Computational protocol: Distribution of Blastocystis subtypes isolated from humans from an urban community in Rio de Janeiro, Brazil

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Protocol publication

[…] Total DNA of each isolate was extracted from each culture of Blastocystis suspension using the Qiamp DNA Stool Mini Kit (Qiagen, Valencia, CA, USA) according to manufacturer’s recommendations. DNA was stored at -20 °C until use. To amplify segments from an approximately 500 bp fragment of genus-specific small subunit ribosomal DNA (SSU rDNA), we used the following primers: forward Blast 505-532 (5′-GGA GGT AGT GAC AAT AAA TC-3′) [] and reverse Blast 998-1017 (5′-TGC TTT CGC ACT TGT TCA TC-3′), following the protocol described by Santin et al. []. The PCR reaction was performed in a final volume of 50 μl and each reaction contained 100 mM of Tris-HCl (pH 9.0), 500 mM of KCl, 1.5 mM of MgCl2, 200 μM of each dATP, dGTP, dCTP, and dTTP; 0.2 μM of primer; 1.5 U of Taq DNA polymerase (Invitrogen Life Technologies, Carlsbad, CA, USA), 0.05% of bovine serum albumin (BSA); and 5 μl of the DNA sample. Cycle conditions were an initial activation step at 95 °C for 5 min, followed by 35 cycles at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 2 min, with a final extension step at 72 °C for 7 min. PCR amplification reactions were performed in a Veriti™ 96-well thermal cycler (AB Applied Biosystems, Foster City, CA, USA). PCR products were electrophoresed in 1.5% agarose gels in a tris-borate EDTA buffer, stained with Gelred (Biotium Inc., Hayward, CA, USA), and photographed under UV transillumination. Amplicons were purified using the Wizard® SV gel and PCR Clean-Up System kit (Promega, Madison, WI, USA) and sequenced in both directions using the PCR primers. DNA cycle sequencing reactions were performed using the BigDye® Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems) and loaded in the ABI 3730 Sequencing Platform. Bi-directional sequences were assembled and edited using SeqMan (DNASTAR software package, DNASTAR Inc., Madison, WI, USA). DNA sequences of Blastocystis subtypes were downloaded from GenBank and a multiple sequence alignment was performed using the ClustalW algorithm of MEGA software version 6.0 []. Bayesian Inference (BI), Maximum Likelihood (ML), and Neighbor-joining analyses of SSU rDNA data were conducted to explore the relationships between taxa using MrBayes [], PHYML [] and MEGA software [], respectively. Nucleotide substitution models were chosen based on the Akaike Information Criterion in jModelTest [] and MrModeltest []. ML analysis was conducted using the Hasegawa-Kishino-Yanov Model with gamma distribution (HKY + G) [] with four parameters and unequal base frequencies including rate variation among sites (-lnL = 24,599.056), which was predicted as the best model of nucleotide substitutions by the Akaike Information Criterion (AIC). For Bayesian analysis, the HKY + G model was determined by the Bayesian Information Criterion (BIC) as the best-fit substitution model. Proteromonas lacerate (U37108) was used as an out-group [, ].Subtype sequences with mixed traces indicative of mixed infections were analyzed by PCR using subtype-specific sequence-tagged-site (STS) primers (Table ), according to Yoshikawa et al. []. […]

Pipeline specifications

Software tools Clustal W, MEGA, MrBayes, PhyML, jModelTest, MrModelTest
Application Phylogenetics
Organisms Homo sapiens, Toxoplasma gondii, Trichuris trichiura, Enterobius vermicularis
Diseases Taeniasis, Blastocystis Infections