Materials and Methods

Sequencing

1 Sample collection

Family members provided fully informed consent to participate in the study by providing samples and related information. Saliva samples were collected by directly expectorating into a 15 ml tube and immediately frozen for storage.

2 Extraction of genome DNA

Extracted total genome DNA from samples using Nucleo spin DNA stool kit (cat No. 740472.50) according to the manufacturer’s instructions. DNA concentration and purity were monitored on 1% agarose gels. According to the concentration, DNA was diluted to 1ng/μL using sterile water.

3 Amplicon Generation

Amplified 16S rRNA genes of 16SV4 region used specific primer(16S V4: 515F-806R) with the barcode. All PCR reactions were carried out with Phusion® High-Fidelity PCR Master Mix (New England Biolabs).

4 PCR Products quantification and qualification

Mix the same volume of 1X loading buffer (contained SYB green) with PCR products and operate electrophoresis on 2% agarose gel for detection. Samples with a bright main strip between 400-450bp were chosen for further experiments.

5 PCR Products Mixing and Purification

PCR products were mixed in equal density ratios. Then, mixture PCR products were purified with the Qiagen Gel Extraction Kit (Qiagen, Germany).

6 Library preparation and sequencing

Sequencing libraries were generated using TruSeq® DNA PCR-Free Sample Preparation Kit (Illumina, USA) following the manufacturer’s recommendations and index codes were added. The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system. At last, the library was sequenced on an Illumina HiSeq 2500 platform (Illumina, USA)  and 250 bp paired-end reads were generated.

Data analysis

1 Paired-end reads assembly and quality control

1.1 Data split

Assigned paired-end reads to samples based on their unique barcode and truncated by cutting off the barcode and primer sequence.

1.2 Sequence assembly

Paired-end reads were merged using FLASH (V1.2.7) (Magoc and Salzberg, 2011), a very fast and accurate analysis tool, which was designed to merge paired-end reads when at least some of the reads overlap the read generated from the opposite end of the same DNA fragment, and the splicing sequences were called raw tags.

1.3 Data Filtration:

Quality filtering on the raw tags were performed under specific filtering conditions to obtain the high-quality clean tags according to the Qiime (V1.7.0) (Kuczynski et al., 2011) quality controlled process.

1.4 Chimera removal:

The tags were compared with the reference database(Gold database using UCHIME algorithm (Edgar et al., 2011) to detect chimera sequences, and then the chimera sequences were removed. Then the Effective Tags finally obtained.

2 OTU cluster and Species annotation

2.1 OTU Production:

Sequences analysis were performed by Uparse software (Uparse v7.0.1001) (Edgar, 2013)Sequences with ≥97% similarity were assigned to the same OTUs. Representative sequence for each OTU was screened for further annotation.

2.2 Species annotation:

For each representative sequence, the GreenGene Database (McDonald et al., 2012) was used based on RDP classifier(Version 2.2)  (Wang et al., 2007) algorithm to annotate taxonomic information.

2.3 Phylogenetic relationship Construction:

In order to study the phylogenetic relationship of different OTUs, and the difference of the dominant species in different samples(groups), multiple sequence alignment were conducted using the PyNAST software(Version 1.2)  (Caporaso et al., 2010) against the “Core Set” dataset in the GreenGene database.

2.4 Data Normalization:

OTUs abundance information were normalized using a standard of sequence number corresponding to the sample with the least sequences. Subsequent analysis of alpha diversity and beta diversity were all performed basing on this output normalized data.

3 Alpha Diversity

Alpha diversity is applied in analyzing complexity of species diversity for a sample through6 indices, including Observed-species, Chao1, Shannon, Simpson, ACE, Good-coverage. All these indices in our samples were calculated with QIIME(Version 1.7.0) (Kuczynski et al., 2011) and displayed with R software(Version 2.15.3).

Two indices were selected to identify Community richness:

Chao – the Chao1 estimator (Chao, 1984);

ACE – the ACE estimator (Chao et al., 1992) ;

Two indices were used to identify Community diversity:

Shannon – the Shannon index (Seaby R. M. & Henderson, 2006);

Simpson – the Simpson index (Harper, 1999);

One index to characterized Sequencing depth:

Coverage – the Good’s coverage (Seaby R. M. & Henderson, 2006)

4 Beta Diversity

Beta diversity analysis was used to evaluate differences of samples in species complexity, Beta diversity on both weighted and unweighted unifrac were calculated by QIIME software (Version 1.7.0) (Kuczynski et al., 2011).

Non-metric multidimensional scaling(NMDS) was performed to get principal coordinates and visualize from complex, multidimensional data. A distance matrix of weighted or unweighted unifrac among samples obtained before was transformed to a new set of orthogonal axes, by which the maximum variation factor is demonstrated by first principal coordinate, and the second maximum one by the second principal coordinate, and so on. NMDS analysis was displayed by the WGCNA package, stat packages, and ggplot2 package in R software(Version 2.15.3).

Unweighted Pair-group Method with Arithmetic Means(UPGMA) Clustering was performed as a type of hierarchical clustering method to interpret the distance matrix using average linkage and was conducted by QIIME software (Version 1.7.0) (Kuczynski et al., 2011).

Reference

Abeles, S.R., Jones, M.B., Santiago-Rodriguez, T.M., Ly, M., Klitgord, N., Yooseph, S., Nelson, K.E., and Pride, D.T. (2016). Microbial diversity in individuals and their household contacts following typical antibiotic courses. Microbiome. 4(1), 39. Published online 2016/07/31 DOI: 10.1186/s40168-016-0187-9.

Arbes, S.J., Jr., and Matsui, E.C. (2011). Can oral pathogens influence allergic disease? J Allergy Clin Immunol. 127(5), 1119-1127. Published online 2011/05/03 DOI: 10.1016/j.jaci.2011.03.023.

(The above study point to the right place, oral cavity. However, Allergies are not caused by the pathogens but by lack of probiotics.)

Atkinson, E.R. (1979). Assessment of current hyperthermia technology. Cancer Res. 39(6 Pt 2), 2313-2324. Published online 1979/06/01.

Blacher, E., Levy, M., Tatirovsky, E., and Elinav, E. (2017). Microbiome-Modulated Metabolites at the Interface of Host Immunity. J Immunol. 198(2), 572-580. Published online 2017/01/11 DOI: 10.4049/jimmunol.1601247.

Caporaso, J.G., Bittinger, K., Bushman, F.D., DeSantis, T.Z., Andersen, G.L., and Knight, R. (2010). PyNAST: a flexible tool for aligning sequences to a template alignment. Bioinformatics. 26(2), 266-267. Published online 2009/11/17 DOI: 10.1093/bioinformatics/btp636.

Carlier, J.-P., 2015. Veillonella Bergey’s Manual of Systematics of Archaea and Bacteria, 14 SEP 2015 ed. John Wiley & Sons, Inc., in association with Bergey’s Manual Trust., pp. 1-11.

Chao, A. (1984).Non-parametric estimation of the number of classes in a population. Scandinavian Journal of Statistics. 11, 265-270.

Chao, A., Lee, S.M., and Jeng, S.L. (1992). Estimating population size for capture-recapture data when capture probabilities vary by time and individual animal. Biometrics. 48(1), 201-216. Published online 1992/03/01.

Chung, K.F. (2017). Airway microbial dysbiosis in asthmatic patients: A target for prevention and treatment? J Allergy Clin Immunol. 139(4), 1071-1081. Published online 2017/04/10 DOI: 10.1016/j.jaci.2017.02.004.

(The above article emphasizes gut microbiota where is not the cause of allergies.)

Correa-Oliveira, R., Fachi, J.L., Vieira, A., Sato, F.T., and Vinolo, M.A. (2016). Regulation of immune cell function by short-chain fatty acids. Clin Transl Immunology. 5(4), e73. Published online 2016/05/20 DOI: 10.1038/cti.2016.17.

Edgar, R.C. (2013). UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nature methods. 10(10), 996-998. Published online 2013/08/21 DOI: 10.1038/nmeth.2604.

Edgar, R.C., Haas, B.J., Clemente, J.C., Quince, C., and Knight, R. (2011). UCHIME improves sensitivity and speed of chimera detection. Bioinformatics. 27(16), 2194-2200. Published online 2011/06/28 DOI: 10.1093/bioinformatics/btr381.

Ege, M.J., Mayer, M., Schwaiger, K., Mattes, J., Pershagen, G., van Hage, M., Scheynius, A., Bauer, J., and von Mutius, E. (2012). Environmental bacteria and childhood asthma. Allergy. 67(12), 1565-1571. Published online 2012/09/22 DOI: 10.1111/all.12028.

Foliaki, S., Pearce, N., Bjorksten, B., Mallol, J., Montefort, S., and von Mutius, E. (2009). Antibiotic use in infancy and symptoms of asthma, rhinoconjunctivitis, and eczema in children 6 and 7 years old: International Study of Asthma and Allergies in Childhood Phase III. J Allergy Clin Immunol. 124(5), 982-989. Published online 2009/11/10 DOI: 10.1016/j.jaci.2009.08.017.

(Antibiotic usage may be one of the biggest reasons that cause child allergies.)

Gensollen, T., Iyer, S.S., Kasper, D.L., and Blumberg, R.S. (2016). How colonization by microbiota in early life shapes the immune system. Science (New York, N.Y. 352(6285), 539-544. Published online 2016/04/30 DOI: 10.1126/science.aad9378.

Han, S., 2017. Oral microbiota promoting method. Knoze Jr Corp, United States of America.

(The above is our invention to correct the cause of allergies)

Harper, D.A.T. (1999). Numerical Palaeobiology (John Wiley & Sons).

Jatzlauk, G., Bartel, S., Heine, H., Schloter, M., and Krauss-Etschmann, S. (2017). Influences of environmental bacteria and their metabolites on allergies, asthma, and host microbiota. Allergy. Published online 2017/06/11 DOI: 10.1111/all.13220.

Kolderman, E., Bettampadi, D., Samarian, D., Dowd, S.E., Foxman, B., Jakubovics, N.S., and Rickard, A.H. (2015). L-arginine destabilizes oral multi-species biofilm communities developed in human saliva. PLoS ONE. 10(5), e0121835. Published online 2015/05/07 DOI: 10.1371/journal.pone.0121835.

Kuczynski, J., Stombaugh, J., Walters, W.A., Gonzalez, A., Caporaso, J.G., and Knight, R. (2011). Using QIIME to analyze 16S rRNA gene sequences from microbial communities. Curr Protoc Bioinformatics. Chapter 10, Unit 10 17. Published online 2011/12/14 DOI: 10.1002/0471250953.bi1007s36.

Lombardi, C., Penagos, M., Senna, G., Canonica, G.W., and Passalacqua, G. (2008). The clinical characteristics of respiratory allergy in immigrants in northern Italy. Int Arch Allergy Immunol. 147(3), 231-234. Published online 2008/07/03 DOI: 10.1159/000142046. 

(This article about immigrants is important for me to think that something can be changed in adulthood and cause allergies.)

Magoc, T., and Salzberg, S.L. (2011). FLASH: fast length adjustment of short reads to improve genome assemblies. Bioinformatics. 27(21), 2957-2963. Published online 2011/09/10 DOI: 10.1093/bioinformatics/btr507.

McDonald, D., Price, M.N., Goodrich, J., Nawrocki, E.P., DeSantis, T.Z., Probst, A., Andersen, G.L., Knight, R., and Hugenholtz, P. (2012). An improved Greengenes taxonomy with explicit ranks for ecological and evolutionary analyses of bacteria and archaea. The ISME journal. 6(3), 610-618. Published online 2011/12/03 DOI: 10.1038/ismej.2011.139.

Seaby R. M. & Henderson, P.A. (2006). Species Diversity and Richness Version 4 (Pisces Conservation Ltd.: Lymington, England).

Strachan, D.P. (1989). Hay fever, hygiene, and household size. Bmj. 299(6710), 1259-1260. Published online 1989/11/18.

(this article by Strachan is the original proposal of hygiene hypothesis to explain the cause of allergies.)

Takei, M., Fukatsu, T., and Furukawa, S. (1968). [Fundamental studies on the dynamic movement of oral microbes. 5. Relationship of Streptococcus and Veillonella]. Aichi Gakuin Daigaku Shigakkai Shi. 5(4), 312-317. Published online 1968/03/01.

van der Hoeven, J.S., Toorop, A.I., and Mikx, R.H. (1978). Symbiotic relationship of Veillonella alcalescens and Streptococcus mutans in dental plaque in gnotobiotic rats. Caries Res. 12(3), 142-147. Published online 1978/01/01.

von Mutius, E. (2007). Allergies, infections and the hygiene hypothesis–the epidemiological evidence. Immunobiology. 212(6), 433-439. Published online 2007/06/05 DOI: 10.1016/j.imbio.2007.03.002.

(von Mutius and many others’ study confirms hygiene as potential cause of allergies.)

Wang, Q., Garrity, G.M., Tiedje, J.M., and Cole, J.R. (2007). Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Applied and environmental microbiology. 73(16), 5261-5267. Published online 2007/06/26 DOI: 10.1128/AEM.00062-07.

West, C.E., Jenmalm, M.C., Kozyrskyj, A.L., and Prescott, S.L. (2016). Probiotics for treatment and primary prevention of allergic diseases and asthma: looking back and moving forward. Expert Rev Clin Immunol. 12(6), 625-639. Published online 2016/01/30 DOI: 10.1586/1744666X.2016.1147955.

Willcox, M.D. (1996). Identification and classification of species within the Streptococcus sanguis group. Aust Dent J. 41(2), 107-112. Published online 1996/04/01.