Restriction Fragment Length Polymorphism Data Research Articles

Genetic data confirm the presence of juvenile Alosa alosa in the estuary of the River Tamar

AbstractWithin the UK, allis shad (Alosa alosa) are classified as Critically Endangered and are known to breed in only a single river, the Tamar. Despite evidence of spawning within the lower freshwater reaches of the river and at the tidal limit within the estuary, juvenile allis shad have never been found. Genetic analysis, based on mitochondrial DNA haplotype and nuclear Polymerase Chain Reaction ‐ Restriction Fragment Length Polymorphism (PCR‐RFLP) data, confirmed that juvenile shad found within the lower Tamar estuary in autumn 2022 were A. alosa.

Determination of Genetic variation among Beauveria bassiana and Metarhizium anisopliae Isolates from Palestine and other Geographic regions using ITS and β-tubulin Sequences, RFLP and ISSR Data

In this work we report the genetic variation of ten isolates of entomopathogenic fungi, four of which were isolated from Palestine while the remaining six were from other geographic regions. The variation was determined using molecular-based techniques including; sequence analysis of β-tubulin (BT) gene, the Internal Transcribed Spacers (ITS) 1 & 2 of the rDNA, Restriction Fragment Length Polymorphism (RFLP), and Inter-Simple Sequence Repeat (ISSR) analysis. The sequencing data of ITS regions confirmed the taxonomic identity of six isolates as Metarhizium anisopliae while the identity of the other four as Beauveria bassiana. These data also revealed that the two Palestinian Beauveria b. isolates are probably new strains since theyshowed 99% homology with the best match sequences reported in the gene bank. The restriction digestion of the ITS2 sequences produced a fingerprint for the PAL-M01 isolate. The ITS and BT sequences were able to distinguish the B-Bot genotype from the other Beauveria b. isolates. In contrast, the ITS could discriminate the three Metarhizium isolates: Ma-7, PAL-M01, and PPRC. Moreover, the ISSR analysis detected high levels of polymorphism among the Beauveria b. (80%) and the Metarhizium a. (90%) isolates. The fact that the PPRC and B-Bot isolates areobviously genetically different from their Metarhizium a. and Beauveria b. counterparts could be attributed to their distant geographic origins. The molecular data obtained in this work can be utilized for specific identification of each ofthese isolates which, would facilitate the local use of these fungi as biocontrol agents. To the best of our knowledge, this is the first study, in Palestine, attempting to discriminate between EPF isolates using advanced molecular techniques.

Determination of mucopolysaccharidosis IIID in some goat breeds

Mucopolysaccharidosis IIID (MPS IIID) or Sanfilippo D syndrome is an inherited lysosomal storage disease and caused by a deficiency of the N-acetylglucosamine-6-sulfatase activity, which involves in the catabolism of heparan sulfate. In addition to humans, canine, feline and murine, goats have been reported as possessing this lysosomal storage disease. The molecular defect in the MPS IIID goat has been previously identified. It has been observed that a molecular base for this defect is a nonsense mutation at nucleotide 322 (C→T) results in the change of the arginine codon to a stop codon. This mutation is not only to be related to stop enzyme function but also introduces a recognition site for AluI that will enable carrier detection. The goal of this study is to find the presence of MPS IIID genetic disorder in some goat breeds. A total of 80 goat blood samples selected randomly from 4 different countries including Kyrgyzstan, Iraq, Northern Cyprus and Bosnia-Herzegovina were used as a sample. Polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) and DNA sequence data were utilized to identify this disorder in the goat populations. The result of this study demonstrates nonsense mutation which causes MPS IIID genetic disorder was not found in the goat breeds studied, while a silent mutation was found at nucleotide 354 (T→C) when compared with reference sequence.

Rapid, inexpensive methods for exploring SARS CoV-2 D614G mutation

A common mutation has occurred in the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS CoV-2), known as D614G (A23403G). There are discrepancies in the impact of this mutation on the virus's infectivity. Also, the whole genome sequencings are expensive and time-consuming. This study aims to develop three fast economical assays for prompt identifications of the D614G mutation including Taqman probe-based real-time reverse transcriptase polymerase chain reaction (rRT PCR), an amplification refractory mutation system (ARMS) RT and restriction fragment length polymorphism (RFLP), in nasopharyngeal swab samples. Both rRT and ARMS data showed G614 mutants indicated by the presence of HEX probe and 176 bp, respectively. Additionally, the results of the RFLP data and DNA sequencings confirmed the prevalence of the G614 mutants. These methods will be important, in epidemiological, reinfections and zoonotic aspects, through detecting the G614 mutant in retro-perspective samples to track its origins and future re-emergence of D614 wild type.

Biocontrol of Melolontha spp. Grubs in Organic Strawberry Plantations by Entomopathogenic Fungi as Affected by Environmental and Metabolic Factors and the Interaction with Soil Microbial Biodiversity.

Simple SummaryThe study aimed at relating some metabolic characteristics of two entomopathogenic fungi (Beauveria bassiana and B. brongniartii), critical for their virulence and persistence in soil, to the strain’s capacity to reduce the damage of Melolontha sp. grubs in two organic strawberry plantations. Combination of the two species was also tested to achieve a higher efficacy, due to their different living behaviors. This hypothesis was not confirmed in the study, probably due to the B. bassiana metabolic competitive advantage emerged from the phenotypic characterization. The strong in vitro metabolic activity of the B. bassiana strain could be also associated to the higher abundance of this species in the inoculated soils in comparison to B. brongniartii strain. Considering the impact on soil biodiversity, the inoculation with both strains or the co-inoculum did not affect the natural fungal and bacteria communities in the soil, according to terminal restriction fragment length polymorphism (TRFLPs) analysis and qPCR data. The study provides a complex view of the effects of bioinocula to plant protection and soil biodiversity, taking into consideration the mechanisms of fungal virulence and the effect of environmental conditions on them.The efficacy of two strains of two Beauveria species (B. bassiana and B. brongniartii), individually or as co-inoculants, to control Melolontha sp. grubs was assessed in two organic strawberry plantations in relation to the environmental conditions, their abundance after soil inoculation, and their in vitro chitinolytic activity, thereby also verifying their impact on soil microbial communities. A reduction of the grubs’ damage to strawberry plants was observed when compared to the untreated control in one plantation, irrespective of the strain used and whether they were applied as single or as co-inoculum. The metabolic pattern expressed by the two fungi in vitro was different: B. bassiana showed a higher metabolic versatility in the use of different carbon sources than B. brongniartii, whose profile was partly overlapped in the co-inoculum. Similar differences in the chitinolytic activity of each of the fungi and the co-inoculum were also pointed out. A higher abundance of B. bassiana in the soils receiving this species in comparison to those receiving B. brongniartii, together with its in vitro metabolic activity, could account for the observed diverse efficacy of pest damage control of the two species. However, environmental and climatic factors also affected the overall efficacy of the two bioinocula. According to the monitoring of the two species in soil, B. bassiana could be considered as a common native species in the studied locations in contrast to B. brongniartii, which seemed to be a non-endemic species. Nevertheless, the inoculation with both species or the co-inoculum did not consistently affect the soil microbial (fungi and bacteria) biodiversity, as expressed by the operational taxonomic unit (OTU) number and Shannon–Wiener diversity index based on terminal restriction fragment length polymorphism (TRFLP) data. A small transient increase of the share of the inoculated species to the total fungal community was noted by the analysis of genes copy numbers only for B. brongniartii at the end of the third growing season.

On the Origin of Two Species of Charrs (Salvelinus, Salmonidae) in Lake Elgygytgyn: Data on the ATPase6–NADH4L Region of Mitochondrial DNA

To test the reliability of Restriction Fragment Length Polymorphism data for ATPase6–NADH4L region of mitochondrial DNA (2162 bp), which unambiguously support the allopatric origin of the small-mouth charr Salvelinus elgyticus and “Boganida” charr S. “boganidae” in Lake Elgygytgyn, this region was sequenced in some members of the S. alpinus–S. malma complex. It is shown that RFLP data on the number of restriction sites and the size of restriction fragments for the 2162 bp region are poorly consistent with data on nucleotide sequences for the majority of forms and species of charr. Based on the identified errors, data on the MvaI restriction sites for ATPase6–NADH4L region of mitochondrial DNA cannot be considered as unambiguous evidence in favor of the allopatric origin of the two charr species in Lake Elgygytgyn. Nevertheless, the nucleotide data for this fragment suggest with a high probability that the small-mouth and “Boganida” charrs of Lake Elgygytgyn have an allopatric origin.

Thiopurine S-methyltransferase genetic polymorphisms in adult patients with inflammatory bowel diseases in the Latvian population.

Background:Thiopurine methyltransferase (TPMT) plays a significant role in the metabolism of thiopurines, and, for patients with inflammatory bowel disease (IBD), it is useful to perform TPMT genotyping prior to azathioprine (AZA) treatment. In this study, we determined TPMT gene polymorphisms in a cohort of IBD patients in Latvia.Methods:DNA samples were obtained from 244 IBD patients, and qPCR was performed for detection of rs1800462, rs1800460, and rs1142345 single-nucleotide polymorphisms (SNPs). Three common, non-functional TPMT alleles (TPMT*2, *3B, and *3C) were identified (women, 51%; men, 49%). TPMT*2, *3A, *3B, and *3C allelic variants detected using qPCR were consistent with restriction fragment length polymorphism (RFLP) data.Results:Among patients, 78% had ulcerative colitis and 22% had Crohn’s disease, with 93.9% of the former carrying a wild-type homozygous TPMT*1/*1 genotype and 6.1% carrying heterozygous genotypes. The most frequent polymorphisms were TPMT*1/*3A (5.3%: two variants: TPMT*3B and TPMT*3C), TPMT*1/*3C (0.4%), and TPMT*1/*2 (0.4%). None of the patients carried a TPMT*3B polymorphism and no patients were homozygous for any mutation.Conclusion:This is the first study to identify TPMT gene polymorphisms in adult IBD patients in Latvia. The results indicate that the frequency of common TPMT alleles is similar to that of other European populations.

Characteristics of Gut Microbiota in Patients With Diabetes Determined by Data Mining Analysis of Terminal Restriction Fragment Length Polymorphisms.

BackgroundThis study was performed to clarify whether gut microbiota obtained from fecal samples could identify the type of diabetes in patients of each gender by using a combination of terminal restriction fragment length polymorphism (T-RFLP) analysis and data mining.MethodsA cross-sectional study was performed at three centers. Fecal samples were collected from 12 Japanese patients with type 1 diabetes mellitus (T1D), 18 patients with type 2 diabetes mellitus (T2D), and 31 subjects without diabetes mellitus (non-DM). Amplification of fecal 16S rRNA was carried out. After digestion of the amplification products with restriction enzymes (AluI, BslI, HaeIII, and MspI), terminal restriction fragments (T-RFs) of DNA were detected. A data mining algorithm (classification and regression tree (CART) modeling system) provides a decision tree that classifies subjects into various groups according to pre-assigned characteristics.ResultsAmong men, the error rate was 2.4% with MspI, while error rates were 0.0% with other restriction enzymes. Among women, the error rate was 0.0% with all restriction enzymes. The operational taxonomic units (OTUs) incorporated into the decision tree differed between men and women.ConclusionsWe were able to classify the 16SrRNA gene amplification products obtained from fecal samples of T1D patients, T2D patients, and non-DM subjects with a high level of precision by combining T-RFLP analysis and data mining. Specific gut microbiota patterns were found for T1D and T2D patients, as well as a sex difference of the patterns.

Impacts of invasive plant species on soil biodiversity: a case study of dog-strangling vine (Vincetoxicum rossicum) in a Canadian National Park

Soil microbial diversity is expected to be altered by the establishment of invasive plant species, such as dog-strangling vine (DSV) [Vincetoxicum rossicum (Apocynaceae)]. However, in urban ecosystems where DSV invasion is high, there is little research evaluating the impacts of DSV and other anthropogenic disturbances on microbial diversity. Our study was based in Rouge National Urban Park, Canada, where we used terminal restriction fragment length polymorphism data to evaluate (i) if DSV has a detectable impact on soil bacterial community composition and (ii) if these impacts occur independently of other anthropogenic change or soil characteristics. Variation in soil bacterial communities was greatly reduced in DSV-invaded sites vs. less-invaded sites. The degree of DSV invasion independently explained 23.8% of variation in bacterial community composition: a value similar to the explanatory power of proximity to roadways (which explained 22.6% of the variation in community composition), and considerably greater than soil parameters (pH, moisture, carbon, and nitrogen concentrations) which explained only between 6.0% and 10.0% of variation in bacterial community composition. Our findings indicate that DSV influences soil bacterial community composition independent of other anthropogenic disturbances and soil parameters, with potential impacts on multiple facets of plant–soil interactions and plant invasion dynamics.

Time estimations by network of beta globin gene cluster haplotypes linked with Hb D-Los Angeles [β121 (GH4) Glu→Gln GAA→CAA] mutation in the world populations.

Backgroundβ‐Globin gene cluster haplotypes associated with the Hb D‐Los Angeles mutation have been reported in many different locations in different populations including Italy, Iran, Thailand, Belgium, Mexico, Holland, and Turkey. In this study, we have identified genetic relationships and formation periods between the haplotypes reported in the world regarding the Hb D‐Los Angeles.MethodsWe comparatively analyzed the RFLP (restriction fragment length polymorphism) data in Denizli region and world populations using Arlequin 3.5 statistical software program. The data obtained from the Arlequin software were then entered into the Phylogenetic Network software to calculate the age estimates and to discover possible links between the haplotypes.ResultsWe observed the frequencies of the β‐globin gene haplotypes for the seven polymorphic restriction sites around the world and calculated the estimated time of haplotypes using Network software on the basis of ancestral haplotypes. We performed the phylogenetic network analysis of the haplotypes linked with Hb D‐Los Angeles mutation by processing the data of frequency and age estimations with Network software.ConclusionOur period of time results suggests that HAP1 was formed before modern human migration to Asia and/or independent origin of the Hb D‐Los Angeles mutation from other populations. Considering that the population in Denizli region started the Hb D‐Los Angeles mutation past about 40,000 years ago, it can be said that HAP1, HAP15, and HAP21 belong to the gene pool with an external effect. Our period of time results of HAP6 is compatible with published dating results.

Are Sclerotinia sclerotiorum populations from the tropics more variable than those from subtropical and temperate zones?

The genetic variability of Sclerotinia sclerotiorum populations has been investigated in many countries and, consequently, under different ecological conditions. The first studies were conducted to gather information about the genetic structure of populations sampled in temperate climate areas. Based on DNA fingerprinting analysis of restriction fragment length polymorphism (RFLP) data and mycelial compatibility groups, most studies revealed a clonal population structure. Later, using DNA sequences, populations from subtropical regions of North America were reported to be more variable than those from temperate regions. In the past few years, new analyses in temperate, sub-tropical and tropical areas were conducted using microsatellite (SSR) markers. Most studies using SSR reported both high variability and evidence of outcrossing in S. sclerotiorum populations, but evidence of clonality has also been found. Some authors argue that populations from subtropical and tropical areas are more diverse. We argue that the reports of high variability of S. sclerotiorum in tropical or subtropical regions may be due to an increase in the number of studies performed with SSR markers in these regions and in some cases due to inappropriate interpretations of the results.

Technical Aspects of Nominal Partitions on Accuracy of Data Mining Classification of Intestinal Microbiota - Comparison between 7 Restriction Enzymes.

The application of data mining analyses (DM) is effective for the quantitative classification of human intestinal microbiota (HIM). However, there remain various technical problems that must be overcome. This paper deals with the number of nominal partitions (NP) of the target dataset, which is a major technical problem. We used here terminal restriction fragment length polymorphism data, which was obtained from the feces of 92 Japanese men. Data comprised operational taxonomic units (OTUs) and subject smoking and drinking habits, which were effectively classified by two NP (2-NP; Yes or No). Using the same OTU data, 3-NP and 5-NP were examined here and results were obtained, focusing on the accuracies of prediction, and the reliability of the selected OTUs by DM were compared to the former 2-NP. Restriction enzymes for PCR were further affected by the accuracy and were compared with 7 enzymes. There were subjects who possess HIM at the border zones of partitions, and the greater the number of partitions, the lower the obtained DM accuracy. The application of balance nodes boosted and duplicated the data, and was able to improve accuracy. More accurate and reliable DM operations are applicable to the classification of unknown subjects for identifying various characteristics, including disease.

Effects of soil organic matter and bacterial community shift on bioremediation of diesel-contaminated soil

Bioremediation of diesel-contaminated soils were applied to investigating effects of soil organic matter (SOM) and bacterial community shift. Soil samples were artificially contaminated with diesel oil, ranging from 4000 to 12000 mg/kg soil, remediated with laboratory-scale landfarming batch applications. The SOM levels in our experiment were 2.3% (presented as SOM15), 8.9% (SOM092), and 11.8% (SOM125). Based on each of the SOM levels, bioremediation approaches of bioaugmentation (BA015, BA092, and BA125) and using indigenous microorganisms as control groups (CT015, CT092, and CT125) were tested. After about 300-day operation, total petroleum hydrocarbon (TPH) degradation efficiency became 73%, 63%, and 59% in SOM015, SOM092, and SOM125, respectively. Their 1st order degradation rates also reduced with the increase of SOM. We preliminarily concluded that SOM affected the TPH degradation efficiency and 1st degradation rates. With a logarithm transformation, the degradation pattern of SOM092 and SOM125 found to resemble each other. No apparent improvement was found from the BA batches. Our Intergenic spacer (ITS) microarray result indicated the existence of diesel-degrading bacteria in the indigenous communities. Nonmetric multidimensional scaling (MDS) based on terminal restriction fragment length polymorphism (T-RFLP) data indicated that 1) CT community became similar to BA community, once the 1st degradation stage started, impling an activation of the indigenous bacteria; 2) the degradation stage affected the community dynamics more than the SOM or the remediation approaches could do, and 3) both BA092 and BA125 located in the same cluster on the MDS plot all the time, revealing the similar communities. The similar communities might cause the comparable degradation patterns in SOM092 and SOM125. The bacteria community shift found useful in explaining the TPH degradation performance.

Cellulolytic bacterial biodiversity in long-term manure experimental sites

To study the Influence of long-term application of manure on cellulolytic bacterial diversity, 54 efficient cellulolytic bacterial cultures were isolated from two long-term manure experimental field sites of Berlin-Dahlem (established in 1923) and BadLauchstadt (established in 1902). The sequence divergence of highly conserved region of 16S rDNA was exploited by restriction analysis of PCR-amplified 16S rDNA using restriction enzyme ScrFI to assay the evolutionary relatedness of isolates. Restriction analysis identified 10 genetically diverse pattern groups comprising five groups each of bacterial and actinomycetal domain. Irrespective of manuring, a dominant pattern group (H2) was identified, containing 31.48% of total isolates. On the other hand, two site-specific pattern groups highly specific for the brown soil (H2, 9.25% of total isolates) and for black soil (J2, 11.11% of total isolates), respectively were identified. In general, the composition of cellulolytic isolates in two sites displayed differences with respect manure application and soil properties. Manure strongly influenced the abundance of cellulolytic bacterial diversity in brown soil. The terminal restriction fragment length polymorphism (T-RFLP) data revealed a distinct relationship of total bacterial diversity with long-term manure application. This influence is more prominent in nutrient poor brown soil. Based on 16S rDNA sequence analysis, isolates of the dominant as well as the specific pattern groups could be assigned to the genus Streptomyces comprising species of diverse phylogenetic affiliation. Furthermore, sequencing of 16S rDNA of isolates of five bacterial pattern groups revealed a high phylogenetic diversity among these isolates, including Streptococcus, Paenibacillus, Bacillus, Bacillus megaterium and Bacillus pumilus. Key words: Cellulolytic bacteria, 16SrDNA gene, restriction analysis, restriction fragment length polymorphism (T-RFLP), terminal restriction fragment length polymorphism (T-RFLP), phylogenetic diversity.

Molecular and pathogenic characterization of new Xanthomonas oryzae pv. oryzae strains from the coastline region of Fangchenggang city in China

Virulence assays and DNA polymorphism analyses were used to characterize 33 Xanthomonas oryzae pv. oryzae (Xoo) strains collected from the coastline region of Fangchenggang city in China. Two new pathogenic races (FXP1 and FXP2), were determined by leaf-clipping inoculation of 12 near-isogenic International Rice-Bacterial Blight (IRBB) rice lines, each containing a single resistance gene. Race FXP1 consisted of twenty-eight strains that were incompatible on IRBB5 and IRBB7, while race FXP2 included five strains that were incompatible on IRBB5 and IRBB7 and moderately virulent on IRBB8 containing the xa8 gene. Restriction fragment length polymorphism (RFLP) analysis revealed that each probe of avrXa10 and IS1112 resolved two haplotypes. In a dendrogram generated from the combined RFLP data, the 33 Xoo strains were resolved into two clusters. There was a weak correlation (r = 0.53) between race and haplotype. All of the rice cultivars planted in the coastline region of Fangchenggang city were susceptible to the representative Xoo strains tested above. However, we found that four rice cultivars used as breeding materials in the laboratory could fully resist infection by the Xoo strains, suggesting that the isolated Xoo strains could be used to detect resistant rice cultivars suitable for planting in the local rice field.

Phylogeographic Diversity of the Lower Central American Cichlid Andinoacara coeruleopunctatus (Cichlidae).

It is well appreciated that historical and ecological processes are important determinates of freshwater biogeographic assemblages. Phylogeography can potentially lend important insights into the relative contribution of historical processes in biogeography. However, the extent that phylogeography reflects historical patterns of drainage connection may depend in large part on the dispersal capability of the species. Here, we test the hypothesis that due to their relatively greater dispersal capabilities, the neotropical cichlid species Andinoacara coeruleopunctatus will display a phylogeographic pattern that differs from previously described biogeographic assemblages in this important region. Based on an analysis of 318 individuals using mtDNA ATPase 6/8 sequence and restriction fragment length polymorphism data, we found eight distinct clades that are closely associated with biogeographic patterns. The branching patterns among the clades and a Bayesian clock analysis suggest a relatively rapid colonization and diversification among drainages in the emergent Isthmus of Panama followed by the coalescing of some drainages due to historical connections. We also present evidence for extensive cross-cordillera sharing of clades in central Panama and the Canal region. Our results suggest that contemporary phylogeographic patterns and diversification in Lower Central American fishes reflect an interaction of historical drainage connections, dispersal, and demographic processes.

Tissue-specific regulatory T cells: biomarker for acute graft-vs-host disease and survival

Regulatory T cells (Tregs) are a subset of CD4(+) T cells that are characterized by the expression of CD25 and Foxp3 and are capable of suppressing alloimmune responses. We assessed whether high frequencies of circulating skin or gut tissue-specific Tregs at engraftment could predict acute graft-vs-host disease (aGVHD) incidence and survival in a cohort of hematopoietic cell transplant (HCT) recipients. Tregs were analyzed at engraftment in 74 patients receiving HCT. Treg skin-homing (CLA(+)) or gut-homing (α(4)β(7)(+)) subsets were identified by flow cytometry, and patients were divided into high CLA(+) Tregs or high α(4)β(7)(+) Tregs groups, using the 75(th) percentile of tissue-specific Treg percentages as a threshold. At day +100 post-HCT, the cumulative incidence of any stage skin or gut aGVHD was significantly lower in those patients with high CLA(+) Tregs or high α(4)β(7)(+) Tregs at engraftment, respectively (high CLA(+) Tregs, 24.0% vs low CLA(+) Tregs, 55.1%; p = 0.011 for skin aGVHD or high α(4)β(7)(+) Tregs, 47.3% vs low α(4)β(7)(+) Tregs, 74.5%; p = 0.029 for gut aGVHD). The 2-year probabilities of overall survival and nonrelapse mortality were 73.4% and 7.5% among patients with high frequencies of tissue-specific Tregs vs 49.4% and 36.1% for those with both low CLA(+) Tregs and low α(4)β(7)(+) Tregs (p = 0.039, p = 0.010). These results suggest that a threshold value forCLA(+) or α(4)β(7)(+) Tregs could be used to predict important HCT outcomes, and to direct the rationale use of tissue-specific pre-emptive therapies to decrease clinical aGVHD and improveHCT survival.

Seasonal Diversity of Planktonic Protists in Southwestern Alberta Rivers over a 1-Year Period as Revealed by Terminal Restriction Fragment Length Polymorphism and 18S rRNA Gene Library Analyses

The temporal dynamics of planktonic protists in river water have received limited attention despite their ecological significance and recent studies linking phagotrophic protists to the persistence of human-pathogenic bacteria. Using molecular-based techniques targeting the 18S rRNA gene, we studied the seasonal diversity of planktonic protists in Southwestern Alberta rivers (Oldman River Basin) over a 1-year period. Nonmetric multidimensional scaling analysis of terminal restriction fragment length polymorphism (T-RFLP) data revealed distinct shifts in protistan community profiles that corresponded to season rather than geographical location. Community structures were examined by using clone library analysis; HaeIII restriction profiles of 18S rRNA gene amplicons were used to remove prevalent solanaceous plant clones prior to sequencing. Sanger sequencing of the V1-to-V3 region of the 18S rRNA gene libraries from spring, summer, fall, and winter supported the T-RFLP results and showed marked seasonal differences in the protistan community structure. The spring library was dominated by Chloroplastidae (29.8%), Centrohelida (28.1%), and Alveolata (25.5%), while the summer and fall libraries contained primarily fungal clones (83.0% and 88.0%, respectively). Alveolata (35.6%), Euglenozoa (24.4%), Chloroplastida (15.6%), and Fungi (15.6%) dominated the winter library. These data demonstrate that planktonic protists, including protozoa, are abundant in river water in Southwestern Alberta and that conspicuous seasonal shifts occur in the community structure.

Barcoded Primers Used in Multiplex Amplicon Pyrosequencing Bias Amplification

"Barcode-tagged" PCR primers used for multiplex amplicon sequencing generate a thus-far-overlooked amplification bias that produces variable terminal restriction fragment length polymorphism (T-RFLP) and pyrosequencing data from the same environmental DNA template. We propose a simple two-step PCR approach that increases reproducibility and consistently recovers higher genetic diversity in pyrosequencing libraries.

Barcoded Primers Used in Multiplex Amplicon Pyrosequencing Bias Amplification

"Barcode-tagged" PCR primers used for multiplex amplicon sequencing generate a thus-far-overlooked amplification bias that produces variable terminal restriction fragment length polymorphism (T-RFLP) and pyrosequencing data from the same environmental DNA template. We propose a simple two-step PCR approach that increases reproducibility and consistently recovers higher genetic diversity in pyrosequencing libraries.