Computational protocol: The musk chemical composition and microbiota of Chinese forest musk deer males

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Protocol publication

[…] QIIME pipeline was mostly used for data quality controls and analyses. Raw sequence reads were filtered to meet minimum and maximum length of 200 bp and 450 bp with no ambiguous base calls. Sequences that contained more than one ambiguous base call (N) and did not also have a complete barcode and primer at one end were eliminated. A read was discarded if it was identified as a putative chimera by UCHIME. The sequences that passed the above procedure were denoised in order to correct for potential sequencing errors at 99% level using UCLUST. Operational Taxonomic Unit (OTUs) were defined using UCLUST v1.1.579, which have been proven to generate satisfactory and comparable numbers of OTU. The degree of similarity between sequences was defined as ≥97% as a threshold to obtain OTU identity at the species level. RDP classifier (version 2.10) software was used to classify the sequences according to the taxonomy proposed by Garrity et al., maintained at the Ribosomal Database Project (RDP 10 database, Update18). Richness, Shannon Index, ACE index, Chao1 and Coverage were included in Alpha diversity analysis by using MOTHUR. Mann-Whitney U test was used for significance test. Weighted unifrac and unweighted unifrac metric distances, each with 10× subsampling, were calculated to obtain beta diversity index, which used phylogenetic tree to compute phylogenetic relatedness of the bacterial community between samples. These results were viewed by Principal Coordinate Analysis (PCoA). A two-sided Student’s t-test was used for significance test of beta diversity difference between mated and unmated musk deer. Metagenome functional content from 16S rRNA was predicted using PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States). The R package ‘pheatmap’ was used for data analysis and plotting. […]

Pipeline specifications

Software tools QIIME, UCHIME, UCLUST, RDP Classifier, mothur, PICRUSt, PHeatmaps
Applications Phylogenetics, 16S rRNA-seq analysis, Transcriptome data visualization
Organisms Moschus berezovskii
Chemicals Aldosterone, Cholesterol, Sodium, Terpenes, Polyketides