CN117701690A - Method for screening potential probiotics by combining high-throughput sequencing with multiple-mathematic technology - Google Patents
Method for screening potential probiotics by combining high-throughput sequencing with multiple-mathematic technology Download PDFInfo
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- CN117701690A CN117701690A CN202311732586.6A CN202311732586A CN117701690A CN 117701690 A CN117701690 A CN 117701690A CN 202311732586 A CN202311732586 A CN 202311732586A CN 117701690 A CN117701690 A CN 117701690A
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- 239000006041 probiotic Substances 0.000 title claims abstract description 38
- 235000018291 probiotics Nutrition 0.000 title claims abstract description 38
- 238000000034 method Methods 0.000 title claims abstract description 33
- 238000005516 engineering process Methods 0.000 title claims abstract description 18
- 238000012216 screening Methods 0.000 title claims abstract description 17
- 238000012165 high-throughput sequencing Methods 0.000 title claims abstract description 15
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 30
- 241000894006 Bacteria Species 0.000 claims abstract description 27
- 238000010171 animal model Methods 0.000 claims abstract description 18
- 201000010099 disease Diseases 0.000 claims abstract description 10
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 10
- 238000012404 In vitro experiment Methods 0.000 claims abstract description 9
- 230000000694 effects Effects 0.000 claims abstract description 9
- 239000000126 substance Substances 0.000 claims abstract description 9
- 238000001727 in vivo Methods 0.000 claims abstract description 8
- 230000007246 mechanism Effects 0.000 claims abstract description 7
- 238000010230 functional analysis Methods 0.000 claims abstract description 5
- 238000010201 enrichment analysis Methods 0.000 claims description 15
- 238000012163 sequencing technique Methods 0.000 claims description 15
- 238000002474 experimental method Methods 0.000 claims description 7
- 238000012049 whole transcriptome sequencing Methods 0.000 claims description 6
- 206010067484 Adverse reaction Diseases 0.000 claims description 3
- 230000006838 adverse reaction Effects 0.000 claims description 3
- 238000002705 metabolomic analysis Methods 0.000 claims description 3
- 230000001431 metabolomic effect Effects 0.000 claims description 3
- 238000012174 single-cell RNA sequencing Methods 0.000 claims description 3
- 238000012268 genome sequencing Methods 0.000 abstract description 6
- 230000009286 beneficial effect Effects 0.000 description 6
- 244000005700 microbiome Species 0.000 description 5
- 230000001105 regulatory effect Effects 0.000 description 4
- 230000001737 promoting effect Effects 0.000 description 3
- 239000002689 soil Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 230000004110 gluconeogenesis Effects 0.000 description 2
- 230000034659 glycolysis Effects 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 239000002028 Biomass Substances 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000004879 molecular function Effects 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 235000015816 nutrient absorption Nutrition 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000000611 regression analysis Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A90/00—Technologies having an indirect contribution to adaptation to climate change
- Y02A90/10—Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation
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Abstract
The invention relates to the field of high-throughput sequencing, and particularly discloses a method for screening potential probiotics by combining high-throughput sequencing with a multi-group technology; according to the method, an animal model or a clinical case sample is firstly established, macro genome sequencing is carried out on the model or the sample to obtain differential bacteria, the range of functional strains is firstly narrowed, then functional analysis is carried out on the differential bacteria to obtain related potential probiotics, then the effect of the differential bacteria on diseases is specifically verified through in-vivo and in-vitro experiments, the experimental results and the gene function prediction results of macro genome sequencing are compared, and the potential probiotics efficacy and mechanism of the differential bacteria are confirmed by combining a plurality of groups of chemical technologies.
Description
Technical Field
The invention belongs to the technical field of high-throughput sequencing, and particularly relates to a method for screening potential probiotics by combining high-throughput sequencing with a multiple-mathematic technology.
Background
Probiotics are a class of active microorganisms beneficial to a host by colonizing the human body and altering the flora composition of a part of the host. By regulating the immune function of host mucous membrane and system or regulating the balance of flora in intestinal canal, the effect of promoting nutrient absorption and keeping intestinal canal healthy is achieved, so that single microorganism or mixed microorganism with definite composition which is beneficial to health is produced, and extremely abundant flora exists in human body.
In Chinese patent publication No. CN111793680A, a method for screening growth-promoting strain based on high-throughput sequencing and application thereof are disclosed. According to the invention, the gradient of effective soil function screening is constructed so as to form the difference of gradient change of the growth of the test plants and the composition of soil microbial communities, the composition of the microbial communities in the soil is analyzed by combining the widely used high-throughput sequencing technology with low cost and high accuracy, regression analysis is carried out by the absolute abundance of the dominant strain OTU and the biomass of the test plants, whether the dominant strain is a potential growth-promoting strain is judged, the dominant and potential growth-promoting strain with high absolute abundance is separated, and a tieback test is carried out to verify the growth-promoting effect. The method directly depends on the growth promoting effect to screen the growth promoting strain, and can effectively avoid uncertainty and false positive of the strain screened by the functional gene and the functional selection culture medium in practical application; in addition, the method has definite target and higher success rate, and has wide application prospect in screening growth-promoting strains.
The applicant found in the application process that although the technology disclosed in the above application can be applied to functional screening of microorganism fungus, the technology cannot be directly applied to screening of probiotics in animal models, and the technology lacks related experimental steps, and does not verify and analyze functions and effects of related animal probiotics.
Disclosure of Invention
The invention aims to provide a method for screening potential probiotics by combining high-throughput sequencing with a multi-group technology so as to solve the problems in the background technology.
In order to achieve the above purpose, the present invention provides the following technical solutions:
the method for screening the potential probiotics by combining high-throughput sequencing with multi-mathematic technology specifically comprises the following steps:
s1, establishing a disease animal model or collecting clinical case samples;
s2, performing metagenome sequencing on the model or the sample to find differential bacteria;
s3, analyzing the function of the differential bacteria to obtain potential probiotics;
s4, performing in-vivo and in-vitro experiments, verifying the effect of the differential bacteria on the diseases through experimental animals, deeply discussing related mechanisms by combining a multi-genetics method, and analyzing whether the experimental results correspond to gene function prediction results of metagenome sequencing;
s5, verifying the efficacy and adverse reaction by combining single probiotics with the existing treatment method and combining multiple probiotics to prove that the traditional clinical treatment method can be supplemented.
Preferably, in S2, the model or sample is subjected to metagenomic sequencing, and one or more techniques selected from whole transcriptome sequencing, whole exon sequencing, whole transcriptome sequencing in combination with whole exon sequencing, single cell RNA sequencing, epigenomic, metabolomic and microbiome are selected according to the purpose of the experiment.
Preferably, in the step S3, the functional analysis method of the differential bacteria is a gene enrichment analysis, including KEGG enrichment analysis, GO enrichment analysis and GSEA enrichment analysis.
Preferably, in S4, in-vivo and in-vitro experiments are performed, a blank control group and an experimental group are required to be set, probiotics are not applied to experimental animals in the blank control group, and the experimental animals in the experimental group are divided into a plurality of groups of probiotics which are used singly or combined with the existing treatment mode, and the probiotics are in different dosages and different combinations.
Compared with the prior art, the invention has the beneficial effects that:
according to the method, an animal model or a clinical case sample is firstly established, macro genome sequencing is carried out on the model or the sample to obtain differential bacteria, the range of functional strains is firstly narrowed, then functional analysis is carried out on the differential bacteria to obtain related potential probiotics, then the effect of the differential bacteria on diseases is specifically verified through in-vivo and in-vitro experiments, the experimental results and the gene function prediction results of macro genome sequencing are compared, and the potential probiotics efficacy and mechanism of the differential bacteria are confirmed by combining a plurality of groups of chemical technologies.
Drawings
Fig. 1 is a block diagram of the method of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Examples:
referring to fig. 1, the method for screening potential probiotics by combining high-throughput sequencing with multi-group technology specifically comprises the following steps:
s1, establishing a disease animal model or collecting clinical case samples;
s2, performing metagenome sequencing on the model or the sample to find differential bacteria;
s3, analyzing the function of the differential bacteria to obtain potential probiotics;
s4, performing in-vivo and in-vitro experiments, verifying the effect of the differential bacteria on the diseases through experimental animals, deeply discussing related mechanisms by combining a multi-genetics method, and analyzing whether the experimental results correspond to gene function prediction results of metagenome sequencing;
s5, verifying the efficacy and adverse reaction by combining single probiotics with the existing treatment method and combining multiple probiotics to prove that the traditional clinical treatment method can be supplemented.
From the above, the experimenter can establish a disease animal model or directly collect clinical case samples according to a general method, then conduct macro genome sequencing on the animal model or the case samples, determine the differential bacteria, analyze the functions of the differential bacteria after determining the differential bacteria, determine potential probiotics according to beneficial functions, conduct in-vitro experiments on the potential probiotics, verify the relevant effects of the differential bacteria on diseases through the conditions of experimental animals, further analyze and discuss whether experimental results correspond to gene function prediction results of macro genome sequencing, effectively screen the differential bacteria from a plurality of hybrid bacteria, and then effectively screen the differential bacteria, verify and analyze the beneficial effects of the probiotics while screening, and rapidly correspond the probiotics and the beneficial effects thereof.
In S2, the model or sample is subjected to metagenomic sequencing, and one or more techniques of whole transcriptome sequencing, whole exon sequencing, whole transcriptome sequencing in combination with whole exon sequencing, single cell RNA sequencing, epigenomics, metabolomics, and microbiology are selected according to the purpose of the experiment.
In the step S3, the functional analysis method of the differential bacteria is gene enrichment analysis, including KEGG enrichment analysis, GO enrichment analysis and GSEA enrichment analysis.
The difference genes or substances obtained by the histology data are very large, and the mechanism of the phenomenon to be researched cannot be clearly analyzed by carrying out research and verification one by one on the mass data. By enrichment analysis, we can classify different genes or substances according to their functions, so that genes or substances with similar functions are put together, thereby reducing the workload and realizing the functional and phenotypic correlation.
KEGG is a metabolic pathway map, so we have a network that shows the relationship of individual genes or substances. For example, we obtained that one of the enrichment pathways was numbered map00010 (glycolysis/gluconeogenesis) after KEGG analysis, we could consider the mechanism of the phenomenon we studied to be related to glycolysis/gluconeogenesis. Also, it is possible that many genes are enriched in the same channel, but it is also possible that many genes are not enriched, so that the number of differential genes is not in one-to-one correspondence with the KEGG enrichment result.
GO enrichment is the collection of genes that we find to be differential genes or whether the substance is enriched at three levels, molecular function (e.g. catalytically active), cellular components (e.g. localized on the cell membrane), involved in biological processes (e.g. involved in substance transport, etc.). For example, if we performed GO enrichment analysis on the obtained differential gene to obtain the term enrichment, we can consider that the phenomenon we studied might be related to iron ion binding, where GO is the term of GO: 0005506. From the above information, it can also be seen that the number of differential genes obtained by us is not in one-to-one correspondence with the number of enriched GO term, there are multiple genes in one GO term, and one gene can be enriched in multiple term.
The GSEA enrichment analysis is to firstly sequence the differential expression genes from big to small according to the expression difference multiple, then see if all genes under a certain gene set are mainly positioned at the front part or the rear part of the sequence, if the genes are positioned at the front part, the genes are up-regulated; if later it is indicated that the gene is down-regulated, GSEA enrichment is primarily concerned with genes at both ends.
In the step S4, when in-vivo and in-vitro experiments are carried out, a blank control group and an experiment group are required to be arranged, probiotics are not applied to experimental animals in the blank control group, and the experimental animals in the experiment group are subdivided into a plurality of groups of probiotics which are singly used or combined with the existing treatment mode, and have different dosages and different combination forms.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (4)
1. The method for screening the potential probiotics by combining high-throughput sequencing with multi-mathematic technology is characterized by comprising the following steps of:
s1, establishing a disease animal model or collecting clinical case samples;
s2, performing metagenome sequencing on the model or the sample to find differential bacteria;
s3, analyzing the function of the differential bacteria to obtain potential probiotics;
s4, performing in-vivo and in-vitro experiments, verifying the effect of the differential bacteria on the diseases through experimental animals, deeply discussing related mechanisms by combining a multi-genetics method, and analyzing whether the experimental results correspond to gene function prediction results of metagenome sequencing;
s5, verifying the efficacy and adverse reaction by combining single probiotics with the existing treatment method and combining multiple probiotics to prove that the traditional clinical treatment method can be supplemented.
2. The method for screening potential probiotics by combining high-throughput sequencing with multi-mathematic technology according to claim 1, characterized in that: in S2, the model or sample is subjected to metagenomic sequencing, and one or more techniques of whole transcriptome sequencing, whole exon sequencing, whole transcriptome sequencing in combination with whole exon sequencing, single cell RNA sequencing, epigenomics, metabolomics, and microbiology are selected according to the purpose of the experiment.
3. The method for screening potential probiotics by combining high-throughput sequencing with multi-mathematic technology according to claim 2, characterized in that: in the step S3, the functional analysis method of the differential bacteria is gene enrichment analysis, including KEGG enrichment analysis, GO enrichment analysis and GSEA enrichment analysis.
4. The method for screening potential probiotics by combining high throughput sequencing with multiple sets of chemical technology according to claim 3, characterized in that: in the step S4, when in-vivo and in-vitro experiments are carried out, a blank control group and an experiment group are required to be arranged, probiotics are not applied to experimental animals in the blank control group, and the experimental animals in the experiment group are subdivided into a plurality of groups of probiotics which are singly used or combined with the existing treatment mode, and have different dosages and different combination forms.
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| CN202311732586.6A CN117701690A (en) | 2023-12-17 | 2023-12-17 | Method for screening potential probiotics by combining high-throughput sequencing with multiple-mathematic technology |
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| CN202311732586.6A CN117701690A (en) | 2023-12-17 | 2023-12-17 | Method for screening potential probiotics by combining high-throughput sequencing with multiple-mathematic technology |
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- 2023-12-17 CN CN202311732586.6A patent/CN117701690A/en active Pending
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