Computational protocol: DGGE Identification of Microorganisms Associated with Borrelia burgdorferi Sensu Lato- or Anaplasma phagocytophilum-Infected Ixodes ricinus Ticks from Northwest Norway

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Protocol publication

[…] 16S rDNA fragments from DGGE bands were cut out from the gel and placed into tubes containing 20 μL of ddH2O; these fragments were then used as templates for PCR using 341f/518r primers () and the same reaction protocol as the original amplification. Purification of the amplified products was performed in a two-step process using ExoSAP-IT (Affymetrix, Santa Clara, USA) according to the manufacturer's protocol. The sequencing reactions were carried out in 10 μL volumes containing 1 μL of Big Dye Terminator 3.1 (Applied Biosystems, Carlsbad, USA), 1 μL of 5x Sequencing Buffer (Applied Biosystems, Carlsbad, USA), 0.32 μL of 341f primer (10 μM), 4.68 μL of ddH2O, and 3 μL of template DNA. Amplification was performed in a 2720 Thermal Cycler (Applied Biosystems, Carlsbad, USA) with 1 cycle of denaturation (6 min, 96°C), followed by 25 cycles of denaturation (10 sec, 96°C), annealing (5 sec, 50°C), and extension (4 min, 60°C). Next, 10 μL of ddH2O was added to each well upon completion of the sequencing reaction as previously described []. The source of each sequence was identified using BLAST to compare sequences from the DGGE analysis to sequences of known bacterial species in the GenBank database []. Sequences that exhibited less than 95% identification by BLAST search were classified by the RDP classifier []. [...] Statistical analysis was utilised to study the statistical coherence between the selection of microorganisms identified within each group and the specific pathogenic bacteria. Statistical comparison of the variance between the groupings was calculated by a one-way ANOVA analysis using IBM SPSS Statistics 20 (SPSS Inc., Chicago, USA). The significance level was set to 0.05. Analyses were performed to test whether there were significant differences between the microorganisms in Borrelia burgdorferi sensu lato- or Anaplasma phagocytophilum-infected I. ricinus ticks. In addition, it was determined whether there were significant differences in the presence of microorganisms within the DGGE profiles of Borrelia burgdorferi sensu lato- or Anaplasma phagocytophilum-infected I. ricinus ticks. Statistical analysis was performed to analyse the variance between the microbial contents from groups G1, G2, and G3. A scatter plot was created to study the distribution of bacterial species between the three groups. […]

Pipeline specifications

Software tools RDP Classifier, SPSS
Applications Miscellaneous, 16S rRNA-seq analysis
Organisms Borreliella burgdorferi, Anaplasma phagocytophilum, Ixodes ricinus, Candidatus Midichloria mitochondrii