Computational protocol: Air-Dried Brown Seaweed, Ascophyllum nodosum, Alters the Rumen Microbiome in a Manner That Changes Rumen Fermentation Profiles and Lowers the Prevalence of Foodborne Pathogens

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Protocol publication

[…] Total DNA was extracted from each sample (1.3 to 1.7 g/sample) by bead beating, followed by phenol-chloroform extraction (). Each sample was adjusted to a DNA concentration of 50 ng/μl and used to amplify partial bacterial 16S rRNA gene fragments with primers A (5′-TGCTGCCTCCCGTAGGAGT-3′) and B (5′-AGAGTTTGATCCTGGCTCAG-3′) () and partial archaeal 16S rRNA gene fragments with Arc915aF (5′-AGGAATTGGCGGGGGAGCAC-3′) and Arc1386R (5′-GCGGTGTGTGCAAGGAGC-3′) (, ). All of the DNA samples were sent to Genome Quebec (Montreal, QC) for amplification of bacterial and archaeal 16S rRNA gene fragments. Briefly, bacterial 16S rRNA genes were amplified by using the thermocycler settings described by Malmuthuge et al. (). Archaeal 16S rRNA gene fragments were amplified (50-μl reaction mixture volume) by using 50 ng of template, 5 μl of 10× buffer (Invitrogen, Carlsbad, CA), 1.5 mM MgCl2, 0.4 mM each primer, 10 mM each deoxynucleoside triphosphate, 0.5 U of Taq DNA polymerase (Invitrogen), and nuclease-free water. Amplifications were performed with an initial denaturation at 94°C for 5 min and 30 cycles of 94°C for 30 s, 58°C for 30 s, and 68°C for 1 min, followed by a final extension at 68°C for 7 min. The reaction products were run on an agarose gel, and amplicons with the proper size were trimmed and purified with the QIAquick gel extraction kit (Qiagen, ON, Canada). All of the purified amplicons were then subjected to sequencing on an Illumina MiSeq platform. Reads were processed by using Quantitative Insights into Microbial Ecology (QIIME, version 1.8.0) () to assess the composition of the microbial community for each sample. To obtain suitable output for functional prediction, taxonomic analyses assigned the reads to different OTUs at various taxonomic levels (phylum, family, genus, and species) on the basis of the Greengenes database (gg-13-5 version, chosen as compatible to run functional predictions) () and employing uclust, chimera check, and singleton removal. The species richness of each sample was estimated with the Chao1 index at 97% sequence similarity. OTUs were assigned on the basis of unique OTU reads. Shannon and Simpson indexes were calculated to indicate community diversity through QIIME. OTUs from each sample were normalized with the lowest number identified from the entire sample set prior to analysis for beta diversity (). The relative abundance of each microbial phylotype was compared among the different levels of Tasco. […]

Pipeline specifications

Software tools QIIME, UCLUST
Databases Greengenes
Application Amplicon sequencing analysis
Organisms Escherichia coli, Bacteria
Chemicals Butyrates, Carbon