WO2022148309A1 - 一种rRNA沉默的RNA文库构建方法及试剂盒 - Google Patents

一种rRNA沉默的RNA文库构建方法及试剂盒 Download PDF

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WO2022148309A1
WO2022148309A1 PCT/CN2021/143546 CN2021143546W WO2022148309A1 WO 2022148309 A1 WO2022148309 A1 WO 2022148309A1 CN 2021143546 W CN2021143546 W CN 2021143546W WO 2022148309 A1 WO2022148309 A1 WO 2022148309A1
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rrna
oligonucleotides
oligonucleotide
specific
optionally
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French (fr)
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曹林
聂俊伟
瞿志鹏
叶廷跃
韩锦雄
吴恒
景雅
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南京诺唯赞生物科技股份有限公司
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms

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  • the present application belongs to the field of biotechnology, and relates to a method for preparing an rRNA-silencing RNA library and a reagent or kit therefor.
  • NGS High-throughput sequencing technology
  • NGS second-generation sequencing technology
  • RNA sequencing RNA sequencing
  • RNA-seq RNA sequencing
  • maximizing the amount of information received from a sequencing run is of great interest.
  • RNA sequencing RNA sequencing
  • the presence of ribosomal RNA may result in a low signal-to-noise ratio, making it difficult to detect RNA species of interest. Therefore, removing rRNA and/or other unwanted RNA increases the value of downstream sequencing.
  • several methods have been developed in the prior art.
  • the first thing to note is that for eukaryotes, because their mRNA has a poly(A) tail structure, it is easy to enrich and purify with poly(T) to remove other RNAs, or directly use poly(T) primers for Synthesize cDNA; however, prokaryotic mRNA does not have such a structure, so it is more technically difficult to remove non-target RNA from prokaryotic total RNA. Therefore, the rRNA removal methods described in detail below are universally applicable methods for both prokaryotes and eukaryotes.
  • PolyA RNA can be isolated by common methods, such as magnetic beads functionalized with poly(T) oligonucleotides, which can then capture PolyA RNA accordingly.
  • the advantage of preparing sequencing libraries from polyA RNA is that RNAs that do not carry polyA tails, such as rRNA, are not recovered from the total RNA and accordingly are not carried over into the sequencing reaction.
  • the majority of sequences obtained from sequencing libraries generated using polyA RNA correspond to protein-coding mRNAs that do carry polyA tails.
  • using purified polyA RNA to prepare sequencing libraries also has disadvantages. There are several types of RNAs that do not have polyA tails and are therefore lost in polyA enrichment, but are still of interest for transcriptome sequencing.
  • PolyA enrichment results in the loss of non-polyadenylated mRNA sequences that are essential components of the transcriptome. Certain eukaryotic mRNAs, such as those encoding histones, also do not carry polyA tails, others carry polyA tails that are too short to be efficiently captured by oligomeric dT. Furthermore, this method cannot be used for prokaryotic mRNAs because they are not polyadenylated. A further disadvantage is that polyA enrichment requires high-quality intact total RNA as input material. PolyA enrichment is not feasible for degraded RNA samples because only fragments carrying polyA tails can be captured.
  • rRNA hydrolysis technology utilizes the ability of RNase H to specifically degrade DNA: RNA in RNA hybrid molecules without degrading single strands of RNA (Hausen P, Stein H, Ribonuclease H. An enzyme degrading the RNAmoiety of DNA - RNA hybrids. Eur J Biochem/FEBS 1970;14(2):278-83).
  • total RNA is first hybridized or reverse transcribed using a series of primer mixtures specific for rRNA, then RNase H is added to degrade rRNA in the DNA:RNA hybrid duplex, followed by DNase I to degrade residual DNA.
  • subtractive hybridization uses the antisense sequence of rRNA as a specific probe, so that after the rRNA hybridizes with the probe bound to the microsphere or magnetic bead, the hybridization molecule can be removed from the solution.
  • This method is currently the most widely used technology, such as Ambion's MICROB Express kit, which uses two-step sequential hybridization to capture rRNA on magnetic beads.
  • Ambion's MICROB Express kit which uses two-step sequential hybridization to capture rRNA on magnetic beads.
  • the kit requires high integrity of RNA samples - degraded rRNA is often lost Hybridization sites cannot be effectively removed.
  • the relatively recent Ribo-Zero kit developed by Epicentre uses a specific probe coupled to biotin, so rRNA can be hybridized with the corresponding streptavidin-coated affinity chromatography magnet. Bead removal.
  • the present application provides a simple and rapid rRNA silencing method and kit to prepare RNA sequencing libraries. Compared with the RNase H digestion method, the steps are reduced, and the time is shortened from 3 hours to 15 minutes, thereby reducing the damage to RNA.
  • the application provides a 5'-end blocked rRNA-specific oligonucleotide having a 5'-end blocking module and a 3'-end rRNA-specific sequence (with or without additional sequences), the blocking module Blocks ligation (eg, ligation of an adaptor) at the 5' end that is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary.
  • ligation eg, ligation of an adaptor
  • the 5' end blocking means that the oligonucleotides of the present application cannot be ligated to the adapters described herein at the 5' end, so that dsDNA derived from rRNA cannot be efficiently amplified with primers specific for the adapter due to the lack of adapter ligation.
  • augmentation which in turn forms a library, eg, for sequencing.
  • the 5' end blocking module is a modification of the 5'-phosphate group or the 5'-hydroxyl group of the 5' terminal nucleoside.
  • the modification is esterification or amidation of the 5'-phosphate group or esterification or etherification of the 5'-hydroxyl group.
  • the modification is the attachment of a spacer sequence at the 5' position, eg, 5' spacer 18, 5' spacer 9, 5'C3-spacer, 5'C6-spacer, 5'nobase base residues (d spacer, r spacer), 5'-5' reverse nucleotides.
  • the base of the 5' terminal nucleotide is not any of thymine, adenine, cytosine, guanine and uracil.
  • one or both of the 2' hydrogens of the deoxyribose sugar of the 5' terminal nucleotide are replaced by another atom or blocking moiety.
  • the oligonucleotide comprises a 5' terminal nucleotide having a pentose sugar whose steric conformation is different from that of ribose or deoxyribose in RNA or DNA.
  • 5' blocking see WO2017/032808 or CN107849561A, which are incorporated herein by reference for all purposes.
  • the 3'-end rRNA-specific sequence and/or the rRNA target sequence has, for example, a length of 4nt to 100nt, 6nt to 75nt, 10nt to 60nt, 20nt to 50nt, or 30nt to 40nt, including The length of any integer in the range, such as 4nt, 5nt, 6nt, 7nt, 8nt, 9nt, 10nt, 11nt, 12nt, 13nt, 14nt, 15nt, 20nt, 25nt, 30nt, 35nt, 40nt, 45nt, 50nt, 55nt , 60nt, 65nt, 70nt or 75nt in length.
  • the rRNA-specific sequence is specific for 28S, 26S, 25S, 18S, 5.8S or 5S eukaryotic cytoplasmic rRNA or 16S or 12S eukaryotic mitochondrial rRNA or 23S, 16S or 5S prokaryotic rRNA
  • the rRNA target sequence is from 28S, 26S, 25S, 18S, 5.8S or 5S eukaryotic cytoplasmic rRNA or 16S or 12S eukaryotic mitochondrial rRNA or 23S, 16S or 5S prokaryotic rRNA.
  • the rRNA target sequence is a conserved or consensus sequence of rRNA.
  • the 3' end rRNA-specific sequence comprises degenerate nucleotides.
  • the bases of the oligonucleotides are modified, such as 5-Methyl dC, Super T and/or 2,6-Amino-dA, to stably anneal to the rRNA into a hybrid strand and extend .
  • all Cs in the oligonucleotide are modified.
  • all Ts in the oligonucleotide are modified.
  • all A's in the oligonucleotide are modified.
  • the rRNA is from a species of interest, including but not limited to mammals, eg, primates or rodents, eg, humans, cynomolgus monkeys, mice, rats, rabbits, dogs, cats , cattle, horses, sheep, pigs.
  • mammals eg, primates or rodents, eg, humans, cynomolgus monkeys, mice, rats, rabbits, dogs, cats , cattle, horses, sheep, pigs.
  • the rRNA is from a microorganism of interest, such as a bacterium or fungus, including but not limited to Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Saccharomyces cerevisiae, Pichia pastoris.
  • a microorganism of interest such as a bacterium or fungus, including but not limited to Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Saccharomyces cerevisiae, Pichia pastoris.
  • the oligonucleotide comprises any one selected from the group consisting of SEQ ID NO. 1 to SEQ ID NO. 119.
  • the present application provides a collection of oligonucleotides comprising a plurality of oligonucleotides of the present application, and for the same type of rRNA, such as 28S, 26S, 25S, 18S, 5.8S or 5S eukaryotic Specific to one of cytoplasmic rRNA or 16S or 12S eukaryotic mitochondrial rRNA or 23S, 16S or 5S prokaryotic rRNA.
  • rRNA such as 28S, 26S, 25S, 18S, 5.8S or 5S eukaryotic Specific to one of cytoplasmic rRNA or 16S or 12S eukaryotic mitochondrial rRNA or 23S, 16S or 5S prokaryotic rRNA.
  • the rRNA target sequences of the plurality of oligonucleotides overlap, adjoin, discontinue, or any combination thereof on the rRNA.
  • the rRNA target sequences of the plurality of oligonucleotides are uniform, nearly uniform (eg, less than about 25%, 20%, 15%, 10%, 5% relative to uniformity) across the rRNA %, 3%, 2% or 1%, calculated based on the length of the rRNA target sequence) or unevenly distributed.
  • the (average) length of the overlapping portion is at least about 5%, 10%, 20%, 25%, 50%, 75%, 80%, 90%, 95%, 100%.
  • the (average) length of the non-target rRNA sequence is at least about 5 times the (average) length of the rRNA target sequence. %, 10%, 20%, 25%, 50%, 75%, 80%, 90%, 95%, 100%, 125%, 150%, 175%, 200%.
  • the 3' end rRNA-specific sequence and/or rRNA target sequence of the set of oligonucleotides has, for example, about 4nt to 100nt, 6nt to 75nt, 10nt to 60nt, 20nt to 50nt or 30nt to 40nt
  • the average length of including the average length of any value within the range, for example about 4nt, 5nt, 6nt, 7nt, 8nt, 9nt, 10nt, 11nt, 12nt, 13nt, 14nt, 15nt, 20nt, 25nt, 30nt, 35nt , 40nt, 45nt, 50nt, 55nt, 60nt, 65nt, 70nt or 75nt average length.
  • the rRNA target sequences of the set of oligonucleotides cover at least about 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60% of the full length of the rRNA %, 70%, 75%, 80%, 90% or 100%. Coverage refers to the percentage of the set of rRNA target sequences over the full length of the rRNA sequence.
  • the set of oligonucleotides comprises a set selected from the group consisting of SEQ ID NO. 1 to SEQ ID NO. 2, the group consisting of SEQ ID NO. 3 to SEQ ID NO. 5, the group consisting of SEQ ID NO. The group consisting of ID NO.6 to SEQ ID NO.19, or any one of the group consisting of SEQ ID NO.20 to SEQ ID NO.119.
  • the present application provides an oligonucleotide mixture comprising at least two, three, four, five, six, seven, eight or more oligonucleotides of the present application
  • a collection, the first collection of oligonucleotides is for a first type of rRNA, such as 28S, 26S, 25S, 18S, 5.8S or 5S eukaryotic cytoplasmic rRNA or 16S or 12S eukaryotic mitochondrial rRNA or 23S, 16S or 5S prokaryotic rRNA
  • a specific, second set of oligonucleotides is directed against a second type of rRNA, such as 28S, 26S, 25S, 18S, 5.8S or 5S eukaryotic cytoplasmic rRNA or 16S or 12S eukaryotic mitochondrial rRNA or 23S, 16S or 5S Specific to one of the prokaryotic rRNAs, the first type of rRNA is different from the second type of rRNA
  • the mixture of oligonucleotides comprises detection of all eukaryotic rRNAs and/or all prokaryotic rRNAs, ie each of 28S, 26S, 25S, 18S, 5.8S and 5S eukaryotic cytoplasmic rRNAs and/or Multiple sets of oligonucleotides specific for each class in 16S and 12S eukaryotic mitochondrial rRNA and/or each class in 23S, 16S and 5S prokaryotic rRNA.
  • the oligonucleotide mixture comprises a mixture selected from the group consisting of SEQ ID NO. 1 to SEQ ID NO. 2, the group consisting of SEQ ID NO. 3 to SEQ ID NO. 5, the Any two or more groups of the group consisting of ID NO. 6 to SEQ ID NO. 19, and/or the group consisting of SEQ ID NO. 20 to SEQ ID NO. 119.
  • the oligonucleotide, collection of oligonucleotides or mixture of oligonucleotides is used as a primer.
  • the application provides a method of preparing an rRNA-silenced RNA library (e.g., for sequencing) comprising one or more of the following steps:
  • RNA sample optionally, fragmenting the RNA sample
  • rRNA-specific oligonucleotides or oligonucleotide pools or oligonucleotide mixtures of the present application as primers for rRNA, random oligonucleotides (eg random hexamers) as primers for other RNAs Perform reverse transcription with reverse transcriptase to obtain the first strand of cDNA;
  • the ligation product is amplified.
  • the adaptor is a sequencing adaptor. In one embodiment, the sequencing adapter is a Y-shaped sequencing adapter. In one embodiment, the sequencing adapter is the sequencing adapter of the illumina platform, the sequencing adapter of the Ion Torrent platform, or the sequencing adapter of the MGI platform made by MGI.
  • the amount of 5'-end blocked rRNA-specific oligonucleotide or pool of oligonucleotides or mixture of oligonucleotides added (in moles) is the amount of rRNA or fragment thereof (measured or estimated) at least about 0.01x, 0.02x, 0.05x, 0.1x, 0.2x, 0.5x, 1x, 1.5x, 2x, 2.5x, 3x, 3.5x, 4x, 4.5x, 5x, 10x, 20x, 50x, 100x.
  • the amount (by mass) of the 5' end-blocked rRNA-specific oligonucleotide or pool of oligonucleotides or mixture of oligonucleotides added is the amount (measured or estimated) of the rRNA or fragment thereof of) at least about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 33%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 1x, 2x, 3x, 5x, 10x, 20x, 30x, 35x, 40x, 45x, 50x, 55x, 60x, 65x, 70x, 75x, 80x, 85x, 90x, 95x, 100x, 200x, 500x, 1000x.
  • the present application provides a kit comprising the 5'-end blocked rRNA-specific oligonucleotides or oligonucleotide collections or oligonucleotide mixtures of the present application.
  • the kit further comprises other reagents required for carrying out the methods of the present application.
  • the kit is used to practice the methods of the present application, eg, for the preparation of rRNA-silenced RNA libraries.
  • the RNA library is used for sequencing.
  • the methods or constructed libraries of the present application are strand-specific.
  • the present application can distinguish between forward and reverse transcripts, eg, by the sequence of an adaptor. That is, the present application can construct a forward-transcribed library or a reverse-transcribed library.
  • Fragmentation of total RNA into fragments of suitable length or length range usually non-sequence or site specific.
  • the length or length range of the fragments can be about 50-1000 nt, such as 100-500 nt, such as 100-200, 200-300, 300-400, 400-500, 100 nt -300, 200-400 or 300-500nt.
  • Fragmentation reaction temperature and time can be adjusted to obtain different lengths or length ranges.
  • the fragmented rRNA fragments are bound to 5'-end blocked rRNA-specific oligonucleotides. A closed 5' end cannot be ligated with the adapters described herein.
  • free random primers e.g. random hexamers
  • RNAs e.g. mRNA
  • the free 5' end can be ligated with adaptors described herein.
  • the hybrid product in step (2) is subjected to an extension reaction by reverse transcriptase to obtain a DNA-RNA hybrid.
  • an extension reaction by reverse transcriptase to obtain a DNA-RNA hybrid.
  • purification and/or digestion of the RNA strand in the DNA-RNA hybrid can be performed.
  • a DNA polymerase e.g. high fidelity DNA polymerase, i.e. with proofreading activity, i.e. 3'-5' exonucleation activity, e.g. Pfu and T4 DNA polymerase
  • random primers free at the 5' end e.g. random hexamers
  • Carry out double-stranded DNA synthesis e.g. polynucleotide kinases are also included.
  • enzymes with terminal transferase activity eg, Taq
  • the resulting terminus comprises adenosine for ligation with a thymidine bearing adapter.
  • U can be used for second strand synthesis, and the U-containing second strand is digested at an appropriate time.
  • purification can be performed.
  • a corresponding adapter eg, Y-shaped sequencing adapter
  • the 5'-end of the double-stranded double-stranded derivatized with rRNA as a template using rRNA-specific oligonucleotides blocked at the 5'-end cannot be connected to the adapter, while the 5'-end free random primers are derived from other RNAs as templates.
  • the 5' end of the double strand can be ligated with an adaptor.
  • the ligation product is purified, eg, using magnetic beads.
  • unpurified or purified ligation products are amplified, eg, by PCR.
  • the above steps can be arbitrarily combined according to the situation.
  • the product thus obtained can be used for sequencing, eg, second generation sequencing.
  • only the first strand is sequenced, particularly the first strand derived from RNA other than rRNA.
  • the present application provides a kit for preparing an rRNA-silencing RNA library, characterized in that it includes one or more of the following reagents: interruption buffer, rRNA-specific primers (such as 5'-blocked ) and random primers (e.g. 5' free), reverse transcriptase, reverse transcription buffer, 2-strand synthase mix, 2-strand synthesis buffer, RNA purification beads, DNA purification beads, adaptors (e.g. sequencing adaptors) head), ligase, ligation reaction buffer, high-fidelity DNA polymerase, amplification buffer, amplification primers, and nuclease-free water.
  • interruption buffer rRNA-specific primers (such as 5'-blocked ) and random primers (e.g. 5' free)
  • reverse transcriptase reverse transcription buffer
  • 2-strand synthase mix 2-strand synthase mix
  • 2-strand synthesis buffer RNA purification beads
  • DNA purification beads DNA purification beads
  • adaptors e.g.
  • reagents can be arbitrarily combined according to the situation.
  • reverse transcriptase reverse transcription buffer solution can be mixed into one mixture.
  • the amplification buffer and the amplification primers can be mixed into a mixture.
  • RNA purification magnetic beads, DNA purification magnetic beads, amplification primers, nuclease-free water may or may not be present in the kit, ie provided externally.
  • This kit can subtract rRNA from total RNA, retain mRNA and other non-coding RNA, and can be used for the analysis of non-coding RNA such as LncRNA.
  • Degraded RNA samples can also be used for library construction with this kit.
  • This application applies to total RNA samples with a starting amount of 1-1000ng.
  • Figure 1 shows the distribution of rRNA target sequences of the present application.
  • Panel A shows a schematic representation of the target rRNA.
  • Panel B shows a schematic diagram of rRNA target sequences overlapping each other.
  • Panel C shows a schematic diagram of rRNA target sequences adjacent to each other.
  • Panel D shows a schematic representation of rRNA target sequences discontinuing each other.
  • Figure 2 shows a flow chart of the method of the present application.
  • Figure 3A and Figure 3B show the results of Agilent 2100 Bioanalyzer analysis of libraries prepared in the present application and controls, respectively.
  • RNA removal with Illumina's Ribo-zero rRNA removal kit and Stranded Total RNA Sample Preparation kits were used for library construction as controls.
  • RNA extracted from conventionally cultured HEK293 cells was used as the starting sample to perform rRNA silencing and construct a transcriptome library for sequencing.
  • the specific process is as follows:
  • each primer was 50 ⁇ M. All primers were modified with a C3 spacer at the 5' end. All A, T, C bases are modified: C, 5-Methyl dC; T, Super T; A, 2,6-Amino-dA.
  • the second strand synthesis reaction proceeds immediately.
  • the ligation product was purified with VAHTS DNA Clean Beads (Nanjing Novizan Biotechnology Co., Ltd.) according to the instructions.
  • VAHTS DNA Clean Beads to perform 0.6 ⁇ /0.1 ⁇ two rounds of magnetic beads size sorting according to the instructions, and a library of 450-550bp can be obtained.
  • Ligation products were purified using VAHTS DNA Clean Beads according to the manufacturer's instructions.
  • This example uses the primer mix listed in the appendix to achieve a reduction in the proportion of rRNA from over 90% in total RNA samples to 1.01% in sequencing results. In contrast, the control method dropped to 5.32%.
  • optimizing the library construction conditions such as increasing the number of primers, improving the coverage of target sequences, optimizing the selection of target sequences, and increasing the amount of primers added relative to the sample, the proportion of rRNA in the sequencing results can be further reduced or even completely eliminated.
  • This application provides a new rRNA-removed RNA library construction scheme, the operation of which is not much different from the standard library construction process, without introducing additional enzymatic reactions, and a high-quality library can be obtained. rRNA removal efficiency.

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Abstract

提供一种制备rRNA沉默的RNA文库的方法及其使用的试剂或试剂盒。

Description

一种rRNA沉默的RNA文库构建方法及试剂盒 技术领域
本申请属于生物技术领域，涉及一种制备rRNA沉默的RNA文库的方法及其使用的试剂或试剂盒。
背景技术
高通量测序技术又称为第二代测序技术，可简写为NGS，是指一次并行对几十万到几百万条DNA分子进行序列测定的技术，其测定序列长度一般较短。
转录组测序的一个主要问题是存在干扰性RNA分子。例如，核糖体RNA(rRNA)是总RNA中最丰富的分子，其中通常超过90％的总RNA为rRNA。然而，核糖体RNA提供的关于转录的信息很少。在应用中，如RNA测序(RNA-seq)时，最大程度提高从测序运行接收到的信息量是极其令人感兴趣的。如果文库构建涉及丰富的rRNA，大部分的测序能力将用于测序这些无所不在的分子，从而减少研究其余转录组可用的能力。因此，宝贵的测序资源被浪费。此外，存在核糖体RNA可能会导致低信噪比，从而难以检测感兴趣的RNA种类。因此，除去rRNA和/或其它不需要的RNA提高了下游测序的价值。为了提供不含rRNA或其它不需要的RNA种类的测序文库，现有技术中开发了几种方法。
近年来，科研工作者们已经开发出多种方法以去除以rRNA为代表的非目标RNA。这些方法使用不同RNA之间的方方面面区别设计去除手段，如大小、序列特点、5’磷酸基、二级结构、丰度等，都可作为去除方法的依据；同时，也有若干商业化的试剂盒可用于去除非目标RNA。
首先要说明的是，对于真核生物，因其mRNA具有poly(A)尾的结构，因此很容易用poly(T)加以富集纯化进而去除其它RNA，或是直接用poly(T)引物进行合成cDNA；而原核生物的mRNA并不具备这样的结构，因此去除原核生物总RNA中非目标RNA，具有更高的技术难度。因此下文要详述的rRNA去除方法，均是针对包括原核生物与真核生物在内的，普遍适用的方法。
PolyA RNA可采用普通方法分离，例如利用以poly(T)寡核苷酸作官能化，从而可相应捕获PolyA RNA的磁性珠。从polyA RNA制备测序文库的优点在于， 不携带polyA尾的RNA，例如rRNA不会从总RNA中回收，并且相应地不会被带入测序反应。因此，从利用polyA RNA产生的测序文库中获得的大多数序列对应于确实携带polyA尾的蛋白编码mRNA。然而，利用纯化的polyA RNA制备测序文库也有缺点。有几种类型的RNA不带有polyA尾，并因此在polyA富集中丢失，但仍是转录组测序所感兴趣的。polyA富集导致作为转录组重要组成部分的非聚腺苷酸化mRNA序列损失。某些真核mRNA，例如那些编码组蛋白的，也不携带polyA尾，其它携带polyA尾太短，从而无法通过寡聚dT有效捕获。此外，该方法不能用于原核mRNA，因为它们未聚腺苷酸化。再一缺点是，polyA富集需要高品质的完整的总RNA作为输入材料。PolyA富集对于降解的RNA样本不可行，因为只有携带polyA尾的片段才能被捕获。
另一种rRNA酶解技术则是利用了RNA酶H特异性降解DNA：RNA杂交分子中RNA、而不降解RNA单链的能力(Hausen P,Stein H,Ribonuclease H.An enzyme degrading the RNAmoiety of DNA-RNA hybrids.Eur J Biochem/FEBS 1970；14(2):278-83)。该方法中，总RNA首先使用一系列针对rRNA的特异性引物混合物进行杂交或反转录，然后加入RNA酶H以降解DNA：RNA杂交双链中的rRNA，继而再加入DNA酶I以降解残留的DNA。John Morlan利用此原理设计是针对人/大鼠/小鼠的5S、18S、28S rRNA的短的反义DNA探针(50-80bp)。将这些探针与人体RNA杂交后用RNA酶H和DNA酶I处理，该方法可去除98％的rRNA。基于RNA酶H，DNA酶I消化技术的缺点是十分显著的：第一，由于利用的引物是针对目标RNA设计的，要达到这些引物不与非目标RNA杂交的可能性是极低的，所以极有可能在最终的目标RNA中存在部分非目标RNA；第二，目标RNA是在经过RNA酶H，DNA酶I等一系列处理后得到的，而RNA是极易被污染、被降解，目标RNA去除步骤越多，其被降解的可能性越大。
利用特异性探针的消减杂交技术，消减杂交利用rRNA的反义序列作为特异性探针，因而rRNA与结合在微球或磁珠上的探针杂交后，杂交分子可以从溶液中去除。该方法是目前使用最为广泛的技术，如Ambion公司的MICROB Express kit，使用两步连续杂交将rRNA捕获于磁珠上。试剂盒使用上有明确的物种局限，所有古生菌样本均不适用；且受限于所用探针种类与数量，该试剂盒对RNA样品的完整性要求很高——降解的rRNA往往会丢失杂交位点而无法 被有效去除。另有较为晚近的Epicentre公司研发的Ribo-Zero kit，使用的则是偶联生物素的特异性探针，因而rRNA与之杂交后可用相应的链霉亲合素包被的亲和层析磁珠去除。
发明内容
本申请提供了一种简单快速的rRNA沉默的方法和试剂盒来制备RNA测序文库，相比于RNA酶H消化法，减少步骤，时间由3小时缩短到了15分钟，从而降低对RNA的破坏。
在一个方面，本申请提供一种5’端封闭的rRNA特异性寡核苷酸，其具有5’端封闭模块和3’端rRNA特异性序列(有或无别的序列)，所述封闭模块阻断5’末端的连接(例如连接衔接头)，所述rRNA特异性序列与rRNA靶序列至少约75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％互补。5’端封闭意味着本申请的寡核苷酸不能在5’端连接本文所述衔接头，使得自rRNA衍生的dsDNA因连接不上衔接头而无法利用对衔接头特异性的引物进行有效扩增，继而形成文库，例如用于测序。
在一个实施方案中，所述5’端封闭模块是对5’末端核苷(酸)的5’-磷酸基团或5’-羟基的修饰。在进一步的实施方案中，所述修饰是5’-磷酸基团的酯化或酰胺化或5’-羟基的酯化或醚化。在进一步的实施方案中，所述修饰是5’位连接间隔子序列，例如5’间隔子18、5’间隔子9、5’C3-间隔子、5’C6-间隔子、5’无碱基残基(d间隔子、r间隔子)、5’-5’反向核苷酸。在一个实施方案中，所述5’端核苷酸的碱基不是胸腺嘧啶、腺嘌呤、胞嘧啶、鸟嘌呤和尿嘧啶中的任一者。在一个实施方案中，所述5’端核苷酸的脱氧核糖的2’氢中的一个或两个被另一个原子或封闭模块取代。在一个实施方案中，所述寡核苷酸包含具有戊糖的5’端核苷酸，所述戊糖的立体构象不同于RNA或DNA中的核糖或脱氧核糖的立体构象。关于5’封闭，参见WO2017/032808或CN107849561A，通过援引将其引入本文用于所有目的。
在另一个实施方案中，所述3’端rRNA特异性序列和/或所述rRNA靶序列具有例如4nt至100nt、6nt至75nt、10nt至60nt、20nt至50nt或30nt至40nt的长度，包括所述范围内的任一整数的长度，例如4nt、5nt、6nt、7nt、8nt、9nt、10nt、11nt、12nt、13nt、14nt、15nt、20nt、25nt、30nt、35nt、40nt、45nt、50nt、 55nt、60nt、65nt、70nt或75nt的长度。
在另一个实施方案中，所述rRNA特异性序列是对28S、26S、25S、18S、5.8S或5S真核细胞质rRNA或16S或12S真核线粒体rRNA或23S、16S或5S原核rRNA特异性的，和/或所述rRNA靶序列来自28S、26S、25S、18S、5.8S或5S真核细胞质rRNA或16S或12S真核线粒体rRNA或23S、16S或5S原核rRNA。
在另一个实施方案中，所述rRNA靶序列是rRNA的保守序列或共有序列。
在另一个实施方案中，所述3’端rRNA特异性序列包含简并核苷酸。
在另一个实施方案中，所述寡核苷酸的碱基经过修饰，例如5-Methyl dC、Super T和/或2,6-Amino-dA，能够与rRNA稳定退火成杂合链，并延伸。在进一步的实施方案中，所述寡核苷酸中的所有C均为经过修饰的。在进一步的实施方案中，所述寡核苷酸中的所有T均为经过修饰的。在进一步的实施方案中，所述寡核苷酸中的所有A均为经过修饰的。
在另一个实施方案中，所述rRNA来自感兴趣的物种，包括但不限于哺乳动物，例如灵长动物或啮齿动物，又例如人、食蟹猴、小鼠、大鼠、兔、犬、猫、牛、马、羊、猪。
在另一个实施方案中，所述rRNA来自感兴趣的微生物，例如细菌或真菌，包括但不限于大肠杆菌、金黄色葡萄球菌、铜绿假单胞菌、酿酒酵母、巴斯德毕赤酵母。
在另一个实施方案中，所述寡核苷酸包含选自SEQ ID NO.1至SEQ ID NO.119的任意一种。
在另一个方面，本申请提供一种寡核苷酸集合，其包含多种本申请的寡核苷酸，且是对同一类rRNA，例如28S、26S、25S、18S、5.8S或5S真核细胞质rRNA或16S或12S真核线粒体rRNA或23S、16S或5S原核rRNA之一特异性的。
在一个实施方案中，所述多种寡核苷酸的rRNA靶序列在所述rRNA上彼此交叠、毗邻、断续或其任意组合。在一个实施方案中，所述多种寡核苷酸的rRNA靶序列在所述rRNA上均匀、接近均匀(例如相对于均匀而言偏差小于约25％、20％、15％、10％、5％、3％、2％或1％，基于rRNA靶序列的长度计算)或不均匀分布。在rRNA靶序列交叠的一个实施方案中，交叠部分的(平均)长度 为rRNA靶序列的(平均)长度的至少约5％、10％、20％、25％、50％、75％、80％、90％、95％、100％。在rRNA靶序列断续的一个实施方案中，非靶rRNA序列(即rRNA上相邻两个rRNA靶序列之间的序列)的(平均)长度为rRNA靶序列的(平均)长度的至少约5％、10％、20％、25％、50％、75％、80％、90％、95％、100％、125％、150％、175％、200％。
在另一个实施方案中，所述寡核苷酸集合的3’端rRNA特异性序列和/或rRNA靶序列具有例如约4nt至100nt、6nt至75nt、10nt至60nt、20nt至50nt或30nt至40nt的平均长度，包括所述范围内的任一数值的平均长度，例如约4nt、5nt、6nt、7nt、8nt、9nt、10nt、11nt、12nt、13nt、14nt、15nt、20nt、25nt、30nt、35nt、40nt、45nt、50nt、55nt、60nt、65nt、70nt或75nt的平均长度。
在另一个实施方案中，所述寡核苷酸集合的rRNA靶序列覆盖所述rRNA的全长的至少约10％、15％、20％、25％、30％、40％、50％、60％、70％、75％、80％、90％或100％。覆盖率是指rRNA靶序列的集合占rRNA序列的全长的百分比。
在另一个实施方案中，所述寡核苷酸集合包含选自由SEQ ID NO.1至SEQ ID NO.2组成的组，由SEQ ID NO.3至SEQ ID NO.5组成的组，由SEQ ID NO.6至SEQ ID NO.19组成的组，或由SEQ ID NO.20至SEQ ID NO.119组成的组的任意一组。
在另一个方面，本申请提供一种寡核苷酸混合物，其包含至少两种、三种、四种、五种、六种、七种、八种或更多种本申请的寡核苷酸集合，第一寡核苷酸集合是对第一类rRNA，例如28S、26S、25S、18S、5.8S或5S真核细胞质rRNA或16S或12S真核线粒体rRNA或23S、16S或5S原核rRNA之一特异性的，第二寡核苷酸集合是对第二类rRNA，例如28S、26S、25S、18S、5.8S或5S真核细胞质rRNA或16S或12S真核线粒体rRNA或23S、16S或5S原核rRNA之一特异性的，第一类rRNA不同于第二类rRNA，以此类推。
在一个实施方案中，所述寡核苷酸混合物包含对全部真核rRNA和/或全部原核rRNA，即28S、26S、25S、18S、5.8S和5S真核细胞质rRNA中每一类和/或16S和12S真核线粒体rRNA中每一类和/或23S、16S和5S原核rRNA中每一类特异性的多种寡核苷酸集合。
在另一个实施方案中，所述寡核苷酸混合物包含选自由SEQ ID NO.1至 SEQ ID NO.2组成的组，由SEQ ID NO.3至SEQ ID NO.5组成的组，由SEQ ID NO.6至SEQ ID NO.19组成的组，和/或由SEQ ID NO.20至SEQ ID NO.119组成的组的任意两组或更多组。
在任何上述方面的一个实施方案中，所述寡核苷酸、寡核苷酸集合或寡核苷酸混合物用作引物。
在另一个方面，本申请提供一种制备rRNA沉默的RNA文库(例如用于测序)的方法，其包括以下一个或多个步骤：
任选地，将RNA样品片段化；
使用本申请的5’端封闭的rRNA特异性寡核苷酸或寡核苷酸集合或寡核苷酸混合物作为rRNA的引物、随机寡核苷酸(例如随机六聚物)作为其它RNA的引物和逆转录酶进行逆转录，获得cDNA第一链；
使用随机寡核苷酸(例如随机六聚物)和DNA聚合酶合成DNA第二链；
连接衔接头；
任选地，回收并纯化连接产物；以及
任选地，扩增连接产物。
在一个实施方案中，所述衔接头是测序衔接头。在一个实施方案中，所述测序衔接头是Y型测序衔接头。在一个实施方案中，所述测序衔接头是illumina平台的测序衔接头、Ion Torrent平台的测序衔接头或华大智造MGI平台的测序衔接头。
在一个实施方案中，添加5’端封闭的rRNA特异性寡核苷酸或寡核苷酸集合或寡核苷酸混合物的量(按摩尔数计)是rRNA或其片段的量(测量的或估算的)的至少约0.01x、0.02x、0.05x、0.1x、0.2x、0.5x、1x、1.5x、2x、2.5x、3x、3.5x、4x、4.5x、5x、10x、20x、50x、100x。在一个实施方案中，添加5’端封闭的rRNA特异性寡核苷酸或寡核苷酸集合或寡核苷酸混合物的量(按质量计)是rRNA或其片段的量(测量的或估算的)的至少约1％、2％、3％、4％、5％、10％、15％、20％、25％、30％、33％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％、1x、2x、3x、5x、10x、20x、30x、35x、40x、45x、50x、55x、60x、65x、70x、75x、80x、85x、90x、95x、100x、200x、500x、1000x。
在另一个方面，本申请提供一种试剂盒，其包含本申请的5’端封闭的rRNA 特异性寡核苷酸或寡核苷酸集合或寡核苷酸混合物。在一个实施方案中，所述试剂盒还包含用于实施本申请的方法需要的其它试剂。在另一个实施方案中，所述试剂盒用于实施本申请的方法，例如用于制备rRNA沉默的RNA文库。在进一步的实施方案中，所述RNA文库用于测序。
在另一个实施方案中，本申请的方法或构建的文库是链特异性的。在另一个实施方案中，本申请可区分正向转录本与反向转录本，例如通过衔接头的序列。也就是说，本申请可构建正向转录本文库或反向转录本文库。
本申请实施方案步骤如下：
(1)RNA片段化
将总RNA断裂成合适长度或长度范围的片段，通常是非序列或位点特异性的。例如，采用二价阳离子在高温条件下断裂总RNA，片段的长度或长度范围可以是约50-1000nt，例如100-500nt，例如100-200、200-300、300-400、400-500、100-300、200-400或300-500nt。可调整片段化的反应温度和时间，获得不同长度或长度范围。
(2)引物杂交
断裂后的rRNA片段与5’端封闭的rRNA特异性寡核苷酸结合。封闭的5’端无法连接本文描述的衔接头。
同时，5’端游离的随机引物(例如随机六聚物)与其它RNA(例如mRNA)的片段结合。游离的5’端能够连接本文描述的衔接头。
(3)一链合成
通过逆转录酶，对步骤(2)中的杂交产物进行延伸反应，获得DNA-RNA杂合物。任选地，合成第一链后，可以进行纯化，和/或消化掉DNA-RNA杂合物中的RNA链。
(4)二链合成
利用DNA聚合酶(例如高保真DNA聚合酶，即具有校对活性，即3’-5’外切活性，例如Pfu和T4 DNA聚合酶)和5’端游离的随机引物(例如随机六聚物)进行二链DNA合成。任选地，还包括多核苷酸激酶。任选地，还包括具有末端转移酶活性的酶(例如Taq)。优选地，所得末端包含腺苷，用于与带有胸苷的衔接头连接。任选地，第二链合成可以使用U，并在合适的时机消化掉含U的第二链。任选地，合成第二链后，可以进行纯化。
(5)衔接头连接
根据需要(例如测序使用的固相和引物)，选择相应的衔接头(例如Y型测序衔接头)与步骤(4)中得到的双链DNA连接。此时，利用5’端封闭的rRNA特异性寡核苷酸以rRNA为模板衍生的双链的5’端无法与衔接头连接，而利用5’端游离的随机引物以其它RNA为模板衍生的双链的5’端能够与衔接头连接。任选地，对连接产物进行纯化，例如使用磁珠。任选地，对未经纯化或经过纯化的连接产物进行扩增，例如通过PCR。
上述步骤可以根据情况任意合并。由此得到的产物可以用于测序，例如第二代测序。又例如，仅对第一链进行测序，特别是对自除了rRNA以外的RNA衍生的第一链进行测序。
在另一个方面，本申请提供一种用于制备rRNA沉默的RNA文库的试剂盒，其特征在于，包括以下一种或多种试剂：打断缓冲液、rRNA特异性引物(例如5’封闭的)和随机引物(例如5’游离的)、逆转录酶、逆转录缓冲溶液、二链合成酶混合液、二链合成缓冲液、RNA纯化磁珠、DNA纯化磁珠、衔接头(例如测序衔接头)、连接酶、连接反应缓冲液、高保真DNA聚合酶、扩增缓冲液、扩增引物和无核酸酶水。
上述试剂可以根据情况任意合并。例如，逆转录酶、逆转录缓冲溶液可以混合为一个混合物。又例如，扩增缓冲液、扩增引物可以混合为一个混合物。另外/或者，RNA纯化磁珠、DNA纯化磁珠、扩增引物、无核酸酶水可以存在或者不存在于试剂盒中，即外部提供。
本试剂盒可以自总RNA中消减rRNA，保留mRNA和其它非编码RNA，可用于LncRNA等非编码RNA的分析。
降解的RNA样品也可用本试剂盒进行文库构建。
本申请适用起始量为1-1000ng的总RNA样本。
附图说明
图1显示本申请的rRNA靶序列的分布方式。小图A显示靶rRNA的示意图。小图B显示rRNA靶序列彼此交叠的示意图。小图C显示rRNA靶序列彼此毗邻的示意图。小图D显示rRNA靶序列彼此断续的示意图。
图2显示本申请方法的流程图。
图3A和图3B分别显示本申请及对照制备的文库的Agilent 2100 Bioanalyzer分析结果。
具体实施方式
以Illumina的Ribo-zero rRNA removal kit进行rRNA去除及以
Figure PCTCN2021143546-appb-000001
Stranded Total RNA Sample Preparation kits进行建库，作为对照。
本实施例以1000ng自常规培养的HEK293细胞提取的总RNA作为起始样品，进行rRNA沉默并构建转录组文库用于测序，具体流程如下：
1、RNA片段化和引物杂交
将1000ng的总RNA用RNase-free ddH 2O稀释到7μL。
在RNase-free离心管中配制如下混合液：
表1
Figure PCTCN2021143546-appb-000002
a南京诺唯赞生物科技有限公司(vazyme，#N402)，含随机引物。
b见序列表。每种引物的浓度为50μM。所有引物5’端均修饰有C3间隔子。所有A、T、C碱基均被修饰：C，5-Methyl dC；T，Super T；A，2,6-Amino-dA。
进行如下的反应：
表2
Figure PCTCN2021143546-appb-000003
Figure PCTCN2021143546-appb-000004
2、逆转录
配制逆转录反应体系：
表3
Figure PCTCN2021143546-appb-000005
a南京诺唯赞生物科技有限公司VAHTS Universal V6 RNA-seq library Prep Kit for Illumina试剂盒。
逆转录反应条件：
表4
温度 时间
25℃ 10min
42℃ 15min
70℃ 15min
4℃ 保持
立刻进行第二链合成反应。
3、二链合成
配制混合液：
表5
Figure PCTCN2021143546-appb-000006
Figure PCTCN2021143546-appb-000007
a南京诺唯赞生物科技有限公司VAHTS Universal V6 RNA-seq library Prep Kit for Illumina试剂盒。
进行如下反应：
表6
温度 时间
16℃ 30min
65℃ 15min
4℃ 保持
4、衔接头连接
将Adapter从-20℃取出，解冻后颠倒混匀，配制连接反应液：
表7
Figure PCTCN2021143546-appb-000008
a南京诺唯赞生物科技有限公司VAHTS Universal V6 RNA-seq library Prep Kit for Illumina试剂盒。
b以Illumina平台的衔接头为例。
进行如下反应：
表8
Figure PCTCN2021143546-appb-000009
Figure PCTCN2021143546-appb-000010
5、连接产物纯化和片段大小分选
用VAHTS DNA Clean Beads(南京诺唯赞生物科技有限公司)依照说明书纯化连接产物。
用VAHTS DNA Clean Beads依照说明书进行0.6×/0.1×两轮磁珠进行片段大小分选，可获得450-550bp的文库。
6、文库扩增
配制PCR反应液。
表9
Figure PCTCN2021143546-appb-000011
a南京诺唯赞生物科技有限公司VAHTS Universal V6 RNA-seq library Prep Kit for Illumina试剂盒。
将样品置于PCR仪中，进行下述反应
表10
Figure PCTCN2021143546-appb-000012
7.扩增产物纯化
用VAHTS DNA Clean Beads依照说明书纯化连接产物。
8.Qubit检测文库浓度
使用Qubit对所得文库进行浓度测定，计算文库产出。结果见下表。
表11
样品制备方法 文库产出(ng/μL)
本申请 38.6
对照(Ribo-zero) 37.9
9、用Agilent 2100 Bioanalyzer评价文库质量
取1μL纯化后的PCR产物，用Agilent DNA 1000 kit(Agilent,Cat.No.5067-1504)进行分析。结果见图3(A：本申请，B：Ribo-zero)。
10、测序
Illumina平台上机测序，并进行数据分析，得到下表数据。
表12：具体的测序数据情况
Figure PCTCN2021143546-appb-000013
由表12中数据可知，从Reads数、GC含量、Q20、Q30数等信息来看，两个文库的数据基本一致，证明文库测序指标都正常；将测序数据比对到人类参考基因组上，两个文库都具有高百分比的比对率(Mapping Rate)；证明文库质量良好；从Duplication Rate、检测基因数等信息来看，本申请得到的文库有更低的冗余率和更高的基因检出数；从rRNA ratio可以看出，本申请有不俗的rRNA去除效率，减少数据浪费，并可获得更多的数据信息。
本实施例使用附录所列引物混合物实现将rRNA的比例从总RNA样品中的超过90％降至测序结果中的1.01％。与之对比，对照方法降至5.32％。通过优化建库条件，例如增加引物的数目、提高靶序列的覆盖率、优化靶序列的选择、 提高引物相对于样品的添加量能进一步降低，甚至完全消除测序结果中rRNA的比例。
综上：本申请提供了一种新型的rRNA去除的RNA建库方案，其操作与标准的建库流程相差不大，不引入额外的酶反应，可获得高质量的文库，并且有很高的rRNA去除效率。

Claims (13)

  1. 一种5’端封闭的rRNA特异性寡核苷酸，其具有5’端封闭模块和3’端rRNA特异性序列，所述封闭模块阻断5’末端的连接，所述rRNA特异性序列与rRNA靶序列至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％互补。
  2. 根据权利要求1所述的寡核苷酸，其中，所述5’端封闭模块是对5’末端核苷(酸)的5’-磷酸基团或5’-羟基的修饰。
  3. 根据权利要求2所述的寡核苷酸，其中，所述修饰是5’-磷酸基团的酯化或酰胺化或5’-羟基的酯化或醚化，或者，所述修饰是5’位连接间隔子序列。
  4. 根据权利要求1所述的寡核苷酸，其中，所述3’端rRNA特异性序列和/或所述rRNA靶序列具有例如4nt至100nt、6nt至75nt、10nt至60nt、20nt至50nt或30nt至40nt的长度，包括所述范围内的任一整数的长度，例如4nt、5nt、6nt、7nt、8nt、9nt、10nt、11nt、12nt、13nt、14nt、15nt、20nt、25nt、30nt、35nt、40nt、45nt、50nt、55nt、60nt、65nt、70nt或75nt的长度。
  5. 根据权利要求1-4任一项所述的寡核苷酸，其中，所述rRNA特异性序列是对28S、26S、25S、18S、16S、12S、5.8S或5S真核rRNA或23S、16S或5S原核rRNA特异性的，和/或所述rRNA靶序列来自28S、26S、25S、18S、16S、12S、5.8S或5S真核rRNA或23S、16S或5S原核rRNA。
  6. 根据权利要求1-5任一项所述的寡核苷酸，其中，所述rRNA靶序列是rRNA的保守序列或共有序列。
  7. 根据权利要求1-6任一项所述的寡核苷酸，其中，所述3’端rRNA特异性序列包含简并核苷酸和/或经过修饰的碱基。
  8. 一种寡核苷酸集合，其包含多种根据权利要求1-7任一项所述的寡核苷酸，且是对同一类rRNA，例如28S、26S、25S、18S、16S、12S、5.8S或5S真核rRNA或23S、16S或5S原核rRNA之一特异性的，
    任选地，所述多种寡核苷酸的rRNA靶序列在所述rRNA上彼此交叠、毗邻、断续或其任意组合，
    任选地，所述多种寡核苷酸的rRNA靶序列在所述rRNA上均匀或不均匀分布，
    任选地，所述寡核苷酸集合的3’端rRNA特异性序列和/或rRNA靶序列具有例如约4nt至100nt、6nt至75nt、10nt至60nt、20nt至50nt或30nt至40nt 的平均长度，包括所述范围内的任一数值的平均长度，例如约4nt、5nt、6nt、7nt、8nt、9nt、10nt、11nt、12nt、13nt、14nt、15nt、20nt、25nt、30nt、35nt、40nt、45nt、50nt、55nt、60nt、65nt、70nt或75nt的平均长度，
    任选地，所述寡核苷酸集合的rRNA靶序列覆盖所述rRNA的全长的至少约10％、15％、20％、25％、30％、40％、50％、60％、70％、75％、80％、90％或100％。
  9. 一种寡核苷酸混合物，其包含至少两种、三种、四种、五种、六种、七种、八种或更多种根据权利要求5所述的寡核苷酸集合，第一寡核苷酸集合是对第一类rRNA，例如28S、26S、25S、18S、16S、12S、5.8S或5S真核rRNA或23S、16S或5S原核rRNA之一特异性的，第二寡核苷酸集合是对第二类rRNA，例如28S、26S、25S、18S、16S、12S、5.8S或5S真核rRNA或23S、16S或5S原核rRNA之一特异性的，第一类rRNA不同于第二类rRNA，以此类推，
    任选地，所述寡核苷酸混合物包含对全部真核rRNA和/或全部原核rRNA特异性的多种寡核苷酸集合。
  10. 根据权利要求1-7任一项所述的寡核苷酸或根据权利要求8所述的寡核苷酸集合或根据权利要求9所述的寡核苷酸混合物，其用作引物。
  11. 一种制备rRNA沉默的RNA测序文库的方法，其包括以下一个或多个步骤：
    任选地，将RNA测序样品片段化；
    使用根据权利要求5所述的寡核苷酸集合或根据权利要求6所述的寡核苷酸混合物、随机引物(例如随机六聚物)和逆转录酶进行逆转录，获得cDNA第一链；
    使用随机寡核苷酸(例如随机六聚物)和DNA聚合酶合成DNA第二链；
    连接测序衔接头，任选地，所述测序衔接头是Y型测序衔接头，任选地，所述测序衔接头是illumina平台的测序衔接头、Ion Torrent平台的测序衔接头或华大智造MGI平台的测序衔接头；
    任选地，回收并纯化连接产物；以及
    任选地，扩增连接产物。
  12. 根据权利要求11所述的方法，其特征在于，还包括对连接产物进行测序。
  13. 一种试剂盒，其包含根据权利要求8所述的寡核苷酸集合或根据权利要 求9所述的寡核苷酸混合物，任选地，其用于实施根据权利要求11所述的方法。
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