Computational protocol: EPSP of L. casei BL23 Protected against the Infection Caused by Aeromonas veronii via Enhancement of Immune Response in Zebrafish

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Protocol publication

[…] Total bacteria DNA was extracted from intestinal contents samples by using Power FecalTM DNA Isolation kit (MO BIO Laboratories, Carlsbad, CA, United States) according to manufacturer’s instruction. Sequencing was performed at the Novogene Bioinformatics Technology Co., Ltd. Briefly, DNA was amplified by using the 515F/806R primer set (341F: 5′-CCTAYGGGRBGCASCAG-3′ 806R: 5′-XXXXXXGGACTACHVGGG TWTCTAAT-3′), which targets the V-V43 region of the bacterial 16S rDNA, with the reverse primer containing a 6-bp error-correcting barcode unique to each sample. PCR reaction was performed using phusion high-fidelity PCR Mastermix (New England Biolabs LTD., Beijing, China) with the following condition: 94°C for 5 min (1 cycle), 94°C for 20 s/55°C for 20 s/72°C for 30 s (30 cycles), and a last step of 72°C for 10 min. PCR products were purified by using the QIAquick Gel Extraction Kit (QIAGEN, Dusseldorf, Germany). Pyrosequencing was conducted on an Illumina HiSeq 2500 platform according to protocols described by . Paired-end reads were merged using FLASH (V1.2.7) (). Sequences were then demultiplexed and quality filtered using the default parameters of the Quantitative Insights into Microbial Ecology (QIIME) software package (). The operational taxonomic unit (OTU) clustering pipeline UPARSE was used to select OTUs at 97% similarity (). The representative sequence sets were aligned and given a taxonomic classification using Ribosomal Database Project (). The similarity among microbial communities was determined using histograms, UniFrac principal coordinates analysis (PCoA), and the unweighted pair-group method with arithmetic mean (UPGMA). […]

Pipeline specifications

Software tools QIIME, UPARSE
Application 16S rRNA-seq analysis
Organisms Lactobacillus casei BL23, Aeromonas veronii, Danio rerio, Escherichia coli, Bacteria, Caenorhabditis elegans, Mus musculus