CN109487345B - Nanopore sequencing platform-based metagenome sample library building method, identification method and kit - Google Patents
Nanopore sequencing platform-based metagenome sample library building method, identification method and kit Download PDFInfo
- Publication number
- CN109487345B CN109487345B CN201811528417.XA CN201811528417A CN109487345B CN 109487345 B CN109487345 B CN 109487345B CN 201811528417 A CN201811528417 A CN 201811528417A CN 109487345 B CN109487345 B CN 109487345B
- Authority
- CN
- China
- Prior art keywords
- sample
- final concentration
- host
- host dna
- saponin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
-
- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
- C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a metagenome sample library construction method, an identification method and a kit based on a nanopore sequencing platform. The method is based on the combination of a nanopore sequencing platform and incomplete dephospitation, and carries out dephospitation pretreatment and sample library construction on human and animal metagenome samples, thereby being used for sequencing identification of metagenome. Compared with the traditional process, the method is simpler and faster, meets the reasonable detection requirement, omits the processes of establishing a library PCR and the like in the identification process, eliminates the bias caused by PCR amplification, and can identify the metagenome microorganisms more truly and accurately.
Description
Technical Field
The invention relates to the field of metagenome identification, in particular to a metagenome sample library construction method, an identification method and a kit based on a nanopore sequencing platform.
Background
Compared with the traditional culture identification method, the metagenome sequencing identification method has the advantages of short identification period, low requirements on technical levels of operators and identification personnel and the like. And the metagenome sequencing overcomes the identification defects of a plurality of microbes which can not be cultured, and is increasingly applied to the identification of the microbes, in particular to the identification of pathogenic microbes with unknown causes. At present, the mainstream metagenome sequencing method is mainly a high-throughput sequencing method, and comprises second-generation sequencing and third-generation sequencing. The second generation sequencing identification has many problems, such as: the identification of next generation sequencing is usually from sample to result with a long cycle (>72 h); the sequencing reading length increases the difficulty and the time of letter generation splicing; the sequencer is large in investment, and multiple samples are generally needed to be combined for sequencing so as to reduce the cost; third generation sequencing such as nanopore sequencing seems to be a fast and convenient metagenome research method at present due to its excellent characteristics including ultra-long sequence read length, real-time data generation and raw information analysis, and small and portable equipment.
However, high-throughput sequencing, whether second-generation sequencing or third-generation sequencing, is based on the genome of the sample, but in most clinical samples, due to inflammatory reactions and the like, a large amount of host DNA often exists, and pathogen DNA accounts for only a small portion. Therefore, only a small portion of the obtained sequencing data can be used for true species and drug resistance gene identification, and the filtering of host sequences from a large amount of raw data by bioinformatics methods requires a large amount of computational power and is time-consuming, and also affects the sensitivity of low-abundance pathogen identification in clinical samples.
Among the current rapid identification procedures developed based on the nanopore sequencing platform, the wet experiment for infected samples developed by professor Grady in the UK (DOI: https:// doi.org/10.1101/387548) group to host and rapid PCR barcode merging and library construction is the most mature technical route at present (see FIG. 1). The method of Grady firstly frees host DNA by destroying host cell membranes, then degrades the host DNA by salt-dependent HL-SAN enzyme, centrifugally collects microbial thalli mainly containing bacteria, and extracts the bacterial DNA, and the amount of nucleic acid obtained by the process is usually very low. Therefore, the rapid PCR library building kit with low initial quantity requirement (1-5ng) is adopted, the total amount of DNA is increased by adding a bar code to transposase and then by one-step whole genome PCR amplification, the library building can be completed by connecting a rapid joint, the identification of bacterial pathogens can be successfully completed by real-time baseloading of on-board sequencing data and total data of 2 h.
However, this method has several disadvantages:
firstly, some necessary purification steps exist in the whole wet experiment process, so the whole process can be completed within 4 hours unlike the process presented in the article, and the whole process needs 5.5 hours according to actual inspection; secondly, the step of removing host DNA removes most of the host DNA in the sample, so that the extraction concentration of the sample is very low. For example, the library cannot be continuously built due to PCR failure in the process of building the library for the sample; more importantly, amplification bias is inevitably introduced due to the PCR step in the library building process, so that a serious problem occurs in the identification of some bacteria with high GC content, and the abundance ratio of the strains is seriously unbalanced.
Therefore, there is still a need in the art for a more efficient and convenient method for identifying metagenome. In view of the above, the present invention is particularly proposed.
Disclosure of Invention
In order to accurately obtain the sequence information of the metagenome of the sample and identify the species, the field needs to carry out host-free pretreatment on the sample. The technical prejudice in the prior art is often to remove the host components as much as possible during the host-removing process, and then to select samples without host DNA contamination to be subjected to a library-building process, such as the Grady method mentioned above and some mature and commercial kits (MolYsis)TMHost DNA removal kit) is this concept. In the research process, the complete host removal ensures the purity of the sample, but also brings certain challenges to later experiments, for example, the sudden reduction of the host removal sample amount requires PCR amplification to enrich the DNA content, so that the process time is increased, and the amplification bias is introduced. Considering that the whole process of metagenome sequencing and identification comprises not only the steps of removing a sample from a host, but also multiple steps of nucleic acid extraction, nucleic acid library construction, on-machine sequencing, biological analysis and the like, onlyThe most rapid and accurate comprehensive coordination of all steps is realized, and the process with the lowest cost seems to be the most ideal metagenome identification scheme.
According to the invention, host removal and sequencing optimization are carried out on different types of clinical samples, so that optimal systems suitable for the different types of samples are respectively obtained, and the optimal systems have the characteristics of short running time, accurate result, high cost and the like of the whole process. And surprisingly, the optimal systems have a common characteristic that the prejudice of complete host removal is overcome, and the methods of incomplete host removal and non-PCR library building are adopted. By incompletely derogating the sample, the host proportion of the sample is lower than that of the original sample, but higher than that of the conventional library establishing process of Grady. Therefore, the difficulty of letter generation analysis caused by PCR bias can be avoided, the total amount of DNA is ensured to be enough for building a library and operating, the library is built without a step of building a library and PCR, joint sequencing can be directly added, the library building time is shortened, more importantly, the composition and abundance of strains in a sample are more truly reflected, and the accuracy/goodness of fit is far higher than that of Grady by processing clinical samples, so that the method has great commercial popularization value.
The first purpose of the invention is to provide a metagenome sample library construction method based on a nanopore sequencing platform, which realizes the library construction without PCR amplification by optimizing incomplete host removal in the sample library construction process and meets the sequencing requirement.
The second purpose of the invention is to provide a metagenome identification method based on a nanopore sequencing platform, which realizes the completion of library construction without PCR amplification by optimizing the incomplete removal of a host in the process of sample library construction, and meets the sequencing requirement.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
a metagenome sample library construction method based on a nanopore sequencing platform comprises a step of incompletely removing host DNA and a step of directly constructing a library without PCR amplification.
In some embodiments, the incomplete removal of host DNA comprises: dissociating and degrading the host DNA, and separating the degraded host DNA from the sample.
In some embodiments, the host DNA episomally comprises: adding saponin into the sample to a final concentration of 0.08-0.25%, and incubating for 5-15 mm, preferably, the final concentration of the saponin in the sample is 0.8%, 0.11%, 0.13%, 0.2%, 0.22% or 0.25%, and the incubation time of the saponin is 5min, 7min, 10min, 12min or 15 min.
In some embodiments, the host DNA degradation comprises: adding HL-SAN enzyme into the sample after the host DNA is dissociated to a final concentration of 10-55U/mL, and incubating for 10-20 min, preferably, the final concentration of the HL-SAN enzyme is 10U/mL, 15.6U/mL, 20U/mL, 25U/mL, 30U/mL, 35U/mL, 40U/mL, 45U/mL, 47.9U/mL, 50U/mL or 55U/mL, and the incubation time of the HL-SAN enzyme is 10min, 12min, 15min, 17min or 20 min.
In some specific embodiments, the sample is a viscous material at the time of collection (optionally, the viscous material is sputum, pus, or nasal secretions), or the sample is a non-viscous material at the time of collection (optionally, the non-viscous material is alveolar lavage fluid).
In some specific embodiments, when the sample is a viscous substance at the time of collection, the saponin has a final concentration of 0.2-0.25%, preferably 0.22%, and the HL-SAN enzyme has a final concentration of 30-55U/mL, preferably 35U/mL, 40U/mL, 45U/mL, 47.9U/mL, or 50U/mL.
In some specific embodiments, when the sample is a non-viscous substance at the time of collection, the final concentration of the saponin is 0.08-0.13%, preferably 0.11%, and the final concentration of the HL-SAN enzyme is 10-30U/mL, preferably 15.6U/mL, 20U/mL or 25U/mL.
In some embodiments, the sample size of the viscous material is 150 to 250 μ L, preferably 200 μ L; the sampling amount of the non-viscous substance is 0.5-1.5 mL, and preferably 1 mL.
In some embodiments, the method further comprises, prior to dissociating the host DNA, pre-treating the sample, the pre-treating comprising: centrifuging, discarding the supernatant, and resuspending and centrifuging the obtained precipitate to prepare a resuspension solution; preferably, the centrifugal force is 12000-14000g, the centrifugation time is 3-7 min (optionally, 5min), and the volume of the resuspension solution is 200-300 μ L, preferably 250 μ L.
In some specific embodiments, the isolation of the host DNA from the sample comprises: after host DNA is degraded, adding a buffer solution into a sample, uniformly mixing, centrifuging, removing a supernatant, and keeping a precipitate; preferably, the buffer solution is PBS, the volume of the buffer solution is 500-100 μ L, the centrifugal force is 12000-14000g, the centrifugal time is 3-5 min, and the precipitate at most 50 μ L of supernatant is retained.
In some specific embodiments, the centrifuging step comprises: taking 200 mu L-1mL metagenome sample, and centrifuging until precipitation appears; preferred centrifugation steps: 200 mu L-1mL human or animal metagenome sample is taken to be placed in a 2mL round-bottom centrifuge tube, and is centrifuged for 5min at 12000-14000g until precipitation appears.
In some embodiments, the host DNA episomal step comprises: resuspend with 250 μ L PBS, add saponin solution, mix well by aspiration, and incubate at room temperature for 10min by rotation. Preferred host DNA episomes include: resuspending with 250 μ L PBS, adding 200 μ L saponin solution, and mixing by blowing and sucking up and down; performing rotary incubation on a rotary mixer at room temperature for 10min, blowing and mixing uniformly every 2min in the process, adding 350 μ L of water, blowing and mixing uniformly by using a gun, adding 12 μ L of 5M NaCl after 30s, and immediately shaking; the preparation method of the saponin solution comprises the following steps: 500mg saponin +10ml PBS, filtering, sterilizing, storing at room temperature in dark place, and diluting to working solution concentration before use.
In some embodiments, the host DNA degradation comprises: adding HL-SAN enzyme, and incubating for 15 min; preferably, the host DNA degradation comprises: adding 800 μ L HL-SAN buffer solution, and blowing and mixing uniformly by using a gun, wherein the HL-SANbbuffer solution is 5MNaCl and 100mM MgCl2An aqueous solution; adding 1-3 μ L HL-SAN enzyme 25U/μ L, mixing immediately, incubating at 37 deg.C and 800rpm for 15 min.
In some specific embodiments, said isolating the degraded host DNA from the sample comprises: PBS is mixed evenly, centrifuged and the supernatant is discarded; preferably comprising: adding 800 μ L PBS, blowing and mixing uniformly by a gun, centrifuging for 3min at 12000-14000g, discarding the supernatant, collecting without touching the precipitate, and keeping less than or equal to 50 μ L of the supernatant.
In some specific embodiments, the library building is performed by using an ONT library building kit (SQK-RBK004), and the specific method is as follows:
1) breaking and adding the barcode: sample loading system (200 μ L PCR vial):
remove host DNA 400ng, make up volume to 7.5. mu.L
Bar code RB01-12 (one for each sample) 2.5 μ L
Reaction system: 1min at 30 ℃;
80℃1min；
2) mixing and purifying: the total mass of the mixed sample is 1-1.8 mug, the dosage of each sample depends on the specific concentration, the sample is purified by 1 multiplied by magnetic beads, and 13 mul 10mM Tris-HCl pH 7.5-8.0 is eluted by 50mM NaCl;
3) adding a joint: mu.L of RAP was added to the above 10. mu.L sample, and the reaction was carried out at room temperature for 10 min.
In some embodiments, the sample is an infectious sample, preferably a non-viral infectious sample, and more preferably a bacterial infectious sample.
In some specific embodiments, the infection sample is a respiratory infection sample; preferably, the respiratory infection sample is derived from sputum and alveolar lavage fluid.
In some embodiments, the sample is derived from a human or animal subject.
In some aspects, the invention also relates to a metagenome identification method based on the nanopore sequencing platform, and the method further comprises the step of performing on-machine sequencing and credit-generation analysis on the library building sample by using the nanopore sequencing platform on the basis of the library building method.
In some aspects, the invention also relates to a metagenomic sample off-host kit based on a nanopore sequencing platform, the kit comprising a saponin solution with a final concentration of 0.08-0.25% and HL-SAN enzyme with a final concentration of 10-55U/mL.
Preferably, the final concentration of the saponin is 0.8%, 0.11%, 0.13%, 0.2%, 0.22% or 0.25%, and the final concentration of the HL-SAN enzyme is 0U/mL, 15.6U/mL, 20U/mL, 25U/mL, 30U/mL, 35U/mL, 40U/mL, 45U/mL, 47.9U/mL, 50U/mL or 55U/mL.
Preferably, the kit is used for viscous samples, and comprises a saponin solution with a final concentration of 0.2-0.25% and HL-SAN enzyme with a final concentration of 30-55U/mL, more preferably, the final concentration of the saponin is 0.22%, and the final concentration of the HL-SAN enzyme is 15.6U/mL, 20U/mL or 25U/mL.
Preferably, the kit is used for non-viscous samples, and comprises a saponin solution with a final concentration of 0.08-0.13% and HL-SAN enzyme with a final concentration of 10-30U/mL, more preferably, the final concentration of the saponin is 0.11%, and the final concentration of the HL-SAN enzyme is 15.6U/mL, 20U/mL or 25U/mL.
In some aspects, the metagenomic identification methods described herein can be applied in fields including, but not limited to, clinical research and scientific research.
The invention has the following beneficial effects:
(1) the method is simple and rapid, meets the detection requirement, omits the processes of library construction, PCR and the like through the method of incomplete host removal and the optimization and improvement of the host removal process, eliminates the bias caused by PCR amplification, and can more truly and accurately identify the metagenomic microorganisms.
(2) The invention carries out the system optimization of the host removal and the library establishment aiming at the clinical samples, verifies a plurality of different pretreatment processes, and obviously reduces the failure probability of the sample library establishment through the improvement and the adjustment of system parameters.
(3) Based on the process optimization, the single sample identification cost is reduced, and the purchase cost of PCR related reagents and the like is saved.
(4) The identification time of the whole process is shortened, PCR steps are saved, 2.5h is saved, and the whole wet experiment process is shortened to 3 h.
(5) Compared with the Grady professor process and the direct library building process without pretreatment, the incomplete host removing scheme of the invention has the highest conformity with the clinical culture gold standard, and greatly improves the accuracy and reliability of the sequencing result.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1: developing a metagenome-based identification flow pipeline schematic diagram by a professor Grady;
FIG. 2: the invention relates to a macro-genome identification process pipeline schematic diagram based on a nanopore creep-measuring platform.
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 illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by manufacturers, and are all conventional products available on the market.
The following examples and experimental examples relate to apparatus comprising: biological safety cabinet, GridION, nucleic acid automatic extraction workstation, high-speed centrifuge, Qubit 3.0, breaking appearance, vibration metal bath, pipettor, magnetic frame, refrigerator etc..
The related reagent comprises: saponin, PBS buffer solution, Promega nucleic acid extraction kit, ONT library construction kit, AMPure XP purified magnetic bead and QubitTMA detection kit, an ONT sequencing chip and the like.
The experimental process of the invention is shown in fig. 2, the invention optimizes the pretreatment process according to different sample types, so that the host proportion is lower than that of the original sample but higher than that of the Grady original process, thus the nucleic acid after extraction and purification can be directly built and operated on a database by using a quick bar code kit, and the data is analyzed in real time, and the specific experimental steps are as follows:
first, pathogen enrichment process
1. Sputum host removing process
1.1. Adding 200 mu L of sample into a 2ml round-bottom centrifuge tube, and adding 1ml of de-thickening agent;
1.2. after vortex shaking, incubating at 42 ℃ and 400rpm for 5min, and centrifuging at 12000g for 5 min;
1.3. the supernatant was discarded (no pellet was touched and ≤ 50 μ L of supernatant could be retained) and 250 μ L of PBS was resuspended. Blowing and beating up and down by a gun to be fully mixed (stirring and precipitating by using the tip of the gun, blowing and beating up and down-neglecting small particles which are difficult to resuspend);
1.4. adding 200 μ L of 0.5% saponin, and mixing by blowing and sucking up and down to obtain final concentration of 0.22%. Rotating on a rotary blending instrument at room temperature for 10 min;
note: 5% of saponin: 500mg saponin +10ml PBS. Filtering, sterilizing, and storing at room temperature in dark place. The effective period is one week. Diluting to working solution concentration before use;
1.5. add 350. mu.L of water, blow and mix well with a gun, add 12. mu.L of 5M NaCl after 30s, shake immediately.
Note: all solutions must be sterile filtered before use.
1.6. Add 800. mu.L HL-SAN buffer (5M NaCl, 100mM MgCl)2Aqueous solution) was blown with a gun and mixed well. (the pellet comparison solution at this step was resuspended);
1.7. adding 3 μ L HL-SAN, mixing immediately, the final concentration of HL-SAN is 47.9U/mL, incubating at 37 deg.C and 800rpm for 15 min;
1.8. adding 800 μ L PBS, blowing with a gun, mixing, centrifuging at 12000g for 3 min;
1.9. discard the supernatant (leave ≦ 50 μ l without touching the pellet).
2. Alveolar lavage fluid-host removal procedure
2.1. Taking 1ml of sample to be put into a 2ml round-bottom centrifuge tube for the following host removal process;
2.2.14000g, centrifuging for 5 min;
note 1 that if the sample does not settle, the time for centrifugation can be increased;
2.3. resuspend with 250 μ Ι _ of PBS;
2.4. adding 200 μ L of 0.25% saponin with final concentration of 0.11%, blowing, sucking, and mixing. Rotating on a rotary blending instrument at room temperature for 10min, and blowing and blending uniformly every 2min in the incubation process;
note: 5% of saponin: 500mg saponin +10ml PBS. Filtering for sterilization, and storing at room temperature in dark place; the effective period is one week, and the solution is diluted to the concentration of working solution before use;
2.5. adding 350 μ L of water, blowing with a gun, mixing, adding 12 μ L of 5M NaCl after 30s, and shaking immediately;
2.6.800 μ L PBS was resuspended (by pipetting with a gun) and 100 μ L HL-SAN buffer (5M NaCl, 100mM MgCl)2Aqueous solution) was blown with a gun and mixed well. (the pellet comparison solution at this step was resuspended);
2.7. adding 1 μ L HL-SAN, mixing immediately, HL-SAN final concentration of 15.6U, mL, incubating at 37 deg.C and 800rpm for 15 min;
2.8. adding 800 μ L PBS, blowing with a gun, mixing, centrifuging at 14000g for 3 min;
2.9. discard the supernatant (leave ≦ 50 μ l without touching the pellet).
Second, nucleic acid extraction
2.1. The samples were resuspended in 700. mu.L PBS and the fluid transferred to a Lysing Matrix E disruption tube;
2.2. setting crushing parameters on a fastprep-5G instrument: the operation is carried out once at 6m/s for 40 s;
2.3.14000g for 10min, and aspirating 500. mu.L of the supernatant using Promega nucleic acid extraction kit:
RSC white Blood DNA Kit extraction.
Thirdly, building a library and operating the machine:
3.1. purification of
3.1.1. Taking 35-50 mu L of the sample extracted by the nucleic acid in the previous step, and adding water to supplement the sample to 100 mu L;
3.1.2. adding 1.2 xbeads balanced to room temperature, rotating and mixing uniformly at room temperature, incubating for 5min, and removing supernatant on a magnetic frame instantly;
3.1.3.500 μ L of freshly prepared 70% alcohol for 2 times of beads;
3.1.4. adding 11 μ L of water, rotating at room temperature, mixing, incubating for 2min, and placing on magnetic frame to clarify;
3.1.5 carefully suck 10. mu.L of supernatant into a 1.5ml centrifuge tube with low adsorption, and take 1. mu.L of qubit to detect the concentration;
3.2. building a library: take the ONT library construction kit (SQK-RBK004) as an example.
3.2.1. Breaking and adding the barcode:
sample loading system (200 μ L PCR vial):
remove host DNA 400ng make up to 7.5. mu.L
RB01-12 (one for each sample) 2.5. mu.L
Reaction system:
30℃ 1min；
80℃ 1min；
3.2.2. sample mixing and purification
The total mass of the mixed sample is 1-1.8 mug, and the dosage of each sample depends on the specific concentration. Purification was carried out using 1 Xmagnetic beads as described above, 13. mu.L of 10mM Tris-HCl pH 7.5-8.0with 50mM NaCl (note: 1. mu.L of Qubit was taken for quantification).
3.2.3. Add the piecing
mu.L of RAP was added to the above 10. mu.L sample, and the reaction was carried out at room temperature for 10 min.
3.3. Upper machine
3.3.1. Melting at room temperature: sequencing buffers (SQB), Loading Beads (LB), Flush Tether (FLT), Flush Buffer (FLB), melting and immediately placing into an ice box.
3.3.2. mixing the Sequencing Buffer (SQB) and Flush Buffer (FLB) by vortex, centrifuging for a short time, and placing into an ice box.
3.3.3.Flush Tether (FLT) after brief centrifugation, blow evenly with a pipette and put into an ice box.
3.3.4. Open the lid of the Nanopore sequencing instrument and open the lid of the Priming port of the Flow cell clockwise.
3.3.5. Check the Priming port for bubbles under the cap, pipette small volumes to remove bubbles:
(1) adjusting a pipettor with the range of 1000 mu L to 200 mu L;
(2) inserting the lance tip into the Priming port
(3) The knob was slowly adjusted until a small volume of buffer was seen entering the tip, and the pipette was removed.
3.3.6. Preparing a Priming mix: to the thawed and blended Flush Buffer (FLB) was added 30. mu.L of thawed and blended Flush (FLT) and pipetted up and down.
3.3.7. Add 800. mu.L of prime mix to the Flow cell via prime port, avoid introducing air bubbles, and wait 5 min.
3.3.8. And fully and uniformly blowing the SQB and the LB by using a liquid transfer device.
3.3.9. In a new tube, a loading library was prepared:
3.3.10. the SpotON sample port is opened.
3.3.11. Adding 200 mu L of prime mix into the Flow cell through a prime port to avoid introducing air bubbles;
3.3.12. mixing the library, and blowing and beating the library by using a pipettor to mix the library uniformly;
3.3.13. add 75. mu.L of sample, one drop by one, to the flow cell via the SpotoN sample port, ensuring that the next drop is added to the flow port.
3.3.14. Carefully cover the SpotON sample port, ensure that the stopper enters the SpotON port, close the priming port.
3.4. Sequencing runs
And (3) sequencing by adopting a third-generation sequencing platform GridION/MinION.
3.5. Analysis of letter of birth
And performing letter generation analysis on the sequencing off-line data.
The technical effects of the present invention are demonstrated below with reference to specific examples.
Example 1 optimization of host reaction System
In the invention, through setting different saponin concentrations (5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25% … …) and the dosage of HL-SAN enzyme (10 muL, 5 muL, 3 muL, 1 muL … …), a host removing experiment is carried out on a plurality of clinical samples, and the results show that although the heterogeneity of the samples is very large, the effect of the treatment of the saponin concentration of more than 1% is similar to the host removing effect of 5% saponin used by professor Grady, and the finally extracted nucleic acid amount cannot be obviously improved. Finally, in order to meet the requirement of establishing a library, the processing conditions of sputum and alveolar lavage fluid are determined respectively by comprehensively considering multi-factors such as host removing effect, bacterial load loss balance and the like, and then the randomly-collected clinical samples are used for pretreatment according to the conditions, and then the library establishing kit is used for establishing a library and computer, so that the feasibility, the stability and the high efficiency of the process are determined.
Example 2 identification of clinically positive pathogen samples
Aiming at 30 cases of randomly collected clinical culture positive bacterial infection sputum and alveolar lavage fluid samples, the method is used for metagenome library construction and sequencing identification, and clinical culture results are used as controls (the clinical culture results are from microbial culture and mass spectrum identification). Results all the libraries were successfully built, and table 1 below is a comparison of detailed identification results with the cultured gold standards.
Table 1, detailed comparison table of pathogen detection result and culture gold standard in the process of the invention
The results show that 20 cases of alveolar lavage fluid and 26 cases of sputum can be detected by the process of the invention. The undetected corynebacterium striatum and burkholderia polyphagi are further discovered through subsequent bioinformatics optimization, and the undetected corynebacterium striatum and burkholderia polyphagi are not detected due to the fact that the adopted comparison database does not contain corynebacterium striatum and burkholderia polyphagi, and therefore the undetected corynebacterium striatum and burkholderia polyphagi are not detected due to the wet test process. Therefore, the positive detection rates for alveolar lavage fluid and sputum in the corrected procedure of the present invention were 87% (26/30) and 97% (29/30), respectively, as shown in Table 2.
TABLE 2 pathogen detection results and positive detection rate in the procedure of the invention
| Alveolar lavage fluid (30 cases) | Sputum (30 cases) | |
| Detection of pathogens | 20 examples of | 26 examples of |
| Detection of pathogens after correction | 26 examples of | 29 examples of |
| The positive detection rate% | 87％ | 97％ |
Example 3 comparison with Grady procedure
The invention further randomly collects 27 clinical culture positive bacterial infection sputum samples and 23 alveolar lavage fluid samples, and carries out pathogen identification strictly according to Grady development (figure 1), and the result shows that: in 1 sputum sample and 12 alveolar lavage fluid samples, the library was failed (since the nucleic acid concentration after PCR was <4 ng/. mu.g, the sequencing on the machine was not possible). Table 3 below shows the detailed identification of successful sample by Grady banking against the culture gold standard.
Table 3 detailed comparison of successful results of pathogen banking in Grady's process with culture gold standards
TABLE 4 successful result and positive detection rate table for Grady process pathogen banking
| Alveolar lavage fluid (23 cases) | Sputum (27 cases) | |
| Detection of all pathogens cultured | 9 examples of | 11 examples of |
| The positive detection rate% | 39％ | 41％ |
Table 4 the results show that: for positive clinical samples, the positive detection rates of Grady are 39% and 41% respectively, and the failure rates of reservoir building of sputum samples and alveolar lavage fluid samples are 3.7% and 52.2%, while the positive detection rates of sputum and alveolar lavage fluid by adopting the host removal and reservoir building process of the invention are 67% and 87% (the detection ratio after correction is 87% and 97%), and the reservoir building power is 100%. In contrast, the invention has stronger universality on samples and obviously improves the sequencing detection rate of clinically positive samples.
And for 1 sputum and 12 alveolar lavage fluids which can not be subjected to library building by the Grady process, further jumping to a host pretreatment process to directly extract total DNA, and then continuing to build a library according to the Grady process, so that the library is successfully built and the positive detection rate is verified. Table 5 below compares the detailed identification of this sample with the culture gold standard.
Table 5 detailed comparison of pathogen detection results from direct extraction of the on-board library with the gold culture standards in Grady's procedure without pretreatment
TABLE 6 Grady's procedure without pretreatment, direct extraction of the library, machine, pathogen detection results and positive detection tables
| Alveolar lavage fluid (12 cases) | Sputum (1 case) | |
| Detection of all pathogens cultured | 6 examples of | Example 0 |
| Rate of positive detection | 50％ | 0％ |
The results in tables 5-6 show that: even if the samples which cannot be subjected to library construction according to Grady process pretreatment are directly extracted and subjected to library construction and sequencing, the actual detection rate is only 50%, which indicates that the detection of the microbiome is very difficult to complete quickly and accurately under the condition of not removing a host at all based on a nanopore sequencing platform.
And (4) conclusion: the invention is obviously superior to the best macro-genome-based-host sequencing identification method developed by Grady in the field based on a nanopore sequencing platform and the traditional identification method for direct library construction and sequencing without host.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (11)
1. A metagenome sample library construction method based on a nanopore sequencing platform is characterized by comprising a step of incompletely removing host DNA and a step of directly constructing a library without PCR amplification;
the incompletely removed host DNA includes: dissociating and degrading host DNA, and separating the degraded host DNA from the sample;
the host DNA episomally comprises: adding saponin into the sample, and incubating for 5-15 mim;
the host DNA degradation comprises: adding HL-SAN enzyme into the sample after the DNA of the host is dissociated, and incubating for 10-20 min;
when the sample is a viscous substance during collection, the final concentration of the saponin is 0.2-0.25%, and the final concentration of the HL-SAN enzyme is 30-55U/mL;
when the sample is a non-viscous substance during collection, the final concentration of the saponin is 0.08-0.13%, and the final concentration of the HL-SAN enzyme is 10-30U/mL.
2. The method of claim 1, wherein the viscous material is sputum, pus, or nasal secretions; the non-viscous substance is alveolar lavage fluid.
3. The method of claim 1, wherein the final concentration of the saponin is 0.22% and the final concentration of the HL-SAN enzyme is 35U/mL, 40U/mL, 45U/mL, 47.9U/mL, or 50U/mL when the sample is a viscous substance at the time of collection;
when the sample is a non-viscous substance at the time of collection, the final concentration of the saponin is 0.11%, and the final concentration of the HL-SAN enzyme is 15.6U/mL, 20U/mL or 25U/mL.
4. The method of claim 1, wherein the viscous material is sampled at a rate of 150 to 250 μ L; the sampling amount of the non-viscous substance is 0.5-1.5 mL.
5. The method of any one of claims 1 to 4, further comprising a pretreatment of the sample prior to the liberation of the host DNA, the pretreatment comprising: centrifuging, discarding the supernatant, and resuspending and centrifuging the obtained precipitate to prepare a resuspension solution; the centrifugal force is 12000-14000g, the centrifugal time is 3-7 min, and the volume of the resuspension liquid is 200-300 muL.
6. The method of any one of claims 1 to 4, wherein the isolation of the host DNA from the sample comprises: after host DNA is degraded, adding a buffer solution into a sample, uniformly mixing, centrifuging, removing a supernatant, and keeping a precipitate; the buffer solution is PBS, the volume of the buffer solution is 500-100 mu L, the centrifugal force is 12000-14000g, the centrifugal time is 3-5 min, and the precipitate at most 50 mu L of supernatant is reserved.
7. The method of any one of claims 1 to 4, wherein the sample is an infected sample, and the sample is derived from a human or animal body.
8. The method of claim 7, wherein the infected sample is a respiratory infected sample.
9. The method of claim 7, wherein the infected sample is a non-viral infected sample.
10. The method of claim 9, wherein the non-viral infection is a bacterial infection.
11. A method for metagenomic identification based on a nanopore sequencing platform for non-disease diagnostic purposes, comprising the method of banking according to any one of claims 1-10, and further comprising the steps of on-machine sequencing and credit-generating analysis of the banked sample using the nanopore sequencing platform.
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911343507.6A CN110964795B (en) | 2018-12-13 | 2018-12-13 | Nanopore sequencing platform based alveolar lavage fluid sample library building method, identification method and kit |
| CN201911343496.1A CN111304287B (en) | 2018-12-13 | 2018-12-13 | Sputum sample library building method, identification method and kit based on nanopore sequencing platform |
| CN201811528417.XA CN109487345B (en) | 2018-12-13 | 2018-12-13 | Nanopore sequencing platform-based metagenome sample library building method, identification method and kit |
| PCT/CN2019/081931 WO2020118978A1 (en) | 2018-12-13 | 2019-04-09 | Method for constructing metagenomic sample library on basis of nanopore sequencing platform, identification method and kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811528417.XA CN109487345B (en) | 2018-12-13 | 2018-12-13 | Nanopore sequencing platform-based metagenome sample library building method, identification method and kit |
Related Child Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911343496.1A Division CN111304287B (en) | 2018-12-13 | 2018-12-13 | Sputum sample library building method, identification method and kit based on nanopore sequencing platform |
| CN201911343507.6A Division CN110964795B (en) | 2018-12-13 | 2018-12-13 | Nanopore sequencing platform based alveolar lavage fluid sample library building method, identification method and kit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109487345A CN109487345A (en) | 2019-03-19 |
| CN109487345B true CN109487345B (en) | 2020-03-10 |
Family
ID=65710077
Family Applications (3)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911343507.6A Active CN110964795B (en) | 2018-12-13 | 2018-12-13 | Nanopore sequencing platform based alveolar lavage fluid sample library building method, identification method and kit |
| CN201911343496.1A Active CN111304287B (en) | 2018-12-13 | 2018-12-13 | Sputum sample library building method, identification method and kit based on nanopore sequencing platform |
| CN201811528417.XA Active CN109487345B (en) | 2018-12-13 | 2018-12-13 | Nanopore sequencing platform-based metagenome sample library building method, identification method and kit |
Family Applications Before (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911343507.6A Active CN110964795B (en) | 2018-12-13 | 2018-12-13 | Nanopore sequencing platform based alveolar lavage fluid sample library building method, identification method and kit |
| CN201911343496.1A Active CN111304287B (en) | 2018-12-13 | 2018-12-13 | Sputum sample library building method, identification method and kit based on nanopore sequencing platform |
Country Status (2)
| Country | Link |
|---|---|
| CN (3) | CN110964795B (en) |
| WO (1) | WO2020118978A1 (en) |
Families Citing this family (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB201917101D0 (en) * | 2019-11-25 | 2020-01-08 | Uea Enterprises Ltd | Method for digesting nucleic acid in a sample |
| CN111304285B (en) * | 2019-12-13 | 2020-09-29 | 北京先声医学检验实验室有限公司 | Urinary metagenome sample library building and detecting method based on nanopore sequencing platform |
| CN111304286B (en) * | 2019-12-23 | 2020-12-18 | 北京先声医学检验实验室有限公司 | Urinary metagenome sample library building and detecting method based on nanopore sequencing |
| WO2021114501A1 (en) * | 2019-12-13 | 2021-06-17 | 北京先声医学检验实验室有限公司 | Nanopore sequencing-based urinary metagenomic sample library contstruction and detection method |
| CN113249436A (en) * | 2020-02-12 | 2021-08-13 | 广州微远基因科技有限公司 | Method and application for reducing host nucleic acid in biological sample |
| CN111471676A (en) * | 2020-03-13 | 2020-07-31 | 广州市达瑞生物技术股份有限公司 | Preparation method of database building sample for metagenome next generation sequencing |
| CN111394486A (en) * | 2020-04-09 | 2020-07-10 | 复旦大学附属儿科医院 | Child infectious disease pathogen detection and identification method based on metagenome sequencing |
| CN112322701A (en) * | 2020-11-09 | 2021-02-05 | 绍兴迅敏康生物科技有限公司 | Pathogen microorganism metagenome de-hosting method and kit |
| CN112481254B (en) * | 2020-12-01 | 2022-09-23 | 杭州杰毅生物技术有限公司 | Method and kit for removing host DNA and enriching microorganisms by one-step method |
| CN114369640A (en) * | 2021-03-08 | 2022-04-19 | 南京诺唯赞生物科技股份有限公司 | Method for quickly and efficiently removing animal-derived host nucleic acid |
| CN113322304B (en) * | 2021-06-19 | 2022-06-07 | 江苏先声医学诊断有限公司 | Clinical sample processing method applied to second-generation infection metagenome detection |
| CN114277092B (en) * | 2021-12-02 | 2023-03-24 | 江苏先声医疗器械有限公司 | RNA virus macro transcriptome database building method based on nanopore sequencing platform and application |
| CN115678966A (en) * | 2022-11-14 | 2023-02-03 | 北京金匙医学检验实验室有限公司 | Multi-sample type universal metagenome rapid detection method |
| CN115806971A (en) * | 2022-11-22 | 2023-03-17 | 北京金匙医学检验实验室有限公司 | Method for removing host of blood sample pathogen microorganism metagenome |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2980769A1 (en) * | 2015-04-02 | 2016-10-06 | The Jackson Laboratory | Method for detecting genomic variations using circularised mate-pair library and shotgun sequencing |
| US10724110B2 (en) * | 2015-09-01 | 2020-07-28 | Seven Bridges Genomics Inc. | Systems and methods for analyzing viral nucleic acids |
| CN106148513A (en) * | 2016-06-22 | 2016-11-23 | 杭州杰毅麦特医疗器械有限公司 | A kind of dissociative DNA library constructing method and test kit |
| CN106244580B (en) * | 2016-08-23 | 2019-05-24 | 厦门基源医疗科技有限公司 | A kind of extracting method of the macro genome of bronchoalveolar lavage fluid |
| GB2557654A (en) * | 2016-12-14 | 2018-06-27 | Uea Enterprises Ltd | Method for nucleic acid depletion |
| CN108048450B (en) * | 2017-12-21 | 2021-10-29 | 广州赛哲生物科技股份有限公司 | Sputum microorganism metagenome de-hosting extraction and library building method |
-
2018
- 2018-12-13 CN CN201911343507.6A patent/CN110964795B/en active Active
- 2018-12-13 CN CN201911343496.1A patent/CN111304287B/en active Active
- 2018-12-13 CN CN201811528417.XA patent/CN109487345B/en active Active
-
2019
- 2019-04-09 WO PCT/CN2019/081931 patent/WO2020118978A1/en active Application Filing
Also Published As
| Publication number | Publication date |
|---|---|
| CN111304287B (en) | 2020-09-15 |
| WO2020118978A1 (en) | 2020-06-18 |
| CN110964795A (en) | 2020-04-07 |
| CN110964795B (en) | 2020-09-18 |
| CN109487345A (en) | 2019-03-19 |
| CN111304287A (en) | 2020-06-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109487345B (en) | Nanopore sequencing platform-based metagenome sample library building method, identification method and kit | |
| CN111304285B (en) | Urinary metagenome sample library building and detecting method based on nanopore sequencing platform | |
| CN112481254B (en) | Method and kit for removing host DNA and enriching microorganisms by one-step method | |
| CN115232866A (en) | Sequencing method for target enrichment of 16S rRNA gene of bacteria based on nanopore sequencing | |
| CN115927565A (en) | Establishment and application of method for detecting pathogenic microorganisms in CAR-T cell product based on mNGS | |
| CN111304286B (en) | Urinary metagenome sample library building and detecting method based on nanopore sequencing | |
| CN111518869B (en) | Nucleic acid enrichment method for fragile microorganisms on cell surface and application of nucleic acid enrichment method in high-throughput sequencing | |
| CN110656037B (en) | Micro-fluidic chip for pathogen nucleic acid detection and detection method | |
| CN113265452A (en) | Bioinformatics pathogen detection method based on Nanopore metagenome RNA-seq | |
| CN114277092B (en) | RNA virus macro transcriptome database building method based on nanopore sequencing platform and application | |
| CN115386573A (en) | Method and kit for removing host nucleic acid in blood plasma and application | |
| CN108977436A (en) | Biotinylated nucleic acid DNA quick release extracts reagent and preparation method and application | |
| RU2808832C1 (en) | Method of isolating viral ribonucleic acid from cerebrospinal fluid for molecular biological research | |
| CN116949154B (en) | Macrotranscriptome-based pathogen detection method for non-therapeutic purpose | |
| WO2021114501A1 (en) | Nanopore sequencing-based urinary metagenomic sample library contstruction and detection method | |
| CN117051129B (en) | Method for setting background bacteria threshold value for microorganism detection and application thereof | |
| CN116926065B (en) | Nucleic acid extraction kit suitable for detecting pathogenic microorganisms and host residues and extraction method thereof | |
| US20220049321A1 (en) | Method, system and apparatus for blood processing unit | |
| CN117701678A (en) | Method for efficiently removing host nucleic acid of infected sample | |
| CN115678966A (en) | Multi-sample type universal metagenome rapid detection method | |
| CN117737049A (en) | Treatment method of cervical exfoliated cells and cervical exfoliated cell lysis kit | |
| CN115747204A (en) | Method for extracting and purifying mycobacteria microscopic examination positive sample DNA | |
| CN116574782A (en) | Lesion tissue sample dissociation host method applied to mNGS pathogen detection | |
| WO2022108728A1 (en) | Method, system and apparatus for blood processing unit | |
| CN116218966A (en) | Method for rapidly detecting umbilical cord blood bacteria and fungi |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |