CN111455102A - Preparation method of capture probe for target sequencing of new coronavirus SARS-CoV-2 genome - Google Patents
Preparation method of capture probe for target sequencing of new coronavirus SARS-CoV-2 genome Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
Abstract
A process for preparing the capture probe used for target sequencing of SARS-CoV-2 genome of new coronavirus includes downloading the reference genome sequence NC-045512.2 of SARS-CoV-2 virus from NCBI database, screening 75-120nt probes according to GC content, Tm value and homology, quantitatively evaluating the probe library, filtering out unqualified probe sequences, covering the whole genome of new coronavirus with 249 probes, preparing DNA template of 249 probe library, PCR amplifying, purifying PCR product, digestion of purified PCR product, smooth compensation of T4DNA polymerase end, digestion of single-chain DNA with 5' -end phosphorylation by L ambda exonuclease to obtain single-chain DNA with biotin label, purifying the single-chain DNA with biotin label by magnetic bead method, and obtaining modified probe library.
Description
Technical Field
The present invention relates to genome-targeted sequencing, and in particular to sequencing for targeted NGS against the genome of the novel coronavirus SARS-CoV-2.
Background
In the field of nucleic acid detection methods, subsequent to PCR and sequencing of one generation, Next Generation Sequencing (NGS) is widely used in various fields of application of nucleic acid molecular diagnostics. As technology advances, NGS is increasingly data throughput and sequencing costs are increasingly low. However, whole genome sequencing of large sample sizes of different species even whole genome sequencing of a single sample is relatively difficult to practice, both from a cost standpoint and from a data analysis standpoint.
The target region is targeted and captured and then subjected to NGS sequencing (as shown in figure 1), and the DNA fragments of the target gene or the genome region are directionally enriched from the whole genome and then subjected to NGS sequencing, so that the cost is greatly reduced, and the pressure of subsequent data analysis is also relieved. Compared with the conventional NGS method, the sequencing is carried out after the target capture, so that the sequencing cost and the data analysis cost are greatly reduced, and the detection sensitivity is improved. At present, the kit is widely applied to clinical detection of human full exome, and is rarely applied to pathogenic microorganisms.
In the diagnosis and treatment plan for coronavirus pneumonia (trial fifth edition), the method for detecting nucleic acid mainly relies on RT-PCR to confirm whether suspected cases are positive cases, but in the subsequent practice, the method has high undetected rate, and the reasons are as follows:
1) generally, 3 target regions, namely ORF1b of SARS-CoV-2 genome, N end and nucleic acid sequence coding core shell are amplified by RT-PCR method, and the kits approved by national drug administration on the market detect more than 1 target region;
2) usually, the target region of SARS-CoV-2 genome amplified by RT-PCR primer design is conserved sequence, but the new coronavirus is RNA virus, once mutation and recombination occur, the RT-PCR primer is difficult to amplify effectively;
3) the viral load of new coronary patients of symptom representatives or asymptomatic carriers is unstable, and even if the detection result obtained by the RT-PCR method is positive, the situation of bacterial infection or virus co-infection cannot be eliminated;
4) the detection of the novel coronavirus SARS-CoV-2 in the peripheral blood and urine of a patient by amplifying the N-terminal spiegene is unstable and reliable.
The NGS method plays an irreplaceable role in the detection and identification of the new coronavirus SARS-CoV-2, and has higher accuracy and sensitivity compared with the widely applied RT-PCR method nucleic acid detection technology. Therefore, the diagnosis and treatment plan for coronavirus pneumonia (trial seventh edition) increased "detection of nucleic acids by RT-PCR or/and NGS method", and emphasized "detection of lower respiratory tract specimens (sputum or airway extracts) was more accurate. "NGS differs significantly from RT-PCR from a methodological point of view. The existing RT-PCR method is mainly used for targeted amplification of 3 target regions in a novel coronavirus genome, and NGS can cover the whole genome region of the novel coronavirus, so that on one hand, false negative caused by mutation of an RT-PCR target region can be prevented, on the other hand, more sequence information can be obtained, more comprehensive whole genome sequence information is assembled, and the method is used for source-tracing evolution analysis, virus propagation modes, discovery of new mutation sites and the like.
For biological samples from mass sources, if the pathogen load is low, the NGS still has difficulty in ensuring the sensitivity of clinical detection after effectively removing the data of human hosts.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a preparation method of a capture probe for targeted sequencing of a novel coronavirus SARS-CoV-2 genome. The invention greatly reduces the sequencing data volume, simplifies the data analysis process and further improves the sensitivity and the accuracy of the NGS method.
The invention relates to a method for preparing a capture probe for the targeted sequencing of a novel coronavirus SARS-CoV-2 genome, which comprises the following steps:
downloading a SARS-CoV-2 virus reference genome sequence NC-045512.2 from an NCBI database, adopting a design strategy of imbricated coverage, and according to the length of a quantitative parameter probe of 75-120 nt;
step two, filtering out unqualified probe sequences and reserving 249 probe libraries;
step three, preparing DNA templates of 249 probe libraries, and purifying and quantifying PCR products after PCR amplification;
step four, carrying out enzyme digestion and T4DNA polymerase end smooth filling on the purified PCR product, and then digesting a single strand with 5' end phosphorylation in the double-strand DNA by using L ambda exonuclease so as to obtain single-strand DNA with biotin labels;
and step five, purifying the single-stranded DNA with the biotin label by a magnetic bead enrichment method to obtain a modified probe library.
The method for preparing the capture probe for the targeted sequencing of the genome of the novel coronavirus SARS-CoV-2 comprises the following steps of:
GC%, if < 25% or > 55%, removed;
tm value, if Tm <55 ℃ or >85 ℃, removed;
b L ASTN alignment, in all species, especially in the host owner's GeneBank genome sequence, more than or equal to 80% of the homology sequence probe, remove.
In the invention, for biological samples from mass sources, if the pathogen capacity is low, the NGS still has difficulty in ensuring the sensitivity of clinical detection after effectively removing the data of human hosts. In order to improve the deficiency of the NGS process, a probe covering SARS-CoV-2 virus genome is designed, SARS-CoV-2 virus genome nucleic acid sequence is directionally captured from a biological sample with mass sources, and then deep sequencing is carried out, so that the sequencing data volume is expected to be greatly reduced, the data analysis process is simplified, the detection period is shortened, and the sensitivity and the accuracy of the NGS method are further improved.
Drawings
FIG. 1 is a schematic flow diagram of the present invention;
FIG. 2 is a schematic diagram of the shingle overlay design strategy.
Detailed Description
The invention relates to a method for preparing a capture probe for the targeted sequencing of a new coronavirus SARS-CoV-2 genome, which comprises the following steps:
downloading a SARS-CoV-2 virus reference genome sequence NC-045512.2 from an NCBI database, adopting a design strategy of overlapping coverage, namely covering a probe from top to bottom, and adjusting the length of the probe to 75-120nt according to GC content;
step two, carrying out quantitative evaluation on the probe library, and filtering out unqualified probe sequences; removing unqualified probes, and then leaving 249 probe libraries to cover 99.23% of the whole genome of the new coronavirus;
step three, preparing DNA templates of 249 probe libraries, carrying out PCR amplification, and purifying PCR products;
step four, carrying out enzyme digestion and T4DNA polymerase end smooth filling on the purified PCR product, and then digesting a single strand with 5' end phosphorylation in the double-strand DNA by using L ambda exonuclease so as to obtain single-strand DNA with biotin labels;
and step five, purifying the single-stranded DNA with the biotin label by a magnetic bead enrichment method to obtain a modified probe library.
And (3) optimally adjusting the proportion of the concentration of each probe in the probe library through experiments to form a final capture probe product.
The method for preparing the capture probe for the targeted sequencing of the genome of the novel coronavirus SARS-CoV-2 comprises the following steps of:
GC%, if < 25% or > 55%, removed;
tm value, if Tm <55 ℃ or >85 ℃ is removed;
b L ASTN alignment, in all species, especially in the host owner's GeneBank genome sequence, remove more than 80% of the homology sequence probe.
Claims (2)
1. The preparation method of the capture probe for the genome targeted sequencing of the novel coronavirus SARS-CoV-2 is characterized by comprising the following steps:
downloading a SARS-CoV-2 virus reference genome sequence NC-045512.2 from an NCBI database, adopting a design strategy of imbricated coverage, and according to the length of a quantitative parameter probe of 75-120 nt;
step two, filtering out unqualified probe sequences and reserving 249 probe libraries;
step three, preparing DNA templates of 249 probe libraries, and purifying and quantifying PCR products after PCR amplification;
step four, carrying out enzyme digestion and T4DNA polymerase end smooth filling on the purified PCR product, then digesting a single strand with 5' end phosphorylation in the double-strand DNA by using L ambda exonuclease, thereby obtaining single-strand DNA with biotin label;
and step five, purifying the single-stranded DNA with the biotin label by a magnetic bead enrichment method to obtain a modified probe library.
2. The method for preparing capture probe for targeted sequencing of the genome of the novel coronavirus SARS-CoV-2 according to claim 1, wherein the quantitative evaluation rule for the probe library in the second step is:
GC%, if < 25% or > 55%, removed;
tm value, if Tm <55 ℃ or >85 ℃, removed;
b L ASTN alignment, in all species, especially in the host owner's GeneBank genome sequence, more than or equal to 80% of the homology sequence probe, remove.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112195281A (en) * | 2020-11-12 | 2021-01-08 | 武汉凯德维斯生物技术有限公司 | Probe for detecting human papilloma virus HPV56 and kit thereof |
| CN112322788A (en) * | 2020-11-24 | 2021-02-05 | 杭州杰毅生物技术有限公司 | mNGS primer group and kit for detecting SARS-CoV-2 |
| CN113327646A (en) * | 2021-06-30 | 2021-08-31 | 南京医基云医疗数据研究院有限公司 | Sequencing sequence processing method and device, storage medium and electronic equipment |
| CN115346606A (en) * | 2022-10-17 | 2022-11-15 | 南京诺因生物科技有限公司 | Method and system for designing targeting probe based on species sequence |
| CN113327646B (en) * | 2021-06-30 | 2024-04-23 | 南京医基云医疗数据研究院有限公司 | Sequencing sequence processing method and device, storage medium and electronic equipment |
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2020
- 2020-04-09 CN CN202010272077.XA patent/CN111455102A/en active Pending
Patent Citations (2)
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| GB2401175A (en) * | 2003-05-02 | 2004-11-03 | Hong Kong Dna Chips Ltd | Detection of SARS virus by PCR |
| CN105647907A (en) * | 2016-03-04 | 2016-06-08 | 杭州联川生物技术有限公司 | Preparation method of modified DNA (deoxyribonucleic acid) hybridization probe for targeted hybrid capture |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112195281A (en) * | 2020-11-12 | 2021-01-08 | 武汉凯德维斯生物技术有限公司 | Probe for detecting human papilloma virus HPV56 and kit thereof |
| CN112322788A (en) * | 2020-11-24 | 2021-02-05 | 杭州杰毅生物技术有限公司 | mNGS primer group and kit for detecting SARS-CoV-2 |
| CN112322788B (en) * | 2020-11-24 | 2021-07-06 | 杭州杰毅生物技术有限公司 | mNGS primer group and kit for detecting SARS-CoV-2 |
| CN113327646A (en) * | 2021-06-30 | 2021-08-31 | 南京医基云医疗数据研究院有限公司 | Sequencing sequence processing method and device, storage medium and electronic equipment |
| CN113327646B (en) * | 2021-06-30 | 2024-04-23 | 南京医基云医疗数据研究院有限公司 | Sequencing sequence processing method and device, storage medium and electronic equipment |
| CN115346606A (en) * | 2022-10-17 | 2022-11-15 | 南京诺因生物科技有限公司 | Method and system for designing targeting probe based on species sequence |
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