Computational protocol: Evaluation of the impact of refrigeration on next generation sequencing-based assessment of the canine and feline fecal microbiota

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Protocol publication

[…] Fecal samples were collected from seven dogs and ten cats from a local animal shelter immediately after defecation. Dogs were clinically normal with no history of antimicrobial exposure or gastrointestinal disease, although medical histories were limited based on the nature of the population. Samples were stored in plastic fecal containers at 4°C for up to 2 hours prior to arrival at the laboratory. Immediately after arrival, samples were manually homogenized and separated into five aliquots. One aliquot was processed immediately while the other four were stored in a refrigerator at 4°C. One of each aliquots was then tested after 3, 7, 10 and 14 days of refrigeration.DNA was extracted using a commercial kita, and DNA quantity and quality were assessed by spectrophotometryb. The V4 region of the 16S rRNA gene was then amplified using the primers S-D-Bact-0564-a-S-15 (5'-AYTGGGYDTAAAGNG-3') and S-D-Bact-0785-b-A-18 (5'-TACNVGGGTATCTAATCC-3') []. The amplicon library was purified with Agencourt AMPure XP beadsc with slight modification to the manufacturer’s protocol. Briefly, 72 μL of AMPure beads was added to 20 μL of library and incubated for 10 min at room temperature. Samples were washed twice with 80% ethanol, and eluted with 20 μL of PCR-grade H2O. Purified samples were quantified by spectrophotometry, evaluated by electrophoresis on a 1% agarose gel, and diluted to 5 ng/μL. Sequencing was performed using an Illumina MiSeq with 2X250 chemistry.dMothur v33.3 was used for analysis []. After paired end reads were assembled, sequences were aligned with the Silva 16S rRNA reference database [] and any sequences not consistent with the target amplicon size (240 bp), containing any ambiguous base calls or long runs (>8 bp) of holopolymers, or that did not align with the correct 16S rRNA gene region were removed. Chimeras were detected using uchime [] and removed. Taxonomy was assigned using the RDP taxonomy database []. Sequences were then binned into operational taxon units (OTUs) at a 3% dissimilarity level.Subsampling was performed to normalize sequence number for analyses []. This involved random selection of a number of sequences from each sample that corresponded to the smallest number of sequences from an individual sample. Population diversity (inverse Simpson’s index), evenness (Shannon’s evenness index) and richness (Chao1) were calculated and compared between groups using Wilcoxon and Steel-Dwass tests. Linear discriminatory analysis effective size (LefSe) [] analysis and indicator analysis [] were performed.Community membership was described using the classical Jaccard index, while population structure (evaluation of membership and relative abundance of members) was evaluated using the Yue & Clayton index of dissimilarity and Bray-Curtis index. Unifrac was used to compare these indices between groups []. Principal coordinate analysis (PCoA) and random forest analysis were also performed. The relative abundance of all phyla were compared between groups, along with the relative abundance of all genera accounting for ≥ 1% of sequences in the Day 0 or Day 14 samples using the Steel-Dwass test. A P <0.05 was considered significant for all comparisons. […]

Pipeline specifications

Software tools UCHIME, LEfSe, UniFrac
Applications Metagenomic sequencing analysis, 16S rRNA-seq analysis
Organisms Canis lupus familiaris, Felis catus