Computational protocol: The gut microbiome of hooded cranes (Grus monacha) wintering at Shengjin Lake, China

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Protocol publication

[…] All sequences acquired using the Illumina–MiSeq were saved in the raw fastq files. Initial processing of the raw dataset included screening to remove short and low‐quality reads; only high‐quality sequences without primer sequences were retained. Using USEARCH (version 7.1 http://drive5.com/uparse/), these high‐quality sequences were clustered into operational taxonomic units (OTUs) at a 97% identity threshold. The taxonomic classification of OTUs was performed using the Ribosomal Database Project Classifier with the QIIME bioinformatics pipeline (http://qiime.org/scripts/assign_taxonomy.html) to determine the community composition of each sample at each taxonomic level (Wang et al., ) at a 70% confidence level. Rarefaction curves and alpha diversity calculations were based on OTUs with >97% identity. Rarefaction analysis and alpha‐diversity indices (abundance‐based coverage estimation (ACE), Chao1, Shannon and Simpson) were calculated using Mothur. R language tools were used to generate rarefaction curves (Amato et al., ) and Shannon–Wiener curves (Wang et al., ). Heatmaps were generated using the R package (Jami et al., ). The core fecal microbiome of the hooded cranes was assigned if it comprised >90% of samples and represented ≤0.1% of the reads. […]

Pipeline specifications

Software tools USEARCH, UPARSE, RDP Classifier, QIIME, mothur
Application 16S rRNA-seq analysis
Organisms Grus monacha, Hemisus marmoratus, Firmicutes
Chemicals Nucleotides