Method for screening donor easy to colonize for intestinal flora transplantation

Abstract

The invention relates to a method for screening FMT (intestinal flora transplantation) donors easy to colonize. Comprising the following steps: collecting excrement of a receptor before intestinal flora transplantation, performing full-length 16s DNA sequencing on the excrement and a donor library, calculating the abundance and satisfaction degree of the donor and the receptor, and the like. The method provided by the invention can be used for screening a donor sample library easy to colonize for the FMT and screening and matching proper donors for patients, so that the colonization of donor strains after the FMT and the effectiveness of the FMT are ensured.

Description

Technical Field

[0001] The invention relates to the field of bioinformatics analysis and clinical medical technology, and in particular to a method for screening intestinal flora transplantation easy-to-colonize donors. Background Art

[0002] FMT is a transplantation of functional intestinal flora from the feces of healthy people into the intestines of patients to rebuild new intestinal flora and achieve the treatment of intestinal and extraintestinal diseases. FMT has been used to treat a variety of diseases. Clinically, it has been found that the efficacy of FMT in treating recurrent Clostridium difficile infection is 90%, but the efficacy for other diseases ranges from 30% to 70%. Studies have found that the degree of disease remission after FMT varies among different individuals with the same disease, and the disease remission rate of different patients with the same donor also varies. The key factors affecting the efficacy of FMT in treating diseases have not been effectively explained. Whether there are "super donors" for FMT and how to choose FMT donors are still under debate and research.

[0003] Therefore, analyzing the factors that affect the effectiveness of FMT, identifying donors that are easy to colonize, and matching patients with suitable donors to improve disease remission rates are difficult problems that FMT urgently needs to solve and are also the key to the applicability of FMT. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a method for screening donors who are prone to colonization of intestinal flora transplantation.

[0005] According to one aspect of the present invention, a method for screening intestinal flora transplantation easy-to-colonize donors is provided, comprising the following steps:

[0006] 1) Obtain donor pool samples and sequence the donors separately to obtain full-length 16s DNA sequences;

[0007] 2) Collecting the feces of the recipient before intestinal flora transplantation and sequencing to obtain the full-length 16s DNA sequence;

[0008] 3) Perform strain-level analysis on the full-length 16s DNA sequences of steps 1) and 2) to calculate the strain abundance of the recipient and donor;

[0009] 4) For the key colonization bacteria, the sum of the abundances of the key colonization bacteria is calculated according to the strain abundances obtained in step 3). If the sum of the abundances is greater than 20%, the donor is identified as an easy colonization donor;

[0010] 5) According to the strain abundance obtained in step 3) and the easy-to-colonize donor obtained in step 4), the bacterial colony distance between the donor and the recipient is calculated using the following formula:

[0011]

[0012] Among them, S R,i and S D,i represents the abundance of the ith strain in the two samples R and D, min means taking the minimum value of the two, R is the receptor, and D is the donor;

[0013] 6) For the key colonization bacteria, the satisfaction degree of the donor to the recipient is calculated according to the strain abundance obtained in step 3), and the formula is as follows:

[0014]

[0015] Among them, S R,i and S D,i represents the abundance of the ith strain in samples R and D, min means taking the minimum value of the two, S i represents the value of the i-th strain, ∑Si represents the cumulative value of all strains, R is the receptor, and D is the donor;

[0016] 7) According to the bacterial colony distance obtained in step 5) and the satisfaction degree obtained in step 6), the matching score is calculated, and the formula is as follows:

[0017] M score =xD bc +yF score , x,y are between 0.1 and 0.9;

[0018] Therefore, according to M score The top three donors with the highest scores are recommended as easy-to-colonize donors. Preferably, the easy-to-colonize donor is M score The top two donors with the highest scores, preferably the easy-to-colonize donor is M score The donor with the highest score.

[0019] Preferably, the strain level analysis in step 2) includes:

[0020] A. Bacterial species annotation: annotate the full-length 16s DNA sequence at the bacterial species level;

[0021] B. Multiple sequence alignment: extract multiple sequences annotated to the same bacterial species and perform multiple sequence alignment to discover SNP polymorphic sites;

[0022] C Calculate Shannon entropy: Calculate the Shannon entropy of each SNP polymorphic site, the formula is: Shannon entropy = -∑(Pi+logPi), where Pi is the proportion of each base, there are four bases: A, T, G, and C;

[0023] D. Calculate FASTID and proportion: Select sites with Shannon entropy > 0.4, use the base combinations of these sites to form different FASTIDs, and calculate the proportions of different FASTIDs, thereby filtering out sequencing errors and low-frequency SNPs;

[0024] E Filter out FASTIDs that appear only in one sample and have a sequence number <5 and a proportion less than 0.001%, and further obtain high-confidence FASTIDs.

[0025] Preferably, the key bacterial species for colonization in step 4) include any multiple of fecal Rosenblumeria, beneficial rectal rod-shaped bacteria, butyrate-producing beneficial small rod-shaped bacteria, anaerobic butyric acid bacteria of Hall, anaerobic butyric acid bacteria of Soth, anaerobic rod-shaped bacteria of Pet, Bifidobacterium adolescentis, Bifidobacterium longum, Bifidobacterium pseudosmall chain, healthy fecal Blautia, sludge Blautia, Weymouth Blautia, butyric acid bacteria, associated coprococcus, regular coprococcus, formic acid-producing Dole's bacteria, long chain Dole's bacteria, Clostridium prausnitzii, sucrose clostridium, fastidious Lachnospira, rumen host-associated Lactobacillus, human Rosenblumeria, intestinal Rosenblumeria and glucosinolate Rosenblumeria.

[0026] Preferably, the recipient is a patient suitable for intestinal flora transplantation.

[0027] Preferably, the subject suffers from any one of a gastrointestinal disease, a metabolic disease and a nervous system disease.

[0028] The beneficial effects of the present invention are:

[0029] Without being limited to any theory, the inventors analyzed the structure of the donor and recipient flora and the improvement effect during the FMT process and found that a high colonization rate of the flora after FMT was highly correlated with disease remission, and innovatively proposed key bacteria for FMT colonization and conditions for easily colonized donors. This method was used to screen a sample library of easily colonized donors for FMT, and at the same time, suitable donors were screened and matched for patients, thereby ensuring the colonization of donor strains after FMT and the effectiveness of FMT. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0031] Figure 1 Schematic diagram of the process of strain analysis (FASTID) for 16s DNA full-length data;

[0032] Figure 2 The strain-level colonization of Faecalibacterium prausnitzii was identified by FASTID, which depicts the colonization of Faecalibacterium prausnitzii strains in two samples, Recipient 1 and Recipient 2, before and after transplantation with Donor 1. The horizontal axis represents the sample, and the vertical axis represents the abundance %.

[0033] Figure 3 for recipient and donor colonization rates;

[0034] Figure 4 The abundance of key bacterial species for FMT colonization before and after FMT;

[0035] Figure 5 The abundance distribution of the total abundance of key bacterial species for FMT colonization before and after FMT and in donors;

[0036] Figure 6 recipient and donor colonization rates for patients who selected different donors;

[0037] Figure 7 The changes of bacterial flora before and after FMT. DETAILED DESCRIPTION

[0038] In the present invention, the donor pool samples are preferably selected from healthy college students in the hospital according to the "Donor Screening Criteria" in the "Chinese Expert Consensus on the Clinical Application Management of Intestinal Flora Transplantation".

[0039] In the present invention, the method for full-length 16s DNA sequencing is not particularly limited, and those skilled in the art may use conventional methods.

[0040] In the present invention, the full-length 16s DNA sequence information of the donors in the donor library sample can be retained for use in the next screening of easily colonized donors, without the need to perform the donor full-length 16s DNA sequence sequencing step each time.

[0041] In the present invention, the key bacterial species for colonization preferably include Agathobacter faecis (fecal Rosebaceous Bacteria), Agathobacter rectalis (beneficial rod-shaped bacteria in the rectum), Agathobaculum butyriciproducens (butyric acid-producing beneficial small bacillus), Anaerobutyricum hallii (anaerobic butyric acid bacteria of Hall), Anaerobutyricumsoehngenii (anaerobic butyric acid bacteria of Soehngenii), Anaerostipes hadrus (anaerobic rod-shaped bacteria of pet), Bifidobacterium adolescentis (adolescent Bifidobacterium), Bifidobacterium longum (long Bifidobacterium), Bifidobacterium pseudocatenulatum (pseudo small chain Bifidobacterium), Blautia stercoris (healthy fecal Blautia), Blautia luti (sludge Blautia), Blautia wexlerae (Wexlerae), Butyribacter intestini (butyric acid bacteria), Coprococcus eutactus (Associated Coprococcus), Coprococcus catus (Regular Coprococcus), Dorea formicigenerans (Dorea formicigenerans), Dorealongicatena (Dorea longicatena), Faecalibacterium prausnitzii (Faecalibacterium prausnitzii), Fusicatenibacter saccharivorans (Fusicatenibacter saccharivorans), Lachnospira eligens (Lachnospira eligens), Ligilactobacillus ruminis (Rumen Host-Associated Lactobacillus), Roseburia hominis (Roseburia hominis), Roseburia intestinalis (Roseburia inulinivorans), and any multiple thereof.

[0042] In the present invention, the recipient is preferably a patient suitable for intestinal flora transplantation.

[0043] In the present invention, the recipient is preferably a patient suffering from any one of a gastrointestinal disease, a metabolic disease and a nervous system disease.

[0044] In order to further illustrate the present invention, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0045] Example 1

[0046] This example provides a group of key colonization strains and identification of easy-to-colonize donors, including:

[0047] 1. Collect data on subjects who underwent FMT, including 16s DNA full-length data and phenotypic data from stool samples.

[0048] 1.1 The basic personal information, gastrointestinal symptoms, unhealthy lifestyle habits, and disease information of the 14 subjects before and after FMT were investigated by questionnaire (see Table 1 for details).

[0049] 1.2 The stool samples collected before and after FMT were sequenced using the library construction and sequencing method for detecting the full-length 16s DNA of bacteria disclosed in CN201910790501.7. The data from the sequencing machine was processed and analyzed by bioinformatics technology to obtain the full-length 16s DNA sequence of the bacterial species.

[0050] 1.3 Obtain the donor capsule, perform sequencing, and obtain the full-field 16s DNA sequence.

[0051] The donor capsules come from Shanghai Tenth People's Hospital. They are made from feces from healthy donors that have undergone separation and purification processes through an intelligent system, leaving only metabolites and bacteria. After strict quality control testing, they are finally made into live bacteria freeze-dried powder capsules "Shaoyi Zhang, Rong Yang, et al. Donor Screening for Fecal Microbiota Transplantation in China: Evaluation of 8483 Candidates, Gastroenterology, Volume 162, Issue 3, 2022".

[0052] 2. Strain-level (FASTID) analysis of 16s DNA full-length data

[0053] The present invention develops a supervised computational method to identify the full-length 16s DNA sequence at the strain level. The method implements strain-level annotation by combining and analyzing the SNP (Single-nucleotide polymorphism) polymorphic sites on 16s DNA, and defines FASTID to represent a strain. The specific method is as follows:

[0054] 1) Species annotation: The full-length 16s DNA sequence was annotated at the species level.

[0055] 2) Multiple sequence alignment: Extract multiple sequences annotated to the same bacterial species and perform multiple sequence alignment to discover SNP polymorphic sites.

[0056] 3) Calculate Shannon entropy: Calculate the Shannon entropy of each SNP polymorphic site, the formula is: Shannon entropy = -∑(Pi+logPi). Where Pi is the proportion of each base, and there are four bases in total.

[0057] 4) Calculation of FASTID and proportion: In order to filter out sequencing errors and low-frequency SNPs, sites with Shannon entropy > 0.4 were selected, and the base combinations of these sites were used to form different FASTIDs, and the proportions of different FASTIDs were calculated.

[0058] 5) In order to further obtain highly reliable FASTIDs, FASTIDs that only appeared in one sample and had a sequence <5 and a proportion less than 0.001% were filtered out.

[0059] 3. FASTID enables identification of colonization at the strain level

[0060] The analysis of 16s DNA full-length data by FASTID enabled high-resolution discovery across the species level and identified the occurrence of colonization events in donor-recipient paired samples. Figure 2 The data showed that FASTID identified different strains of the bacteria Faecalibacterium prausnitzii and found that both the donor and the recipient contained Faecalibacterium prausnitzii before and after FMT. However, after FMT, only the donor strain Fast-ID3 colonized in donor 1, while recipient 2 did not.

[0061] 4. Strain-level colonization assessment

[0062] The present invention defines the presence of strains in donor samples (abundance greater than 0), not present in subject samples before FMT (abundance is 0), and present in subject samples after FMT as strain colonization; defines the recipient colonization rate as the ratio of the number of colonized strains to the total number of strains in the recipient after FMT, and evaluates the colonization results after FMT. The donor colonization rate is defined as the ratio of the number of colonized strains to the total number of strains in the donor, reflecting the colonization quality of the donor. The calculation results are as follows: Figure 3 We found that the correlation between the recipient colonization rate and the donor colonization rate reached 0.85, and speculated that the high donor quality of the donor was the key influencing factor for the high colonization rate.

[0063] 5. Donor colonization rate matches FMT effect

[0064] Based on the above collected phenotypic data of the subjects before and after FMT, the improvement effect was defined as: almost no improvement, mild improvement, and effective improvement, mainly based on the improvement of digestive tract symptoms before and after FMT. Among them, effective improvement: the digestive tract symptoms before transplantation disappeared; mild improvement: partial improvement of digestive tract symptoms before transplantation; almost no improvement: no improvement of digestive tract symptoms before transplantation.

[0065] The results showed that the higher the colonization rate, the better the tendency of improving results (see Table 1).

[0066] Table 1 Phenotype and colonization rate of subjects before and after FMT

[0067]

[0068]

[0069] 6. Identification of key bacterial species for colonization

[0070] 6.1 Aggregation of colonizing strains

[0071] The examples found that the strains colonized at the FASTID strain level were clustered at the species level, and the abundance increased significantly before and after FMT. These bacteria were defined as key strains for FMT colonization. The results are shown in Figure 4 .

[0072] 6.2 The abundance of key colonizing bacteria increased significantly

[0073] The content of key colonizing bacteria in subjects before FMT was significantly lower than that in the donor pool (19.6±12.7 vs 28.2±10.8, p=0.002), but it increased significantly after FMT (27.7±14.5, p=0.003), almost reaching the level of the donor. The results are shown in Figure 5 .

[0074] 6.3 High content of key colonization bacteria in donors and high colonization rate

[0075] The embodiment found that the content of key colonization bacteria in the donor was high, and the colonization rate of the recipient selected for FMT was also high. Figure 6 .

[0076] 7. Identification of easily colonized donors

[0077] In the examples, donors with a key bacteria content greater than 20% were defined as easy-to-colonize donors, and the improvement rate of easy-to-colonize donors was significantly higher than that of other donors (OR=32.00, P=0.025).

[0078] Table 2 Donor classification and improvement effect statistics

[0079] Effective improvement Slight improvement Almost no improvement Easy to colonize donor 5 3 1 Non-colonization-prone donors 0 1 4

[0080] Example 2

[0081] This embodiment provides a method for matching FMT donors and recipients, comprising:

[0082] 1. Collect data of donors and recipients, including full-length 16s DNA data of feces;

[0083] Table 3 Receptor data

[0084]

[0085] 2. Perform strain level (FASTID) analysis on the 16s DNA data, refer to Example 1, and calculate the strain abundance in the donor and recipient.

[0086] 3. Calculate the distance between bacterial colonies

[0087] At the whole strain level, the distance between the recipient sample and the easily colonized donor library was calculated (Bray-

[0088] Curtis difference), the formula is as follows:

[0089]

[0090] S R,i and S D,i Represents the abundance of the ith strain in the two samples R (recipient) and D (donor), and min means taking the minimum value of the two.

[0091] 4. Calculate satisfaction

[0092] 4.1 For the list of key colonization strains, the satisfaction score of the donor to the recipient is calculated at the strain level. The formula is as follows:

[0093]

[0094] S R,i and S D,i Represents the abundance of the ith strain in the two samples R (recipient) and D (donor), and min means taking the minimum value of the two. S i represents the value of the ith strain. ∑Si represents the sum of all strain values.

[0095] 4.2 List of key bacterial species for colonization

[0096] Table 3 List of key bacterial species for colonization

[0097]

[0098] 5. Calculate the matching score

[0099] The matching is the comprehensive score of bacterial colony distance and satisfaction, and the formula is as follows:

[0100] M score =xD bc +yF score , x,y are weights, ranging from 0.1 to 0.9.

[0101] The calculation results are shown in Table 4. According to the score values, the first three easy-to-colonize donors are recommended to the recipient (in case of insufficient donor capsules). Preferably, donors 1, 2, and 3 in Table 4 are easy-to-colonize donors. Preferably, donor 1 is a matched and selected easy-to-colonize donor.

[0102] Table 4 Matching calculation score

[0103] Donor Dbc Fscore M score Donor 1 0.94 0.42 0.306 Donor 2 0.94 0.35 0.272 Donor 3 0.86 0.28 0.223 Donor 4 0.90 0.18 0.182 Donor 5 0.92 0.16 0.171 Donor 6 0.89 0.19 0.184 Donor 7 0.87 0.26 0.218

[0104] 6. Evaluation of planting effect

[0105] The recipient took donor 1 as recommended in 5). One month after FMT, feces were collected and 16s DNA full-length sequencing was performed. The flora structure before and after FMT was compared, and it was found that the flora diversity was significantly improved (4.16 before transplantation, 5.79 after transplantation); at the species level, it can be seen that the flora structure has changed significantly; the donor colonization rate is as high as 38%, and the recipient colonization rate is 34%. According to the questionnaire survey, gastrointestinal symptoms almost disappeared. It can be seen that the FMT effect of the recipient sample is effective, and the donor recommended in 5) is reliable.

[0106] Example 3

[0107] This example reviews the samples that underwent FMT in Example 1, and compares the consistency of the donors actually used with the easy-to-colonize donors recommended by the FMT donor-recipient matching method described in Example 2, and the impact on the improvement effect. The specific process is as follows:

[0108] 1. The method in Example 2 was used to calculate the easily colonized donors for the samples in Example 1, see Table 5.

[0109] 2. Compared with the actual donors used in the FMT samples and the improvement effects, it was found that the donors actually used in the 4 samples with almost no improvement were not the recommended easy-to-colonize donors, 2 of the 5 samples with slight improvement did not use the recommended easy-to-colonize donors, and the remaining 3 samples used the top 2 or top 3 of the recommended easy-to-colonize donors; all 5 samples with effective improvement used the recommended easy-to-colonize donors, including top 1, top 2, or top 3, as shown in Table 6.

[0110] 3. Based on the above findings, it can be seen that if the easy-to-colonize donors recommended by the FMT donor-recipient matching method of the present invention are not used, the colonization effect may be poor. The FMT donor-recipient matching method provided by the present invention is effective.

[0111] Table 5 Recommended easy-to-colonize donors and actual donors for FMT

[0112] serial number Improved results Receptor colonization rate Actual donor Recommended easy-to-colonize donors Consistency R1 Effective improvement 35.7％ Donor 3 Donor 2, 3, 6 yes R2 Effective improvement 24.3％ Donor 1 Donor 5, 1, 2 yes R3 Slight improvement 17.5％ Donor 1 Donor 5, 2, 1 yes R4 Slight improvement 12.5％ Donor 3 Donor 2, 3, 6 yes R5 Almost no improvement 6.3％ Donor 3 Donor 4, 5, 2 no R6 Slight improvement 11.7％ Donor 3 Donor 2, 6, 7 no R7 Effective improvement 25.6％ Donor 5 Donor 4, 5, 7 yes R8 Effective improvement 26.0％ Donor 1 Donor 5, 2, 1 yes R9 Almost no improvement 10.7％ Donor 4 Donor 2, 3, 6 no R10 Almost no improvement 9.9％ Donor 1 Donor 3, 2, 6 no R11 Slight improvement 12.9％ Donor 2 Donor 6, 3, 2 yes R12 Effective improvement 39.8％ Donor 2 Donor 2, 3, 7 yes R13 Almost no improvement 11.1％ Donor 5 Donor 2, 3, 6 no R14 Slight improvement 13.1％ Donor 4 Donor 2, 6, 3 no

[0113] Table 6 FMT effect and donor

[0114] Effective improvement Slight improvement Almost no improvement Consistency 5 3 0 Inconsistency 0 2 4 Consistency 100％ 60％ 0％

[0115] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for screening intestinal flora transplantation easy to colonize donors, characterized in that: The following steps are involved: 1) Obtain donor pool samples and sequence the donors separately to obtain full-length 16s DNA sequences; 2) Collect the feces of the recipient before intestinal flora transplantation and sequence it to obtain the full-length 16s DNA sequence; 3) Perform strain-level analysis on the full-length 16s DNA sequences of steps 1) and 2) to calculate the strain abundance of the recipient and donor; 4) For the key colonization bacteria, the sum of the abundances of the key colonization bacteria is calculated according to the strain abundances obtained in step 3). If the sum of the abundances is greater than 20%, the donor is identified as an easy colonization donor; 5) According to the strain abundance obtained in step 3) and the easy-to-colonize donor obtained in step 4), the bacterial colony distance between the donor and the recipient is calculated using the following formula: Among them, S R,i and S D,i represents the abundance of the ith strain in the two samples R and D, min means taking the minimum value of the two, R is the receptor, and D is the donor; 6) For the key colonization bacteria, the satisfaction degree of the donor to the recipient is calculated according to the strain abundance obtained in step 3), and the formula is as follows: Among them, S R,i and S D,i represents the abundance of the ith strain in samples R and D, min means taking the minimum value of the two, S i represents the value of the i-th strain, ∑Si represents the cumulative value of all strains, R is the receptor, and D is the donor; 7) According to the bacterial colony distance obtained in step 5) and the satisfaction degree obtained in step 6), the matching score is calculated, and the formula is as follows: M score =xD bc +yF score , x,y are between 0.1 and 0.9; Therefore, according to M score The top three donors with the highest scores are recommended as easy-to-colonize donors.

2. The method according to claim 1, characterized in that The strain level analysis in step 2) includes: A. Bacterial species annotation: annotate the full-length 16s DNA sequence at the bacterial species level; B. Multiple sequence alignment: extract multiple sequences annotated to the same bacterial species and perform multiple sequence alignment to discover SNP polymorphic sites; C Calculate Shannon entropy: Calculate the Shannon entropy of each SNP polymorphic site, the formula is: Shannon entropy = -∑(Pi+logPi), where Pi is the proportion of each base of A, T, G, and C; D. Calculate FASTID and proportion: Select sites with Shannon entropy > 0.4, use the base combinations of these sites to form different FASTIDs, and calculate the proportions of different FASTIDs; E Filter FASTIDs that appear only in one sample and have a sequence number > 5 and a proportion less than 0.001%, thereby obtaining high-confidence FASTIDs.

3. The method according to claim 1, characterized in that The key bacterial species for colonization in step 4) include any multiple of fecal Rosenblum, beneficial rectal rod-shaped bacteria, butyrate-producing beneficial small rod-shaped bacteria, anaerobic butyric acid bacteria of Hall, anaerobic butyric acid bacteria of So, anaerobic rod-shaped bacteria of Pet, Bifidobacterium adolescentis, Bifidobacterium longum, Bifidobacterium pseudosmall chain, healthy fecal Blautia, sludge Blautia, Weymouth Blautia, butyric acid bacteria, associated coccobacilli, regular coccobacilli, formic acid-producing Dole's bacteria, long-chain Dole's bacteria, Clostridium prausnitzii, sucrose-producing Clostridium, fastidious Lachnospira, rumen host-associated Lactobacillus, human Rosenblum, intestinal Rosenblum and glucosinolate Rosenblum.

4. The method according to claim 1, characterized in that: The recipient is a patient suitable for intestinal flora transplantation.

5. The method according to claim 1 or 4, characterized in that: The recipient is a patient suffering from any one of a gastrointestinal disease, a metabolic disease and a nervous system disease.

6. The method according to claim 1, characterized in that The easy-to-colonize donor is M score The top two scoring donors.

7. The method according to claim 6, characterized in that The easy-to-colonize donor is M score The donor with the highest score.

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