CN116426696A - Plasma virus detection and analysis method based on sequencing technology - Google Patents
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Abstract
The invention provides a plasma virus detection and analysis method based on a sequencing technology, which comprises the following steps: enriching plasma sample virus particles, and virus detection analysis; wherein the enriching of the plasma sample virus particles is performed as follows: a centrifugation step; a nuclease treatment step; a filtering step; ultrafiltration, centrifugation and concentration; the performing a virome stain removal assay on the sequencing result comprises: filtering the pollution in the batch; filtering nonspecific virus sequences; filtering the non-human host virus. The method can reduce false positive results and improve virus detection capability, and experimental results show that the average LoD detection limit of the method is improved by about 100 times, even can be reduced to 5 Copies/ml detection limit, and is suitable for detecting extremely low-abundance virus pathogens in clinical samples.
Description
Technical Field
The invention relates to the field of pathogen detection and analysis, in particular to a plasma virus enrichment and decontamination detection and analysis method based on a sequencing technology.
Background
Clinical blood flow infection detection always has the difficulties of large influence of human hosts and low pathogen abundance, and a large number of sequences which are highly consistent with viruses in species sequences such as experimental cross contamination, human sources and the like can cause false positive detection of the viruses, which brings great challenges for detection of clinical viruses. Although ultrafiltration centrifugation and random anchored amplification can be used for library-building sequencing aiming at low initial quantity of nucleic acid, the content of human cfDNA and human cells in a clinical conventional plasma sample is extremely high, usually more than 99%, so that pathogenic data are severely interfered by the human data, and as virus particles are extremely tiny, the abundance of virus genes is extremely low, usually less than 1% and even less than 0.1%, and the virus genes are difficult to effectively enrich, so that the conventional plasma mNSS sequencing is not friendly to detect trace pathogenic nucleic acid in blood, especially viruses with extremely low blood content.
In the aspect of pathogen detection analysis, the conventional sequencing technology usually judges and reads a sequence (environmental pollution, reagent pollution and aerosol pollution) polluted by a sequencing result by adding negative control in the same batch of data, and then calculates the RPM-Ratio through the number of samples ready/the number of negative control ready after calculating the standardized RPM later. However, many contaminating sequences cannot be removed only by the method of negative control, and cross-contamination between samples and species specificity in samples are still difficult to solve, which causes great interference to interpretation of the final result.
In view of this, the present invention has been proposed.
Disclosure of Invention
The invention provides a plasma virus detection and analysis method based on a sequencing technology, which comprises the following technical scheme:
embodiment 1. A method for detecting and analyzing plasma virus based on a sequencing technique, comprising: enriching plasma sample virus particles, and virus detection analysis; wherein the enriching of the plasma sample virus particles is performed as follows:
and (3) centrifuging: centrifuging the collected blood to prepare centrifuged plasma;
nuclease treatment step: treating the centrifuged plasma with a omnipotent nuclease;
and (3) filtering: filtering the centrifuged plasma treated with the omnipotent nuclease to obtain filtered plasma;
ultrafiltration and centrifugal concentration steps: centrifuging the filtered plasma by using an ultrafiltration centrifugal filter device to obtain concentrated plasma;
the virus detection analysis was performed as follows: carrying out virus sequencing on the concentrated plasma to obtain a sequencing result; carrying out virus sequencing analysis on the sequencing result to obtain a sequencing analysis result; performing a virome decontamination assay on the sequencing assay result, wherein the performing a virome decontamination assay on the sequencing result comprises:
and (3) filtering the pollution in the batch: removing intra-batch contamination sequences in the same-batch sequencing sample;
non-specific viral sequence filtration: removing non-specific viral sequences shared by the virus and other microorganisms and human sources;
filtering non-human host virus: and removing viruses of non-human hosts to obtain a virus detection result.
Embodiment 2. The method for analyzing plasma virus detection based on sequencing technology according to embodiment 1, wherein the centrifugation step comprises: removing precipitate by low-speed centrifugation, centrifuging at high speed, and retaining supernatant to obtain centrifugal plasma.
Embodiment 3. The method for detecting and analyzing plasma viruses according to embodiment 1, wherein in the filtering step, the centrifuged plasma treated with the omnipotent nuclease is filtered by a PES filter.
Embodiment 4. The method for analyzing plasma virus detection based on the sequencing technology according to embodiment 1, wherein the virus sequencing is performed as follows: extracting virus nucleic acid and pre-amplifying; constructing a library by using a library constructing kit; sequencing was performed on a sequencing platform.
Embodiment 5. The method for analyzing plasma virus detection based on the sequencing technology according to embodiment 1, wherein the virus sequencing analysis is performed as follows: (1) Performing data quality control on the original data obtained by sequencing, and removing low-quality and shorter sequences to obtain clean data; (2) Comparing the clean data with the human genome by using comparison software, and removing the sequence of the human after comparison to obtain a sequence after removing the human; (3) The sequence after human source removal is subjected to virus database comparison by using comparison software, and then virus species annotation is carried out; (4) And obtaining reads information, depth information and genome coverage of the virus species in each sample according to the comparison result statistics.
Embodiment 6. The method for detecting and analyzing plasma viruses based on the sequencing technology according to embodiment 1 is characterized in that the sequence with the occurrence frequency of more than 50% of the positions of the same genome of the same species in the same sequencing batch is selected from the sequence contaminated in the batch.
Embodiment 7. The method for detecting and analyzing plasma viruses according to embodiment 1, wherein the non-specific viral sequences comprise a consensus sequence of a virus with other species comprising one or more species selected from the group consisting of human, bacterial, fungal, and parasitic species, and wherein the consensus sequence of the virus with other species is obtained by aligning a database of viruses with one or more of a database of human, bacterial, fungal, and parasitic species, respectively, and selecting a sequence or a region of a sequence in any of the database of human, bacterial, fungal, and parasitic viral sequences.
Embodiment 8. The method for analyzing plasma virus detection based on sequencing technology according to embodiment 1, further comprising: adding a negative control sample to the same batch of sequencing samples for virome decontamination analysis; the virome decontamination assay further comprises: sequencing each sample in the same batch removes virus species detected in the negative control sample that are more abundant than the sequencing sample in the same batch.
Embodiment 9. A computer-readable medium storing a computer program which, when executed by a processor, implements the method of any one of embodiments 1 to 8.
Embodiment 10. An electronic device comprising a processor and a memory having stored thereon one or more readable instructions which, when executed by the processor, implement the method of any of embodiments 1 to 8.
The beneficial technical effects of the invention include: 1. aiming at the characteristics of small volume and weight of virus particles, low relative content of genome and difficult enrichment in clinical samples, the invention firstly provides a four-step enrichment method of virus group to effectively enrich the virus particles, thereby greatly improving the detection capability and the detection accuracy of the virus. 2. The invention provides a non-specific viral sequence construction method, which is applied to the virus identification process, so that false positives caused by exogenous species are greatly reduced, and the detection accuracy is improved. 3. The invention provides a method for filtering in-batch pollution, which can filter the pollution of a pathogen sequence detected by samples in the same batch under the condition that blank control samples are not needed. 4. The invention skillfully utilizes the virus host information to filter the non-human host virus, further improves the accuracy of virus detection, and is more suitable for clinical application. The method can greatly improve the abundance of virus components in the sample, and through a reasonable pollution treatment method, the virus detection capacity is improved, meanwhile, the preference and false positive result introduced in enrichment, library building and amplification are reduced, and the clinical pathogen detection capacity is improved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a flow chart of the "four-step enrichment method of viral group" and related extraction and library construction of the present invention.
FIG. 2 is a flow chart of the data analysis quality control and "virome decontamination" decontamination process of the present invention.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention, and that the examples are some examples of the present invention but not all examples. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Partial term definition
Unless defined otherwise hereinafter, all technical and scientific terms used in the detailed description of the invention are intended to be identical to what is commonly understood by one of ordinary skill in the art. While the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the present invention.
As used herein, the terms "comprising," "including," "having," "containing," or "involving" are inclusive or open-ended and do not exclude additional unrecited elements or method steps. The term "consisting of …" is considered to be a preferred embodiment of the term "comprising". If a certain group is defined below to contain at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists of only these embodiments.
The terms "about" and "substantially" in this invention mean the range of accuracy that one skilled in the art can understand yet still guarantee the technical effect of the features in question. The term generally means a deviation of + -10%, preferably + -5%, from the indicated value.
The indefinite or definite article "a" or "an" when used in reference to a singular noun includes a plural of that noun.
Furthermore, the terms first, second, third, (a), (b), (c), and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.
The following terms or definitions are provided solely to aid in the understanding of the invention. These definitions should not be construed to have a scope less than understood by those skilled in the art.
Some technical terms in the present invention are explained as follows:
the "reads" in the present invention refers to a sequencing sequence obtained after sequencing;
the clean data refers to a sequencing sequence obtained after quality control;
the invention discloses a method for preparing a database, which comprises the steps of (1) setting a database, wherein 'taxi' or 'tax_id' refers to an id number in a tax database;
"depth" as used herein refers to the depth of a sequencing sequence in a genome or region of a genome of a species being aligned;
the invention relates to a coverage of a sequencing sequence on a certain species genome in comparison result;
it should be understood that any application including the above-described design method, such as flow, program, software, system, etc., is within the scope of the present invention.
The invention provides a plasma virus detection and analysis method based on a sequencing technology, which is characterized by comprising the following steps of: enriching plasma sample virus particles, and virus detection analysis; wherein the enriching of the plasma sample virus particles is performed as follows: (1) a centrifugation step: centrifuging the collected blood, for example, clinically freshly collected EDTA anticoagulated, to prepare a centrifuged plasma; (2) nuclease treatment step: treating the centrifuged plasma with a omnipotent nuclease; (3) a filtering step: filtering the centrifuged plasma treated with the omnipotent nuclease to obtain filtered plasma; (4) ultrafiltration centrifugal concentration step: centrifuging the filtered plasma by using an ultrafiltration centrifugal filter device to obtain concentrated plasma;
the virus detection analysis was performed as follows: carrying out virus sequencing on the concentrated plasma to obtain a sequencing result; carrying out virus sequencing analysis on the sequencing result to obtain a sequencing analysis result; performing a virome decontamination assay on the sequencing assay result, wherein the performing a virome decontamination assay on the sequencing result comprises:
(1) And (3) filtering the pollution in the batch: removing intra-batch pollution sequences in the sequencing samples in the same batch to obtain a filtered sequence; and the intra-batch pollution sequences are selected and analyzed together with sequencing samples in the same batch, and sequences with the occurrence frequency of the same genome position of the same species larger than a threshold value are compared according to the sequencing samples in the same batch. The threshold may be set by a reasonable choice by one skilled in the art, for example 30%, or 40%, or 50%, or 60%, or 70%; (2) non-specific viral sequence filtration: removing non-specific viral sequences shared by the virus and other microorganisms and human sources, wherein the non-specific viral sequences comprise the shared sequences of the virus and other species, and the non-specific viral sequences are described in the application, or 60 percent or bacteria, fungi, parasites and other microorganisms; (3) non-human host virus filtration: and removing viruses of non-human hosts to obtain a virus detection result.
The method can effectively enrich the virus particles, solve the problems of low virus content and difficult enrichment in the blood plasma, greatly improve the virus content in the concentrated blood plasma, and is suitable for carrying out subsequent sequencing analysis and decontamination analysis, and greatly improve the virus detection capability and detection accuracy. Further, the virus group decontamination method is used for filtering the contamination of samples, namely, filtering the contamination in batches, filtering non-specific virus sequences and filtering non-human host viruses, subtracting the contamination sequences and the consensus sequences in batches from original virus sequences with qualified ready lengths in samples to obtain species information after decontamination, and can carry out the contamination filtering on the pathogen sequences detected by samples in the same batch under the condition that blank control samples are not needed, meanwhile, the false positive caused by foreign species is greatly reduced, the detection accuracy is improved, and the final virus group detection result is obtained through virus host information filtering, so that the accuracy of virus detection is further improved, and the virus group decontamination method is more suitable for clinical application.
In some embodiments, the centrifuging step comprises: removing precipitate by low-speed centrifugation, centrifuging at high speed, and retaining supernatant to obtain centrifugal plasma. The low-speed centrifugation and the high-speed centrifugation described herein have meanings commonly understood by those skilled in the art, and a reasonable centrifugation speed and centrifugation time can be set by those skilled in the art based on empirical values known for a specific sample type, for example, the low-speed centrifugation has a speed of 2000-5000r/min, preferably 3000r/min, and the high-speed centrifugation has a speed of 10000r/min-20000r/min, preferably 12000r/min; for example, the centrifugation time is 5 minutes to 2 hours, preferably 10 minutes. Through two-step centrifugation, human cell interference can be removed to the greatest extent for subsequent processing.
In some embodiments, the filtration step uses a PES filter to filter the centrifuged plasma after treatment with the omnipotent nuclease. Preferably, the PES filter is a 0.45 μm PES filter.
In some embodiments, the viral sequencing is performed as follows: (1) extracting viral nucleic acid and performing pre-amplification; (2) Library construction is performed with a library construction kit, preferably with a vazyme5ng initiation amount genome fragmentation library construction kit; (3) On-machine sequencing is performed using a sequencing platform, preferably using the NGS sequencing platform PE150 strategy.
In some embodiments, the viral nucleic acid comprises only DNA virus, and the DNA procedure may be used to amplify the virus, for specific procedures, reference may be made to the following: taking a certain amount of extracted nucleic acid, carrying out one-strand synthesis and two-strand synthesis by using a random anchor primer and Takara enzyme, and carrying out 20-25 rounds of amplification and enrichment by using the anchor primer and the Takara enzyme;
in some embodiments, the viral nucleic acid comprises only RNA virus and the virus is amplified using an RNA procedure, specific procedures can be referenced as follows: taking a certain amount of extracted nucleic acid, carrying out one-strand synthesis by using a random anchor primer and reverse transcriptase, carrying out two-strand synthesis by using Klenow fragment, and further carrying out 20-25 rounds of amplification and enrichment by using the anchor primer and Takara enzyme;
in some embodiments, library construction with the library building kit proceeds as follows: library construction was performed using a vazyme5ng starting amount genome fragmentation library building kit according to the kit instructions; then, adopting an NGS sequencing platform PE150 strategy to carry out on-machine sequencing, and preferably, each sample is used for measuring 6G original data;
further, third generation sequencing platforms, such as Pacbio or ONT sequencing platforms, are used for sequencing.
In some embodiments, the viral sequencing analysis is performed as follows: (1) Performing data quality control on the original data obtained by sequencing, and removing low-quality and shorter sequences to obtain clean data; (2) Comparing the clean data with the human genome by using comparison software, and removing the sequence of the human after comparison to obtain a sequence after removing the human; (3) The sequence after human source removal is subjected to virus database comparison by using comparison software, then virus species annotation is carried out, and preferably, a taxonomy relationship is adopted for virus species annotation; (4) And obtaining reads information, depth information and genome coverage of the virus species in each sample according to the comparison result statistics, and preferably adopting samtools, bedtools for the result statistics.
After virus particles are enriched by a virus group four-step enrichment method, nucleic acid extraction, random amplification, library building and on-machine sequencing are carried out to obtain a sequencing sequence. And removing human sequences, virus identification analysis and statistical calculation after quality control of the sequencing sequences, and further removing pollution by a virus group pollution removal method to obtain a final virus detection result.
In some embodiments, the within-lot contaminating sequences are selected from sequences that occur more than 50% frequently at the same genomic location in the same aligned species in the same lot of sequencing samples.
In some embodiments, the non-specific viral sequences comprise consensus sequences of viruses with other species including one or more selected from the group consisting of human, bacterial, fungal, and parasitic species obtained by aligning a database of viruses with one or more of the database of human, bacterial, fungal, and parasitic species, respectively, and selecting a sequence or region of sequence in any of the database of human, bacterial, fungal, and parasitic sequences aligned above.
In some embodiments, the method further comprises: adding a negative control sample to the same batch of sequencing samples for virome decontamination analysis; the virome decontamination assay further comprises: sequencing each sample in the same batch removes virus species detected in the negative control sample that are more abundant than the sequencing sample in the same batch. The negative control sample can remove the pollution introduced by reagents, laboratory background and the like, and reduce false positive results.
The application also discloses a computer readable medium storing a computer program which, when executed by a processor, implements a method according to any of the preceding claims.
The application also discloses an electronic device, which is characterized by comprising a processor and a memory, wherein one or more readable instructions are stored on the memory, and the method of any one of the preceding claims is realized when the one or more readable instructions are executed by the processor.
Through the virus group four-step enrichment method, the LoD detected by the blood flow virus can be effectively improved by about 100 times, the detection effect of the clinical blood flow virus group is greatly improved, and the detection application of the sequencing technology in clinical infection is forcefully promoted. The virus pollution removal method can reduce the false positive result, avoid the false negative result judged by the conventional analysis method, improve the accurate detection of the positive result, effectively reduce the false positive result, ensure the accuracy in clinical application and be more beneficial to the clinical accurate diagnosis and detection of the blood flow virus infection.
The invention is further described by the accompanying drawings and the following examples, which are provided to illustrate specific embodiments of the invention and are not to be construed as limiting the scope of the invention in any way. Unless otherwise indicated, all experimental procedures disclosed herein employ techniques conventional in the art, and reagents and starting materials used in the examples are commercially available.
It is to be understood that the core concept of the present invention is not limited to a sequencing platform, since enrichment and subsequent sequence analysis of plasma viruses is not limited by the sequencing platform, and thus the sequencing platforms suitable for the "virome four-step enrichment method" and the "virome decontamination method" of the present invention include first-, second-, third-or fourth-generation sequencing data; preferably, the sequencing data is second generation sequencing data.
Examples
Example 1 viral detection LoD evaluation after four-step enrichment of viral groups
Adding CMV, HCV, HBV and Sars-Cov-2 virus standard into clinical negative blood plasma in spike-in manner, and dividing into 5 copies/ml, 5×10 copies/ml, and 5×10 copies/ml 2 copies/ml、5×10 3 copies/ml、5×10 4 copies/ml、5×10 5 The number of copies/ml was 5 and 10 times the number of gradients, each with 3 replicates, were used as control replicates for Lod evaluation using the methods of the invention and the traditional extraction and pooling methods, respectively.
The method comprises the following steps:
1. the "four-step enrichment of viral groups" enriches viral particles (see fig. 1):
1) Two-step centrifugation: collecting EDTA anticoagulation 4ml clinically and freshly, taking at least 2ml plasma, centrifuging at 3000r/min for 10 min, centrifuging at 12000r/min for 10 min, and reserving supernatant plasma to obtain centrifugal plasma;
2) Nuclease treatment: treating the centrifugal plasma with UniversalBenzo Nuclease omnipotent nuclease, and incubating for 1h at 37 ℃ to obtain cfDNA-removed plasma;
3) And (3) filtering by a filter membrane: filtering the cfDNA removed plasma obtained in the previous step by using a 0.45 μm PES filter (Millex) to obtain filtered plasma;
4) Ultrafiltration and centrifugal concentration: and (3) carrying out high-speed centrifugation on the plasma filtered by the filter membrane by using an Amicon Ultra ultrafiltration centrifugal filter device to obtain concentrated plasma, and further enriching virus particles.
2. Viral nucleic acid extraction
200ul of concentrated plasma was used to extract viral nucleic acid using the PureLinkTM Viral RNA/DNA Mini Kit according to the Kit instructions.
3. Pre-amplification of viral nucleic acids
Random anchored amplification is divided into two workflows, DNA and RNA
1) DNA flow
Taking 20ul of extracted nucleic acid, and carrying out one-strand synthesis and two-strand synthesis by using Takara enzyme by using random anchor primers (added with primer sequences);
2) RNA flow
Taking 11ul of extracted nucleic acid, carrying out one-strand synthesis by using a random anchor primer (added primer sequence), carrying out two-strand synthesis by using Klenow fragment;
the DNA and RNA flow products were mixed and subjected to 20-25 rounds of pre-amplification enrichment using the anchor primer and Takara enzyme.
4. Library construction and sequencing
Library construction was performed using a vazyme5ng starting amount genome fragmentation library-building kit (TD 501/TD 502) according to the kit instructions; then, an NGS sequencing platform PE150 strategy is used to perform on-machine sequencing, and each sample is used to test 6G raw data.
5. Sequencing data analysis
1) And (3) data quality control: performing data quality control on the original data obtained by library building sequencing by using fastp, and removing low-quality and shorter sequences according to default parameters to obtain clean data;
2) Removal of human sequences: comparing the clean data with the GRCH38 of the humanized genome by using comparison software, and removing sequences on the comparison to remove the humanized sequence to obtain a sequence after removing the humanized;
3) Virus identification: performing virus database comparison on the sequence after human source removal by using comparison software, and then performing taxonomy relation annotation on virus species by using a taxonomy zrlibrary package in R;
4) Processing samtools, bedtools according to the comparison result to obtain reads information, depth information and genome coverage of each sample identified virus species.
"virome decontamination method" to remove contaminating sequences
1) And (3) filtering the pollution in the batch: analyzing the sequencing samples in the same batch together, extracting base sequences with the occurrence frequency of more than 50% of the positions of the same genome of the same species in the same batch of samples according to the comparison result, namely, in-batch pollution sequences, and removing sequences (in-batch pollution sequences) with high occurrence probability in the same batch to obtain filtered sequences;
2) Non-specific viral sequence filtration: performing blast comparison on the virus database and human, bacteria, fungi and parasites databases respectively to obtain a common sequence of the virus, other microorganisms and human sources, namely a non-specific virus sequence, and removing the sequence of the non-specific virus sequence region on the comparison part;
3) Filtering non-human host virus: and (3) removing the virus of the non-human host by comparing the filtered sequences with host information of the virus species to obtain a virus detection result.
The results of the LoD test for virus detection according to the present invention obtained in the above steps are shown in table 1 below:
TABLE 1 four-step enrichment of the virosomes LoD
After library construction by conventional methods, the same data analysis steps (i.e., 5. Sequencing data analysis) and decontamination steps (i.e., 6."virome decontamination" to remove contaminating sequences) were performed according to this example to obtain the results of the virus detection LoD experiments as shown in table 2 below:
TABLE 2 LoD for conventional mNGS Virus detection group
The detection results of the viruses can show that the invention can greatly improve the virus detection capability of both DNA viruses and RNA viruses, the average LoD detection limit is improved by about 100 times, even the LoD detection limit can be as low as 5 Copies/ml, and the invention is greatly applicable to the detection of extremely low-abundance virus pathogens in clinical samples.
Example 2 clinical application of Virus enrichment and decontamination analysis methods
The method of the present invention was compared with a conventional method using clinical whole blood, and the conventional mNGS method was carried out by centrifuging whole blood, and then was set up and put on machine, and the analytical procedure was the same as that of the data analysis method in this example, and the detailed processing in this example is described below.
1. Constructing a database of human sources and various species comparison:
1) Human source database: GRCh38 whole genome was downloaded at NCBI.
2) Bacterial, fungal, parasite database: the taxonomic information of the taxonomic species (containing the taxonomic information of the taxonomic genus of the phylum family) is downloaded at the NCBI, and the data is downloaded from the micro-cal-nt database. The taxid of each major class is obtained by classification according to bacteria/archaea, fungi, parasites. According to the aligned seqid2 axid file (nt library), seqid of each sequence is obtained, and the following filtering is further carried out on the sequences in the database: i) Deleting sequences below 1 k; ii) bacteria/archaea and fungi: deleting unclassified and environmental microbial sequences; iii) Parasite: deletion of unclassified and environmental sequences and ectoparasite/insect sequences; iv) data de-redundant, with only one genome per species selected, ordered by reference genome, representative genome, complex genome, and preferentially selected in order.
3) Virus database: the Viralzone downloads the eukaryotic virus database Complete virus sequences.
Based on the three kinds of databases, a database and a virus identification database are constructed by respectively constructing a humanized alignment database and a consensus sequence.
2. The "virome four-step enrichment method" enriches virosomes:
1) Two-step centrifugation: collecting EDTA anticoagulation 4ml clinically and freshly, taking at least 2ml plasma, centrifuging at 3000r/min for 10 min, centrifuging at 12000r/min for 10 min, and reserving supernatant plasma to obtain centrifugal plasma;
2) Nuclease treatment: treating the centrifugal plasma with UniversalBenzo Nuclease omnipotent nuclease, and incubating for 1h at 37 ℃ to obtain cfDNA-removed plasma;
3) And (3) filtering by a filter membrane: filtering the cfDNA removed plasma obtained in the previous step by using a 0.45 μm PES filter (Millex) to obtain filtered plasma;
4) Ultrafiltration and centrifugal concentration: and (3) carrying out high-speed centrifugation on the plasma filtered by the filter membrane by using an Amicon Ultra ultrafiltration centrifugal filter device to obtain concentrated plasma, and further enriching virus particles.
3. Viral nucleic acid extraction
200ul of concentrated plasma was used to extract viral nucleic acid using the PureLinkTM Viral RNA/DNA Mini Kit according to the Kit instructions.
4. Pre-amplification of viral nucleic acids
Random anchored amplification is divided into two workflows, DNA and RNA
1) DNA flow
Taking 20ul of extracted nucleic acid, and carrying out one-strand synthesis and two-strand synthesis by using a random anchor primer and Takara enzyme;
2) RNA flow
Taking 11ul of extracted nucleic acid, carrying out one-strand synthesis by using random anchor primers and reverse transcriptase, and carrying out two-strand synthesis by using Klenow fragment;
the DNA and RNA flow products were mixed and subjected to 20-25 rounds of pre-amplification enrichment using the anchor primer and Takara enzyme.
5. Library construction and sequencing
Library construction was performed using a vazyme5ng starting amount genome fragmentation library-building kit (TD 501/TD 502) according to the kit instructions; then, an NGS sequencing platform PE150 strategy is used to perform on-machine sequencing, and each sample is used to test 6G raw data.
6. Sequencing data analysis
1) And (3) data quality control: performing data quality control on the original data obtained by library building sequencing by using fastp, and removing low-quality and shorter sequences according to default parameters to obtain clean data;
2) Removal of human sequences: comparing the clean data with the GRCH38 of the humanized genome by using comparison software, and removing sequences on the comparison to remove the humanized sequence to obtain a sequence after removing the humanized;
3) Virus identification: performing virus database comparison on the sequence after human source removal by using comparison software, and then performing taxonomy relation annotation on virus species by using a taxonomy zrlibrary package in R;
4) Processing the sample according to the comparison result by samtools, bedtools to obtain reads and depth information of each sample for identifying virus species;
5) And analyzing the virus species detected in the sample according to the comparison result to obtain the numbers of reads of each species and genome coverage thereof.
"virome decontamination method" to remove contaminating sequences
1) And (3) filtering the pollution in the batch: analyzing the sequencing samples in the same batch together, extracting base sequences with the occurrence frequency of more than 50% of the positions of the same genome of the same species in the same batch of samples according to the comparison result, namely, in-batch pollution sequences, and removing sequences (in-batch pollution sequences) with high occurrence probability in the same batch to obtain filtered sequences;
2) Non-specific viral sequence filtration: performing blast comparison on the virus database and human, bacteria, fungi and parasites databases respectively to obtain a common sequence of the virus, other microorganisms and human sources, namely a non-specific virus sequence, and removing the sequence of the non-specific virus sequence region on the comparison part;
3) Filtering non-human host virus: and (3) removing the virus of the non-human host by comparing the filtered sequences with host information of the virus species to obtain a virus detection result.
The results of analysis by conventional plasma mNGS sequencing and the methods of the examples of the invention are shown in table 3 below:
TABLE 3 comparison of conventional mNGS sequencing and viral four-step enrichment method mNGS sequencing
The table shows that the ratio of the viral sequences enriched by the method in the final embodiment of the invention is improved by more than 100 times compared with that of the conventional method, the viruses are remarkably enriched, and the detection capability of clinical virus groups is greatly improved.
Example 3 comparison of virome decontamination method with traditional filtration method
Clinical whole blood is collected, subjected to virus enrichment by the method of the invention, and then subjected to on-machine sequencing in a warehouse, and then the advantages of the virus decontamination method are compared in parallel by adopting the virus decontamination method of the invention and a conventional mNGS data filtering analysis method according to the results, and the detailed treatment of the embodiment is described below (refer to figure 2).
1. Constructing a database of human sources and various species comparison:
1) Human source database: GRCh38 whole genome was downloaded at NCBI.
2) Bacterial, fungal, parasite database: the taxonomic information of the taxonomic species (containing the taxonomic information of the taxonomic genus of the phylum family) is downloaded at the NCBI, and the data is downloaded from the micro-cal-nt database. The taxid of each major class is obtained by classification according to bacteria/archaea, fungi, parasites. According to the aligned seqid2 axid file (nt library), seqid of each sequence is obtained, and the following filtering is further carried out on the sequences in the database: i) Deleting sequences below 1 k; ii) bacteria/archaea and fungi: deleting unclassified and environmental microbial sequences; iii) Parasite: deletion of unclassified and environmental sequences and ectoparasite/insect sequences; iv) data de-redundant, with only one genome per species selected, ordered by reference genome, representative genome, complex genome, and preferentially selected in order.
3) Virus database: the Viralzone downloads the eukaryotic virus database Complete virus sequences.
Based on the three kinds of databases, a database and a virus identification database are constructed by respectively constructing a humanized alignment database and a consensus sequence.
The "virome four-step enrichment method" enriches virosomes:
1) Two-step centrifugation: collecting EDTA anticoagulation 4ml clinically and freshly, taking at least 2ml plasma, centrifuging at 3000r/min for 10 min, centrifuging at 12000r/min for 10 min, and reserving supernatant plasma to obtain centrifugal plasma;
2) Nuclease treatment: treating the centrifugal plasma with UniversalBenzo Nuclease omnipotent nuclease, and incubating for 1h at 37 ℃ to obtain cfDNA-removed plasma;
3) And (3) filtering by a filter membrane: filtering the cfDNA removed plasma obtained in the previous step by using a 0.45 μm PES filter (Millex) to obtain filtered plasma;
4) Ultrafiltration and centrifugal concentration: and (3) carrying out high-speed centrifugation on the plasma filtered by the filter membrane by using an Amicon Ultra ultrafiltration centrifugal filter device to obtain concentrated plasma, and further enriching virus particles.
3. Viral nucleic acid extraction
200ul of concentrated plasma was used to extract viral nucleic acid using the PureLinkTM Viral RNA/DNA Mini Kit according to the Kit instructions.
4. Pre-amplification of viral nucleic acids
Random anchored amplification is divided into two workflows, DNA and RNA
1) DNA flow
Taking 20ul of extracted nucleic acid, and carrying out one-strand synthesis and two-strand synthesis by using a random anchor primer and Takara enzyme;
2) RNA flow
Taking 11ul of extracted nucleic acid, carrying out one-strand synthesis by using random anchor primers and reverse transcriptase, and carrying out two-strand synthesis by using Klenow fragment;
the DNA and RNA flow products were mixed and subjected to 20-25 rounds of pre-amplification enrichment using the anchor primer and Takara enzyme.
5. Library construction and sequencing
Library construction was performed using a vazyme5ng starting amount genome fragmentation library-building kit (TD 501/TD 502) according to the kit instructions; then, an NGS sequencing platform PE150 strategy is used to perform on-machine sequencing, and each sample is used to test 6G raw data.
6. Sequencing data analysis
1) And (3) data quality control: performing data quality control on the original data obtained by library building sequencing by using fastp, and removing low-quality and shorter sequences to obtain clean data;
2) Removal of human sequences: comparing the clean data with the constructed human sequence database by using comparison software bowtie2, and removing the human sequence after comparison to obtain a sequence after removing human;
3) Virus identification: performing virus database comparison on the sequence after human source removal by using comparison software, and then performing taxonomy relation annotation on virus species by using a taxonomy zrlibrary package in R;
4) And processing according to the comparison result by samtools, bedtools to obtain reads information, depth information and genome coverage of each sample.
"virome decontamination method" to remove contaminating sequences
1) And (3) filtering the pollution in the batch: analyzing the sequencing samples in the same batch together, extracting base sequences with the occurrence frequency of more than 50% of the positions of the same genome of the same species in the same batch of samples according to the comparison result, namely, in-batch pollution sequences, and removing sequences (in-batch pollution sequences) with high occurrence probability in the same batch to obtain filtered sequences;
2) Non-specific viral sequence filtration: performing blast comparison on the virus database and human, bacteria, fungi and parasites databases respectively to obtain a common sequence of the virus, other microorganisms and human sources, namely a non-specific virus sequence, and removing the sequence of the non-specific virus sequence region on the comparison part;
3) Filtering non-human host virus: and (3) removing the virus of the non-human host by comparing the filtered sequences with host information of the virus species to obtain a virus detection result.
The results of analysis data filtered by the "virome decontamination method" in this example with conventional according to reads or RPM-Ratio values are shown in Table 4 below:
TABLE 4 alignment of traditional filtration methods and virome decontamination methods
* The conventional RPM-Ratio method threshold is 10; "+": a positive result; "-": negative results
As can be seen from the above table, the CMV virus that is clinically positive in Sample1 in the conventional method was judged to be negative, but positive could be accurately judged by the "virome decontamination method", and the CMV, CMV and EBV clinical positive results corresponding to Sample2, sample3 and Sample4 respectively were judged to be positive (false positive results) in the conventional method, and the "virome decontamination method" could be accurately judged to be negative. The above shows that the virus group pollution-removing method can more accurately judge the detection of clinical sample virus than the conventional method, and greatly reduces the false positive of the judging result.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (10)
1. A method for detecting and analyzing plasma viruses based on a sequencing technology, which is characterized by comprising the following steps:
enriching plasma sample virus particles, and virus detection analysis;
wherein the enriching of the plasma sample virus particles is performed as follows:
and (3) centrifuging: centrifuging the collected blood to prepare centrifuged plasma;
nuclease treatment step: treating the centrifuged plasma with a omnipotent nuclease;
and (3) filtering: filtering the centrifuged plasma treated with the omnipotent nuclease to obtain filtered plasma;
ultrafiltration and centrifugal concentration steps: centrifuging the filtered plasma by using an ultrafiltration centrifugal filter device to obtain concentrated plasma;
the virus detection analysis was performed as follows:
carrying out virus sequencing on the concentrated plasma to obtain a sequencing result;
carrying out virus sequencing analysis on the sequencing result to obtain a sequencing analysis result;
performing a virome decontamination assay on the sequencing assay results,
wherein said performing a virome decontamination assay on said sequencing result comprises:
and (3) filtering the pollution in the batch: removing intra-batch contamination sequences in the same-batch sequencing sample;
non-specific viral sequence filtration: removing non-specific viral sequences shared by the virus and other microorganisms and human sources;
filtering non-human host virus: and removing viruses of non-human hosts to obtain a virus detection result.
2. The sequencing-based plasma virus detection assay of claim 1, wherein the centrifugation step comprises: removing precipitate by low-speed centrifugation, centrifuging at high speed, and retaining supernatant to obtain centrifugal plasma.
3. The method according to claim 1, wherein in the step of filtering, a PES filter is used to filter the centrifuged plasma treated with the omnipotent nuclease.
4. The method of claim 1, wherein the viral sequencing is performed as follows:
extracting virus nucleic acid and pre-amplifying;
constructing a library by using a library constructing kit;
sequencing was performed on a sequencing platform.
5. The method of claim 1, wherein the viral sequencing analysis is performed as follows:
(1) Performing data quality control on the original data obtained by sequencing, and removing low-quality and shorter sequences to obtain clean data;
(2) Comparing the clean data with the human genome by using comparison software, and removing the sequence of the human after comparison to obtain a sequence after removing the human;
(3) The sequence after human source removal is subjected to virus database comparison by using comparison software, and then virus species annotation is carried out;
(4) And obtaining reads information, depth information and genome coverage of the virus species in each sample according to the comparison result statistics.
6. The method of claim 1, wherein the sequence of contamination in the batch is selected from sequences with a frequency of occurrence of more than 50% at the same genomic position of the same species in the same batch of sequencing samples.
7. The method for detecting and analyzing plasma viruses based on the sequencing technology according to claim 1, wherein,
the non-specific viral sequences include consensus sequences of viruses with other species,
the other species comprises one or more selected from human sources, bacteria, fungi, parasites,
the consensus sequences of the viruses and other species are obtained by comparing the virus database with one or more of human, bacterial, fungal and parasite databases respectively, and selecting the sequence or sequence region in any of the human, bacterial, fungal and parasite databases to which the virus sequences are compared.
8. The sequencing technology based plasma virus detection and analysis method according to claim 1, wherein the method further comprises: adding a negative control sample to the same batch of sequencing samples for virome decontamination analysis;
the virome decontamination assay further comprises: sequencing each sample in the same batch removes virus species detected in the negative control sample that are more abundant than the sequencing sample in the same batch.
9. A computer readable medium storing a computer program which, when executed by a processor, implements the method of any one of claims 1 to 8.
10. An electronic device comprising a processor and a memory having stored thereon one or more readable instructions which, when executed by the processor, implement the method of any of claims 1 to 8.
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