first strand synthesis utilizes a portion of the tag as a priming site to provide one or more DNA / RNA hybrid molecules; (e) ligating of adapters to the one or more DNA / RNA hybrid molecules to provide one or more adapter ligated DNA / RNA hybrid molecules; (f) ligating adapters to the one or more fragmented DNA molecules in the sample to provide one or more adapter ligated DNA molecules; (g) extending each of the one or more adapter ligated DNA / RNA hybrid molecules and the one or more adapter ligated DNA molecules in the sample; and (h) amplifying each of the extended one or more adapter ligated DNA / RNA hybrid molecules and the one or more adapter ligated DNA molecules in the sample to provide adapter ligated double stranded cDNA molecules and double stranded DNA molecules. In some embodiments, the method further comprises sequencing the one or more adapted ligated double stranded cDNA molecules and the one or more adapter ligated DNA molecules in the library. In some embodiments, the tag selectively ligated to the one or moreAttorney Docket No P39431-WO-1

fragmented RNA molecules includes a primer binding site. In some embodiments, the tag selectively ligated to the one or more fragmented RNA molecules includes (i) a 5' sequence index; (ii) an adapter sequence; and (iii) a 3' terminal di-deoxyribonucleotide. In some embodiments, the tag is ligated to the one or more fragmented RNA molecules using an RNA ligase or a T4 RNA ligase II. In some embodiments, wherein the sequencing comprises nextgeneration sequencing.

BRIEF DESCRIPTION OF THE FIGURES

[0014] For a general understanding of the features of the disclosure, reference is made to the drawings. In the drawings, like reference numerals have been used throughout to identify identical elements.

[0015] FIG. 1 provides for an overview of methods of tagging RNA molecules within a sample.

[0016] FIG. 2 depicts a method of selectively tagging the 5' terminal ends of fragmented RNA molecules and preparing a library including double stranded cDNA and DNA, in accordance with embodiment of the present disclosure.

[0017] FIG. 3A, 3B, 3C, 3D, 3E, 3F, 3G, and 3H outline the steps of preparing double stranded cDNA and DNA in accordance with an embodiment of the present disclosure.

[0018] FIG. 4 sets forth a method of selectively incorporating a poly(A) tail into the terminal ends of fragmented RNA molecules and preparing a library including double stranded cDNA and DNA, in accordance with embodiment of the present disclosure.

[0019] FIG. 5A, 5B, 5C, 5D, 5E, 5F, 5G, and 5H outline the steps of preparing double stranded cDNA and DNA in accordance with an embodiment of the present disclosure.

[0020] FIG. 6 sets forth a method of selectively ligating a tag to fragmented RNA molecules in a total nucleic acid sample.

[0021] FIG. 7A, 7B, 7C, 7D, 7E, 7F, 7G, 7H, and 71 outline the steps of preparing double stranded cDNA and DNA in accordance with an embodiment of the present disclosure.

[0022] FIG. 8 depicts the specificity of polyA tagging via Terminal Deoxynucleotidyl Transferase (TdT) of RNA. The percentage of total reads (y-axis) attributed to tagged DNA (diagonal hatch), tagged RNA (squares), and tagged rRNA (circles) is shown for RNA-only (Samples 1-19) and TNA (Samples 20-38) inputs. Samples 15, 16, 18, 19, 32, 33, 37, and 38 were treated with TdT to add adenines to the 3'-end. Within this group, samples 15, 16, 32, and 33 were amplified with a 22 bp polyT oligo, while samples 18, 19, 37, and 38 wereAttorney Docket No P39431-WO-1

amplified with a 20 bp polyT oligo. The remaining samples utilized alternative tagging methods, serving as a control to demonstrate the specificity of the poly A methodology.

[0023] FIG. 9 illustrates the specificity of low complexity poly A tagging of RNA. The percentage of total reads (y-axis) attributed to tagged DNA (diagonal hatch), tagged RNA (squares), and tagged rRNA (circles) is shown across RNA-only (Samples 1-19) and TNA (Samples 20-38) inputs. Samples 7, 8, 26, and 27 were treated with a low complexity polyA tag during first strand synthesis, demonstrating specificity compared to alternative tagging methodologies used in the remaining samples.

DETAILED DESCRIPTION

[0024] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0025] As used herein, the singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "includes" is defined inclusively, such that "includes A or B" means including A, B, or A and B.

[0026] 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 exclusivity⁷, 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.

[0027] The terms "comprising," "including," "having," and the like are used interchangeably and have the same meaning. Similarly, "comprises," "includes," "has," and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of "comprising" and is therefore interpreted to be an open term meaning "at least the following," and is alsoAttorney Docket No P39431-WO-1

interpreted not to exclude additional features, limitations, aspects, etc. Thus, for example, "a device having components a, b, and c" means that the device includes at least components a, b, and c. Similarly, the phrase: "a method involving steps a, b, and c" means that the method includes at least steps a, b, and c. Moreover, while the steps and processes may be outlined herein in a particular order, the skilled artisan will recognize that the ordering steps and processes may vary'.

[0028] 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.

[0029] As used herein, the term "about" refers to a range of values including the specified value, which a person of ordinary skill in the art would consider reasonably similar to the specified value. In some embodiments, the term "about" means within a standard deviation using measurements generally acceptable in the art. In some embodiments, about means a range extending to +/— 10% of the specified value.

[0030] As used herein, the terms "P5" and "P7" refer to amplification primers, e.g., universal primer extension primers. It will be understood that any suitable amplification primers can be used in the methods presented herein. Uses of amplification primers such as P5 and P7 are know n in the art, as exemplified by the disclosures of WO 2007/010251, WO 2006/064199, WO 2005/065814, WO 2015/106941, WO 1998/044151, and WO 2000/018957. For example, any suitable forward amplification primer, whether immobilized or in solution, can be useful in the methods presented herein for hybridization to a complementary sequenceAttorney Docket No P39431-WO-1

and amplification of a sequence. Similarly, any suitable reverse amplification primer, whether immobilized or in solution, can be useful in the methods presented herein for hybridization to a complementary sequence and amplification of a sequence. One of skill in the art will understand how to design and use primer sequences that are suitable for capture, and amplification of nucleic acids as presented herein.

[0031] As used herein, the term "adapter" refers to a nucleotide sequence that may be added to another sequence to import additional properties to that sequence. An adapter can be single- or double-stranded or may have both a single-stranded portion and a double-stranded portion. The ligation of an adapter to a target polynucleotide or a target polynucleotide strand of interest enables the generation of amplification-ready products of the target polynucleotide or the target polynucleotide strand of interest. The target polynucleotide molecules may be fragmented or not prior to the addition of adaptors. In some embodiments, the adapter is substantially non-complementary to the 3' end or the 5' end of any target sequence present in the sample. In some embodiments, suitable adapter lengths are in the range of about 10 - 100 nucleotides, about 12 - 60 nucleotides and about 15 - 50 nucleotides in length. Generally, the adapter can include any combination of nucleotides and/or nucleic acids. In some embodiments, the adapter can include one or more cleavable groups at one or more locations. In another aspect, the adapter can include a sequence that is substantially identical, or substantially complementary', to at least a portion of a primer, for example a universal primer. In some embodiments, the adapter can include a barcode or tag to assist with downstream error correction, identification or sequencing.

[0032] As used herein "amplification" refers to a process in which a copy number increases. Amplification may be a process in which replication occurs repeatedly over time to form multiple copies of a template. Amplification can produce an exponential or linear increase in the number of copies as amplification proceeds. Exemplary amplification strategies include polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), rolling circle replication (RCA), cascade-RCA, nucleic acid-based amplification (NASBA), and the like. Also, amplification can utilize a linear or circular template. Amplification can be performed under any suitable temperature conditions, such as with thermal cycling or isothermally. Furthermore, amplification can be performed in an amplification mixture (or reagent mixture), which is any composition capable of amplifying a nucleic acid target, if any, in the mixture. PCR amplification relies on repeated cycles of heating and cooling (i.e., thermal cycling) to achieve successive rounds of replication. PCR can be performed by thermal cy clingAttorney Docket No P39431-WO-1

between two or more temperature setpoints, such as a higher denaturation temperature and a lower annealing/ extension temperature, or among three or more temperature setpoints, such as a higher denaturation temperature, a lower annealing temperature, and an intermediate extension temperature, among others. PCR can be performed with a thermostable polymerase, such as Taq DNA polymerase. PCR produces an exponential increase in the amount of a product amplicon over successive cycles. PCR is described, for example, in U.S. Pat. No.

4,683,202; U.S. Pat. No. 4,683.195; U.S. Pat. No. 4.000,159; U.S. Pat. No. 4,965.188; U.S. Pat. No. 5,176,995), the disclosures of each are hereby incorporated by reference herein in their entirety .

[0033] As used herein, the term "end" or "ends" refer to the regions of sequence at (or proximal to) either end of a nucleic acid sequence. As used herein, the term "3' region" refers to a region of a nucleotide strand that includes the 3' end of the strand. As used herein, the term "3' end" designates the end of a nucleotide strand that has the hydroxyl group of the third carbon in the sugar-ring of the deoxyribose at its terminus. As used herein, the term "5' region" refers to a region of a nucleotide strand that includes the 5' end of the strand. As used herein, the term "5' end" designates the end of a nucleotide strand that has the fifth carbon in the sugar-ring of the deoxyribose at its terminus.

[0034] As used herein, a "library" refers to a collection of nucleic acids. A library can contain one or more fragments of nucleic acid molecules. In some embodiments, the fragments are amplified nucleic acid molecules. In other embodiments, the fragments are nucleic acid molecules that are not amplified. In some embodiments, the fragments are target nucleic acid molecules. In some embodiments, a library may include a nucleic acid molecule that has one or more known oligonucleotide sequence(s) added to the 3' end, the 5' end, or both the 3' and 5' end (e.g., adapters, y-adapters, etc.). In some embodiments, the library may be prepared so that the fragments can contain a known oligonucleotide sequence that identifies the source of the library (e.g., a molecular identification barcode identifying a patient or DNA source).

[0035] As used herein, the term " ligation" refers to a condensation reaction j oining tw o nucleic acid strands wherein a 5'-phosphate group of one molecule reacts with the 3'-hydroxyl group of another molecule. Uigation is ty pically an enzy matic reaction catalyzed by a ligase or a topoisomerase. Ligation may join two single strands to create one single-stranded molecule. Ligation may also join two strands each belonging to a double-stranded molecule thus joining two double-stranded molecules. Ligation may also join both strands of a doublestranded molecule to both strands of another double-stranded molecule thus joining twoAttorney Docket No P39431-WO-1

double-stranded molecules. Ligation may also join two ends of a strand within a doublestranded molecule thus repairing a nick in the double-stranded molecule.

[0036] As used herein, the term "nanopore" refers to a pore, channel, or passage formed or otherwise provided in a membrane or other barrier material that has a characteristic width or diameter of about 0.1 nm to about 1000 nm. A nanopore can be made of a naturally occurring pore-forming protein, such as a-hemolysin from S. aureus, or a mutant or variant of a wildtype pore-forming protein, either non-naturally occurring (i.e., engineered) such as a-HL-C46, or naturally occurring. A membrane may be an organic membrane, such as a lipid bilayer, or a synthetic membrane made of a non-naturally occurring polymeric material. The nanopore may be disposed adjacent or in proximity to a sensor, a sensing circuit, or an electrode coupled to a sensing circuit, such as, for example, a complementary metal-oxide semiconductor (CMOS) or field effect transistor (FET) circuit.

[0037] As used herein, the terms "nucleic acid" or "nucleic acid molecule" as used herein, refer to a high-molecular-w eight biochemical macromolecule composed of nucleotide chains that convey genetic information. The most common nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The monomers from which nucleic acids are constructed are called nucleotides. Each nucleotide consists of three components: a nitrogenous heterocyclic base, either a purine or a pyrimidine (also known as a nucleobase); and a pentose sugar. Different nucleic acid types differ in the structure of the sugar in their nucleotides; DNA contains 2-deoxyribose while RNA contains ribose.

[0038] As used herein, the term "next generation sequencing" refers to sequencing technologies having high-throughput sequencing as compared to traditional Sanger- and capillary electrophoresis-based approaches, wherein the sequencing process is performed in parallel, for example producing thousands or millions of relatively small sequence reads at a time. Some examples of next generation sequencing techniques include, but are not limited to, sequencing by synthesis, sequencing by ligation, and sequencing by hybridization. These technologies produce shorter reads (anywhere from about 25 - about 500 bp) but many hundreds of thousands or millions of reads in a relatively short time. Examples of such sequencing devices available from Illumina (San Diego, CA) include, but are not limited to iSEQ, MiniSEQ, MiSEQ, NextSEQ, NoveSEQ.

[0039] It is believed that the Illumina next-generation sequencing technology uses clonal amplification and sequencing by synthesis (SBS) chemistry' to enable rapid sequencing. The process simultaneously identifies DNA bases while incorporating them into a nucleic acid chain. Each base emits a unique fluorescent signal as it is added to the growing strand, whichAttorney Docket No P39431-WO-1

is used to determine the order of the DNA sequence. A non-limiting example of a sequencing device available from ThermoFisher Scientific (Waltham, MA) includes the Ion Personal Genome Machine™ (PGM™) System.

[0040] It is believed that Ion Torrent sequencing measures the direct release of H+ (protons) from the incorporation of individual bases by DNA polymerase. A non-limiting example of a sequencing device available from Pacific Biosciences (Menlo Park, CA) includes the PacBio Sequel Systems. A non-limiting example of a sequencing device available from Roche (Pleasanton, CA) is the Roche 454. Next-generation sequencing methods may also include nanopore sequencing methods. In general, three nanopore sequencing approaches have been pursued: strand sequencing in which the bases of DNA are identified as they pass sequentially through a nanopore, exonuclease-based nanopore sequencing in which nucleotides are enzymatically cleaved one-by-one from a DNA molecule and monitored as they are captured by and pass through the nanopore, and a nanopore sequencing by synthesis (SBS) approach in which identifiable polymer tags are attached to nucleotides and registered in nanopores during enzyme-catalyzed DNA synthesis. Common to all these methods is the need for precise control of the reaction rates so that each base is determined in order.

[0041] Strand sequencing requires a method for slowing down the passage of the DNA through the nanopore and decoding a plurality of bases within the channel, ratcheting approaches, taking advantage of molecular motors, have been developed for this purpose. Exonuclease-based sequencing requires the release of each nucleotide close enough to the pore to guarantee its capture and its transit through the pore at a rate slow enough to obtain a valid ionic current signal. In addition, both methods rely on distinctions among the four natural bases, two relatively similar purines and two similar pyrimidines.

[0042] The nanopore SBS approach utilizes synthetic polymer tags attached to the nucleotides that are designed specifically to produce unique and readily distinguishable ionic current blockade signatures for sequence determination. In some embodiments, sequencing of nucleic acid molecules includes via nanopore sequencing includes preparing nanopore sequencing complexes and determining polynucleotide sequences. Methods of preparing nanopores and nanopore sequencing are described in U.S. Patent Application Publication No.

2017/0268052, and PCT Publication Nos. WO2014/074727, W02006/028508, WO2012/083249, and WO/2014/074727, the disclosures of which are hereby incorporated by reference herein in their entireties. In some embodiments, tagged nucleotides may be used in the determination of the polynucleotide sequences (see, e.g., PCT Publication No.Attorney Docket No P39431-WO-1

WO/2020/131759, WO/2013/191793, and WO/2015/148402, the disclosures of which are hereby incorporated by reference herein in their entireties).

[0043] Analysis of the data generated by sequencing is performed using software and/or statistical algorithms that perform various data conversions, e.g., conversion of signal emissions into base calls, conversion of base calls into consensus sequences for a nucleic acid template, etc. Such software, statistical algorithms, and the use of such are described in detail, in U.S. Patent Application Publication Nos. 2009/0024331 2017/0044606 and in PCT Publication No. WO/2018/034745, the disclosures of which are hereby incorporated by reference herein in their entireties.

[0044] As used herein, the term "nucleotide" refers to a nucleoside-5'-oligophosphate compound, or structural analog of anucleoside-5'-oligophosphate, which can act as a substrate or inhibitor of a nucleic acid polymerase. Exemplar}' nucleotides include, but are not limited to, nucleoside-5 '-triphosphates (e.g., dATP, dCTP, dGTP, dTTP, and dUTP); nucleosides (e.g., dA, dC, dG, dT, and dU) with 5'-oligophosphate chains of 4 or more phosphates in length (e.g., 5'-tetraphosphosphate, 5'-pentaphosphosphate, 5'-hexaphosphosphate, 5'-heptaphosphosphate, 5'-octaphosphosphate); and structural analogs of nucleoside-5 '-triphosphates that can have a modified base moiety (e.g., a substituted purine or pyrimidine base), a modified sugar moiety (e.g., an O-alkylated sugar), and/or a modified oligophosphate moiety (e.g., an oligophosphate comprising a thio-phosphate, a methylene, and/or other bridges between phosphates).

[0045] As used herein, the "polymerase" as used herein, refers to an enzyme that catalyzes the process of replication of nucleic acids. More specifically, DNA polymerase catalyzes the polymerization of deoxyribonucleotides alongside a DNA strand, which the DNA polymerase "reads" and uses as a template. The newly polymerized molecule is complementary to the template strand and identical to the template's partner strand.

[0046] As used herein, the term "sample" or the like refers to any sample including a biomolecule (such as a protein, a peptide, a nucleic acid, a lipid, a carbohydrate, or a combination thereof) that is obtained from any organism including viruses. Other examples of organisms include mammals (such as humans; veterinary animals like cats, dogs, horses, cattle, and swine; and laboratory animals like mice, rats, and primates), insects, annelids, arachnids, marsupials, reptiles, amphibians, bacteria, and fungi. Biological samples include tissue samples (such as tissue sections and needle biopsies of tissue), cell samples (such as cytological smears such as Pap smears or blood smears or samples of cells obtained by microdissection), or cell fractions, fragments, or organelles (such as obtained by lysing cells and separating theirAttorney Docket No P39431-WO-1

components by centrifugation or otherwise). Other examples of biological samples include blood, serum, urine, semen, fecal matter, cerebrospinal fluid, interstitial fluid, mucous, tears, sweat, pus, biopsied tissue (for example, obtained by a surgical biopsy or a needle biopsy), nipple aspirates, cerumen, milk, vaginal fluid, saliva, swabs (such as buccal swabs), or any material containing biomolecules that is derived from a first biological sample. In certain embodiments, the term "biological sample" as used herein refers to a sample (such as a homogenized or liquefied sample) prepared from a tumor or a portion thereof obtained from a subject.

[0047] As used herein, the term "sequence," when used in reference to a nucleic acid molecule, refers to the order of nucleotides (or bases) in the nucleic acid molecules. In cases, where different species of nucleotides are present in the nucleic acid molecule, the sequence includes an identification of the species of nucleotide (or base) at respective positions in the nucleic acid molecule. A sequence is a property of all or part of a nucleic acid molecule. The term can be used similarly to describe the order and positional identity of monomeric units in other polymers such as amino acid monomeric units of protein polymers.

[0048] As used herein, the term "sequence complementarity " refers to a property shared between two nucleic acid sequences, such that when they are aligned antiparallel to each other, the nucleotide bases at each position will be complementary.

[0049] As used herein, the term "sequencing" refers to the determination of the order and position of bases in a nucleic acid molecule. More particularly, the term "sequencing" refers to biochemical methods for determining the order of the nucleotide bases, adenine, guanine, cytosine, and thymine, in a DNA oligonucleotide. Sequencing, as the term is used herein, can include without limitation parallel sequencing or any other sequencing method known of those skilled in the art, for example, chain-termination methods, rapid DNA sequencing methods, wandering-spot analysis, Maxam-Gilbert sequencing, dye- terminator sequencing, or using any other modem automated DNA sequencing instruments.

[0050] OVERVIEW

[0051] Disclosed herein is a method for preparing sequencing libraries from DNA and RNA simultaneously, i.e., providing genomic and transcriptomic information in a single tube without the need for separate DNA and RNA library preparation workflows. When information is desired from both DNA and RNA, it is especially advantageous to have a combined workflow as described herein so a single source (e g., a single patient specimen) could be used. This, it is believed, reduces the need for sample material as well as eliminatesAttorney Docket No P39431-WO-1

errors. This is especially advantageous for precious samples, such as clinical plasma or formalin fixed paraffin embedded tissue (FFPET) samples, forensic samples or historical or archival samples. Moreover, for these precious samples, there may not even be enough material to perform two separate isolations for DNA and RNA. With simultaneous analysis of DNA and RNA from a single source as described herein, the additional information gleaned from the second type of nucleic acid can be significant. For example, DNA holds information about mutations, including single nucleotide variants (SNVs) and copy number variations (CNVs). In addition, the information derived from the DNA can be quantitative, i.e., reflect not only the type of mutation but also the mutation burden in the tumor sample. By contrast, RNA provides qualitative information about mutations as the varying expression levels obscure the mutation burden in the genome. At the same time, gene transcription amplifies the signal from a rare mutation event making it easier to detect. Analysis of RNA is especially useful for detecting gene fusions in the background of wild-type DNA sequences from both fusion partners.

[0052] The present disclosure is also directed to different methods for selectively tagging an RNA fraction within a total nucleic acid sample, i.e., a sample which includes both DNA and RNA). Also disclosed herein are methods of preparing a library including double stranded DNA and double stranded cDNA, where the double stranded cDNA is derived from selectively tagged RNA molecule present in a total nucleic acid sample. The methods disclosed herein are intended for use in total nucleic acid as well as target enrichment sequencing methodologies. The selective labeling (e g., tagging) aims to discriminate between RNA from DNA molecules in sequencing libraries.

[0053] Also disclosed herein are kits, such as kits including one or more components for selectively tagging RNA molecules within a total nucleic acid sample; and/or generating double stranded cDNA from selectively tagged RNA molecules.

[0054] SELECTIVE TAGGING OF TOTAL NUCLEIC ACID SAMPLES [0055] Disclosed herein are various methods of selectively tagging RNA molecules within a total nucleic acid a sample. In some embodiments, the present disclosure provides a method for preparing a nucleic acid library including cDNA and DNA derived from a total nucleic acid sample, the method comprising (a) obtaining a sample including at least an RNA fraction and a DNA fraction; and (b) selectively modifying the RNA fraction to generate a modified RNA fraction including an oligonucleotide tag and an unmodified DNA component. In some embodiments, the modified RNA fraction is converted to double stranded cDNA. such as double stranded cDNA which includes an adapter sequence. In some embodiments, theAttorney Docket No P39431-WO-1

prepared nucleic acid library is sequenced, such as using a next-generation sequencing technique. The methods of the present disclosure do not require physical separation of the DNA and RNA from the sample.

[0056] An overview of the methods of the present disclosure is set forth in FIG. 1. In some embodiments, a total nucleic acid sample is obtained, i.e., a sample is obtained that includes DNA and RNA molecules (step 100). Next, (i) RNA; or (ii) RNA and DNA in the total nucleic acid sample is fragmented (step 101). Subsequently, the nucleic acid molecules in the sample are prepared for the ligation of an oligonucleotide tag to the fragmented RNA molecules. In some embodiments, an oligonucleotide tag, such as one having a known sequence, is ligated to the fragmented RNA molecules (step 102). Next, double stranded cDNA molecules and double stranded DNA molecules, each including an adapter sequence, are prepared (step 103). The double stranded cDNA molecules and double stranded DNA molecules, each including an adapter sequence, may then be sequenced (step 104). Variations of the aforementioned mentioned method are set forth in FIGS. 2 - 6 and described in further detail herein.

[0057] The methods of the present disclosure permit for simultaneous transcriptome analysis and genomic or epigenomic analysis of a total nucleic acid sample, while preventing or mitigating the loss of any nucleic acid material from the sample. In some embodiments, the methods of the present disclosure permit the analysis of copy number variation, the concurrent measurement of mutations in the RNA and DNA to identify tumor neoantigens, etc. In some embodiments, the methods of the present disclosure permit for the detection of variants in the genome (e.g., SNPs, the transcriptome-like fusions and splice variants post translation).

[0058] SAMPLE PREPARATION

[0059] In some embodiments, a sample (e.g., a total nucleic acid sample) comprising one or more nucleic acid molecules is obtained and prepared for downstream processing. In some embodiments, samples may be obtained from any source including a target nucleic acid molecule having one or more modified nucleotides, e.g., tissue (including tumor tissue or formalin-fixed paraffin-embedded (FFPE) tissue), blood, skin, swab (e.g., buccal, vaginal), urine, saliva, etc. In some embodiments, the sample is derived from a subject or a patient, such as a subject or a patient diagnosed with a disease or suspected of having a disease. In some embodiments, the sample may include a fragment of a solid tissue, or a tumor sample derived from the subject or the patient, e.g., by biopsy. As used herein, the term "tumor sample" encompasses samples prepared from a tumor or from a sample potentially including orAttorney Docket No P39431-WO-1

suspected of comprising cancer cells, or to be tested for the potential presence of cancer cells, such as a lymph node. As used herein, the term "tumor" refers to a mass or a neoplasm, which itself is defined as an abnormal new growth of cells that usually grow more rapidly than normal cells and will continue to grow if not treated sometimes resulting in damage to adjacent structures. Tumor sizes can vary' widely. A tumor may be solid, or fluid filled. A tumor can refer to benign (not malignant, generally harmless), or malignant (capable of metastasis) growths. Some tumors can include neoplastic cells that are benign (such as carcinoma in situ) and, simultaneously, contain malignant cancer cells (such as adenocarcinoma). This should be understood to include neoplasms found in multiple locations throughout the body. Therefore, for purposes of the present disclosure, tumors include primary tumors, lymph nodes, lymphatic tissue, and metastatic tumors. In some embodiments, the sample is a fresh sample, such as a fresh-frozen sample. In some embodiments, the sample is a fixed sample. In some embodiments, the sample is a formalin-fixed paraffin embedded sample. In some embodiments, the sample comprises ctDNA or cfDNA.

[0060] Methods for isolating nucleic acid molecules from obtained samples and/or purifying the obtained samples are known (see. e.g., Sambrook et al.. Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989; Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Press, 2001) and several kits are commercially available (e.g., High Pure RNA Isolation Kit, High Pure Viral Nucleic Acid Kit, and MagNA Pure LC Total Nucleic Acid Isolation Kit, DNA Isolation Kit for Cells and Tissues, DNA Isolation Kit for Mammalian Blood, High Pure FFPET DNA Isolation Kit, available from Roche). In the context of the presently disclosed methods, nucleic acid molecules, including genomic DNA, can be collected, purified, and/or isolated.

[0061] In some embodiments, the nucleic acid molecules within the obtained sample are selected from DNA molecules, genomic DNA molecules, cfDNA molecules, cDNA molecules, RNA molecules, mRNA molecules, rRNA molecules, mtDNA, siRNA molecules, or any combination thereof, provided that the sample includes an RNA fraction and a DNA component. In some embodiments, a ratio between RNA and DNA in a sample may range from between about 0.2:1 to about 25:1, such as from about 0.4 to about 22:1, such as from about 0,5:1 to about 20:1, etc.

[0062] Selective Tagging of 5¹ Terminal Ends of Fragmented RNA Molecules [0063] A first method of selectively tagging total nucleic acid molecules comprises the selective tagging of the 5' terminal ends of fragmented RNA molecules with an oligonucleotide tag having a predetermined sequence. With reference to FIG. 2, a first step of this particularAttorney Docket No P39431-WO-1

method comprises obtaining a total nucleic acid sample comprising DNA molecules and RNA molecules (step 200), such as described above.

[0064] Next, the RNA molecules in the total nucleic acid sample are selectively fragmented (step 201), i.e., the RNA molecules in the sample are fragmented but the DNA molecules are not. In some embodiments, the RNA molecules are selectively fragmented using an RNA endonuclease (also referred to an "endoribonuclease"). In some embodiments, the RNA endonuclease is RNase III (see FIG. 3A).

[0065] In some embodiments, the RNA endonuclease provides fragmented RNA molecules having 5' phosphate ends and 3' hydroxyl ends. Thus, the sample will include fragmented RNA molecules and unfragmented DNA molecules both having 5' phosphate and 3' hydroxyl ends (see FIGS. 3B and 3C). In these embodiments, the 5' phosphate is removed by introducing an enzy me that dephosphorylates the 5' ends of RNA and DNA molecules. In some embodiments, the 5' phosphate is removed (i.e., dephosphorylated) with a phosphatase, such as calf-intestinal alkaline phosphatase. Dephosphorylation of the 5' phosphate of the RNA and DNA molecules results in the formation of RNA and DNA molecules having 5' hydroxyl ends.

[0066] Alternatively, the RNA molecules may be fragmented using a RNaseT2 endonuclease. In these embodiments, the RNAaseT2 fragments the RNA molecules within the sample, providing fragmented RNA molecules and unfragmented DNA molecules each including a 5' hydroxyl end and a 3' phosphate end.

[0067] In some embodiments, the RNA fragments have a length ranging from about 50 nucleotides to about 1500 nucleotides. In other embodiments, the RNA fragments have a length ranging from about 80 nucleotides to about 1200 nucleotides. In yet other embodiments, the RNA fragments have a length ranging from about 100 nucleotides to about 1000 nucleotides.

[0068] Following the selective fragmentation of the RNA molecules within the sample, an oligonucleotide tag is ligated to the 5' ends of the RNA fragments (step 202). In some embodiments, the oligonucleotide tag has a predetermined nucleotide sequence, or at least a portion of the oligonucleotide tag has a predetermine nucleotide sequence. In some embodiments, the oligonucleotide tag comprises at least 5 nucleotides, such as at least 10 nucleotides, such as at least 15 nucleotides, such as at least 20 nucleotides, such as at least 25 nucleotides, such as at least 30 nucleotides, etc. In some embodiments, the oligonucleotide tag is single stranded. In some embodiments, the tag is any sequence that can be distinguishable and not occurring in the genome under investigation.Attorney Docket No P39431-WO-1

[0069] In some embodiments, the oligonucleotide tag permits identification of the fragmented RNA molecule to which it is ligated. In embodiments where the only feature to be determined based on the presence of the oligonucleotide tag (or the sequence of the oligonucleotide tag ) is the type of polynucleotide (e.g., where presence indicates the sequence corresponds to the sequence of an RNA from the sample), an oligonucleotide tag having the same tag sequence can be joined to polynucleotides from multiple different samples. If two or more different samples are to be distinguished based on tag sequences, then oligonucleotide tags having differing nucleotide sequences will, in some embodiments, differ between the two or more different samples. In some embodiments, oligonucleotide tags are of sufficient length and comprise sequences that are sufficiently different to allow the identification of samples based on tag sequences with which they are associated.

[0070] In some embodiments, the oligonucleotide tag includes one or more indexes, barcodes, or UMIs. In some embodiments, UMIs may be incorporated as part of an overall DNA amplification and sequencing workflow to perform error correction. In some embodiments, errors are introduced (1) by the polymerase during amplification, and (2) during sequencing (i.e., reading) of the amplified molecules. In some embodiments, UMIs ligated to nucleic acid molecules reduce the impact of one or both sources of error. For instance, UMIs incorporate a unique barcode onto each molecule within a given sample library . By incorporating individual barcodes on each original DNA fragment, variant alleles present in the original sample (true variants) can be distinguished from errors introduced during library preparation, target enrichment, or sequencing.

[0071] As noted above, the oligonucleotide tag is ligated to the 5' hydroxyl ends of the fragmented RNA molecules (step 202). In some embodiments, the ligation is performed using a RtcB ligase (see FIG. 3C). RtcB ligase is an atypical RNA ligase that joins either 2',3'-cyclic phosphates or 3'-phosphate termini to 5' hydroxyl termini. Without wishing to be bound by any particular, tit is believed that the RtcB ligase is specific for this function and may only ligate the tag in this conformation to the DNA.

[0072] In some embodiments, the tag is an RNA tag. In other embodiments, the tag is a DNA tag.

[0073] Next, the DNA molecules in the sample are fragmented, providing a sample including tagged RNA molecules and fragmented DNA molecules (see FIG. 3D). The DNA molecules in the sample may be selectively fragmented using an enzymatic technique. In some embodiments, the DNA molecules in the sample are enzymatically fragmented using a DNase, such as DNasel, shrimp DNase, and salt tolerant DNase I-XT.Attorney Docket No P39431-WO-1

[0074] In some embodiments, the DNA fragments have a length ranging from about 100 nucleotides to about 1500 nucleotides. In other embodiments, the DNA fragments have a length ranging from about 125 nucleotides to about 1250 nucleotides. In yet other embodiments, the DNA fragments have a length ranging from about 180 nucleotides to about 1000 nucleotides.

[0075] Following the fragmentation of the DNA in the sample, in some embodiments, the fragmented nucleic acid molecules are end repaired and then a "tailing" reaction is performed (see FIG. 3D). Tailing is an enzymatic method for adding a non-templated nucleotide to the 3' end of a blunt, double-stranded DNA molecule. In some embodiments, a Taq polymerase is utilized for A-tailing.

[0076] Next, a double stranded cDNA molecule is prepared from the tagged RNA molecules in the sample. In some embodiments, a first-strand synthesis is first performed, which generates a DNA / RNA hybrid (step 204; see also FIG. 3E). In some embodiments, the first-strand synthesis is performed using a reverse transcriptase and random primers. In some embodiments, the random primers comprise 5 to 7 nucleotides. In some embodiments, the random primers comprise 6 nucleotides, i.e., the random primer is a random hexamer primer. In some embodiments, the random hexamer primers are synthesized entirely randomly by using a mixture of A, G, C and T at every’ coupling step to give a numerous range of all possible 6-mer sequences (4096 sequences). In some embodiments, the reverse transcriptase primers will anneal to the tagged RNA molecules and extend to generate a DNA compliment (i.e., a reverse transcription of the target). In some embodiments, the primers are oligo(dT)N primers or anchored oligo(dT)N primers. Without wishing to be bound by any particular theory', it is believed that an anchored primer may help to control the length of the Atail tag. In some embodiments, using a primer that is not tagged will result in more variable Atail tag lengths.

[0077] Following first-strand synthesis, a second-strand synthesis is performed to provide double stranded cDNA (step 205, see also FIG. 3F). Any method of forming cDNA may be utilized. In some embodiments, second-strand synthesis is performed using an RNase H (which nicks the RNA strands of cDNA:RNA complexes, providing 3'-OH priming sites for DNA synthesis), a DNA polymerase I (which extends the nicked RNA strands by 5' to 3' polymerase activity and replaces the RNA strand in the direction of synthesis by 5' to 3' exonuclease activity', in a process known as nick translation), a DNA ligase (which seals the nicks between the newly synthesized cDNA segments), and a T4 DNA polymerase (which blunts the termini of the double-stranded cDNA). In some embodiments, second strandedAttorney Docket No P39431-WO-1

synthesis performed according to methods known in the art, such as by following the procedures established in the Kapa RNA HyperPrep kit (available from Roche).

[0078] Finally, adapters are ligated to the double stranded cDNA and DNA molecules in the sample (step 206, see also FIG. 3G). In some embodiments, the adapters are Y-adapters. The term "Y-adapter" refers to an adapter formed by two DNA strands (see for example, U.S. Pat. No. 6.346,399, U.S. Pat. No. 7,741,463. US Patent application US 2007/0172839, and International Publication No. W02007/111937; the disclosures of which are hereby incorporated by reference herein in their entireties). A Y-adapter includes single stranded non-complementary regions (also referred to herein as "arms," "single-stranded arms," or "arm regions") and a double stranded complementary' region (also referred to herein as "stem regions"). In particular, a first portion (a 3' region) of the first strand and a first portion (a 5' region) of a second strand form a double stranded region by sequence complementarity. The ends of the double stranded region formed by the 3' region of the first DNA strand and the 5' region of the second DNA strand of the Y-adapter are compatible with the ends of the double stranded nucleic acid molecules within the obtained sample. A second portion of the first strand and a second portion of the second strand comprise non-self-complementary single stranded regions. In some embodiments, the Y-adapter is configured for ligation to a double stranded nucleic acid molecule. In some embodiments, a double stranded region of aY-adapter comprises a 5'-overhang or a 3'-overhang that is complementary to a 3'-overhang or a 5'-overhang of an end of a double stranded nucleic acid molecule.

[0079] In some embodiments, the Y-adapters include one or more regions which permit binding of adapter ligated nucleic acid molecules to flow cells. In some embodiments, the Y-adapters include a P5 region and a P7 region.

[0080] In some embodiments, the adapter ligated double stranded cDNA and DNA molecules in the sample may be amplified prior to sequencing. In some embodiments, the sample may be amplified using an isothermal amplification technique.

[0081] In some embodiments, the adapter ligated double stranded cDNA and DNA molecules (see FIG. 3H) in the sample may then be sequenced, such as with a next-generation sequencing technique.

[0082] Polyadenylation Tagging of RNA Fraction of Total Nucleic Acid Samples [0083] A second method of selectively tagging total nucleic acid molecules comprises the polyadenylation tagging of an RNA fraction within a total nucleic acid sample. With reference to FIG. 4, a first step of this particular method comprises obtaining a total nucleicAttorney Docket No P39431-WO-1

acid sample comprising DNA molecules and RNA molecules (step 400), such as described above.

[0084] Next, both the RNA molecules and DNA molecules in the total nucleic acid sample are fragmented (step 401; see also FIG. 5 A). In some embodiments, the RNA molecules and DNA molecules in the sample are fragmented using such procedures as mechanical force, sonication, restriction endonuclease cleavage, or any method known in the art. In some embodiments, the fragmented RNA and DNA molecules within any obtained sample have a size ranging from between about 20 mer to about 70 mer. In other embodiments, the fragmented RNA and DNA molecules within any obtained sample have a size ranging from between about 30 mer to about 50 mer. Following the fragmentation of the sample, in some embodiments, the fragmented nucleic acid molecules are end repaired (see FIG. 5B). In some embodiments, only DNA is end repaired.

[0085] Following the fragmentation of the RNA and DNA molecules in the sample, the RNA molecules are selectively polyadenylated to incorporate a polyA tail (step 402, see also FIG. 5C). In some embodiments, the fragmented RNA molecules are polyadenylated using a Poly(A) Polymerase, such as E. coh Poly(A) Polymerase, where the Poly(A) Tail incorporated serves as an oligonucleotide tag. In some embodiments, the Poly(A) Polymerase is any template-independent enzyme capable of polyadenylating the 3' end of an RNA molecule. Other Poly(A) Polymerases are described in International Publication No. WO/1993/011227; and in U.S. Patent Nos. 11,384.375, 8,088.574, and 6.511,832; the disclosures of which are hereby incorporated by reference herein in their entireties. Another suitable enzyme includes E.coli with Yeast.

[0086] In some embodiments, the polyA tail includes a random number of A bases, e.g., (A)_n, where n indicates a random number of bases. For instance, the polyA tail may include between 5 and 45 A bases, such as between 8 and 30 bases, such as between 10 and 30 bases, etc.

[0087] Next, the polyA-tailed RNA molecules in the sample are converted to double stranded cDNA molecules, to provide a sample including double stranded cDNA molecules and fragmented DNA molecules. In some embodiments, a first-strand synthesis is first performed, which generates a cDNA / RNA hybrid (step 403). In some embodiments, the first-strand synthesis is performed using a reverse transcriptase and a primer specific to at least a portion of the polyA tail incorporated into the polyA-tailed RNA molecules (see FIGS. 5D and 5E). Said another way, at least a portion of the polyA tail incorporated into the polyA-tailed RNA molecules is utilized to prime an RNA first-strand synthesis, described further herein. InAttorney Docket No P39431-WO-1

some embodiments, the number of nucleotides of the poly A tail that are used for priming depends on the length of A-tail extension. In some embodiments, the primer has the structure: NV(T)_m, where N represents any base, V represents any base except T, and m represents an empirically determined number of T bases. An example of a primer that may be used is a NV base with 15 T bases.

[0088] Following first-strand synthesis, a second-strand synthesis is performed to provide double stranded cDNA (step 404, see also FIG. 5F). Any method of forming cDNA may be utilized. In some embodiments, second-strand synthesis is performed using an RNase H (which nicks the RNA strands of cDNA:RNA complexes, providing 3'-OH priming sites for DNA synthesis), a DNA polymerase I (which extends the nicked RNA strands by 5'3' polymerase activity and replaces the RNA strand in the direction of synthesis by 5'3' exonuclease activity, in a process known as nick translation), a DNA ligase (which seals the nicks between the newly synthesized cDNA segments), and a T4 DNA polymerase (which blunts the termini of the double-stranded cDNA). In some embodiments, second stranded synthesis performed according to methods known in the art. such as by following the procedures established in the Kapa RNA HyperPrep kit (available from Roche).

[0089] Finally, adapters (including any of those described herein) are ligated to the double stranded cDNA and DNA molecules in the sample (step 405, see also FIGS. 5G and 5H). The adapter ligated double stranded cDNA and DNA molecules (see FIG. 5H) in the sample may then be sequenced, such as with a next-generation sequencing technique.

[0090] Selective Indexed Adapter Ligation of an RNA Fraction of Total Nucleic Acid Samples

[0091] A third method of selectively tagging total nucleic acid molecules comprises the selective incorporation of an indexed adapter to an RNA fraction of a total nucleic acid sample. With reference to FIG. 6, a first step of this particular method comprises obtaining a total nucleic acid sample comprising DNA molecules and RNA molecules (step 600), such as described above.

[0092] Next, both the RNA molecules and DNA molecules in the total nucleic acid sample are fragmented (step 601; see also FIG. 7A). In some embodiments, the RNA molecules and DNA molecules in the sample are fragmented using such procedures as mechanical force, sonication, restriction endonuclease cleavage, or any method know n in the art. In some embodiments, the fragmented RNA and DNA molecules within any obtained sample have a size ranging from between about 50 mer to about 1500 mer, such as about from 80 mer to about 1200 mer, such as about from 100 mer to about 1000 mer. Following theAttorney Docket No P39431-WO-1

fragmentation of the sample, in some embodiments, the fragmented nucleic acid molecules are end repaired (see FIG. 7B).

[0093] Subsequently, an oligonucleotide tag is ligated to the RNA fragments within the sample (step 602, see also FIG. 7C). In some embodiments, the oligonucleotide tag serves as a priming site for a reverse oligonucleotide used during first-strand synthesis (described herein).

[0094] In some embodiments, the oligonucleotide tag comprises: (i) a 5' sequence index; (ii) an adapter sequence; and (iii) a 3' terminal di-deoxyribonucleotide (such as to prevent 3'extension and/or prevent or mitigate the octamerization of the adapter). In some embodiments, the sequence index comprises between 4 and 8 nucleotides. In some embodiments, the sequence index comprises 4, 5, 6, 7. or 8 nucleotides. In some embodiments, the sequence index is TAGTAG. In some embodiments, the adapter sequence is a P7 adapter sequence. In other embodiments, the adapter sequence is a P5 adapter sequence. In some embodiments, the 3' terminal di-deoxyribonucleotide is ddCTP. A non-limiting example of an oligonucleotide is 5'-TAGTAG-P7-ddCTP-3'.

[0095] In some embodiments, the oligonucleotide tag is ligated to the fragmented RNA molecules in the sample using an RNA ligase or a T4 RNA ligase II.

[0096] Following ligation of the oligonucleotide tag to the RNA molecules in the sample, a first-strand synthesis is performed to convert the tagged RNA molecules to RNA/DNA hybrids (step 603, see also FIG. 7E). In some embodiments, the first-strand synthesis is performed using a reverse transcriptase and a primer specific to at least a portion of the oligonucleotide tag incorporated into the tagged RNA molecules (see FIGS. 7D and 7E). Said another way, at least a portion of the oligonucleotide tag incorporated into the tagged RNA molecules is utilized to prime an RNA first-strand synthesis. In some embodiments, the primer is complementary to a portion of sequence index and/or a portion of the adapter sequence. For example, for the oligonucleotide tag 5'-TAGTAG-P7-ddCTP-3', a primer may be 3'-ATCATC-P7'. In some embodiments, the primer is a random hexamer.

[0097] Next, adapters are ligated to the hybrid RNA/DNA molecules and DNA molecule sin the same (step 604, see also FIGS. 7F and 7G). In some embodiments, the DNA molecules are A-tailed prior to ligation of the adapters (see FIG. 7E). In some embodiments, the adapters are Y-adapters, including any of those disclosed herein.

[0098] In some embodiments, the adapter-ligated RNA/DNA hybrid molecules and the DNA molecules in the sample are extended (step 605). In some embodiments, the adapter-ligated RNA/DNA hybrid molecules and the DNA molecules are extended with a polymeraseAttorney Docket No P39431-WO-1

and by incorporating dNTPs. In other embodiments, the adapter-ligated RNA/DNA hybrid molecules and the DNA molecules are extended with Kapa HiFi Uracil+ Kit (available from Roche), which can extend across uracil bases of the RNA/DNA hybrids (see FIG. 7G and 7H).

[0099] The extended RNA/DNA hybrid molecules and the extended DNA molecules are then amplified to provide adapter ligated (and tagged) double stranded cDNA molecules and adapter ligated double stranded DNA molecules (step 606, see also FIGS. 7H and 71). The sample may then be sequenced, such as with a next-generation sequencing platform.

[0100] Optional Amplification

[0101] In some embodiments, the sample including double stranded cDNA and double stranded DNA is amplified. For instance, the sample including the double stranded cDNA and double stranded DNA is contacted with a polymerase and/or other amplification reagents to provide one or more amplified nucleic acid molecules.

[0102] Non-limiting examples of polymerases include prokaryotic DNA polymerases (e.g., Pol I, Pol II, Pol III, Pol IV, and Pol V), eukary otic DNA polymerase, archaeal DNA polymerase, etc. In some embodiments, suitable polymerases may be derived from: archaea (e.g.. Thermococcus litoralis (Vent, GenBank: AAA72101), Pyrococcus furiosus (Pfu, GenBank: D12983, BAA02362), Pyrococcus woesii, Pyrococcus GB-D (Deep Vent, GenBank: AAA67131), Thermococcus kodakaraensis KODI (KOD, GenBank: BD175553, BAA06142; Thermococcus sp. strain KOD (Pfx, GenBank: AAE68738)), Thermococcus gorgonarius (Tgo, Pdb: 4699806), Sulfolobus solataricus (GenBank: NC002754, P26811), Aeropyrum pemix (GenBank: BAA81109), Archaeglobus fulgidus (GenBank: 029753), Pyrobaculum aerophilum (GenBank: AAL63952), Pyrodictium occultum (GenBank: BAA07579, BAA07580), Thermococcus 9 degree Nm (GenBank: AAA88769, Q56366), Thermococcus fumicolans (GenBank: CAA93738. P74918), Thermococcus hydrothermalis (GenBank: CAC 18555), Thermococcus sp. GE8 (GenBank: C AC 12850), Thermococcus sp. JDF-3 (GenBank: AX135456; WO0132887), Thermococcus sp. TY (GenBank: CAA73475), Pyrococcus abyssi (GenBank: P77916), Pyrococcus glycovorans (GenBank: CAC12849), Pyrococcus horikoshii (GenBank: NP 143776), Pyrococcus sp. GE23 (GenBank: CAA90887), Pyrococcus sp. ST700 (GenBank: CAC 12847), Thermococcus pacificus (GenBank: AX411312.1), Thermococcus zilligii (GenBank: DQ3366890), Thermococcus aggregans, Thermococcus barossii, Thermococcus celer (GenBank: DD259850.1), Thermococcus profundus (GenBank: E14137), Thermococcus siculi (GenBank: DD259857.1), Thermococcus thioreducens, Thermococcus onnurineus NA1, Sulfolobus acidocaldarium, Sulfolobus tokodaii, Pyrobaculum calidifontis, Pyrobaculum islandicum (GenBank: AAF27815),Attorney Docket No P39431-WO-1

Methanococcus jannaschii (GenBank: Q58295), Desulforococcus species TOK, Desulforococcus, Pyrolobus, Pyrodictium, Staphylothermus, Vulcanisaeta, Methanococcus (GenBank: P52025) and other archaeal B polymerases, such as GenBank AAC62712, P956901, BAAA07579)), thermophilic bacteria Thermus species (e.g., flavus, ruber, thermophilus, lacteus, rubens, aquaticus), Bacillus stearothermophilus, Thermotoga maritima, Methanothermus fervidus, KOD polymerase, TNA1 polymerase, Thermococcus sp. 9 degrees N-7, T4, T7, phi29, Pyrococcus furiosus, P. abyssi, T. gorgonarius, T. litoralis, T. zilligii, T. sp. GT, P. sp. GB-D, KOD, Pfu, T. gorgonarius, T. zilligii, T. litoralis and Thermococcus sp.

9N-7 polymerases.

[0103] To effectuate amplification, the double stranded cDNA and double stranded DNA in the sample are heat denatured. Melting temperatures for heat denaturation are dependent upon several variables including the GC content of the nucleic acid molecule and/or the size of the nucleic acid molecule, but in general may be about 95°C or higher, such as for about 15 seconds to about 2 minutes. Following heat denaturation, oligonucleotide primers are annealed to the template sequence of the double stranded cDNA and double stranded DNA at a lower temperature, (typically between about 40°C and about 60°C, such as for about 30 to about 60 seconds). The annealing temperature, like the heat denaturation temperature, is dependent upon the GC content and/or length of the primers. The oligonucleotides form stable associations ('anneal') with the single stranded DNA (hereinafter referred to as the template strand) and thus serve as primers for nucleic acid synthesis by a polymerase. Subsequently, a corresponding nucleic acid strand to the template is synthesized from the primer oligonucleotide through use of the polymerase and deoxynucleotide triphosphates (dNTPs) (also referred to as "primer extension"). In some embodiments, the temperature is raised for the polymerase, which in the case of commonly used thermostable polymerases is about 74° C, primer extension then lasts approximately 1 to 2 minutes. Reactions take place in a PCR master mixture which includes the nucleic acid molecule, a polymerase, oligonucleotide primers, deoxynucleotide triphosphates (dNTPs), reaction buffer, magnesium and/or optional additives.

[0104] In some embodiments, the sample including the double stranded cDNA and double stranded DNA is isothermally amplified. As used herein, the term "isothermal amplification reaction" means that the temperature does not significantly change during the reaction. In some embodiments, the temperature of the isothermal amplification reaction does not deviate by more than 10° C.. preferably by not more than about 5°C, even more preferably not more than about 2°C. Any method of isothermal amplification (now known or laterAttorney Docket No P39431-WO-1

discovered or optimized) may be utilized to isothermally amplify the one or more nucleic acid molecules in the sample. Non-limiting examples of isothermal amplification techniques include, but are not limited to. Rolling Circle Amplification (RCA), Recombinase Polymerase Amplification (RPA), Strand Displacement Amplification (SDA), Loop-Mediated Isothermal Amplification (LAMP), and Hybridization Chain Reaction (HCR). Other examples of isothermal amplification include nucleic acid sequence-based amplification (NASBA), transcription mediated amplification (TMA), and helicase dependent amplification (HD A). A further example of isothermal amplification includes multiple displacement amplification (MCDA) or a "simple method for amplify ing RNA targets" (SMART). Yet another example of isothermal amplification is cross-priming amplification (CPA). CPA is described in more detail in Fang et al. (Cross-Priming Amplification for Rapid Detection of Mycobacterium tuberculosis in Sputum Specimens, Journal of Clinical Microbiology, March 2009, p. 845-847 Vol. 47, No. 3) and Xu et al. (Cross Priming Amplification: Mechanism and Optimization for Isothermal DNA Amplification, Scientific Reports, February 2012, Vol. 2 No. 246), the disclosures of which are hereby incorporated by reference in their entirety.

[0105] It is believed that isothermal amplification methods provide for the detection of a nucleic acid target sequence in a streamlined, exponential manner, and are not limited by the constraint of thermal cycling. Indeed, isothermal amplification conditions do not include any thermocycling steps in the amplification process. Instead, the reaction is kept at a substantially constant temperature, such as a temperature that does not fluctuate more than about 5°C. such as more than about 4° C, such as more than about 3°C, such as more than about 2°C, etc.

[0106] In some embodiments, isothermal amplification may be conducted at a predetermined temperature ranging from between about 35°C to about 75°C, such as a predetermined temperature ranging from between about 35°C to about 65°C, such as a predetermined temperature ranging from between about 37°C to about 65°C, such as a predetermined temperature ranging from between about 37°C to about 60°C, such as a predetermined temperature ranging from between about 40°C to about 65°C, such as a predetermined temperature ranging from between about 40°C to about 60°C, etc. In some embodiments, the predetermined temperature at which to conduct the isothermal amplification depends on the type of isothermal amplification conducted (e.g., LAMP may be conducted at a temperature of about 60°C to 65°C; TMA may be conducted at a temperature of about 35°C to 45°C, such as at about 42°C; RPA may be conducted at a temperature of about 37°C to 42°C, such as at a temperature of about 40°C; SDA may be conducted at a temperature of about 37°CAttorney Docket No P39431-WO-1

to 60°C; RCA may be conducted at a temperature of about 35°C to 40°C, such as at a temperature of about 37°C; and NASBA may be conducted at a temperature of about 41 °C).

[0107] Optional Target Enrichment

[0108] Following the optional amplification, the sample is optionally enriched for one or more target nucleic acid molecules (step 104). During the step of enrichment, non-target nucleic acid molecules are removed from the amplified sample to provide for an enriched sample, namely a sample enriched for the presence of target nucleic acid molecules.

[0109] Any method may be utilized to enrich the prepared sample for the presence of one or more target nucleic acid molecules. In some embodiments, a hybridization-based target enrichment workflow may be utilized to enrich the prepared sample. In hybridization-based target enrichment workflows, a target area of a target nucleic acid molecule is captured by one or more hybridization probes that can selectively bind to a capture surface. This capture allows the removal of non-target nucleic acids and subsequent release and collection of captured target molecules. Hybridization of target regions may occur either on a solid surface (microarray) or in solution. Hybridization-based target enrichment workflows are described in United States Patent No. 8,383,338, the disclosure of which is hereby incorporated by reference herein in its entirety. Commercial hybridization-based target enrichment workflows are available from Roche Sequencing Solutions, Inc. (e.g., KAPA HyperCap Workflow). Other commercial hybridization-based target enrichment workflows include SECAP EZ Target Enrichment System (ROCHE) and SURESELECT Target Enrichment System (AGILENT).

[0110] By way of example, hybridization-based target enrichment may be performed by capturing the target nucleic acid molecules in a sample with one or more introduced targetspecific probes. In some embodiments, the one or more target nucleic molecules in an obtained sample may be denatured and contacted with single-stranded target-specific probes. In some embodiments, the single-stranded target-specific probes may comprise a ligand for an affinity capture moiety such that following the formation of hybridization complexes, the hybridization complexes are captured by contacting the sample with the affinity capture moiety. In some embodiments, the affinity capture moiety is avidin or streptavidin and the ligand is biotin. In some embodiments, the moiety is bound to solid support. In some embodiments, the solid support may comprise superparamagnetic spherical polymer particles such as DYNABEADS™ magnetic beads or magnetic glass particles.

[OHl] In other embodiments, a primer extension target enrichment (PETE) workflow may be utilized to enrich the prepared input sample (see, e.g., FIGS. 10 and 11). PETE workflows are described in United Patent Application Publication Nos. 2021/0207211 andAttorney Docket No P39431-WO-1

2020/0392483; in United States Patent Nos. 10,907,204 and 11,499,180; and in International Publication Nos. WO/2018/013710 and WO/2022/008578. the disclosures of which are each incorporated by reference herein in their entireties. Commercial PETE workflows are available from Roche (e.g., HAPA HyperPETE Workflow). By way of example only, a PETE workflow may be utilized to enrich a sample with one or more target nucleic acid molecules by: a) providing a reaction mixture comprising the sample and a first target-specific primer, wherein the sample comprises single-stranded target nucleic acid molecule having a 3' and a 5' end and non-target nucleic acid molecules; b) hybridizing a first target-specific primer to the singlestranded target nucleic acid molecules in the reaction mixture, wherein the first target-specific primer hybridizes at least 6 nucleotides from the 3' end of the single-stranded target nucleic acid molecule and comprises an affinity ligand; c) extending the hybridized first target-specific primer with a DNA polymerase to form a first double-stranded product comprising the target nucleic acid molecule hybridized to the extended first target-specific primer, wherein the hybridized target nucleic acid molecule comprises a single-stranded overhang region of at least 6 consecutive nucleotides at the 3' end; d) removing single-stranded target and non-target nucleic acid molecules from the reaction mixture by capturing the affinity ligand of the first double-stranded product; e) hybridizing a second target-specific primer to the single-stranded overhang region at the 3' end of the hybridized target polynucleotide of the captured first double stranded product, wherein the second target-specific primer comprises a 3' hybridizing region and a barcode region; and f) extending the hybridized second target-specific primer with a DNA polymerase, wherein the DNA polymerase comprises strand displacement activity, 5'-3' double stranded DNA exonuclease activity, or a combination thereof, thereby displacing or degrading the extended first target-specific primer and forming a second double-stranded product comprising a barcode, wherein the second double-stranded product comprises the target nucleic acid molecule hybridized to an extended second target-specific primer, wherein the extended second target-specific primer comprises: i) a complement of at least a portion of the target nucleic acid molecule; and, ii) a single-stranded 5' overhang region comprising the barcode.

[0112] Sequencing of cDNA and DNA Molecules in the Formed Libraries [0113] Following the preparation of the libraries including the double stranded cDNA molecules and double stranded DNA molecules, the double stranded cDNA and double stranded DNA within the library are sequenced. In some embodiments, the double stranded cDNA and double stranded DNA within the library may be sequenced and analyzed using methods known to those of skill in the art, e.g., by next-generation sequencing (NGS). In someAttomey Docket No P39431-WO-1

embodiments, the double stranded cDNA and double stranded DNA within the library are sequenced by any suitable method or with nay suitable instrument including SMRT (singlemolecule real-time sequencing), ion semiconductor, pyrosequencing, sequencing by synthesis, combinatorial probe anchor synthesis, and SOLiD sequencing (sequencing by ligation). Nonlimiting sequencing platforms include those provided by Illumina® (e.g., the MiniSeq™, MiSeq™, NextSeq™, and/or NovaSeq™ sequencing systems); Ion Torrent™ (e g., the Ion PGM™, Ion S5™, and/or Ion Proton™ sequencing systems); Pacific Biosciences (e.g., the PACBIO RS II and/or Sequel II System sequencing system); ThermoFisher (e.g., a SOLID® sequencing system); or BGI Genomics (e.g., DNBSeq™ sequencing systems). See, for example U.S. Pat. Nos. 7,211,390; 7,244,559; 7,264,929; 6,255,475; 6,013,445; 8,882,980; 6,664,079; and 9,416,409; the disclosures of which are hereby incorporated by reference herein in their entireties.

[0114] In some embodiments, determination of the sequence of a nucleic acid sequence of interest can be performed using a variety' of sequencing methods know n in the art including, but not limited to, sequencing by synthesis (SBS), sequencing by hybridization (SBH), sequencing by ligation (SBL) (Shendure et al. (2005) Science 309:1728), quantitative incremental fluorescent nucleotide addition sequencing (QIFNAS), stepwise ligation and cleavage, fluorescence resonance energy transfer (FRET), molecular beacons, TaqMan reporter probe digestion, pyrosequencing, fluorescent in situ sequencing (FISSEQ). FISSEQ beads (U.S. Pat. No. 7,425.431), wobble sequencing (PCT/US05/27695), multiplex sequencing (U.S. Ser. No. 12/027,039, filed Feb. 6, 2008; Porreca et al (2007) Nat. Methods 4:931), polymerized colony (POLONY) sequencing (U.S. Pat. Nos. 6,432,360, 6,485,944 and 6,511,803, and PCT/US05/06425); nanogrid rolling circle sequencing (ROLONY) (US2009/0018024), allele-specific oligo ligation assays (e.g., oligo ligation assay (OLA), single template molecule OLA using a ligated linear probe and a rolling circle amplification (RCA) readout, ligated padlock probes, and/or single template molecule OLA using a ligated circular padlock probe and a rolling circle amplification (RCA) readout) and the like. High-throughput sequencing methods, e.g., using platforms such as Roche 454, Illumina Solexa, AB-SOLiD, Helicos, Complete Genomics, Polonator platforms and the like, can also be utilized. A variety of light-based sequencing technologies are known in the art (Landegren et al. (1998) Genome Res. 8:769-76; Kwok (2000) Pharmacogenomics 1:95-100; and Shi (2001) Clin. Chem. 47:164-172).

[0115] In some embodiments, the cDNA molecules in the library can be sequenced by any suitable screening method. In particular, the cDNA library can be sequenced using ahigh-Attorney Docket No P39431-WO-1

throughput screening method, such as Applied Biosystems' SOLiD sequencing technology, or Illumina's Genome Analyzer. In one aspect of the invention, the cDNA library can be shotgun sequenced. The number of reads can be at least 10,000, at least 1 million, at least 10 million, at least 100 million, or at least 1000 million. In another aspect, the number of reads can be from 10,000 to 100,000, or alternatively from 100,000 to 1 million, or alternatively from 1 million to 10 million, or alternatively from 10 million to 100 million, or alternatively from 100 million to 1000 million. A "read" is a length of continuous nucleic acid sequence obtained by a sequencing reaction.

[0116] In some embodiments, analysis of the generated sequencing data can be used for DNA variant detection, copy number analysis, fusion gene detection and structural variant detection, etc. In some embodiments, analysis of sequencing data derived from the cDNA in the library may be used for RNA variant detection, gene expression analysis, fusion gene detection, etc. In some embodiments, the double stranded cDNA and the double stranded DNA in the library can be used for paired DNA and RNA profiling.

[0117] KITS

[0118] The present disclosure also provides for kits including selectively tagging RNA molecules within a total nucleic acid sample. In some embodiments, the kit includes enzymes for fragmenting RNA and DNA molecules within the total nucleic acid sample. In some embodiments, the kit includes: (i) one of an RNA endonuclease or a RNaseT2; and (ii) an enzyme that selectively and specifically fragments DNA. In some embodiments, the kit further includes an oligonucleotide tag. In some embodiments, the kit further includes reagents for the preparation of a double stranded cDNA molecule. For instance, in some embodiments, the kit may include one or more of a reverse transcriptase, an RNase H, a DNA polymerase I, a DNA ligates, and/or a T4 DNA polymerase. In other embodiments, the kit may include (i) a reverse transcriptase, and (ii) one or more of an RNase H, a DNA polymerase I, a DNA ligates, and/or a T4 DNA polymerase. In some embodiments, the kit further includes one or more primers. In some embodiments, the kit further includes one or more adapters.

[0119] EXAMPLES

[0120] Example 1: Specific PolyA-tailing of RNA by Enzymatic Activity [0121] Sample Preparation and Fragmentation

[0122] Input material consisted of 100 ng of E. coli K12 (ATCC) and 200 ng of human Universal Human Reference (UHR) RNA (Agilent). RNA was chemically fragmented using a modified KAPA RNA HyperPrep 2x Fragment, Prime, and Elute Buffer (formulated without hexamers) to achieve a size distribution approximating that of the size distribution of aAttorney Docket No P39431-WO-1

formalin-fixed paraffin-embedded tissue (FFPET) (-200 bp). Fragmentation was performed prior to combining the RNA with DNA for TNA samples.

[0123] Enzymatic Repair and Poly A Tailing

[0124] To prepare the samples (35 pL RNA or TNA), an initial repair step was performed by adding 25 pL of KAPA EvoPlus FragTail Ready Mix. The mixture was vortexed thoroughly and incubated for 15 minutes at 35°C, followed by 30 minutes at 55°C. A 2x beadbased cleanup was then performed using 120 pL KAPA HyperPure beads in the 60 pL reaction volume. The sample was resuspended in 17 pL.

[0125] Following repair, RNA was specifically polyA-tailed using E. coli Poly(A) Polymerase (NEB) according to the manufacturer's standard protocol. The reaction was assembled using 2 pL 10X E. coli Poly(A) Polymerase Reaction Buffer, 2 pL 10 mM ATP, 1 pL E. coli Poly(A) Polymerase, and 16 pL of the sample. After incubation, a 2x cleanup was performed (40 pL KAPA HyperPure beads into the 20 pL reaction), and the library was eluted in 11 pL 10 mM Tris pH 8.0.

[0126] Library Construction via PolyT Priming

[0127] Priming was performed on the eluted sample using specific polyT primers to target the newly-added polyA tails. Two primer variants were utilized: a 20 bp polyT primer (5'-T{20]VN-3') and a 22 bp polyT primer (5'-T{22)VN-3'). A primer mix was prepared by combining 2.2 pL of the specific primer with 8.8 pL of KAPA Fragment and Elute (FE) buffer (excluding standard primers). Ten microliters (10 pL) of this mix were added to the sample, vortexed, and incubated for 1 minute at 65°C.

[0128] First strand synthesis was performed by preparing a master mix of 3 pL KAPA Script and 8 pL First Strand Synthesis Buffer; 10 pL of this mix was added to the primed reaction. Incubation was performed at 25°C for 10 minutes. 42°C for 15 minutes, and 70°C for 15 minutes.

[0129] Second strand synthesis and A-tailing were carried out by adding 30 pL of a master mix containing 2 pL Second Strand/ A-tailing Enzyme Mix and 31 pL Second Strand Marking Buffer. The reaction was incubated for 5 minutes at 42°C followed by 10 minutes at 62°C.

[0130] Adapter Ligation and Cleanup

[0131] Adapter ligation was performed by adding 5 pL of 15 pM KAPA Universal Adapter and 10 pL KAPA Ev oPrep LRM to the reaction mixture. The reaction was incubated for 5 minutes at 20°C. A 0.9x cleanup was subsequently performed using 110 pL KAPAAttorney Docket No P39431-WO-1

HyperPure beads in a 125 pL total reaction volume, and the library' was eluted in 25 pL 10 mM Tris pH 8.0.

[0132] Library Amplification

[0133] Libraries were amplified using KAPA HiFi HotStart ReadyMix (HS RM). Twenty microliters (20 pL) of the eluted library' were combined with 5 pL Universal Primer Mix (UPM) and 25 pL KAPA HiFi HS RM. The cycling conditions were: initial denaturation at 98°C for 45 seconds; 9 cycles of denaturation (98°C, 15 s), annealing (60°C, 30 s), and extension (72°C, 30 s); and a final extension at 72°C for 1 minute. A final lx cleanup was performed using 50 pL KAPA HyperPure beads, and the library' was resuspended in 25 pL 10 mM Tris pH 8.0.

[0134] Analysis of PolyA-Tailed Libraries (see FIG. 8)

[0135] Sequence reads were first aligned to their respective reference genomes to classify origin: E. coli K12 (ATCC) for DNA content and Homo sapiens (hg38) for RNA content. To evaluate the specificity of the E. coli polyA polymerase tailing and subsequent polyT priming, the 5'-end of Read 1 was interrogated. Specifically, the presence of a polyA signature was identified by filtering, where the polyA signature was defined as a stretch of 9 adenines allowing for a single mismatch. This orientation was anticipated because the library preparation preserves strand information; thus, the polyA tail sequence was expected at the start of Read 1, while its absence in Read 2 confirms the directionality and stranded-ness of the sequencing protocol.

[0136] Thus, as can be seen in FIG. 8, the methods described in Example 1 exhibit high specificity' for polyA tagging of RNA and subsequent polyT priming, compared to alternative tagging methodologies.

[0137] Example 2: Specific PolyA tagging of RNA by Low-Complexity PolyA- Tagging

[0138] Sample Preparation and Fragmentation

[0139] Input material consisted of 100 ng of A. coli K12 (ATCC) and 200 ng of human Universal Human Reference (UHR) RNA (Agilent). RNA was chemically fragmented prior to mixing with DNA to achieve a size distribution approximating that of the size distribution of a formalin-fixed paraffin-embedded tissue (FFPET) (-200 bp). For TNA samples, the fragmented RNA was combined with DNA to a total volume of 35 pL.

[0140] Library Preparation via Low-Complexity PolyA TaggingAttorney Docket No P39431-WO-1

[0141] Library preparation was initiated by combining 35 pL of the input sample (RNA or TNA) with 25 pL of KAPA EvoPlus FragTail ReadyMix. The reaction was vortexed thoroughly and incubated for 15 minutes at 35°C, followed by 30 minutes at 55°C.

[0142] To evaluate the specificity of the tagging method, priming was performed using a custom low-complexity tag (5'-AAAAAAAANNNNNN-3'). A mixture containing 2.2 pL of the randomer and 8.8 pL of KAPA RNA HyperPrep Fragment, Prime, and Elute (FPE) buffer (modified to exclude standard primers) was prepared. Ten microliters (10 pL) of this primer mix were added to the EvoPlus reaction, vortexed, and incubated for 1 minute at 65°C.

[0143] First and Second Strand Synthesis

[0144] First strand synthesis utilized standard KAPA HyperRNA components. A master mix comprising 3 pL KAPA Script and 8 pL First Strand Synthesis Buffer was prepared, and 10 pL was added to the primed reaction. The mixture was incubated for 10 minutes at 25°C, 15 minutes at 42°C, and 15 minutes at 70°C.

[0145] Subsequently, second strand synthesis and A-tailing were performed by adding 30 pL of a master mix containing 2 pL Second Strand/ A- tailing Enzyme Mix and 31 pL Second Strand Marking Buffer. The reaction was incubated for 5 minutes at 42°C and 10 minutes at 62°C.

[0146] Adapter Ligation and Cleanup

[0147] Adapter ligation was carried out by combining the reaction mixture with 5 pL of 15 pM KAPA Universal Adapter and 10 pL KAPA EvoPrep LRM. The reaction was incubated for 5 minutes at 20°C. Following ligation, a 0.9x bead-based cleanup was performed using 110 pL KAPA HyperPure beads. The library was eluted in 25 pL 10 mM Tris pH 8.0.

[0148] Library Amplification

[0149] Libraries were amplified using KAPA HiFi HotStart ReadyMix (HS RM). The 25 pL eluted library (20 pL used) was combined with 5 pL Universal Primer Mix (UPM) and 25 pL KAPA HiFi HS RM. Amplification was performed with the following cycling conditions: initial denaturation at 98°C for 45 seconds; 9 cycles of denaturation (98°C, 15 s), annealing (60°C, 30 s), and extension (72°C, 30 s); and a final extension at 72°C for 1 minute. A final lx cleanup was performed using 50 pL KAPA HyperPure beads, and the final library was resuspended in 25 pL 10 mM Tris pH 8.0.

[0150] Analysis of Low Complexity Tag Priming (see FIG. 9)

[0151] For libraries generated via low-complexity' gene-specific priming, reads were similarly mapped to E. coli and human reference genomes. Detection of the tag relied on identifying a homopolymer repeat of 8 adenines at the 5'-end of Read 1, corresponding to theAttorney Docket No P39431-WO-1

randomer primer design (5'-AsN6-3'). Consistent with the stranded nature of the library , this signature was expected exclusively on Read 1. However, in instances where the insert size is shorter than the sequencing read length, the tag sequence may occasionally be observed at the 3'-end of Read 1 due to read-through, though it remains absent from the start of Read 2.

[0152] Thus, as can be seen in FIG. 9, the methods described in Example 2 exhibit high specificity for poly A tagging of RNA, compared to alternative tagging methodologies.

[0153] Example 3: Specific 5'-tagging of RNA using RtcB ligase

[0154] Sample Preparation and Fragmentation

[0155] Input material consisted of 100 ng of E. coli K12 (ATCC) and 200 ng of human Universal Human Reference (UHR) RNA (Agilent). RNA was fragmented using chemical fragmentation to obtain a size distribution similar to that of FFPET (-200 bp); notably, alternative methods such as RNase II digestion or Covaris shearing provide comparable results. Fragmentation was performed prior to combining the RNA with DNA for TNA samples.

[0156] Enzymatic Repair and 5 ' Dephosphorylation

[0157] To initiate library preparation, 35 pL of the sample (RNA or TNA) was combined with 25 pL of KAPA EvoPlus FragTail ReadyMix. The mixture was vortexed thoroughly and incubated for 15 minutes at 35°C, followed by 30 minutes at 55°C. A 2x beadbased cleanup was performed (120 pL KAPA HyperPure beads in the 60 pL reaction volume), and the sample as resuspended in 18 pL of 10 mM Tris (pH 8.0).

[0158] Subsequently, the 5'-phosphate was removed from the nucleic acids using Calf Intestinal Alkaline Phosphatase (CIP). The reaction included 2 pL rCutSmart Buffer, 1 pL CIP, and 17 pL of the repaired sample. Following incubation, a 2x cleanup w as performed (40 pL KAPA HyperPure beads in the 20 pL reaction volume), and the library' was resuspended in 20 pL of 10 mM Tris (pH 8.0).

[0159] RtcB Ligation o f Tag

[0160] Tagging was performed using RtcB RNA Ligase to ligate the 5' end of the input molecules to the 3' end of a custom tag (5'-GCTATAG-PO4-3'). The tag w as designed with a dephosphorylated 5'-end and a phosphorylated 3'-end. The ligation reaction was assembled using 2 pL RtcB Reaction Buffer, 2 pL 1 mM GTP, 2 pL 10 mM MnCh, 1 pL RtcB RNA Ligase, 7 pL of the tag, and 7 pL of the input sample. Post-ligation, a 2x cleanup was performed (40 pL KAPA HyperPure beads in the 20 pL reaction), and the library⁷ was eluted in 11 pL of 10 mM Tris (pH 8.0).

[0161] Library Construction via Random PrimingAttorney Docket No P39431-WO-1

[0162] Priming was performed using a random hexamer (5'-NNNNNN-3'). A primer mix was prepared by combining 2.2 pL of the randomer with 8.8 pL of KAPA Fragment and Elute (FE) buffer (excluding standard primers). Ten microliters (10 pL) of this mix were added to the EvoPlus reaction, vortexed, and incubated for 1 minute at 65°C.

[0163] First strand synthesis utilized standard KAPA HyperRNA components: 3 pL KAPA Script and 8 pL First Strand Synthesis Buffer were combined, and 10 pL of this master mix was added to the reaction. Incubation occurred at 25°C for 10 minutes, 42°C for 15 minutes, and 70°C for 15 minutes.

[0164] Second strand synthesis and A-tailing were performed by adding 30 pL of a master mix containing 2 pL Second Strand/ A-tailing Enzyme Mix and 31 pL Second Strand Marking Buffer. The reaction was incubated for 5 minutes at 42°C followed by 10 minutes at 62°C.

[0165] Adapter Ligation and Amplification

[0166] Adapter ligation was performed by adding 5 pL of 15 pM KAPA Universal Adapter and 10 pL KAPA Ev oPrep LRM to the reaction mixture, followed by incubation for 5 minutes at 20°C. A lx cleanup was then performed using 75 pL KAPA HyperPure beads in the 75 pL reaction volume. The library was resuspended in 25 pL of 10 rnM Tris.

[0167] Libraries were amplified using KAPA HiFi HotStart ReadyMix (HS RM). Twenty microliters (20 pL) of the eluted library were combined with 5 pL Universal Primer Mix (UPM) and 25 pL KAPA HiFi HS RM. Amplification conditions were: initial denaturation at 98°C for 45 seconds; 9 cycles of denaturation (98°C, 15 s), annealing (60°C, 30 s), and extension (72°C, 30 s); and a final extension at 72°C for 1 minute. A final lx cleanup was performed (50 pL beads into 50 pL PCR reaction), and the final library was resuspended in 25 pL of 10 mM Tris (pH 8.0).

[0168] Analysis of 5 '-Ligated Tagging

[0169] The bioinformatics strategy for the 5'-ligation method required a distinct search parameter due to the library⁷ topology⁷. Since the molecular tag (5'-GCTATAG-3') was ligated to the 5'-end of the RNA and the original RNA was depleted during second-strand synthesis, the tag sequence was preserved as the reverse complement (5'-CTATAGC-3') at the 3'-end of the generated cDNA. Consequently, the specific tag sequence within the first 8 bases of Read 2 was screened for. The absence of this tag in Read 1 served as an internal control, validating the strand specificity of the ligation and library⁷ preparation workflow (data not shown).

[0170] Although the present disclosure has been described with reference to several illustrative embodiments, it should be understood that numerous other modifications andAttorney Docket No P39431-WO-1

embodiments can be devised by those skilled in the art that will fall within the spirit and scope of the principles of this disclosure. More particularly, reasonable variations and modifications are possible in the component parts and/or arrangements of the subject combination arrangement within the scope of the foregoing disclosure, the drawings, and the appended claims without departing from the spirit of the disclosure. In addition to variations and modifications in the component parts and/or arrangements, alternative uses will also be apparent to those skilled in the art.

Claims

Attorney Docket No P39431-WO-1 CLAIMS

1. A method of preparing a library comprising one or more cDNA molecules and one or more DNA molecules, comprising:

a. obtaining a sample comprising one or more DNA molecules and one or more RNA molecules;

b. fragmenting the one or more RNA molecules in the sample and optionally fragmenting the one or more DNA molecules in the same to provide a sample including at least one or more fragmented RNA molecules;

c. selectively ligating a tag to the one or more fragmented RNA molecules in the sample to provide one or more tagged RNA molecules; and

d. generating one or more double stranded cDNA molecules from the one or more tagged RNA molecules, wherein the one or more generated double stranded cDNA molecules each include an adapter sequence.

2. The method of claim 1, further comprising sequencing the library comprising the one or more cDNA molecules and the one or more DNA molecules.

3. The method of claim 1, wherein the one or more DNA molecules and the one or more RNA molecules are not physically separated prior to the fragmentation of the one or more RNA molecules in the sample.

4. The method of any one of claims 1-3, wherein the one or more RNA molecules in the obtained sample are selectively fragmented.

5. The method of claim 4, wherein the one or more RNA molecules in the obtained sample are selectively fragmented using an RNA endonuclease.

6. The method of claim 5, wherein the RNA endonuclease is RNase III.

7. The method of claim 4, wherein the one or more RNA molecules in the obtained sample are selectively fragmented using a RNaseT2 endonuclease.

8. The method of any one of claims 4-7, wherein the tag is selectively ligated to the 5' ends of the one or more fragmented RNA molecules.

9. The method of any one of claims 4-8, wherein the tag comprises at least 5 nucleotides.

10. The method of any one of claims 4-8, wherein the ligation is performed using an RNA ligase.

11. The method of claim 10, wherein the RNA ligase is RtcB ligase.

12. The method of any one of claims 4-11, wherein the one or more DNA molecules in the obtained sample are separately fragmented.Attorney Docket No P39431-WO-1

13. The method of claim 12, wherein the one or more DNA molecules are fragmented enzymatically.

14. The method of claim 13, wherein the enzymatic fragmentation of the one or more DNA molecules comprises contacting the sample with a DNase.

15. The method of any one of claims 12-14, wherein the one or more fragmented DNA molecules are end repaired.

16. The method of any one of claims 12-15, wherein the one or more cDNA molecules are prepared by (i) performing a first-strand synthesis on the one or more tagged RNA molecules to provide one or more DNA / RNA hybrid molecules; (ii) performing a second- strand synthesis on the provided one or more DNA / RNA hybrid molecules to generate the one or more cDNA molecules; and (iii) ligating adapters to the one or more generated cDNA molecules.

17. The method of claim 16, further comprising ligating an adapter to the one or more fragmented DNA molecules or the one or more end repaired fragmented DNA molecules.

18. The method of any one of claims 1-3, wherein the one or more RNA molecules and the one or more DNA molecules in the sample are each fragmented.

19. The method of claim 18, wherein the fragmentation is selected from the group consisting of the application of mechanical force, sonication, and restriction endonuclease cleavage.

20. The method of any one of claims 18-20, wherein the one or more fragmented RNA molecules are selectively tagged by polyadenylating the one or more fragmented RNA molecules to provide one or more fragmented RNA molecules each including a polyA tail.

21. The method of claim 20, wherein the poly adenylating of the one or more fragmented RNA molecules comprises contacting the sample with a Poly(a) Polymerase.

22. The method of any one of claims 18-21, wherein the one or more cDNA molecules are prepared by (i) performing a first-strand synthesis on the one or more tagged RNA molecules to provide one or more DNA / RNA hybrid molecules; (ii) performing a second- strand synthesis on the provided one or more DNA / RNA hybrid molecules to generate the one or more cDNA molecules; and (iii) ligating adapters to the one or more generated cDNA molecules.

23. The method of claim 22, further comprising ligating an adapter to the one or more fragmented DNA molecules or the one or more end repaired fragmented DNA molecules.

24. The method of claim 18, wherein the tag selectively ligated to the one or more fragmented RNA molecules includes a primer binding site.Attorney Docket No P39431-WO-1

25. The method of claim 18, wherein the tag selectively ligated to the one or more fragmented RNA molecules includes (i) a 5' sequence index; (ii) an adapter sequence; and (iii) a 3' terminal di-deoxyribonucleotide.

26. The method of any one of claims 24-25, wherein the tag is ligated to the one or more fragmented RNA molecules using an RNA ligase or a T4 RNA ligase II.

27. The method of any one of claims 24-26, wherein the one or more cDNA molecules are generated by (i) performing a first-strand synthesis on the one or more tagged RNA molecules to provide one or more DNA / RNA hybrid molecules; (ii) ligating adapters to the one or more DNA / RNA hybrid molecules; (iii) extending the one or more adapter ligated DNA / RNA hybrid molecules; and (iv) amplifying the one or more adapter ligated DNA / RNA hybrid molecules.

28. The method of claim 27, wherein the first-strand synthesis is performed using a reverse transcriptase and a primer specific to at least a portion of the tag selectively ligated to the one or more fragmented RNA molecules.

29. The method of claim 28. further comprising ligating an adapter to the one or more fragmented DNA molecules.

30. The method of claim 29, further comprising extending the one or more adapter ligated DNA molecules to provide one or more extended DNA molecules.

31. The method of claim 30, further comprising amplifying the one or more extended DNA molecules.

32. A method of sequencing a sample comprising both RNA and DNA molecules, comprising:

(a) obtaining a sample comprising one or more RNA molecules and one or more DNA molecules;

(b) selectively fragmenting the one or more RNA molecules within the sample; (c) ligating a tag to the 5' ends of the one or more RNA molecules within the sample;

(d) fragmenting the DNA molecules within the sample to provide one or more fragmented DNA molecules;

(e) performing a first strand synthesis on the one or more fragmented tagged RNA molecules in the sample to provide one or more hybrid DNA/RNA molecules;Attorney Docket No P39431-WO-1

(f) performing a second strand synthesis on the one or more provided hybrid DNA/RNA molecules to provide one or more double stranded cDNA molecules;

(g) ligating adapters to the one or more double stranded cDNA molecules and the one or more DNA molecules in the sample to provide a library including the one or more adapter ligated double stranded cDNA molecules and the one or more adapter ligated DNA molecules; and

(h) sequencing the one or more adapted ligated double stranded cDNA molecules and the one or more adapter ligated DNA molecules in the library.

33. The method of claim 32, wherein the one or more RNA molecules in the obtained sample are selectively fragmented using an RNA endonuclease.

34. The method of claim 33, wherein the RNA endonuclease is RNase III.

35. The method of claim 32, wherein the one or more RNA molecules in the obtained sample are selectively fragmented using a RNaseT2 endonuclease.

36. The method of any one of claims 33-35, wherein the tag is selectively ligated to the 5' ends of the one or more fragmented RNA molecules.

37. The method of any one of claims 32-36, wherein the tag comprises at least 5 nucleotides.

38. The method of any one of claims 32-37, wherein the ligation is performed using an RNA ligase.

39. The method of claim 38. wherein the RNA ligase is RtcB ligase.

40. The method of any one of claims 32-39, wherein the one or more DNA molecules in the obtained sample are separately fragmented.

41. The method of claim 40, wherein the one or more DNA molecules are fragmented enzymatically.

42. The method of claim 41, wherein the enzymatic fragmentation of the one or more DNA molecules comprises contacting the sample with a DNase.

43. The method of any one of claims 41-42, wherein the one or more fragmented DNA molecules are end repaired.

44. The method of any one of claims 32-43, wherein the sequencing comprises next-generation sequencing.

45. A method of sequencing a sample comprising both RNA and DNA molecules, comprising:

(a) obtaining a sample comprising one or more RNA molecules and one or more DNA molecules;Attorney Docket No P39431-WO-1

(b) fragmenting both the one or more RNA molecules and the one or more DNA molecules in the obtained sample;

(c) selectively polyadenylating the one or more fragmented RNA molecules to provide a sample including one or more polyA tagged fragmented RNA molecules;

(d) performing a first strand synthesis on the one or more polyA tagged fragmented RNA molecules in the sample using a primer which anneals to the polyA tail to provide one or more DNA/RNA hybrid molecules;

(e) performing a second strand synthesis on the one or more DNA / RNA hybrid molecules to provide one or more double stranded cDNA molecules;

(f) ligating adapters to the one or more double stranded cDNA molecules and the one or more DNA molecules in the sample to provide the library including the one or more adapter ligated double stranded cDNA molecules and the one or more adapter ligated DNA molecules; and

(g) sequencing the one or more adapted ligated double stranded cDNA molecules and the one or more adapter ligated DNA molecules in the library.

46. The method of claim 45, wherein the fragmentation of both the one or more RNA molecules and the one or more DNA molecules in the obtained sample is selected from the group consisting of the application of mechanical force, sonication, and restriction endonuclease cleavage.

47. The method of claim 45. wherein the polyadenylating of the one or more fragmented RNA molecules comprises contacting the sample with a Poly(a) Polymerase.

48. The method of any one of claims 45-47, wherein the sequencing comprises next-generation sequencing.

49. A method of sequencing a sample comprising both RNA and DNA molecules, comprising:

(a) obtaining a sample comprising one or more RNA molecules and one or more DNA molecules;

(b) fragmenting both the one or more RNA molecules and the one or more DNA molecules in the obtained sample;

(c) selectively ligating a tag to the 3' ends of the one or more fragmented RNA molecules in the sample;

(d) performing a first strand synthesis of the one or more tagged RNA molecules in the sample, wherein the first strand synthesis utilizes a portion of the tag as a priming site to provide one or more DNA / RNA hybrid molecules;Attorney Docket No P39431-WO-1

(e) ligating of adapters to the one or more DNA / RNA hybrid molecules to provide one or more adapter ligated DNA / RNA hybrid molecules;

(f) ligating adapters to the one or more fragmented DNA molecules in the sample to provide one or more adapter ligated DNA molecules;

(g) extending each of the one or more adapter ligated DNA / RNA hybrid molecules and the one or more adapter ligated DNA molecules in the sample;

(h) amplifying each of the extended one or more adapter ligated DNA / RNA hybrid molecules and the one or more adapter ligated DNA molecules in the sample to provide adapter ligated double stranded cDNA molecules and double stranded DNA molecules; and

(h) sequencing the one or more adapted ligated double stranded cDNA molecules and the one or more adapter ligated DNA molecules in the library.

50. The method of claim 49, wherein the tag selectively ligated to the one or more fragmented RNA molecules includes a primer binding site.

51. The method of claim 49, wherein the tag selectively ligated to the one or more fragmented RNA molecules includes (i) a 5' sequence index; (ii) an adapter sequence; and (iii) a 3' terminal di-deoxyribonucleotide.

52. The method of any one of claims 49-51, wherein the tag is ligated to the one or more fragmented RNA molecules using an RNA ligase or a T4 RNA ligase II.

53. The method of any one of claims 49-52, wherein the sequencing comprises next-generation sequencing.

54. A kit comprising (i) one or more enzymes for fragmenting RNA; and (ii) an oligonucleotide tag.

55. The kit of claim 54, further comprising a sequencing instrument.