Computational protocol: Profiling bacterial diversity in a limestone cave of the western Loess Plateau of China

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Protocol publication

[…] Analysis of 454 pyrosequencing data was conducted using the Mothur software v1.30.2 () combined with RDP II for taxonomic identification. All reads obtained were processed by removing tags and primer, with only reads with an average quality score above 30 and read lengths above 250 nt deemed acceptable. The trimmed sequences were aligned against the SILVA bacterial 16S rRNA gene databases using the Needleman algorithm. Chimeric sequences were identified and removed using the implementation of Chimera-uchime (). Potential chloroplasts and mitochondria sequences were identified and discarded by an assignment against respective sequence references. The high quality sequences were aligned to generate a distance matrix and clustering with the average neighbor algorithm. Representative sequences for each operational taxonomic unit (OTU), as defined by a 97% sequence identity, were obtained for further taxonomic analysis. Taxonomic placement of each OTU was carried out with RDP Classifier () with a confidence threshold of >80% selected.Operational taxonomic unit richness and Shannon and Simpson diversity of each library were calculated at the cutoff of 0.03. Principal coordinate analysis (PCoA) was performed based on Bray–Curtis dissimilarity distances between the libraries with Mothur. Maximum likelihood (M-L) trees were calculated based on the Jukes–Cantor correction with Mega version 5.2 () with representative cave sequences and the closest relatives deposited in the Genbank database. Bootstrap support was calculated (1000 replications). […]

Pipeline specifications

Software tools mothur, UCHIME, RDP Classifier, MEGA
Application 16S rRNA-seq analysis
Diseases Bacterial Infections