Computational protocol: Microbiome Dynamics in a Shrimp Grow-out Pond with Possible Outbreak of Acute Hepatopancreatic Necrosis Disease

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Protocol publication

[…] Raw sequence reads were sorted according to their respective barcodes into individual libraries. The sequences of primers, barcodes, and adaptors were trimmed. The sequence reads shorter than 300 bp, chimerical sequences, and those containing ambiguous nucleotides were filtered to obtain quality reads and then clustered for OTUs (3% sequence cutoff) with USEARCH. Paired-end reads were merged using FLASH. The OTU sequences were taxonomically assigned to the SILVA 16S rRNA gene database and taxonomy. After excluding the chloroplast-related sequences, rank abundance, species richness (Chao1), and species diversity (Shannon–Weiner index) indices were calculated using QIIME. To normalize the uneven sequencing effects, the OTU table was 3X randomly rarefied subset of 14,000 sequences per sample. A weighted Unifrac-PCoA was performed to evaluate the structural similarities of the bacterial communities among the samples. A Tukey test and T-test were used to evaluate the differences of sample groups. Bacterial interaction network analysis was performed with the read abundance data at the OTU-level by using a stand-alone tool, MetaMIS. The topology of the resulting bacterial network was visualised using Gephi. […]

Pipeline specifications

Software tools USEARCH, QIIME, MetaMIS, Gephi
Application Metagenomic sequencing analysis
Organisms Vibrio parahaemolyticus, Bacteria