CN116355990A - mNGS library building method based on micro-fluidic technology - Google Patents
mNGS library building method based on micro-fluidic technology Download PDFInfo
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Abstract
The invention relates to a mNGS library building method based on a microfluidic technology. S1, developing and building a library building reagent micro system adapting to a microfluidic library building instrument, wherein the library building reagent micro system comprises the following reagents and preparation methods, (1) BE: is prepared from DNA purified magnetic beads and reinforcing agent; (2) EB: is prepared from an absorption buffer and an enhancer; (3) FEA: is prepared from enzyme VA, nucleic-Free Water and reinforcing agent; (4) LIG: is prepared from enzyme VB, buffer VB and reinforcing agent; (5) UDB: is prepared from UDB Adapter, buffer VB, nuclear-Free Water and reinforcing agent; (6) PCR-MIX: the PCR primer is prepared from enzyme VC, UDBxx PCR primer containing a double index sequence and an enhancer; (7) enhancer diluent: is prepared from reinforcing agent and Nuclear-Free Water. S2, loading a sealing liquid, a sample and a reagent on the microfluidic chip; s3, loading the microfluidic chip into a microfluidic library-building instrument; s4, automatically executing a library building process by the microfluidic library building instrument; s5, collecting the established library by the microfluidic chip.
Description
Technical Field
The invention belongs to the technical field of metagenome new generation sequencing, and particularly relates to a mNSS library building method based on a microfluidic technology.
Background
The incidence and mortality of pulmonary infections caused by bacteria, viruses or fungi continue to rise worldwide, being the third leading cause of total life loss. Timely etiology diagnosis is critical for proper clinical treatment. The traditional etiology diagnosis technology mainly depends on a laboratory to culture microorganisms, and has long time consumption, the traditional microorganism culture technology means have certain limitations, and partial bacteria, viruses, fungi and the like cannot be cultured, so that research and cognition of scientific researchers on various and huge-number microorganisms is limited. In addition, traditional microorganism detection techniques have lower sensitivity, and clinical diagnosis is difficult and limited especially for patients who have previously used anti-infective drugs.
Currently, the era of precise public health and personalized pathogen treatment has entered a new stage of metagenomic next generation sequencing. Sequence reads generated by mNGS can be linked to a precise reference genome (or marker) database to identify pathogens. mNGS is unbiased and non-targeted and thus allows broad-spectrum detection of known and unexpected pathogens, even unknown organisms. mNGS, which has been introduced in clinical practice in recent years as a rapid diagnostic method for infectious disease microorganisms, provides a large amount of etiology information including species, strains, drug resistance, and even virulence characteristics. The mNGS is independent of traditional microorganism culture, directly carries out high-throughput sequencing on nucleic acid in a clinical sample, can rapidly and objectively detect various pathogenic microorganisms (including viruses, bacteria, fungi and parasites) in the clinical sample, and is particularly suitable for diagnosing acute and serious diseases and difficult infections.
Most library construction in the current mNGS field is mainly manual library construction, and an automatic workstation is used as an auxiliary. While automation workstations are favored, they are not friendly to small laboratories because of their large and cumbersome equipment, their large space requirement; the application times of each reagent is more, a plurality of reagents and plates are manually prepared, and the manual sample adding amount is large; multiple times of manual intervention are needed in the operation process, the occurrence of problems is manually handled, and the degree of automation is reduced; the library construction process, the equipment run time is long. Drawbacks of manual library building: 1) The amount of the manual operation sample is small; the sample flux is low when the sample is operated by a single person, so that the delivery time is long, the delivery time period is tension, and the illness state is easy to delay; 2) The artificial operation time is long; the manual operation time is prolonged, and the delivery time period is tense; 3) The risk of human misoperation is high; the detection result of pathogenic microorganisms is needed to guide the administration of drugs, which is easy to cause risks such as wrong administration of drugs for patients; automated workstation shortcoming: 1) The equipment occupies large space; the layout and arrangement of laboratory instruments and equipment are affected, and the occupied space is not friendly to a small laboratory; 2) The experiment cost is high; the special consumable is needed, the sample adding times of each reagent are more, the sample adding amount is large, and the consumable usage amount is large; the sample adding reagent has large volume, and the dead volume of the reagent is easy to cause reagent waste because of the error volume of the instrument; 3) The degree of automation is low; multiple times of manual intervention are needed, and the occurrence of problems needs to be manually treated; the manpower cannot be completely removed; the running time of the equipment is long; the method is limited by a pipetting workstation, pipetting logic and the time for library construction is generally more than 3 hours, which affects the lead time.
At the same time, microfluidic technology has been developed in recent years. Microfluidic technology (Microfluidics) refers to the science and technology involved in systems that use microchannels (tens to hundreds of microns in size) to process or manipulate minute fluids (nanoliters to attic volumes) and is an emerging intersection discipline involving chemical, fluid physics, microelectronics, new materials, biology and biomedical engineering. The microfluidic device mainly comprises a microfluidic chip, an integrated circuit board and an imaging system. The integrated circuit board is the most core part of the microfluidic technology, and the on-off of the electrodes on the microfluidic chip is controlled by the command of the written program, so that the changes of movement, combination and the like of the liquid drops on the electrodes are realized. The microfluidic technology is not only used in the aspect of rapid detection of nucleic acid, but also successfully applied to complex library establishment processes such as single cell capture and lysis, genome amplification and purification.
The characteristics of the micro-fluidic technology enable the micro-fluidic technology to have application possibility in the aspect of mNGS library establishment, no feasible mNGS library establishment method based on the micro-fluidic technology exists at present, and the design needs to be developed in a targeted manner.
Disclosure of Invention
The invention provides a mNGS library construction method based on a micro-fluidic technology for solving the technical problems in the prior art, which fully plays the characteristics of the micro-fluidic technology, improves the convenience of operation, reduces the operation time and the library construction time, reduces the cross contamination rate and reduces the human intervention.
The invention adopts the technical proposal for solving the technical problems in the prior art that: a mNGS library building method based on micro-fluidic technology,
s1, developing and building a library building reagent micro system adapting to a microfluidic library building instrument, wherein the library building reagent micro system comprises reagents and a preparation method as follows,
(1) BE: the DNA purification magnetic bead is prepared from DNA purification magnetic beads and an enhancer, wherein the volume of the enhancer is 0.5-1.5% of the volume of the DNA purification magnetic beads;
(2) EB: the reinforcing agent is prepared from an Elutrionbuffer and a reinforcing agent, and the volume of the reinforcing agent is 0.5-1.5% of the volume of the Elutrionbuffer;
(3) FEA: the enzyme is prepared from enzyme VA, nucleic-Free Water and an enhancer, wherein the volume ratio of the enzyme VA to the nucleic-Free Water is 2-2.5:1, and the enhancer is added in an amount of 0.5-1% after the enzyme VA and the nucleic-Free Water are uniformly mixed;
(4) LIG: the enzyme VB is prepared from enzyme VB, buffer VB and reinforcing agent, wherein the volume ratio of the enzyme VB to the buffer VB is 0.5-1.5:1, and the reinforcing agent with the volume of 0.5-1.5% is added after the enzyme VB and the buffer VB are uniformly mixed;
(5) UDB: the preparation method comprises the steps of preparing a UDB Adapter, a buffer VB, a nucleic-Free Water and an enhancer, wherein the specification of the UDB Adapter is 1 mu M, the volume ratio of the UDB Adapter to the nucleic-Free Water is 0.1-1:1, the volume ratio of the buffer VB to the UDB Adapter is 5-10:1, and 0.5-1.5% of the enhancer is added after the three components are uniformly mixed;
(6) PCR-MIX: the kit is prepared from enzyme VC, UDBxx PCR primer containing a double index sequence and an enhancer, wherein the specification of the UDBxx PCR primer is 20 mu M, the volume ratio of the enzyme VC to the UDBxx PCR primer is 5-6:1, and the enhancer with the volume of 0.5-1.5% is added after the two components are uniformly mixed;
(7) Reinforcing agent diluent: the reinforcing agent is prepared from the reinforcing agent and the Nuclear-Free Water, and the volume ratio of the reinforcing agent to the Nuclear-Free Water is 1:9;
s2, loading a sealing liquid, a sample and a reagent on the microfluidic chip;
s3, loading the microfluidic chip into a microfluidic library-building instrument;
s4, automatically executing a library building process by the microfluidic library building instrument;
s5, collecting the established library by the microfluidic chip.
Preferably: the reinforcing agent is Tween 20 with the concentration of 1-10 percent; the volume of the reinforcing agent added to BE was 1%, the volume of the reinforcing agent added to EB was 1%, the volume of the reinforcing agent added to FEA was 0.77%, the volume of the reinforcing agent added to LIG was 1%, the volume of the reinforcing agent added to UDB was 1%, and the volume of the reinforcing agent added to PCR-MIX was 1%.
Preferably: the volume ratio of enzyme VA to nucleic-Free Water in FEA is 2.25:1; the volume ratio of enzyme VB to buffer VB in LIG is 1:1; the volume ratio of UDB Adapter to nucleic-Free Water in UDB is 0.5:1, and the volume ratio of buffer VB to UDB Adapter is 7:1; the volume ratio of enzyme VC to UDBxx PCR primer in PCR-MIX was 5.5:1.
The invention has the advantages and positive effects that:
compared with the existing manual library construction and the scheme of adopting an automatic workstation to construct libraries, the library construction method is realized based on the micro-fluidic technology, and the characteristics of the micro-fluidic technology are fully exerted.
The microfluidic library-building instrument has high integration degree, small occupied area and laboratory space saving. The microfluidic library-building instrument device can be fully automatically operated, and can be operated by one key to complete library building after the microfluidic chip is loaded, manual intervention is not needed in the middle, the requirement on manpower is reduced, the operation convenience is improved, and the operation time is reduced. The microfluidic library-building instrument realizes a totally-enclosed library-building process and can effectively avoid aerosol pollution. The total time of the microfluidic library-building instrument can be reduced, and the library-building process can be completed within 2 hours.
The invention creatively applies the microfluidic technology to the stock building of the mNGS, greatly shortens the total duration of the detection flow of the pathogenic microorganisms and improves the detection sensitivity of the pathogenic microorganisms. According to the invention, a library-building reagent micro system which is matched with a micro-fluidic library-building instrument to perform mNGS library building is developed and designed, so that the feasibility of the micro-fluidic technology applied to mNGS library building is ensured. The development and design of the database creation reagent microsystem enable the sample processing of the database creation method to be compatible with samples with different sample qualities, and can be suitable for DNA samples with better quality, DNA samples with poor quality (such as few human hosts), cDNA samples subjected to reverse transcription and the like, so that the response capability of a microfluidic database creation instrument to different samples when mNGS database creation is carried out is improved.
Drawings
FIG. 1 is a schematic diagram of the operation flow of a microfluidic library creator of the present invention;
FIG. 2 is a block diagram of a microfluidic chip of a microfluidic library creator;
FIG. 3 is a graph of library peaks for experiment group 1 in experiment one;
FIG. 4 is a graph of library peaks for experiment group 2 in experiment one;
FIG. 5 is a graph of library peaks for experiment group 3 in experiment one;
FIG. 6 is a graph of library peaks for experiment group 1 in experiment two;
FIG. 7 is a graph of library peaks for experiment group 2 in experiment two.
Detailed Description
In order to further understand the summary, features and advantages of the present invention, the following examples are set forth in detail.
The mNGS library construction method is realized based on a microfluidic technology, and the specific mNGS library construction operation is performed by adopting the existing microfluidic library construction instrument and the microfluidic chip.
Step S1: the invention develops and establishes a library-building reagent micro-system adapting to a microfluidic library-building instrument, and the library-building reagent micro-system comprises the following reagents and preparation methods:
(1) BE: the DNA purification magnetic bead is prepared from DNA purification magnetic beads and an enhancer, wherein the volume of the enhancer is 0.5-1.5% of the volume of the DNA purification magnetic beads;
in this example, the volume of the enhancer added to BE was 1% of the volume of the DNA purification magnetic beads.
(2) EB: the reinforcing agent is prepared from an Elutrionbuffer and a reinforcing agent, and the volume of the reinforcing agent is 0.5-1.5% of the volume of the Elutrionbuffer;
in this example, the volume of the reinforcing agent added to EB was 1% of the volume of the solution buffer.
(3) FEA: the enzyme is prepared from enzyme VA, nucleic-Free Water and an enhancer, wherein the volume ratio of the enzyme VA to the nucleic-Free Water is 2-2.5:1, and the enhancer is added in an amount of 0.5-1% after the enzyme VA and the nucleic-Free Water are uniformly mixed;
in this example, the volume ratio of enzyme VA to nucleic-Free Water in FEA is 2.25:1;
in this example, the volume of enhancer added to FEA was 0.77% of the combined volume of enzyme VA and Nuclear-Free Water.
(4) LIG: the enzyme VB is prepared from enzyme VB, buffer VB and reinforcing agent, wherein the volume ratio of the enzyme VB to the buffer VB is 0.5-1.5:1, and the reinforcing agent with the volume of 0.5-1.5% is added after the enzyme VB and the buffer VB are uniformly mixed;
in this example, the volume ratio of enzyme VB to buffer VB in LIG is 1:1;
in this example, the volume of the enhancer added to LIG was 1% of the volume of the mixture of enzyme VB and buffer VB.
(5) UDB: the preparation method comprises the steps of preparing a UDB Adapter, a buffer VB, a nucleic-Free Water and an enhancer, wherein the specification of the UDB Adapter is 1 mu M, the volume ratio of the UDB Adapter to the nucleic-Free Water is 0.1-1:1, the volume ratio of the buffer VB to the UDB Adapter is 5-10:1, and 0.5-1.5% of the enhancer is added after the three components are uniformly mixed;
in the embodiment, the volume ratio of UDB Adapter to Nuclear-Free Water in UDB is 0.5:1, and the volume ratio of buffer VB to UDB Adapter is 7:1;
in this example, the volume of the enhancer added to UDB was 1% of the volume of the buffer VB, UDB Adapter and Nuclear-Free Water.
(6) PCR-MIX: the kit is prepared from enzyme VC, UDBxx PCR primer containing a double index sequence and an enhancer, wherein the specification of the UDBxx PCR primer is 20 mu M, the volume ratio of the enzyme VC to the UDBxx PCR primer is 5-6:1, and the enhancer with the volume of 0.5-1.5% is added after the two components are uniformly mixed;
in this example, the volume ratio of enzyme VC to UDBxx PCR primer in PCR-MIX is 5.5:1;
in this example, the volume of the enhancer added to the PCR-MIX was 1% of the volume of the mixture of the enzyme VC and the UDBxx PCR primer.
(7) Reinforcing agent diluent: the reinforcing agent is prepared from the reinforcing agent and the Nuclear-Free Water, and the volume ratio of the reinforcing agent to the Nuclear-Free Water is 1:9.
In this example, the enhancer is selected to be tween 20 at a concentration of 1 to 10%, preferably at a concentration of 7.5%.
The components of the library-building reagent microsystem of the microfluidic library-building instrument are explained as follows:
1) BE: BE (DNA Clean Beads) the main components are DNA purification magnetic beads and reinforcing agents; the function is to adapt to a microfluidic library-building instrument for purifying nucleic acids or libraries; the manufacturer of the DNA purification magnetic beads is Nanjinopran biotechnology Co., ltd (Nuo pran, vazyme), the product number is N411-01/02/03, and the product specification is 5ml/60ml/450ml; the enhancer manufacturer is Shenzhen Huazhi Dazhisha science and technology Co., ltd (Hua Dazhi, MGI), the product number is 090-000620-00, and the product specification is 500 μl;
2) DNA purification magnetic beads: DNA purification magnetic beads (VAHTS DNA Clean Beads) which are mainly composed of DNA purification magnetic beads; acting as a means for purifying nucleic acids or libraries; the manufacturer is Nanjinouzan biotechnology Co., ltd (Vazyme), the product number is N411-01/02/03, and the product specification is 5ml/60ml/450ml;
3) EB: EB (Elutionbuffer) the main ingredients are Elutronbuffer and reinforcing agent; the function is to adapt to nucleic acid or library solvent after the microfluidic library-building instrument purifies nucleic acid or library; elutionbuffer manufacturer is Kaijia organism (Qiagen), product number is 19086, and product specification is 250ml; the enhancer manufacturer is Shenzhen Huazhi Dazhisha science and technology Co., ltd (Hua Dazhi, MGI), the product number is 090-000620-00, and the product specification is 500 μl;
4) An execution buffer: the main component of the Elutionbuffer is Elutionbuffer; acting as a purified nucleic acid or library solvent; the manufacturer is Kaijia organism (Qiagen), the product number is 19086, and the product specification is 250ml;
5) FEA: FEA (segmentation/End repair/dA-labeling Reagent) mainly comprises enzyme VA and reinforcing agent; the kit is used as a fragmentation reagent which is suitable for a microfluidic library-building instrument; the source of the enzyme VA is VAHTS Universal DNA Library Prep Kit for MGI kit components, the manufacturer is Nanjinouzan biotechnology Co., ltd (Vazyme), the product number is NDM607, and the product specification is 24rxns/96rxns; the enhancer manufacturer is Shenzhen Huazhi Dazhisha science and technology Co., ltd (Hua Dazhi, MGI), the product number is 090-000620-00, and the product specification is 500 μl;
6) Enzyme VA: enzyme VA, the main component is End Prep Mix 4; acting as a fragmenting agent; the source is VAHTS Universal DNA Library Prep Kit for MGI kit components, the manufacturer is Nanjinouzan biotechnology Co., ltd (Vazyme), the product number is NDM607, and the product specification is 24rxns/96rxns;
7) Nuclear-Free Water: non-enzyme Water (Nuclease-Free Water) with its main ingredient being Nuclease-Free Water; acting as a diluent or formulation reagent; the manufacturer is Ambion, the product number is AM9930, and the product specification is 500ml;
8) LIG: LIG (Ligation Reagent) the main components are enzyme VB, buffer VB and reinforcing agent; the function is to adapt to the connecting reagent of the microfluidic database builder; the source of the enzyme VB and the buffer VB is VAHTS Universal DNA Library Prep Kit for MGI kit components, the manufacturer is Nanjinouzan biotechnology Co-Ltd (Vazyme), the product number is NDM607, and the product specification is 24rxns/96rxns; the enhancer manufacturer is Shenzhen Huazhi Dazhisha science and technology Co., ltd (Hua Dazhi, MGI), the product number is 090-000620-00, and the product specification is 500 μl;
9) Enzyme VB: enzyme VB, the main ingredient of which is rapidDNA enzyme; an enzyme acting as a ligation in the ligation reagent; the source is VAHTS Universal DNA Library Prep Kit for MGI kit components, the manufacturer is Nanjinouzan biotechnology Co., ltd (Vazyme), the product number is NDM607, and the product specification is 24rxns/96rxns;
10 Buffer VB): buffer VB, the main component of which is Rapid Ligation Buffer 2; a buffer solution which serves to provide a buffer environment in the linking reagent; the source is VAHTS Universal DNA Library Prep Kit for MGI kit components, the manufacturer is Nanjinouzan biotechnology Co., ltd (Vazyme), the product number is NDM607, and the product specification is 24rxns/96rxns;
11 UDB): UDB, wherein the main components are UDB Adapter, nuclear-Free Water and reinforcing agent with the specification of 1 mu M; the function is a short joint in a connecting reagent which is adapted to a microfluidic library-building instrument; the UDB Adapter with the specification of 1 mu M is obtained from Shenzhen Dazhisha technology Co., ltd (Hua Dazhi, MGI), the product number is 1000022801/1000022802, and the product specification is 480 mu l/branch; the manufacturer of the clear-Free Water is Ambion, the product number is AM9930, and the product specification is 1 x 500ml; the enhancer manufacturer is Shenzhen Huazhi Dazhisha science and technology Co., ltd (Hua Dazhi, MGI), the product number is 090-000620-00, and the product specification is 500 μl;
12 UDB Adapter with specification of 1 μm): UDB Adapter with specification of 1 mu M comprises main component of UDB Adapter with specification of 1 mu M; a linker acting as a linking agent; the source is Shenzhen Dazhisha manufactured technology Co., ltd (Hua Dazhi manufactured, MGI), the product number is 1000022801/1000022802, and the product specification is 480 mu l/branch;
13 Buffer VB): buffer VB, the main component of which is Rapid Ligation Buffer 2; a buffer solution which serves to provide a buffer environment in the linking reagent; the source is VAHTS Universal DNA Library Prep Kit for MGI kit components, the manufacturer is Nanjinouzan biotechnology Co., ltd (Vazyme), the product number is NDM607, and the product specification is 24rxns/96rxns;
14 PCR-MIX: PCR-MIX, the main components are UDBxx PCR primer containing double index sequence, nucleic-Free Water, enzyme VC and reinforcing agent; the amplification reagent is used as an amplification reagent which is suitable for a microfluidic library-building instrument; the UDBxx PCR primer containing the double index sequence is prepared from Shenzhen Dazhisha technology and technology Co., ltd (Hua Dazhi, MGI), the product number is 1000022801/1000022802, and the product specification is 96rxn; the manufacturer of the clear-Free Water is Ambion, the product number is AM9930, and the product specification is 1 x 500ml; the source of the enzyme VC is VAHTS Universal DNA Library Prep Kit for MGI kit components, the manufacturer is Nanjinouzan biotechnology Co., ltd (Vazyme), the product number is NDM607, and the product specification is 24rxns/96rxns; the enhancer manufacturer is Shenzhen Huazhi Dazhisha science and technology Co., ltd (Hua Dazhi, MGI), the product number is 090-000620-00, and the product specification is 500 μl;
15 Enzyme VC: an enzyme VC mainly comprising
HiFi Amplification Mix; the reaction enzyme acts as about amplification in the amplification reagent; the source is VAHTS Universal DNA Library Prep Kit for MGI kit components, the manufacturer is Nanjinouzan biotechnology Co., ltd (Vazyme), the product number is NDM607, and the product specification is 24rxns/96rxns;
16 UDBxx PCR primers containing a double index sequence: the main component of the UDBxx PCR primer containing the double index sequence is the UDBxx PCR primer containing the double index sequence; specific index sequences which act to distinguish between different libraries in the amplification reagents; the source is Shenzhen Dazhisha manufactured technology Co., ltd (Hua Dazhi manufactured, MGI), the product number is 1000022801/1000022802, and the product specification is 96rxn;
17 Reinforcing agent): the reinforcing agent mainly comprises tween 20 and acts as a surfactant which can obviously increase the surface tension of liquid drops; the manufacturer is Shenzhen Dazhihua manufactured technology and technology Co., ltd (Hua Dazhi manufactured, MGI), the product number is 090-000620-00, and the product specification is 500 μl;
18 Sealing liquid: a sealing liquid which mainly comprises low-viscosity silicone oil; the silicone oil with the viscosity of 5 is generally used for controlling the movement of liquid drops in an electric field, the source is a sample preparation card kit component, the manufacturer is Shenzhen Dazhihua Zhizhen science and technology Co., ltd (Hua Dazhi manufacturer, MGI), the product number is 930-000063-00, and the product specification is 4 ml/count.
S2, loading a sealing liquid, a sample and a reagent on the microfluidic chip;
s3, loading the microfluidic chip into a microfluidic library-building instrument;
s4, automatically executing a library building process by the microfluidic library building instrument;
and S5, collecting the established library by the microfluidic chip.
Please refer to fig. 1 and fig. 2 for a mNGS library building flow and operation mode based on the microfluidic technology, wherein:
in fig. 1, a schematic operation flow diagram of the microfluidic library builder is shown, wherein the content of a solid line box represents manual operation, and the content of a dotted line box represents automatic operation of the microfluidic library builder. Steps S2 to S5 of the mNGS library construction method of the present invention are shown in fig. 1, and the operations in the first three solid frames correspond to step S2 and step S3, i.e., loading a blocking liquid, a sample, and a reagent onto a microfluidic chip, and then loading the microfluidic chip into a microfluidic library builder. The operation in the dashed box corresponds to step S4, namely, the microfluidic library-building instrument automatically executes a library-building process, specifically including sample fragmentation & end repair, linker ligation, linker product bead purification, library amplification (PCR), library bead purification. The operation within the last solid line box corresponds to step S5, i.e. the library after the build up is collected by the microfluidic chip.
The structure diagram of the microfluidic chip of the microfluidic library-building instrument is shown in fig. 2, the adding position marks of each reagent, sealing liquid and sample of the library-building reagent microsystem are shown on the structure diagram of the microfluidic chip, and the arrow mark shown in the structure diagram is the direction of the inclination of the gun head when the reagent and the sample are added.
Step S2 is specifically described below with reference to the marks in fig. 2:
injecting sealing liquid into a hole site in a round frame at the rightmost side of the microfluidic chip; (1) EB is injected into the number hole site, the injection amount is 30 μl, (2) BE is injected into the number hole site, the injection amount is 30 μl, (3) FEA is injected into the number hole site, the injection amount is 10 μl, (4) LIG is injected into the number hole site, the injection amount is 10 μl, (5) UDB is injected into the number hole site, the injection amount is 10 μl, (6) PCR-MIX is injected into the number hole site, the injection amount is 5 μl, and four holes need to BE careful to avoid index repetition.
After steps S3 and S4 are performed, that is, after the microfluidic library creator is operated, the corresponding library is sucked out from the hole sites in each circular frame in the middle, and the suction amount of each hole site is about 6 μl.
The invention optimizes the proportion of each reagent of the database-building reagent microsystem by designing the following experiment.
Experiment one
FEA ratio optimization test, experimental purposes: and determining the optimal ratio of the FEA microsystem reagent.
Experimental protocol:
experimental group 1FEA ratio: the volume ratio of the enzyme VA to the nucleic-Free Water is 1, and 0.77% of reinforcing agent is added after the enzyme VA and the nucleic-Free Water are uniformly mixed;
experimental group 2FEA ratio: the volume ratio of the enzyme VA to the nucleic-Free Water is 2.25, and 0.77% of reinforcing agent is added after the enzyme VA and the nucleic-Free Water are uniformly mixed;
experimental group 3FEA ratio: the volume ratio of the enzyme VA to the nucleic-Free Water is 3, and 0.77% of reinforcing agent is added after the enzyme VA and the nucleic-Free Water are uniformly mixed;
the other reagents are prepared by the same preparation method and are prepared in the same batch, and after library construction is performed by using a microfluidic library-building instrument according to different preparation methods, a library peak pattern diagram is analyzed by using an Agligent 2100 bioanalyzer (5067-4626#2100High sensitivity DNAkit).
Experimental results:
the peak pattern of experimental group 1 in this experiment is shown in fig. 3, the peak pattern of experimental group 2 in this experiment is shown in fig. 4, and the peak pattern of experimental group 3 in this experiment is shown in fig. 4. The concrete explanation is as follows:
Experiment two
UDB ratio optimization test, experimental purpose: determining the optimal ratio of the reagent of the UDB microsystem.
Experimental protocol:
experiment group 1UDB ratio: UDB Adapter (1. Mu.M) to nucleic-Free Water volume ratio of 0.5, buffer VB to UDB Adapter (1. Mu.M) volume ratio of 1; adding 1% of reinforcing agent after uniformly mixing the three components;
experiment group 2UDB ratio: UDB Adapter (1. Mu.M) to nucleic-Free Water volume ratio 0.5, buffer VB to UDB Adapter (1. Mu.M) volume ratio 7; adding 1% of reinforcing agent after uniformly mixing the three components;
experiment group 3UDB ratio: UDB Adapter (1. Mu.M) to nucleic-Free Water volume ratio of 0.5, buffer VB to UDB Adapter (1. Mu.M) volume ratio of 15; adding 1% of reinforcing agent after uniformly mixing the three components;
the other reagents are prepared by the same preparation method and are prepared in the same batch, and after library construction is performed by using a microfluidic library-building instrument according to different preparation methods, a library peak pattern diagram is analyzed by using an Agligent 2100 bioanalyzer (5067-4626#2100High sensitivity DNA kit).
Experimental results:
the library concentration profile data in this experiment are given in table 1.
TABLE 1 library concentration cases for Experimental groups 1-3
The concrete explanation is as follows:
both experimental group 1 and experimental group 2 meet the library construction quality control standard (library concentration is greater than 1 ng/. Mu.l) of the microfluidic library construction instrument, and the experimental group 3 cannot meet the joint amount of the library construction nucleic acid and the joint due to the fact that the joint content is too low, so that library delivery fails.
The peak pattern for experimental group 1 in this experiment is given in fig. 6, and the peak pattern for experimental group 2 in this experiment is given in fig. 7, where library peak pattern is not shown because experimental group 3 was not successfully ex-warehouse; from the above graph, the obvious joint peak (130 bp) exists in the experimental group 1, the obvious joint peak does not exist in the experimental group 2, the joint proportion of the experimental group 2 is proper, and the condition of obvious joint excess is avoided on the premise of ensuring normal ex-warehouse.
Experiment three
PCR-MIX ratio optimization test, experimental purpose: and determining the optimal ratio of the PCR-MIX microsystem reagent.
Experimental protocol:
experiment group 1PCR-MIX ratio: the volume ratio of enzyme VC to UDBxx PCR primer (containing double index sequence) is 11; adding 1% of reinforcing agent after uniformly mixing;
experiment group 2PCR-MIX ratio: the volume ratio of enzyme VC to UDBxx PCR primer (containing double index sequence) is 5.5; adding 1% of reinforcing agent after uniformly mixing;
experimental group 3PCR-MIX ratio: the volume ratio of enzyme VC to UDBxx PCR primer (containing double index sequence) is 3.67; adding 1% of reinforcing agent after uniformly mixing;
experiment group 4PCR-MIX ratio: the volume ratio of enzyme VC to UDBxx PCR primer (containing double index sequence) is 2.75; adding 1% of reinforcing agent after uniformly mixing;
the other used reagents are prepared by the same preparation method and are prepared in the same batch, and after library construction is performed by using a microfluidic library construction instrument according to different preparation methods, the library concentration and library construction stability are concerned.
Experimental results:
the library concentration profile data in this experiment is given in table 2.
TABLE 2 library concentration conditions for Experimental groups 1-4
As can be seen from the experimental results, the library concentration of the experimental group 2 is obviously improved compared with that of the experimental group 1, which indicates that the primer concentration greatly influences the amplification efficiency and thus the library concentration; the library concentration difference between the experimental groups 2 and 4 is not large, which indicates that the primer concentration reaches the plateau period, and the influence of increasing the primer concentration proportion on the library concentration is not large; the experimental group 2 has better database stability than other experimental groups.
The invention verifies the effect of developing and designing a database-building reagent microsystem applied to mNGS database building by designing the following experiments.
Experiment four
Microfluidic library construction cross contamination test, experimental purposes: and testing the cross contamination condition in the library construction process by using the microfluidic library builder.
Experimental protocol:
experiment group 1: using 1 microfluidic chip, marking 4 hole sites as S1-S4 from top to bottom, and putting 2ng of negative quality control material into the S1 hole site; s2, putting 2ng of sample tag (tag number T31) into the hole site; s3, putting 2ng of negative quality control material into the hole site; s4, inputting a 2ng sample tag (tag number T32) into the hole site, and operating a microfluidic library-building instrument to build a library;
experiment group 2: using 1 microfluidic chip, marking 4 hole sites as S1-S4 from top to bottom, and putting 2ng sample labels (label number T6) into the S1 hole site; s2, putting 2ng of negative quality control material into the hole site; s3 hole site inputs 2ng sample label (label number T16), S4 hole site inputs 2ng negative quality control; operating a microfluidic library-building instrument to build a library;
note that: the sample labels are fixed base sequences, and the fixed base sequences of different label numbers are different, so that the sample labels are used as positive standard substances for testing;
sequencing the library after library establishment on a machine, and loading negative samples and positive samples in different batches; the number of reads detected by the sample tag in the sequencing data analysis was not removed and 20M homogenization was performed.
Experimental results:
the library construction for experiments 1 and 2 in this experiment is given in Table 3, and the cross-contamination is illustrated in Table 4.
TABLE 3 library construction for experiments 1 and 2
Table 4 Cross contamination of experiments 1 and 2
The concrete explanation is as follows:
the cross-contamination rate of experiment 1 is below 1.06 ppm and the cross-contamination rate of experiment 2 is below 1 ppm. The cross contamination rate of the microfluidic library-building instrument can be controlled to be 1 kilomillion, and the cross contamination condition is far lower than that of manual library building.
Experiment five
Testing the stability of microfluidic library-building batch-to-batch, and testing the purpose: the microfluidic library builder was tested for intra-batch and inter-batch differences within the same sample batch.
Experimental protocol: 3 chips (12 hole sites) are respectively taken by using a negative quality control product experiment, two experimenters A, B use different microfluidic library-building instruments to build libraries in different time periods, the input amount is 3ng, and the CV value in batches (same Zhang Xinpian) and between batches (different chips) is calculated.
Experimental results:
the experimental results are given in table 5.
Table 5 microfluidic library-building intra-lot stability testing
The concrete explanation is as follows:
the CV value in the batch is between 5.71 and 17.86 percent, the CV value between batches is 11.8 percent, and the microfluidic library establishment has good stability.
Experiment six
Testing the detection conditions of different library building methods of positive samples, and testing the purpose: and testing the detection difference of positive samples of the manual library construction method and the microfluidic library construction method.
Experimental protocol: and selecting 34 samples such as cerebrospinal fluid, alveolar lavage fluid, peripheral blood, sputum and the like, and respectively carrying out manual DNA library construction flow and microfluidic library construction flow library construction by using the same nucleic acid and the same initial quantity, so as to compare the detected difference of positive pathogenic bacteria.
The experimental results are given in table 6.
Table 6 detection of Positive samples by different database methods
The concrete explanation is as follows:
the difference of the two processes for detecting the positive pathogenic bacteria is not great, and the microfluidic library construction process for detecting part of pathogenic bacteria is superior to the manual DNA library construction process.
Experiment seven
Poor quality (few humanized hosts) DNA and cDNA samples were pooled for experimental purposes: microfluidic banking apparatus is used to create libraries in poor quality (fewer human hosts) DNA samples and cDNA samples.
Experimental protocol:
10 DNA samples with poor quality (few human hosts) and 10 cDNA samples were selected, according to the DNA samples with poor quality (few human hosts): amplifying for 20cycles, wherein the upper limit of the concentration of the nucleic acid is 0.5 ng/. Mu.l; after dilution, 10. Mu.l of the sample and 0.5. Mu.l of Q-buffer (Q-buffer is a human DNA standard substance) were mixed, and the intermediate product was sampled to a volume of 5. Mu.l, and 0.5. Mu.l of the 10-fold diluted enhancer was added. RNA flow reverse transcription cDNA samples: amplifying the cDNA after 20cycles and manual reverse transcription; after 5. Mu.l of each sample volume, 0.5. Mu.l of 10-fold diluted enhancer was added.
Library construction was performed, library concentrations were assessed and differences were detected against positive pathogens. The experimental results are given in tables 7 and 8.
TABLE 7 poor quality and cDNA library concentration
| Sample species | Sample type | Library numbering | PCR cycle number | Concentration of 20cycles library (ng/. Mu.l) | |
| cDNA | Reference disc | FZW11 | 20 | 1.68 | |
| cDNA | Reference disc | FZW12 | 20 | 1.71 | |
| cDNA | Reference disc | FZW13 | 20 | 1.97 | |
| cDNA | Reference disc | FZW14 | 20 | 4.36 | |
| cDNA | Reference disc | FZW15 | 20 | 6.96 | |
| cDNA | Reference disc | FZW16 | 20 | 11.3 | |
| cDNA | Reference disc | FZW17 | 20 | 7.82 | |
| cDNA | Reference disc | FZW18 | 20 | 1.78 | |
| cDNA | Reference disc | FZW19 | 20 | 1.924 | |
| cDNA | Reference disc | FZW20 | 20 | 1.652 |
TABLE 8 concentration of poor quality (few humanized hosts) DNA sample library
| Sample species | Sample type | Library numbering | PCR cycle number | Library concentration (ng/. Mu.l) | |
| DNA samples of poor quality | DNAPCR | HB1 | 20 | 1.42 | |
| DNA samples of poor quality | DNAPCR | HB2 | 20 | 1 | |
| DNA samples of poor quality | | HB3 | 20 | 2.36 | |
| DNA samples of poor quality | | HB4 | 20 | 6.5 | |
| DNA samples of poor quality | Sputum (sputum) | |
20 | 8.32 | |
| DNA samples of poor quality | Sputum (sputum) | |
20 | 8.6 | |
| DNA samples of poor quality | Sputum (sputum) | |
20 | 10.8 | |
| DNA samples of poor quality | | HB8 | 20 | 10.8 | |
| DNA samples of poor quality | Alveolar | HB9 | 20 | 10.9 | |
| DNA samples of poor quality | Alveolar | HB10 | 20 | 8.78 |
The concrete explanation is as follows:
the library can be normally taken out of the library and meets the quality control standard (the library concentration is more than 1 ng/. Mu.l).
A summary of the detection is given in table 9 and table 10.
Table 9 summary of detection cases one
Table 10 shows two summary of detection conditions
The concrete explanation is as follows:
the difference between the two processes of positive pathogenic bacteria detection is not great.
Claims (3)
1. A mNGS library building method based on a microfluidic technology is characterized in that:
s1, developing and building a library building reagent micro system adapting to a microfluidic library building instrument, wherein the library building reagent micro system comprises reagents and a preparation method as follows,
(1) BE: the DNA purification magnetic bead is prepared from DNA purification magnetic beads and an enhancer, wherein the volume of the enhancer is 0.5-1.5% of the volume of the DNA purification magnetic beads;
(2) EB: the reinforcing agent is prepared from an Elution buffer and a reinforcing agent, and the volume of the reinforcing agent is 0.5-1.5% of the volume of the Elution buffer;
(3) FEA: the enzyme is prepared from enzyme VA, nucleic-Free Water and an enhancer, wherein the volume ratio of the enzyme VA to the nucleic-Free Water is 2-2.5:1, and the enhancer is added in an amount of 0.5-1% after the enzyme VA and the nucleic-Free Water are uniformly mixed;
(4) LIG: the enzyme VB is prepared from enzyme VB, buffer VB and reinforcing agent, wherein the volume ratio of the enzyme VB to the buffer VB is 0.5-1.5:1, and the reinforcing agent with the volume of 0.5-1.5% is added after the enzyme VB and the buffer VB are uniformly mixed;
(5) UDB: the preparation method comprises the steps of preparing a UDB Adapter, a buffer VB, a nucleic-Free Water and an enhancer, wherein the specification of the UDB Adapter is 1 mu M, the volume ratio of the UDB Adapter to the nucleic-Free Water is 0.1-1:1, the volume ratio of the buffer VB to the UDB Adapter is 5-10:1, and 0.5-1.5% of the enhancer is added after the three components are uniformly mixed;
(6) PCR-MIX: the kit is prepared from enzyme VC, UDBxx PCR primer containing a double index sequence and an enhancer, wherein the specification of the UDBxx PCR primer is 20 mu M, the volume ratio of the enzyme VC to the UDBxx PCR primer is 5-6:1, and the enhancer with the volume of 0.5-1.5% is added after the two components are uniformly mixed;
(7) Reinforcing agent diluent: the reinforcing agent is prepared from the reinforcing agent and the Nuclear-Free Water, and the volume ratio of the reinforcing agent to the Nuclear-Free Water is 1:9;
s2, loading a sealing liquid, a sample and a reagent on the microfluidic chip;
s3, loading the microfluidic chip into a microfluidic library-building instrument;
s4, automatically executing a library building process by the microfluidic library building instrument;
s5, collecting the established library by the microfluidic chip.
2. The micro-fluidic technology-based mNGS library building method according to claim 1, wherein: the reinforcing agent is Tween 20 with the concentration of 1-10 percent; the volume of the reinforcing agent added to BE was 1%, the volume of the reinforcing agent added to EB was 1%, the volume of the reinforcing agent added to FEA was 0.77%, the volume of the reinforcing agent added to LIG was 1%, the volume of the reinforcing agent added to UDB was 1%, and the volume of the reinforcing agent added to PCR-MIX was 1%.
3. The micro-fluidic technology-based mNGS library building method according to claim 2, wherein: the volume ratio of enzyme VA to nucleic-Free Water in FEA is 2.25:1; the volume ratio of enzyme VB to buffer VB in LIG is 1:1; the volume ratio of UDB Adapter to nucleic-Free Water in UDB is 0.5:1, and the volume ratio of buffer VB to UDB Adapter is 7:1; the volume ratio of enzyme VC to UDBxx PCR primer in PCR-MIX was 5.5:1.
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