CN112646868A - Method for detecting pathogenic molecules based on nanopore sequencing - Google Patents
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Abstract
The invention belongs to the technical field of biology, and discloses a method for detecting pathogenic molecules based on nanopore sequencing, which comprises the following steps: obtaining a nasopharyngeal swab sample or a nasopharyngeal swab sample; extracting and purifying nucleic acid from the obtained nasopharyngeal swab or nasopharyngeal swab sample; preparing cDNA; amplifying and purifying cDNA; preparing and sequencing a nanopore library; and (5) performing bioinformatics analysis to obtain a pathogen molecule detection result. The invention can directly detect the pathogenic molecules based on clinical samples, has high sequencing speed and can carry out real-time monitoring; meanwhile, the sensitivity is high, and the detection limit is low. The addition of the universal joint and the one-step PCR amplification during the construction of reverse transcription of the nanopore library can realize the unbiased amplification of all sequences in a clinical sample, truly reflect the composition and abundance of pathogenic molecules in the sample, and more importantly, greatly improve the sensitivity and accuracy of detection.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for detecting pathogenic molecules based on nanopore sequencing.
Background
At present: influenza is a major global public health threat because of its highly pathogenic variants, large zoonotic pool and pandemic potential. Genomic virus sequencing offers the possibility to provide insight into the diagnostic testing of influenza viruses as to transmission, evolution and resistance, while detecting other viruses.
Most of the current clinical diagnostic tests for influenza viruses rely on antigen detection of respiratory viral nucleic acids or PCR amplification of the virus; antigen testing is generally rapid, but less sensitive; while PCR is more time consuming, but more sensitive. However, regardless of the test results, most clinical diagnostic devices report non-quantitative (binary) diagnostic results, and the ability of data routinely generated for influenza diagnosis provides limited insight into epidemiological associations, vaccine efficacy, or susceptibility to antiviral drugs.
Through the above analysis, the problems and defects of the prior art are as follows: the existing pathogenic molecule detection methods have low sensitivity and long detection time, and the data capability for influenza diagnosis provides limited knowledge on epidemiological associations, vaccine efficacy or susceptibility to antiviral drugs.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a method for detecting pathogenic molecules based on nanopore sequencing.
The invention is realized in such a way that a method for detecting pathogenic molecules based on nanopore sequencing comprises the following steps:
step one, obtaining a nasopharyngeal swab or a nasopharyngeal swab sample; RNA nucleic acid extraction and purification are carried out on the obtained nasopharyngeal swab or nasopharyngeal swab sample;
preparing cDNA by a single primer amplification method independent of sequence to prepare reverse-transcribed cDNA with a universal joint;
amplifying and purifying the cDNA, namely performing PCR amplification on a cDNA random fragment by using the cDNA as a template and a joint sequence as a primer, and purifying a PCR product by using AMPure XP magnetic beads;
step four, preparing and sequencing a nanopore sequencing library: constructing a technical nanopore sequencing library by using a library based on a bar code, wherein the total sequencing DNA amount of each sample is kept equal; adding the prepared nanopore sequencing library into a nanopore sequencing chip and performing on-machine sequencing
Step five, bioinformatics analysis of pathogenic microorganisms: and (3) carrying out quantitative analysis on the sequence obtained by sequencing, and then carrying out comparison on a pathogenic microorganism database to obtain the relative abundance and composition of pathogenic microorganisms, namely the detection result of pathogenic molecules.
Further, in the first step, the extracting nucleic acid from the obtained nasopharyngeal swab sample or nasopharyngeal swab sample comprises:
(1) taking a fresh nasopharyngeal swab or a nasopharyngeal swab sample into a 1.5ml centrifuge tube without RNase; simultaneously, adding 800 mu L of physiological saline into the centrifuge tube by using a sterile RNA enzyme-free pipette tip, rinsing for 20 seconds, drying liquid on a swab by sticking the wall of the centrifuge tube, centrifuging for 2min at 16,000rpm, discarding 700 mu L of supernatant, and fully oscillating and uniformly mixing the residual 100 mu L of supernatant;
(2) adding 200 μ L lysate and 20 μ L digestive juice, shaking, mixing, and water-bathing for 10min at 56 deg.C;
(3) adding 500 μ L of the precipitation solution, and slightly reversing and mixing;
(4) transferring the solution into an adsorption column with a collecting pipe, standing for 2min, centrifuging at 4 ℃ and 12000rpm for 1min, and discarding waste liquid in the collecting pipe;
(5) adding 500 μ L of washing solution into adsorption column, centrifuging at 4 deg.C and 12000rpm for 1min, and discarding the waste liquid in the collection tube;
(6) placing the adsorption column back into the collection tube, centrifuging at 12000rpm for 2min at 4 deg.C;
(7) taking out the adsorption column, putting into a new 1.5mL RNase-free centrifuge tube, adding 30-50 μ L RNase-free water, standing for 3min, centrifuging at 4 deg.C and 12000rpm for 2min, and collecting RNA solution to obtain RNA nucleic acid of pathogenic microorganism in the sample;
(8) RNA of the pathogenic microorganism to be extracted was purified using RNA Clean & concentrate-5 (Zymo Research) kit.
Further, in the step (1), the centrifugal force is 16000rpm, and the centrifugal time is 2 min. Further, the lysis solution is one of guanidinium isothiocyanate, lithium iodide, citric acid and Tween 20.
Further, in step two, the preparation of the cDNA comprises:
(1) mu.l of RNA and 1. mu.l of primer A (5 '-GTTTCCCACTGGAGGATA-N9-3', 40 pmol/. mu.l) were mixed and incubated at 65 ℃ for 5min, then cooled to room temperature.
(2) To the above product was added 2. mu.L of SuperScript IV first-strand buffer (Thermo Fisher), 1. mu.L of 12.5mM trinucleoside diphosphate (dNTPs), 0.5. mu.L of 0.1M Dithiothreitol (DTT), 1. mu. L H2O and 0.5 mu L SuperScript IV Reverse Transcriptase (Thermo Fisher) Reverse Transcriptase are incubated at 42 ℃ for 10min, and cDNA of pathogenic microorganisms in the sample is obtained.
Further, in step three, the amplification and purification of the cDNA comprises:
(1) configuration of amplification System for cDNA: mu.L of the above cDNA template was added with 5. mu.L of AccuTaq LA (Sigma) reaction mix and 1. mu.L of primer B (5'-GTTTCCCACTGGAGGATA-3') for cDNA amplification.
(2) PCR amplification conditions: 30s at 98 ℃, 30 cycles (94 ℃ for 15s, 50 ℃ for 20s, 68 ℃ for 2min), and 10min at 68 ℃.
(3) The amplification products were purified using AMPure XP magnetic beads (Beckman Coulter, Brea, CA) and quantified using the Qubit high sensitivity double stranded DNA (dsDNA) kit (Thermo Fisher).
Further, in step four, the preparation and sequencing of the nanopore sequencing library comprises:
(1) and repairing and dA tail adding through a NEBNext terminal repairing/dA tail adding module, purifying S-dsDNA-A by using AMPure XP magnetic beads, connecting the purified S-dsDNA-A in different samples with corresponding bar codes, and purifying by using AMPure XP magnetic beads again to obtain the nanopore sequencing library.
(2) And loading the prepared nanopore sequencing library into a nanopore sequencing chip, and sequencing by using a MinION sequencing platform.
Further, in step five, the bioinformatics analysis of the pathogenic microorganisms includes:
firstly, filtering and sequencing a linker sequence, reads with a filtering quality value of less than 7 and reads with a filtering length of less than 500bp on nanopore sequencing data; meanwhile, mapping clean data to a reference genome, and carrying out statistics on comparison efficiency, whole genome reads coverage and sequencing depth;
secondly, carrying out overall comparison quality evaluation on nanopore sequencing data, judging that the third-generation data is abnormal, and carrying out downstream analysis if the comparison efficiency is higher than 70% and the whole genome coverage is higher than 50%; mining information of within-aligned reads and split reads based on the comparison result;
then, defining structural variation and typing based on reads support information; integrating the variation types of the multi-sample data; meanwhile, predicting the base type and quality value of the maximum possibility of the variation position of the nanopore sequencing data based on the integration result;
finally, the plurality of variant fragments are integrated into one read length based on the most probable base type and quality values.
Further, in step five, the bioinformatics analysis includes:
step one, establishing a database containing 300 Refseq standard viruses, and classifying the obtained sequences according to the Refseq database;
secondly, mapping the sequence obtained by sequencing to the selected reference sequence, determining the nucleotide at each position by using a generated consistent sequence draft, and obtaining a negotiated consistent draft sequence;
thirdly, performing BLAST search on a virus sequence database; reading the remapped reference sequence; the percentage and the depth of coverage of the mapping are read.
Another object of the present invention is to provide a terminal for implementing the nanopore sequencing-based pathogenic molecule detection method.
The invention also aims to provide an application of the pathogenic molecule detection method based on nanopore sequencing in detection of influenza viruses.
By combining all the technical schemes, the invention has the advantages and positive effects that: the invention can directly detect the pathogenic molecules based on clinical samples, has high sequencing speed and can carry out real-time monitoring; meanwhile, the sensitivity is high, and the detection limit is low. According to the nanopore library construction method, the library is constructed through a single primer PCR amplification method independent of sequences, joint sequencing can be directly added, the abundance of all pathogenic microorganisms in a sample is improved, more importantly, the pathogenic molecule composition and abundance in the sample are more truly reflected, and the accuracy/goodness of fit is high.
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In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiments of the present application will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present application, and it is obvious for those skilled in the art that other drawings can be obtained from the drawings without creative efforts.
Fig. 1 is a flowchart of a method for detecting pathogenic molecules based on nanopore sequencing according to an embodiment of the present invention.
FIG. 2 is a flow chart of a method for extracting nucleic acid from a captured nasopharyngeal swab sample or a nasopharyngeal swab sample according to an embodiment of the present invention.
FIG. 3 is a flow chart of the steps for amplification and purification of cDNA provided in the examples of the present invention.
FIG. 4 is a flow chart of nanopore library construction provided by an embodiment of the invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Aiming at the problems in the prior art, the invention provides a method for detecting pathogenic molecules based on nanopore sequencing, and the invention is described in detail below with reference to the accompanying drawings.
As shown in fig. 1, the method for detecting pathogenic molecules based on nanopore sequencing provided by the embodiment of the present invention includes:
s101: obtaining a nasopharyngeal swab or nasopharyngeal swab sample; extracting and purifying nucleic acid from the obtained nasopharyngeal swab or nasopharyngeal swab sample;
s102: preparing reverse-transcribed cDNA with a universal linker by a single primer amplification method independent of sequence;
s103: amplifying and purifying cDNA, namely performing PCR amplification of a cDNA random fragment by using the cDNA as a template and a joint sequence as a primer, and purifying a PCR product by using AMPure XP magnetic beads;
s104: preparing a nanopore sequencing library and sequencing: constructing a technical nanopore sequencing library by using a library based on a bar code, wherein the total sequencing DNA amount of each sample is kept equal; adding the prepared nanopore sequencing library into a nanopore sequencing chip and performing machine sequencing;
s105: bioinformatics analysis of pathogenic microorganisms: and (3) carrying out quantitative analysis on the sequence obtained by sequencing, and then carrying out comparison on a pathogenic microorganism database to obtain the relative abundance and composition of pathogenic microorganisms, namely the detection result of pathogenic molecules.
As shown in fig. 2, in step S101, the nucleic acid extraction from the acquired nasopharyngeal swab sample or nasopharyngeal swab sample according to the embodiment of the present invention includes:
s201, taking a fresh pharyngeal swab or a nasopharyngeal swab sample into a 1.5ml centrifuge tube, and adding the hazara virus into the centrifuge tube; simultaneously, 300ul of physiological saline and 50ul of protease treatment solution are added into the centrifuge tube by using a sterile pipette tip, uniformly mixed by shaking for 5min, and kept stand in water bath at 60 ℃ for 5 min;
s202, putting 50-200ul of sample treatment solution into a 1.5ml centrifuge tube, adding 320ul of lysis solution, 310ul of isopropanol and 25ul of nano magnetic beads into the centrifuge tube by using an aseptic pipette tip, uniformly mixing for 2min by shaking, and standing for 15min at room temperature;
s203, placing the centrifuge tube on a magnetic frame for 40S, and after the nano magnetic beads are completely absorbed to the tube wall, absorbing and discarding the supernatant solution by using a sterile pipette tip; adding 800ul of 300 plus cleaning solution into the centrifuge tube by using the sterile pipette tip, shaking and uniformly mixing the centrifuge tube for 2min, placing the centrifuge tube on a magnetic frame for 40s, and after the nano magnetic beads are completely adsorbed to the tube wall, absorbing and discarding the supernatant solution by using the sterile pipette tip;
s204, repeating the step S203 once, and then opening the cover to dry for 25min at room temperature until no liquid remains in the tube; adding 100-;
s205, shaking and uniformly mixing the centrifugal tube for 2min, placing the centrifugal tube on a magnetic frame for 40S, and after the nano magnetic beads are completely absorbed to the tube wall, absorbing the supernatant to a new centrifugal tube by using a sterile pipette tip, thereby obtaining the pathogen nucleic acid in the sample.
In step S201, the concentration of the protease treatment solution provided by the embodiment of the present invention is 60 mg/ml; the protease treatment solution comprises proteinase K.
The lysis solution provided by the embodiment of the invention is one of guanidinium isothiocyanate, lithium iodide, citric acid and Tween 20.
As shown in fig. 3, in step S102, preparing a nanopore library according to an embodiment of the present invention includes:
s301, obtaining a nasopharyngeal swab or a nasopharyngeal swab sample, centrifuging the sample, discarding supernatant, and carrying out resuspension and centrifugation on the obtained precipitate to prepare a resuspension solution;
s302, adding saponin with the concentration of 0.36% into the resuspension, and incubating for 20 mim; after the incubation is finished, adding 60U/mL HL-SAN enzyme, incubating for 10-25 min, and performing DNA degradation;
s303, adding a PBS buffer solution into the degraded sample, uniformly mixing, centrifuging at 16000rpm for 2min, removing the supernatant, and keeping the precipitate; obtaining separated DNA; purifying the separated DNA; and carrying out quantification and dilution;
s304, repairing and dA tail adding are carried out through a NEBNext terminal repairing/dA tail adding module, S-dsDNA-A is purified through magnetic beads, a barcode is added to the purified S-dsDNA-A and amplified, and DNA is sequenced through a sequencing platform, so that the nanopore library can be obtained.
In step S301, the centrifugal force provided by the embodiment of the present invention is 16000rpm, and the centrifugal time is 2 min.
As shown in fig. 4, the nanopore library construction provided by the embodiment of the present invention includes:
s401, filtering and sequencing a linker sequence, reads with a filtering quality value less than 7 and reads with a filtering length less than 500bp to nanopore sequencing data; meanwhile, mapping clean data to a reference genome, and carrying out statistics on comparison efficiency, whole genome reads coverage and sequencing depth;
s402, carrying out overall comparison quality evaluation on nanopore sequencing data, judging that the third-generation data is abnormal, and carrying out downstream analysis if the comparison efficiency is higher than 70% and the whole genome coverage is higher than 50%; mining information of within-aligned reads and split reads based on the comparison result;
s403, defining structural variation and typing based on reads support information; integrating the variation types of the multi-sample data; meanwhile, predicting the base type and quality value of the maximum possibility of the variation position of the nanopore sequencing data based on the integration result;
s404, integrating the variant fragments into a read length according to the base type and the quality value of the maximum possibility;
s405, establishing a database containing 300 Refseq standard viruses, and classifying the obtained sequences according to the Refseq database;
s406, mapping the sequence obtained by sequencing to the selected reference sequence, determining the nucleotide at each position by using a generated consistent sequence draft, and obtaining a negotiated consistent draft sequence;
s407, performing BLAST search on a virus sequence database; reading the remapped reference sequence; the percentage and the depth of coverage of the mapping are read.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention, and the scope of the present invention is not limited thereto, and any modification, equivalent replacement, and improvement made by those skilled in the art within the technical scope of the present invention disclosed herein, which is within the spirit and principle of the present invention, should be covered by the present invention.
Claims (10)
1. A method for detecting pathogenic molecules based on nanopore sequencing is characterized by comprising the following steps:
step one, obtaining a nasopharyngeal swab or a nasopharyngeal swab sample; RNA nucleic acid extraction and purification are carried out on the obtained nasopharyngeal swab or nasopharyngeal swab sample;
preparing cDNA by a single primer amplification method independent of sequence to prepare reverse-transcribed cDNA with a universal joint;
amplifying and purifying the cDNA, namely performing PCR amplification on a cDNA random fragment by using the cDNA as a template and a joint sequence as a primer, and purifying a PCR product by using AMPure XP magnetic beads;
step four, preparing and sequencing a nanopore sequencing library: constructing a technical nanopore sequencing library by using a library based on a bar code, wherein the total sequencing DNA amount of each sample is kept equal; adding the prepared nanopore sequencing library into a nanopore sequencing chip and performing machine sequencing;
step five, bioinformatics analysis of pathogenic microorganisms: and (3) carrying out quantitative analysis on the sequence obtained by sequencing, and then carrying out comparison on a pathogenic microorganism database to obtain the relative abundance and composition of pathogenic microorganisms, namely the detection result of pathogenic molecules.
2. The method for detecting pathogenic molecules based on nanopore sequencing according to claim 1, wherein in the first step, the step of extracting nucleic acid from the obtained nasopharyngeal swab sample or nasopharyngeal swab sample comprises:
(1) taking a fresh nasopharyngeal swab or a nasopharyngeal swab sample into a 1.5ml centrifuge tube without RNase; simultaneously, adding 800 mu L of physiological saline into the centrifuge tube by using a sterile RNA enzyme-free pipette tip, rinsing for 20 seconds, drying liquid on a swab by sticking the wall of the centrifuge tube, centrifuging for 2min at 16,000rpm, discarding 700 mu L of supernatant, and fully oscillating and uniformly mixing the residual 100 mu L of supernatant;
(2) adding 200 μ L lysate and 20 μ L digestive juice, shaking, mixing, and water-bathing for 10min at 56 deg.C;
(3) adding 500 μ L of the precipitation solution, and slightly reversing and mixing;
(4) transferring the solution into an adsorption column with a collecting pipe, standing for 2min, centrifuging at 4 ℃ and 12000rpm for 1min, and discarding waste liquid in the collecting pipe;
(5) adding 500 μ L of washing solution into adsorption column, centrifuging at 4 deg.C and 12000rpm for 1min, and discarding the waste liquid in the collection tube;
(6) placing the adsorption column back into the collection tube, centrifuging at 12000rpm for 2min at 4 deg.C;
(7) taking out the adsorption column, putting into a new 1.5mL RNase-free centrifuge tube, adding 30-50 μ L RNase-free water, standing for 3min, centrifuging at 4 deg.C and 12000rpm for 2min, and collecting RNA solution to obtain RNA nucleic acid of pathogenic microorganism in the sample;
(8) RNA of the pathogenic microorganism to be extracted was purified using RNA Clean & concentrate-5 (Zymo Research) kit.
3. The method for detecting pathogenic molecules based on nanopore sequencing according to claim 1, wherein the lysis solution is one of guanidinium isothiocyanate, lithium iodide, citric acid and Tween 20.
4. The method for detecting pathogenic molecules based on nanopore sequencing according to claim 1, wherein in step (1), the centrifugal force is 16000rpm, and the centrifugal time is 2 min.
5. The method for detecting pathogenic molecules based on nanopore sequencing according to claim 1, wherein in step two, the preparation of cDNA comprises:
(1) mu.l of RNA and 1. mu.l of primer A (5 '-GTTTCCCACTGGAGGATA-N9-3', 40 pmol/. mu.l) were mixed and incubated at 65 ℃ for 5min, then cooled to room temperature.
(2) To the above product was added 2. mu.L of SuperScript IV first-strand buffer (Thermo Fisher), 1. mu.L of 12.5mM trinucleoside diphosphate (dNTPs), 0.5. mu.L of 0.1M Dithiothreitol (DTT), 1. mu. L H2O and 0.5 mu L SuperScript IV Reverse Transcriptase (Thermo Fisher) Reverse Transcriptase are incubated at 42 ℃ for 10min, and cDNA of pathogenic microorganisms in the sample is obtained.
6. The method for detecting pathogenic molecules based on nanopore sequencing according to claim 1, wherein in step three, the amplification and purification of the cDNA comprises:
(1) configuration of amplification System for cDNA: 5. mu.L of the above cDNA template was added with 5. mu.L of AccuTaq LA (Sigma) reaction mix and 1. mu.L of primer B (5'-GTTTCCCACTGGAGGATA-3') for cDNA amplification;
(2) PCR amplification conditions: 30s at 98 ℃, 30 cycles (94 ℃ for 15s, 50 ℃ for 20s, 68 ℃ for 2min), and 10min at 68 ℃;
(3) the amplification products were purified using AMPure XP magnetic beads (Beckman Coulter, Brea, CA) and quantified using the Qubit high sensitivity double stranded DNA (dsDNA) kit (Thermo Fisher).
7. The method for detecting pathogenic molecules based on nanopore sequencing according to claim 1, wherein in step four, the preparation and sequencing of the nanopore sequencing library comprises:
(1) repairing and dA tail adding are carried out through a NEBNext terminal repairing/dA tail adding module, S-dsDNA-A is purified by AMPure XP magnetic beads, the S-dsDNA-A purified in different samples is connected with corresponding bar codes, and then the S-dsDNA-A is purified by AMPure XP magnetic beads again, so that a nanopore sequencing library can be obtained;
(2) and loading the prepared nanopore sequencing library into a nanopore sequencing chip, and sequencing by using a MinION sequencing platform.
8. The method for detecting pathogenic molecules based on nanopore sequencing according to claim 1, wherein in step five, the nanopore sequencing pathogenic microorganism bioinformatics analysis comprises:
firstly, filtering and sequencing a linker sequence, reads with a filtering quality value of less than 7 and reads with a filtering length of less than 500bp on nanopore sequencing data; meanwhile, mapping clean data to a reference genome, and carrying out statistics on comparison efficiency, whole genome reads coverage and sequencing depth;
secondly, carrying out overall comparison quality evaluation on nanopore sequencing data, judging that the third-generation data is abnormal, and carrying out downstream analysis if the comparison efficiency is higher than 70% and the whole genome coverage is higher than 50%; mining information of within-alignment reads and split reads based on the comparison result;
then, defining structural variation and typing based on reads support information; integrating the variation types of the multi-sample data; meanwhile, predicting the base type and quality value of the maximum possibility of the variation position of the nanopore sequencing data based on the integration result;
finally, integrating the plurality of variant fragments into a read length according to the base type and quality value of the maximum probability;
in step three, the bioinformatics analysis includes:
step one, establishing a database containing 300 Refseq standard viruses, and classifying the obtained sequences according to the Refseq database;
secondly, mapping the sequence obtained by sequencing to the selected reference sequence, determining the nucleotide at each position by using a generated consistent sequence draft, and obtaining a negotiated consistent draft sequence;
thirdly, performing BLAST search on a virus sequence database; reading the remapped reference sequence; the percentage and the depth of coverage of the mapping are read.
9. A terminal for performing the method for detecting pathogenic molecules according to any one of claims 1 to 8 based on nanopore sequencing.
10. Use of a nanopore sequencing based pathogenic molecule detection method as claimed in any one of claims 1 to 8 for detecting influenza virus.
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| CN113025761A (en) * | 2021-05-27 | 2021-06-25 | 广州赛哲生物科技股份有限公司 | Multi-amplification matched high-throughput sequencing method and kit for pathogenic microorganism identification |
| CN113265452A (en) * | 2021-05-14 | 2021-08-17 | 北京大学人民医院 | Bioinformatics pathogen detection method based on Nanopore metagenome RNA-seq |
| CN113667729A (en) * | 2021-07-29 | 2021-11-19 | 杭州圣庭医疗科技有限公司 | Rapid microorganism identification method based on nanopore sequencer |
| CN113718023A (en) * | 2021-08-23 | 2021-11-30 | 普瑞基准生物医药(苏州)有限公司 | Prokaryotic ribonucleic acid direct sequencing method based on bovine nanopore sequencing technology |
| CN117594130A (en) * | 2024-01-19 | 2024-02-23 | 北京普译生物科技有限公司 | Nanopore sequencing signal evaluation method and device, electronic equipment and storage medium |
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| CN113718023A (en) * | 2021-08-23 | 2021-11-30 | 普瑞基准生物医药(苏州)有限公司 | Prokaryotic ribonucleic acid direct sequencing method based on bovine nanopore sequencing technology |
| CN117594130A (en) * | 2024-01-19 | 2024-02-23 | 北京普译生物科技有限公司 | Nanopore sequencing signal evaluation method and device, electronic equipment and storage medium |
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