Computational protocol: Variation in Raw Milk Microbiota Throughout 12 Months and the Impact of Weather Conditions

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Protocol publication

[…] The raw data were separated by barcode using the MiSeq system program, and each sample was assigned paired FASTQ files. The paired FASTQ files were assembled using Mothur (version 1.37.0) with the command “make.contigs”. After removing sequences with ambiguous bases and shorter sequences (<350 bp), sequences were also excluded if they were identified as chimeric sequences or contaminants. The high-quality DNA sequences were grouped into OTUs with 97% by comparing them to the SILVA reference database (V119). Data normalization was carried out with the command “sub.sample” based on the minimum sample size (7,780). Community richness and diversity analysis (Shannon, ACE, Chao1 and Good’s coverage) were performed using the command “single.summary”. Taxonomy was assigned using the online software RDP classifier at an 80% threshold based on the Ribosomal Database Project. The functional predictions based on the 16 S rRNA genes were performed using PICRUSt software. [...] A t-test with 2 tails was used to determine whether the means of the Shannon diversity indices between June and Dec were significantly different using an R package, and p < 0.01 was considered a significant difference.Abundance differences in genera between months were analyzed by Metastats. Differences with q-values <1E-5 and fold changes >2 between months were considered significant, and the detailed results are listed in Tables –. The complex relations among the different genera were drawn using Circos software based on the major genera (percentage >1% in at least one month) and the phylum percentage. The coefficients of the relationships among genus abundances and between genera and milk quality parameters were analyzed using the non-parametric Spearman rank correlation with an R package. The p-value and adjusted p-value (q-value) were both calculated for finding relationships with a multiple test procedure. Because most q-values were over 0.05 (some even 0.5), we chose p-value (p < 0.01) as a less stringent cutoff. The software Genesis (version 1.7.7) was used to draw heatmaps using the taxonomy information (genus information) of the representative OTU sequences, and hierarchical clustering was calculated for the monthly distributions. More differences between major genera are listed in Tables –. An RDA analysis based on the value of “axis lengths” (<3) was performed using the Vegan package in R. The identification of species was determined through BlastN based on the SILVA database and the NCBI NT database, and the parameters were set as identity ≥99% and alignment ≥97% based on a previous study. To analyze the functional differences between months, a two-sided Welch’s test was performed using STAMP software, and the resultant P-values were multiply adjusted by the Benjamini-Hochberg procedure (q < 0.05); then, ratios of proportions higher than 2 were retained. […]

Pipeline specifications

Software tools mothur, RDP Classifier, PICRUSt, Metastats, Circos, BLASTN
Applications Metagenomic sequencing analysis, 16S rRNA-seq analysis, Genome data visualization
Organisms Bacteria, Firmicutes, Lactococcus