CN113699277A - Novel coronavirus metagenome sequencing primer, sequencing method and application - Google Patents

Abstract

The invention relates to the technical field of gene detection, and discloses a novel coronavirus metagenome sequencing primer, a sequencing method and application, wherein the sequencing primer comprises 60 pairs of specific PCR amplification primers designed aiming at the complete genome sequence of a novel coronavirus isolated strain, and the 60 pairs of specific PCR amplification primer sequences are SED ID No.1-SED ID No.120 in sequence; the sequencing method comprises the following steps: sequencing sample RNA reverse transcription; sequencing the specific amplification of sample cDNA, and purifying PCR amplification products by using a purified magnetic bead reagent; preparing a sample library by using a third-generation library building kit; and performing on-machine sequencing on the constructed library sample. The invention can solve the problems of low accuracy and long time of the metagenome sequencing process of the conventional novel coronavirus metagenome.

Description

Novel coronavirus metagenome sequencing primer, sequencing method and application

Technical Field

The invention relates to the technical field of gene detection, in particular to a novel coronavirus metagenome sequencing primer, a sequencing method and application.

Background

The novel coronavirus pneumonia is acute infectious pneumonia, and the pathogen of the novel coronavirus is a new emerging coronavirus which is named as '2019-nCoV' by the world health organization. The novel coronavirus belongs to the genus coronavirus beta, is an nonsegmented single-stranded positive-strand RNA virus, has an envelope, is mainly transmitted through droplets, has strong infectious capacity, is mainly characterized by fever, hypodynamia and dry cough after infecting people, gradually shows severe manifestations such as dyspnea and the like, has good prognosis for most patients, and can cause pneumonia, severe acute respiratory syndrome, renal failure and even death for some severe cases. Currently, there is still a lack of effective antiviral drugs against new coronaviruses.

At present, the variation trend and the propagation rule of the novel coronavirus in the epidemic situation process are still unknown, so that rapid virus genome data are obtained, and the genome variation condition of the virus in the epidemic situation development process is monitored in real time; determining key nodes of chain propagation, and tracking possible superpropagators; can provide effective data support for accurate prevention and control of epidemic situations. The novel coronavirus whole genome sequence information is obtained through a high-throughput sequencing platform, which is not only beneficial to the detection of the novel coronavirus, but also can monitor the virus variation aiming at the type and the position of the virus genome variation and the influence of the virus genome variation on a nucleic acid detection method, determine a virus propagation chain, and analyze the virus variation monitoring and propagation evolution.

The currently used high throughput sequencing platforms include the second generation high throughput sequencers, such as the illumina sequencing platform, Ion Torrent platform from Thermo Fisher, and the third generation high throughput sequencing platform, NanoPore. Compared with the third-generation sequencing technology, the second-generation sequencing technology platform needs to finish the processes of reverse transcription, PCR specific amplification, library building and computer data analysis of the whole sample, and is not beneficial to quickly obtaining the sequencing result. The third generation sequencing technology is also a single molecule sequencing technology, PCR amplification is not needed, the library construction is simple, and the sequencing time is obviously shorter than that of the second generation sequencing platform. Meanwhile, the third-generation NanoPore sequencing instrument is portable and can be used in areas far away from laboratories, such as epidemic areas, pastures and the like. However, for the third-generation Nanopore sequencing platform, the initial nucleic acid amount is required to be high (generally more than or equal to 500ng) when virus RNA is directly used for library building, and the concentration of the nucleic acid sample of the pharyngeal swab virus genome of a new coronavirus patient is difficult to meet the requirement, so that the sequencing accuracy is low, the acquisition of genome data is not facilitated, and the difficulty of bioinformatics analysis is increased.

Disclosure of Invention

In view of the above, the invention aims to provide a novel coronavirus metagenome sequencing primer, a sequencing method and an application, which can solve the problems that the sequencing accuracy of the current novel coronavirus metagenome is low, the metagenome sequencing process time is long, the acquisition of genome data is not facilitated, and the difficulty of bioinformatics analysis is increased.

The invention solves the technical problems by the following technical means:

a novel coronavirus metagenome sequencing primer comprises 60 pairs of specific PCR amplification primers designed aiming at a complete genome sequence of a novel coronavirus isolated strain, wherein the 60 pairs of specific PCR amplification primers are SED ID No.1-SED ID No.120 in sequence, and specific sequence information is shown in example 1.

The invention also discloses a novel coronavirus metagenome sequencing method, which comprises the following steps:

s1, reverse transcription of sequencing sample RNA;

s2, specific amplification of sequencing sample cDNA: carrying out PCR amplification on sample cDNA by using 60 pairs of specific PCR amplification primers aiming at the self-designed new coronavirus and QIAseq SARS-CoV-2Primer Panel kit primers, wherein the sequences of the 60 pairs of self-designed specific PCR amplification primers are SED ID No.1-SED ID No.120 in sequence;

s3, purifying the PCR amplification product in the step S2 by using a purified magnetic bead reagent;

s4, preparing a sample library by using a third library construction kit;

s5, sequencing: and performing on-machine sequencing on the constructed library sample, wherein the sequencing method is a non-disease diagnosis method.

Further, the specific step of step S1 is: accurately quantifying the initial sequencing sample by using a fluorescence quantifier, taking 5 mu L of the initial sequencing sample with the total RNA amount less than 100ng, and carrying out reverse transcription of the RNA nucleic acid of the sequencing sample according to the QIAseq SARS-CoV-2Primer Panel instruction; wherein the RP primer is diluted by 11 times; sample mixed solution reaction system: sample 5. mu.l, diluted RP primer 1. mu.l, RNase-free water 6. mu.l; reacting for 5min under the reaction condition of 65 ℃; 20 μ l reverse transcription reaction: RNA/RP mixture 12. mu.l, M. mu.l multimodal buffer 4. mu.l, RNase-free water 1. mu.l, RI 1. mu.l, EZ Reverse Transcriptase 1. mu.l, reaction conditions: the reaction is carried out at 42 ℃ for 50min and at 70 ℃ for 15 min.

Further, the specific step of step S2 is: carrying out PCR amplification on the sample cDNA by using SED ID No.1-SED ID No.120 primers; wherein 25. mu.l of PCR reaction system: reverse transcription sample 5. mu.l, Pool1/Pool 2primer 3. mu.l, 2 XQIAGEN HiFi PCR MM 12.5. mu.l, RNase-free water 4.5. mu.l; and (3) PCR reaction conditions: the reaction is carried out for 2min at 98 ℃, for 20s at 98 ℃ and for 5min at 65 ℃.

Further, in the step S4, 10 μ l of the reaction system, sample: 7.5. mu.l, Barcoded RB: 2.5 mul; reaction conditions are as follows: reacting at 30 ℃ for 1min and at 80 ℃ for 1 min; all barcoded samples were mixed together in proportion, gently mixed and then centrifuged, 10 μ l was pipetted into a new 0.2ml PCR tube, 1 μ l rap to 10 μ l barcoded DNA was added, gently mixed and then centrifuged, and left at room temperature for 5 min.

Further, the upper machine sequencing in the step S5 uses a Nanopore third generation high throughput sequencer.

The invention also discloses application of the novel coronavirus metagenome sequencing primer in sequencing of the novel coronavirus metagenome, wherein the application is non-disease diagnosis application, the novel coronavirus metagenome sequencing primer comprises 60 pairs of specific PCR amplification primers which are self-designed aiming at the complete genome sequence of a novel coronavirus isolated strain, and the 60 pairs of specific PCR amplification primer sequences are sequentially SED ID No.1-SED ID No. 120.

The invention has the beneficial effects that:

1. the invention directly utilizes the novel coronavirus RNA to carry out reverse transcription, utilizes a specific primer aiming at the novel coronavirus to carry out PCR amplification on the sample cDNA, and finally utilizes a third-generation sequencing platform NanoPore to carry out sequencing library construction and high-throughput sequencing analysis; therefore, the sequencing time can be shortened, and the whole process of reverse transcription, PCR specific amplification, library building and on-computer data analysis of the sample can be completed within 6 hours; background information in the pharyngeal swab RNA nucleic acid sample is reduced, effective data of high-throughput sequencing is increased, and sequencing accuracy can be improved.

2. Compared with the currently adopted method for directly performing third-generation sequencing without PCR amplification, the method can obtain more whole genome sequence information after the same sample subjected to the process optimization of the invention is subjected to specific PCR amplification.

Drawings

FIG. 1 is a graph showing the effect of PCR amplification on sequencing effect according to the present invention;

FIG. 2 is a plot of percentage and fit of new coronavirus sequences in sequencing data from different samples according to the present invention;

FIG. 3 is a graph showing the effect of different PCR-specific primers of the present invention on PCR amplification in ng/ul concentration units.

Detailed Description

The invention will be described in detail below with reference to examples and the accompanying drawings:

1. the samples and reagents used in the following examples are as follows:

9 samples of RNA nucleic acids that have been identified as new corona positive pharyngeal swabs, of which 4 samples of known CT values, labeled a: CT value 11.99, B: CT value 18.31, C: CT value 34.6, D: a CT value of 42; the remaining positive sample 5 examples, labeled E, F, G, H, I; specific PCR self-contained primers are designed and stored by the laboratory of the applicant, and specific sequence information is shown in example 1; QIAseq SARS-CoV-2Primer Panel from Qiagen (cat # 333896); purified magnetic beads AMPure XP from Beckman Co. mu. lter (cat # A63881); the third generation library construction Kit Rapid coding Kit is purchased from Oxford NANOPORE Technologies (cat # SQK-RBK004), and the third generation sequencing chip Flow cell (R9.4.1) is purchased from Oxford NANOPORE Technologies (cat # FLO-MIN 106D).

2. The apparatus used in the following examples is as follows:

nanopore third generation high throughput sequencers were purchased from Oxford Nanopore Technologies, inc;

the Qubit 2.0 fluorescence quantifier was purchased from Life Technologies.

Examples 1,

The present example is the design of specific PCR amplification primers for the complete genome sequence 60 of a novel coronavirus isolated strain, and the specific sequence information is shown in table 1 below:

TABLE 1 sequence information of primers SED ID No.1-SED ID No.120 of the present invention

Examples 2,

In this example, 9 new coronary positive pharyngeal swab RNA nucleic acid samples were sequenced as follows:

1. sequencing sample PCR amplification

1.1 reverse transcription of sequenced sample RNA

The initial sample was accurately quantified using a Qubit 2.0 fluorescence quantifier and 5. mu.L (total RNA <100ng) was taken for reverse transcription of the sequenced sample RNA nucleic acids according to the QIAseq SARS-CoV-2Primer Panel instructions. Wherein the RP primers were diluted 11-fold. Sample mixed solution reaction system: sample 5. mu.l, diluted RP primer 1. mu.l, RNase-free water 6. mu.l; the reaction was carried out at 65 ℃ for 5 min. 20 μ l reverse transcription reaction: RNA/RP mixture 12. mu.l, M. mu.l multimodal buffer 4. mu.l, RNase-free water 1. mu.l, RI 1. mu.l, EZreverse Transcriptase 1. mu.l; reaction conditions are as follows: the reaction is carried out at 42 ℃ for 50min and at 70 ℃ for 15 min.

1.2 specific amplification of sequenced sample cDNA

For sample E, F, G, H, I, PCR amplification was performed on sample cDNA using the self-designed primers and QIAseq SARS-CoV-2Primer Panel kit for the novel coronavirus specific primers Pool1 and Pool2, respectively. The reaction conditions and the reaction system are the same. 25 μ l PCR reaction: reverse transcription sample 5. mu.l, Pool1/Pool 2primer 3. mu.l, 2 XQIAGEN HiFi PCR MM 12.5. mu.l, RNase-free water 4.5. mu.l; and (3) PCR reaction conditions: reacting at 98 ℃ for 2min, at 98 ℃ for 20s, and at 65 ℃ for 5min, wherein the steps are carried out for 35 cycles in 2-3 steps. Sample A, B, C, D sample cDNA was PCR amplified using QIAseq SARS-CoV-2Primer Panel kit alone against the novel coronavirus specific primers Pool1 and Pool 2. The reaction conditions and the reaction system are the same as the previous steps.

1.3 sequencing sample PCR product purification

Samples from Pool1 and Pool2 tubes were mixed together and the sequencing sample PCR products were purified using AMPure XP reagent. Adding 50 mu L of AMPure XP reagent into each library, and repeatedly blowing and sucking for 5 times to fully and uniformly mix the magnetic bead suspension and the DNA; the mixture was incubated at room temperature for 5 minutes; the sample was then placed on a magnetic stand and allowed to stand for 2 minutes. Then carefully aspirating and removing the supernatant; then 200. mu.L of freshly prepared 80% ethanol was added to wash the beads, followed by careful aspiration and removal of the supernatant; the Ep tube was placed on a magnetic stand and dried for 5 minutes at room temperature. The Ep tube was removed from the magnetic frame and the beads were resuspended in 30. mu.l water. Vortex to mix well and then centrifuge to collect droplets. The Ep tube was placed on a magnetic stand for 2 minutes. Aspirate 28. mu.l of supernatant into a new 1.5ml EP tube. And accurately quantifying the purified sample by using a Qubit 2.0 fluorescence quantifier.

2. Third Generation library preparation of sequencing samples

And preparing a sample library by using a three-generation library construction Kit Rapid Barcoding Kit. 10 μ l reaction: samples 7.5 μ l, Barcoded RB (one for each sample) 2.5 μ l; reaction conditions are as follows: the reaction is carried out at 30 ℃ for 1min and at 80 ℃ for 1 min. All barcoded samples were mixed together in proportion, gently mixed, then briefly centrifuged, and 10 μ l was pipetted into a new 0.2ml PCR tube. Mu.l RAP was added to 10. mu.l barcoded DNA, gently mixed, and then briefly centrifuged, and left at room temperature for 5 min.

3. Nanopore three-generation sequencing machine

Preparing a built library sample for computer sequencing, rotating clockwise and leftwards by 90 degrees to open a Nanopore P hole of a third-generation sequencing instrument, and sucking yellow liquid by a tip of a gun head; preparing a prime Mix: adding 30uL Flush Tether (FLT) into a Flush Buffer (FB), uniformly mixing, pushing 800uL of uniformly mixed Priming Mix into a P hole by using a P1000 hole at a constant speed, placing for 5min, fully mixing the library with loading Mix, turning a rubber cover upwards to open an S hole, pushing 200uL of uniformly mixed Priming Mix into the P hole at a constant speed, dropwise adding 75 mul of mixed library to be detected and loading Mix into the S hole, closing the S hole and the P hole, and clicking to start sequencing.

The sequencing results of example 2 are shown below:

1. the virus nucleic acid content of clinical throat swab samples is very low, and the throughput of third generation sequencers is low (the sequencing capability is not higher than that of the second generation sequencers, and the single output data is less than 1 million). For samples with low viral load, PCR amplification is firstly required, and the whole genome sequence of the virus can be obtained through third-generation sequencing only by directionally increasing the nucleic acid of the virus. Therefore, we used specific new coronavirus genome sequence PCR primers to treat sample A with the method of non-amplification and PCR amplification respectively. Mapping the sequences obtained by sequencing to the genome of the new coronavirus respectively, and analyzing the difference of the virus nucleic acid content in the sequencing result. The statistics of the sequencing data for the two specific treatments are shown in table 2 below and fig. 1:

TABLE 2 statistical tables of sequencing data for the two processing methods

As seen in FIG. 1, A is the mapping of reads to new coronavirus obtained without sequencing by PCR amplification, covering 49.8% of the whole genome. And B is mapping of reads sequenced by a whole genome PCR amplification method to the new coronavirus, and covers the whole genome sequence.

As can be seen from the results of Table 2 and FIG. 1, the third generation of sequencing without PCR amplification did not result in the full genome sequence, which was obtained after PCR amplification.

2. The effect of specific PCR amplification on sequencing effect of samples with different CT values is shown in the following table 3 and the attached FIG. 2:

TABLE 3 sequencing data statistics of samples with different CT values

As can be seen from the results in Table 3 and FIG. 2, the CT value is the most direct quantitative indicator in nucleic acid detection, and the magnitude of the CT value reflects the load of the target virus in the sample. The third generation sequencer has requirements on the nucleic acid concentration of a sample, and effective third generation sequencing data cannot be obtained due to the overhigh CT value. In this study, we selected 4 samples with different nucleic acid detection CT values, and used them after target amplification by specific PCR and three generations of high throughput sequencing. The results show that the novel coronavirus positive sequences can be obtained even when the CT value is more than or equal to 40 by adopting the experimental process.

3. In the invention, in order to select the optimized PCR specific targeting Primer, the self-designed Primer and the QIAseq SARS-CoV-2Primer clone kit Primer are selected to carry out PCR reaction on the same sample. As can be seen from FIG. 3 (the self-contained primers in FIG. 3 refer to the primers designed in example 1), for the same sample, the concentration of the nucleic acid amplified by QIAseq SARS-CoV-2Primer Pane kit primers is obviously higher than that of the nucleic acid amplified by the self-contained primers, and the nano-pore library building requirement is basically met.

Although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the spirit and scope of the invention as defined in the appended claims. The techniques, shapes, and configurations not described in detail in the present invention are all known techniques.

Claims (7)

1. A novel coronavirus metagenome sequencing primer is characterized in that: comprises 60 pairs of specific PCR amplification primers which are self-designed aiming at the complete genome sequence of a novel coronavirus isolated strain, wherein the 60 pairs of specific PCR amplification primers have SED ID No.1-SED ID No.120 in sequence.

2. A novel method for sequencing a coronavirus metagenome is characterized by comprising the following steps:

s1, reverse transcription of sequencing sample RNA;

s2, specific amplification of sequencing sample cDNA: carrying out PCR amplification on sample cDNA by using 60 pairs of specific PCR amplification primers aiming at the self-designed new coronavirus and QIAseq SARS-CoV-2Primer Panel kit primers, wherein the sequences of the 60 pairs of self-designed specific PCR amplification primers are SED ID No.1-SED ID No.120 in sequence;

s3, purifying the PCR amplification product in the step S2 by using a purified magnetic bead reagent;

s4, preparing a sample library by using a third library construction kit;

s5, sequencing: and performing on-machine sequencing on the constructed library sample, wherein the sequencing method is a non-disease diagnosis method.

3. The novel method of coronavirus metagenomic sequencing as claimed in claim 2, wherein: the step S1 specifically includes: accurately quantifying the initial sequencing sample by using a fluorescence quantifier, taking 5 mu L of the initial sequencing sample with the total RNA amount less than 100ng, and carrying out reverse transcription of the RNA nucleic acid of the sequencing sample according to the QIAseq SARS-CoV-2Primer Panel instruction; wherein, RP primer dilution; sample mixed solution reaction system: sample 5. mu.l, diluted RP primer 1. mu.l, RNase-free water 6. mu.l; reacting for 5min under the reaction condition of 65 ℃; 20 μ l reverse transcription reaction: RNA/RP mixture 12. mu.l, M. mu.l multimodal buffer 4. mu.l, RNase-free water 1. mu.l, RI 1. mu.l, EZ Reverse Transcriptase 1. mu.l, reaction conditions: the reaction is carried out at 42 ℃ for 50min and at 70 ℃ for 15 min.

4. The novel method of coronavirus metagenomic sequencing as claimed in claim 3, wherein: the step S2 specifically includes: carrying out PCR amplification on the sample cDNA by using SED ID No.1-SED ID No.120 primers; wherein 25. mu.l of PCR reaction system: reverse transcription sample 5. mu.l, Pool1/Pool 2primer 3. mu.l, 2 XQIAGEN HiFi PCR MM 12.5. mu.l, RNase-free water 4.5. mu.l; and (3) PCR reaction conditions: the reaction is carried out for 2min at 98 ℃, for 20s at 98 ℃ and for 5min at 65 ℃.

5. The novel method of coronavirus metagenomic sequencing as claimed in claim 4, wherein: in step S4, 10 μ l of the reaction system, sample: 7.5. mu.l, Barcoded RB: 2.5 mul; reaction conditions are as follows: reacting at 30 ℃ for 1min and at 80 ℃ for 1 min; all barcoded samples were mixed together in proportion, gently mixed and then centrifuged, 10. mu.l was pipetted into a new 0.2ml PCR tube, 1. mu.l RAP to 10. mu.l barcoded DNA was added, mixed, centrifuged and left at room temperature.

6. The novel method of coronavirus metagenomic sequencing as claimed in claim 5, wherein: the Nanopore third generation high throughput sequencer is used for the on-line sequencing in step S5.

7. The application of the novel coronavirus metagenome sequencing primer in sequencing the novel coronavirus metagenome is non-disease diagnosis, the novel coronavirus metagenome sequencing primer comprises 60 pairs of specific PCR amplification primers which are self-designed aiming at the whole genome sequence of a novel coronavirus isolated strain, and the 60 pairs of specific PCR amplification primer sequences are sequentially SED ID No.1-SED ID No. 120.

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