Computational protocol: Blastocystis Isolates from Patients with Irritable Bowel Syndrome and from Asymptomatic Carriers Exhibit Similar Parasitological Loads, but Significantly Different Generation Times and Genetic Variability across Multiple Subtypes

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Protocol publication

[…] High Resolution Melting (HRM) analysis was conducted in order to support the ST identification, in some DNA samples that were amplified in 96-well plates, and HRM curves acquisition was performed, similarly to qPCR, under the same amplification conditions and system, but using a Light Cycler HRM master kit (Roche, Germany) to assign a melting temperature. DNA samples with ST identified by sequencing (ST1, ST2, ST3 and ST7) were included as references for wild-type curves. According to Wittwer et al. [] and Gonzalez-Bosquet et al. [], normalization and background subtraction were first performed by fitting an exponential to the background surrounding the HRM transitions of interest; the normalized HRM curves were temperature-overlaid, to eliminate slight temperature errors between wells or runs; after, different plots of these normalized and temperature-overlaid curves were obtained by deducting the fluorescence difference of each curve from the average wild-type curve at all temperature points. Thus, HRM profile with a plot interpreted by the software to be different from the averaged wild-type curve, were considered to be suspicious of harboring a nucleotide change, mutation or variant that, in our particular case, corresponded to each ST. Direct sequencing was performed to identify the ST in DNA samples. Direct qPCR product sequencing was performed according to Poirier et al [], after purification with a QIAquick PCR Purification Kit (Qiagen, Germany). Sequencing was performed by a commercial supplier using the same primers as those used for qPCR (BL18SPPF1 and BL18SR2PP) that amplify a DNA fragment of 320 to 342bp, depending on the subtype.All sequences were subjected to a BLAST search in the GenBank database; multiple alignments were performed using the CLUSTAL W [] and Muscle [] programmes with manual adjustment in MEGA 5.05 software []. Hasegawa Kishino Yano model with gamma distribution and invariable sites was the best fit model of nucleotide substitution for SSUrDNA, determined using the Akaike Information Criterion in Modeltest version 3.7 software []. The phylogenetic reconstruction using Bayesian inference was performed with the Mr. Bayes 3.1.2 program [–]. The analysis was done for 10 million generations with sampling trees every 100 generations. Trees with scores lower than those at the stationary phase (burn-in) were discarded, and the trees that reached the stationary phase were collected and used to build majority consensus trees. Other sequences were obtained from GenBank and used as subtype references. A genetic diversity analysis within and between populations was performed using DnaSPv4 [] and included nucleotide diversity (π), haplotype polymorphism (θ), gene flow (Nm), genetic differentiation index (FST) and Tajima’s D test. These indexes refer to the following: π, the average proportion of nucleotide differences between all possible pairs of sequences in the sample; θ, the proportion of nucleotide sites that are expected to be polymorphic in any suitable sample from this region of the genome. Both indexes are used to assess polymorphisms at the DNA level and to monitor diversity within or between ecological populations and examine the genetic variation in related species or their evolutionary relationships. FST is a typical genetic statistic used to measure differentiation between or among populations. The common used values for genetic differentiation are as follows: 0 to 0.05, small; 0.05 to 0.15, moderate; 0.15 to 0.25, great; values above 0.25 indicate huge genetic differentiation. The gene flow or migration index (Nm) refers to the movement of organisms among subpopulations; those strongly differentiated have an Nm<<1, whereas an Nm>4 behaves as a single panmictic unit []. […]

Pipeline specifications

Software tools Clustal W, MUSCLE, MEGA, ModelTest-NG
Applications Phylogenetics, Nucleotide sequence alignment
Organisms Homo sapiens
Diseases Blastocystis Infections, Irritable Bowel Syndrome