Blocking oligonucleotides for selective depletion of unwanted fragments from an amplified library

Abstract

The present disclosure relates to methods, compositions, and kits for the selective depletion of undesired fragments from an amplified library using blocking oligonucleotides.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. 63 / 169,185, filed March 31, 2021, the disclosure of which is incorporated herein by reference.

[0002] FIELD OF THEINVENTION The present disclosure relates to methods, compositions, and kits for the selective depletion of undesired fragments from an amplified library using blocking oligonucleotides. [Background technology]

[0003] Library preparation aims to build a collection of DNA fragments for next-generation sequencing (NGS). High-quality DNA libraries ensure uniform and consistent genome coverage, and therefore deliver comprehensive and reliable sequencing data. However, library preparations contain many undesired sequences, such as sequences for rRNA, sequences for housekeeping genes, mitochondrial sequences, etc. Therefore, elimination of these undesired sequences in library preparations can provide more focused and data-rich next-generation sequencing (NGS) libraries. Summary of the Invention

[0004] Current methods for depletion of abundant sequences, such as hybridization pull-down of rRNA (e.g., RiboZero, RiboMinus) or enzymatic digestion (e.g., RNaseH, CRISPR), perform well on high-quality, high-input samples but often perform poorly on the low-quality, low-abundance inputs encountered in clinically relevant sample types such as formalin fixed / paraffin-embedded (FFPE) tissues and plasma-derived circulating RNA (C-RNA). Alternatively, sequence-specific enrichment approaches (e.g., exome capture) perform better on low-input samples but are limited by the need to pre-specify a set of targets. This limits their utility for detecting rare transcript isoforms and non-coding RNAs that could be useful biomarkers.

[0005] The present disclosure provides an alternative depletion strategy, "PCR blocking," and related methods that use long, tightly binding oligonucleotides to block polymerase extension in PCR. The approach described herein is expected to improve library conversion in low-input applications by eliminating the time-consuming and inefficient incubation and purification steps characteristic of existing approaches and allowing abundant sequences to act as built-in "carriers" during pre-amplification steps.

[0006] In certain embodiments, the disclosure provides a method for selectively depleting undesired fragments from an amplified DNA or cDNA library by using one or more blocking oligonucleotides, the method comprising: amplifying a plurality of library fragments comprising a double-stranded template sequence comprising an adapter sequence in a polymerase chain reaction (PCR) reaction, where a portion of the fragments comprises undesired fragments that are not to be analyzed, the PCR reaction comprising the plurality of fragments, a polymerase, dNTPs, PCR primers, and one or more blocking oligonucleotides, where the one or more blocking oligonucleotides comprise: (i) one or more nucleotides comprising a phosphorothioate bond at the 5' end, and / or (ii) one or more nucleotides comprising a phosphorothioate bond at the 3' end, and (iii) a 3'-block that prevents polymerase extension at the 3' end of the blocking oligonucleotide, where the one or more blocking primers bind to the template sequence of the undesired fragments, thereby blocking amplification of the undesired fragments by PCR. In further embodiments, one or more of the blocking oligonucleotides are between 15 nt and 100 nt in length. In still further embodiments, if the polymerase has 5'-3' exonuclease activity, one or more of the blocking oligonucleotides comprises 1-5 nucleotides at the 5' end that contain phosphorothioate linkages. In another embodiment, if the polymerase has 3'-5' proofreading activity, one or more of the blocking oligonucleotides comprises 1-5 nucleotides at the 3' end that contain phosphorothioate linkages. In yet another embodiment, one or more of the blocking oligonucleotides comprises (i), (ii), and (iii): (i) 2-5 nucleotides at the 5' end that contain phosphorothioate linkages, (ii) 2-5 nucleotides at the 3' end that contain phosphorothioate linkages, and (iii) a 3'-block that prevents polymerase extension at the 3' end of the blocking oligonucleotide.In another embodiment, the 3'-block is selected from a C3-spacer, a 3' inverted base, a 3' phosphorylation, a 3' dideoxy base, or a 3' non-complementary overhanging base. In yet another embodiment, the amplified library comprises template sequences from cDNA. In a further embodiment, the amplified library comprises template sequences from gDNA. In certain embodiments, the adaptor sequences are derived from Y-shaped adaptors that are ligated to each end of the template sequences. In another embodiment, one or more blocking oligonucleotides bind to template sequences from rRNA and / or globin. In yet another embodiment, one or more blocking oligonucleotides comprise a pool of blocking oligonucleotides that bind to template sequences from 18S rRNA, 5.8S rRNA, and / or 28S RNA. In a further embodiment, one or more of the blocking oligonucleotides bind to template sequences from mtDNA. In still a further embodiment, the amplified DNA or cDNA library is analyzed by using next generation sequencing. In certain embodiments, the PCR amplification step is preceded by the following steps: obtaining an RNA sample, fragmenting the RNA, reverse transcribing the RNA fragments into cDNA, blunt-ending the cDNA and adding an A nucleotide to the 3' end of the blunt-ended cDNA, and ligating the A-tailed cDNA to an adaptor that contains a non-complementary T nucleotide at its 3' end. In further embodiments, prior to reverse transcribing the RNA fragments into cDNA, the RNA sample is treated to deplete rRNA sequences from the RNA sample.

[0007] In certain embodiments, the disclosure further provides a method for selectively depleting undesired fragments from an amplified DNA or cDNA library by using one or more blocking oligonucleotides, the method comprising amplifying a plurality of library fragments comprising a double-stranded template sequence comprising an adapter sequence in a polymerase chain reaction (PCR) reaction, a portion of the fragments comprising undesired fragments containing template sequences that are not to be analyzed, the PCR reaction comprising a plurality of fragments, a polymerase, dNTPs, PCR primers, and a pool of blocking oligonucleotides, a portion of the pool of blocking oligonucleotides binds to each strand of the template sequence of the undesired fragments, and one or more blocking primers bind to the template sequence of the undesired fragments, thereby blocking amplification of the undesired fragments by PCR. In a further embodiment, the pool of blocking oligonucleotides is between 15 nt and 100 nt in length. In yet a further embodiment, the pool of blocking oligonucleotides comprises blocking oligonucleotides that bind to strands of the template in a non-overlapping and adjacent manner. In another embodiment, the pool of blocking oligonucleotides comprises blocking oligonucleotides that are reverse complementary to other blocking oligonucleotides. In yet another embodiment, the pool of blocking oligonucleotides comprises (i), and / or (ii), and (iii): (i) one or more nucleotides comprising a phosphorothioate bond at the 5' end, and / or (ii) one or more nucleotides comprising a phosphorothioate bond at the 3' end, and (iii) a 3'-block that prevents polymerase extension at the 3' end of the blocking oligonucleotide. In a further embodiment, if the polymerase has 5'-3' exonuclease activity, one or more of the blocking oligonucleotides comprises 1-5 nucleotides comprising a phosphorothioate bond at the 5' end. In a still further embodiment, if the polymerase has 3'-5' proofreading activity, one or more of the blocking oligonucleotides comprises 1-5 nucleotides comprising a phosphorothioate bond at the 3' end.In certain embodiments, one or more blocking oligonucleotides comprise (i), (ii), and (iii): (i) 2-5 nucleotides at the 5' end that comprise a phosphorothioate bond, (ii) 2-5 nucleotides at the 3' end that comprise a phosphorothioate bond, and (iii) a 3'-block that prevents polymerase extension on the 3' end of the blocking oligonucleotide. In further embodiments, the 3'-block is selected from a C3-spacer, a 3' inverted base, a 3' phosphorylation, a 3' dideoxy base, or a 3' non-complementary overhanging base. In another embodiment, the amplified library comprises template sequences from cDNA. In yet another embodiment, the amplified library comprises template sequences from gDNA. In a further embodiment, the adaptor sequences are derived from Y-shaped adaptors that are ligated to each end of the template sequences. In still further embodiments, the pool of blocking oligonucleotides binds to template sequences from rRNA and / or globin. In another embodiment, the pool of blocking oligonucleotides binds to template sequences derived from 18S rRNA, 5.8S rRNA, and / or 28S RNA. In a further embodiment, the pool of blocking oligonucleotides binds to template sequences derived from mtDNA. In yet a further embodiment, the amplified DNA or cDNA library is analyzed by using next generation sequencing. In another embodiment, the PCR amplification step is preceded by the following steps: obtaining an RNA sample, fragmenting the RNA, reverse transcribing the RNA fragments into cDNA, blunt-ending the cDNA and adding an A nucleotide to the 3' end of the blunt-ended cDNA, and ligating the A-tailed cDNA with an adaptor that contains a non-complementary T nucleotide at the 3' end. In still a further embodiment, prior to reverse transcribing the RNA fragments into cDNA, the RNA sample is treated to deplete rRNA sequences from the RNA sample.

[0008] In certain embodiments, the present disclosure further provides an RNA-Seq-based library preparation kit comprising one or more blocking oligonucleotides, the blocking oligonucleotides comprising (i), and / or (ii), and (iii): (i) one or more nucleotides comprising a phosphorothioate bond at the 5' end, and / or (ii) one or more nucleotides comprising a phosphorothioate bond at the 3' end, and (iii) a 3'-block that prevents polymerase extension at the 3' end of the blocking oligonucleotide, wherein the blocking oligonucleotides bind to template sequences of undesired library fragments, thereby blocking amplification of undesired library fragments by PCR. In further embodiments, the library preparation kit comprises an A-tailing mix, an enhanced PCR mix, a ligation mix, a resuspension buffer, a stop ligation buffer, an Elute, Prime, Fragment High Concentration Mix, a First strand Synthesis Act D Mix, a reverse transcriptase, and a second strand master mix. In still further embodiments, one or more of the blocking oligonucleotides are between 15 nt and 100 nt in length.

[0009] In certain embodiments, the present disclosure provides an RNA-Seq-based library preparation kit comprising a pool of blocking oligonucleotides, a portion of which binds to each strand of the template sequence of the undesired fragment in a non-overlapping and adjacent manner, thereby blocking amplification of the undesired library fragments by PCR. In further embodiments, the library preparation kit comprises an A-tailing mix, an enhanced PCR mix, a ligation mix, a resuspension buffer, a stop ligation buffer, an Elute, Prime, Fragment High Concentration Mix, a First strand Synthesis Act D Mix, a reverse transcriptase, and a second strand master mix. In further embodiments, the pool of blocking oligonucleotides is between 15 nt and 100 nt in length. In yet further embodiments, the pool of blocking oligonucleotides comprises (i), and / or (ii), and (iii): (i) one or more nucleotides comprising a phosphorothioate linkage at the 5' end, and / or (ii) one or more nucleotides comprising a phosphorothioate linkage at the 3' end, and (iii) a 3'-block that prevents polymerase extension at the 3' end of the blocking oligonucleotide. In further embodiments, the 3'-block is selected from a C3-spacer, a 3' inverted base, a 3' phosphorylation, a 3' dideoxy base, or a 3' non-complementary overhanging base.

[0010] The details of one or more embodiments of the disclosure are set forth in the accompanying drawings and the following description. Other features, objects, and advantages will become apparent from the description and drawings, and from the claims. [Brief description of the drawings]

[0011] [Figure 1] We present a workflow overview of a traditional total RNA workflow compared to the use of PCR clamps to deplete RNA-Seq libraries for rRNA fragments. [Figure 2A]FIG. 1 provides an illustration of how PCR clamps can be used to deplete a sequencing library of unwanted fragments. Key reagents in the reaction: a sequencing library composed of desired and undesired fragments, PCR clamps, and PCR amplification primers. For simplicity, only two library fragment types are shown: one undesired fragment (red) that is targeted by a PCR clamp, and one fragment that is not targeted by a PCR clamp. The dark grey ends of the library fragments represent universal adaptor sequences. [Figure 2B] An example is provided of how PCR clamps can be used to deplete a sequencing library of unwanted fragments. Hybridization of PCR clamps and PCR primers: After high temperature denaturation in PCR, the reaction is cooled to allow annealing of the PCR primers. At the same time, undesired library fragments are targeted for removal by hybridizing with the PCR clamps, while desired library fragments remain unbound by any PCR clamps. An important feature is that complete end-to-end hybridization of the PCR clamp to its target is not required. Thus, many undesired library fragments can be targeted for depletion without a priori knowledge of their specific nature within the library. [Figure 2C] An example is provided of how PCR clamps can be used to deplete a sequencing library of unwanted fragments. Extension: A thermostable polymerase extends from the PCR primer to generate copies of the library fragments. PCR clamps bound to undesired fragments cannot be completely copied due to blocking by the bound PCR clamp. Desirable library fragments are copied unhindered by the PCR clamps. [Figure 2D]An example is provided of how PCR clamps can be used to deplete a sequencing library of unwanted fragments. Final library: The final library is generated from the exponential amplification of the desired library fragments (gray), while the undesired library fragments (red) were amplified inefficiently. The result is a library that is "depleted" of the undesired library fragments. [Diagram 3] An overview of exemplary PCR clamps designed to block amplification of rRNA genes is provided. Design 1 provides antiparallel and adjacent PCR clamps. Design 1+2 provides non-overlapping PCR clamps that incorporate features of Design 1 with the addition of an additional reverse complementary PCR clamp. Design 3 provides overlapping antiparallel PCR clamps. [Figure 4] Figure 1 shows that the PCR clamps designed in Design1 or Design1_2 significantly reduced rRNA amplified transcripts when non-depleted total RNA was used. rRNA was reduced from approximately 85% to 30% using the PCR clamps compared to the control (no PCR clamps). [Diagram 5] This shows that PCR clamps designed in Design1 or Design1_2 further reduced rRNA in RPO enriched and non-depleted total RNA samples. DesignOffSet (Design3) did not significantly affect rRNA enrichment in RPO samples. Using Design1 or Design1_2 PCR clamps reduced rRNA enrichment from approximately 20% to 1%. [Figure 6] We demonstrate that PCR clamps designed with Design1 or Design1_2 reduced targeted rRNA in mRNA-selected samples. Designs 1 and 2 could further reduce % rRNA in mRNA-selected samples from about 1.5% rRNA to about 0.25% rRNA. [Figure 7]A comparison of Fragments Per Kilobase of transcript per Million mapped reads (FPKM) between PCR clamp and RiboZero methods is provided. [Figure 8] We demonstrate that samples using PCR clamps have a high level of expression correlation across different depletion methods with FPKM R2 values ​​>0.95. [Figure 9] Figure 1 shows a trace of data generated from a probe panel without optimization.Additional benefits may be possible by optimizing probe design and workflow biochemistry. [Figure 10] 1 provides an exemplary embodiment of a PCR clamp (blocking oligo) of the present disclosure. [Figure 11] Provided are examples of PCR clamps that can be generated from 28S rRNA, 18S rRNA, 5.85 rRNA, Mt12S rRNA, and mt16S sequences, with PCR clamps designed to have melting temperatures of 75° C. or 80° C. Circles indicate gaps in the sequence where an 80° C. PCR clamp cannot be generated from the rRNA sequence (as indicated in the table). [Figure 12] Data from rRNA-content RNAseq data are shown. The majority of reads were blocked with a PCR clamp with a melting temperature of 80°C. [Figure 13]An overview of the PCR clamp study is presented. (Top panel) Overview of the 42 kbp human ribosomal DNA perfect repeat unit (GenBank U13359.1). The three loci encoding highly abundant ribosomal RNAs (18S, 5.8S, and 28S) are marked in red. Additional features are shown in dark grey. (Bottom panel) Zoom-in view of the region containing the loci encoding 18S, 5.8S, and 28S rRNAs. The rRNA genes are marked in red. Two designs of PCR clamps are shown: Design1 with alternating 80-mer PCR clamps tiled end-to-end. Every other PCR clamp is in an alternating 5'→3' orientation relative to the targeted rRNA gene (either light grey or dark grey). Design2 contains PCR clamps in the same relative positions as Design1, but each clamp is the reverse complementary sequence of Design1.

[0012] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one or more embodiments of the present disclosure and, together with the detailed description, serve to explain the principles and implementations of the disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to an "oligonucleotide" includes a plurality of such oligonucleotides, reference to "the target sequence" includes reference to one or more target sequences, and so forth.

[0014] Also, unless otherwise stated, the use of "or" means "and / or." Similarly, "comprise," "comprises," "comprising," "include," "includes," "including," "have," "haves," and "having" are interchangeable and are not intended to be limiting.

[0015] It is further understood that where the descriptions of various embodiments use the term "comprising," those of ordinary skill in the art will understand that in some specific instances, an embodiment can be alternatively described using the language "consisting essentially of" or "consisting of."

[0016] Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the methods and compositions of the present disclosure, exemplary methods, devices, and materials are described herein.

[0017] The term "amplification" or "amplifying" refers to a process in which extra or multiple copies of a particular polynucleotide are formed. Amplification includes methods such as PCR, ligation amplification (or ligase chain reaction, LCR), and amplification methods. These methods are known and widely practiced in the art. See, for example, U.S. Pat. Nos. 4,683,195 and 4,683,202, and Innis et al., "PCR protocols: a guide to method and applications," Academic Press, Incorporated (1990) (for PCR) and Wu et al. (1989) Genomics 4:560-569 (for LCR). In general, the PCR procedure describes a method of gene amplification that consists of (i) sequence-specific hybridization of primers to specific genes within a DNA sample (or library), (ii) subsequent amplification with multiple rounds of annealing, extension, and denaturation using DNA polymerase, and (iii) screening of the PCR products for bands of the correct size. The primers used are oligonucleotides of sufficient length and appropriate sequence to provide for the initiation of polymerization, i.e., each primer is specifically designed to be complementary to each strand of the genomic locus to be amplified.

[0018] Reagents and hardware for carrying out amplification reactions are commercially available. Primers useful for amplifying sequences from specific gene regions are preferably complementary to and specifically hybridize to sequences in the target region or its adjacent regions, and can be prepared using the polynucleotide sequences provided herein. Nucleic acid sequences generated by amplification can be sequenced directly.

[0019] "Blocking oligonucleotide" as used herein refers to a nucleic acid molecule that can specifically bind to at least one of one or more undesired nucleic acid species, whereby the binding between the blocking oligonucleotide and one or more undesired nucleic acid species can reduce or prevent the amplification or extension (e.g., reverse transcription) of one or more undesired nucleic acid species. For example, the blocking oligonucleotide can include a nucleic acid sequence that can hybridize to one or more undesired nucleic acid species. In some embodiments, multiple blocking oligonucleotides can be provided. The multiple blocking oligonucleotides can specifically bind to at least one, at least two, at least five, at least ten, at least one hundred, at least one thousand or more of the one or more undesired nucleic acid species. Furthermore, the multiple different blocking oligonucleotides can specifically bind to at least one, at least two, at least five, at least ten, at least twenty, at least one hundred different sites on the same undesired nucleic acid species, at parallel, antiparallel, spaced, or consecutive sites on the undesired nucleic acid species. The location at which the blocking oligonucleotide specifically binds to the undesired nucleic acid species can vary. For example, the blocking oligonucleotide may specifically bind to a sequence near the 5' end of an undesired nucleic acid species. In some aspects, the blocking oligonucleotide may specifically bind within 10nt, 20nt, 30nt, 40nt, 50nt, 100nt, 200nt, 300nt, 400nt, 500nt, or 1,000nt of the 5' end of at least one of one or more undesired nucleic acid species. In some embodiments, the blocking oligonucleotide may specifically bind to a sequence near the 3' end of an undesired nucleic acid species. For example, the blocking oligonucleotide may specifically bind within 10nt, 20nt, 30nt, 40nt, 50nt, 100nt, 200nt, 300nt, 400nt, 500nt, or 1,000nt of the 3' end of at least one of one or more undesired nucleic acid species. As another example, the blocking oligonucleotide may specifically bind to a sequence in the middle of an undesired nucleic acid species.In some aspects, the blocking oligonucleotide may specifically bind within 10 nt, 20 nt, 30 nt, 40 nt, 50 nt, 100 nt, 200 nt, 300 nt, 400 nt, 500 nt, 1,000 nt of the midpoint of at least one of one or more undesired nucleic acid species, hi some embodiments, the blocking oligonucleotide may bind at multiple positions between the 5' and 3' ends of the undesired nucleic acid species.

[0020] In some embodiments, binding between the blocking oligonucleotide and the undesired nucleic acid species may reduce amplification and / or extension of the undesired nucleic acid species by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%.

[0021] It is contemplated that blocking oligonucleotides may reduce the amplification and / or extension of undesired nucleic acid species, for example, by forming a hybridization complex with the undesired nucleic acid species, whereby the complex has a high melting temperature (T m ) and thus does not allow the blocking oligonucleotide to function as a primer for a reverse transcriptase or a polymerase, or a combination thereof. In some embodiments, the blocking oligonucleotide has a T of 48° C., 49° C., 50° C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58° C., 59° C., 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 70° C., 75° C., 80° C., or a range including or between any two of the above temperatures (e.g., 50° C. to 60° C.). m may have:

[0022] The blocking oligonucleotide may, in some embodiments, comprise one or more non-naturally occurring nucleotides. The non-naturally occurring nucleotide may be, for example, a photosensitive or triggerable nucleotide. Examples of non-naturally occurring nucleotides may include, but are not limited to, peptide nucleic acid (PNA), morpholino and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA). In some embodiments, the blocking oligonucleotide is a chimeric oligonucleotide, such as an LNA / PNA / DNA chimera, an LNA / DNA chimera, a PNA / DNA chimera, a GNA / DNA chimera, a TNA / DNA chimera, or a combination thereof.

[0023] The blocking oligonucleotide may have a length of about 10nt, 11nt, 12nt, 13nt, 14nt, 15nt, 16nt, 17nt, 18nt, 19nt, 20nt, 21nt, 22nt, 23nt, 24nt, 25nt, 26nt, 27nt, 28nt, 29nt, 30nt, 35nt, 40nt, 45nt, 50nt, 60nt, 70nt, 80nt, 90nt, 100nt, 200nt, or a range including or between any two of the above nucleotide lengths (e.g., 17nt to 30nt).

[0024] The melting temperature (T m ) may be modified in some embodiments by adjusting the length of the blocking oligonucleotide. In some embodiments, the T mis modified by the number of DNA residues in the blocking oligonucleotide comprising an LNA / DNA chimera or a PNA / DNA chimera. For example, a blocking oligonucleotide comprising an LNA / DNA chimera or a PNA / DNA chimera can have a percentage of DNA residues that is about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or a range between any two of the above values.

[0025] In some embodiments, blocking oligonucleotides may be designed to be unable to function as primers or probes for amplification and / or extension reactions. For example, blocking oligonucleotides may be unable to function as primers for reverse transcriptase or polymerase. For example, blocking oligonucleotides comprising LNA / DNA chimeras or PNA / DNA chimeras may be designed to have a specific percentage of LNA or PNA residues, or to have LNA or PNA at a specific location, such as near the 3' end, 5' end, or at the 3' end, 5' end, or middle portion of the oligonucleotide. In some embodiments, blocking oligonucleotides comprising LNA / DNA chimeras or PNA / DNA chimeras may have a percentage of LNA or PNA residues that is about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or a range between any two of the above values.

[0026] The term "cDNA library" refers to a collection of cloned complementary DNA (cDNA) fragments that together constitute some portion of the transcriptome of a single cell or multiple single cells. cDNA is generated from the fully transcribed mRNA found in a cell and therefore contains only the expressed genes of a single cell or, when pooled together, the expressed genes from multiple single cells.

[0027] As used herein, the term "complementary" may refer to the ability of precise pairing between two nucleotides. For example, if a nucleotide at a given position of a nucleic acid can hydrogen bond with a nucleotide of another nucleic acid, the two nucleic acids are considered to be complementary to each other at that position. Complementarity between two single-stranded nucleic acid molecules may be "partial," where only a portion of the nucleotides bind (e.g., there are one or more mismatches between the blocking oligo and the complementary target), or it may be complete, where there is complete complementarity between the single-stranded molecules (e.g., there are no mismatches between the blocking oligo and the complementary target). A first nucleotide sequence may be said to be the "complement" of a second sequence if the first nucleotide sequence is complementary to the second nucleotide sequence. A first nucleotide sequence may be said to be the "reverse complement" of a second sequence if the first nucleotide sequence is complementary to a sequence that is the reverse of the second sequence (i.e., the order of the nucleotides is reversed). As used herein, the terms "complement," "complementary," and "reverse complement" may be used interchangeably. It is understood from the present disclosure that when a molecule can hybridize to another molecule, it can be the complement of the hybridizing molecule.

[0028] A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). The following six groups each contain amino acids that are conservative substitutions for one another: 1) serine (S), threonine (T), 2) aspartic acid (D), glutamic acid (E), 3) asparagine (N), glutamine (Q), 4) arginine (R), lysine (K), 5) isoleucine (I), leucine (L), methionine (M), alanine (A), valine (V), and 6) phenylalanine (F), tyrosine (Y), tryptophan (W).

[0029] As used herein, "expression" refers to the process by which a polynucleotide is transcribed into mRNA and / or the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0030] The term "homolog" as used in reference to an original enzyme or gene of a first family or species refers to a distinct enzyme or gene of a second family or species that is determined by functional, structural or genomic analysis to be an enzyme or gene of the second family or species that corresponds to the original enzyme or gene of the first family or species. In most cases, homologs will have functional, structural or genomic similarity. Techniques are known by which homologs of enzymes or genes can be readily cloned using genetic probes and PCR. The identity of the cloned sequence as a homolog can be confirmed using functional assays and / or by genomic mapping of the gene.

[0031] As used herein, two polynucleotides, oligonucleotides, peptides, polypeptides, or proteins (or fragments of any of the above) are substantially homologous when the nucleic acid or amino acid sequences have at least about 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. To determine the percent identity of two amino acid sequences, or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can be disregarded for comparison purposes). In one embodiment, the length of the reference sequence aligned for comparison purposes is at least 30%, typically at least 40%, more typically at least 50%, even more typically at least 60%, and even more typically at least 70%, 80%, 90%, or 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position (as used herein, amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences.

[0032] When hybridization occurs between two single-stranded polynucleotides in an antiparallel configuration, the reaction is called "annealing" and these polynucleotides are described as "complementary." A double-stranded polynucleotide can be complementary or homologous to another polynucleotide if hybridization can occur between one of the strands of the first polynucleotide and the second polynucleotide. Complementarity or homology (the degree to which one polynucleotide is complementary to another) can be quantified in terms of the percentage of bases in opposing strands that are expected to form hydrogen bonds with each other according to commonly accepted base-pairing rules.

[0033] The terms "oligonucleotide" and "polynucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or their analogs. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. Polynucleotides (e.g., blocking oligonucleotides) can contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. The terms also refer to both double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of the present disclosure containing a polynucleotide encompasses both the double-stranded form and each of the two complementary single-stranded forms that are known or predicted to constitute the double-stranded form.

[0034] Nucleic acids useful in the methods and compositions disclosed herein may contain non-natural sugar moieties in the backbone. Exemplary sugar modifications include, but are not limited to, 2' modifications such as the addition of halogens, alkyls, substituted alkyls, -SH, -SCH3, -OCN, -Cl, -Br, -CN, -CF3, -OCF3, -SO2CH3, -OSO2, -SO3, -CH3, -ONO2, -NO2, -N3, -NH2, substituted silyls, and the like. Similar modifications may also be made at other positions on the sugar, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides, and the 5' position of the 5' terminal nucleotide. Nucleic acids, nucleoside analogs, or nucleotide analogs having sugar modifications may be further modified to include a reversible blocking group, a peptide bond label, or both. In those embodiments in which the above 2' modifications are present, the base may bear a peptide bond label.

[0035] Nucleic acids useful in the methods and compositions disclosed herein may also include native or non-native bases. In this regard, native deoxyribonucleic acids can have one or more bases selected from the group consisting of adenine, thymine, cytosine, or guanine, and ribonucleic acids can have one or more bases selected from the group consisting of uracil, adenine, cytosine, or guanine. Exemplary non-native bases that may be included in a nucleic acid, whether or not it has a native backbone or an analog structure, include, without limitation, inosine, xanthate, hypoxanthate, isocytosine, isoguanine, 5-methylcytosine, 5-hydroxymethylcytosine, 2-aminoadenine, 6-methyladenine, 6-methylguanine, 2-propylguanine, 2-propyladenine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 15-halouracil, 15-halocytosine, 5-propylaminouracil ... nyluracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-uracil, 4-thiouracil, 8-halo adenine or guanine, 8-amino adenine or guanine, 8-thiol adenine or guanine, 8-thioalkyl adenine or guanine, 8-hydroxyl adenine or guanine, 5-halo substituted uracil or cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine. 3-deazaguanine, 3-deazaadenine, etc. Certain embodiments may utilize isocytosine and isoguanine in nucleic acids to reduce non-specific hybridization, as generally described in U.S. Pat. No. 5,681,702.

[0036] The non-native base used in the nucleic acid of the present disclosure can have universal base pairing activity and can base pair with any other naturally occurring base.Exemplary bases with universal base pairing activity include 3-nitropyrrole and 5-nitroindole.Other bases that can be used include those that have base pairing activity with a subset of naturally occurring bases, such as inosine, which base pairs with cytosine, adenine, or uracil.

[0037] A polynucleotide is composed of a specific sequence of the four nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), and, when the polynucleotide is RNA, uracil (U) in place of thymine. Thus, the term polynucleotide sequence is an alphabetical representation of a polynucleotide molecule. This alphabetical representation can be entered into a database in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching.

[0038] The term "library" refers to a collection of template molecules or a plurality of template molecules that typically include adapter sequences added to the 5' and 3' ends. The use of the term "library" to refer to a collection of template molecules or a plurality of template molecules should not be construed to imply that the templates that make up the library are derived from a particular source or that the "library" has a particular composition. By way of example, the use of the term "library" should not be construed to imply that individual templates within a library must be of different nucleotide sequence or that the templates are related in terms of sequence and / or source.

[0039] As used herein, the term "locked nucleic acid" or "LNA" refers to a modified RNA nucleotide. The ribose moiety of the LNA nucleotide is modified with an extra bridge connecting the 2' oxygen and the 4' carbon. The bridge "locks" the ribose in a 3'-endo(North) conformation. Some of the advantages of using LNA in the methods of the present disclosure include increased thermal stability of the duplex, increased target specificity, and resistance from exo- and endo-nucleases.

[0040] In various embodiments, the present disclosure encompasses the formation of so-called "single template" libraries, which contain multiple copies of a single type of template molecule, each with an adapter sequence attached to their 5' end and their 3' end, and "composite" libraries, in which many, but not all, of the individual template molecules contain different target sequences (as defined below), each template molecule having an adapter sequence attached to their 5' end and their 3' end. Such composite template libraries can be prepared using the methods of the present disclosure, starting from a complex mixture of target polynucleotides, such as, but not limited to, random genomic DNA fragments, cDNAs, etc. The present disclosure also extends to "composite" libraries formed by mixing together several individual "single template" libraries, each of which has been separately prepared using the methods of the present disclosure, starting from a single type of target molecule (i.e., mono-template). In certain embodiments, more than 50%, or more than 60%, or more than 70%, or more than 80%, or more than 90%, or more than 95% of the individual polynucleotide templates in a composite library can contain different target sequences.

[0041] As used herein, "plurality" refers to a population of molecules and can include any number of molecules desired to be analyzed.

[0042] As used herein, "peptide nucleic acid" or "PNA" refers to an artificially synthesized polymer similar to DNA or RNA, where the backbone is composed of repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. The backbone of PNA is substantially non-ionic under neutral conditions, in contrast to the highly charged phosphodiester backbone of naturally occurring nucleic acids. This provides two non-limiting advantages. First, PNA backbones exhibit improved hybridization kinetics. Second, PNAs have a larger change in melting temperature (Tm) of mismatched to perfectly matched base pairs. DNA and RNA typically exhibit a 2-4°C drop in Tm for internal mismatches. With non-ionic PNA backbones, the drop is closer to 7-9°C. This may provide better sequence discrimination. Similarly, due to their non-ionic nature, hybridization of bases attached to these backbones is relatively insensitive to salt concentration.

[0043] A "primer" is generally a short polynucleotide with a free 3'-OH group that hybridizes to the target and subsequently binds to a target or template potentially present in a sample of interest by promoting polymerization of a polynucleotide complementary to the target. Primers of the present disclosure are comprised of nucleotides ranging from 17 to 30 nucleotides. In one embodiment, the primer is at least 17 nucleotides, or alternatively at least 18 nucleotides, or alternatively at least 19 nucleotides, or alternatively at least 20 nucleotides, or alternatively at least 21 nucleotides, or alternatively at least 22 nucleotides, or alternatively at least 23 nucleotides, or alternatively at least 24 nucleotides, or alternatively at least 25 nucleotides, or alternatively at least 26 nucleotides, or alternatively at least 27 nucleotides, or alternatively at least 28 nucleotides, or alternatively at least 29 nucleotides, or alternatively at least 30 nucleotides, or alternatively at least 50 nucleotides, or alternatively at least 75 nucleotides, or alternatively at least 100 nucleotides.

[0044] As used herein, "single cell" refers to one cell. Single cells useful in the methods described herein can be obtained from a tissue of interest or from a biopsy, blood sample, or cell culture. In addition, cells from a particular organ, tissue, tumor, neoplasm, etc. can be obtained and used in the methods described herein. Furthermore, cells from any population, such as a population of prokaryotic or eukaryotic single-cell organisms, including bacteria or yeast, in general, can be used in the methods. In some embodiments, the method of preparing a cDNA library can include a step of obtaining single cells. Single cell suspensions can be obtained using standard methods known in the art, including, for example, enzymatically using trypsin or papain to bind cells in a tissue sample, or releasing adherent cells in culture, or mechanically separating cells in a sample. Single cells can be placed in any suitable reaction vessel in which the single cells can be individually processed, such as a 96-well plate such that each single cell is placed in a single well.

[0045] Methods for manipulating single cells are known in the art and include fluorescence activated cell sorting (FACS), micromanipulation, and the use of semi-automated cell pickers (e.g., the Quixell™ Cell Transfer System manufactured by Stoelting Co.). Individual cells can be individually selected based on characteristics detectable by microscopy, such as location, morphology, or reporter gene expression.

[0046] The use of the term "template" to refer to individual polynucleotide molecules in a library simply indicates that one or both strands of a polynucleotide in a library are capable of acting as a template for template-dependent nucleic acid polymerization catalyzed by a polymerase. Use of this term should not be construed as limiting the scope of the present disclosure to libraries of polynucleotides that are actually used as templates in a subsequent enzyme-catalyzed polymerization reaction.

[0047] The term "unmatched region" refers to a region of an adapter where the sequences of the two polynucleotide strands forming the adapter exhibit a degree of non-complementarity such that the two strands cannot anneal to each other under standard annealing conditions for a PCR reaction. The two strands in the unmatched region may exhibit some degree of annealing under standard reaction conditions for an enzyme-catalyzed ligation reaction, provided that the two strands are reversed to single-stranded form under annealing conditions.

[0048] The pooled cDNA samples can be amplified by polymerase chain reaction (PCR), including emulsion PCR and single primer PCR, in the manner described herein. For example, the cDNA samples can be amplified by single primer PCR. The cDNA synthesis primer can include a 5' amplification primer sequence (APS), which allows the first strand of the cDNA to be subsequently amplified by PCR using a primer that is complementary to the 5' APS. The template switch oligonucleotide can also include a 5' APS, which can be at least 70% identical, at least 80% identical, at least 90% identical, at least 95% identical, or 70%, 80%, 90%, or 100% identical to the 5' APS in the cDNA synthesis primer. This means that the pooled cDNA samples can be amplified by PCR using a single primer (i.e., by single-primer PCR), which takes advantage of the PCR suppression effect to reduce the amplification of short contaminating amplicons and primer dimers (Dai et al., J Biotechnol 128(3):435-43(2007)). Since the two ends of each amplicon are complementary, short amplicons will form stable hairpins, which are poor templates for PCR. This reduces the amount of truncated cDNA and improves the yield of longer cDNA molecules. The 5'APS can be designed to facilitate downstream processing of the cDNA library. For example, if the cDNA library is to be analyzed by a particular sequencing method, such as Life Technology's SOLiD sequencing technology or Illumina's Genome Analyzer, the 5'APS can be designed to be identical to the primers used in these sequencing methods. For example, the 5' APS can be identical to the SOLiD P1 primer and / or the SOLiD P2 sequence inserted into the cDNA synthesis primer, such that the P1 and P2 sequences required for SOLiD sequencing are essential to the amplified library.

[0049] Another exemplary method for amplifying pooled cDNA includes PCR. PCR is a reaction in which duplicate copies are made from a target polynucleotide using a pair or set of primers consisting of an upstream and downstream primer, a catalyst for polymerization such as DNA polymerase, and typically a thermostable polymerase enzyme. Methods of PCR are well known in the art and are taught, for example, in MacPherson et al. (1991) PCR1: A Practical Approach (IRL Press at Oxford University Press). All processes that generate duplicate copies of a polynucleotide, such as PCR or gene cloning, are collectively referred to herein as duplication. Primers can also be used as probes in hybridization reactions, such as Southern or Northern blot analysis.

[0050] For emulsion PCR, the emulsion PCR reaction occurs by vigorously shaking or stirring the "water-in-oil" mixture to generate millions of micron-sized aqueous compartments. The DNA library is mixed with the beads prior to emulsification or directly into the emulsion mixture at limiting dilution. A combination of compartment size and limiting dilution of beads and target molecules is used to generate compartments that contain, on average, only one DNA molecule and bead (at optimal dilution, many compartments will have beads that do not contain any target). To promote amplification efficiency, both upstream (low concentration, matching the primer sequence on the beads) and downstream PCR primers (high concentration) are included in the reaction mixture. Depending on the size of the aqueous compartments generated during the emulsification step, up to 3 x 10 per μl can be used. 9 Individual PCR reactions can be carried out simultaneously in the same tube. In essence, each small compartment in the emulsion forms a micro PCR reactor. The average size of the compartments in the emulsion ranges from submicron to over 100 microns in diameter, depending on the emulsification conditions.

[0051] "Identity," "homology," or "similarity" are used interchangeably and refer to sequence similarity between two nucleic acid molecules. Identity can be determined by comparing a position in each sequence that can be aligned for purposes of comparison. When a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of identity between sequences is a function of the number of matching or identical positions shared by the sequences. An unrelated or non-homologous sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences described herein.

[0052] A polynucleotide having a certain percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of "sequence identity" to another sequence means that, when aligned, that percentage of bases are the same when comparing the two sequences. This alignment and percent sequence identity or homology can be determined using software programs known in the art, such as those described in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, (1993). Preferably, default parameters are used for the alignment. One alignment program is BLAST, using default parameters. In particular, the programs are BLASTN and BLASTP using the following default parameters: Genetic code=standard, filter=none, strand=both, cutoff=60, expect=10, Matrix=BLOSUM62, Descriptions=50 sequences, sort by=HIGH SCORE, Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein SPupdate+PIR. Details of these programs can be found at the National Center for Biotechnology Information.

[0053] Sequence homology for polypeptides, which may also be referred to as percent sequence identity, is typically measured using sequence analysis software. See, for example, the Sequence Analysis Software Package of the Genetics Computer Group (GCG), University of Wisconsin Biotechnology Center, 910 University Avenue, Madison, Wis. 53705. Protein analysis software matches similar sequences using measures of homology assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG contains programs such as "Gap" and "Bestfit," which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from organisms of different species, or between a wild-type protein and its mutant proteins. See, for example, GCG version 6.1.

[0054] A typical algorithm used to compare molecular sequences with databases containing a large number of sequences from different organisms is the computer program BLAST (Altschul, 1990; Gish, 1993; Madden, 1996; Altschul, 1997; Zhang, 1997), in particular blastp or tblastn (Altschul, 1997). Typical parameters for BLASTp are: Expectation value: 10 (default), Filter: seg (default), Cost to open a gap: 11 (default), Cost to extend a gap: 1 (default), Max. alignments: 100 (default), Word size: 11 (default), No. of descriptions: 100 (default), Penalty Matrix: BLOWSUM62.

[0055] When searching a database containing sequences from many different organisms, it is typical to compare amino acid sequences. Database searches using amino acid sequences can be measured by algorithms other than blastp known in the art. For example, polypeptide sequences can be compared using FASTA, a program in GCG version 6.1. FASTA provides alignment and percent sequence identity of the best overlapping regions between the query sequence and the search sequence (Pearson, 1990, incorporated herein by reference). For example, percent sequence identity between amino acid sequences can be determined using FASTA with its default parameters (word size of 2 and PAM250 scoring matrix), as provided in GCG version 6.1, incorporated herein by reference.

[0056] The method of preparing a cDNA library described herein may further include processing the cDNA library to obtain a library suitable for sequencing. As used herein, a library is suitable for sequencing when the complexity, size, purity, etc. of the cDNA library is suitable for the desired screening method. In particular, the cDNA library may be processed to generate samples suitable for any high-throughput screening method, such as Life Technology's SOLiD sequencing technology, Oxford's Nanopore DNA sequencing technology, or Illumina's cluster generation and sequencing technology. Thus, the cDNA library may be processed by fragmenting the cDNA library (e.g., with DNase) to obtain a 5'-end library of short fragments. Adapters may be added to the cDNA, for example, at one or both ends, to facilitate sequencing of the library. The cDNA library may be further amplified, for example, by PCR, to obtain a sufficient amount of cDNA for sequencing.

[0057] An embodiment of the present disclosure provides a cDNA library generated by any of the methods described herein, which can be sequenced to provide an analysis of gene expression in a single cell or in a plurality of single cells.

[0058] An embodiment of the present disclosure also provides a method for analyzing gene expression in a plurality of single cells, the method comprising preparing a cDNA library using the methods described herein and sequencing the cDNA library. A "gene" refers to a polynucleotide that contains at least one open reading frame (ORF) that can encode a particular polypeptide or protein after being transcribed and translated. Any of the polynucleotide sequences described herein can be used to identify larger fragments or full-length coding sequences of the genes associated with them. Methods for isolating larger fragment sequences are known to those skilled in the art.

[0059] The cDNA library may be sequenced by any suitable screening method. In particular, the cDNA library may be sequenced using any high-throughput screening method, such as Life Technology's SOLiD sequencing technology, Oxford's Nanopore DNA sequencing technology, or Illumina's cluster generation and sequencing technology. In one embodiment, the cDNA library may be shotgun sequenced. The number of reads may be at least 10,000, at least 1 million, at least 10 million, at least 100 million, or at least 1 billion. In another embodiment, the number of reads may be 10,000 to 100,000, or alternatively 100,000 to 1 million, or alternatively 1 million to 10 million, or alternatively 10 million to 100 million, or alternatively 100 million to 100 million. A "read" is the length of a continuous nucleic acid sequence obtained by a sequencing reaction.

[0060] Next generation sequencing (NGS) libraries often contain abundant sequences with little biological significance, such as ribosomal RNA sequences in transcriptome libraries, host sequences in microbiome or metagenomic libraries, or majority allele sequences in somatic variant detection applications. In RNA-seq libraries, for example, ribosomal RNA (rRNA) sequences may constitute more than 95% of all reads, and in most applications, these reads are uninformative and are discarded during secondary analysis. The "real estate" of the flow cell taken up by these sequences can significantly increase the cost of sequencing, especially for count-based applications or detection of rare fragments where greater sequencing depth is required to adequately sample the species of interest.

[0061] In all organisms, ribosomal RNA (rRNA), a highly abundant structural component of the ribosome, constitutes the majority of all RNA. Without selectively depleting RNA samples of these ribosomal RNAs, the resulting NGS libraries will be composed of fragments that represent mostly rRNA, which is of little use or scientific interest to the end user. Thus, rRNA must be depleted from samples prior to library construction. Current methods for depletion of abundant sequences, such as hybridization pull-down (e.g., RiboZero, RiboMinus) or enzymatic digestion (e.g., RNaseH, CRISPR) of rRNA, perform well on high-quality, high-input samples, but often perform poorly with the low-quality, low-abundance inputs encountered in clinically relevant sample types such as formalin-fixed / paraffin-embedded (FFPE) tissues and plasma-derived circulating RNA (C-RNA). Alternatively, sequence-specific enrichment approaches (e.g., exome capture) perform better on low-input samples, but are limited by the need to pre-specify a set of targets. This limits their usefulness for detecting rare transcript isoforms and non-coding RNAs that may be useful biomarkers. In addition, these treatments to remove rRNA act directly on samples composed of chemically labile RNA, introducing the risk of sample damage. Furthermore, these methods for reducing rRNA are only applicable to the RNA sample itself; once the sample is converted into a library, the same methods are not applicable for rRNA capture or depletion.

[0062] The use of one or more blocking oligonucleotides to reduce the abundance of undesired library fragments is described herein.The method of the present disclosure is very easy for end users, and does not require additional library preparation steps and the addition of one or more oligonucleotides.The method described herein acts on the library created, not on the sample directly, and reduces the risk of damage to the original polynucleotide sample.

[0063] As shown in the study presented herein, the method of the present disclosure significantly reduced rRNA for RNA-Seq technology. Similar results would be expected when the method of the present disclosure is applied to other library preparations where undesired library fragments are generated (e.g., ds DNA libraries). Other examples of potential uses include, but are not limited to, globin RNA, mitochondrial DNA fragments, housekeeping gene fragments from libraries, removal of non-host genetic material, and other scenarios where depletion of host or other abundant nucleic acids is desirable for generating more focused data-rich NGS libraries.

[0064] Thus, the disclosed methods, compositions, and kits can be used with DNA libraries generated from gDNA or other DNA sources. In such cases, library generation will utilize standard methodologies, except for the PCR amplification step to generate DNA sequencing libraries from adapter / template constructs. In particular, one or more blocking oligonucleotides of the present disclosure will be added as components to the PCR amplification step to generate DNA sequencing libraries.

[0065] Various non-limiting specific embodiments of the method disclosed herein will now be described in more detail with reference to the accompanying drawings, in which: Features described as preferred with respect to one specific embodiment apply mutatis mutandis to other specific embodiments of the present disclosure, unless otherwise stated.

[0066] Figure 1 illustrates the process traditionally used to generate a template library for sequencing from total RNA. Library preparation from total RNA is common to all major sequencing platforms, including those from Illumina™, Life Technologies™, and Oxford Nanopore™.

[0067] As shown in Figure 1, total RNA samples are isolated from samples using methodologies such as those described herein. Total RNA is typically treated to remove rRNA by performing an rRNA depletion step. Current methods for rRNA depletion include hybridization pull-down of rRNA (e.g., RiboZero™, RiboMinus™), or enzymatic digestion (e.g., RNaseH, CRISPR). The rRNA depletion methods described above can be very long (1.5-2 hours) and involve multiple subcomponents and steps. These depletion methods work well for high-quality, high-input samples, but often perform poorly with low-quality, low-abundance inputs encountered in clinically relevant sample types such as formalin-fixed / paraffin-embedded (FFPE) tissues and plasma-derived circulating RNA (C-RNA). Alternatively, sequence-specific enrichment approaches (e.g., exome capture) perform better for low-input samples but are limited by the need to pre-specify a set of targets. This limits their usefulness for detecting rare transcript isoforms and non-coding RNAs that may be useful biomarkers. Furthermore, depletion methods to remove rRNA and other undesired RNAs must be performed on the RNA sample itself. RNA is an unstable nucleic acid and is sensitive to handling, storage conditions, and RNase activity. It should be noted that incomplete depletion of rRNA and other undesired RNAs using the above methods cannot be improved in subsequent steps once converted into a library.

[0068] In stark contrast, the present disclosure provides new and innovative methods for depleting undesired nucleotide sequences using one or more blocking oligonucleotides (i.e., PCR clamps). Considerations for designing blocking oligonucleotides are further described herein.

[0069] Figure 1 illustrates the RNA-Seq process that is typically used to generate a template library for sequencing from RNA. Figure 1 further illustrates the RNA-Seq process that is modified to incorporate one or more blocking oligonucleotides of the present disclosure. RNA-Seq (named as an abbreviation of "RNA sequencing") is a sequencing technology that uses next-generation sequencing (NGS) to reveal the presence and amount of RNA in a biological sample at a given moment, and analyzes the continuously changing cellular transcriptome.

[0070] Specifically, RNA-Seq facilitates the ability to look at alternative gene spliced ​​transcripts, post-transcriptional modifications, gene fusions, mutations / SNPs, and changes in gene expression over time, or differences in gene expression in different groups or treatments. In addition to mRNA transcripts, RNA-Seq can look at different populations of RNA to include total RNA, small RNAs such as miRNAs, tRNAs, and ribosome profiling. RNA-Seq can also be used to determine exon / intron boundaries and verify or revise already annotated 5' and 3' gene boundaries. Recent advances in RNA-Seq include single cell sequencing and in situ sequencing of fixed tissues.

[0071] Prior to RNA-Seq, gene expression studies were performed using hybridization-based microarrays. Issues with microarrays include cross-hybridization artifacts, poor quantification of low- and high-expressing genes, and the need to know the sequence a priori. Due to these technical issues, transcriptomics moved to sequencing-based methods. These progressed from Sanger sequencing of expressed sequence tag libraries, to chemical tag-based methods (e.g., continuous analysis of gene expression), and finally to the current technology of next-generation sequencing of cDNA (specifically RNA-Seq). Next-generation sequencing (NGS) typically requires library preparation in which a known adapter DNA sequence is added to the target nucleotides to be sequenced. Traditionally, this requires that RNA is converted to cDNA, fragmented, end-repaired, and then ligated to the adapter DNA (see, e.g., Figure 1). This library preparation is common to all major sequencing platforms, including those from Illumina™, Pacific Biosciences™, and Oxford Nanopore™.

[0072] As shown in FIG. 1, RNA is isolated from a sample. In certain embodiments, RNA can be isolated from cells by lysing the cells. Lysis can be achieved, for example, by heating the cells or by using detergents or other chemical methods, or by a combination thereof. However, any suitable lysis method known in the art can be used. A gentle lysis procedure can be advantageously used to prevent the release of nuclear chromatin, thereby avoiding genomic contamination of the cDNA library and minimizing degradation of mRNA. For example, heating the cells at 72° C. for 2 minutes in the presence of Tween-20 is sufficient to lyse the cells without resulting in detectable genomic contamination from the nuclear chromatin. Alternatively, cells can be heated to 65° C. for 10 minutes in water (Esumi et al., Neurosci Res 60(4):439-51 (2008)), or to 70° C. for 90 seconds in PCR buffer II (Life Technology) suspended in 0.5% NP-40 (Kurimoto et al., Nucleic Acids Res 34(5):e42 (2006)), or lysis can be achieved with a protease such as proteinase K, or by the use of a chaotropic salt such as guanidine isothiocyanate (U.S. Patent Application Publication No. 2007 / 0281313).

[0073] DNase is typically added to the RNA sample. DNase reduces the amount of genomic DNA. The amount of RNA degradation is checked by gel and capillary electrophoresis and is used to assign an RNA integrity number to the sample. This RNA quality and the total amount of starting RNA is considered during the subsequent library preparation, sequencing, and analysis steps. RNA can be isolated with good yield and high quality using any number of commercially available kits, such as kits from Qiagen or Ambion, Lucigen MasterPure Kits, or using specific RNA isolation reagents such as TRIzol. The RNA integrity number should be greater than 8. RNA can be quantified using fluorometric based methods, such as Ribo-green.

[0074] As shown in Figure 1, RNA is then typically enriched by polyA selection or treated to deplete RNA from rRNA samples. Current methods for depletion of abundant sequences, such as hybridization pull-down (e.g., RiboZero, RiboMinus) or enzymatic digestion (e.g., RNaseH, CRISPR) of rRNA, work well for high-quality, high-input samples but often perform poorly with the low-quality, low-abundance inputs encountered in clinically relevant sample types such as formalin-fixed / paraffin-embedded (FFPE) tissues and plasma-derived circulating RNA (C-RNA). Alternatively, sequence-specific enrichment approaches (e.g., exome capture) perform better for low-input samples but are limited by the need to pre-specify a set of targets. This limits their utility for detecting rare transcript isoforms and non-coding RNAs that may be useful biomarkers. Typically, it takes 1-2 hours to deplete an RNA sample of rRNA.

[0075] After the RNA is processed to enrich the RNA sample with the desired template, the RNA is reverse transcribed into cDNA. Optionally, the RNA can be fragmented and size-selected before conversion to cDNA. Fragmentation and size selection are performed to purify sequences that are of a suitable length for sequencing machines. The RNA, cDNA, or both are fragmented with an enzyme, sonication, or nebulizer. RNA fragmentation reduces the effect of the 5' bias and primer binding sites of randomly primed reverse transcription, which has the disadvantage that the 5' and 3' ends are converted to cDNA less efficiently. Fragmentation is followed by size selection to remove small sequences or select a narrow range of sequence lengths. Small RNAs such as miRNAs are lost, so they are analyzed separately.

[0076] As shown in FIG. 1, the processed RNA is converted to cDNA. cDNA is typically synthesized from mRNA by reverse transcription. Methods for synthesizing cDNA from small amounts of mRNA, including from single cells, have been described previously (Kurimoto et al., Nucleic Acids Res 34(5):e42(2006); Kurimoto et al., Nat Protoc 2(3):739-52(2007); and Esumi et al., Neurosci Res 60(4):439-51(2008)). To generate amplifiable cDNA, these methods introduce primer annealing sequences at both ends of each cDNA molecule in such a way that a cDNA library can be amplified using a single primer. The Kurimoto method uses a polymerase to add a 3' polyA tail to the cDNA strand, which can then be amplified using a universal oligo-T primer. In contrast, the Esumi method uses template switching to introduce any sequence into the 3' end of the cDNA, which is designed to be reverse-complementary to the 3' tail of the cDNA synthesis primer. Again, the cDNA library can be amplified by a single PCR primer. Single primer PCR utilizes the PCR suppression effect to reduce the amplification of short contaminating amplicons and primer dimers (Dai et al., J Biotechnol 128(3):435-43(2007)). Since the two ends of each amplicon are complementary, short amplicons will form stable hairpins, which are poor templates for PCR. This reduces the amount of truncated cDNA and improves the yield of longer cDNA molecules.

[0077] In certain embodiments, the synthesis of the first strand of cDNA may be guided by a cDNA synthesis primer (CDS) that includes an RNA complementary sequence (RCS). In another embodiment, the RCS is at least partially complementary to one or more mRNAs in an individual mRNA sample. This allows the primer, typically an oligonucleotide, to hybridize to at least some mRNAs in an individual mRNA sample to guide cDNA synthesis using the mRNA as a template. The RCS may include oligo(dT) or be gene family specific, such as a sequence of nucleic acid present in all or most related genes, or may be composed of a random sequence, such as a random hexamer. To avoid the cDNA synthesis primer priming itself and thus generating undesired by-products, a non-self-complementary quasi-random sequence may be used. For example, one letter of the genetic code may be eliminated, or a more complex design may be used, while constraining the cDNA synthesis primer to be non-self-complementary.

[0078] The RCS can also be at least partially complementary to a portion of the first strand of the cDNA, thereby allowing the first strand of the cDNA to be used as a template to guide the synthesis of the second strand of the cDNA. Thus, following first strand synthesis, an RNase enzyme (e.g., an enzyme with RNase H activity) can be added after the synthesis of the first strand of the cDNA to degrade the RNA strand and allow the cDNA synthesis primer to reanneal on the first strand to guide the synthesis of the second strand of the cDNA. For example, the RCS can include random hexamers, or non-self-complementary quasi-random sequences (which minimize self-annealing of the cDNA synthesis primer).

[0079] A template switch oligonucleotide (TSO) containing a portion at least partially complementary to a portion of the 3' end of the first strand of the cDNA can be added to each individual RNA sample in the manner described herein. Such a template switching method is described in (Esumi et al., Neurosci Res 60(4):439-51 (2008)) and allows full-length cDNA to be synthesized that contains the complete 5' end of the RNA. The terminal transferase activity of reverse transcriptase typically incorporates 2-5 cytosines at the 3' end of the first strand of cDNA synthesized from mRNA, so that the first strand of cDNA can contain multiple cytosines or cytosine analogs at its 3' end that base pair with guanosine (see U.S. Pat. No. 5,962,272). In one embodiment, the first strand of the cDNA may include a 3' portion that includes at least two, at least three, at least four, at least five, or two, three, four, or five cytosines or cytosine analogs that base pair with guanosine. A non-limiting example of a cytosine analog that base pairs with guanosine is 5-aminoallyl-2'-deoxycytidine.

[0080] In one embodiment, the template switch oligonucleotide can include a 3' portion that includes multiple guanosines or guanosine analogs that base pair with cytosines. Non-limiting examples of guanosines or guanosine analogs useful in the methods described herein include, but are not limited to, deoxyriboguanosine, riboguanosine, locked nucleic acid-guanosine, and peptide nucleic acid-guanosine. The guanosines can be ribonucleosides or locked nucleic acid monomers.

[0081] In certain embodiments, the template switch oligonucleotide may include a 3' portion that includes at least two, at least three, at least four, at least five, or two, three, four, or five, or two to five guanosines or guanosine analogs that base pair with cytosines. The presence of multiple guanosines (or guanosine analogs that base pair with cytosines) allows the template switch oligonucleotide to transiently anneal to exposed cytosines at the 3' end of the first strand of the cDNA. This allows the reverse transcriptase to switch templates and continue synthesizing a strand complementary to the template switch oligonucleotide. In one embodiment, the 3' end of the template switch oligonucleotide may be blocked, e.g., by a 3' phosphate group, to prevent the template switch oligonucleotide from functioning as a primer during cDNA synthesis.

[0082] In another embodiment, RNA is released from cells by cell lysis. If lysis is partially accomplished by heating, cDNA synthesis primers and / or template switch oligonucleotides can be added to each individual RNA sample during cell lysis, which will aid in oligonucleotide hybridization. In some embodiments, reverse transcriptase can be added after cell lysis to avoid denaturation of the enzyme.

[0083] In some embodiments of the present disclosure, a tag may be incorporated into the cDNA during synthesis of the cDNA. For example, the cDNA synthesis primer and / or template switch oligonucleotide may include a tag, such as a specific nucleotide sequence that may be at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 nucleotides in length. For example, the tag may be a nucleotide sequence that is 4-20 nucleotides in length, e.g., 4, 5, 6, 7, 8, 9, 10, 15, or 20 nucleotides in length. The tag, because it is present in the cDNA synthesis primer and / or template switch oligonucleotide, becomes incorporated into the cDNA during its synthesis and thus may act as a "barcode" to identify the cDNA. Both the cDNA synthesis primer and the template switch oligonucleotide may include a tag. The cDNA synthesis primer and the template switch oligonucleotide may each include a different tag such that a tagged cDNA sample includes a combination of tags. Each cDNA sample generated by the above methods may have a distinct tag or distinct combination of tags, such that once the tagged cDNA samples are pooled, the tags can be used to identify which single cell each cDNA sample is derived from. Thus, each cDNA sample may be associated with a single cell even after the tagged cDNA samples are pooled by the methods described herein.

[0084] Before the tagged cDNA samples are pooled, cDNA synthesis can be stopped, for example, by removing or inactivating the reverse transcriptase. This prevents cDNA synthesis by reverse transcription from continuing in the pooled samples. The tagged cDNA samples can optionally be purified prior to amplification, either before or after they are pooled.

[0085] If the RNA was not fragmented prior to conversion to cDNA, the cDNA is fragmented and size selection is performed. The cDNA can be fragmented with an enzyme, sonication, or a nebulizer. Fragmentation is followed by size selection to remove small sequences or to select for a narrow range of sequence lengths.

[0086] After the cDNA reaction, an end-repair reaction is then performed using T4 polynucleotide kinase, rATP, and T4 DNA polymerase, dNTPs to form a blunt-ended double-stranded template. After end-repair completion and size selection, an A-tailing reaction is performed using Klenow exo-, dNTPs (e.g., dATP) (see FIG. 1) to facilitate adapter ligation. The adapter is formed by annealing two single-stranded oligonucleotides prepared by conventional automated oligonucleotide synthesis. The oligonucleotides are partially complementary such that the 3' end of the first oligonucleotide is complementary to the 5' end of the second oligonucleotide. The 5' end of the first oligonucleotide and the 3' end of the second oligonucleotide are not complementary to each other. When the two strands are annealed, the resulting structure is double-stranded at one end (the double-stranded region) and single-stranded at the other end (the unmatched region), and is referred to herein as a "Y-shaped adapter." The double-stranded region of the Y-shaped adaptor may be blunt-ended or may have an overhang. In the latter case, the overhang may be a 3' overhang or a 5' overhang and may contain a single nucleotide or two or more nucleotides. The Y-shaped adaptor is phosphorylated at its 5' end and the double-stranded portion contains a single-base 3' overhang that includes a "T" deoxynucleotide. The adaptor is then ligated to the end of a double-stranded template molecule that contains a single base 5' to the "A" nucleotide using T4 ligase, rATP.

[0087] The Y-shaped adaptor is phosphorylated at its 5' end and the double-stranded portion of the duplex contains a single-base 3' overhang containing a "T" deoxynucleotide (see FIG. 1). The adaptor is then ligated to the end of a double-stranded template molecule containing a single base 5' to the "A" nucleotide using T4 ligase, rATP.

[0088] Libraries are generally formed by ligating adapter polynucleotide molecules to the 5' and 3' ends of one or more target polynucleotide duplexes (which may be of known, partially known, or unknown sequence) to form an adapter-target construct, and then performing PCR amplification to form a library of template polynucleotides. The library of template polynucleotides can then be sequenced using next generation sequencing. To conserve resources, multiple libraries can be pooled together and sequenced in the same run, a process known as multiplexing. During adapter ligation, a unique index sequence, or "barcode," is added to each library. These barcodes are used to distinguish libraries during data analysis.

[0089] The adaptors added onto the double-stranded template using the disclosed non-homologous end-joining agents and methods typically contain a double-stranded region of complementary sequence and a single-stranded region of sequence mismatch. In certain embodiments, the adaptors have a Y-shape, with the region of sequence mismatch separating the arms of the adaptor from each other. The "double-stranded region" of the adaptor is a short double-stranded region, typically comprising five or more consecutive base pairs, formed by the annealing of two partially complementary polynucleotide strands. This term simply refers to the double-stranded region of the nucleic acid where the two strands are annealed, and does not imply any particular structural conformation. In an alternative embodiment, instead of having a Y-shape, the adaptors are U-shaped, so that once the adaptors are added to the ends of the template using the disclosed non-homologous end-joining agents, the disclosed methods form continuous loops at the 5' and 3' ends of the template. Thus, the resulting DNA library template can be amplified using rolling circle amplification.

[0090] Generally, it is advantageous for the double-stranded region to be as short as possible without compromising function. "Functional" in this context means that the double-stranded region forms a stable duplex under the reaction conditions for the prokaryotic end-joining and repairing agents described herein, so that the two strands forming the adaptor remain partially annealed during the ligation of the adaptor to the target molecule. It is not absolutely necessary that the double-stranded region is stable under the conditions typically used in the annealing step of a PCR reaction.

[0091] In another embodiment, identical adaptors are added to both ends of each template molecule, and the target sequence in each adaptor-target construct is flanked by complementary sequences derived from the double-stranded region of the adaptor. The longer the double-stranded region, and thus the complementary sequence derived from the double-stranded region in the adaptor-target construct, the more likely it is that the adaptor-target construct can fold back and base-pair with itself at these regions of internal self-complementarity under the annealing conditions used in PCR. In general, to reduce this effect, it is preferred that the double-stranded region is 20 or less, 15 or less, or 10 or less base pairs in length. The stability of the double-stranded region can be increased, and thus potentially reduced in length, by including non-natural nucleotides that exhibit stronger base pairing than standard Watson-Crick base pairs.

[0092] In a preferred embodiment, the two strands of the adapter are 100% complementary in the double-stranded region. However, it will be understood that one or more nucleotide mismatches can be tolerated within the double-stranded region, provided that the two strands are capable of forming a stable duplex under standard ligation conditions.

[0093] Alternatively, the adapters added onto the double-stranded templates using the disclosed non-homologous end-joining agents and methods contain double-stranded complementary sequences. The resulting adapter / template molecules can then be amplified by PCR to form the DNA library templates. In further embodiments, splint oligonucleotides can be used to join the ends of the DNA library templates to form circles. An exonuclease is added to remove all remaining linear single-stranded and double-stranded DNA products. The result is a completed circular DNA template.

[0094] Adapters for use in the methods disclosed herein will generally include a double-stranded region adjacent to the "ligatable" end of the adapter, i.e., the end that is joined to the target polynucleotide using a ligase or non-homologous end joining agent. The ligatable end of the adapter may be blunt, or in other embodiments, a short 5' or 3' overhang of one or more nucleotides may be present to facilitate / promote ligation. The 5' terminal nucleotide at the ligatable end of the adapter should be phosphorylated to allow for a phosphodiester bond to the 3' hydroxyl group on the target polynucleotide.

[0095] The portions of the two strands that form the double-stranded region typically comprise at least 10, or at least 15, or at least 20 consecutive nucleotides on each strand. The lower limit of the length of the unmatched region will typically be determined by the need to provide a suitable sequence for the binding of primers for function, e.g., PCR and / or sequencing. In theory, there is no upper limit to the length of the unmatched region, except in cases where it is generally advantageous to minimize the total length of the adapter, e.g., to facilitate the separation of unbound adapters from the adapter-target construct after one or more ligation steps. Thus, it is preferred that the unmatched region is less than 50, or less than 40, or less than 30, or less than 25 consecutive nucleotides in length on each strand.

[0096] The total length of the two strands forming the adapter typically ranges from 25 to 100 nucleotides, more typically from 30 to 55 nucleotides.

[0097] The portions of the two strands that form the unmatched region should preferably be of similar length, but this is not absolutely essential, provided that the length of each portion is sufficient to perform its desired function (e.g., primer binding).Experiments have shown that the portions of the two strands that form the unmatched region can differ by up to 25 nucleotides without excessively affecting adapter function.

[0098] In certain embodiments, the portions of the two polynucleotide strands that form the unmatched region are completely mismatched or 100% non-complementary. However, some sequence "matching", i.e., a lower degree of non-complementarity, can be tolerated in this region without affecting function to a substantial degree. As mentioned above, the degree of sequence mismatch or non-complementarity is such that the two strands in the unmatched region remain in single-stranded form under the annealing conditions defined above.

[0099] The exact nucleotide sequence of the adaptor is generally not critical to the present disclosure and may be selected by the user such that the desired sequence elements are included in the consensus sequence of the library of templates ultimately derived from the adaptors, e.g., to provide binding sites for a particular set of universal extension primers and / or sequencing primers (e.g., P7 or P5 primers). Additional sequence elements may be included, e.g., to provide binding sites for sequencing primers that will ultimately be used in sequencing the template molecules in the library, or for products derived from amplification of the template library, e.g., on a solid support. The adaptors may further include a "barcode" sequence, which may be used to barcode template molecules derived from a particular source.

[0100] The exact nucleotide sequence of the adapters is generally non-limiting to the present disclosure, but the sequences of the individual strands in the unmatched regions should be such that none of the individual strands exhibits any internal self-complementarity that may lead to self-annealing, formation of hairpin structures, etc. under standard annealing conditions. Self-annealing of a strand in an unmatched region should be avoided, as this may prevent or reduce specific binding of an amplification primer to this strand.

[0101] Mismatch adaptors are preferably formed from two strands of DNA, but may contain a mixture of natural and non-natural nucleotides (e.g., one or more ribonucleotides) linked by a mixture of phosphodiester and non-phosphodiester backbone linkages. As discussed in more detail below, other non-nucleotide modifications may be included, such as, for example, biotin moieties, blocking groups, and capture moieties for attachment to a solid surface.

[0102] The one or more "target polynucleotide duplexes" to which the adaptors are ligated can be any polynucleotide molecule that can be used with additional methodologies, including amplification by solid-phase PCR, next generation sequencing, subcloning, and the like. The target polynucleotide duplexes can occur in double-stranded DNA form (e.g., genomic DNA fragments) or can occur in single-stranded form as DNA or RNA and be converted to dsDNA form prior to ligation. As an example, an mRNA molecule can be copied into a double-stranded cDNA suitable for use in the methods of the present disclosure using standard methodologies known in the art. The precise sequence of the target molecule is generally not critical to the present disclosure and can be known or unknown. Modified DNA molecules containing non-natural nucleotides and / or non-natural backbone linkages can serve as targets, provided that the modifications do not prevent the addition to the adaptors, the tagging of the adaptors to the DNA molecules, and / or the copying by PCR.

[0103] As used herein, the terms "tagmentation," "tagment," or "tagmenting" refer to the conversion of a nucleic acid, e.g., DNA, into an adaptor-modified template such that the nucleic acid is modified to contain 5' and 3' adaptor molecules. This process often involves modification of the nucleic acid with a transposon complex that includes a transposase enzyme complexed with an adaptor that contains a transposon end sequence. Tagging results in simultaneous fragmentation of the nucleic acid and ligation of adaptors to the 5' ends of both strands of the double-stranded fragment. Following a purification step to remove the transposase enzyme, additional sequences can be added to the ends of the adapted fragments by PCR.

[0104] "Transposase" refers to an enzyme that can form a functional complex with a transposon end-containing composition (e.g., a transposon, a transposon end, a transposon end composition) and catalyze the insertion or transposition of the transposon end-containing composition into a double-stranded target nucleic acid with which it is incubated, e.g., in an in vitro transposition reaction. The transposases presented herein can also include integrases from retrotransposons and retroviruses. Transposases, transposomes and transposome complexes are generally known to those of skill in the art, as exemplified by the disclosure of U.S. Patent Application Publication No. 2010 / 0120098, the contents of which are incorporated herein by reference in their entirety. Although many embodiments described herein refer to Tn5 transposase and / or hyperactive Tn5 transposase, it is understood that any transposition system capable of inserting transposon ends with sufficient efficiency to 5' tag and fragment the target nucleic acid for the intended purpose can be used in the present invention. In certain embodiments, a preferred transposition system is capable of inserting transposon ends in a random or near random manner and 5' tagging and fragmenting the target nucleic acid.

[0105] As used herein, the term "transposition reaction" refers to a reaction in which one or more transposons are inserted into a target nucleic acid, for example, at random or near-random sites. The essential components of a transposition reaction are a transposase and a DNA oligonucleotide that represents the nucleotide sequence of the transposon, including the transferred transposon sequence and its complement (the transposon end sequence that is not transferred), as well as other components required to form a functional transposition or transposon complex. The DNA oligonucleotide may further include additional sequences (e.g., adapter or primer sequences) as needed or desired. In some embodiments, the methods provided herein are exemplified by using transposition complexes formed by hyperactive Tn5 transposase and Tn5-type transposon ends (Goryshin and Reznikoff, 1998, J. Biol. Chem., 273:7367), or by MuA transposase and Mu transposon ends containing R1 and R2 end sequences (Mizuuchi, 1983, Cell, 35:785; Savilahti et al., 1995, EMBO J., 14:4893). However, any transposition system capable of inserting transposon ends in a random or near-random manner with sufficient efficiency to 5'-tag and fragment target DNA for the intended purpose may be used in the present invention.Examples of transposition systems known in the art that may be used in the methods of the invention include Staphylococcus aureus Tn552 (Colegio et al., 2001, J Bacterid., 183:2384-8; Kirby et al., 2002, MoI Microbiol, 43:173-86), TyI (Devine and Boeke, 1994, Nucleic Acids Res., 22:3765-72 and International Patent Application No. 95 / 23875), transposon Tn7 (Craig, 1996, Science. 271:1512; Craig, 1996, Review in: Curr Top Microbiol Immunol, 204:27-48), TnIO and ISL0 (Kleckner et al., 1996, Curr Top Microbiol Immunol, 204:27-48). Immunol, 204:49-82), mariner transposase (Lampe et al., 1996, EMBO J., 15:5470-9), Tci (Plasterk, 1996, Curr Top Microbiol Immunol, 204:125-43), P elements (Gloor, 2004, Methods MoI Biol, 260:97-114), TnJ (Ichikawa and Ohtsubo, 1990, J Biol Chem. 265:18829-32), bacterial insertion sequences (Ohtsubo and Sekine, 1996, Curr. Top. Microbiol. Immunol. 204:1-26), retroviruses (Brown et al., 1989, Proc Natl Acad Sci USA, 86:2525-9), and yeast retrotransposons (Boeke and Corces, 1989, Annu Rev Microbiol. 43:403-34). Methods for inserting transposon ends into target sequences can be performed in vitro using any suitable transposon system for which a suitable in vitro transposition system is available or which can be developed based on knowledge in the art.In general, a suitable in vitro transposition system for use in the methods provided herein requires at least a transposase enzyme of sufficient purity, sufficient concentration, and sufficient in vitro transposition activity, and a transposase end that forms a functional complex with the respective transposase that can catalyze a transposition reaction. Suitable transposase transposon end sequences that can be used in the present invention include, but are not limited to, wild-type, derivative, or mutant transposon end sequences that form a complex with a transposase selected from a wild-type, derivative, or mutant of the transposase.

[0106] As used herein, the term "transposome complex" refers to a transposase enzyme that is non-covalently bound to double-stranded nucleic acid. For example, the complex can be a transposase enzyme pre-incubated with double-stranded transposon DNA under conditions that support non-covalent complex formation. The double-stranded transposon DNA can include, but is not limited to, Tn5 DNA, a portion of Tn5 DNA, a transposon end composition, a mixture of transposon end compositions, or other double-stranded DNA that can interact with a transposase, such as a hyperactive Tn5 transposase.

[0107] The term "transposon end" (TE) refers to a double-stranded nucleic acid, e.g., a double-stranded DNA that exhibits only the necessary nucleotide sequences ("transposon end sequences") to form a complex with a transposase or integrase enzyme that functions in an in vitro transposition reaction. In some embodiments, the transposon end is capable of forming a functional complex with a transposase in a transposition reaction. As a non-limiting example, the transposon end may include a 19-bp outer end ("OE") transposon end, an inner end ("IE") transposon end, or a "mosaic end" ("ME") transposon end recognized by a wild-type or mutant Tn5 transposase, or an R1 and R2 transposon end, as described in the disclosure of U.S. Patent Application Publication No. 2010 / 0120098, which is incorporated herein by reference in its entirety. Transposon ends may comprise any nucleic acid or nucleic acid analogue suitable for forming a functional complex with a transposase or integrase enzyme in an in vitro transposition reaction. For example, transposon ends may comprise DNA, RNA, modified bases, unnatural bases, modified backbones, and may contain nicks in the single or double strands. Although the term "DNA" is sometimes used in this disclosure in connection with transposon end compositions, it should be understood that any suitable nucleic acid or nucleic acid analogue may be utilized in the transposon ends.

[0108] "Ligation" of the adapter to the 5' and 3' ends of each target polynucleotide involves binding the two polynucleotide strands of the adapter to the double-stranded target polynucleotide such that a covalent bond is formed between both strands of the two double-stranded molecules. In this context, "binding" refers to the covalent binding of two polynucleotide strands that were not previously covalently linked. Preferably, such "binding" will occur by the formation of a phosphodiester linkage between the two polynucleotide strands, although other covalent binding means (e.g., non-phosphodiester backbone linkages) can be used. However, the covalent bond formed in the ligation reaction should allow polymerase read-through so that the resulting construct can be copied in a PCR reaction using primers that bind to sequences in the region of the adapter-target construct that originates from the adapter molecule.

[0109] The ligation reaction will typically be enzymatically catalyzed. In certain embodiments, the ligation reaction will be catalyzed by a ligase or a non-homologous end joining agent. Non-enzymatic ligation techniques (e.g., chemical ligation) can also be used, provided that the non-enzymatic ligation leads to the formation of a covalent bond that allows polymerase read-through, so that the resulting construct can be copied by PCR.

[0110] The desired product of the ligation reaction is an adaptor-target construct, where adaptors are ligated at both ends of each target polynucleotide, giving the structure adaptor-target-adaptor. Thus, the conditions of the ligation reaction should be optimized to maximize the formation of this product, in preference to targets with an adaptor at only one end.

[0111] Before the adapter-target construct is further processed, the products of the tagging or ligation reaction may be subjected to a purification step to remove unbound adapter molecules. Any suitable technique may be used to remove excess unbound adapters, preferred examples of which are described in more detail below.

[0112] The adaptor-target constructs are then amplified by PCR, as described in further detail below. The products of such further PCR amplifications can be collected to form a library of templates. In certain embodiments, the primers used for PCR amplification will anneal to different primer binding sequences on opposite strands in the unmatched regions of the adaptors. However, other embodiments can be based on the use of a single type of amplification primer that anneals to a primer binding sequence in the double-stranded region of the adaptor.

[0113] As shown in FIG. 1, a novel and improved method for depleting undesired sequences to form a template library provides for the inclusion of one or more blocking oligonucleotides in the adapter construct PCR amplification reaction. Thus, unlike standard RNA-Seq protocols, there is no need to process the RNA sample to deplete the RNA sample for rRNA transcripts or enrich the RNA sample for mRNA prior to conversion to cDNA. The simplicity of using one or more blocking oligonucleotides of the present disclosure to reduce undesired fragments is advantageous in automated library preparation systems where reducing the number of reagents and steps is paramount to a simple and robust workflow. The use of one or more blocking oligonucleotides of the present disclosure facilitates the depletion of undesired fragments after library construction, allowing for reduced hands-on time with labile RNA. In addition, the use of PCR clamps can be combined with traditional rRNA depletion approaches for more challenging samples known to have biologically abundant rRNA, globin transcripts, or other undesired transcripts.

[0114] It is generally advantageous for adapter-target constructs amplified by PCR in solution or on a solid support to contain regions of "different" sequence at their 5' and 3' ends, despite being common to all template molecules in the library, particularly if the amplified products are ultimately to be sequenced. For example, the presence of a common unique sequence at only one end of each template in a library can provide a binding site for a sequencing primer, allowing one strand of each template in the amplified form of the library to be sequenced in a single sequencing reaction using a single type of sequencing primer.

[0115] The conditions encountered during the annealing step of a PCR reaction are generally known to those of skill in the art, although the precise annealing conditions will vary from reaction to reaction (Sambrook et al., 2001, Molecular Cloning, A Laboratory Manual, 3rd Ed, Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory Press, NY; Current Protocols, eds Ausubel et al.). Typically, such conditions may include, but are not limited to, exposure to a temperature ranging from about 40° C. to 72° C. (preferably 50-68° C.) for about 1 minute in a standard PCR reaction buffer (followed by a denaturation step at a temperature of about 94° C. for about 1 minute).

[0116] The inclusion of PCR amplification to form complementary copies of the adaptor-target constructs is advantageous for several reasons. First, the inclusion of a primer extension step and subsequent PCR amplification acts as an enrichment step to select adaptor-target constructs with adaptors ligated at both ends, particularly in the case of the disclosed method, since undesired transcripts are not amplified in the PCR reaction. Only target constructs with adaptors ligated at both ends provide valid templates for PCR using common or universal primers specific to the primer binding sequences in the adaptors, and therefore it is advantageous to generate a template library that contains only dual-ligated targets prior to PCR amplification.

[0117] Secondly, the inclusion of PCR amplification allows the length of the common sequence at the 5' and 3' ends of the target to be increased prior to sequencing. As outlined above, it is generally advantageous to keep the length of the adapter molecule as short as possible to maximize the efficiency of ligation and subsequent removal of unbound adapters. However, for sequencing purposes, it may be advantageous to have longer sequence common or "universal" sequences at the 5' and 3' ends of the template to be amplified. The inclusion of PCR amplification means that the length of the common sequence at one (or both) ends of the polynucleotides in the template library can be increased after ligation by the inclusion of additional sequences at the 5' ends of the primers used for PCR amplification.

[0118] The template library prepared according to the method disclosed herein can be used in any method of nucleic acid analysis, such as sequencing the template or its amplification product.Exemplary uses of the template library include, but are not limited to, providing templates (either single template or multiple template library) for whole genome amplification, sequencing, subcloning, and PCR amplification.

[0119] Template libraries prepared according to the disclosed methods from a complex mixture of genomic DNA fragments representing an entire genome or substantially an entire genome provide suitable templates for so-called "whole genome" amplification. The term "whole genome amplification" refers to a nucleic acid amplification reaction (e.g., PCR) in which the template to be amplified comprises a complex mixture of nucleic acid fragments that are representative of an entire (or substantially an entire) genome.

[0120] The library of templates prepared according to the methods described herein can be used for solid-phase nucleic acid amplification. As used herein, the term "solid-phase amplification" refers to any nucleic acid amplification reaction carried out on or in association with a solid support such that all or a portion of the amplification product is immobilized on the solid support when formed. Specifically, the term encompasses solid-phase polymerase chain reaction (solid-phase PCR), which is a reaction similar to standard solution-phase PCR, except that one or both of the forward and reverse amplification primers are immobilized on a solid support.

[0121] In "solid phase" amplification methods, one amplification primer may be immobilized (the other primer is usually in free solution). Alternatively, both the forward and reverse primers may be immobilized. In practice, since the PCR process requires an excess of primers to support amplification, there will be "multiple" identical forward primers and / or "multiple" identical reverse primers immobilized on a solid support. References herein to forward and reverse primers should be construed as encompassing "multiple" such primers, unless the context dictates otherwise.

[0122] Solid-phase amplification can be performed using only one type of primer, and such single primer method is included within the scope of the present disclosure.Other embodiments can use forward and reverse primers that contain the same template-specific sequence but differ in some other structural features.For example, one type of primer can contain non-nucleotide modifications that are not present in the other.In other embodiments, forward and reverse primers can contain template-specific portions of different sequences.

[0123] Amplification primers for solid-phase PCR are preferably immobilized by covalent attachment to a solid support at or near the 5' end of the primer, leaving the template-specific portion of the primer free for annealing to its cognate template and the 3' hydroxyl group free for primer extension. Any suitable covalent attachment means known in the art can be used for this purpose. The attachment chemistry selected will depend on the nature of the solid support and any derivatization or functionalization applied to it. The primer itself may contain moieties that may be non-nucleotidic chemical modifications to facilitate attachment.

[0124] It is preferred to use the library of templates prepared according to the method disclosed herein to prepare clustered arrays of nucleic acid colonies by solid-phase PCR amplification. The terms "cluster" and "colony" are used interchangeably herein and refer to distinct sites on a solid support that are composed of a plurality of identical immobilized nucleic acid strands and a plurality of identical immobilized complementary nucleic acid strands. The term "clustered array" refers to an array formed from such clusters or colonies. In this context, the term "array" should not be understood as requiring an ordered arrangement of clusters.

[0125] In certain embodiments, the present disclosure further provides a method of sequencing amplified nucleic acids produced by PCR amplification. Accordingly, the present disclosure provides a method of nucleic acid sequencing comprising amplifying a library of nucleic acid templates using PCR as described above, and performing a nucleic acid sequencing reaction to determine the sequence of all or a portion of at least one amplified nucleic acid strand produced by PCR.

[0126] Sequencing can be carried out using any suitable "sequencing by synthesis" technique, in which nucleotides are added sequentially to a free 3' hydroxyl group, resulting in the synthesis of a polynucleotide chain in the 5' to 3' direction. The nature of the added nucleotide is preferably determined after each nucleotide addition.

[0127] The starting point for the sequencing reaction may be provided by annealing of a sequencing primer to the product of the whole genome or a solid-phase amplification reaction. In this regard, one or both of the adapters added during the formation of the template library may contain a nucleotide sequence that allows annealing of a sequencing primer to the amplification product derived by solid-phase amplification of the whole genome or the template library.

[0128] The product of a solid-phase amplification reaction, in which both forward and reverse amplification primers are covalently immobilized on a solid surface, is a so-called "bridged" structure formed by paired annealing of an immobilized polynucleotide strand and an immobilized complementary strand, both strands attached at their 5' ends to a solid support (e.g., a flow cell). Arrays composed of such bridged structures provide inefficient templates for nucleic acid sequencing, since hybridization of a conventional sequencing primer to one of the immobilized strands is unfavorable compared to annealing of this strand to its immobilized complementary strand under standard conditions for hybridization.

[0129] To provide a template more suitable for nucleic acid sequencing, it is preferred to remove substantially all or at least a portion of one of the immobilized strands of the "bridged" structure to generate a template that is at least partially single-stranded. Thus, the portion of the template that is single-stranded is available for hybridization to a sequencing primer. The process of removing all or a portion of one of the immobilized strands in the "bridged" double-stranded nucleic acid structure may be referred to herein as "linearization."

[0130] The bridged template structure may be linearized by cleavage of one or both strands with a restriction endonuclease, or by cleavage of one strand with a nicking endonuclease. Other methods of cleavage may be used as alternatives to restriction or nicking enzymes, including, inter alia, chemical cleavage (e.g., cleavage of diol bonds with periodic acid), cleavage of abasic sites by cleavage with endonucleases or by exposure to heat or alkali, cleavage of ribonucleotides otherwise incorporated into the amplification products composed of deoxyribonucleotides, photochemical cleavage, or cleavage of peptide linkers.

[0131] It will be appreciated that if the solid phase amplification reaction is performed with only one primer covalently immobilized and the other in free solution, the linearization step may not be necessary.

[0132] To generate a linearized template suitable for sequencing, it is necessary to remove "unequal" amounts of the complementary strand in the bridge structure formed by amplification to leave a linearized template for sequencing that is completely or partially single-stranded. Most preferably, one strand of the bridge structure is substantially or completely removed.

[0133] Following the cleavage step, regardless of the method used for cleavage, the product of the cleavage reaction can be subjected to denaturing conditions to remove the portion of the cleaved strand that is not attached to the solid support. Suitable denaturing conditions will be clear to the skilled reader with reference to standard molecular biology protocols (Sambrook et al., 2001, Molecular Cloning, A Laboratory Manual, 3rd Ed, Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory Press, NY; Current Protocols, eds Ausubel et al.).

[0134] Denaturation (and subsequent reannealing of the cleaved strand) results in the generation of a sequencing template that is partially or substantially single-stranded. The sequencing reaction can then be initiated by hybridization of a sequencing primer to the single-stranded portion of the template.

[0135] Thus, a nucleic acid sequencing reaction can involve hybridizing a sequencing primer to a single-stranded region of the linearized amplification product, sequentially incorporating one or more nucleotides into a polynucleotide strand complementary to the region of the amplified template strand to be sequenced, and identifying a base present in one or more of the incorporated nucleotides, thereby determining the sequence of the region of the template strand.

[0136] One preferred sequencing method that can be used by the present disclosure relies on the use of modified nucleotides that can act as chain terminators. When a modified nucleotide is incorporated into a growing polynucleotide strand that is complementary to the region of the template being sequenced, there is no free 3'-OH group available to induce further sequence extension, and therefore the polymerase cannot add additional nucleotides. Once the nature of the base incorporated into the growing strand is determined, the 3' block can be removed to allow the addition of the next successive nucleotide. By sequencing the products derived using these modified nucleotides, it is possible to deduce the DNA sequence of the DNA template. If each of the modified nucleotides is attached with a different label known to correspond to a particular base, to facilitate discrimination between the bases added at each incorporation step, such reactions can be performed in a single experiment. Alternatively, separate reactions can be performed, containing each of the modified nucleotides separately.

[0137] The modified nucleotides may carry a label to facilitate their detection. Preferably, this is a fluorescent label. Each type of nucleotide may carry a different fluorescent label. However, the detectable label does not have to be a fluorescent label. Any label that allows the detection of the incorporated nucleotide may be used.

[0138] One method for detecting fluorescently labeled nucleotides involves the use of laser light of a wavelength specific to the labeled nucleotide, or other suitable illumination source. Fluorescence from the label on the nucleotide can be detected by a CCD camera or other suitable detection means.

[0139] The present disclosure is not limited to the use of the sequencing methods outlined above, since essentially any sequencing methodology that relies on the sequential incorporation of nucleotides into a polynucleotide chain may be used.Suitable alternative techniques include, for example, Pyrosequencing™, FISSEQ (fluorescent in situ sequencing), MPSS (massively parallel signature sequencing), and ligation-based methods of sequencing.

[0140] The target polynucleotide sequenced using the method of the present disclosure can be any polynucleotide that is desired to be sequenced.Using the template library preparation method described in detail herein, it is possible to prepare a template library starting from essentially any double-stranded or single-stranded target polynucleotide of known, unknown or partially known sequence.The use of clustered arrays prepared by solid-phase amplification allows for parallel sequencing of multiple targets of the same or different sequences.

[0141] Various non-limiting specific embodiments of the method of the present disclosure will now be described in more detail with reference to the accompanying drawings, in which: Features described as preferred with respect to one specific embodiment of the present disclosure apply mutatis mutandis to other specific embodiments of the present disclosure, unless otherwise stated.

[0142] Figure 1 provides the RNA-Seq technique for the generation of sequencing libraries from RNA samples, as described in detail above. Unlike traditional RNA workflows, the workflow, enabled by the addition of one or more blocking oligonucleotides specific for undesired rRNA fragments, does not require a long 1-2 h depletion of rRNA before conversion of the RNA to cDNA, as is the case for on-market technologies. This allows for faster workflow times and, in some implementations, easier automation due to the reduced need for various reagents.

[0143] FIG. 2 provides an illustration and overview of an exemplary method of the present disclosure. As shown, PCR clamps selectively block amplification of targeted undesired library fragments (see FIG. 2A). Following denaturation of the library in the initial heat denaturation step of PCR, amplification primers bind to the ends of the library fragments. PCR clamps designed to be complementary to the undesired fragments also hybridize to the selected library fragments (see FIG. 2B). A thermostable polymerase can extend the primers and copy the desired library fragments. However, because typical thermostable polymerases used in PCR lack 5'-3' exonuclease and strand displacement activity, the PCR clamps effectively block the copying of undesired fragments (see FIG. 2C). After several cycles of PCR, the desired library fragments have been exponentially amplified, while the amplification of undesired fragments has been suppressed. The result is a final amplified library with reduced representation of undesired library fragments (see FIG. 2D). The methods of the disclosure have been found to work well with Kapa HiFi polymerase due to the lack of 5' to 3' exonuclease activity and strand displacement of Kapa HiFi polymerase.

[0144] Figure 3 provides various designs of pools of blocking oligonucleotides (i.e., PCR clamps) for depleting undesired transcripts from a template library. Design 1 provides a pool of antiparallel and adjacent PCR clamps. Design 1+2 provides the same pool of PCR clamps as Design 1, but with an inverse complementary PCR clamp added to the pool. Design 3 provides antiparallel overlapping PCR clamps.

[0145] Figure 4 shows that the Design1 PCR clamp pool and the Design1_2 PCR clamp pool reduced the percentage of rRNA transcripts from 80% to 30% in an RNA-seq protocol using non-depleted RNA. No additional work-up steps were required.

[0146] FIG. 5 shows that the pool of PCR clamps from Design1 and the pool of PCR clamps from Design1_2 further reduced the percentage of rRNA transcripts from 20% to 1% in the RNA-seq protocol using RPO-depleted RNA samples (left panel). The RPO-depleted RNA samples are enriched with the library fragments of interest, but some undesired ribosomal rRNA is still observed (20%). (RPO=RNA Pan-Cancer Oligos (i.e., oligos from Illumina™ TruSight RNA Pan-Cancer products). Furthermore, the pool of PCR clamps from Design1 and the pool of PCR clamps from Design1_2 were able to deplete rRNA transcripts in non-depleted RNA samples to a level comparable to the RPO-depleted RNA samples (right panel). Design3 (DesignOffSet) was unable to deplete the sample of rRNA transcripts. It is hypothesized that the PCR clamps were priming off each other to form rRNA artifact secondary structures.

[0147] FIG. 6 shows that the Design1 pool of PCR clamps and the Design1_2 pool of PCR clamps further reduced the percentage of rRNA transcripts from 1.5% to 0.25% in an RNA-seq protocol using mRNA-selected samples.

[0148] Figure 8 shows that samples depleted with Design1 PCR clamp or Design1_2 PCR clamp exhibited high levels of gene expression, as measured by fragments per kilobase of transcript per million mapped reads (FPKM), with values ​​of >0.95, which were comparable to other depletion methods.

[0149] FIG. 9 provides traces showing that rRNA transcripts were significantly reduced in samples that were rRNA depleted versus non-depleted using blocking oligonucleotides.

[0150] Figure 10 presents an exemplary blocking oligonucleotide of the present disclosure. Blocking oligonucleotides are designed to hybridize with the internal (i.e. non-overlapping primer binding site) regions of target fragments. Because most DNA polymerases used in PCR lack significant strand displacement activity, the presence of sufficiently tightly bound blocking oligonucleotides should physically impede the progression of polymerase and prevent the synthesis of full-length amplicons. Considerations for blocking nucleotides include, but are not limited to: (1) Have a melting temperature (Tm) higher than the temperature of the extension step in the PCR reaction, which ensures that the blocking oligonucleotide remains bound throughout the PCR extension step. (2) The blocking oligonucleotide may contain a 3'-block at its 3' end to prevent polymerase extension. This 3'-block prevents the blocking oligonucleotide from acting as a primer and generating undesired PCR by-products. Several methods can be used to achieve this, including 3' spacer modifications (e.g., C3), 3' inverted bases, 3' phosphorylation, 3' dideoxy bases, or 3' non-complementary overhanging bases. (3) If a proofreading DNA polymerase (i.e., a polymerase with strong 3'->5' exonuclease activity) is used in the PCR reaction, the blocking oligo should be resistant to exonuclease activity at the 3' end to prevent degradation. This can be achieved by the blocking oligonucleotide containing one or more phosphorothioate linkages at the 3' end of the blocking oligonucleotide. (4) When a polymerase with strong 5'->3' exonuclease activity (e.g., Taq DNA polymerase) is used, the blocking oligo should be resistant to exonuclease degradation at its 5' end, which can be achieved by the blocking oligonucleotide containing one or more phosphorothioate linkages at the 5' end of the blocking oligonucleotide.

[0151] Due to the sequence dependence of Tm, the length of the oligo required to achieve consideration (1) can be quite long, especially for AT-rich sequences. Additional oligo modifications, such as locked nucleic acid (LNA) bases or peptide nucleic acid (PNA) linkages, can be used in this situation to increase the Tm of the blocking oligonucleotide without changing its length or sequence.

[0152] Figures 11-12 demonstrate the use of blocking oligonucleotides to deplete ribosomal sequences from an RNA-seq library. A pool of blocking oligos can be designed such that a majority of potential library fragments from each of the five major rRNA sequences (18S, 28S, 5S, mitochondrial 12S, and mitochondrial 16S) are targeted by one or more blocking oligonucleotides. The pool of blocking oligos can then be added to the sample during the PCR amplification step of library preparation, resulting in specific depletion of rRNA amplicons in the final library.

[0153] In addition to the general blocking oligonucleotide considerations outlined above, several additional parameters need to be considered for rRNA blocking oligonucleotide pool design. (1) The length of the blocking oligonucleotide should be minimized as much as possible while maintaining the target T. This allows the maximum number of possible rRNA library fragments to be covered by end-to-end matches with the blocking oligo. (2) Blocking oligonucleotide spacing should be selected to minimize the number of gaps larger than the insert size of the target library. (3) Blocking oligonucleotides may need to be designed to target both the sense and antisense strands of the targeted rRNA fragment.

[0154] To design a pool of rRNA blocking oligos for use with human RNA-seq libraries, a computational strategy was implemented that included the following steps. (1) Starting from the 5' end of each rRNA sequence, a 90 bp window (approximately 0.5 times the average insert size of the RNA library) was designated and scanned for oligos with a Tm above 80° C. The oligo length was initially set at 15 bp and increased iteratively until either (a) an oligo with the desired Tm was found or (b) the oligo length was greater than 90 bp. (2) Once an oligo is identified within a window, a new 90 bp window is set starting from the 3' end of the oligo and the search procedure from step (1) is repeated. If the oligo is not found within a given window, a new window is set starting from the 3' end of the previous window. (3) Steps (1) and (2) are repeated until the end of the sequence is reached.

[0155] Using this approach, a set of blocking oligos was designed that covered nearly the entire length of five human rRNAs with only 11 gaps longer than 90 bp across all sequences (see Figures 11 and 12). Simulations using a non-depleted RNAseq library (i.e., consisting mostly of rRNA) showed that nearly 90% of the rRNA library fragments would be targeted for depletion by one or more of the blocking oligonucleotides from the designed pools. This suggests that the blocking oligonucleotide approach described herein can provide comparable depletion efficiency to commercially available rRNA depletion kits (e.g., about 95% depletion for RiboMinus), with a greatly simplified workflow and better performance on low-input RNA samples. This approach to pool design can also be applied to other NGS methods where contamination by abundant sequences is problematic, such as rare somatic mutation detection, NIPT, metagenomics, or pathogen detection.

[0156] Thus, the studies presented herein demonstrated that a pool of blocking oligonucleotides (i.e., PCR clamps) selectively prevented PCR amplification of undesired library fragments. Depletion of undesired transcripts from libraries does not require extra work-up steps by the user, and only one or more blocking polynucleotides need to be added to the PCR amplification reaction. The studies clearly demonstrate that one can selectively reduce rRNA content in an amplified RNA-Seq library by using one or more blocking oligonucleotides (i.e., PCR clamps) of the present disclosure. Furthermore, in samples treated with rRNA depleting agents (RPO treatment) and mRNA selected samples, the use of one or more blocking oligonucleotides significantly further reduced rRNA content in these samples. For example, in RPO treated samples, the use of one or more blocking oligonucleotides (i.e., PCR clamps) of the present disclosure reduced rRNA content from approximately 10-15% to <1% rRNA.

[0157] Compared to other rRNA depletion techniques, the disclosed compositions, methods, and kits provide for more rapid preparation of depleted RNA libraries using an RNA-Seq workflow. Furthermore, the disclosed compositions, methods, and kits depleted rRNA content from 80% to 30%, which was comparable to existing rRNA depletion techniques. The disclosed compositions, methods, and kits are fully compatible with existing rRNA depletion techniques and can be used with them to further reduce rRNA content to barely detectable levels. Few off-target effects were observed, and the disclosed compositions, methods, and kits maintained a high correlation of gene-level expression that was comparable to Ribozero and RNase H depletion methods. The number of cycles in the PCR reaction correlates to the level of reduction of undesired transcripts in the resulting library. In other words, the higher the number of PCR cycles, the greater the reduction of undesired transcripts in the resulting library.

[0158] It should be noted that studies were performed with blocking oligonucleotides where a 3'-block was not utilized (i.e., PCR clamps). It is anticipated that blocking oligonucleotides will likely provide further improvement in depleting samples of undesired transcripts and greatly reduce the formation of concatemers at overlapping blocking nucleotides (Design 3). If it is necessary to increase the Tm of the blocking nucleotide without increasing the length of the blocking oligonucleotide, modified bases such as LNA or PNA can be used.

[0159] Although the study was directed to depleting rRNA transcripts from total RNA samples, it is expected that the disclosed methods, compositions, and kits are generally applicable to reducing undesired transcripts in library preparations. For example, one or more blocking oligonucleotides can be used to reduce undesired mtDNA in ATAC-Seq preparations, or to reduce host transcripts for epidemiological samples.

[0160] The present disclosure further provides kits comprising one or more blocking oligonucleotides disclosed herein. The kits may be tailored for use in a particular application. For example, the kits may be directed to the use of one or more blocking oligonucleotides in preparing a library of template polynucleotides using the methods of the present disclosure. Such kits may include at least a supply of adaptors as defined herein, and a supply of at least one amplification primer that can anneal to the adaptor to initiate synthesis of an extension product, which will include any target sequence ligated to the adaptor when the adaptor is used. The structure and properties of amplification primers will be well known to those of skill in the art. Suitable primers of appropriate nucleotide sequence for use with the adaptors included in the kits can be readily prepared using standard automated nucleic acid synthesis equipment and reagents routinely used in the art. The kits may include pairs of PCR primers suitable for PCR amplification of templates modified with mismatch adaptors (i.e., solid-phase PCR) in solution phase and / or on a suitable solid support, either as a supply of one single type of primer or as separate supplies of two different primers (or even in a mixture).

[0161] The adaptors, PCR primers, and one or more blocking oligonucleotides may be provided in a ready-to-use kit, or more preferably as concentrates that require dilution before use, or in lyophilized or dried form that requires reconstitution before use. If necessary, the kit may further include a supply of suitable diluents for diluting or reconstituting the primers. Optionally, the kit may further include supplies of reagents, buffers, enzymes, dNTPs, etc. for use in performing PCR amplification. Additional components that may optionally be provided in the kit include "universal" sequencing primers suitable for sequencing templates prepared using the adaptors and primers.

[0162] The present disclosure further provides that the methods and compositions described herein can be further defined by the following aspects (Aspects 1-43). Embodiment 1. A method for selectively depleting undesired fragments from an amplified DNA or cDNA library by using one or more blocking oligonucleotides, comprising: amplifying a plurality of library fragments comprising double-stranded template sequences including adapter sequences in a polymerase chain reaction (PCR) reaction, a portion of the fragments including unwanted fragments that are not to be analyzed; The PCR reaction comprises a plurality of fragments, a polymerase, dNTPs, PCR primers, and one or more blocking oligonucleotides, the one or more blocking oligonucleotides being selected from (i), and / or (ii), and (iii): (i) at the 5' end, one or more nucleotides containing a phosphorothioate bond, and / or (ii) at the 3' terminus, one or more nucleotides containing a phosphorothioate bond; and (iii) a 3'-block that prevents polymerase extension at the 3' end of the blocking oligonucleotide; A method in which one or more blocking primers bind to the template sequence of the undesired fragments, thereby blocking the amplification of the undesired fragments by PCR. Embodiment 2. The method of embodiment 1, wherein one or more of the blocking oligonucleotides are between 15 nt and 100 nt in length, preferably the blocking nucleotides are between 15 nt and 80 nt, 15 nt and 70 nt, 15 nt and 60 nt, 15 nt and 50 nt, 15 nt and 40 nt, 15 nt and 30 nt, 17 nt and 30 nt, or 20 nt and 30 nt in length. Embodiment 3. The method of embodiment 1 or embodiment 2, wherein when the polymerase has 5'-3' exonuclease activity, one or more of the blocking oligonucleotides comprises, at the 5' end, from 1 to 5 nucleotides that contain phosphorothioate linkages, preferably, the 5' end comprises from 2 to 5, 3 to 5, 4 to 5, 2 to 4, or 2 to 3 nucleotides that contain phosphorothioate linkages. Embodiment 4. The method of any one of embodiments 1-3, wherein when the polymerase has 3'-5' exonuclease activity, one or more of the blocking oligonucleotides comprises, at the 3' end, from 1 to 5 nucleotides that contain phosphorothioate linkages, preferably, the 3' end comprises 2 to 5, 3 to 5, 4 to 5, 2 to 4, or 2 to 3 nucleotides that contain phosphorothioate linkages.

[0163] Embodiment 5. One or more blocking oligonucleotides comprise (i), (ii), and (iii): (i) The 5'-terminus contains 2 to 5 nucleotides including phosphorothioate bonds, preferably 2 to 5, 3 to 5, 4 to 5, 2 to 4, or 2 to 3 nucleotides including phosphorothioate bonds. and / or (ii) The 3'-terminus contains 2 to 5 nucleotides including phosphorothioate bonds, preferably 2 to 5, 3 to 5, 4 to 5, 2 to 4, or 2 to 3 nucleotides including phosphorothioate bonds. and (iii) a 3'-block that prevents polymerase extension on the 3' end of the blocking oligonucleotide. Embodiment 6. The method of any one of embodiments 1 to 5, wherein the 3'-block is selected from a C3-spacer, a 3' inverted base, a 3' phosphorylation, a 3' dideoxy base, or a 3' non-complementary overhanging base, preferably the 3'-block is a C3-spacer. Embodiment 7 The method of any one of embodiments 1 to 6, wherein the amplified library comprises template sequences derived from cDNA. Embodiment 8. The method of any one of embodiments 1 to 7, wherein the amplified library comprises template sequences derived from gDNA. Embodiment 9. The method of any one of embodiments 1-8, wherein the adaptor sequences are derived from Y-shaped adaptors that are ligated to each end of the template sequence. Embodiment 10. The method of any one of embodiments 1 to 9, wherein the one or more blocking oligonucleotides bind to a template sequence derived from rRNA and / or globin. Embodiment 11. The method of any one of embodiments 1 to 10, wherein the one or more blocking oligonucleotides comprise a pool of blocking oligonucleotides that bind to a template sequence derived from 18S rRNA, 5.8S rRNA, and / or 28S RNA. Embodiment 12 The method of any one of embodiments 1 to 11, wherein one or more of the blocking oligonucleotides binds to a template sequence derived from mtDNA. Embodiment 13. The method of any one of embodiments 1 to 12, wherein the amplified DNA or cDNA library is analyzed by using next generation sequencing. 14. The PCR amplification process comprises the following steps: obtaining an RNA sample; Fragmenting the RNA, preferably by sonication, use of enzymes, heat alone, or exposure to divalent cations at elevated temperature; reverse transcribing the RNA fragments into cDNA; blunting the cDNA and adding an A nucleotide to the 3' end of the blunt-ended cDNA; 14. The method of any one of embodiments 1-13, preceded by a step of ligating the A-tailed cDNA to an adaptor comprising a non-complementary T nucleotide at its 3' end. Embodiment 15. The method of embodiment 14, wherein the RNA sample is treated to deplete rRNA sequences from the RNA sample prior to reverse transcribing the RNA fragments into cDNA. Embodiment 16. The method of any one of embodiments 1 to 13, wherein the PCR amplification step is preceded by a tagging reaction step to generate a plurality of library fragments comprising double-stranded template sequences that include adapter sequences. Embodiment 17. A method for selectively depleting undesired fragments from an amplified DNA or cDNA library by using one or more blocking oligonucleotides, comprising: amplifying a plurality of library fragments comprising double-stranded template sequences ligated to adapter sequences in a polymerase chain reaction (PCR) reaction, a portion of the fragments including unwanted fragments containing template sequences that are not to be analyzed; the PCR reaction comprises a plurality of fragments, a polymerase, dNTPs, PCR primers, and a pool of blocking oligonucleotides, a portion of the pool of blocking oligonucleotides binding to each strand of a template sequence of the unwanted fragments; A method in which one or more blocking primers bind to the template sequence of the undesired fragments, thereby blocking the amplification of the undesired fragments by PCR. Embodiment 18. The method of embodiment 17, wherein the pool of blocking oligonucleotides is between 15 nt and 100 nt in length, preferably the blocking nucleotides are between 15 nt and 80 nt, 15 nt and 70 nt, 15 nt and 60 nt, 15 nt and 50 nt, 15 nt and 40 nt, 15 nt and 30 nt, 17 nt and 30 nt, or 20 nt and 30 nt in length. Embodiment 19. The method of embodiment 17, wherein the pool of blocking oligonucleotides comprises blocking oligonucleotides that bind to the strands of the template in a non-overlapping and contiguous manner, preferably in the manner of Design1 in FIG. 3. Embodiment 20. The method of embodiment 19, wherein the pool of blocking oligonucleotides comprises blocking oligonucleotides that are reverse complementary to other blocking oligonucleotides, preferably in the format of Design1+2 in FIG. 3. Embodiment 21. A pool of blocking oligonucleotides comprising (i), and / or (ii), and (iii): (i) at the 5' end, one or more nucleotides containing a phosphorothioate bond, and / or (ii) at the 3' terminus, one or more nucleotides containing a phosphorothioate bond; and (iii) a 3'-block that prevents polymerase extension on the 3' end of the blocking oligonucleotide. Embodiment 22. The method of embodiment 21, wherein one or more of the blocking oligonucleotides comprises, at the 5' end, 1 to 5 nucleotides comprising phosphorothioate linkages, when the polymerase has 5'-3' exonuclease activity. Embodiment 23. The method of embodiment 21, wherein one or more of the blocking oligonucleotides comprises, at the 3' end, 1 to 5 nucleotides that contain phosphorothioate linkages, if the polymerase has 3'-5' proofreading activity. Embodiment 24. One or more blocking oligonucleotides comprise (i), (ii), and (iii): (i) 2 to 5 nucleotides containing phosphorothioate bonds at the 5' end; (ii) 2 to 5 nucleotides containing phosphorothioate bonds at the 3' end, and (iii) a 3'-block that prevents polymerase extension on the 3' end of the blocking oligonucleotide. Embodiment 25. The method of any one of embodiments 21 to 24, wherein the 3'-block is selected from a C3-spacer, a 3' inverted base, a 3' phosphorylation, a 3' dideoxy base, or a 3' non-complementary overhanging base. Embodiment 26 The method of any one of embodiments 17 to 25, wherein the amplified library comprises template sequences derived from cDNA. Embodiment 27 The method of any one of embodiments 17 to 25, wherein the amplified library comprises template sequences derived from gDNA. Embodiment 28 The method of any one of embodiments 17 to 27, wherein the adaptor sequences are derived from Y-shaped adaptors that are ligated to each end of the template sequence. Embodiment 29 The method of any one of embodiments 17 to 28, wherein the pool of blocking oligonucleotides binds to a template sequence derived from rRNA and / or globin. Embodiment 30 The method of any one of embodiments 17 to 29, wherein the pool of blocking oligonucleotides binds to a template sequence derived from 18S rRNA, 5.8S rRNA, and / or 28S RNA. Embodiment 31 The method of any one of embodiments 17 to 30, wherein the blocking pool of blocking oligonucleotides binds to a template sequence derived from mtDNA. Embodiment 32. The method of any one of embodiments 17 to 31, wherein the amplified DNA or cDNA library is analyzed by using next generation sequencing. 33. The PCR amplification process comprises the following steps: obtaining an RNA sample; Fragmenting the RNA, preferably by sonication, use of enzymes, heat alone, or exposure to divalent cations at elevated temperature; reverse transcribing the RNA fragments into cDNA; blunting the cDNA and adding an A nucleotide to the 3' end of the blunt-ended cDNA; 33. The method of any one of embodiments 17-32, preceded by a step of ligating the A-tailed cDNA to an adaptor comprising a non-complementary T nucleotide at its 3' end. Embodiment 34 The method of embodiment 33, wherein the RNA sample is treated to deplete rRNA sequences from the RNA sample prior to reverse transcribing the RNA fragments into cDNA. Embodiment 35. The method of any one of embodiments 17 to 34, wherein the PCR amplification step is preceded by a tagging reaction step to generate a plurality of library fragments comprising double-stranded template sequences that include adapter sequences. Embodiment 36. An RNA-Seq based library preparation kit comprising one or more blocking oligonucleotides, the one or more blocking oligonucleotides comprising (i), and / or (ii), and (iii): (i) at the 5' end, one or more nucleotides containing a phosphorothioate bond, and / or (ii) at the 3' terminus, one or more nucleotides containing a phosphorothioate bond; and (iii) a 3'-block that prevents polymerase extension at the 3' end of the blocking oligonucleotide; An RNA-Seq-based library preparation kit in which one or more blocking oligonucleotides bind to template sequences of undesired library fragments, thereby blocking amplification of the undesired library fragments by PCR. 37. A library preparation kit comprising: A-tailing mix; Enhanced PCR mix and Ligation mix, Resuspension buffer; Stop Ligation Buffer; Elute, Prime, Fragment High Concentration Mix, First strand Synthesis Act D Mix and Reverse transcriptase and 37. The RNA-Seq-based library preparation kit of embodiment 36, further comprising a second strand master mix. Embodiment 38. The RNA-Seq based library preparation kit of embodiment 37, wherein one or more of the blocking oligonucleotides are between 15 nt and 100 nt in length, preferably the blocking nucleotides are between 15 nt and 80 nt, 15 nt and 70 nt, 15 nt and 60 nt, 15 nt and 50 nt, 15 nt and 40 nt, 15 nt and 30 nt, 17 nt and 30 nt, or 20 nt and 30 nt in length. Embodiment 39. An RNA-Seq based library preparation kit comprising a pool of blocking oligonucleotides, wherein a portion of the pool of blocking oligonucleotides binds to each strand of a template sequence of an undesired fragment in a non-overlapping and adjacent manner, thereby blocking amplification of the undesired library fragments by PCR. 40. A library preparation kit comprising: A-tailing mix; Enhanced PCR mix and Ligation mix, Resuspension buffer; Stop Ligation Buffer; Elute, Prime, Fragment High Concentration Mix, First strand Synthesis Act D Mix and Reverse transcriptase and 40. The RNA-Seq-based library preparation kit of embodiment 39, further comprising a second strand master mix. Embodiment 41. The RNA-Seq based library preparation kit of embodiment 39 or embodiment 40, wherein the pool of blocking oligonucleotides is between 15 nt and 100 nt in length, preferably the blocking nucleotides are between 15 nt and 80 nt, between 15 nt and 70 nt, between 15 nt and 60 nt, between 15 nt and 50 nt, between 15 nt and 40 nt, between 15 nt and 30 nt, between 17 nt and 30 nt, or between 20 nt and 30 nt in length. Embodiment 42. A pool of blocking oligonucleotides comprising (i), and / or (ii), and (iii): (i) at the 5' end, one or more nucleotides containing a phosphorothioate bond, and / or (ii) at the 3' terminus, one or more nucleotides containing a phosphorothioate bond; and (iii) a 3'-block that prevents polymerase extension on the 3' end of the blocking oligonucleotide. Embodiment 43. The RNA-Seq based library preparation kit of embodiment 42, wherein the 3'-block is selected from a C3-spacer, a 3' inverted base, a 3' phosphorylation, a 3' dideoxy base, or a 3' non-complementary overhanging base.

[0164] A number of embodiments of the present disclosure have been described. However, it should be understood that various modifications can be made without departing from the spirit and scope of the disclosure. Accordingly, other embodiments are within the scope of the following claims.

Claims

1. A method for selectively depleting undesired fragments from an amplified DNA or cDNA library by using a pool of blocking oligonucleotides, comprising: amplifying a plurality of library fragments comprising double-stranded template sequences that include adapter sequences in a polymerase chain reaction (PCR) reaction, a portion of the fragments comprising unwanted fragments that are not to be analyzed; The PCR reaction comprises a plurality of fragments, a polymerase, dNTPs, PCR primers, and a pool of blocking oligonucleotides, the pool of blocking oligonucleotides comprising (i), and / or (ii), and (iii): (i) at the 5'-terminus, one or more nucleotides containing a phosphorothioate bond; and / or (ii) at the 3' terminus, one or more nucleotides containing a phosphorothioate bond; and (iii) a 3'-block that prevents polymerase extension at the 3' end of the blocking oligonucleotide; The pool of blocking oligonucleotides binds to the template sequence of an undesired fragment at two or more positions in anti-parallel, adjacent, and reverse complementary orientations, thereby blocking amplification of the undesired fragment by PCR.

2. A method for selectively depleting undesired fragments from an amplified DNA or cDNA library by using a pool of blocking oligonucleotides, comprising: amplifying a plurality of library fragments comprising double-stranded template sequences including adapter sequences in a polymerase chain reaction (PCR) reaction, a portion of the fragments including unwanted fragments containing template sequences that are not to be analyzed; the PCR reaction comprises a plurality of fragments, a polymerase, dNTPs, PCR primers, and a pool of blocking oligonucleotides, a portion of the pool of blocking oligonucleotides binding to each strand of a template sequence of an undesired fragment; A method in which blocking oligonucleotides of a pool of blocking oligonucleotides bind to said template sequence of an undesired fragment at two or more positions, thereby blocking amplification of said undesired fragment by PCR.

3. The method of claim 1 or 2, wherein the oligonucleotides of the pool of blocking oligonucleotides are between 15 nt and 100 nt in length.

4. 3. The method of claim 2, wherein the pool of blocking oligonucleotides comprises blocking oligonucleotides that bind to the strands of the template in a non-overlapping and adjacent manner, preferably wherein the pool of blocking oligonucleotides comprises blocking oligonucleotides that are reverse complementary to other blocking oligonucleotides.

5. The pool of blocking oligonucleotides comprises (i), and / or (ii), and (iii): (i) at the 5'-terminus, one or more nucleotides containing a phosphorothioate bond; and / or (ii) at the 3' terminus, one or more nucleotides containing a phosphorothioate bond; and 3. The method of claim 2, comprising: (iii) a 3'-block that prevents polymerase extension on the 3' end of the blocking oligonucleotide.

6. If the polymerase has 5'-3' exonuclease activity, one or more of the blocking oligonucleotides comprises 1-5 nucleotides at the 5' end that contain phosphorothioate linkages, or if the polymerase has 3'-5' proofreading activity, one or more of the blocking oligonucleotides comprises 1-5 nucleotides at the 3' end that contain phosphorothioate linkages, or The one or more blocking oligonucleotides include (i), (ii), and (iii): (i) 2 to 5 nucleotides containing phosphorothioate bonds at the 5' end; (ii) 2 to 5 nucleotides containing phosphorothioate linkages at the 3' terminus, and (iii) a 3'-block that prevents polymerase extension on the 3' end of the blocking oligonucleotide; or The 3'-block is C ３ The method of claim 1 or 2, wherein the base is selected from a 3' spacer, a 3' inverted base, a 3' phosphorylation, a 3' dideoxy base, or a 3' non-complementary overhanging base.

7. The method of claim 1 or 2, wherein the amplified library comprises a cDNA-derived template sequence or a gDNA-derived template sequence.

8. 3. The method of claim 1 or 2, wherein the adaptor sequences are derived from Y-shaped adaptors that are ligated to each end of a template sequence.

9. Oligonucleotides of the pool of blocking oligonucleotides bind to template sequences derived from rRNA and / or globin, or Oligonucleotides of the pool of blocking oligonucleotides bind to a template sequence derived from 18S rRNA, 5.8S rRNA, and / or 28S RNA; or The method of claim 1 or 2, wherein the oligonucleotides of the pool of blocking oligonucleotides bind to a template sequence derived from mtDNA.

10. The method of claim 1 or 2, wherein the amplified DNA or cDNA library is analyzed by using next generation sequencing.

11. The PCR amplification step comprises the steps of: Obtaining an RNA sample; fragmenting the RNA; reverse transcribing the RNA fragments into cDNA; blunt-ending the cDNA and adding an A nucleotide to the 3' end of the blunt-ended cDNA; 3. The method of claim 1 or 2, wherein the method is preceded by a step of ligating the A-tailed cDNA with an adaptor comprising a non-complementary T nucleotide at the 3' end, and preferably wherein the RNA sample is treated to deplete rRNA sequences from the RNA sample prior to reverse transcribing the RNA fragments into cDNA.

12. An RNA-Seq-based library preparation kit comprising a pool of blocking oligonucleotides, the pool of blocking oligonucleotides comprising: (i), and / or (ii), and (iii): (i) at the 5'-terminus, one or more nucleotides containing a phosphorothioate bond; and / or (ii) at the 3' terminus, one or more nucleotides containing a phosphorothioate bond; and (iii) a 3'-block that prevents polymerase extension at the 3' end of the blocking oligonucleotide; An RNA-Seq-based library preparation kit, wherein the pool of blocking oligonucleotides binds to template sequences of undesired library fragments at two or more positions in anti-parallel, adjacent, and reverse-complementary orientations, thereby blocking amplification of the undesired library fragments by PCR.

13. 1. An RNA-Seq-based library preparation kit comprising a pool of blocking oligonucleotides, a portion of said pool of blocking oligonucleotides binding in a non-overlapping and adjacent manner to each strand of a template sequence of an undesired fragment, said pool of blocking oligonucleotides comprising: (i), and / or (ii), and (iii): (i) at the 5'-terminus, one or more nucleotides containing a phosphorothioate bond; and / or (ii) at the 3' terminus, one or more nucleotides containing a phosphorothioate bond; and (iii) a 3'-block that prevents polymerase extension at the 3' end of the blocking oligonucleotide; An RNA-Seq-based library preparation kit in which one or more blocking primers block amplification of undesired library fragments by PCR by binding to template sequences of the undesired fragments.

14. 14. The RNA-Seq-based library preparation kit of claim 12 or 13, wherein the oligonucleotides of the pool of blocking oligonucleotides are between 15 nt and 100 nt in length.

15. The pool of blocking oligonucleotides comprises (i), and / or (ii), and (iii): (i) at the 5'-terminus, one or more nucleotides containing a phosphorothioate bond; and / or (ii) at the 3' terminus, one or more nucleotides containing a phosphorothioate bond; and (iii) a 3'-block that prevents polymerase extension on the 3' end of the blocking oligonucleotide, preferably the 3'-block is ３ 15. The RNA-Seq-based library preparation kit of claim 14, wherein the base is selected from the group consisting of a 3' spacer, a 3' inverted base, a 3' phosphorylated, a 3' dideoxy base, or a 3' non-complementary overhanging base.

Images