Computational protocol: The Impact of Environmental Heterogeneity and Life Stage on the Hindgut Microbiota of Holotrichia parallela Larvae (Coleoptera: Scarabaeidae)

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Protocol publication

[…] For each clone library, a representative clone from each DGGE group was sequenced. All sequences were checked for chimeric artifacts using the check-chimera program of the Ribosomal Database Project (RDP) , and aligned against those found in the GenBank nr database, in the RDP II database , and on the EzTaxon server using the Basic Local Alignment and Search Tool (BLAST) algorithm . These nonchimeric sequences data have been submitted to the GenBank database under accession numbers JF964265–JF964858 and JN006162–JN006258.All the sequences from the bacterial clone libraries were aligned with ClustalX(,and distance matrices were calculated according to the Jukes-Cantor distance Model in the PHYLIP 3.69 software package ( to group the sequences into operational taxonomic units (OTUs). Sequences were clustered by the program MOTHUR into OTUs using the furthest neighbor method and identity cut-offs of 0.03 representing approximately species-level clustering . The OTU was also represented by the name of the representative clone in further phylogenetic analyses. In each clone library, if several groups were classified as the same OTU finally, the number of clones in these groups were added up to calculate the number of clones that this OTU contained. The output files from OTU clustering(distance matrices, file of representative OTU sequences) had been deposited to Data Dryad ( (doi:10.5061/dryad.h3r16). [...] Phylogenetic analysis of these partial 16S rRNA gene sequences was performed using the MEGA4.0 software . For phylogenetic analysis, a representative clone of each OTU was chosen from the clone libraries, and their sequences were aligned with the nearest neighbors found in the NCBI database and SILVA web server [http://www.arbsilva. de/] . Clones obtained from the larval gut of other scarab beetles that were available in NCBI database were also added to the alignment. Neighbour-joining (NJ) analysis was performed by using MEGA4.0 . The NJ tree was constructed from the distance matrix calculated by the algorithm of Kimura's two-parameter model. Bootstrap confidence values were obtained with 1000 resamplings. [...] The richness estimations and diversity indices were calculated using EstimateS (Version 8.2, software. The bias-corrected Chao1 estimator of species richness was calculated after 1,000 randomizations of sampling without replacement. The diversity of the sampled sequence set was estimated using the Simpson and Shannon indices (H′) in the EstimateS application. The coverage of the clone library is given as: C = 1−(n1/N), where n1 is the number of clones that occurred only once in the library, and N is the total number of clones examined . Rarefaction curves were produced using the Analytic Rarefaction 1.3 program, which is available online at correlation between the Chao1 estimates and environmental factors was analyzed using the Spearman nonparametric correlation test available in the SPSS 16.0 software (SPSS Inc., Chicago, IL).∫-LIBSHUFF, a tool in the mothur software package that implements the Cramer-von Mises test statistic, was used to compare the clone libraries and determine the degree of similarity between them . The relationship between the hindgut microbial community composition and environmental factors was analyzed by redundancy discriminate analysis (RDA) using CANOCO software (Microcomputer Power, Ithaca, New York). The data matrices containing the species data were log(x+1) transformed before analysis. Out of all of the environmental variables, the environmental factors that best described the most influential gradients were identified by manual forward selection and subjected to further analysis. We used a Monte Carlo permutation test based on 499 random permutations to test the significance (P<0.05) of the relationship between the explanatory variables and the community composition. […]

Pipeline specifications

Software tools Clustal W, PHYLIP, mothur, MEGA, SPSS
Databases EzBioCloud
Applications Miscellaneous, Phylogenetics
Chemicals Carbon, Nitrogen