CN115927566A - Sequencing method, sequencing reagent and application for Small RNA - Google Patents
Sequencing method, sequencing reagent and application for Small RNA Download PDFInfo
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Abstract
The application discloses a sequencing method, a sequencing reagent and application for Small RNA. The sequencing method for Small RNA comprises the steps of adding a normal sequencing primer and a blocking sequencing primer into a primer solution when sequencing an annular library of a mixed sample, and sequencing by adopting a mixed primer; the blocking sequencing primer is formed by chemically modifying the hydroxyl at the 3' terminal of a normal sequencing primer. According to the sequencing method, the blocking sequencing primer is added, so that Small RNA sequencing of the mixed sample annular library is realized, sequencing of a tag sequence can be effectively guaranteed, the data utilization rate is further improved, and a good splitting rate is obtained. The Small RNA sequencing method and the sequencing reagent provide a new idea and scheme for sequencing a Small RNA mixed sample, and solve the problems of sequencing resource waste and the like of the conventional circular library Small RNA sequencing.
Description
The application is a divisional application, and the application numbers of the original application are as follows: 201710855713.X, filed 2017, 09 and 20.X, and is named as a sequencing method, a sequencing reagent and application for Small RNA.
Technical Field
The application relates to the field of gene sequencing, in particular to a sequencing method, a sequencing reagent and application for Small RNA.
Background
Small RNAs including micro RNAs, siRNAs and pi RNAs are a large class of regulatory molecules, exist in almost all organisms, are important regulatory factors for life activities, and play an important role in physiological processes such as gene expression regulation, biological ontogenesis, metabolism and disease occurrence. Small RNA maps of the whole genome level of the species can be obtained from Small RNA large-scale sequencing analysis, and scientific applications including excavation of new Small RNA molecules, prediction and identification of Small RNA action target genes, differential expression analysis among samples, small RNA clustering, expression profile analysis and the like are realized. Small RNA regulates the growth and development of organisms and the occurrence of diseases through various action pathways including mRNA degradation, translation inhibition, heterochromatin formation, DNA removal and the like, so that the Small RNA has high scientific research value, researchers are keen on the related research of the Small RNA, and the sequencing of the Small RNA becomes a high-interest hotspot.
Current methods for sequencing Small RNAs by each sequencing platform are, in one case, sequencing through a chain library and, in another case, sequencing through a circular library. Wherein, the sequencing of the circular library is to perform the cyclization of Small RNA to form the circular library; then generating a DNA nanosphere by taking the annular library as a template, wherein the DNA nanosphere is a multi-copy chain-shaped chain to be detected, and each copy is complementary with the annular library; sequencing the multiple copies of the chain to be detected to obtain the sequence of the target region. One important drawback of circular library Small RNA sequencing is that when multiple samples are sequenced in a mixed manner, different tag sequences are usually added to different samples, and due to the Small fragmentation of Small RNA, which is only 18-30nt, in order to obtain more data, the sequencing read length is usually set to 50 cycles; therefore, when the target fragment region is sequenced, most of the tag sequences including the primer regions can be covered by the newly generated strands of the sequenced target fragments, so that the primers of the tag sequences cannot be combined when the tag sequences are sequenced again, namely, the sequencing of the tag sequences cannot be completed; firstly, because the sequencing of the tag sequence cannot be completed and different samples cannot be distinguished, the data can be filtered and discarded, namely most of the data are unavailable data, so that the sequencing resource is greatly wasted; second, because most of the data is unusable, a better resolution rate cannot be obtained.
Because of the above problems and disadvantages, the current sequencing of Small RNA in circular library only can sequence one sample at a time if more data is needed, and can not sequence multiple samples at the same time. However, the existing sequencing platform generally has a high-throughput sequencing capability, and only one sample is sequenced at a time, which also causes a great waste of sequencing resources.
Disclosure of Invention
The application aims to provide an improved sequencing method for Small RNA, a sequencing reagent adopted by the sequencing method and application of the sequencing reagent.
The following technical scheme is adopted in the application:
one aspect of the application discloses a sequencing method for Small RNA, which comprises the steps of adding a normal sequencing primer and a blocking sequencing primer into a primer solution to form a mixed primer when sequencing an annular library of a mixed sample, and sequencing by adopting the mixed primer; wherein, the blocking sequencing primer is formed by chemically modifying hydroxyl at the 3' tail end of the normal sequencing primer.
It should be noted that, in the present application, the normal sequencing primer is a sequencing primer used in an existing sequencing platform, the blocking sequencing primer is a primer formed by chemically modifying a hydroxyl group at the 3 'end of the normal sequencing primer, and the normal sequencing primer is a primer that is normal and has no chemically modified hydroxyl group at the 3' end. The key point of the method is that a conventional sequencing primer and a blocking sequencing primer are used simultaneously in the sequencing process; normally, as shown in fig. 4, under the condition that 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 detected, the last base, namely the hydroxyl group at the 3' end is exposed and can be combined with the phosphate group of free dNTPs in the solution through chemical reaction, specific free dNTPs are selected according to the base complementary pairing principle with the template to be detected, and newly-accessed dNTPs also carry a hydroxyl group to be combined with the phosphate group of the next dNTPs to realize extension; in the sequencing-blocking primer of the present application, since the hydroxyl group at the 3' end is chemically modified as shown in fig. 5, extension cannot be achieved, and the sequencing-blocking primer functions. It is understood that the blocking sequencing primer of the present application is chemically modified according to the sequencing primer, and as to the specific sequencing primer sequence, the specific sequencing primer sequence of different sequencing platforms are different, but the method of the present application can be used for sequencing the Small RNA circular library of the mixed sample, and therefore, the sequences of the normal sequencing primer and the blocking sequencing primer are not specifically limited herein. For another example, in the case of Illumina sequencing platform, the principle is bridge amplification, and the primers are two primers designed based on I5 and I7 linkers, and the two primers are suitable for all platforms in the same series. The blocking primers of the two primers are designed based on the principle of the invention, and can also be used for an Illumina sequencing platform. In addition, the chemical modification has the functions of avoiding the combination of hydroxyl and phosphate groups and blocking extension, so that the existing hydroxyl modification method can be used for the application, for example, hydroxyl is eliminated and then hydrogenated, and the hydroxyl is replaced by H; if the hydroxyl is replaced by alkali metal, modifying-OH into-OY, wherein Y is alkali metal element; or the alkyl or other groups are adopted for substitution reaction, and the ether is obtained by dehydration; and is not particularly limited herein.
It should be further noted that the object of circular library sequencing is a DNA nanosphere, and the DNA nanosphere is a multi-copy chain-like chain to be sequenced, that is, there are a plurality of binding sites for normal sequencing primers on the sequencing template chain of the DNA nanosphere, and certainly there are a plurality of binding sites for blocking sequencing primers, and the blocking of random binding of the sequencing primers actually occupies a vacancy, because the blocking sequencing primers do not extend after binding to the sequencing template chain, and therefore, no covering is performed on the tag sequence thereafter, so that at least one copy of the tag sequence is retained, so that the tag sequence can be subjected to normal sequencing. Therefore, when the sequencing method is used for Small RNA sequencing of the annular library of the mixed sample, the tag sequence can be sequenced normally, the data utilization rate is effectively improved, and good resolution rate can be obtained, so that Small RNA sequencing of the annular library of the mixed sample is realized.
Preferably, the chemical modification comprises the replacement of the hydroxyl group as a whole or H in the hydroxyl group.
It should be noted that the chemical modification is to block the hydroxyl group at the 3' end to prevent the hydroxyl group from binding to a phosphate group, thereby achieving the purpose of blocking extension, and therefore, any chemical modification capable of reacting with a hydroxyl group can be used in the present application.
Preferably, the blocking sequencing primer accounts for 10% -70% of the total amount of the mixed primers.
It should be noted that the more the amount of the blocking sequencing primer is added, the more the sequencing data of the final tag primer, and correspondingly, the less the sequencing data of the Small RNA target region is blocked because the blocking sequencing primer occupies a gap. Therefore, in general, the specific dosage of the blocking sequencing primer can be determined according to the number of the target sequences, and more blocking sequencing primers can be added to obtain more sequencing data of the tag primer under the condition that more target sequences exist; conversely, if the target sequence is Small, the amount of the blocking sequencing primer added is Small in order to ensure sequencing data of the Small RNA target region. In this respect, the number of each sample in the mixed sample is large enough, and if the number is too Small, sufficient sequencing data of the Small RNA target region or sequencing data of the tag primer cannot be obtained. In addition, in general, the amount of the blocking sequencing primer is 10-70% of the total mixed primers, and the sequencing result is not influenced in the range.
The application also discloses a sequencing reagent for Small RNA, which comprises a blocking sequencing primer, wherein the blocking sequencing primer is formed by chemically modifying a hydroxyl group at the 3' terminal of a normal sequencing primer.
The key to the Small RNA sequencing method of the present application is to realize circular library sequencing of a mixed sample, and the blocking sequencing primer is used, so that a sequencing reagent containing the blocking sequencing primer is separately provided for convenience of use. Similarly, the key point of the blocking sequencing primer is that the hydroxyl group at the 3' end is chemically modified, and as for the specific sequence, the sequencing primer of each sequencing platform can be referred to, and the specific chemical modification can also refer to the existing hydroxyl modification scheme, which is not specifically limited herein.
Preferably, the chemical modification comprises the replacement of the hydroxyl group as a whole or H in the hydroxyl group.
Preferably, the sequencing reagent of the present application further comprises a normal sequencing primer, the normal sequencing primer is a primer in which the hydroxyl group at the 3' end 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.
The key point of the Small RNA sequencing method is that a normal sequencing primer and a blocking sequencing primer are added at the same time, so that the sequencing reagent of the Small RNA sequencing method simultaneously comprises the sequencing primer and the corresponding blocking sequencing primer for convenience in use. The normal sequencing primer and the blocking sequencing primer in the sequencing reagent of the application are corresponding, namely, the blocking sequencing primer is chemically modified by the hydroxyl at the 3' terminal of the normal sequencing primer. It can be understood that the normal sequencing primer and the blocking sequencing primer in the sequencing reagent can be matched according to a preset proportion, and a primer mixed solution is directly added when the sequencing reagent is used; or can be respectively and independently packaged according to the proportion, and the proportion can be flexibly adjusted according to the requirements when in use, and is not particularly limited.
In yet another aspect, the present application discloses a sequencing kit for Small RNA, comprising the sequencing reagents of the present application.
It is understood that a sequencing kit may comprise other reagents besides the sequencing reagents of the present application, such as buffers for sequencing reaction, dNTPs, etc., and is not limited herein.
Still another aspect of the present application discloses the use of the sequencing reagent of the present application or the sequencing kit of the present application in circular library sequencing of small fragment nucleic acid mixed samples.
It should be noted that the key to the sequencing of the circular library of Small RNA mixed samples is that Small RNA fragments are Small and read longer than the Small RNA fragments, so that most of the tag sequences are covered and tag sequence sequencing cannot be completed; based on the same principle, the sequencing method, the sequencing reagent or the sequencing kit are not limited to sequencing of the circular library of the Small RNA mixed sample, and the circular library sequencing of Small fragment nucleic acid mixed samples in other similar cases is also applicable. It is understood that a Small fragment nucleic acid sample, specifically a Small fragment nucleic acid sample with a fragment length smaller than the read length, includes, but is not limited to, small RNA.
The beneficial effect of this application lies in:
according to the Small RNA sequencing method, the Small RNA sequencing of the annular library of the mixed sample is realized by adding the blocking sequencing primer, the sequencing of the tag sequence can be effectively guaranteed, the data utilization rate is further improved, and the good splitting rate is obtained. The Small RNA sequencing method and the sequencing reagent provide a new idea and scheme for sequencing a Small RNA mixed sample, and solve the problems of sequencing resource waste and the like of the conventional circular library Small RNA sequencing.
Drawings
FIG. 1 is a graph of the statistical results of the quality values of various pooled samples sequenced using different sequencing methods in the examples of the present application;
FIG. 2 is a graph showing the statistical results of the number of effective nucleotide sequences obtained by sequencing different sequencing methods for each mixed sample in the examples of the present application;
FIG. 3 is a graph of the statistical results of the resolution ratio of each mixed sample when sequenced by different sequencing methods according to the example of the present application;
FIG. 4 is a schematic diagram of the structure of a normal sequencing primer without chemical modification at the 3' end of the primer;
FIG. 5 is a structural diagram of the blocked sequencing primer of the present application after chemical modification of the 3' end, wherein R is a modification group.
Detailed Description
Herein, the resolution ratio (SplitRate) means: can correspond to the number of sequences in the barcode list, as a ratio of the total fastq sequences.
The inventors of the present application have made extensive studies and experiments to propose that Small RNA sequencing of a circular library of mixed samples cannot sequence tag sequences well, but the key is that Small RNA has a short target region and the sequencing read length is larger than the target region, so that the tag sequence is covered at the set read length, which is set to obtain more data.
Based on the above knowledge, the inventors of the present application have creatively proposed that if some gaps can be reserved, i.e., some positions are not used for sequencing the target region, the tag sequences corresponding to these positions will not be covered, and the tag sequence sequencing can be normally performed, thereby solving the problem of tag sequence sequencing. In view of the above, the inventors of the present application further propose that a normal sequencing primer and a blocking sequencing primer can be added at the same time during sequencing, and both primers are randomly bound to a template strand, while the position where the blocking sequencing primer is bound does not undergo amplification extension, thereby playing a role of space occupation and reserving space for sequencing a tag sequence.
The present application will be described in further detail with reference to specific examples. The following examples are merely illustrative of the present application and should not be construed as limiting the present application.
Examples
In this example, small RNA was sequenced on a BGISEQ-500RS platform, a sequencer developed autonomously from Huada. The normal sequencing primer is a sequence shown in Seq ID No.1,
Seq ID No.1：5’-AAGT CGGA GGCC AAGC GGTC TTAG GAAG ACAA-3’
the blocking sequencing primer of the embodiment is formed by chemically modifying the 3' end of a normal sequencing primer of the sequence shown in Seq ID No.1, and the blocking sequencing primer of the embodiment which adopts-H substituted hydroxyl group and-H substituted hydroxyl group is synthesized by Shanghai.
Other reagents for sequencing adopted in the embodiment are derived from a library building kit and a single-end computer sequencing kit which are matched with the sequencer, and are hereinafter referred to as SE50 kits. Please refer to the application method of the BGISEQ-500RS platform in the procedures of sequencing instrument, reagent operation and the like used in the verification process.
The example adopts nucleic acid samples from three sources of human, saffron and mouse, which are combined in the following way to form a mixed sample, wherein human serum samples adopt serum samples from six different people respectively, and all the samples are provided by Huada gene storage:
i. the first group of test samples was derived from a pooled sample of mouse and human serum # 1, both added at a volume ratio of 1;
a second set of test samples was formed by equal volume mixing of human serum 1 from source No. two and No. three, labeled human serum 1;
the third group of test samples was formed by equal volume mixing of human serum 1 from source No. four and No. five, labelled human serum 2;
a fourth set of test samples was a mixed sample of saffron and human serum No. six;
and preparing the samples from different sources according to a BGISEQ-500RS library construction method and a principle of preparing the on-machine samples. The library construction kit adopts MGIEasy Small RNA library preparation kit (manufacturer: shenzhen Hua Da Zhi manufacturing science and technology, inc., abbreviated as "Hua Da Zhi manufacturing", english abbreviated as MGI), and the specific library construction process is detailed in the kit application instruction.
When sequencing reaction solution is prepared, the sequencing primers in the loading reagent plate in the SE50 kit are taken out and replaced by mixed primers of normal sequencing primers and the sequencing blocking primers, wherein in the mixed primers, the original normal sequencing primers account for 70% of the total mixed primers, and the sequencing blocking primers account for 30% of the total mixed primers. And then, preparing the chip according to a BGISEQ-500RS chip preparation mode, and performing machine sequencing.
For comparison, for the same circular library of mixed samples, the normal sequencing primer is directly used in this example, and the blocking sequencing primer is not added, and the mixed samples are sequenced by using the same sequencing platform and method. The conditions were the same except that no blocking sequencing primer was added.
In this example, four sets of test and four sets of comparative sequencing data were statistically analyzed, and the four sets of test and four sets of comparative sequencing quality values, effective nucleotide sequence numbers, and resolution were compared, and the statistical analysis was performed by basecall software carried by the sequencing platform. The results are shown in FIGS. 1 to 3. FIG. 1 is a graph of the statistical results of mass values for four tests and four comparative sequencing runs, where in FIG. 1 the abscissa is the different pooled samples, each pooled sample being subjected to the sequencing test and comparative sequencing run for this example; the ordinate is the quality value obtained when each sample is sequenced in a different manner; as can be seen from the figure, the same mixed sample does not reduce the sequencing quality when the sequencing method of the embodiment is adopted, and the same effect is obtained among different samples, namely the sequencing method of the embodiment does not influence the sequencing result; that is, blocking the addition of sequencing primers does not affect the normal sequencing quality. FIG. 2 is a graph showing the statistical results of the number of effective nucleotide sequences in four sets of tests and four sets of comparative sequencing, in FIG. 2, the abscissa represents different mixed samples, each of which is subjected to the sequencing test and comparative sequencing of the present example; the ordinate is the number of effective nucleic acid sequences obtained when each sample is subjected to different sequencing modes; as can be seen from the figure, the number of effective nucleic acid sequences obtained by the same mixed sample when the sequencing method of the embodiment is adopted is not obviously different, and the same effect is obtained among different samples, namely the sequencing method of the embodiment does not influence the sequencing result. FIG. 3 is a graph of the statistical results of the splitting for four sets of tests and four sets of comparative sequencing, in FIG. 3 the abscissa is the different mixed samples, each mixed sample is subjected to the sequencing test and comparative sequencing of the present example; the ordinate is the resolution ratio obtained when each sample is in different sequencing modes; as can be seen from the figure, in the sequencing method of the embodiment, the same mixed sample is obviously higher in resolution rate due to the addition of the blocking sequencing primer, namely different samples which are mixed together for sequencing can be distinguished, and the four groups of mixed samples are higher in resolution rate, so that the resolution problem of Small RNA mixed sample sequencing is effectively solved.
The foregoing is a more detailed description of the present application in connection with specific embodiments thereof, and it is not intended that the present application be limited to the specific embodiments thereof. For those skilled in the art to which the present application pertains, several simple deductions or substitutions can be made without departing from the concept of the present application, which should be considered as belonging to the protection scope of the present application.
Claims (7)
1. A sequencing reagent for Small RNA characterized by: the sequencing reagent comprises a blocking sequencing primer, and the blocking sequencing primer is formed by chemically modifying hydroxyl at the 3' tail end of a normal sequencing primer.
2. The sequencing reagent of claim 1, wherein: the chemical modification includes replacement of the hydroxyl group as a whole or H in the hydroxyl group.
3. The sequencing reagent of claim 1, wherein: the sequencing reagent also comprises a normal sequencing primer, the blocking sequencing primer accounts for 10-70% of the total amount of the mixed primer, and the mixed primer is obtained by mixing the blocking sequencing primer and the normal sequencing primer.
4. The sequencing reagent of claim 3, wherein: the normal sequencing primer is a primer of which the hydroxyl at the 3' tail end of the blocking sequencing primer is not chemically modified.
5. A sequencing kit for Small RNA, characterized in that: the sequencing kit contains the sequencing reagent of any one of claims 1-4.
6. Use of the sequencing reagent of any one of claims 1 to 4 or the sequencing kit of claim 5 for circular library sequencing of small fragment nucleic acid mixed samples.
7. Use according to claim 6, characterized in that: the small fragment is 18-30 nt.
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