Construction method and application of V4 region sequence database of vaginal flora 16S rRNA (ribosomal Ribonucleic Acid) gene

Abstract

The invention discloses a construction method and application of a human vaginal flora 16S rRNA gene V4 region sequence database, and the construction method mainly comprises the following steps: (1) retrieving and downloading 16S rRNA sequences in the database, (2) carrying out sequence comparison and correction, (3) constructing a human vaginal flora 16S rRNA full-length database, (4) obtaining human vaginal flora 16S rRNA gene V4 region fragments, and (5) constructing the human vaginal flora 16S rRNA gene V4 region sequence database. (5) forming a V4 region sequence database of the human vaginal flora 16S rRNA gene; based on the construction method, the human vaginal flora is identified, community type classification is carried out, and a new community type is named as CST6. Human vaginal flora detection can be annotated to the genus or species level, the diversity and richness of the human vaginal flora are disclosed, and the data can be used for analyzing whether the human vaginal flora is disordered or not, discovering whether known pathogenic bacteria or potential pathogenic bacteria exist or not, and determining whether the human vaginal flora is disordered or not. Further, the correlation between the vaginal flora disorder and diseases such as female health conditions and cervical cancer is explored.

Description

Technical Field

[0001] The invention belongs to the field of microbial ecology and microbial taxonomy, and specifically relates to a method for constructing a vaginal flora 16S rRNA gene V4 region sequence database and an application thereof. Background Art

[0002] A large number of microorganisms live in the human body. As one of the most important microbial flora in the human body, the vaginal microbiome has an important impact on women's physiological and reproductive health. In recent years, many studies have shown that vaginal microecological dysbiosis is closely related to the occurrence and development of persistent HPV infection and cervical cancer. Persistent HPV infection can lead to vaginal microecological dysbiosis. Disturbance of vaginal microecology can reduce the barrier function of the cervix and vagina, and promote HPV infection and the occurrence of cervical prelesions / cancer. Cervical cancer is a common malignant tumor that seriously threatens women's health and has become a major global public health issue. The incidence of cervical cancer in my country has been increasing year by year. In 2021, there were 111,820 new cases of cervical cancer in China, accounting for 18% of the world's new cases, and 61,579 deaths, making it the world's second largest country with the burden of cervical cancer. The average age of onset is gradually decreasing, with a trend of younger age.

[0003] 16S rRNA is a component of the 30S small subunit of the prokaryotic ribosome, and the gene encoding 16S rRNA is called the 16S rRNA gene. The 16S rRNA gene is commonly found in bacteria and archaea, with multiple copies and a total length of about 1500bp. Its structure is composed of 9 variable regions and 10 conservative regions alternating. The conservative regions are conducive to the design of primer amplification, and the variable regions reflect the evolutionary differences between species. 16S rRNA sequencing can analyze the diversity and distribution of microbial communities, reveal the types and relative abundance of microorganisms, etc., and it takes less time and has lower sequencing costs.

[0004] Currently, the existing databases have the following drawbacks: (1) The taxonomic hierarchy and scientific names of some species have changed, and the gene annotation information database has not been updated for a long time, which affects the accuracy of the annotation; (2) The gene annotation information of some species is only at the "genus" level, and the annotation information at the "species" level is unclear, which affects the accuracy of the analysis. Summary of the invention

[0005] To solve the above problems, the present invention provides a method for constructing a 16S rRNA gene V4 region sequence database based on human vaginal flora and its application. By analyzing the 16S sequencing data of local genital tract bacteria, the miscellaneous bacteria contaminated by environmental factors and the like are eliminated, and a multi-sequence global alignment of the 16S rRNA gene hypervariable region V4 sequence is performed to reconstruct a vaginal flora 16S rRNA hypervariable region V4 database, providing analysis data at the "genus" or "species" level, and exploring the correlation between vaginal flora disorders and women's health conditions, cervical cancer and other diseases.

[0006] To achieve the above object, the technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a method for constructing a human vaginal flora 16S rRNA gene V4 region sequence database, which specifically comprises the following steps:

[0008] S1-1: Obtain 16S rRNA gene sequences. Based on the vaginal microbiome species in EzBioCloud, mBodyMap, and VMGC databases, retrieve and download the corresponding 16S rRNA gene sequences;

[0009] S1-2: Sequence alignment and correction. The above sequences were uploaded to the NCBI RefSeq 16S rRNA database, and the sequences were aligned and corrected using BLAST software to confirm the species taxonomic nomenclature and delete erroneous sequences;

[0010] S1-3: Construct a full-length 16S rRNA database of human vaginal flora. Supplement and update the EzBioCloud database (2018 version) based on new discoveries and literature publications on vaginal flora.

[0011] S1-4: Obtain the V4 region fragment of the 16S rRNA gene of the human vaginal flora. The gene sequence in the human vaginal flora 16S rRNA full-length sequence database constructed in step S1-3 was extracted using the extract-reads method in the feature-classifier plug-in of QIIME 2, and the V4 region primers (515F: 5'-GTGYCAGCMGCCGCGGTAA-3'; 806R: 5'-GGACTACNVGGGTWTTAAT-3') were used to obtain the V4 region fragment of the 16S rRNA gene, and the fragments with a length of less than 120 bp and greater than 300 bp were filtered out.

[0012] S1-5: Form a human vaginal flora 16S rRNA gene V4 region sequence database. Based on the vaginal flora 16S rRNA gene V4 region fragments obtained above, delete environmental pollution and non-genital tract colonization flora from the database, delete all flora sequences that may originate from the intestine or the environment (such as Ruminococcus, Bacteroides, etc.), merge the species annotation information with completely consistent V4 region sequences, and renumber these species to obtain the vaginal flora 16S rRNA gene V4 region sequence database.

[0013] In an embodiment of the present invention, the sequence correction step in step S1-2 is as follows:

[0014] (1) Statistics were collected for the sequences in the local 16S rRNA gene V4 region sequence database constructed above and the NCBI RefSeq 16S rRNA database with 100% identity and coverage and the same species name;

[0015] (2) Statistics were collected for sequences in the local 16S rRNA gene V4 region sequence database constructed above and the NCBI RefSeq 16S rRNA database whose identity and coverage were both 100% and whose species names were inconsistent;

[0016] (3) For the sequences whose identity and coverage were not 100% in the local 16S rRNA gene V4 region sequence database constructed above and the NCBI RefSeq 16S rRNA database, they were first filtered according to the rules of identity greater than 80% and coverage greater than 80%. Then, for the species corresponding to one sequence, they were sorted according to the bit score, and the annotated species with the highest bit score was selected as the final result. The total sequence, sequence with consistent name, and sequence with inconsistent name were counted separately.

[0017] (4) The sequence results of the above steps were merged, and the sequences with duplicate NCBI accession numbers were removed. The total number of sequences and species was counted. Then, the corresponding fasta sequences were extracted from NCBI RefSeq 16S rRNA, and BVAB-1, BVAB-2, and BVAB-3 were merged. The Lactobacillus sequences were manually screened through the literature. For low-abundance but vaginal-specific Lactobacillus species, the V4 region sequences of the species were retained as much as possible. All the sequences counted, screened, and retained as above were used as the final reference sequences for the reproductive tract.

[0018] In an embodiment of the present invention, the sequence determination in step S1-1 of the construction method is high-throughput sequencing. The embodiment of the present invention is based on the sequence data obtained by sequencing the V4 region of the 16S rRNA gene of the human vaginal flora sample to be tested using the MG2000 sequencing platform.

[0019] In an embodiment of the present invention, the mBodyMap in step S1-1 of the construction method is a selected database for microorganisms in the human body and their association with health and disease, and VMGC is a vaginal microbial genome library.

[0020] In an embodiment of the present invention, the NCBI RefSeq 16S rRNA database contains annotated non-redundant genomic DNA, transcripts, and protein sequences, all of which are naturally occurring molecules in the central dogma, providing reference sequence standards from chromosomes to mRNAs to proteins.

[0021] In an embodiment of the present invention, when performing the filtering of the fragments in step S1-4, for BVAB and rare lactobacilli, the filtering conditions are relaxed to 100-320 bp to ensure integrity.

[0022] In an embodiment of the present invention, the vaginal flora may be any microorganism, including but not limited to bacteria, fungi, archaea, viruses, etc. In an embodiment of the present invention, the vaginal flora is bacteria.

[0023] In a second aspect, based on the human vaginal flora 16S rRNA gene V4 region sequence database constructed above, the present invention provides an application for identifying human vaginal flora at the "genus" or "species" level and classifying the community type based on the vaginal flora 16S rRNA gene V4 region sequence, and the community type classification steps are as follows:

[0024] S2-1: Classify the human vaginal flora into community types based on publicly published literature;

[0025] S2-2: Based on the constructed human vaginal flora 16S rRNA gene V4 region sequence database and combined with the classification in step S2-1, the local database is annotated with species, the dominant bacteria of each sample are determined, and a CST label is added to each sample;

[0026] S2-3: Statistical analysis of vaginal flora of local data population and calculate the proportion of human vaginal flora;

[0027] S2-4: Classify the newly discovered community type and name it CST6.

[0028] In an embodiment of the present invention, the infectious agents involved in the community include VVC, HPV, CT and mycoplasma.

[0029] The present invention has the following beneficial effects:

[0030] (1) The method for constructing a vaginal flora 16S rRNA gene V4 region sequence database provided by the present invention compares and corrects the sequences of vaginal microbiome species, removes bacteria contaminated by environmental factors, and corrects and updates the existing database, making the classification and annotation information more accurate and comprehensive.

[0031] (2) The use of the ASV region of the V4 region of the 16S rRNA gene helps to identify specific bacterial species in local databases, such as Escherichia coli and Klebsiella pneumoniae.

[0032] (3) Using the V4 variable region, the primers are more universal and can detect more low-abundance species, which helps to identify specific bacterial species in the local database and fully reveal the diversity of the microbial community.

[0033] (4) The use of the vaginal flora 16S rRNA gene V4 region sequence database provided by the present invention is helpful to identify and characterize potential pathogens of the reproductive tract, reveal the diversity and richness of the vaginal flora, and provide data support for exploring the correlation between vaginal flora disorders and female gynecological diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a statistical diagram of the proportion of human vaginal flora in Example 2 of the present invention.

[0035] Figure 2 Figure 2 is a diagram showing the detection of pathogenic bacteria in Example 2 of the present invention.

[0036] Figure 3 Flowchart for constructing the database of the present invention DETAILED DESCRIPTION

[0037] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the following specific embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The materials, reagents, instruments, etc. used in the examples are all commercially available unless otherwise specified. It should be understood that the embodiments described herein are only a part of the embodiments of the present invention and are not intended to limit the present invention. Based on the embodiments of the present invention, all other embodiments of the changes or replacements of the experimental steps and conditions of the present invention belong to the protection scope of the present invention.

[0038] The definitions of the main abbreviations and key terms involved in the present invention are as follows:

[0039] HPV:Human Papilloma Virus

[0040] NCBI: National Center for Biotechnology Information, U.S. National Center for Biotechnology Information;

[0041] ASVs: Amplicon Sequence Variants, amplicon sequence variations;

[0042] VMGC: Vaginal Microbial Genome Collection;

[0043] VVC: Vulvovaginal Candidiasis;

[0044] CT: Chlamydia Trachomatis;

[0045] BVAB: Bacterial vaginosis-associated bacteria, bacterial vaginosis-associated bacteria, is a Gram-negative anaerobic bacterium that is usually not dominant in a healthy vaginal environment, but can multiply under certain conditions.

[0046] Example 1

[0047] A method for constructing a database based on the 16S rRNA gene V4 region sequence of vaginal flora

[0048] 30,800 Chinese vaginal flora samples were sequenced using the MGI2000 sequencing platform, and 100,000 16SrRNA gene sequences were obtained.

[0049] S1-1: Obtain 16S rRNA gene sequences. Based on the vaginal microbiome species in the EzBioCloud database, retrieve and download the corresponding 16S rRNA gene sequences.

[0050] S1-2: Sequence alignment and correction. The sequences obtained in step S1-2 above were uploaded to the NCBI RefSeq 16SrRNA database, and the sequences were aligned and corrected using BLAST software to confirm the species taxonomic nomenclature and delete erroneous sequences, see Table 1. The correction process and results are as follows:

[0051] (1) The identity and coverage of the sequences in the local database and the NCBI RefSeq 16S rRNA database were both 100%, and there were 312 sequences with the same species name;

[0052] (2) The identity and coverage of the sequences in the local database and the NCBI RefSeq 16S rRNA database were both 100%, and there were 73 sequences with inconsistent species names;

[0053] (3) For sequences in the local database whose identity and coverage are not 100% in the NCBI RefSeq 16s rRNA database, we first filter them according to the rule that identity is greater than 80% and coverage is greater than 80%. Then, for multiple species corresponding to one sequence, we sort them according to the bit score and select the annotated species with the highest bit score as the final result.

[0054] A total of 786 sequences were obtained, of which 323 sequences had consistent species names and 463 sequences had inconsistent species names.

[0055] (4) The sequence results of steps (1) to (3) were merged, and the sequences with duplicate NCBI accession numbers were removed, resulting in a total of 983 sequences and 851 species. Then, the corresponding fasta sequences were extracted from NCBI RefSeq 16S rRNA, and then BVAB-1, BVAB-2, and BVAB-3 were merged. The Lactobacillus sequences were manually screened through the literature. For the low-abundance but vagina-specific Lactobacillus species, the V4 region sequence of the species was retained as much as possible. All the sequences counted, screened, and retained above were used as the final reference sequences for the reproductive tract, resulting in a total of 1216 sequences, including 997 species.

[0056] S1-3: Construct a database of full-length sequences of 16S rRNA of human vaginal flora. Based on new discoveries and published literature on vaginal flora, the EzBioCloud database (2018 version) is supplemented and updated. It includes 50,000 16S rRNA data of reproductive tract bacteria, and can detect and identify 1,177 published bacterial species.

[0057] Table 1. Vaginal flora revision record

[0058]

[0059] S1-4: Obtain the V4 region fragments of the 16S rRNA gene of the human vaginal flora. The 50,000 gene sequences in the human vaginal flora 16SrRNA full-length database constructed above were extracted using the extract-reads method in the QIIME 2 plug-in feature-classifier. According to the hypervariable region V4 primers (515F: 5'-GTGYCAGCMGCCGCGGTAA-3'; 806R: 5'-GGACTACNVGGGTWTTAAT-3'), the 16S rRNA gene V4 region fragments were obtained by computer shearing. At the same time, the fragments with lengths less than 120 and greater than 300 were filtered out. A total of 1216 V4 region fragments were obtained, including 997 species and 879 different fragments. There are 781 V4 region-specific fragments (corresponding to only one species) and 98 V4 region-nonspecific fragments.

[0060] S1-5: Form a human vaginal flora 16S rRNA gene V4 region sequence database. Based on the vaginal flora 16S rRNA gene V4 region fragments obtained above, delete environmental pollution and non-genital tract colonization flora from the database, delete all flora sequences that may originate from the intestine or the environment (such as Ruminococcus, Bacteroides, etc.), merge the species annotation information with completely consistent V4 region sequences, and renumber these species to obtain the vaginal flora 16S rRNA gene V4 region sequence database.

[0061] In an embodiment of the present invention, when filtering the fragments in step S1-4, for bacterial vaginosis-associated flora (BVAB) and rare lactobacilli, the filtering conditions are relaxed to 100-320 bp to ensure their integrity.

[0062] In the embodiments of the present invention, by correcting and updating the existing database and using the ASV region of the V4 region of the 16S rRNA gene, it helps to provide more accurate and comprehensive species annotation information and identify specific bacterial species in the local database.

[0063] Example 2

[0064] Identification of flora types and classification of flora types based on human vaginal flora 16S rRNA gene V4 sequence database In this example, 90,000 vaginal flora samples were used for 16S V4 region amplicon sequencing.

[0065] S2-1: According to the published literature (PMID: 20534435), the human vaginal flora is divided into five categories: CST1 (dominated by Lactobacillus crispatus), CST2 (dominated by Lactobacillus gasseri), CST3 (dominated by Lactobacillus iners), CST4 (dominated by non-lactobacillus with a significantly reduced number of Lactobacillus), and CST5 (dominated by Lactobacillus jensenii).

[0066] S2-2: Based on the human vaginal flora 16S rRNA gene V4 region sequence database constructed in Example 1 and combined with the classification standard of step S2-1, the 90,000 vaginal flora sample data are annotated with species, the dominant bacteria of each sample are determined, and a CST label is added to each sample;

[0067] S2-3: Statistical analysis of vaginal flora of people in the local database and calculate the proportion of human vaginal flora (see Figure 1 ). The specific situation is: the population sample data is mainly CST1 and CST3, which account for about 72% of the total population, followed by CST4, which accounts for about 23%, and others such as CST2, which accounts for 3%, and CST5, which accounts for 2%.

[0068] S2-4: Division and naming of new community types. Based on the sequencing data of 90,000 vaginal flora samples, it was found that the vaginal flora of a group of people was dominated by Lactobacillus delbrueckii, which was defined as CST6.

[0069] In an embodiment of the present invention, the division of new community types in the above step S2-4 not only broadens the traditional classification system, but also demonstrates the significant advantages of the adopted database in high-resolution species annotation, providing a powerful tool support for future research on the complexity and diversity of vaginal flora.

[0070] Furthermore, based on the detection of pathogenic bacteria in 90,000 samples and combined with the CST classification results, it was found that the infection rates of four infectious substances in the CST3 population were higher than those in CST1 / CST5 / CST4, respectively. These four infectious substances were VVC, HPV, CT and mycoplasma; three infectious substances in the CST2 population were lower than those in other CST populations, namely HPV, Chlamydia trachomatis (CT) and mycoplasma; and one infectious substance in the CST6 population, namely Chlamydia trachomatis, was lower than that in other CST populations (see Figure 2 ).

[0071] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprises" or any other variations are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0072] It should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. For ordinary technicians in this technical field, several improvements and modifications can be made on the basis of the above embodiments without departing from the principles of the present invention. These improvements and modifications all fall within the scope of protection of the claims of the present invention.

Claims

1. A method for constructing a vaginal flora 16S rRNA gene V4 region sequence database, characterized in that: The method for constructing the database comprises the following steps: S1-1: Obtain 16S rRNA sequences: Based on the vaginal microbiome species in the EzBioCloud database, mBodyMap, and VMGC, retrieve and download the corresponding 16S rRNA gene sequences; S1-2: Sequence alignment and correction: Upload the sequence obtained in step S1-1 to the NCBI RefSeq 16S rRNA database, use BLAST software to align and correct the sequence, confirm the species taxonomic nomenclature, and delete the erroneous sequence; S1-3: Construction of a full-length 16S rRNA database of human vaginal flora: Supplement and update the EzBioCloud database (2018 version) based on new discoveries and literature publications on vaginal flora; S1-4: Obtain the V4 region fragment of the human vaginal flora 16S rRNA gene. The gene sequence of the human vaginal flora 16S rRNA full-length database constructed in S1-3 above was extracted using the extract-reads method in the QIIME 2 plug-in feature-classifier. According to the hypervariable region V4 primers (515F: 5'-GTGYCAGCMGCCGCGGTAA-3'; 806R: 5'-GGACTACNVGGGTWTTAAT-3'), the 16S rRNA gene V4 region fragment was obtained by computer shearing, and the fragments with lengths less than 120 bp and greater than 300 bp were filtered out. S1-5: Form a human vaginal flora 16S rRNA gene V4 region sequence database. Based on the vaginal flora 16S rRNA gene V4 region fragment obtained in step S1-4, delete environmental pollution and non-genital tract colonization flora from the database, delete all flora sequences that may originate from the intestine or the environment (including Ruminococcus and Bacteroides), merge the species annotation information with completely consistent V4 region sequences, and renumber these species to obtain the vaginal flora 16S rRNA gene V4 region sequence database.

2. A method for constructing a vaginal flora 16S rRNA gene V4 region sequence database according to claim 1, characterized in that: The database for searching the 16S rRNA gene V4 region sequence in step S1-1 comes from a publicly available sequence library, including but not limited to: EzBioCloud database, mBodyMap and VMGC database.

3. The method for constructing a vaginal flora 16S rRNA gene V4 region sequence database according to claim 1, characterized in that: The specific steps of sequence correction in step S1-2 are as follows: (1) Statistics were collected for the sequences in the local 16S rRNA gene V4 region sequence database constructed above and the NCBI RefSeq 16S rRNA database with 100% identity and coverage and the same species name; (2) Statistics were collected for sequences in the local 16S rRNA gene V4 region sequence database constructed above and the NCBI RefSeq 16S rRNA database whose identity and coverage were both 100% and whose species names were inconsistent; (3) For the sequences whose identity and coverage were not 100% in the local 16S rRNA gene V4 region sequence database constructed above and the NCBI RefSeq 16S rRNA database, we first filtered them according to the rules of identity greater than 80% and coverage greater than 80%. Then, for sequences corresponding to multiple species, we sorted them according to the bit score, and selected the species with the highest bit score as the final result. We then counted the total sequences, sequences with consistent names, and sequences with inconsistent names respectively. (4) The sequence results in steps (1)-(3) were merged, and the sequences with duplicate NCBI accession numbers were removed. The total number of sequences and species was counted. Then, the corresponding fasta sequences were extracted from NCBI RefSeq 16S rRNA, and BVAB-1, BVAB-2, and BVAB-3 were merged. The Lactobacillus sequences were manually screened through the literature. For low-abundance but vaginal-specific Lactobacillus species, the V4 region sequences of the species were retained as much as possible. All the sequences counted, screened, and retained as described above were used as the final reference sequences for the reproductive tract.

4. The method for constructing a vaginal flora 16S rRNA gene V4 region sequence database according to claim 1, characterized in that: When filtering the fragments in step S1-4, for bacterial vaginosis-associated flora (BVAB) and rare lactobacilli, the filtering conditions are relaxed to 100-320 bp to ensure integrity.

5. The method for constructing a vaginal flora 16S rRNA gene V4 region sequence database according to claim 1, characterized in that: The vaginal flora may be any microorganism, including but not limited to bacteria, fungi, archaea and viruses.

6. An application of a vaginal flora 16S rRNA gene V4 region sequence database, characterized in that: Based on the 16S rRNA gene V4 region sequence, human vaginal flora is identified at the "genus" or "species" level and the flora type classification is performed. The flora type classification specifically includes the following process: S2-1: Classify the human vaginal flora into community types based on publicly published literature; S2-2: Based on the vaginal flora 16S rRNA gene V4 region sequence database constructed in claim 1 and combined with the classification in step S2-1, perform species annotation on the local database, determine the dominant bacteria of each sample, and add a CST tag to each sample; S2-3: Statistical analysis of vaginal flora of local data population and calculate the proportion of human vaginal flora; S2-4: Classify the newly discovered community type and name it CST6.

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