Computational protocol: Exploring the microbial community (microflora) associated with ovine Haemonchus contortus (macroflora) field strains

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Protocol publication

[…] After trimming the adaptor and primer sequences from Illumina reads, the raw sequences were assembled for each sample according to the unique barcode using Qiime (V1.7.0, http://qiime.org/scripts/split_libraries_fastq.html).Paired-end reads from the original DNA fragments were merged using FLASH (V1.2.7, http://ccb.jhu.edu/software/FLASH/).Quality filtering was performed under specific filtering conditions to obtain the high-quality clean tags according to the pipeline tool quantitative insights into microbial ecology Qiime (V1.7.0, http://qiime.org/scripts/split_libraries_fastq.html).The clean tags were compared against the reference database (Gold database, http://drive5.com/uchime/uchime_download.html) employing UCHIME algorithm (UCHIME Algorithm, http://www.drive5.com/usearch/manual/uchime_algo.html) to detect and remove chimeric sequences. Paired-end reads were assigned to each sample according to the unique barcodes. Sequence analysis was performed by UPARSE software package (Uparse v7.0.1001, http://drive5.com/uparse/) using the UPARSE-OTU and UPARSE-OTU ref algorithms. Alpha (within samples) and beta (among samples) diversities were analyzed employing in-house Perl scripts. Sequences with ≥97% identities were assigned to the same OTUs. Representative sequences for each OTU were picked up to be used in the ribosomal database project (RDP) classifier to annotate the taxonomic information for each representative sequence. In order to estimate alpha diversity, we rarified the operational taxonomic units (OTUs) and calculated five metrics: Phylogenetic diversity (PD) Whole tree, Good’s coverage, Chao1, Shannon and Observed species. Rarefaction curves were created based on this five metrics.The Kruskal-Wallis test was used to compare diversity metrics, such as genus evenness, Simpson and Shannon-Wiener species diversity indices. The level of significance was determined at P < 0.05.The Principal Coordinate Analysis (PCoA) was conducted to explore the differences in the bacterial community structures and was displayed with the WGCNA, stat and ggplot2 packages in the R software (Version 2.15.3). Weighted and un-weighted unifrac distances were calculated employing QIIME. Graphical representation of the relative abundance of bacterial diversity from phylum to genus was visualized using a bar plot with horizontal bars. […]

Pipeline specifications

Software tools QIIME, UCHIME, USEARCH, UPARSE, RDP Classifier, Ggplot2, UniFrac
Applications Miscellaneous, Phylogenetics, 16S rRNA-seq analysis
Diseases Escherichia coli Infections