Computational protocol: Cleansing effect of acidic L-arginine on human oral biofilm

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Protocol publication

[…] The salivary microbiome of the six healthy volunteers (#4-#9) was characterized by Illumina Miseq 16S rDNA sequencing analysis of DNA extracted from 3 ml saliva. To identify the microbial groups that were sensitive to citrate or L-arginine rinsing, human saliva-derived biofilms formed on 13.5-mm plastic discs were washed with the test reagents described above. The test reagents were then recovered, and the bacteria from the washings were collected by centrifugation (washed-out fraction). The discs were washed three times with 0.5 ml saline, cut into pieces with sterile scissors, and then transferred to 0.5 ml saline for sonication with a Bioruptor (Cosmobio, 3 x 1 min with 1-min interval, output setting H). The detached biofilms were collected by centrifugation (sustained fraction). DNA from both fractions was also purified and the microbial composition determined by 16S rDNA sequencing analysis.DNA extraction was performed according to the method reported by Morita et al. []. Sequencing libraries were prepared by amplifying the V3-V4 region of the 16S rDNA using the primers described by Klindworth et al. []. After initial amplification, a second PCR was performed to attach Illumina adaptors as well as barcodes that allowed for multiplexing. Amplifications were performed in 25 μl reactions containing 2.5 μl diluted template, 12.5 μl 2x KAPA HiFi HotStart Ready Mix and 2.5 μl of each primer. Thermal cycling consisted of an initial denaturation step (3 min at 95 °C), followed by 25 cycles of denaturation (30 s at 95 °C), annealing (30 s at 55 °C) and 30 s extension at 72 °C. The final extension step consisted of 5 min at 72 °C. Amplicons were purified using AMPure XP beads (Beckman Coulter). Sequencing was performed on the Illumina MiSeq platform (MiSeq Reagent Kit ver. 3, 600 cycles) according to the manufacturer’s specifications to generate paired-end reads of 300 bases in each direction. A total of 10,381,210 2 x 300 base pair reads with an average of 432,550 reads per sample was obtained. Primer sequences were trimmed, and the paired-end reads merged using Fastq-join [] with default parameters and processed with the QIIME 1.8.0 pipeline []. After a chimera check by Usearch, 20,000 Illumina reads per sample (average quality score above 20) were randomly selected for further analysis. Using the UCLUST [] algorithm built into the QIIME pipeline, sequences were clustered at > 97 % identity against the Greengenes reference database, producing 635 operational taxonomic units (OTUs). Using the QIIME pipeline, unweighted UniFrac distances were produced and used to investigate beta diversity by plotting PCA coordinates. […]

Pipeline specifications

Software tools ea-utils, QIIME, USEARCH, UCLUST
Application 16S rRNA-seq analysis
Organisms Homo sapiens
Diseases Dental Caries, Dental Plaque, Periodontal Diseases
Chemicals Arginine, Gentian Violet, Sucrose