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WO2012037881A1 - Marqueurs d'acides nucléiques et leurs utilisations - Google Patents

Marqueurs d'acides nucléiques et leurs utilisations Download PDF

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Publication number
WO2012037881A1
WO2012037881A1 PCT/CN2011/079903 CN2011079903W WO2012037881A1 WO 2012037881 A1 WO2012037881 A1 WO 2012037881A1 CN 2011079903 W CN2011079903 W CN 2011079903W WO 2012037881 A1 WO2012037881 A1 WO 2012037881A1
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rna
small
dna
tag
library
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Chinese (zh)
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章文蔚
周妍
张艳艳
徐小红
张秀清
杨焕明
汪建
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BGI Shenzhen Co Ltd
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BGI Shenzhen Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups

Definitions

  • the invention relates to the field of nucleic acid sequencing technology, in particular to the field of small molecule RNA sequencing technology.
  • the present invention relates to nucleic acid tags for small molecule RNA sequencing and uses thereof. More specifically, the present invention provides a nucleic acid tag, a PCR primer, a small RNA library, a preparation method thereof, a method for determining small molecule RN A sequence information, and a plurality of sample small molecules RN A for constructing a small molecular RNA library. Methods for sequence information and kits for constructing small RNA libraries.
  • Small RNAs are important special molecules in organisms that induce gene silencing and are involved in the regulation of many life activities such as cell growth, development, gene transcription and translation.
  • the small-molecule RNA digitization analysis based on Solexa high-throughput sequencing technology, SBS-sequencing by synthesis, can reduce the loss of a region caused by secondary structure. With the required sample volume, high throughput, high accuracy, easy-to-operate automation platform and powerful features, it can acquire millions of small RNA sequences in one time, and can quickly and comprehensively identify the species.
  • the small-molecule RNA in this state and the discovery of new small-molecule RNA construct a differential expression profile of small RNA between samples, providing a powerful tool for small-molecule RNA function research.
  • Illumina's Solexa sequencing platform provides two methods for preparing small-molecule RNA libraries, namely, Method 1 for preparing Samples for analysis of small RNA and Method 2 for replacing vl. 5 Procedures Prepare Samples for Small RNA Sequencing Using the Alternative vl. 5 Protocol.
  • Method 1 for preparing Samples for analysis of small RNA
  • Method 2 for replacing vl. 5 Procedures Prepare Samples for Small RNA Sequencing Using the Alternative vl. 5 Protocol.
  • Method 1 for preparing Samples for analysis of small RNA
  • Method 2 for replacing vl. 5 Procedures Prepare Samples for Small RNA Sequencing Using the Alternative vl. 5 Protocol.
  • RNAs of 18 to 30 nt in length are isolated from total RNA, and then the isolated small RNAs are sequentially combined with 5' adapters, 3 'Terminal junction, wherein each time the linker is connected, it is necessary to cut the target fragment of the linker successfully, and then the target fragment with the known linker is subjected to reverse transcription reaction, and the target fragment with the linker is amplified by PCR reaction. Finally, the gel is used to recover a library of the fragment of interest with a linker. Referring to FIG.
  • RNAs of 18 to 30 nt in length are isolated from total RNA, and then the isolated small RNAs are sequentially connected to the 3' connector and the 5' connector, and then The target fragment of the linker is known to undergo a reverse transcription reaction, the target fragment having the linker is amplified by a PCR reaction, and finally the gel fragment is used to recover a library of the target fragment having the linker.
  • the order of the linker is different from that of the method 1, and in the second method, the 5' end site modification of the 3' linker sequence is different from the method one.
  • the second method after the linker is ligated, it is not necessary to cleave the target fragment, and the target fragment having the known linker can be subjected to a reverse transcription reaction, and the target fragment having the linker can be amplified by a PCR reaction. Finally, the gel cut contains a library of the desired fragment.
  • the inventors of the present invention found that both methods of library preparation can only perform Solexa Single End sequencing on a single library sample, and the Solexa small molecule RNA library sample cannot be mixed and sequenced. However, with the increase in Solexa sequencing throughput, the data generated by one sequencing lane (lane) is much larger than the amount of data required for the target fragment. If the constructed library sample cannot be mixed and sequenced, it will be to some extent. "Waste sequencing resources" and affect sequencing throughput.
  • the present invention is directed to solving at least one of the problems of the prior art.
  • a nucleic acid tag herein, sometimes referred to as a "tag” that can be used to construct a small RNA library is proposed to accurately perform a small RNA library. distinguish.
  • the invention proposes a set of isolated nucleic acid tags.
  • These isolated nucleic acid tags are each composed of the nucleotides shown in SEQ ID NOS: 1-16.
  • the source of the sample of small RNA can be accurately characterized by linking the nucleic acid tag to small RNA or its equivalent.
  • a small RNA library for sequencing (in this case, sometimes referred to as a small RNA tag library) can be constructed simultaneously for a plurality of small RNA samples, and thus can be derived from different samples.
  • the small RNA library is mixed and sequenced, and the small RNA sequence is classified based on the nucleic acid tag to obtain sequence information of various small RNAs. This allows for the full use of high-throughput sequencing technologies, such as Solexa sequencing, to simultaneously sequence multiple small RNAs to improve the efficiency and throughput of small RNA sequencing.
  • the invention also provides a set of isolated tag PCR primers for introducing the above nucleic acid tag into small RNA or equivalents thereof.
  • a set of isolated nucleic acid PCR primers according to an embodiment of the present invention which consists of the nucleotides set forth in SEQ ID NOS: 17-32. These tag PCR primers correspond to nucleic acid tags, respectively, for introducing the corresponding nucleic acid tags into small RNAs or their equivalents.
  • a method of constructing a small molecule RNA library comprises: connecting a 5' connector and a 3' connector at a 5' end and a 3' end of a small molecular RNA having a length of 18 to 30 nt, respectively, to obtain a small 5' connector and a 3' connector.
  • the small RNA having the 5' linker and the 3' linker is sequentially subjected to a reverse transcription reaction and a PCR amplification reaction to obtain a PCR amplification product, wherein the PCR amplification reaction is carried out by using a PCR primer
  • the tag PCR primer comprises a nucleic acid tag selected from the group consisting of the set of isolated nucleic acid tags of claim 1, the PCR amplification product comprising the target fragment, a 5' linker, a 3' linker, and a nucleic acid tag, Wherein the sequence of the target fragment corresponds to the sequence of the small molecule RNA; and the PCR amplification product is isolated and recovered, and the PCR amplification product constitutes the small molecule RNA library.
  • the nucleic acid tag according to the embodiment of the present invention can be efficiently introduced into a small molecule RNA library constructed for small molecule RNA. Therefore, the source of the small RNA can be distinguished by sequencing the molecular RNA library, obtaining sequence information of the molecular RNA, and sequence information of the nucleic acid tag.
  • the present invention also provides a small molecule RNA library obtained by the method of constructing a small RNA library according to an embodiment of the present invention.
  • the present invention also provides a method of determining small molecule RNA sequence information.
  • a method of constructing a small molecule RNA library according to an embodiment of the present invention comprising: a method of constructing a small molecule RNA library according to an embodiment of the present invention, establishing a small molecule RNA library of the small molecule RNA; and sequencing the small molecule RNA library to determine Sequence information of small molecule RNA. Based on this method, the sequence information of the small molecule RNA and the sequence information of the nucleic acid tag can be efficiently obtained, so that the source of the small molecule RNA can be distinguished.
  • the present invention also provides a method of determining a plurality of sample small molecule RNA sequence information.
  • the method comprises the steps of: establishing, for each of the plurality of samples, a small molecule of the small RNA of the sample independently according to the method for constructing a small molecular RNA library according to an embodiment of the present invention An RNA library, wherein small RNAs of different samples are labeled with nucleic acids different from each other and known sequences, wherein the plurality of samples are 2-16; the small RNA libraries of the plurality of samples are combined to obtain a small RNA library mixture; sequencing the small RNA library mixture using Solexa sequencing technology using small molecule RNA sequencing primers and small molecule RNA tag sequencing primers, respectively, to obtain sequence information of the small molecule RNA and the Sequence information of the tag; and classifying the sequence information of the small molecule RNA based on sequence information of the tag to determine small molecule RNA sequence information of the pluralit
  • a small RNA library for sequencing of a plurality of small-molecule RNA samples can be simultaneously constructed, thereby allowing a small RNA library derived from different samples to be mixed and simultaneously sequenced, based on nucleic acid tags.
  • Small RNA sequence Line classification obtaining sequence information of a variety of small molecular RNAs. This allows for the full use of high-throughput sequencing technologies, such as the use of Solexa sequencing technology, while sequencing a variety of small RNAs to improve the efficiency and throughput of small RNA sequencing.
  • a kit for constructing a small molecule RNA library comprises: 16 kinds of tag PCR primers, wherein the 16 tag PCR primers are respectively composed of nucleotides represented by SEQ ID NOS: 17-32, wherein the 16 tag PCRs Primers are placed in separate containers.
  • a nucleic acid tag according to an embodiment of the present invention can be conveniently introduced into a constructed small molecule RNA library.
  • Figure 1 shows a schematic flow diagram of two small-molecule RNA library preparation methods provided by Illumina's Solexa sequencing platform.
  • 1A is a schematic diagram of the process of preparing a sample for analyzing small RNAs (Preparing Samples for analysis of small RNA).
  • Figure IB is a second schematic diagram of the method for preparing a sample for small RNA sequencing using the alternative vl .5 protocol (Preparing Samples for Small RNA Sequencing Using the Alternative vl. 5 Protocol).
  • Figure 2 shows a schematic diagram of a protocol for constructing a small molecule RNA library by embedding a nucleic acid tag in a 5' linker, i.e., protocol 1 or a control scheme in the examples, in accordance with one embodiment of the present invention.
  • Figure 3 shows the construction of a small-molecule RNA library using Scheme 1 and further the Solexa vJfl'J sequence according to one embodiment of the present invention.
  • foldchange represents a multiple of the difference, and the Arabidopsis and human miRNAs are compared with known miRNAs, respectively, and then the correlation analysis is performed by comparing miRNA molecules that are considered to be consistent with known miRNA molecules. owned. Foldchange is less than 2 ( ⁇ 1 ), indicating that when the library is constructed using the same sample, the data is stable (ie, there is no large deviation in the data).
  • FIG. 3A shows the construction of two different small RNA libraries, respectively, based on different nucleic acid tags using the same Arabidopsis samples. Between the two libraries, the expression of 125 genes in 178 miRNA gene expression levels was more than 4 times, that is, 70.2% of miRNA gene expression was significantly different.
  • Figure 3B shows the construction of two different small RNA libraries, based on different nucleic acid tags, using the same human samples. Between the two libraries, 184 genes in 430 miRNA gene expression levels were more than four times different, that is, 42.8% of miRNA gene expression was significantly different.
  • Figure 4 is a flow diagram showing the construction of a small molecule RNA library using a tag PCR primer of an embodiment of the present invention by embedding a tag in a PCR primer, i.e., according to an embodiment of the present invention.
  • Fig. 4A shows a schematic flow chart of constructing an RNA library having a tag sequence at the 3' end of the target fragment
  • Fig. 4B shows a schematic flow chart of constructing an RNA library having a tag sequence at the 5' end of the target fragment.
  • Figure 5 shows the differential expression of rice (Or 3 ⁇ 4a Sativa ) miRNA ( A ) and human miRNA ( B ) obtained by constructing a small RNA library using Scheme 1 and then performing Solexa sequencing according to an embodiment of the present invention.
  • the multiple of the foldchange generation difference is obtained by comparing rice and human miRNAs with known miRNAs, and then correlating the miRNA molecules of the database.
  • the foldchange is less than 2 ( ⁇ 1 ), indicating that when the library is constructed using the same sample, the data results are stable (ie, there is no large deviation in the data).
  • a foldchange greater than 4 indicates a large data deviation and an unstable data result.
  • Figure 5A shows the construction of two different small RNA libraries, respectively, based on different nucleic acid tags using the same rice samples. Among the two libraries, the expression difference of 7 genes in 323 miRNA gene expression levels was more than 4 times, that is, 2.2% of miRNA gene expression was significantly different).
  • Figure 5B shows the use of the same Human samples, based on different nucleic acid tags, were constructed into two different small RNA libraries. Among the two libraries, the expression difference of 11 genes in 375 miRNA gene expression levels was more than 4 times, that is, 2.9% of miRNA gene expression was significantly different.
  • Figure 6 shows a schematic representation of Solexa sequencing Readl of a small molecule RNA library (Scheme 1) constructed by embedding a tag in a 5' linker, in accordance with one embodiment of the present invention.
  • Scheme 1 a small molecule RNA library constructed by embedding a tag in a 5' linker, in accordance with one embodiment of the present invention.
  • Read 1 Seq Primer indicates sequencing primers.
  • Figure 7 shows a schematic representation of Solexa sequencing Readl of a small RNA library constructed using a tag PCR primer (Scheme 2), in accordance with one embodiment of the present invention.
  • Read 1 Seq Primer indicates sequencing primers
  • Index Seq Primer indicates label sequencing primers.
  • Figure 8 shows the results of data correlation analysis of a small RNA library of mouse RNA samples with a control library, in accordance with an embodiment of the present invention.
  • the mouse RNA sample was constructed using the nucleic acid tags Indexl, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16 to construct a small RNA library and pass Solexa sequencing, correlation analysis between small RNA library and control library data.
  • the horizontal coordinate shows the logarithm of the gene expression of different small RNA libraries constructed with different nucleic acid tags. The coordinates show that the gene expression of the same standard small-molecule RNA library is logarithmically based on 2, and then the correlation coefficient between the expression levels of the two genes is calculated.
  • Fig. 8 from top to bottom, from left to right, are the correlation analysis of the small molecule RNA tag library 1-16 and the control library, respectively.
  • mouse-Indexl is a data correlation analysis graph, and the correlation data is represented by dots.
  • the data expression amount of label 1 (indexl) is the abscissa, and the data expression of the control library is the ordinate. The closer the data is, the more relevant. The closer the sex value is to 1 (ie the closer the point is to the diagonal of the coordinate).
  • Figure 9 shows the analysis of the proportion of various RNAs in total data using two mouse control small RNA libraries and 16 mouse small RNA libraries constructed in Examples 2 and 3 of the present invention, respectively.
  • the small RNA library was constructed by using the nucleic acid tags Indexl, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 respectively for mouse RNA, respectively.
  • the invention proposes isolated nucleic acid tags.
  • these isolated nucleic acid tags are each composed of the nucleotide sequences shown in SEQ ID NOS: 1-16.
  • nucleic acid may be any polymer comprising deoxyribonucleotides or ribonucleotides, including but not limited to modified or unmodified DNA, RNA.
  • the sample source of the small molecule RNA can be accurately characterized by linking the nucleic acid tag to the small molecule RNA or its equivalent.
  • a small RNA library for sequencing which can be obtained by mixing small RNA libraries derived from different samples and simultaneously sequencing, and classifying small RNA sequences based on nucleic acid tags to obtain sequence information of various small RNAs. .
  • nucleic acid tag linked to small molecule RNA or its equivalent means that the nucleic acid tag can be directly linked to a small RNA to construct a small RNA library, or can have the same sequence as a small RNA.
  • the nucleic acid (for example, may be DNA obtained by reverse transcription, which has the same sequence as the small RNA) is linked.
  • a set of nucleic acid tags In order to efficiently construct and sequence small RNA libraries, a set of nucleic acid tags must be constructed to ensure reliable results and high reproducibility. That is, for the same RNA sample, a small RNA library constructed using different tags in the set of nucleic acid tags can be ensured, and consistent sequencing results can be obtained, thereby ensuring reliable and reproducible experimental results.
  • the inventors of the present invention found that in the case where the labeling amount is less than 12 (samples), the GT content of each base site on the mixed label must be considered. Because the excitation fluorescence of the bases G and T is the same in the Solexa sequencing process, the excitation lights of the bases A and C are the same, so the "balance" of the base “GT” content and the base “AC” content must be considered.
  • the base base “GT” content is 50%, which guarantees the highest label recognition rate and the lowest error rate.
  • the inventors of the present invention conducted a large number of screening work, and selected a set of isolated nucleic acid tags according to an embodiment of the present invention, i.e., having the nucleotide sequences shown in SEQ ID NOS: 1 to 16, respectively. After verification in the model plant "Rice Sample” and the model animal "Human Blood Sample", the final screening of these nucleic acid tags confirms the accuracy and reproducibility of the data.
  • the inventors have surprisingly found that the use of a set of isolated nucleic acid tags according to embodiments of the present invention, i.e., having the nucleotide sequences set forth in SEQ ID NOS: 1-16, can be flexibly applied not only to the sequencing of small RNA samples, It also improves the sequencing throughput of current small-molecule RNA samples and ensures the repeatability and accuracy of data output.
  • the present invention also provides a set of isolated nucleic acid tags which are nucleotide sequences of 6 bases in length, and the difference between the tag sequences is 3 or more bases.
  • the set of tags comprises or consists of: at least 2, or at least 3, or at least 4, or at least 5 of the 16 tags shown in Table 1 or the tags 1 base apart therefrom, At least 6, or at least 7, or at least 8, or at least 9, or 10, or at least 11, or at least 12, or at least 13, or at least 14, or at least 15, or all 16
  • the set of separated nucleic acid tags includes at least Index 1 and Index 2, or Index 3 and Index 4, or Index 5 and Index 6, or Index 7 and Index 8 , or Index 9 and Index 10 in the 16 tags shown in Table 1 above, or Index 11 and Indexl2, or Index 13 and IndexM, or Indexl5 and Indexl6, or a combination of any two or more of them.
  • a difference of one base comprises substitutions, additions and deletions of one base in the tag.
  • the current sequencing platform has a certain sequencing error rate.
  • the Solexa sequencing error rate is about 2%.
  • this design can correct 6 bases by setting a specific nucleotide sequence of 6 bases in length and ensuring that the difference between the tag sequences is 3 or more bases. Any one of the base errors above the nucleic acid tag sequence, and the output data of a base error is also listed as valid data, ensuring that the tag recognition rate is 98% or more.
  • FIG. 4A A schematic diagram showing the flow of an RNA library having a tag sequence at the 3' end of the target fragment
  • Fig. 4B is a flow chart showing the construction of an RNA library having a tag sequence at the 5' end of the target fragment.
  • a method of constructing a small molecule RNA library containing a nucleic acid tag includes:
  • a 5' linker and a 3' linker are ligated to the 5' end and the 3' end of a small molecule RNA of 18 to 30 nt in length to obtain a small molecule RNA having a 5' linker and a 3' linker.
  • the source of the small molecule RNA having a length of 18 to 30 nt is not particularly limited.
  • the sample includes, but is not limited to, from plants such as rice, Arabidopsis and animals such as mice, humans, and the recovery can be achieved, for example, by electrophoresis, particularly by denaturing PAGE electrophoresis.
  • the small molecule RNA thus obtained is more suitable for the sequencing scheme of the present invention.
  • the order in which the separated small molecule RNAs are respectively linked to the 5' linker and the 3' linker is not particularly limited.
  • a 3' linker is first ligated to the 3' end of a small molecule RNA of 18 to 30 nt in length, and then a 5' linker is ligated to the 5' end of a small molecule RNA of 18 to 30 nt in length.
  • Applicants have surprisingly found that ⁇ using this connection sequence can achieve better results.
  • the types of the 5' linker and the 3' linker are not particularly limited, and those skilled in the art can select according to the sequencing platform.
  • the 5' linker and the 3' linker are the universal 5' linker and 3' linker of Solexa sequencing technology, respectively.
  • Solexa sequencing technology enables high-throughput multi-sample sequencing of small RNAs with high stability and reproducibility.
  • a small molecule RNA having a 5' linker and a 3' linker is sequentially subjected to a reverse transcription reaction and a PCR amplification reaction to obtain a PCR amplification product.
  • a PCR amplification reaction is required by using a PCR primer, and the PCR amplification product PCR thus obtained contains a target fragment, a 5' linker, A 3' linker and a nucleic acid tag, wherein the sequence of the fragment of interest corresponds to the sequence of the small molecule RNA.
  • fragment of interest refers to a fragment obtained after a small molecule RNA has undergone a specific reaction treatment such as a reverse transcription reaction and a PCR amplification reaction, and the sequence of the target fragment corresponds to the sequence of the small molecule RNA, which means It is suggested that the sequence of the small RNA can be directly derived from the sequence of the target fragment.
  • the sequence of the target fragment can be identical to the sequence of the small RNA, or it can be completely complementary, or even increase or decrease the known number. Knowing bases, as long as the sequence of small RNAs can be obtained by limited calculation.
  • the length of the obtained amplification product is not particularly limited, and according to a specific example of the present invention, the length of the amplification product used is about 100 bp, thereby being capable of improving the sequencing recognition efficiency of Solexa, and Can further improve the sequencing throughput and depth of Solexa.
  • reverse transcription reaction and PCR amplification reaction should be understood in a broad sense, which means that the RNA is reversely transcribed into DNA in turn, and the DNA is further amplified in a form that is not particularly
  • the restriction may be carried out in one step in the same reaction system or in multiple steps in different reaction systems, and may be performed by first performing a reverse transcription reaction on a small molecule RNA having a 5' linker and a 3' linker, and then Further, the obtained reverse transcription product is subjected to a PCR reaction for amplification, or a primer and a reagent required for the PCR amplification reaction may be added to the reverse transcription reaction system, thereby performing reverse transcription and simultaneous completion in the same reaction system.
  • PCR amplification may be understood in a broad sense, which means that the RNA is reversely transcribed into DNA in turn, and the DNA is further amplified in a form that is not particularly
  • the restriction may be carried out in one step in the same reaction system or in multiple steps in different reaction systems, and
  • tag PCR primer means that a nucleic acid tag is embedded in a PCR primer sequence, whereby a tag PCR primer can be introduced to a target during amplification reaction using a tag PCR primer. At one end of the fragment, either the nucleic acid tag can be introduced to the 5' end or the tag can be introduced to the 3' end.
  • the PCR amplification reaction typically requires two primers to be performed, and one skilled in the art can select another primer for performing the PCR amplification reaction as needed. For example, referring to FIG.
  • the tag PCR primer when the tag PCR primer is used as an upstream primer, that is, when a nucleic acid tag is introduced at the 5′ end of the target fragment, the tag PCR primer specifically recognizes the sequence of the 5′ linker, and the downstream primer can utilize the specificity. Identify the sequence of the 3' linker.
  • a tag PCR primer that introduces a nucleic acid tag into the 5' end of the fragment of interest is used. Thereby, the introduction efficiency of the nucleic acid tag can be improved.
  • a PCR primer consisting of the nucleotide shown in any one of SEQ ID NO: 17 32 is used as a PCR primer for the tag, that is, a tag PCR primer as shown in Table 2 above, No longer.
  • These tag PCR primers are actually composed of three partial sequences, sequence 1 (also known as the primer sequence on the surface of the Solexa chip), sequence 2 (also known as the tag sequence), and sequence 3 (also known as the linker sequence).
  • sequence 1 also known as the primer sequence on the surface of the Solexa chip
  • sequence 2 also known as the tag sequence
  • sequence 3 also known as the linker sequence
  • the label 1 ie, Indexl: AAGTCG
  • the corresponding PCR primer sequence of the label is Index 1_PCR_2.0. It consists of the following three parts.
  • nucleic acid tags and their associated tag PCR primers can be applied to library construction of any small molecule RNA. And using these tag PCR primers, very good amplification efficiency can be obtained, and the difference between these tags is 3 bases, and when any one of the 6 bases of the tag has a sequencing error or a synthetic error, The final identification of the tags is not affected, and these primers can avoid the phenomenon of primer hairpin structures or the same sequence as the sequencing primers and their reverse complements.
  • the present invention provides a kit for constructing a small molecule RNA library, comprising: 16 tag PCR primers, wherein the 16 tag PCR primers are respectively SEQ ID NO: 17 A nucleotide composition represented by -32, wherein the 16 tag PCR primers are respectively disposed in different containers.
  • the kit can be used to conveniently construct a small RNA library.
  • those skilled in the art can understand that other conventional components for constructing a small molecular RNA library can also be included in the kit, and details are not described herein.
  • the resulting PCR amplification products can be isolated and recovered by any conventional method, and these PCR amplification products constitute a small molecule RNA library.
  • the small-molecule RN A library thus obtained can be directly applied to any conventional sequencing platform, and only the 5' connector and the 3' connector can be selected according to the sequencing platform.
  • the inventors have surprisingly found that small molecule RNA libraries constructed by this method can have significant reproducibility and accuracy when using a Solexa sequencing platform such as the Solexa Single End sequencing platform.
  • the sequencing of the small RNA library requires sequencing of the target fragment with the sequencing primer Readl Seq primer, and then the tag sequencing primer (also known as Index Seq primer). Sequencing the label, the sequencing reaction requires two sequencing primers, but it can avoid the bias connection of different label junctions in the connection reaction, resulting in different connection efficiency between each different label connector and the target segment, ultimately affecting To the stability and repeatability of the data (as disclosed below).
  • a small molecule RNA sequencing primer and a small molecule RNA tag and 5' ATGATACGGCGACCACCGACAGC SEQ ID NO: 56
  • RNAs of 18 to 30 nt in length are first isolated from total RNA, and the isolated small RNAs are first linked to the 3' linker and then to the tagged 5' linker (5) '
  • the nucleic acid tag sequence is embedded in the 3' end of the linker, sometimes referred to as the 5' tag linker, and then the small molecule RNA with the known linker is subjected to reverse transcription reaction, and further amplified by PCR reaction to obtain a tagged linker.
  • the target fragment is finally cut into a library of the target fragment containing the tag.
  • the fragment of interest here corresponds to the sequence of small RNA.
  • the small RNA library thus constructed is in the sequencing process, for example, by means of the Solexa sequencing platform, and only needs one sequencing primer (also called Read l Seq primer ) to carry the label together with the sequence of the target fragment. It is very convenient to measure.
  • Scenario 1 demonstrates the stability and repeatability of poor data.
  • RNA linker-Index2 GUUCAGAGUUCUACAGUCCGACGAUCACGGCU(34) Small RNA linker-Index3 GUUCAGAGUUCUACAGUCCGACGAUCCAUUAG(35) Small RNA linker-Index4 GUUCAGAGUUCUACAGUCCGACGAUCCCUGAU(36) Small RNA linker-Index5 GUUCAGAGUUCUACAGUCCGACGAUCGGACUA(37) Small molecule RNA linker-Index6 GUUCAGAGUUCUACAGUCCGACGAUCGUAAUC(38) Small RNA linker-Index7 GUUCAGAGUUCUACAGUCCGACGAUCUGCCGA(39) Small RNA linker-Index8 GUUCAGAGUUCUACAGUCCGACGAUCUUCAGC(40) Small RNA linker-Index9 GUUCAGAGUUCUACAGUCCGACGAUCUUCAGC(40) Small RNA linker-Index9 GUUCA
  • a method of introducing a nucleic acid tag by a tag PCR primer is to embed a nucleotide sequence of a specific length (ie, a nucleic acid tag) into a PCR primer.
  • a small molecular RNA tag library can be constructed by using different tag PCR primers, as shown in Figures 4A and 4B, and the tag can be embedded in any primer in the PCR primer.
  • the inventors used the model plant "rice sample” and the model animal "human blood sample” to construct a small molecule RNA tag library (see Examples 6 and 7), respectively.
  • the current sequencing platform has a certain sequencing error rate.
  • the Solexa sequencing error rate is about 2%.
  • this design can correct 6 bases by setting a specific nucleotide sequence of 6 bases in length and ensuring that the difference between the tag sequences is 3 or more bases. Any one of the base errors above the nucleic acid tag sequence, and the output data of a base error is also listed as valid data, ensuring that the tag recognition rate is 98% or more.
  • the statistical information of the output data is as follows.
  • the sequencing result is exactly matched with the expected label by 96.76%, and due to sequencing error
  • the error caused the tag to have a base difference of 1.12% from the expected tag sequence. Therefore, the data obtained by 0 mismatch and 1 mismatch accounts for 98.88% of the total data, that is, the tag identification rate of the data is 98.88%.
  • the tag provided by the present invention is used for the construction and sequencing of a small molecule RNA library, wherein the tag is contained in a PCR primer for amplifying a sequence of interest, thereby constituting a corresponding tag.
  • PCR primers PCR primers.
  • the tag is embedded in a PCR primer for amplifying a sequence of interest, or is linked to the 5' or 3' end of the PCR primer for amplifying the sequence of interest with or without a linker
  • the 5' end of the PCR primer for amplifying the sequence of interest is not ligated by a linker to constitute a corresponding corresponding tag PCR primer.
  • the tag is ligated to the 5' end of the PCR primer for amplification of the sequence of interest by a linker, thereby constituting a corresponding corresponding tag PCR primer, wherein the linker is 1 - 10
  • the sequence of one base preferably a sequence of 1 - 5 bases, more preferably a sequence of 1 - 3 bases.
  • Another aspect of the present invention provides a set of tag PCR primers corresponding to the tag, comprising the tag of the present invention, the set of tag PCR primers comprising or consisting of the following: 16 cells shown in Table 2 Labeling PCR primers or at least 2, or at least 3, or at least 4, or at least 5, at least 6, or at least 7, of the PCR primers differing by one base from the tag sequence contained therein, or At least 8, or at least 9, or 10, or at least 11, or at least 12, or at least 13, or at least 14, or at least 15, or all 16 of said set of PCR primers
  • at least the index 1 PCR 2.0 and Index2_PCR_2.0, or Index3_PCR_2.0 and Index4_PCR_2.0, or Index5_PCR_2.0 and Index6_PCR_2.0, or Index7_PCR_2.0 and Index8_PCR_2.0 of the 16 tags shown in Table 2 are included.
  • the tag PCR primers of the present invention are useful for the construction and sequencing of small RNA libraries.
  • the invention provides a small molecule RNA library constructed using the tag PCR primers shown in Table 2.
  • the present invention provides a method for constructing and sequencing a small molecule RN A library, comprising: 1) providing n total RNA samples and recovering 18-30 nt small RNA by recovery, n is an integer, and 1 ⁇ n ⁇ 16, preferably 2 ⁇ n ⁇ 16, and the sample includes, but is not limited to, from plants such as rice, Arabidopsis and animals such as mice, humans, and the recovery can be performed by, for example, electrophoresis.
  • the tag PCR primers in the method comprise or consist of the following: 16 tag PCR primers shown in Table 2 or tag PCR primers differing from the tag sequence contained therein by 1 base At least 2, or at least 3, or at least 4, or at least 5, at least 6, or at least 7, or at least 8, or at least 9, or 10, or at least 1 1 , or at least 12, or at least 13, or at least 14, or at least 15, or all 16 and the set of tag PCR primers preferably comprises at least The 16 labels of Index 1 PCR 2.0 and Index2_PCR_2.0, or Index3_PCR_2.0 and Index4_PCR_2.0, or Index5_PCR_2.0 and Index6_PCR_2.0, or Index7_PCR_2.0 and Index8_PCR_2.0, or Index9_PCR_2.0 and Index 10_PCR_2. 0, or Indexl 1_PCR_2.0 and Index 12_PCR_2.0, or Indexl 3_PCR_2.0 and Indexl 4_PCR_2.0, or Indexl 5_PCR_2.0 and Indexl 6_PCR_2.0 or a combination of
  • the present invention provides a method for determining small molecule RNA sequence information, comprising: a method of constructing a small molecule RNA library according to an embodiment of the present invention, establishing a small small RNA a molecular RNA library; and sequencing the small RNA library to determine sequence information of the small molecule RNA.
  • the sequencing method is not particularly limited.
  • sequencing of small RNAs is performed using Solexa sequencing technology.
  • the inventors of the present invention have surprisingly found that the method of constructing small molecule RNA of the present invention is particularly suitable for sequencing by Solexa sequencing technology, thereby achieving better data repeatability and stability.
  • small molecule RNA sequencing primers and tag sequencing primer pairs, molecular RNA libraries can be separately obtained to obtain sequence information of molecular RNA and sequence information of nucleic acid tags, such as small molecules.
  • the RNA sequencing primer has the nucleotide sequence shown as SEQ ID NO: 55
  • the tag sequencing primer has the nucleotide sequence shown as SEQ ID NO: 56.
  • the sequence information of the small molecule RNA and the sequence information of the tag can be efficiently obtained by sequencing, and thus, another aspect of the present invention provides a small RNA capable of determining a plurality of samples. The method of sequence information.
  • the method comprises the steps of: establishing a small molecule of the small RNA of the sample according to the method for constructing a small molecular RNA library according to an embodiment of the present invention, respectively, for each of the plurality of samples
  • An RNA library wherein small RNAs of different samples are labeled with nucleic acids different from each other and known sequences, wherein the plurality of samples are 2-16; the small RNA libraries of the plurality of samples are combined to obtain a small RNA library mixture; sequencing the small RNA library mixture using Solexa sequencing technology using small molecule RNA sequencing primers and small molecule RNA tag sequencing primers, respectively, to obtain sequence information of the small molecule RNA and the Sequence information of the tag; and classifying the sequence information of the small molecule RNA based on sequence information of the tag to determine small molecule RNA sequence information of the plurality of samples.
  • a small-molecule RNA library for sequencing of a plurality of small-molecule RNA samples can be simultaneously constructed, thereby allowing a small RNA library derived from different samples to be mixed and simultaneously sequenced, based on nucleic acid
  • the tag classifies small RNA sequences to obtain sequence information for a variety of small RNAs. This allows for the use of high-throughput sequencing technologies such as Solexa sequencing, which simultaneously high-throughput sequencing of multiple small RNAs, thereby increasing the efficiency and throughput of small-molecule RNA sequencing.
  • small molecule RNA libraries can be sequenced by small molecule RNA sequencing primers and tag sequencing primers respectively to obtain sequence information of small molecule RN A and sequence of nucleic acid tags.
  • the small molecule RN A sequencing I has the nucleotide sequence set forth in SEQ ID NO: 55
  • the tag sequencing primer has the nucleotide sequence set forth in SEQ ID NO: 56.
  • nucleic acid sequences employed in the following examples of the invention are as follows: Name sequence ( SEQ ID NO: )
  • Index 1_PCR_2.0 e.g. Indexl_PCR_Primer and having the same sequence ⁇ 7.
  • the small RNA RNA 1 linker has the same sequence as the small molecule RNA linker -Indexl.
  • DEPC treated water Add 1000 ⁇ L DEPC (one thousandth of a volume) to 1000 mL of deionized water and shake vigorously for 5 minutes until the bottom oily droplets are evenly dispersed. Leave at 37 °C overnight (at least 2 hours) or overnight at room temperature and autoclave at 121 °C for 30 minutes, mainly for partial RNase-free buffer preparation.
  • NEBuffer 2 Dilute NEBuffer 2 (10 ⁇ ) with DEPC treated water (Ambion) or pure water.
  • the total volume is 10 , mixed and centrifuged, and connected at 20 °C for 6 hours.
  • the total volume is ⁇ , mix and centrifuge, connect at 20 °C for 6 hours and then store at 4 °C (or overnight at 16 °C). 7) An equal volume of 2 ⁇ gel loading dye was added to the ligation product, denatured at 65 ° C for 5 minutes, then the sample was quickly placed on water, and a 1 10 bp gradient was mixed with the loading dye. The samples were electrophoresed in 10% denatured PAGE gel.
  • RT-PCR reaction Enter 10 RT-Primer (100 ⁇ ) into 10 RNA, heat at 65 °C for 10 min, centrifuge to cool to room temperature, and add the following reagents in sequence to perform reverse transcription (RT) reaction:
  • RNA Five micrograms of mouse liver total RNA was taken, mixed with an equal volume of 2 ⁇ gel loading dye, denatured at 65 ° C for 5 minutes, and then placed on water. The sample was then added to a well of a 15% denatured PAGE gel. At the same time, a 1 10 bp DNA gradient and 2 ⁇ L of 14-30 ssRNA gradient markers were mixed with 6 ⁇ loading buffer and added to the wells for electrophoresis.
  • RNA RT-primer 100 ⁇ M was added at 65 °C. Heat lOmin, centrifuge quickly to place water, add the following reagents in sequence, and perform reverse transcription reaction:
  • a small RNA library was constructed for 5 g mouse liver total RNA using the same method as in Example 2, and Solexa sequencing was performed, except that in Example 3, small molecule RNA 3 'linker-1 was used instead of Example 2, step 3. Small molecule RNA3 'linker-1 in ), Small RNA RNA PCR primer 2 (10 ⁇ M) in Example 3 was used instead of the small RNA RNA PCR primer 2 (10 ⁇ M) in Example 10, step 10).
  • Example 2 and Example 3 A non-tagged mouse small RNA library (control library) and a tagged mouse small RNA library were separately constructed and sequenced using the Solexa sequencing method. The results of the data analysis are as follows:
  • RNA tag libraries Using a model organism "mouse" RNA sample, in Examples 2 and 3, a non-tagged and tagged small-molecule RNA tag library was constructed based on essentially the same method, and a non-tag library was constructed.
  • Non-labeled libraries constructed using "mouse” RNA samples were used as data from control libraries, and correlation analysis was performed with data from 16 small-molecule RNA tag libraries carrying different tags. The results are shown in Figure 8.
  • the results in Figure 8 show that the correlation between the data results of the small-molecule RNA tag library constructed using the 16-tag PCR primers according to the embodiment of the present invention and the results of the non-tag library data was 0.94 0.99.
  • the composition ratios of various small-molecule RNAs in the 16 small-molecule RNA tag libraries are not changed, that is, no data bias is generated, and the data stability and repeatability are repeated. The sex is ideal.
  • the small-molecule RNA tag library constructed by 16-tag PCR primers has a stable shield and can be applied to various small RNA tag libraries.
  • a tagged small RNA library was constructed for 5 micrograms of total Arabidopsis leaf total RNA in the same manner as in Example 2, and subjected to Solexa sequencing. Only in Example 4, the small molecule RNA 5' linker in step 2 of Example 2 was replaced with a small molecule RNA linker -Index3 carrying the tag.
  • a tagged small molecule RNA library was constructed for 5 micrograms of human blood RNA in substantially the same manner as in Example 4, and subjected to Solexa sequencing. However, in Example 5, the small molecule RNA linker -Index3 in step 8 of Example 8 was replaced with a small molecule RNA linker -Index8 carrying the tag.
  • the small-molecule RNA library sequencing results constructed using the small-molecule RNA linker -Index8 were compared with those of the human small-molecule RNA library sequence constructed without the tag.
  • a small RNA library carrying Index8 was constructed by Solexa. 5925097 high-profile small RNA sequences were sequenced.
  • the human small RNA library constructed without tags was sequenced by Solexa to sequence 6169468 high-profile small RNA sequences.
  • the inventors selected the miRNA gene and analyzed the degree of difference in its expression level. As shown in Figure 3B, there were 430 miRNA gene expression levels between the two libraries, and the expression difference of 184 genes was more than 4 times (42.8% of miRNA gene expression was significantly different). Therefore, the sequencing results of the small-molecule RNA tag library constructed by this method are quite different from those of the small-molecule RNA non-tag library.
  • Example 6 A small-molecule RNA library having a tag of 5 micrograms of total RNAgo of rice was subjected to Solexa sequencing in substantially the same manner as in Example 4. However, in Example 6, ⁇ used 1000 ⁇ l of 100% ethanol instead of 1000 ⁇ 1500 liters of 100% ethanol in step 2) of Example 5, and used the small molecule RNA 5 'linker instead of the step 4 in Example 4) Small molecule RNA linker - Index3 and using Indexl_PCR Primer 2 instead of small molecule RNA PCR primer 2.
  • the small RNA library sequencing results constructed using Index l_PCR_Primer were compared with the sequencing results of rice small RNA library constructed without using a tag.
  • the constructed small RNA RNA library carrying Indexl was sequenced by Solexa to sequence 693 1837 high-profile small RNA sequences.
  • the rice small RNA library without tag construction was sequenced by Solexa to sequence 8050236 high-profile small RNAs. sequence.
  • the inventors selected the miRNA gene and analyzed the degree of difference in its expression level. As shown in Figure 5A, there were 323 miRNA gene expression levels between the two libraries, and the expression of the seven genes was more than 4 times different (2.2% of miRNA gene expression was significantly different). Therefore, the sequencing results of the small-molecule RNA tag library constructed by this method are less different from those of the small-molecule RNA non-tag library.
  • Example 7 A tagged small RNA library was constructed for 5 micrograms of human blood total RNA in the same manner as in Example 6, and Solexa sequencing was performed. However, in Example 7, Indexl_PCR_Primer was used instead of Indexl_PCR_Primer in step 4) of Example 6.
  • the results of the sequencing of the small-molecule RNA library using the Index5_PCR_Primer and the sequencing results of the human small-molecule RNA library constructed without using the tag were compared.
  • a small human RNA library constructed by Index5 was used to sequence 6295081 high-profile small RNA sequences by Solexa, and 7999586 high-profile small RNA sequences were sequenced by Solexa using a human small RNA library constructed without tags.
  • the inventors selected the miRNA gene and analyzed the degree of difference in its expression level. As shown in Fig. 5B, 375 miRNA gene expression levels were found between the two libraries, and the expression difference of the 1 1 gene was more than 4 times (2.9% of the miRNA gene expression was significantly different). Therefore, the sequencing results of the small-molecule RNA tag library constructed by this method are small compared with the data of the small-molecule RNA non-tag library.
  • Nucleic acid tag, tag PCR primer, small molecule RNA file for constructing small molecular RNA library of the invention Library and its preparation method, method for determining information of small RNA sequence, method for determining information of small sample RNA sequence of various samples, and kit for constructing small RNA library can be applied to sequencing of small RNA and can be effective Improve the sequencing throughput of sequencing platforms such as the Solexa sequencing platform.

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Abstract

La présente invention concerne un groupe de marqueurs d'acides nucléiques pour la construction d'une bibliothèque de petits ARN, un groupe d'amorces de marqueurs d'acides nucléiques, un procédé de construction d'une bibliothèque de petits ARN, la bibliothèque de petits ARN construite et un procédé d'obtention des informations de séquence d'un petit ARN.
PCT/CN2011/079903 2010-09-21 2011-09-20 Marqueurs d'acides nucléiques et leurs utilisations Ceased WO2012037881A1 (fr)

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CN105986020B (zh) * 2015-02-11 2019-08-09 深圳华大智造科技有限公司 构建测序文库的方法及装置
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CN112011834A (zh) * 2019-05-29 2020-12-01 上海京房生物科技有限公司 一种用于miRNA的高通量测序文库制备方法
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