A sequencing method, sequencing reagent and application for Small RNA

Abstract

The present application discloses a sequencing method, sequencing reagent and application for Small RNA. The present application is a sequencing method for Small RNA, comprising adding a normal sequencing primer and a blocking sequencing primer to a primer solution when sequencing a circular library of a mixed sample, and using a mixed primer for sequencing; the blocking sequencing primer is formed by chemically modifying the 3' terminal hydroxyl group of the normal sequencing primer. The sequencing method of the present application, by adding a blocking sequencing primer, enables Small RNA sequencing of a circular library of a mixed sample, and can effectively ensure the sequencing of the label sequence, thereby improving the data utilization rate and obtaining a good resolution rate. The Small RNA sequencing method and sequencing reagent of the present application provide a new idea and solution for Small RNA mixed sample sequencing, solving the current problem of sequencing resource waste in Small RNA sequencing of circular libraries.

Description

[0001] This application is a divisional application. The application number of the original application is: 201710855713.X, the application date is September 20, 2017, and the name of the invention is “A sequencing method, sequencing reagent and application for Small RNA”. Technical Field

[0002] The present application relates to the field of gene sequencing, and in particular to a sequencing method, sequencing reagents and applications for Small RNA. Background Art

[0003] Small RNA, including micro RNAs, siRNAs and pi RNAs, is a large class of regulatory molecules that exist in almost all organisms. It is an important regulatory factor in life activities and plays an important role in physiological processes such as gene expression regulation, biological individual development, metabolism and disease occurrence. Through large-scale sequencing and analysis of Small RNA, a Small RNA map at the whole genome level of the species can be obtained, and scientific applications such as mining new Small RNA molecules, prediction and identification of Small RNA target genes, differential expression analysis between samples, Small RNA clustering and expression spectrum analysis can be realized. Small RNA regulates the growth and development of organisms and the occurrence of diseases through a variety of action pathways, including mRNA degradation, translation inhibition, heterochromatin formation and DNA removal. Therefore, Small RNA has a high scientific research value. Researchers are very keen on related research on Small RNA, so the sequencing of Small RNA has become a hot topic of great concern.

[0004] At present, various sequencing platforms have two methods for sequencing Small RNA: one is to sequence through a chain library, and the other is to sequence through a circular library. Among them, circular library sequencing is to circularize Small RNA to form a circular library; then use the circular library as a template to generate DNA nanoballs, which are multiple copies of the chained test chain, each copy is complementary to the circular library; sequence this multiple copy of the chained test chain to obtain the sequence of the target area. An important flaw of circular library Small RNA sequencing is that when multiple samples are mixed for sequencing, different label sequences are usually added to different samples. Due to the small fragmentation of Small RNA itself, which is only 18-30nt, in order to obtain more data, the sequencing read length is usually set to 50 cycles; this causes most of the label sequences, including their primer regions, to be covered by the newly generated chains of the sequencing target fragments when sequencing the target fragment region, resulting in the inability to bind to the primers of the label sequence when sequencing the label sequence, i.e., the label sequence sequencing cannot be completed; this will lead to two very serious problems. First, because the label sequence sequencing cannot be completed and different samples cannot be distinguished, these data will be filtered out, i.e., most of the data are unusable data, causing a huge waste of sequencing resources; second, because most of the data are unusable data, a good splitting rate cannot be obtained.

[0005] Because of the above problems and defects, the current circular library Small RNA sequencing can only sequence one sample at a time if it wants to obtain more data, and cannot sequence multiple samples simultaneously. The current sequencing platforms usually have the ability of high-throughput sequencing, but only sequence one sample at a time, which will also cause a great waste of sequencing resources. Summary of the invention

[0006] The purpose of the present application is to provide an improved sequencing method for Small RNA, as well as sequencing reagents used in the sequencing method, and applications thereof.

[0007] This application adopts the following technical solutions:

[0008] One aspect of the present application discloses a sequencing method for Small RNA, comprising adding a normal sequencing primer and a blocking sequencing primer to a primer solution to form a mixed primer when sequencing a circular library of a mixed sample, and using the mixed primer for sequencing; wherein the blocking sequencing primer is formed by chemically modifying the hydroxyl group at the 3' end of the normal sequencing primer.

[0009] It should be noted that in this application, the normal sequencing primer is the sequencing primer used in the existing sequencing platform, and the blocking sequencing primer is formed by chemically modifying the 3' terminal hydroxyl of the normal sequencing primer. The normal sequencing primer is a normal primer whose 3' terminal hydroxyl is not chemically modified. The key to this application is to use both the conventional sequencing primer and the blocking sequencing primer during the sequencing process; normally, if Figure 4 As shown, when the hydroxyl group at the 3' end of the normal sequencing primer is not modified, during synthesis, after the normal sequencing primer is bound to the template chain to be tested, the hydroxyl group at the last base, i.e., the 3' end, is exposed to the outside and will chemically react with the phosphate group of the free dNTPs in the solution to combine together. The specific free dNTPs are selected based on the principle of complementary base pairing with the template to be tested. The newly added dNTPs also carry a hydroxyl group and combine with the phosphate group of the next dNTPs to achieve extension. However, the blocking sequencing primer of the present application has a chemically modified hydroxyl group at the 3' end, such as Figure 5 As shown, therefore, extension cannot be achieved, which plays a role in blocking sequencing. It can be understood that the blocking sequencing primer of the present application is based on the chemical modification of the sequencing primer. As for the specific sequencing primer sequence, different sequencing platforms have different specific sequencing primer sequences, but the method of the present application can be used to sequence the SmallRNA circular library of mixed samples. Therefore, the sequences of normal sequencing primers and blocking sequencing primers are not specifically limited here. For another example, for the Illumina sequencing platform, its principle is bridge amplification, and the primers are two primers designed based on I5 and I7 linkers. These two primers are suitable for all platforms in the same series. The blocking primers designed based on the principles of the present invention for the aforementioned two primers can also be used for the Illumina sequencing platform. In addition, the role of chemical modification is to avoid the combination of hydroxyl and phosphate groups and block extension. Therefore, existing hydroxyl modification methods can be used in this application, such as eliminating the hydroxyl group, hydrogenating it, and replacing the hydroxyl group with H; using alkali metals to perform a replacement reaction on the hydroxyl group to modify -OH to -OY, where Y is an alkali metal element; or using hydrocarbon groups or other groups to perform substitution reactions and dehydrate to ethers; no specific limitations are made here.

[0010] It should also be noted that the object of circular library sequencing is DNA nanoballs, and DNA nanoballs are multi-copy chain-like test chains, that is, there are multiple binding sites for normal sequencing primers on the sequencing template chain of the DNA nanoballs, and of course there are multiple binding sites for blocking sequencing primers, and the random binding of blocking sequencing primers is actually equivalent to occupying a vacancy, because the blocking sequencing primer will not extend after binding to the sequencing template chain, and therefore, the subsequent label sequence will not be covered, so that at least one copy of the label sequence is retained, so that the label sequence can be sequenced normally. Therefore, when the sequencing method of the present application is used to perform Small RNA sequencing on the circular library of the mixed sample, the label sequence can be sequenced normally, effectively improving the data utilization rate, and being able to obtain a good splitting rate, so that the Small RNA sequencing of the circular library of the mixed sample can be achieved.

[0011] Preferably, the chemical modification comprises replacing the entire hydroxyl group or H in the hydroxyl group.

[0012] It should be noted that the role of chemical modification is to block the hydroxyl group at the 3' end to prevent it from combining with the phosphate group, thereby achieving the purpose of blocking extension. Therefore, any chemical modification that can react with the hydroxyl group can be used in this application.

[0013] Preferably, the blocking sequencing primer accounts for 10%-70% of the total amount of the mixed primers.

[0014] It should be noted that the more the blocking sequencing primer is added, the more sequencing data of the final index primer will be, and correspondingly, because the blocking sequencing primer occupies the vacant position to block the sequencing of the Small RNA target region, the less sequencing data of the Small RNA target region will be. Therefore, in general, the specific amount of blocking sequencing primer can be determined according to the number of target sequences. When there are relatively many target sequences, more blocking sequencing primers can be added to obtain more sequencing data of the index primer; on the contrary, if the target sequence is small, in order to ensure the sequencing data of the Small RNA target region, the amount of blocking sequencing primer added is small. From this perspective, the number of each sample in the mixed sample itself must be large enough. If the number is too small, it is impossible to obtain enough sequencing data of the Small RNA target region or sequencing data of the index primer. In addition, under normal circumstances, the amount of blocking sequencing primer is 10-70% of the total mixed primer, and this range will not affect the sequencing results.

[0015] Another aspect of the present application discloses a sequencing reagent for Small RNA, wherein the sequencing reagent includes a blocking sequencing primer, wherein the blocking sequencing primer is formed by chemically modifying the hydroxyl group at the 3' end of a normal sequencing primer.

[0016] It should be noted that the reason why the Small RNA sequencing method of the present application can realize the sequencing of the circular library of mixed samples is that the key lies in the use of blocking sequencing primers. Therefore, for the convenience of use, the present application separately proposes a sequencing reagent containing the blocking sequencing primer. Similarly, the key to the blocking sequencing primer is that the hydroxyl group at the 3' end is chemically modified, and as for its specific sequence, it can refer to the sequencing primers of various sequencing platforms, and its specific chemical modification can also refer to the existing hydroxyl modification scheme, which is not specifically limited here.

[0017] Preferably, the chemical modification comprises replacing the entire hydroxyl group or H in the hydroxyl group.

[0018] Preferably, the sequencing reagent of the present application also includes a normal sequencing primer, which is a primer whose 3' end hydroxyl group of the blocking sequencing primer is not chemically modified, and the ratio of the blocking sequencing primer to the normal sequencing primer is 1-7:3-9.

[0019] It should be noted that the key to the Small RNA sequencing method of the present application is to add normal sequencing primers and blocking sequencing primers at the same time. Therefore, for ease of use, the sequencing reagent of the present application includes sequencing primers and their corresponding blocking sequencing primers. The normal sequencing primers and the blocking sequencing primers in the sequencing reagent of the present application correspond to each other, that is, the blocking sequencing primer is chemically modified from the hydroxyl group at the 3' end of the normal sequencing primer. It can be understood that the normal sequencing primers and the blocking sequencing primers in the sequencing reagent of the present application can be matched according to a predetermined ratio, and the primer mixture can be directly added when used; they can also be packaged separately according to the ratio, and the ratio can be flexibly adjusted according to needs when used, and no specific limitation is made here.

[0020] Another aspect of the present application also discloses a sequencing kit for Small RNA, wherein the sequencing kit contains the sequencing reagent of the present application.

[0021] It is understandable that, as a sequencing kit, in addition to the sequencing reagents of the present application, other reagents may also be included, such as a buffer for sequencing reaction, dNTPs, etc., which are not specifically limited here.

[0022] Another aspect of the present application also discloses the use of the sequencing reagent or sequencing kit of the present application in the sequencing of a circular library of a small-fragment nucleic acid mixed sample.

[0023] It should be noted that the reason why the circular library of the Small RNA mixed sample cannot be sequenced is that the Small RNA itself has a small fragment, and the read length is greater than its own fragment, so that most of the label sequences are covered and the label sequence sequencing cannot be completed; based on the same principle, the sequencing method, sequencing reagent or sequencing kit of the present application is not limited to the sequencing of the circular library of the Small RNA mixed sample, but also applies to the sequencing of the circular library of other similar small fragment nucleic acid mixed samples. It can be understood that the small fragment nucleic acid mixed sample, specifically, is a mixed sample composed of small fragment nucleic acids whose fragment length is less than the read length, including but not limited to Small RNA.

[0024] The beneficial effects of this application are:

[0025] The Small RNA sequencing method of the present application enables Small RNA sequencing of a mixed sample circular library by adding blocking sequencing primers, and can effectively ensure the sequencing of the tag sequence, thereby improving data utilization and obtaining a good resolution rate. The Small RNA sequencing method and sequencing reagent of the present application provide a new idea and solution for Small RNA mixed sample sequencing, solving the current problem of sequencing resource waste in Small RNA sequencing of circular libraries. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a statistical result diagram of the quality values ​​of each mixed sample when sequenced using different sequencing methods in the embodiment of the present application;

[0027] Figure 2 It is a statistical result diagram of the number of effective nucleotide sequences when each mixed sample in the embodiment of the present application is sequenced using different sequencing methods;

[0028] Figure 3 It is a statistical result diagram of the splitting rate when each mixed sample in the embodiment of the present application is sequenced using different sequencing methods;

[0029] Figure 4 It is a schematic diagram of the structure of the normal sequencing primer in this application without chemical modification at the 3' end;

[0030] Figure 5 Schematic diagram of the structure of the blocking sequencing primer after chemical modification at the 3' end in the present application, wherein R is a modification group. DETAILED DESCRIPTION

[0031] In this article, the split rate (SplitRate) refers to the ratio of the number of sequences that can be matched to the barcode list to the total number of fastq sequences.

[0032] After extensive research and experiments, the inventors of the present application proposed that the reason why the tag sequence cannot be sequenced well when SmallRNA sequencing is performed on the circular library of a mixed sample is that the target region of the Small RNA is relatively short, and the sequencing read length is greater than the target region, so that the tag sequence will be overwritten under the set read length, and the set read length is intended to obtain more data.

[0033] Based on the above understanding, the inventors of the present application creatively proposed that if some vacancies can be reserved, that is, some positions are left without sequencing the target region, then the label sequences corresponding to these positions will not be covered, and the label sequence sequencing can be performed normally, thereby solving the problem of label sequence sequencing. In view of this, the inventors of the present application further proposed that normal sequencing primers and blocking sequencing primers can be added at the same time during sequencing, and both of them randomly bind to the template chain, and the position of the binding blocking sequencing primer will not undergo amplification and extension, which plays a role of occupying space and reserving space for the sequencing of the label sequence.

[0034] The present application is further described in detail below through specific examples. The following examples are only used to further illustrate the present application and should not be construed as limiting the present application.

[0035] Example

[0036] In this case, Small RNA sequencing was performed on the BGISEQ-500RS platform, a sequencer independently developed by BGI. The normal sequencing primer is the sequence shown in Seq ID No.1.

[0037] Seq ID No.1: 5'-AAGT CGGA GGCC AAGC GGTC TTAG GAAG ACAA-3'

[0038] The blocking sequencing primer in this example is based on the normal sequencing primer of the sequence shown in Seq ID No. 1, and its 3' end is chemically modified. In this example, -H is used to replace the hydroxyl group. The blocking sequencing primer with -H replacing the hydroxyl group is synthesized by Shanghai Bioengineering.

[0039] The other sequencing reagents used in this example are all from the library construction kit and single-end sequencing kit used with the sequencer, hereinafter referred to as the SE50 kit. For the sequencer and reagent operation used in the verification process, please refer to the instructions for use of the BGISEQ-500RS platform.

[0040] This case used nucleic acid samples from three sources: human, saffron, and mouse, which were combined in the following ways to form mixed samples. Human serum samples were serum samples from six different people. All samples were provided by BGI:

[0041] i. The first group of test samples was a mixed sample of mouse and human serum No. 1, and the two were added in a 1:1 volume ratio;

[0042] ii. The second test sample was a mixture of human sera from sources 2 and 3 in equal volumes in a ratio of 1:1, labeled as human serum 1;

[0043] iii. The third group of test samples was prepared by mixing equal volumes of human sera from sources 4 and 5 in a ratio of 1:1, and was labeled as human serum 2;

[0044] iv. The fourth group of test samples is a mixed sample of saffron and human serum No. 6;

[0045] The samples from the above different sources were prepared according to the library construction method of BGISEQ-500RS and the principles of preparing samples for machine. The library construction kit uses MGIEasy Small RNA Library Preparation Kit (manufacturer: Shenzhen MGI Intelligent Manufacturing Technology Co., Ltd., referred to as "MGI", English abbreviation MGI). The specific library construction process is detailed in the kit instruction manual.

[0046] When preparing the sequencing reaction solution, the sequencing primers in the loading reagent plate in the SE50 kit were taken out and replaced with a mixture of normal sequencing primers and blocking sequencing primers in this example. In the mixture, the amount of the original normal sequencing primers accounted for 70% of the total mixed primers, and the amount of the blocking sequencing primers accounted for 30% of the total mixed primers. After that, the chip was prepared in the same way as the BGISEQ-500RS chip, and the sequencing was performed on the machine.

[0047] For comparison, for the same mixed sample circular library, this example directly uses normal sequencing primers without adding blocking sequencing primers, and uses the same sequencing platform and method to sequence the mixed sample. Except for not adding blocking sequencing primers, other conditions are the same.

[0048] In this example, the sequencing data of four groups of tests and four groups of comparisons were statistically analyzed, and the sequencing quality values, effective nucleotide sequence numbers, and splitting rates of the four groups of tests and four groups of comparisons were compared. The above statistical analysis was performed by the basecall software that comes with the sequencing platform. The results are as follows Figures 1 to 3 shown. Figure 1 This is a statistical result chart of the quality values ​​of four groups of tests and four groups of comparative sequencing. Figure 1In the figure, the horizontal axis is different mixed samples, and each mixed sample is subjected to the sequencing test and comparative sequencing of this example respectively; the vertical axis is the quality value obtained when each sample is sequenced in different ways; it can be seen from the figure that the same mixed sample will not reduce the sequencing quality when the sequencing method of this example is used, and the same effect is obtained between different samples, that is, the sequencing method of this example will not affect the sequencing results; that is to say, the addition of blocking sequencing primers will not affect the normal sequencing quality. Figure 2 This is a statistical result chart of the number of effective nucleotide sequences in four groups of tests and four groups of comparative sequencing. Figure 2 In the figure, the horizontal axis is different mixed samples, and each mixed sample is subjected to the sequencing test and comparative sequencing of this example respectively; the vertical axis is the number of effective nucleic acid sequences obtained when each sample is sequenced in different ways; it can be seen from the figure that there is no obvious difference in the number of effective nucleic acid sequences obtained for the same mixed sample when the sequencing method of this example is used, and the same effect is obtained between different samples, that is, the sequencing method of this example will not affect the sequencing results. Figure 3 This is a statistical diagram of the splitting of four groups of tests and four groups of comparative sequencing. Figure 3 In the figure, the horizontal axis is different mixed samples, and each mixed sample is subjected to sequencing test and comparative sequencing in this example; the vertical axis is the splitting rate obtained when each sample is sequenced in different ways; it can be seen from the figure that for the same mixed sample, in the sequencing method of this example, due to the addition of blocking sequencing primers, a higher splitting rate can be obtained, that is, different samples sequenced together can be distinguished, and the four groups of mixed samples all obtained a higher splitting rate, which effectively solved the splitting problem of SmallRNA mixed sample sequencing.

[0049] The above contents are further detailed descriptions of the present application in combination with specific implementation methods, and it cannot be determined that the specific implementation of the present application is limited to these descriptions. For ordinary technicians in the technical field to which the present application belongs, several simple deductions or substitutions can be made without departing from the concept of the present application, which should be deemed to fall within the scope of protection of the present application.

Claims

1. Application of a sequencing kit in circular library sequencing of a small-fragment nucleic acid mixed sample, wherein: The sequencing kit contains sequencing reagents, which include blocking sequencing primers. The blocking sequencing primers are formed by chemically modifying the hydroxyl group at the 3' end of the normal sequencing primer, wherein the chemical modification includes replacing the entire hydroxyl group or H in the hydroxyl group; The sequencing reagent also includes a normal sequencing primer, the blocking sequencing primer accounts for 10%-70% of the total amount of the mixed primer, the mixed primer is obtained by mixing the blocking sequencing primer and the normal sequencing primer, and the normal sequencing primer is a primer whose 3' end hydroxyl group of the blocking sequencing primer has not been chemically modified; The small fragments are fragments of 18-30 nt.

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