Simplified genome analysis method for combined analysis of host and coexisting microorganisms thereof

Abstract

The invention discloses a simplified genome analysis method for combined analysis of hosts and coexisting microorganisms thereof. The method comprises the following steps: constructing a holographic omics database of host and microorganism IIB enzyme digestion; selecting a proper material to extract mixed DNA; mixing DNA and carrying out quality inspection; after the quality inspection is qualified, a 2b-RAD / multi-omics MisRAD library is constructed, and sequencing is carried out; sequencing data is subjected to quality control and enzyme digestion label extraction and then compared to the constructed database, and host genotype analysis and microorganism species and abundance analysis are conducted on the data compared to hosts and microorganisms through different software. According to the method, synchronous conjoint analysis of host genome typing and microbial species and abundance identification can be realized in a single library at low cost and high efficiency; the requirement on the DNA detection quantity is extremely low, destructive sampling is not needed, and the change of holographic omics can be dynamically tracked.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology DNA genetic markers and microbiology, and specifically relates to a simplified genome analysis method that can jointly analyze a host and its coexisting microorganisms. Background Art

[0002] In recent years, the importance of microorganisms in the growth and development of the host has gradually become prominent, affecting the core functions of the host, such as growth, development, immunity, nutrition and reproduction. In addition to being affected by the environment, the composition and abundance of microorganisms are also affected by the genetic background of the host. Studies have reported that the composition of microorganisms shows stable differences between individuals, the characteristics of microbial communities between different sexes are also different, and different genotypes in the population have different microbial community characteristics. These results all indicate the influence of host genetics on microbial composition. Therefore, holographic omics analysis, that is, the joint analysis of hosts and microorganisms, has become a research hotspot in the ecology and evolution of microorganisms and hosts.

[0003] At present, the strategies adopted by holographic omics research mainly combine 16s amplicon sequencing technology with genotyping technology, such as whole genome resequencing, simplified genome sequencing (RAD / 2b-RAD), etc. 16s amplicon sequencing technology is one of the mainstream technologies for microbial analysis. However, it can only obtain the genetic information of bacteria, and the genetic diversity of fungi and viruses cannot be obtained. In addition, in order to obtain the genetic information of the host, additional host sequencing libraries still need to be constructed. Therefore, this strategy requires obtaining tissue materials for host genotyping and microbial analysis separately, and obtaining host and microbial information through two extractions and two library constructions. The process is complicated and the process is cumbersome. The cost of preparing libraries separately and sequencing them separately is also high.

[0004] In addition to the above strategies, a more comprehensive method than 16S amplicon sequencing is shotgun sequencing. It can sequence the genomes of all microorganisms in the sample, and can also perform functional analysis and genome assembly. Now the method of analyzing a variety of biological information through shotgun sequencing libraries has been established, and holographic omics analysis can be achieved from a shotgun sequencing library. However, the shotgun method requires deep coverage of the sequencing library, which means high costs. Moreover, there is no standardized data analysis process and it has not been applied.

[0005] In addition, the huge amount of material required by the above two strategies makes it particularly difficult to dynamically track the changes in hosts and microorganisms in smaller organisms, such as some shellfish and shrimps. Therefore, how to achieve dynamic holographic omics tracking at low cost is an urgent problem that technicians in this field need to solve. Summary of the invention

[0006] In view of the above deficiencies in the prior art, the present invention provides a simplified genome analysis method for jointly analyzing a host and its coexisting microorganisms.

[0007] The technical solution of the present invention is as follows: A simplified genome analysis method for jointly analyzing a host and its coexisting microorganisms comprises the following steps: (1) Based on the host genome sequence, microbial genome sequence and the recognition sequence of type IIB restriction endonuclease, the electronic restriction endonuclease tags of the host and microorganism are extracted to establish a host-microorganism type IIB restriction endonuclease tag database. Sequences specific to microorganisms at different taxonomic levels and host-specific sequences are obtained to establish a host-microorganism type IIB restriction endonuclease holographic omics database; (2) Extraction of mixed DNA: Extraction of mixed DNA from materials containing mixed DNA; (3) Use primer pair 515F and 907R in the V4-5 region of the 16S rDNA variable region to amplify the mixed DNA obtained in step (2). If bands appear at 400 bp and 600 bp, the amplification is considered qualified; The nucleotide sequence of 515F is: 5'-GTGCCAGCAGCGGTAA (SEQ ID NO: 1); The nucleotide sequence of 907R is: 5'-CCGTCAATTCTTGTAGTTTT (SEQ ID NO: 2); Primers in other regions of 16S can only bind to bacterial genome sequences and effectively amplify bacterial bands, but cannot bind to eukaryotic genome sequences. Therefore, only the presence of microbial DNA can be identified, but not the presence of eukaryotic DNA. The primers used in the present invention can not only amplify the V4-5 region of bacteria, with a fragment length of about 400bp; they also have binding sites with scallop and shrimp genome sequences, and can amplify host genome fragments with a length of about 600bp.

[0008] (4) Using the mixed DNA samples qualified in step (3), construct a 2bRAD / Miso-RAD sequencing library and perform high-throughput sequencing; (5) Preprocess the sequencing data obtained in step (4), remove low-quality data, extract sequences containing restriction sites, align the restriction fragments in the extracted sequencing data to the database constructed in step (1), and then perform host and microbial analysis respectively.

[0009] Preferably, step (1) comprises: According to the host genome sequence, microbial genome sequence and the recognition sequence of type IIB restriction endonuclease, all known microbial genomes and the analyzed host genome are simultaneously electronically digested to obtain electronic enzyme tags of all microorganisms and hosts, and a host-microorganism IIB enzyme tag database is established; the enzyme tags are mutually compared at different classification levels of microbial species, and are also compared with the host enzyme tags, and enzyme fragments that belong only to a certain species and have no duplication with other species are screened out, thereby establishing a host-microorganism IIB enzyme cleavage holographic omics database.

[0010] Preferably, in step (2), the material containing mixed DNA includes cotton swabs, feces, intestinal or larval materials after wiping tissue; the tissue includes gill filaments, mantle, skin, and oral cavity.

[0011] Preferably, the tissue is gill filaments or oral cavity.

[0012] Preferably, in step (4), constructing a 2bRAD / Miso-RAD sequencing library includes constructing a single-tag library or constructing a multiple-tag tandem library.

[0013] Preferably, step (4) comprises: The mixed DNA in step (2) is digested with a type IIB restriction endonuclease, and corresponding adapters are selected for ligation according to the type IIB enzyme used. The ligated fragments are enriched by PCR, the PCR products are recovered and purified, and barcode sequence tags are added for PCR amplification.

[0014] Preferably, the type IIB restriction endonuclease includes AlfI, AloI, BaeI, BcgI, BplI, BsaXI, BslFI, CspCI, FalI, Hin4I or PpiI.

[0015] Preferably, step (5) comprises: The sequencing data obtained in step (4) are preprocessed, and low-quality data are removed according to the principle that the sequence does not contain N and the quality value of more than 80% of the bases in the sequence is greater than 20. The sequences containing restriction sites are extracted, and the restriction fragments in the extracted sequencing data are aligned to the host-microorganism IIB restriction holographic database constructed in step (1); the data of the host sequence aligned to the database are subjected to whole genome typing, and the data of the microbial genome aligned to the microbial genome are subjected to microbial composition and abundance analysis.

[0016] Preferably, the host comprises shellfish or shrimp.

[0017] Preferably, the host comprises the Yesso scallop.

[0018] Beneficial effects of the present invention: (1) It can realize the joint analysis of hosts and microorganisms through a single library at low cost and high efficiency: Under the same sequencing depth, the cost of realizing the joint analysis of hosts and microorganisms by this technology is about 5% of that of shotgun sequencing, and 80% of that of the combination of amplicon sequencing and whole genome genotyping technology. In addition, this technology only requires one sampling, one DNA extraction, and one sequencing library to realize the simultaneous joint analysis of hosts and microorganisms; and it requires a small amount of data, occupies less storage space and computing resources, has a standardized analysis process, and can achieve more efficient data analysis.

[0019] (2) Dynamic holographic omics tracking can be achieved. This technology requires an extremely low amount of DNA input and is also applicable to DNA with a high degree of degradation. Therefore, there is no need to destructively sample the organism. Even for some small aquatic organisms, the amount of material obtained by swabbing the tissue can be used for library construction and analysis.

[0020] (3) The DNA extracted by this technology is mixed DNA, which contains both host genetic information and microbial genetic information. Therefore, the relative proportion of host and microbial DNA in different stages or environments can be obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : Schematic diagram of the process and principle of a simplified genome analysis method for joint analysis of the host and its coexisting microorganisms. Figure 2 for Figure 1 A partial enlarged view of Figure 2 The horizontal axis is the chromosome number, and Chromosome means "chromosome". Figure 3 for Figure 1 A partial enlarged view of .

[0022] Figure 4 : Comparison diagram of detection consistency between Example 1 of the present invention and Comparative Example 2. DETAILED DESCRIPTION

[0023] In order to better understand the technical content of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings.

[0024] Example 1 The Yesso scallop was used as the experimental object, and the cotton swab after wiping the gill filaments was used as the material. The Yesso scallop and its microorganisms were simultaneously analyzed by the following methods: (1) Construction of a holographic database of IIB enzyme digestion of scallop and microorganisms The reference genome of the scallop (GCA_002113885.2) and the currently available microbial genomes were downloaded from NCBI, and the restriction tags of the scallop and microorganisms were extracted according to the IIB enzyme recognition site. Through mutual comparison, the specific DNA tag sequences that appeared only once in each genome were retained in these tags. After extraction, a total of 377,566 non-redundant tags were generated in the scallop, and an average of 1231.48 tags were generated for each microbial genome. After alignment and de-redundancy, 314,707 specific tags were obtained in the scallop; about 8218 microbial genomes were deleted, accounting for 4.75% of all microbial genomes. The removed microbial genomes had almost no effect at the phylum and genus levels (only 0.01% to 0.2%), and only affected 3.71% of species at the species level.

[0025] (2) Collection of scallop swab samples and extraction of mixed DNA Use a sterile cotton swab to gently wipe the gill filaments three times to remove them. Place the gill swab in a 1.5 ml centrifuge tube, add 900 μL 1×PBS, and shake at 3,000 rpm for 10 minutes. Centrifuge briefly and slowly, transfer all the solution in the tube (about 1 ml) to a new 2 ml centrifuge tube, and use a pipette to draw the cotton ball several times to dry it to avoid sample loss. Add 500 μL Buffer AHL (AHL buffer, provided by the QIAamp DNA Microbiome Kit) and incubate at room temperature with end-to-end rotation for 30 minutes. Then use the QIAamp DNA Microbiome Kit to extract DNA.

[0026] (3) Mixed DNA quality inspection The mixed DNA sample was amplified using primers in the 16S rDNA V4-5 region. The total PCR reaction system was 20 µL, including 0.5 µM primer 1 (515F: 5'-GTGCCAGCAGCGGTAA) and 0.5 µM primer 2 (907R: 5'-CCGTCAATTCTTGTAGTTTT), 10 ng of genomic DNA, 0.2 U DNA polymerase (Phusion® DNA polymerase, manufacturer: NEB), and 1× HF buffer (HF buffer, manufacturer: NEB). The reaction conditions were: denaturation at 98°C for 20 s, annealing at 55°C for 15 s, extension at 72°C for 10 s, 35 cycles, extension at 72°C for 5 min, and incubation at 4°C. The amplified product was subjected to agarose gel electrophoresis (1% agarose gel run at 100 V for 25 min) to observe whether the product had double bands. If both 400 bp and 600 bp bands were present, proceed to step (4).

[0027] (4) Construction of DNA library (2b-RAD / Miso-RAD sequencing library) 1) Use BsaXI restriction endonuclease (NEB) to digest the mixed DNA extracted above. The digestion system is 15 μL, including 200 ng genomic DNA, 2U endonuclease, and 1× Cutsmart Buffer (endonuclease buffer, manufacturer: NEB). After incubation at 37℃ for 2h, place at -80℃ for 20min to inactivate the endonuclease.

[0028] 2) Ligate the above digested product with the adapter. The ligation reaction system is 20 μL, including 10 μL digested product, 200U T4 DNA ligase (NEB), 1×T4 Ligase Buffer (ligase buffer, NEB), 1.5mM Slx-AdaA, 1.5mM Slx-AdaB. Ligate at 16℃ for 2h.

[0029] The two nucleotide sequences constituting Ada1A are 5'-ACACTCTTTCCCTACACGACGCTGTTCCGATCTNNN-3' (SEQ ID NO:3) and 5'-AGATCGGAACAGC-3' (SEQ ID NO:4), and the nucleotide sequences of Ada1B are 5'- GTGACTGGAGTTCAGACGTGTGCTGTTCCGATCTNNN -3' (SEQ ID NO:5) and 5'-AGATCGGAACAGC -3' (SEQ ID NO:6) 3) PCR amplification of the ligation product was performed. The amplification reaction system was 50 μL, including 18 μL of ligation product, 0.1 mM PrimerA, 0.1 mM PrimerB, 0.3 mM dNTPs (NEB), 0.8 U DNA polymerase (Phusion® DNA polymerase, manufacturer: NEB), and 1× HF buffer. The reaction conditions were denaturation at 98°C for 5 s, annealing at 60°C for 20 s, extension at 72°C for 10 s, 16 cycles, extension at 72°C for 10 min, and insulation at 4°C. The above products were subjected to acrylamide gel electrophoresis, and the target fragment was recovered by gel excision.

[0030] The nucleotide sequence of PrimerA is 5'-ACACTCTTTCCCTACACGACGCT-3' (SEQ ID NO: 7); the nucleotide sequence of PrimerB is 5'-GTGACTGGAGTTCAGACGTGTGCT-3' (SEQ ID NO: 8); 4) The above-mentioned recovered products were amplified by PCR to introduce the Barcode sequence (also called the tag sequence) to obtain a sequencing library. The PCR amplification reaction system was 50 μL, including 15 μL of recovered products, 0.1 mM Primer3, 0.1 mM index Primer, 0.3 mM dNTPs (NEB), 0.8 U DNA polymerase (Phusion® DNA polymerase, manufacturer: NEB), and 1× HF buffer. The reaction conditions were denaturation at 98°C for 5 s, annealing at 60°C for 20 s, extension at 72°C for 10 s, 6-10 cycles, extension at 72°C for 10 min, and insulation at 4°C.

[0031] The nucleotide sequence of primer Primer3 is 5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCT-3′ (SEQ ID NO: 9); The nucleotide sequence of the primer Index Primer is 5'-CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3' (SEQ ID NO: 10), wherein NNNNNN can be changed according to different Barcode sequences.

[0032] 5) Sequencing of DNA library: Illumina Hiseq / Miseq / Nextseq sequencing platform, Illumina Xten / Nova / NovaX plus sequencing platform, Ion torrent sequencing platform, Pacbio sequencing platform and BGIseq sequencing platform can all be selected. This experiment uses the Illunima Hiseq Xten sequencing platform for PE150 sequencing.

[0033] (5) Data analysis: 1) The raw sequencing data obtained by sequencing were first quality controlled. Low-quality data were removed based on the principle that the sequence could not have consecutive Ns and the quality value of more than 80% of the bases in the sequence was >20. The 32bp base fragments with enzyme-specific cutting sites were extracted from the retained high-quality data to form a sequence for subsequent analysis (proc_data).

[0034] 2) Use the sequences in the holographic database of the Yesso scallop-microbe IIB digestion constructed in step (1) as a reference and compare proc_data with it. SOAP2, Centrifuge, BWA, Bowtie1 / 2, Stack, and Mega-balst can be used for comparison. In this case, SOAP2 was used for comparison and the matching of unique sequences was analyzed. It was found that the proportion of data matched to the Yesso scallop-specific tags was 94.74%-97.70%, and the proportion of data matched to the microbiome-specific tags was 2.30% - 5.26%.

[0035] 3) The data was compared with the data of the Japanese scallop to perform SNP loci typing on the whole genome, and the data was compared with the microbiome to analyze the microbial composition and abundance.

[0036] Comparative test 1 The only difference from Example 1 is that the genomic DNA of the gill filaments of the same Yesso scallop was extracted by the traditional phenol-chloroform extraction method, a 2b-RAD / Miso-RAD sequencing library was constructed, and two parallel replicates were performed as controls.

[0037] The analysis shows that the two parallel libraries constructed in Example 1 of the present invention (i.e., the libraries constructed by two parallel experiments using the method of Example 1) have a tag type consistency of 95.46%, and a sequencing depth consistency Pearson r (Pearson correlation coefficient) of 0.9487. Compared with the control group, the tag type consistency reached 94.76%. The SNP typing results are shown in Table 1. A total of 2,271,248 sites shared with the control group were detected in the library constructed in the present invention, of which 2,268,599 sites had consistent typing results, and the typing accuracy was 99.88%.

[0038] Table 1 Statistical table of typing accuracy of the present invention

[0039] Comparative test 2 The difference from Example 1 is that the extracted DNA is amplified by primers in the 16S rDNA V4-5 region, a 400 bp microbial target band is recovered, and the recovered product is re-amplified to introduce a barcode (also called a tag sequence) to construct a 16S amplicon sequencing library as a control. The amplicon sequencing data uses the standard QIIME (v.1.9) pipeline.

[0040] The analysis showed that the consistency of the number of microbial species detected by the two parallel libraries constructed in the present invention was 95.00%, and the consistency of the relative abundance of the detected species was 0.9996. Compared with 16S amplicon sequencing, at the genus level, the consistency of species detection reached 94%, and the consistency of relative abundance was 0.9367. Figure 4 .

[0041] The above descriptions are only some embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall fall within the protection scope of the present invention.

Claims

1. A simplified genome analysis method for jointly analyzing a host and its coexisting microorganisms, characterized in that: The following steps are involved: (1) Based on the host genome sequence, microbial genome sequence and the recognition sequence of type IIB restriction endonuclease, the electronic restriction endonuclease tags of the host and microorganism are extracted to establish a host-microorganism type IIB restriction endonuclease tag database. Sequences specific to microorganisms at different taxonomic levels and host-specific sequences are obtained to establish a host-microorganism type IIB restriction endonuclease holographic omics database; (2) Extraction of mixed DNA: Extraction of mixed DNA from materials containing mixed DNA; (3) Use primer pair 515F and 907R in the V4-5 region of the 16S rDNA variable region to amplify the mixed DNA obtained in step (2). If bands appear at 400 bp and 600 bp, the amplification is considered qualified; The nucleotide sequence of 515F is: 5'-GTGCCAGCAGCGGTAA; The nucleotide sequence of 907R is: 5'-CCGTCAATTCTTGTAGTTTT; (4) Using the mixed DNA samples qualified in step (3), construct a 2bRAD / Miso-RAD sequencing library and perform high-throughput sequencing; (5) Preprocess the sequencing data obtained in step (4), remove low-quality data, extract sequences containing restriction sites, align the restriction fragments in the extracted sequencing data to the database constructed in step (1), and then perform host and microbial analysis respectively.

2. A simplified genome analysis method for jointly analyzing a host and its coexisting microorganisms as claimed in claim 1, characterized in that: Step (1) includes: According to the host genome sequence, microbial genome sequence and the recognition sequence of type IIB restriction endonuclease, all known microbial genomes and the analyzed host genome are simultaneously electronically digested to obtain electronic enzyme tags of all microorganisms and hosts, and a host-microorganism IIB enzyme tag database is established; the enzyme tags are mutually compared at different classification levels of microbial species, and are also compared with the host enzyme tags, and enzyme fragments that belong only to a certain species and have no duplication with other species are screened out, thereby establishing a host-microorganism IIB enzyme cleavage holographic omics database.

3. A simplified genome analysis method for jointly analyzing a host and its coexisting microorganisms as claimed in claim 1, characterized in that: In step (2), the material containing mixed DNA includes cotton swabs, feces, intestinal or larval materials after wiping tissue; the tissue includes gill filaments, mantle, skin or oral cavity.

4. A simplified genome analysis method for jointly analyzing a host and its coexisting microorganisms as claimed in claim 3, characterized in that: The tissue is gill filaments or oral cavity.

5. A simplified genome analysis method for jointly analyzing a host and its coexisting microorganisms according to claim 1, characterized in that: In step (4), the construction of the 2bRAD / Miso-RAD sequencing library includes constructing a single-tag library or constructing a multiple-tag tandem library.

6. A simplified genome analysis method for jointly analyzing a host and its coexisting microorganisms as claimed in claim 1, characterized in that: Step (4) includes: The mixed DNA in step (2) is digested with a type IIB restriction endonuclease, and corresponding adapters are selected for ligation according to the type IIB enzyme used. The ligated fragments are enriched by PCR, the PCR products are recovered and purified, and barcode sequence tags are added for PCR amplification.

7. A simplified genome analysis method for jointly analyzing a host and its coexisting microorganisms according to claim 6, characterized in that: The type IIB restriction endonuclease includes AlfI, AloI, BaeI, BcgI, BplI, BsaXI, BslFI, CspCI, FalI, Hin4I or PpiI.

8. A simplified genome analysis method for jointly analyzing a host and its coexisting microorganisms as claimed in claim 1, characterized in that: Step (5) includes: The sequencing data obtained in step (4) are preprocessed, and low-quality data are removed according to the principle that the sequence does not contain N and the quality value of more than 80% of the bases in the sequence is greater than 20. The sequences containing restriction sites are extracted, and the restriction fragments in the extracted sequencing data are aligned to the host-microorganism IIB restriction holographic database constructed in step (1); the data of the host sequence aligned to the database are subjected to whole genome typing, and the data of the microbial genome aligned to the microbial genome are subjected to microbial composition and abundance analysis.

9. A simplified genome analysis method for jointly analyzing a host and its coexisting microorganisms according to any one of claims 1 to 8, characterized in that: The host includes shellfish or shrimp.

10. A simplified genome analysis method for jointly analyzing a host and its coexisting microorganisms according to claim 9, characterized in that: The host includes the Yesso scallop.

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