CN113718023A - Prokaryotic ribonucleic acid direct sequencing method based on bovine nanopore sequencing technology - Google Patents
Prokaryotic ribonucleic acid direct sequencing method based on bovine nanopore sequencing technology Download PDFInfo
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- CN113718023A CN113718023A CN202110965797.9A CN202110965797A CN113718023A CN 113718023 A CN113718023 A CN 113718023A CN 202110965797 A CN202110965797 A CN 202110965797A CN 113718023 A CN113718023 A CN 113718023A
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Abstract
The invention discloses a prokaryotic ribonucleic acid direct sequencing method based on a bovine nanopore sequencing technology, which comprises the following steps: the method comprises the following steps: adding a poly A (polyA) sequence at the 3' end of the prokaryotic RNA; step two: constructing an RNA direct sequencing library; step three: sequencing using a nanopore sequencer; the invention belongs to the field of biotechnology, in particular to a prokaryotic ribonucleic acid direct sequencing method based on a bovine nanopore sequencing technology, which utilizes escherichia coli poly (A) polymerase to indiscriminately add poly (A) sequences at the 3' end of prokaryotic ribonucleic acid (RNA), thereby enabling the prokaryotic ribonucleic acid direct sequencing reagent to be used for library construction and realizing final direct sequencing. The problem that the Oxford nanopore ribonucleic acid (RNA) direct sequencing kit cannot be adapted to direct sequencing of ribonucleic acid (RNA) without poly A is solved, and the application range of the ribonucleic acid (RNA) direct sequencing kit is expanded.
Description
Technical Field
The invention belongs to the field of biotechnology, and particularly relates to a prokaryotic ribonucleic acid direct sequencing method based on a bovine nanopore sequencing technology.
Background
Sequencing ribonucleic acid (RNA) in biological samples allows for a wealth of information, including bacterial and viral identity, subtleties in alternative splicing, or transcriptional status of the organism. As the importance of ribonucleic acid (RNA) has become increasingly recognized, researchers have also developed a variety of methods to study it. However, most current "ribonucleic acid (RNA) sequencing" techniques only produce relatively short read lengths, and most methods still require reverse transcription of ribonucleic acid (RNA) into complementary deoxyribonucleic acid (cDNA), which not only introduces errors and preferences, but also is inefficient and prevents efficient characterization of the structural complexity of the ribonucleic acid (RNA) itself.
Ribonucleic acid (RNA) sequencing technologies based on second-generation high-throughput sequencing and the American Pacific biology third-generation sequencing platform complete sequencing after reverse transcription of messenger ribonucleic acid (mRNA) into complementary deoxyribonucleic acid (cDNA) and amplification, so that certain sequence preference exists and direct identification of complexity of the messenger ribonucleic acid (mRNA) cannot be realized. The Oxford nanopore technology can directly sequence natural full-length ribonucleic acid (RNA) molecules due to a unique sequencing principle, fundamentally breaks through the limitations of short reading length and reverse transcription or amplification preference in the prior art, and can directly obtain base modification information on a ribonucleic acid (RNA) sequence.
Oxford nanopore sequencing technology can provide a highly flexible and extensible long read-length ribonucleic acid (RNA) sequencing method, i.e., deoxyribonucleic acid (DNA), ribonucleic acid (RNA), proteins, small molecules and the like can cause changes of ionic currents when passing through protein nanopores, and a sensor completes sequencing by recording the changes of the ionic currents. The unique sequencing principle of the Oxford nanopore technology enables the sequencing process to be free from enzymatic synthesis reaction, so that the direct sequencing of deoxyribonucleic acid (DNA)/ribonucleic acid (RNA) molecules becomes a reality. In the sequencing process, the kit for direct sequencing of ribonucleic acid (RNA) provides the only method for sequencing the full-length non-amplified ribonucleic acid (RNA) strand, without conversion to complementary deoxyribonucleic acid (cDNA), and analysis of sequence and base modifications can be performed. The oxford nanopore sequencing technology-direct ribonucleic acid (RNA) sequencing technology has advantages in the identification and functional characterization of new ribonucleic acid (RNA) isomers and ribonucleic acid (RNA) base modifications. However, the direct ribonucleic acid (RNA) sequencing kit proposed by oxford nanopore sequencing technologies at present only adapts ribonucleic acid (RNA) sequencing with poly a (polyA) sequence at the 3' end because the sequence of its built-in component, reverse transcription linker (RTA), only carries a thymine repeat oligonucleotide (oligo dT) sequence. This procedure cannot be used directly for ribonucleic acid samples such as prokaryotic ribonucleic acid (RNA) and viral ribonucleic acid (RNA) that do not contain poly A (poly A) sequences at their 3' ends.
Disclosure of Invention
In order to overcome the defects of the prior art, the prokaryotic ribonucleic acid direct sequencing method based on the bovine nanopore sequencing technology provided by the invention adds a poly (A) sequence to the 3' end of prokaryotic ribonucleic acid (RNA) by using Escherichia coli poly (A) polymerase without difference, so that the prokaryotic ribonucleic acid (RNA) direct sequencing reagent can be used for library construction and final direct sequencing is realized. The problem that the Oxford nanopore ribonucleic acid (RNA) direct sequencing kit cannot be adapted to direct sequencing of ribonucleic acid (RNA) without poly A is solved, and the application range of the ribonucleic acid (RNA) direct sequencing kit is expanded.
The technical scheme adopted by the invention is as follows: the invention relates to a prokaryotic ribonucleic acid direct sequencing method based on a bovine nanopore sequencing technology, which comprises the following steps:
the method comprises the following steps: addition of PolyA (polyA) sequence to the 3' end of prokaryotic RNA
Preparing a reaction system according to table 1 by using escherichia coli poly (A) polymerase (NEB, M0276), adding a poly (A) sequence at the 3' end of total RNA obtained from prokaryotic escherichia coli DH10B, preparing the finished reaction system, incubating for 30 minutes at 37 ℃, directly adding an ethylene diamine tetraacetic acid (EDTA-Na) solution with the final concentration of 10mM after the reaction is finished, stopping the reaction, and adding 24 mu l of RNA purification magnetic beads (Novozam, N412-01) into the system which finishes the reaction for purification;
table 1: component table of reaction system of Escherichia coli poly (A) polymerase
Wherein, each component is provided with a final reaction system with a total volume of 20uL according to the corresponding volume in the table;
step two: RNA direct sequencing library construction
The RNA sample with the 3' end added with poly A (polyA) sequence is constructed by using a commercial RNA direct sequencing kit (cat # SQK-RNA002) of Oxford nanopore company, and the concrete reaction steps are as follows:
(1) ligating reverse transcription linker-RTA to the end of the RNA obtained in the first step using T4 DNA ligase (NEB, M0202);
(2) continuing to perform reverse transcription by using SuperScript III reverse transcriptase (Thermo Fisher Scientific,18080044), and finally obtaining an RNA-DNA hybrid double-stranded product;
(3) finally, connecting the RNA-DNA hybrid double-stranded structure with a sequencing adaptor-RMX for sequencing by using T4 DNA ligase (NEB, M0202) to complete the construction of the pre-machine library;
step three: sequencing Using a nanopore sequencer
The completed sequencing library was loaded into a nanopore chip (PromethION r9.4.1flow Cell), sequenced using a nanopore sequencer PromethION 48 and data collected.
The invention with the structure has the following beneficial effects: the scheme is based on a prokaryotic ribonucleic acid (RNA) direct sequencing method of a bovine nanopore sequencing technology, overcomes the defects of the existing RNA direct sequencing process, provides a prokaryotic ribonucleic acid (RNA) direct sequencing scheme which is suitable for a prokaryotic ribonucleic acid (RNA) without a poly A (poly A) sequence at the 3' end, and greatly expands the sample application range of the RNA direct sequencing.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the attached tables in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments; all other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention relates to a prokaryotic ribonucleic acid direct sequencing method based on a bovine nanopore sequencing technology, which comprises the following steps:
the method comprises the following steps: addition of PolyA (polyA) sequence to the 3' end of prokaryotic RNA
Preparing a reaction system according to table 1 by using escherichia coli poly (A) polymerase (NEB, M0276), adding a poly (A) sequence at the 3' end of total RNA obtained from prokaryotic escherichia coli DH10B, preparing the finished reaction system, incubating for 30 minutes at 37 ℃, directly adding an ethylene diamine tetraacetic acid (EDTA-Na) solution with the final concentration of 10mM after the reaction is finished, stopping the reaction, and adding 24 mu l of RNA purification magnetic beads (Novozam, N412-01) into the system which finishes the reaction for purification;
table 1: component table of reaction system of Escherichia coli poly (A) polymerase
Wherein, each component is provided with a final reaction system with a total volume of 20uL according to the corresponding volume in the table;
step two: RNA direct sequencing library construction
The RNA sample with the 3' end added with poly A (polyA) sequence is constructed by using a commercial RNA direct sequencing kit (cat # SQK-RNA002) of Oxford nanopore company, and the concrete reaction steps are as follows:
(1) ligating reverse transcription linker-RTA to the end of the RNA obtained in the first step using T4 DNA ligase (NEB, M0202);
(2) continuing to perform reverse transcription by using SuperScript III reverse transcriptase (Thermo Fisher Scientific,18080044), and finally obtaining an RNA-DNA hybrid double-stranded product;
(3) finally, connecting the RNA-DNA hybrid double-stranded structure with a sequencing adaptor-RMX for sequencing by using T4 DNA ligase (NEB, M0202) to complete the construction of the pre-machine library;
step three: sequencing Using a nanopore sequencer
The completed sequencing library was loaded into a nanopore chip (PromethION r9.4.1flow Cell), sequenced using a nanopore sequencer PromethION 48 and data collected.
Through the above steps, we finally obtained high quality data with 6.14Gb average quality value (QC Score) of 9.5, sequencing read length N50 index of 1,068bp, and average sequencing read length (reads) of 959.1bp, and the data quality control analysis is shown in Table 2.
Thus, the implementation of this scheme successfully obtained prokaryotic E.coli RNA direct sequencing data.
Table 2: statistical table of direct sequencing data of total RNA of Escherichia coli DH10B
The off-line data are analyzed by Quality Control (QC) to obtain the following general data indexes: average sequencing read length, average mass value, sequencing read length N50, total data amount are presented separately in the table.
Examples
The total RNA obtained from nonpathogenic prokaryotic bacteria Salmonella enterica serovar Cerro 87 and Escherichia coli JM109 is used, the experiment scheme is used for respectively adding a poly (A) sequence to the 3' end of the total RNA to construct an RNA direct sequencing library, the same type of nanopore sequencing chip and a sequencer are used for sequencing, the result is shown in Table 3 after the data are subjected to quality control analysis, the results show that the two cases of prokaryotic bacteria RNA respectively obtain 6.07Gb and 7.02Gb high-quality data after the direct sequencing, and all quality indexes accord with expectations, so that the scheme can really and successfully realize the direct sequencing of the prokaryotic RNA.
Table 3: statistical Table of direct sequencing results for Salmonella enterica serovar Cerro 87 and Escherichia coli JM109
The general data indexes of the offline data after Quality Control (QC) analysis are average sequencing read length, average quality value and sequencing read length N50, and the total data amount is respectively presented in the table.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
The present invention and its embodiments have been described above, but the description is not limitative, and the actual structure is not limited thereto. In summary, those skilled in the art should appreciate that they can readily use the disclosed conception and specific embodiments as a basis for designing or modifying other structures for carrying out the same purposes of the present invention without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (1)
1. A prokaryotic ribonucleic acid direct sequencing method based on a bovine nanopore sequencing technology is characterized by comprising the following steps: the method comprises the following steps:
the method comprises the following steps: addition of PolyA (polyA) sequence to the 3' end of prokaryotic RNA
Using Escherichia coli poly (A) polymerase (NEB, M0276), configuring a reaction system according to a component table of an Escherichia coli poly (A) polymerase reaction system, adding a poly (A) sequence to the 3' end of total RNA obtained from prokaryotic Escherichia coli DH10B, configuring the completed reaction system, incubating at 37 ℃ for 30 minutes, directly adding a solution of ethylenediamine tetraacetic acid (EDTA-Na) with the final concentration of 10mM after the reaction is completed to stop the reaction, and adding 24 mu l of RNA purification magnetic beads (Norzany, N412-01) into the system which finishes the reaction to purify;
step two: RNA direct sequencing library construction
The RNA sample with the 3' end added with poly A (polyA) sequence is constructed by using a commercial RNA direct sequencing kit (cat # SQK-RNA002) of Oxford nanopore company, and the concrete reaction steps are as follows:
1) ligating reverse transcription linker-RTA to the end of the RNA obtained in the first step using T4 DNA ligase (NEB, M0202);
2) continuing to perform reverse transcription by using SuperScript III reverse transcriptase (Thermo Fisher Scientific,18080044), and finally obtaining an RNA-DNA hybrid double-stranded product;
3) finally, connecting the RNA-DNA hybrid double-stranded structure with a sequencing adaptor-RMX for sequencing by using T4 DNA ligase (NEB, M0202) to complete the construction of the pre-machine library;
step three: sequencing Using a nanopore sequencer
The completed sequencing library was loaded into a nanopore chip (PromethION r9.4.1flow Cell), sequenced using a nanopore sequencer PromethION 48 and data collected.
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