Computational protocol: Kappaphycus alvarezii as a Food Supplement Prevents Diet-Induced Metabolic Syndrome in Rats

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Protocol publication

[…] Immediately following euthanasia and organ removal, two or three fecal pellets were collected from the colon of rats and stored at −80 °C in nuclease-free tubes. DNA extraction and diversity profiling were performed by the Australian Genome Research Facility, Brisbane, QLD, Australia. The V3-V4 region of the 16S rRNA gene was selected for amplification. The primers used were F341 (5′-CCTAYGGGRBGCASCAG-3′) and R806 (5′-GGACTACNNGGGTATCTAAT-3′). PCR amplicons were generated using AmpliTaq Gold 360 mastermix (Life Technologies, Scoresby, VIC, Australia) for the primary PCR. A secondary PCR to index the amplicons was performed with TaKaRa Taq DNA Polymerase (Clontech, Mountain View, CA, USA). The resulting amplicons were measured by fluorometry (Invitrogen Picogreen, Mount Waverley, VIC, Australia) and normalized. The equimolar pool was then measured by qPCR (KAPA) followed by sequencing on the Illumina MiSeq (Illumina Inc., San Diego, CA, USA) with 2 × 300 base pairs paired-end chemistry.Paired-ends reads were assembled by aligning the forward and reverse reads using PEAR (version 0.9.5, The Exelixis Lab, Heidelberg, Germany) []. Primers were identified and trimmed. Trimmed sequences were processed using Quantitative Insights into Microbial Ecology (QIIME 1.8) [] USEARCH (version 7.1.1090) [,] and UPARSE software []. Using USEARCH, sequences were quality filtered, full length duplicate sequences were removed and sorted by abundance. Singletons or unique reads in the data set were discarded. Sequences were clustered followed by chimera filtered using “rdp_gold” database as the reference. To obtain the number of reads in each Operational Taxonomic Unit (OTU), reads were mapped back to OTUs with a minimum identity of 97%. Using QIIME, taxonomy was assigned using Greengenes database (version 13_8, August 2013) []. A heat map was constructed using R statistical software according to the developer’s instructions to visualize the relative abundance of each bacterial species and their respective phyla. […]

Pipeline specifications

Application 16S rRNA-seq analysis
Organisms Rattus norvegicus, Homo sapiens, Zea mays subsp. mays, Firmicutes, Bacteroidetes
Diseases Fatty Liver, Liver Diseases, Metabolic Diseases, Neoplasms, Adipose Tissue
Chemicals Fatty Acids, Potassium, Triglycerides