CN114921534B - RNA long fragment targeted sequencing method with high targeting efficiency and data yield - Google Patents
RNA long fragment targeted sequencing method with high targeting efficiency and data yield Download PDFInfo
- Publication number
- CN114921534B CN114921534B CN202210547190.3A CN202210547190A CN114921534B CN 114921534 B CN114921534 B CN 114921534B CN 202210547190 A CN202210547190 A CN 202210547190A CN 114921534 B CN114921534 B CN 114921534B
- Authority
- CN
- China
- Prior art keywords
- sequencing
- cdna
- reaction
- rna
- kong
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A90/00—Technologies having an indirect contribution to adaptation to climate change
- Y02A90/10—Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation
Abstract
The invention provides a high-targeting efficiency and data-yield RNA long fragment targeted sequencing method, and belongs to the field of RNA sequencing. In the RNA long fragment targeted sequencing method, the terminal transferase is utilized to introduce the dideoxyribonucleotide at the 3' -end of the DNA, so that the condition that a sequencing hole is occupied by a joint in the sequencing process is almost completely eliminated, and the data yield is obviously improved. The RNA long fragment targeted sequencing method overcomes the problems of the sequencing method in the prior art, has the advantages of long reading length, high data yield and high targeting efficiency, reduces the sequencing cost, is suitable for targeted sequencing of RNA long fragments, and has wide application prospect.
Description
Technical Field
The invention belongs to the field of RNA sequencing, and particularly relates to an RNA long fragment targeted sequencing method with high targeted efficiency and data yield.
Background
Microorganisms that are pathogenic to humans and animals are referred to as pathogenic microorganisms, also known as pathogens. These pathogenic microorganisms can cause infection, allergy, tumor, dementia and other diseases, and are one of the main factors that endanger food safety. In recent years, SARS, highly pathogenic avian influenza, west Nile virus infection and other diseases have extremely strong infectivity, so that the detection of pathogens must be fast and accurate. The conventional etiology detection method has the advantages of complex operation, long detection period and higher requirements on the technical level of operators. With the continuous development of medical microbiology research technology, etiology diagnosis is no longer limited to pathogen level, and detection means penetrating into molecular level and gene level are continuously developed and applied to clinic and laboratory. The gene fragments of pathogenic microorganisms are specific, and the specific gene fragment sequences can be detected to identify pathogenic microorganisms, unlike other species or genus. With the development of technology, gene detection technology gradually replaces other detection technologies, and becomes a mainstream detection technology of pathogens in clinical laboratory and basic laboratories.
With the development of genomics technology, high-throughput sequencing technology (next-generation sequencing, NGS) is increasingly widely applied to various aspects of tracing, detection, typing, drug resistance evaluation and the like of infectious diseases, and rapidly develops towards a rapid and economic direction. The general process comprises extracting sample nucleic acid sequence, performing polymerase chain reaction amplification of specific gene target or not, constructing sequencing library, detecting sequence, comparing the detected nucleic acid sequence with reference gene or genome sequence of target pathogen, and judging whether the pathogen exists according to the set detection threshold. However, the existing NGS platform has severe requirements on laboratory environment, the sample preparation process is relatively complex, the sequencing operation time is longer, the read length is shorter, and the data analysis can only be performed on high-performance computing equipment after the sequencing is completed, so that the field detection application of the platform in non-laboratory environment is limited.
Nano Kong Ba sequencing technology (nanopore Cas9-targeted sequencing, nCATS) is a representative gene detection technology that is independent of traditional microbial culture and can rapidly and objectively detect suspected pathogenic microorganisms (including bacteria, fungi, and viruses) in clinical samples. The detection project is based on a third generation nanopore sequencing technology (nanopore sequencing) platform, and is a new generation single molecule real-time sequencing technology, which uses single molecule DNA to sequence by presuming base composition through current change of a biological nanopore. The main characteristics are that the reading length is longer, the average reading length can reach 20kb, and the maximum reading length can reach Mb level; by combining efficient genome nucleic acid extraction and library establishment, sequencing results are compared and analyzed with a professional medical microorganism database after bioinformatics processing and analysis, so that species information of all microorganisms in a sample and carried drug resistance and virulence gene information can be obtained. The method has wide detection range, can efficiently detect pathogenic microorganisms in various clinical samples such as venous blood, alveolar lavage fluid, cerebrospinal fluid, sputum and other body fluids and tissues, and is suitable for diagnosis of fever, difficult infection and acute critical diseases caused by unknown reasons. However, on the one hand, nCATS is a method of sequencing for the genome, which is not suitable for transcriptome sequencing; on the other hand, nCATS also has problems of low data yield and low targeting efficiency.
Therefore, there is a need to develop a long-fragment targeted sequencing method for RNA with long read length, high data yield and high targeting efficiency.
Disclosure of Invention
The invention aims to provide a long-fragment targeted sequencing method for RNA, which has long reading length, high data yield and high targeting efficiency.
The invention provides a nano Kong Ba sequencing method of RNA, which comprises the following steps:
(1) taking a sample to be detected, extracting RNA, and constructing cDNA;
(2) ligating a dideoxyribonucleotide to the 3' terminus of the cDNA using a terminal transferase;
(3) adding alkaline phosphatase into the product obtained in the last step, reacting, and incubating at high temperature after the reaction to inactivate the alkaline phosphatase;
(4) adding a Cas9-gRNA complex of a target sequence into the system after the reaction in the last step, and reacting;
(5) connecting the nanopore sequencing joint, and sequencing by using a nanopore sequencing platform.
Further, in the step (1), the cDNA is obtained by reverse transcription and PCR amplification of RNA;
and/or, the RNA is total RNA.
Further, in the step (2), the method for ligating the dideoxyribonucleotide to the 3' -terminus of the cDNA using the terminal transferase comprises the steps of: the cDNA, the dideoxyribonucleotide, the cobalt salt, the terminal transferase and the reaction buffer are mixed and reacted, and after the reaction, the terminal transferase is inactivated by incubation at high temperature.
Further, the dideoxyribonucleotide is ddATP, ddCTP, ddGTP or ddTTP, preferably ddGTP;
the cobalt salt is CoCl 2 ；
The ratio of cDNA, dideoxyribonucleotide, cobalt salt, terminal transferase is (1-3) μg: (3-7) nmol: (10-15) nmol: (6-14) U, preferably 2. Mu.g: 5nmol:12.5nmol:10U;
the reaction time is 1-3h, preferably 2h, and the temperature is 35-40 ℃, preferably 37 ℃;
the incubation time at the high temperature is 10-30min, preferably 20min, and the temperature is 65-85 ℃, preferably 75 ℃.
Further, in the step (3), the reaction time is 20-40min, preferably 30min, and the temperature is 35-40 ℃, preferably 37 ℃;
the incubation time at the high temperature is 3-8min, preferably 5min, and the temperature is 70-90 ℃, preferably 80 ℃;
the ratio of cDNA to alkaline phosphatase was (1-3) μg: (10-20) U, preferably 2. Mu.g: 15U.
Further, in step (4), the ratio of Cas9-gRNA complex to cDNA is (8-12) pmol: (1-3) μg, preferably 10pmol:2 μg;
the reaction time is 10-20min, preferably 15min, and the temperature is 35-40 ℃, preferably 37 ℃.
Further, in step (5), the method of ligating nanopore sequencing adaptors comprises the steps of: adding dATP and DNA polymerase into the system after the reaction in the last step, reacting, then adding a joint connection buffer solution, a nanopore sequencing joint, T4 DNA ligase and water, and reacting to obtain a sequence connected with the nanopore sequencing joint.
Further, in step (5), the method for sequencing using the nanopore sequencing platform comprises the following steps: adding a sequencing buffer solution and microbeads into the sequence connected with the nanopore sequencing connector, uniformly mixing, adding into a nanopore sequencing chip, and sequencing on a nanopore sequencer.
Further, the following step X is included between the step (4) and the step (5): adding thermolabile protease into the system after the reaction in the step (4), reacting, and incubating at high temperature after the reaction to inactivate the thermolabile protease.
Further, in step X, the thermolabile Protease is a Qiagen Protease;
the ratio of thermolabile protease to cDNA is (0.5-2.5) mAU: (1-3) μg, preferably 1.5mAU:2 μg.
AU is the protease activity unit, 1,000mAU is 1AU.
The reaction time is 2-8min, preferably 5min, and the temperature is 45-65 ℃, preferably 56 ℃;
the incubation time at the high temperature is 10-20min, preferably 15min, and the temperature is 60-80 ℃, preferably 70 ℃.
In the RNA long fragment targeted sequencing method, the terminal transferase is utilized to introduce the dideoxyribonucleotide at the 3' -end of the DNA, so that the condition that a sequencing hole is occupied by a joint in the sequencing process is almost completely eliminated, and the data yield is obviously improved.
On the basis of introducing dideoxyribonucleotide at the 3' -end of DNA by utilizing terminal transferase, the invention further increases thermolabile protease treatment after cutting a target sequence by utilizing Cas9, and discovers that the sequencing method can efficiently acquire sequence information of a target region, and compared with the existing method, the targeting efficiency and data yield are obviously improved.
The RNA long fragment targeted sequencing method overcomes the problems of the sequencing method in the prior art, has the advantages of long reading length, high data yield and high targeting efficiency, reduces the sequencing cost, is suitable for targeted sequencing of RNA long fragments, and has wide application prospect.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Drawings
FIG. 1 is a schematic diagram of the RNA long fragment targeted sequencing method in example 2.
FIG. 2 is a comparison of the percent of sequencing adaptors for the sequencing method of example 1 (shown as "experimental set" in the figures), the sequencing method of control 1 (shown as "control 1" in the figures), and the sequencing method of control 2 (shown as "control 2" in the figures).
FIG. 3 is a comparison of the relative data yields of the sequencing method of example 1 (denoted as "experimental set" in the figure), the sequencing method of control example 1 (denoted as "control 1" in the figure), and the sequencing method of control example 2 (denoted as "control 2" in the figure).
FIG. 4 shows a comparison of the status of sequencing wells of the sequencing method of example 2 (denoted as "the present invention"), the sequencing method of control example 1 (denoted as "the original method" in the figure), and the sequencing method of control example 3 (denoted as "whole RNA sequencing" in the figure).
FIG. 5 is a comparison of the relative data yields of the sequencing method of example 2 (denoted as "the present invention" in the figure), the sequencing method of control example 1 (denoted as "the original method" in the figure), and the sequencing method of control example 3 (denoted as "the whole RNA sequencing" in the figure).
FIG. 6 is a comparison of targeting efficiencies of the sequencing method of example 2 (denoted as "the present invention" in the figure), the sequencing method of control example 1 (denoted as "the original method" in the figure), and the sequencing method of control example 3 (denoted as "full RNA sequencing" in the figure).
FIG. 7 is a comparison of the relative effective data yields of the sequencing method of example 2 (denoted as "the present invention" in the figure), the sequencing method of control example 1 (denoted as "the original method" in the figure), and the sequencing method of control example 3 (denoted as "the whole RNA sequencing" in the figure).
Detailed Description
The raw materials and equipment used in the invention are all known products and are obtained by purchasing commercial products.
Example 1: RNA long fragment targeted sequencing method
1 construction of full Length cDNA library
Extracting total RNA, generating cDNA by template conversion technology and PCR amplification, and specifically performing the following steps:
total RNA extraction:
taking 5×10 6 Cells were resuspended in 1mL TRIzol (Invitrogen, 15596026). After 5min incubation at room temperature 200. Mu.L chloroform was added and mixed rapidly. Centrifuge at 12,000g for 10min at 4℃and collect the supernatant. To the collected liquid was added 500. Mu.L of isopropyl alcohol, and after rapid mixing, RNA was precipitated by incubation at room temperature for 10 min. After centrifugation at 12,000g for 10min at 4℃the liquid was removed. 1mL of 75% ethanol was added to suspend the precipitate to rinse the precipitate. After centrifugation at 7,500g at 4℃for 5min, the liquid was removed thoroughly. The pellet was dried at room temperature with the pellet edge transparent and 20. Mu.L of nuclease-free water was addedThe precipitate was dissolved and the liquid was the extracted RNA. The concentration of RNA was measured using Qubit and RNA was stored at-80℃for further use.
Reverse transcription:
into a 12. Mu.L reaction system, 2. Mu.g of RNA extracted from the experimental material, 2. Mu.L of reverse transcription primer (sequence 5' -ACGAGCATCAGCAGCATACGAT) at a concentration of 10. Mu.M were added 30 VN-3' (SEQ ID NO: 1)), 2. Mu.L of dNTP at a concentration of 10mM and water were mixed well. Immediately after reaction at 65℃for 5min, it was placed on ice. Then, 4. Mu.L of reverse transcription reaction buffer 5 XRT buffer (Thermo Scientific, EP 0751), 1. Mu.L of RNaseOUT (Invitrogen, 10777019), 2. Mu.L of template transfer primer (sequence 5 '-AGAGACAGATTGCGCAATGRGRG-3' (SEQ ID NO: 2), rG being ribonucleotide) at a concentration of 10. Mu.M, and 1. Mu.L of reverse transcriptase Maxima H-minus reverse transcriptase (Thermo Scientific, EP 0751) were added to the above reaction mixture to make the total reaction system 20. Mu.L and mixed. The reaction was performed according to the following temperature procedure:
and (3) PCR amplification:
to 50. Mu.L of the reaction system were added 25. Mu.L of DNA polymerase VAHTS HiFi Amplification Mix (Vazyme, N616), 6. Mu.L of the reverse transcription product obtained in the previous step, 5. Mu.L of 5. Mu.M concentration of amplification primer (SEQ ID NO: 3), 5'-ACGAGCATCAGCAGCATACGA-3' (SEQ ID NO: 4)) and water and mixed well. The reaction was performed according to the following temperature procedure:
2 adding dideoxyribonucleotides to the 3' -end of cDNA using terminal transferase
Into a 50. Mu.L system, 2. Mu.g of cDNA, 0.5. Mu.L of ddGTP (Roche, 03732738001) at a concentration of 10mM, 5. Mu.L of CoCl at a concentration of 2.5mM were added 2 (NEB, M0315), 5. Mu.L of 10 Xterminal transferase reaction buffer (NEB, M0315) and 0.5. Mu.L of terminal transferase (NEB, M0315, 2)0U/. Mu.L), at 37℃for 2h, followed by incubation at 75℃for 20min to inactivate the terminal elongase. The reacted cDNA was then purified.
The cDNA purification steps are as follows: 40. Mu.L of AMPure XP magnetic beads (Beckman, A63881) are added into the reacted system and mixed uniformly, incubated at room temperature for 10min, placed on a magnetic rack, and the liquid is removed after the magnetic beads are completely adsorbed on the magnetic rack. The sample was held on a magnetic rack, and after rinsing with 200 μl of 70% ethanol for 30s, the liquid was removed, and this step was repeated once. And (3) uncovering the cover, drying the magnetic beads at room temperature, taking out the sample from the magnetic rack when the magnetic beads are dried until the surface is frosted, and adding 25 mu L of water to resuspend the magnetic beads. After incubating for 10min at room temperature, placing the sample on a magnetic rack, and collecting liquid after the magnetic beads are completely adsorbed on the magnetic rack, wherein the liquid is the purified sample.
3 removal of the phosphate group at the 5' -end of cDNA
To a 30. Mu.L system were added 24. Mu.L of the cDNA purified in the previous step, 3. Mu.L of reaction Buffer Cutsmart Buffer (NEB, B7204) and 3. Mu.L of alkaline phosphatase (NEB, M0525, 5U/. Mu.L), reacted at 37℃for 30min, followed by incubation at 80℃for 5min to inactivate the alkaline phosphatase.
4 cleavage of target sequence with Cas9
mu.L (10 pmol total) of Cas9-gRNA complex targeting the target sequence was added to the previous reaction mixture and reacted at 37℃for 15min to cleave the target DNA.
Wherein, cas9-gRNA complex assembly process is as follows:
mu.L of tracrRNA (GenScript) at a concentration of 100. Mu.M, 1. Mu.L of a crRNA mixture (specific recognition sequence 5'-UCCCAAAGUGCUGGGAUUAC-3' (SEQ ID NO: 5), 5'-GCCUCGGCCUCCCAAAGUGC-3' (SEQ ID NO: 6), 5'-UCUGAGAUCAAACUGCAAGG-3' (SEQ ID NO: 7), 5'-UUUCAUCCAUGUCCCUACAA-3' (SEQ ID NO: 8)) at a concentration of 100. Mu.L and 8. Mu.L of a Buffer Cutsmart Buffer (NEB, B7204) were mixed, incubated at 95℃for 5min and then at room temperature for 5min for the formation of gRNA; mu.L of gRNA was taken and added to 1.4. Mu.L of Buffer Cutsmart Buffer (NEB, B7204), 9.4. Mu.L of water and 1.2. Mu.L of five-fold diluted Cas9 protein HiFi Cas9 nucleic V3 (IDT, 1081060) and mixed well, and incubated at room temperature for 20min to assemble Cas9-gRNA complexes. The assembled Cas9-gRNA complex was 1 μm in concentration and placed on ice for use.
5 adding a sequencing linker and sequencing
To the previous reaction mixture, 1. Mu.L of dATP (NEB, N0440) at a concentration of 10mM and 1. Mu.L of DNA polymerase (Vazyme, P101-d 1-AC) were added and reacted at 72℃for 5min. Then, 20uL of the adaptor ligation buffer 4 XLNB (ONT, SQK-LSK 110), 3.5 uL of the nanopore sequencing adaptor AMX-F (ONT, SQK-LSK 110), 10 uL of T4 DNA ligase (NEB, M2200) and water were added to make the total reaction system 80 uL and mixed well. The reaction was performed at room temperature for 10min to allow ligation of the sequencing adaptors, and then the DNA was purified.
The DNA purification steps are as follows: to the reacted system were added 80. Mu.L of water and 64. Mu.L of AMPure XP magnetic beads (Beckman, A63881) and mixed well. Incubating for 10min at room temperature, placing on a magnetic rack, and removing liquid after the magnetic beads are completely adsorbed on the magnetic rack. The sample was removed from the magnetic rack, 250 μlsfb (ONT, SQK-LSK 110) was added to resuspend the beads, the sample was placed on the magnetic rack, the liquid was removed after the beads were fully adsorbed on the magnetic rack, and the rinsing step was repeated once. The beads were dried at room temperature for 30s after uncapping, and then 17. Mu.L of elution buffer EB (ONT, SQK-LSK 110) was added and mixed well. After incubation for 10min at room temperature, the sample is placed on a magnetic rack, and after the magnetic beads are completely adsorbed on the magnetic rack, liquid is collected, wherein the liquid is the purified sample and is used for the next step of on-machine sequencing.
To the purified samples were added 37.5. Mu.L of sequencing buffer SQB (ONT, SQK-LSK 110) and 20.5. Mu.L of Loading Beads (ONT, SQK-LSK 110) and mixed well. This mixture was added to the nanopore sequencing chip, minion flow cell R9.4.1, and sequenced on the nanopore sequencer Gridion.
Example 2: RNA long fragment targeted sequencing method
1 construction of full Length cDNA library
A cDNA was obtained in the same manner as in step 1 of example 1.
2 adding dideoxyribonucleotides to the 3' -end of cDNA using terminal transferase
Into a 50. Mu.L system, 2. Mu.g of cDNA, 0.5. Mu.L of ddGTP (Roche, 03732738001) at a concentration of 10mM, 5. Mu.L of CoCl at a concentration of 2.5mM were added 2 (NEB, M0315), 5. Mu.L of 10X terminal transferaseThe reaction buffer (NEB, M0315) and 0.5. Mu.L of terminal transferase (NEB, M0315, 20U/. Mu.L) were reacted at 37℃for 2h, followed by incubation at 75℃for 20min to inactivate the terminal elongase. The reacted cDNA was then purified.
The cDNA purification steps are as follows: 40. Mu.L of AMPure XP magnetic beads (Beckman, A63881) are added into the reacted system and mixed uniformly, incubated at room temperature for 10min, placed on a magnetic rack, and the liquid is removed after the magnetic beads are completely adsorbed on the magnetic rack. The sample was held on a magnetic rack, and after rinsing with 200 μl of 70% ethanol for 30s, the liquid was removed, and this step was repeated once. And (3) uncovering the cover, drying the magnetic beads at room temperature, taking out the sample from the magnetic rack when the magnetic beads are dried until the surface is frosted, and adding 25 mu L of water to resuspend the magnetic beads. After incubating for 10min at room temperature, placing the sample on a magnetic rack, and collecting liquid after the magnetic beads are completely adsorbed on the magnetic rack, wherein the liquid is the purified sample.
3 removal of the phosphate group at the 5' -end of cDNA
To a 30. Mu.L system were added 24. Mu.L of the cDNA purified in the previous step, 3. Mu.L of reaction Buffer Cutsmart Buffer (NEB, B7204) and 3. Mu.L of alkaline phosphatase (NEB, M0525, 5U/. Mu.L), reacted at 37℃for 30min, followed by incubation at 80℃for 5min to inactivate the alkaline phosphatase.
4 cleavage of target sequence with Cas9
mu.L (10 pmol total) of Cas9-gRNA complex targeting the target sequence was added to the previous reaction mixture and reacted at 37℃for 15min to cleave the target DNA.
5 removal of Cas9 protein with thermolabile proteases
To the previous reaction mixture was added 1.5. Mu.L of the thermolabile Protease Qiagen Protease (QIAGEN, 19155,1mAU/. Mu.L), reacted at 56℃for 5min, followed by incubation at 70℃for 15min to inactivate the Protease.
6 adding sequencing adapter and sequencing
To the previous reaction mixture, 1. Mu.L of dATP (NEB, N0440) at a concentration of 10mM and 1. Mu.L of DNA polymerase (Vazyme, P101-d 1-AC) were added and reacted at 72℃for 5min. Then, 20uL of the adaptor ligation buffer 4 XLNB (ONT, SQK-LSK 110), 3.5 uL of the nanopore sequencing adaptor AMX-F (ONT, SQK-LSK 110), 10 uL of T4 DNA ligase (NEB, M2200) and water were added to make the total reaction system 80 uL and mixed well. The reaction was performed at room temperature for 10min to allow ligation of the sequencing adaptors, and then the DNA was purified.
The DNA purification steps are as follows: to the reacted system were added 80. Mu.L of water and 64. Mu.L of AMPure XP magnetic beads (Beckman, A63881) and mixed well. Incubating for 10min at room temperature, placing on a magnetic rack, and removing liquid after the magnetic beads are completely adsorbed on the magnetic rack. The sample was removed from the magnetic rack, 250 μlsfb (ONT, SQK-LSK 110) was added to resuspend the beads, the sample was placed on the magnetic rack, the liquid was removed after the beads were fully adsorbed on the magnetic rack, and the rinsing step was repeated once. The beads were dried at room temperature for 30s after uncapping, and then 17. Mu.L of elution buffer EB (ONT, SQK-LSK 110) was added and mixed well. After incubation for 10min at room temperature, the sample is placed on a magnetic rack, and after the magnetic beads are completely adsorbed on the magnetic rack, liquid is collected, wherein the liquid is the purified sample and is used for the next step of on-machine sequencing.
To the purified samples were added 37.5. Mu.L of sequencing buffer SQB (ONT, SQK-LSK 110) and 20.5. Mu.L of Loading Beads (ONT, SQK-LSK 110) and mixed well. This mixture was added to the nanopore sequencing chip, minion flow cell R9.4.1, and sequenced on the nanopore sequencer Gridion.
The following is a control sequencing method.
Comparative example 1: sequencing method for directly applying nCATS to cDNA
1 construction of full Length cDNA library
A cDNA was obtained in the same manner as in step 1 of example 1.
2 removal of the phosphate group at the 5' -end of cDNA
To a 30. Mu.L system, 2. Mu.g of cDNA, 3. Mu.L of reaction Buffer Cutsmart Buffer (NEB, B7204) and 3. Mu.L of alkaline phosphatase (NEB, M0525, 5U/. Mu.L) were added, reacted at 37℃for 30min, followed by incubation at 80℃for 5min to inactivate the alkaline phosphatase.
3 cleavage of target sequence by Cas9
mu.L (10 pmol total) of Cas9-gRNA complex targeting the target sequence was added to the previous reaction mixture and reacted at 37℃for 15min to cleave the target DNA.
4 adding a sequencing linker and sequencing
To the previous reaction mixture, 1. Mu.L of dATP (NEB, N0440) at a concentration of 10mM and 1. Mu.L of DNA polymerase (Vazyme, P101-d 1-AC) were added and reacted at 72℃for 5min. Then, 20uL of the adaptor ligation buffer 4 XLNB (ONT, SQK-LSK 110), 3.5 uL of the nanopore sequencing adaptor AMX-F (ONT, SQK-LSK 110), 10 uL of T4 DNA ligase (NEB, M2200) and water were added to make the total reaction system 80 uL and mixed well. The reaction was performed at room temperature for 10min to allow ligation of the sequencing adaptors, and then the DNA was purified.
The DNA purification steps are as follows: to the reacted system were added 80. Mu.L of water and 64. Mu.L of AMPure XP magnetic beads (Beckman, A63881) and mixed well. Incubating for 10min at room temperature, placing on a magnetic rack, and removing liquid after the magnetic beads are completely adsorbed on the magnetic rack. The sample was removed from the magnetic rack, 250 μlsfb (ONT, SQK-LSK 110) was added to resuspend the beads, the sample was placed on the magnetic rack, the liquid was removed after the beads were fully adsorbed on the magnetic rack, and the rinsing step was repeated once. The beads were dried at room temperature for 30s after uncapping, and then 17. Mu.L of elution buffer EB (ONT, SQK-LSK 110) was added and mixed well. After incubation for 10min at room temperature, the sample is placed on a magnetic rack, and after the magnetic beads are completely adsorbed on the magnetic rack, liquid is collected, wherein the liquid is the purified sample and is used for the next step of on-machine sequencing.
To the purified samples were added 37.5. Mu.L of sequencing buffer SQB (ONT, SQK-LSK 110) and 20.5. Mu.L of Loading Beads (ONT, SQK-LSK 110) and mixed well. This mixture was added to the nanopore sequencing chip, minion flow cell R9.4.1, and sequenced on the nanopore sequencer Gridion.
Comparative example 2: control sequencing method
The sequencing method of reference example 1 differs only in that ddGTP in step 2 is replaced with dGTP.
Comparative example 3: total RNA sequencing method
The procedure for constructing the full-length cDNA library was the same as in step 1 of example 1, and the subsequent sequencing method was performed according to the procedure recommended by the authorities of ONT company, specifically as follows:
1 construction of full Length cDNA library
A cDNA was obtained in the same manner as in step 1 of example 1.
2cDNA end repair
400ng of cDNA, 3.5. Mu.L of DNA repair buffer FFPE DNA Repair Buffer (NEB, M6630), 2. Mu.L of DNA repair enzyme NEBNext FFPE DNA repair Mix (NEB, M6630), 3.5. Mu.L of end repair buffer NEBNext Ultra II End Prep Reaction Buffer (NEB, E7546) and 3. Mu.L of end repair enzyme NEBNext Ultra II End Prep Enzyme Mix (NEB, E7546) were added to a 60. Mu.L system and mixed well. The reaction was carried out at 20℃for 30min, followed by incubation at 65℃for 5min to inactivate the enzyme.
Purifying the DNA product after the reaction. The method comprises the following specific steps: adding 48 mu LAMPure XP magnetic beads (Beckman, A63881) into the reacted system, uniformly mixing, incubating at room temperature for 10min, placing on a magnetic frame, and removing liquid after the magnetic beads are completely adsorbed on the magnetic frame; holding the sample on a magnetic rack, adding 200 mu L of 70% ethanol, rinsing for 30s, removing liquid, and repeating the rinsing step once; the magnetic beads are dried at room temperature after the cover is opened, when the magnetic beads are dried until the surfaces are frosted, the sample is taken out from the magnetic rack, 60 mu L of water is added and mixed uniformly, and the magnetic beads are incubated for 10min at room temperature; and finally, placing the sample on a magnetic rack, and collecting liquid after the magnetic beads are completely adsorbed on the magnetic rack, wherein the liquid is the purified sample.
3 adding a sequencing linker and sequencing
200ng of the end-repaired cDNA, 25uL of linker ligation buffer 4 XLNB (ONT, SQK-LSK 110), 5uL of nanopore sequencing linker AMX-F (ONT, SQK-LSK 110) and 10 uL of T4 DNA ligase (NEB, M2200) were added to the 100uL reaction system and mixed well. The reaction was performed at room temperature for 10min to allow ligation of the sequencing adaptors, followed by purification of the DNA.
The DNA purification steps are as follows: to the reacted system, 40. Mu.L of AMPure XP beads (Beckman, A63881) were added and mixed well. Incubating for 10min at room temperature, placing on a magnetic rack, and removing liquid after the magnetic beads are completely adsorbed on the magnetic rack. The sample was removed from the magnetic rack, 250 μl of SFB (ONT, SQK-LSK 110) was added to resuspend the beads, the sample was placed on the magnetic rack, the liquid was removed after the beads were completely adsorbed on the magnetic rack, and the rinsing step was repeated once. The beads were dried at room temperature for 30s after uncapping, and then 17. Mu.L of elution buffer EB (ONT, SQK-LSK 110) was added and mixed well. After incubating for 10min at room temperature, placing the sample on a magnetic rack, collecting liquid after the magnetic beads are completely adsorbed on the magnetic rack, and detecting the DNA concentration by using Qubit. This liquid was a purified sample and was used for the next step on-machine sequencing.
100ng of sequencing sample was taken and 37.5. Mu.L of sequencing buffer SQB (ONT, SQK-LSK 110) and a volume of Loading Beads (ONT, SQK-LSK 110) were added to make the total volume 75. Mu.L and mixed. This mixture was added to the nanopore sequencing chip, minion flow cell R9.4.1, and sequenced on the nanopore sequencer Gridion.
The following experiments prove the beneficial effects of the invention.
Experimental example 1: effect verification of reducing sequencing adaptors by adding dideoxyribonucleotides to the 3' end of cDNA using terminal transferase
1. Experimental method
Grouping: the experimental set shows the sequencing method of example 1; control 1 represents the sequencing method of control 1, omitting the terminal transferase treatment step compared to the experimental group; control 2 represents the sequencing method of control 2, compared to the experimental group in which the dideoxyribonucleotide in step 2 was replaced by a deoxyribonucleotide.
The percentage of adaptors during sequencing and relative data yield were compared for three sets of experiments:
sequencing linker percentages represent the percentage of time a nanopore is occupied by a sequencing linker during sequencing as a percentage of the total nanopore sequencing time.
Relative data yield refers to the data yield relative to control 1, calculated as the data yield of each group divided by the data yield of control 1, respectively, such that the data yield of control 1 is normalized to 1.
2. Experimental results
As a result, as shown in fig. 2, in control 1, the sequencing linker percentage reached 67.6% during sequencing by only dephosphorylating the 5 'end of DNA without blocking the 3' end of DNA, which has significantly exceeded the normal range of sequencing linker ratios in nanopore sequencing (normal range less than 10%). The 3' -end of the DNA was extended with terminal transferase in control 2 to be protruded and the dideoxyribonucleotide was added to the 3' -end of the DNA with terminal transferase in experimental group to have no more 3' -hydroxyl group for ligation reaction, which significantly reduced the sequencing linker ratio, the sequencing linker percentage of control 2 was 1.4% and the sequencing linker percentage of experimental group was 1.2%, indicating that both control 2 and experimental group methods can effectively reduce the sequencing linker ratio and that the reduction effect of experimental group was better.
However, in terms of data yield (as shown in fig. 3), the data yield in control 2 was significantly reduced compared to control 1, while the data yield in the experimental group was improved by a factor of 2.8.
The experimental result shows that in the RNA long fragment targeted sequencing method, the dideoxyribonucleotide is introduced into the 3' -end of DNA by utilizing terminal transferase, so that the condition that a sequencing hole is occupied by a connector in the sequencing process is almost completely eliminated, and the data yield is obviously improved.
Experimental example 2: compared with the existing sequencing method, the RNA long fragment targeted sequencing method of the embodiment 2 of the invention
1. Experimental method
Grouping: "the invention" means the sequencing method of example 2; "original method" means the sequencing method of comparative example 1; "Total RNA sequencing" refers to the sequencing method of control example 3.
The nanopore status, relative data yield, effective data yield and targeting efficiency of the three experiments were compared.
The nanopore state is largely divided into three types, namely "free", "sequencing adapter", "DNA". "free" means that the nanopore is in a usable state, but not occupied by DNA; "sequencing linker" means: the nanopore is occupied by a sequencing linker; "DNA" means: the nanopore is occupied by DNA, which is being sequenced. High quality sequencing experiments mean that the nanopore state composition is dominated by "DNA", with less "idle" and "sequencing adaptors.
Relative data yield refers to data yield relative to the "original method".
The effective data yield refers to the amount of data containing the target sequence, so the effective data yield relative to the "original method" is the data of each set divided by the "original method".
The target efficiency calculating method comprises the following steps: reads containing the target sequence are a percentage of all reads output.
2. Experimental results
In nanopore sequencing, the occupation of the nanopores of the DNA being sequenced is more than 70%, which means that the sequencing experiment is good in effect, and the data output can reach the normal level of a sequencing chip in theory. The total RNA sequencing method of comparative example 3 is established well, and can achieve good effects on nanopore sequencing state and data yield under the condition of no error of experimental operation, so the method is used as a positive control for measuring the parameters. As shown in FIG. 4, the average percentage of "DNA" nanopores for the total RNA sequencing method of control 3 was 92.0%, indicating that the experiment was operating properly.
As shown in fig. 4, compared with the sequencing method of comparative example 1 in which the nCATS was directly applied to the cDNA, the situation that the sequencing wells were occupied by the adaptors and the sequencing wells were in an idle state was greatly reduced in the RNA long fragment targeted sequencing method of the present invention, and the nanometer Kong Zhanbi of the sequenced DNA was increased from 7.0% to 93.0% of the method of comparative example 1, which is equivalent to 92.0% of the positive control.
As shown in FIG. 5, the data yield of the RNA long fragment targeted sequencing method of the invention is improved by 39.3 times compared with that of the control example 1 on average, and the data yield is equivalent to that of a positive control.
In targeted sequencing, the absolute ideal targeting efficiency theoretical value is 100%. As shown in FIG. 6, in the total RNA sequencing method of comparative example 3, the reads with the target sequence account for 10.4% of all sequencing reads, and the targeting efficiency is only 10.4%; comparative example 1 the targeting efficiency of the sequencing method directly applying nCATS to cDNA was 59.0%; the targeting efficiency of the RNA long fragment targeted sequencing method is up to 89.5%. Compared with the sequencing method of directly applying nCATS to cDNA in comparative example 1, the targeting efficiency of the sequencing method is remarkably improved, and the effective data yield is improved by 59.7 times (FIG. 7).
The above results demonstrate that the RNA long fragment targeted sequencing method of the embodiment 2 of the present invention can efficiently obtain sequence information of a target region, and compared with the existing method, the method has significantly improved targeting efficiency and data yield.
In summary, the invention provides a RNA long fragment targeted sequencing method with high targeting efficiency and data yield, and belongs to the field of RNA sequencing. In the RNA long fragment targeted sequencing method, the terminal transferase is utilized to introduce the dideoxyribonucleotide at the 3' -end of the DNA, so that the condition that a sequencing hole is occupied by a joint in the sequencing process is almost completely eliminated, and the data yield is obviously improved. The RNA long fragment targeted sequencing method overcomes the problems of the sequencing method in the prior art, has the advantages of long reading length, high data yield and high targeting efficiency, reduces the sequencing cost, is suitable for targeted sequencing of RNA long fragments, and has wide application prospect.
SEQUENCE LISTING
<110> Huaxi Hospital at university of Sichuan
<120> an RNA long fragment targeted sequencing method with high targeting efficiency and data yield
<130> GYKH2189-2022P0115185CCR4
<160> 8
<170> PatentIn version 3.5
<210> 1
<211> 24
<212> DNA
<213> artificial sequence
<220>
<221> misc_feature
<222> (24)..(24)
<223> n is a, c, g, t or u
<400> 1
acgagcatca gcagcatacg at30vn 24
<210> 2
<211> 19
<212> DNA
<213> artificial sequence
<400> 2
agagacagat tgcgcaatgrgrgrg 19
<210> 3
<211> 19
<212> DNA
<213> artificial sequence
<400> 3
agagacagat tgcgcaatg 19
<210> 4
<211> 21
<212> DNA
<213> artificial sequence
<400> 4
acgagcatca gcagcatacg a 21
<210> 5
<211> 20
<212> DNA
<213> artificial sequence
<400> 5
<210> 6
<211> 20
<212> DNA
<213> artificial sequence
<400> 6
<210> 7
<211> 20
<212> DNA
<213> artificial sequence
<400> 7
<210> 8
<211> 20
<212> DNA
<213> artificial sequence
<400> 8
Claims (12)
1. A method for nano Kong Ba directional sequencing of RNA, characterized by: the method comprises the following steps:
(1) taking a sample to be detected, extracting RNA, and constructing cDNA;
(2) ligating a dideoxyribonucleotide to the 3' terminus of the cDNA using a terminal transferase; the dideoxyribonucleotide is ddGTP;
(3) adding alkaline phosphatase into the product obtained in the last step, reacting, and incubating at 70-90 ℃ after the reaction to inactivate the alkaline phosphatase;
(4) adding a Cas9-gRNA complex of a target sequence into the system after the reaction in the last step, and reacting;
x: adding thermolabile protease into the system after the reaction in the step (4), reacting, and incubating at 60-80 ℃ after the reaction to inactivate the thermolabile protease; in step X, the thermolabile Protease is Qiagen Protease, and the ratio of the thermolabile Protease to cDNA is (0.5-2.5) mAU: (1-3) μg, wherein the reaction time is 2-8min, the temperature is 45-65 ℃, and the incubation time is 10-20 min;
(5) connecting the nanopore sequencing joint, and sequencing by using a nanopore sequencing platform.
2. The nano Kong Ba sequencing method according to claim 1, wherein: in the step (1), the cDNA is obtained by reverse transcription and PCR amplification of RNA;
and/or, the RNA is total RNA.
3. The nano Kong Ba sequencing method according to claim 1, wherein: in step (2), the method for ligating a dideoxyribonucleotide to the 3' -terminus of a cDNA using a terminal transferase comprises the steps of: the cDNA, the dideoxyribonucleotide, the cobalt salt, the terminal transferase and the reaction buffer are mixed and reacted, and after the reaction, the terminal transferase is inactivated by incubation at 65-85 ℃.
4. The nano Kong Ba sequencing method according to claim 3, wherein: the cobalt salt is CoCl 2 ；
The ratio of cDNA, dideoxyribonucleotide, cobalt salt, terminal transferase is (1-3) μg: (3-7) nmol: (10-15) nmol: (6-14) U;
the reaction time is 1-3h, and the temperature is 35-40 ℃;
the incubation time is 10-30 min.
5. The nano Kong Ba sequencing method according to claim 4, wherein: the ratio of cDNA, dideoxyribonucleotide, cobalt salt, terminal transferase was 2. Mu.g: 5nmol:12.5nmol:10U is provided;
the reaction time is 2h, and the temperature is 37 ℃;
the incubation time was 20min and the temperature was 75 ℃.
6. The nano Kong Ba sequencing method according to claim 1, wherein: in the step (3), the reaction time is 20-40min, and the temperature is 35-40 ℃;
the incubation time is 3-8min, and the temperature is 70-90 ℃;
the ratio of cDNA to alkaline phosphatase was (1-3) μg: (10-20) U.
7. The nano Kong Ba sequencing method according to claim 6, wherein: in the step (3), the reaction time is 30min, and the temperature is 37 ℃;
the incubation time is 5min, and the temperature is 80 ℃;
the ratio of cDNA to alkaline phosphatase was 2. Mu.g: 15U, U.
8. The nano Kong Ba sequencing method according to claim 1, wherein: in step (4), the ratio of Cas9-gRNA complex to cDNA is (8-12) pmol: (1-3) μg;
the reaction time is 10-20 minn, and the temperature is 35-40 ℃.
9. The nano Kong Ba sequencing method according to claim 8, wherein: in step (4), the ratio of Cas9-gRNA complex to cDNA is 10pmol: 2.μg;
the reaction time was 15min and the temperature was 37 ℃.
10. The nano Kong Ba sequencing method according to claim 1, wherein: in step (5), the method of ligating nanopore sequencing adaptors comprises the steps of: adding dATP and DNA polymerase into the system after the reaction in the last step, reacting, then adding a joint connection buffer solution, a nanopore sequencing joint, T4 DNA ligase and water, and reacting to obtain a sequence connected with the nanopore sequencing joint.
11. The nano Kong Ba sequencing method according to claim 10, wherein: in step (5), the method for sequencing by using the nanopore sequencing platform comprises the following steps: adding a sequencing buffer solution and microbeads into the sequence connected with the nanopore sequencing connector, uniformly mixing, adding into a nanopore sequencing chip, and sequencing on a nanopore sequencer.
12. The nano-Kong Ba sequencing method according to any one of claims 1 to 11, wherein: in step X, the ratio of thermolabile protease to cDNA is 1.5mAU: 2.μg;
the reaction time is 5min, and the temperature is 56 ℃;
the incubation time was 15min and the temperature was 70 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210547190.3A CN114921534B (en) | 2022-05-19 | 2022-05-19 | RNA long fragment targeted sequencing method with high targeting efficiency and data yield |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210547190.3A CN114921534B (en) | 2022-05-19 | 2022-05-19 | RNA long fragment targeted sequencing method with high targeting efficiency and data yield |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114921534A CN114921534A (en) | 2022-08-19 |
| CN114921534B true CN114921534B (en) | 2023-05-30 |
Family
ID=82808556
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210547190.3A Active CN114921534B (en) | 2022-05-19 | 2022-05-19 | RNA long fragment targeted sequencing method with high targeting efficiency and data yield |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114921534B (en) |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019161904A1 (en) * | 2018-02-23 | 2019-08-29 | Kamel Wael Sayed Saleh Elsayed | Stable rna molecules |
| GB201808554D0 (en) * | 2018-05-24 | 2018-07-11 | Oxford Nanopore Tech Ltd | Method |
-
2022
- 2022-05-19 CN CN202210547190.3A patent/CN114921534B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN114921534A (en) | 2022-08-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2016082129A1 (en) | Method and reagent for constructing nucleic acid double-linker single-strand cyclic library | |
| CN106987585B (en) | A kind of single stranded DNA two generations sequencing library construction method for cfDNA | |
| CN108251504A (en) | A kind of method and kit of supper-fast structure genomic DNA sequencing library | |
| CN108517567B (en) | Adaptor, primer group, kit and library construction method for cfDNA library construction | |
| CN113046475B (en) | Primer composition and kit for rapidly detecting mutant novel coronavirus | |
| WO2017143866A1 (en) | Kit and method for quantitative detection of dna methylation in rprm genes | |
| CN113322523B (en) | RNA rapid library construction method and application thereof | |
| CN116287357A (en) | Respiratory tract pathogenic bacteria detection kit based on targeted amplicon sequencing | |
| CN115354070A (en) | Method for detecting nucleic acid based on microspheres | |
| CN114921534B (en) | RNA long fragment targeted sequencing method with high targeting efficiency and data yield | |
| WO2023207909A1 (en) | Crispr-based nucleic acid detection kit and use thereof | |
| TW201321520A (en) | Method and system for virus detection | |
| CN114921535B (en) | RNA long fragment targeted sequencing method | |
| CN112410465A (en) | Novel coronavirus SARS-CoV-2ORF1ab and N gene constant temperature amplification primer group and kit | |
| CN113151406B (en) | FMR1 gene CGG repetition number and methylation detection kit and detection method | |
| CN112522792B (en) | Construction method of RNA sequencing library | |
| CN112063696B (en) | Logic loop based on nucleic acid autocatalysis and application thereof | |
| CN113046415A (en) | Construction method and application of RNA sequencing library | |
| CN112501166A (en) | Chemically modified high-stability RNA, kit and method | |
| JPH099967A (en) | Nucleic acid synthesis | |
| CN113667714A (en) | Target area capturing method, kit and sequencing method | |
| CN111926394A (en) | Database building method and detection kit based on metagenomics | |
| CN110541040B (en) | Method for detecting methylation level by using ARMS-PCR technology, primer and kit thereof | |
| CN115058493B (en) | DNA probe for multiple nucleic acid detection, CRISPR-reverse dot hybridization nucleic acid detection system and application | |
| CN115725792B (en) | Universal avian leukosis virus nucleic acid detection kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |