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We identified SNPs in the orthologs, 13 of which in 13 orthologs were present in both Mz-resistant Tr. A few Tr. This study of genetic variation and gene expression related to drug resistance in multiple T. Our dataset of 3, ddRAD-Seq SNP markers also represents a high confidence set of genetic variants, and greatly expands upon the limited number of genetic markers available for this parasite.

Using these markers, we explored the genetic diversity of T. Our study also offers a panel of biomarkers that can be used to advance personalized treatment and prevention of trichomoniasis.

Genetic variation and exchange through recombination is important to consider in parasites as it influences the spread of drug resistance and virulence genes among them. Our study confirms the existence of two genetically distinct T. Genome-wide LD, reported from analysis of a small number of genes in previous studies Conrad et al. Differences in LD decay between the two populations suggest that different degrees of inbreeding and rates of transmission might be responsible for maintenance of the two-type population structure.

Based on previous work Conrad et al. Higher mean values of Mz resistance were found in one population compared to the other A primary goal of generating the SNP markers in this study was to undertake a genome-wide association analysis to identify novel genetic indicators of Mz resistance.

Of our 72 biomarkers, several e. While we were unable to undertake a direct functional analysis of these new putative resistance mutations and genes because of the limitations of T. We also noticed that diversity in gene expression between isolates is primarily due to variation in expression of paralogous genes, as has been noted in previous studies Pal et al.

For this reason, we suggest that multiple isolates should be used as a control panel in expression studies of T. A major finding of this study was the many changes found in genes coding for proteins involved in drug reduction, efflux, and inactivation fig. First, we confirmed that down-regulation of proteins involved in Mz activation such as nitroreductase ntr and flavin reductase 1 FR1 are a primary response of T. We observed that previously identified mutations in the ntr6 gene Paulish-Miller et al.

Our observation of FR1 reduced expression in resistant isolates is in agreement with Leitsch et al. In addition, iron metabolism in T. It is possible that the reduced expression of many cytosolic and hydrogenosomal metabolic pathways that we observed, as well as reduced expression of iron-SOD and iron-sulfur flavoproteins and genes coding for proteinases, was as a consequence of that optimization.

The effect that iron-induced changes have on the transcriptome and proteome of T. As such, these changes in iron-dependent processes represent consequences rather than causes of Mz resistance, and thus may play an important role in parasite fitness. Red color represents upregulated genes, green color represents downregulated genes. Interestingly, we detected 14 BspA -like genes as down-regulated in our three resistant strains compared to the sensitive strains supplementary file S6 ,Supplementary Material online.

This is in contrast to a recent finding by Ansell and colleagues Ansell et al. Because of the possible modulation of various pathways by iron in T. We also confirmed a possible role for certain NimA genes in Mz detoxification. Transfection of the nim1 gene in E. While the nim2 paralog in our studies was upregulated in our two anaerobically resistant lab strains suggesting its importance in anerobic resistance to Mz, nim1 was downregulated in all three resistant strains indicating that its function must be furthered studied.

Finally, our study identified 14 possible membrane transporter genes that were upregulated in resistant strains, indicating that efflux pumps may play a significant role in the phenotype. Previous work surveyed a single-domain P-glycoprotein-like gene Pgp Johnson et al. While our study is the first to provide a genome-wide scan of genetic variation and transcriptomes in tens of T. For example, the PFO-ferredoxin pathway in hydrogenosomes and TrxR pathway in the cytosol have been described as potent activators of Mz and linked to anaerobic resistance Cerkasovova et al.

However, our TrxR gene expression results are the opposite to the expression pattern shown in other studies Leitsch et al. An important question arising from this work is what our analyses reveal about the evolution of Mz resistance in T. We observed mutations in genes shared between several genetically distinct laboratory-derived T. We propose that one of the first means for developing Mz resistance in trichomonads is the development of tolerance to high oxygen levels, which provides a protective mechanism against the drug e.

Other genes may play an important role in developing subsequently higher levels of resistance. For example, several genes in highly resistant isolates show the same mutation recurrently appearing accompanied by transcriptional change; e.

Many genes identified in our study have also been associated with Mz resistance in other microaerophilic parasites such as Giardia and Entamoeba Pal et al. For example, a recent transcriptomic profile of resistance in Giardia suggests also modification of nitroreductase and oxidoreductase gene families Ansell et al.

However, in these other species, it has been proposed that laboratory-evolved resistant strains of those parasites accumulate multiple changes not only beneficial for resistance but also those related to decreased fitness. As a consequence, clinical resistance in those parasites has been purported to be rare. It is tempting to speculate in the case of T. While the details of these mechanisms and their fitness costs should be further explored, we provide here a first insight into adaptation of the unicellular eukaryote T.

We used a set of T. Most of the strains were isolated from women undergoing routine examination in sexual health clinics and then adapted to growth in culture. Additional isolates included the reference lab strain G3, and three in vitro -derived Mz resistant lines and their isogenic parents, which were made Mz resistant using the method of Kulda et al. All three derived lines are stably resistant in the absence of drug pressure. We also obtained three Tritrichomonas foetus lines derived using a similar selection method [47]: Tr.

S3 in supplementary file S1 , Supplementary Material online. Each isolate was grown in culture media in 96 well plates in the presence of serial dilutions of Mz ranging from 0. The lowest concentration at which no motile parasites were observed by microscopy was recorded as the MLC. Each assay was repeated two times. EcoRI was found to cut most frequently in the unique regions of the genome fig. S4 A in supplementary file S1 , Supplementary Material online.

Sequencing adapters containing a combinatorial in-line barcode and standard Illumina multiplexing read index were used to individually barcode samples such that the identity of each isolate was kept intact. S5 in supplementary file S1 , Supplementary Material online. BAM alignment files of all sequenced isolates were merged and locally realigned to the reference assembly using GATK RealignerTargetCreator in order to minimize the number of mismatching bases across all the reads DePristo et al.

Direct SNP comparisons between parental and drug resistant derived pairs were made for each SNPs such that those SNPs found in ancestral pair must match with reference G3 strain sensitive , and must differ from derived stains with at least three reads for each SNP. Correction of population structure was performed by regressing along the first PCA axis. Filtering this set within each population gave 2, SNPs covering genomic contigs in one population, and 2, SNPs covering contigs in the other.

The decay of LD over distance was estimated using the Hill and Weir formula , and a nonlinear model was used in order to fit the data to the decay function. Three Mz-resistant T. All experiments were carried out in triplicate. A total of 0. The libraries were pooled and sequenced on the Illumina HiSeq with cycles, paired-end reads, and multiplexing.

Approximately 10—20 million reads were generated per RNA-seq library. Counts of reads for each gene were obtained using the program HTseq Anders et al. The raw read counts were normalized by rowMeans normalization function based on counts. Quality control of the data was undertaken by comparing the three replicates of each isolate. A statistical test binomialWaldTest was used to detect up- and down-log 2 fold changes in gene expression.

Significantly downregulated and upregulated expression cluster groups identified by DESeq2 R were additionally tested for their association with Mz susceptibility using chi-square and a clustering test in the heatmap3 R package Zhao et al.

Gene Ontology enrichment analyses of genes was performed using TrichDB tools and a P -value cutoff of 0. Whole genome sequences of three Tr. Sequencing produced between Candidate SNPs were then assigned to the appropriate T. Supplementary data are available at Genome Biology and Evolution online. MB designed the study, performed experiments, analyzed the data, and drafted the paper.

SW made the RNA-seq libraries. GT performed Sanger sequencing validation experiments. PS helped with ddRAD sequencing experimental design. WS provided lab lines and training in drug resistance assays. SS undertook data checking, proofreading, and writing the manuscript. JC oversaw all aspects of the research and experimental design, as well as manuscript writing. We thank members of the Carlton lab for critical reading of the manuscript and Jonathan Flowers and Swapna Uplekar for helpful discussions.

We also thank Peter Augostini at CDC for providing training and protocols for the minimum lethal concentration phenotypic assay. Kissinger Tulane University , S. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.

Genome Biol Evol. Published online Jun Martina Bradic , 1 Sally D. Warring , 1 Grace E. Tooley , 1 Paul Scheid , 1 William E. Secor , 2 Kirkwood M. Sullivan , 1 and Jane M. Carlton 1. Sally D. Grace E. William E. Kirkwood M. Steven A. Jane M. Author information Article notes Copyright and License information Disclaimer. Corresponding author. Accepted Jun For commercial re-use, please contact journals.

Associated Data Supplementary Materials Supplementary file 1. Supplementary file 2. Supplementary file 3. Supplementary file 4. Supplementary file 5. Supplementary file 6. Supplementary file 7.

Keywords: Trichomonas vaginalis , sexually transmitted infection, comparative genomics, genetic association study, antimicrobial drug resistance. Introduction Trichomonads are haploid unicellular microaerophilic members of eukaryotic phylum Parabasalia that infect a variety of vertebrates including humans, wildlife, farm animals, and pets Maritz et al.

Open in a separate window. Association Study of T. Mz Resistance Is Associated with Regulation of Genes Involved in Drug Activation, Accumulation, and Detoxification We first asked if Mz resistance is more likely to occur by drug inactivation or cell repair processes by testing if the number of downregulated genes fig. Similar Genetic Changes Associated with Mz Resistance Occur in Two Distantly Related Trichomonad Species To investigate whether there are evolutionarily common mechanisms of Mz resistance in trichomonads, we undertook whole genome shotgun sequencing and SNP detection of three strains of Tritrichomonas foetus , a trichomonad distantly related to T.

Discussion This study of genetic variation and gene expression related to drug resistance in multiple T. Metronidazole Susceptibility Phenotyping T. RNA-seq of T. Authors’ Contributions MB designed the study, performed experiments, analyzed the data, and drafted the paper. Supplementary Material Supplementary file 1 Click here for additional data file. Supplementary file 2 Click here for additional data file. Supplementary file 3 Click here for additional data file.

Supplementary file 4 Click here for additional data file. Supplementary file 5 Click here for additional data file. Supplementary file 6 Click here for additional data file. Supplementary file 7 Click here for additional data file. Acknowledgments We thank members of the Carlton lab for critical reading of the manuscript and Jonathan Flowers and Swapna Uplekar for helpful discussions. HTSeq—a Python framework to work with high-throughput sequencing data.

Bioinformatics 31 — Transcriptomics indicates active and passive metronidazole resistance mechanisms in three seminal giardia lines.

Front Microbiol. Trichomonas vaginalis cysteine proteinases: iron response in gene expression and proteolytic activity.

Biomed Res Int. Nucleic Acids Res. Bulky trichomonad genomes: encoding a Swiss Army knife. Trends Parasitol. Iron-induced changes in the proteome of Trichomonas vaginalis hydrogenosomes. PLoS One 8 :e Draft genome sequence of Tritrichomonas foetus strain K. Genome Announc. Mobile DNA 5 : Alternative 2-keto acid oxidoreductase activities in Trichomonas vaginalis. Mol Biochem Parasitol. Draft genome sequence of the sexually transmitted pathogen Trichomonas vaginalis.

Science — Metabolic differences between metronidazole resistant and susceptible strains of Tritrichomonas foetus. Mol Biochem Parasitol 11 — A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w; iso-2; iso Fly Austin 6 — Microsatellite polymorphism in the sexually transmitted human pathogen Trichomonas vaginalis indicates a genetically diverse parasite.

Extensive genetic diversity, unique population structure and evidence of genetic exchange in the sexually transmitted parasite Trichomonas vaginalis. CDC Fact Sheet. Cornelius DC, et al. Genetic characterization of Trichomonas vaginalis isolates by use of multilocus sequence typing. J Clin Microbiol. Trichomonas vaginalis associated with low birth weight and preterm delivery. Sex Transm Dis. In vitro metronidazole and tinidazole activities against metronidazole-resistant strains of Trichomonas vaginalis.

Antimicrob Agents Chemother. Challenges and persistent questions in the treatment of Trichomoniasis.

Curr Top Med Chem. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat Genet. Influence of oxygen on the fermentative metabolism of metronidazole-sensitive and resistant strains of Trichomonas vaginalis.

Using false discovery rates to benchmark SNP-callers in next-generation sequencing projects. Sci Rep. Review: dairy cattle reproductive physiology research and management—past progress and future prospects.

J Dairy Sci. Tritrichomonas—systematics of an enigmatic genus. Mol Cell Probes 26 — Conservation of transit peptide-independent protein import into the mitochondrial and hydrogenosomal matrix. We identified single nucleotide polymorphisms SNPs in the sequences in order to explore population structure and genome-wide linkage disequilibrium LD in the isolates, and provide a basis for a genome-wide association study of Mz genetic indicators. In addition, we compared laboratory-derived Mz resistant isolates with their isogenic parents to identify SNPs that could indicate Mz resistance pathways common to resistant lines.

Because there is no efficient high-throughput transfection system for large-scale assay of mutational phenotypes in T. Finally, to explore the universality of the genetic bases of Mz resistance among trichomonads, we analyzed genetic changes between a parental strain and in vitro- derived Mz resistant lines of Tr.

S1 in supplementary file S1 , Supplementary Material online. Our final set of 3, high quality SNPs comprised 1, nonsynonymous mutations, silent synonymous mutations, 10 nonsense mutations, and 1, intergenic mutations.

Population structure and linkage disequilibrium LD properties play a key role in determining success of mapping genotypes to phenotypes Mu et al. The existence of genetic subpopulations and the occurrence of LD decay over short distances are strong indicators of genetic recombination and sexual reproduction in a population Tibayrenc and Ayala ; Halkett et al. The first two principal components accounted for the highest variation within the sampled isolates and explained Similar to published findings, these two PCA axes split the global sample into two subpopulations Conrad et al.

Population structure did not correspond to the geographical origin of the isolates. Principal components analysis of T. The x -axis represents principal component one PC1 and explains Individual isolate assignment to each sub-population cluster is summarized in supplementary table S2 , Supplementary Material online.

In asexual populations, LD is maintained over long genomic distances or even over the entire genome, while genetic exchange in sexually reproducing populations constrains LD to shorter distances.

These results, together with the presence of population structure, provide strong evidence that genetic exchange occurs, or occurred sometime in the recent evolutionary past, of both T. The difference in LD decay rates indicates potential differences in recombination, population size, or phenotypic traits e. Our findings suggested 1 that population structure should be accounted for in T.

We first tested for association between T. Our analysis showed partial overlap of alleles between sensitive and resistant groups fig. We identified the SNPs that contribute the most to distinguishing sensitive from resistant phenotypes fig. Along with multiple silent synonymous and intergenic SNPs, we identified 16 nonsynonymous SNPs associated with moderate resistance in 13 genes e. A Discriminant analysis of principal components DAPC representing variation and distribution of markers between drug resistant red and drug sensitive blue isolates.

Ticks on the x -axis represent individual isolates. The inset represents the distribution of eigenvalues, with the black histogram showing all the principle components that were used in the DAPC analysis. B Loadings plot of the SNPs used for association analysis. The distribution of variances for SNP markers within and between resistant and sensitive isolates is represented as a loading value for each marker.

The loading values above the threshold represent the largest between-group variance and the smallest within-group variance and are associated with the resistant phenotype. SNP markers are indicated on the x -axis, and the loadings value for each SNP on the y -axis; a horizontal line indicates the loadings threshold.

Red dots represent 72 SNPs identified as significantly contributing to Mz resistance. While the 72 SNPs we found associated with moderate and high resistance in disparate clinical isolates represent a potentially important set of genetic markers of the resistance phenotype, such association does not demonstrate causation.

To identify SNPs that may be more strongly inferred to be consequences of adaptation to drug treatment, we exploited existing pairs of T. S2, Supplementary Material online. SNPs were identified by comparing the parental and derived line to the reference genome of the Mz-sensitive lab strain G3.

We found 39 nonsynonymous SNPs that were common to all three derived, highly resistant lines, in 36 genes fig. We also identified multiple synonymous and intergenic SNPs in common between the pairs data not shown. Venn diagram circle sizes are based on numbers of genes that have nonsynonymous SNPs.

A nonsynonymous SNP was defined as a position where the nucleotide in the ancestral strain was the same as in the reference G3 strain, but different in the derived more resistant strain.

These five genes represent novel candidates for roles in both clinical and in vitro -derived Mz resistance. A summary of the genes and their SNPs described in this section is shown in supplementary file S4 , Supplementary Material online.

We employed whole transcriptome analysis to survey changes in gene expression associated with Mz resistance. Of the 57, predicted protein-coding genes in the T. A wide range of gene expression was seen between isolates PC1, It is worth mentioning that a large proportion of gene expression diversity was apparent due to variation in paralogous genes data not shown.

A Principal components analysis of 12 isolates and their replicates based on normalized gene read counts. Replicates are indicated as shapes and different isolates as colors. B Venn diagram of number of genes in three Mz-resistant strains whose expression is upregulated in comparison with nine Mz-sensitive strains.

C Venn diagram of number of genes in the same strains whose expression is downregulated in comparison with nine Mz-sensitive strains. All the circles are sized based on marker numbers.

To identify genes whose expression is most strongly associated with Mz resistance, we compared each of the three resistant strains with the group of nine sensitive strains.

Mz resistant strains B and BM shared the highest number of pairwise down- and up-regulated genes supplementary file S6 , Supplementary Material online , as expected given their common ancestry.

We also identified nine genes with a nonsynonymous SNP that were either up- or downregulated in the experimentally derived BM isolate compared to its B parent. We first asked if Mz resistance is more likely to occur by drug inactivation or cell repair processes by testing if the number of downregulated genes fig. We established that the number of downregulated genes was significantly higher in all the comparisons, suggesting that Mz resistance predominantly occurs through processes that restrict drug activation.

Next, we tested for association between the set of downregulated and 28 upregulated genes identified as shared between the three Mz resistant strains and the Mz resistance phenotype by cluster analysis fig.

Heatmap is constructed on normalized gene read counts. Cluster analysis and chi-square analysis were performed on genes that had significant expression changes in each resistant strain in comparison to sensitive strains. Green color represents downregulated genes, red color represent upregulated genes in resistant strains. These processes are involved in electron transfer and thus likely highly relevant to the activation of Mz supplementary file S6 and table S6, Supplementary Material online.

For example, T. It has been suggested that nitrosoimidazol, the reduced form of Mz, can form covalent bonds with cellular proteins such as cytosolic malate dehydrogenase MDH and thioredoxin reductase TrxR Leitsch et al.

The superoxide dismutase SOD gene family is also involved in scavenging oxygen reactive species as part of an oxygen defense mechanism in T. Other proteins that might contribute to removal of reactive oxygen and help in establishing resistance are the thioredoxin genes Trx. The hydrogenosome is a key site of accumulation and activation of Mz. Of T. A summary of the genes and their expression differences described in this section is shown in supplementary files S4 and S6 , Supplementary Material online.

To investigate whether there are evolutionarily common mechanisms of Mz resistance in trichomonads, we undertook whole genome shotgun sequencing and SNP detection of three strains of Tritrichomonas foetus , a trichomonad distantly related to T. S3, Supplementary Material online. Details of the sequencing are shown in table 1. We identified SNPs in the orthologs, 13 of which in 13 orthologs were present in both Mz-resistant Tr.

A few Tr. This study of genetic variation and gene expression related to drug resistance in multiple T. Our dataset of 3, ddRAD-Seq SNP markers also represents a high confidence set of genetic variants, and greatly expands upon the limited number of genetic markers available for this parasite. Using these markers, we explored the genetic diversity of T. Our study also offers a panel of biomarkers that can be used to advance personalized treatment and prevention of trichomoniasis.

Genetic variation and exchange through recombination is important to consider in parasites as it influences the spread of drug resistance and virulence genes among them. Our study confirms the existence of two genetically distinct T. Genome-wide LD, reported from analysis of a small number of genes in previous studies Conrad et al. Differences in LD decay between the two populations suggest that different degrees of inbreeding and rates of transmission might be responsible for maintenance of the two-type population structure.

Based on previous work Conrad et al. Higher mean values of Mz resistance were found in one population compared to the other A primary goal of generating the SNP markers in this study was to undertake a genome-wide association analysis to identify novel genetic indicators of Mz resistance. Of our 72 biomarkers, several e.

While we were unable to undertake a direct functional analysis of these new putative resistance mutations and genes because of the limitations of T. We also noticed that diversity in gene expression between isolates is primarily due to variation in expression of paralogous genes, as has been noted in previous studies Pal et al. For this reason, we suggest that multiple isolates should be used as a control panel in expression studies of T.

A major finding of this study was the many changes found in genes coding for proteins involved in drug reduction, efflux, and inactivation fig. First, we confirmed that down-regulation of proteins involved in Mz activation such as nitroreductase ntr and flavin reductase 1 FR1 are a primary response of T. We observed that previously identified mutations in the ntr6 gene Paulish-Miller et al. Our observation of FR1 reduced expression in resistant isolates is in agreement with Leitsch et al.

In addition, iron metabolism in T. It is possible that the reduced expression of many cytosolic and hydrogenosomal metabolic pathways that we observed, as well as reduced expression of iron-SOD and iron-sulfur flavoproteins and genes coding for proteinases, was as a consequence of that optimization. The effect that iron-induced changes have on the transcriptome and proteome of T. As such, these changes in iron-dependent processes represent consequences rather than causes of Mz resistance, and thus may play an important role in parasite fitness.

Red color represents upregulated genes, green color represents downregulated genes. Interestingly, we detected 14 BspA -like genes as down-regulated in our three resistant strains compared to the sensitive strains supplementary file S6 ,Supplementary Material online. This is in contrast to a recent finding by Ansell and colleagues Ansell et al.

Because of the possible modulation of various pathways by iron in T. We also confirmed a possible role for certain NimA genes in Mz detoxification. Transfection of the nim1 gene in E. While the nim2 paralog in our studies was upregulated in our two anaerobically resistant lab strains suggesting its importance in anerobic resistance to Mz, nim1 was downregulated in all three resistant strains indicating that its function must be furthered studied.

Finally, our study identified 14 possible membrane transporter genes that were upregulated in resistant strains, indicating that efflux pumps may play a significant role in the phenotype. Previous work surveyed a single-domain P-glycoprotein-like gene Pgp Johnson et al. While our study is the first to provide a genome-wide scan of genetic variation and transcriptomes in tens of T. For example, the PFO-ferredoxin pathway in hydrogenosomes and TrxR pathway in the cytosol have been described as potent activators of Mz and linked to anaerobic resistance Cerkasovova et al.

However, our TrxR gene expression results are the opposite to the expression pattern shown in other studies Leitsch et al. An important question arising from this work is what our analyses reveal about the evolution of Mz resistance in T. We observed mutations in genes shared between several genetically distinct laboratory-derived T. We propose that one of the first means for developing Mz resistance in trichomonads is the development of tolerance to high oxygen levels, which provides a protective mechanism against the drug e.

Other genes may play an important role in developing subsequently higher levels of resistance. For example, several genes in highly resistant isolates show the same mutation recurrently appearing accompanied by transcriptional change; e. Many genes identified in our study have also been associated with Mz resistance in other microaerophilic parasites such as Giardia and Entamoeba Pal et al.

For example, a recent transcriptomic profile of resistance in Giardia suggests also modification of nitroreductase and oxidoreductase gene families Ansell et al. However, in these other species, it has been proposed that laboratory-evolved resistant strains of those parasites accumulate multiple changes not only beneficial for resistance but also those related to decreased fitness.

As a consequence, clinical resistance in those parasites has been purported to be rare. It is tempting to speculate in the case of T.

While the details of these mechanisms and their fitness costs should be further explored, we provide here a first insight into adaptation of the unicellular eukaryote T. We used a set of T. Most of the strains were isolated from women undergoing routine examination in sexual health clinics and then adapted to growth in culture. Additional isolates included the reference lab strain G3, and three in vitro -derived Mz resistant lines and their isogenic parents, which were made Mz resistant using the method of Kulda et al.

All three derived lines are stably resistant in the absence of drug pressure. We also obtained three Tritrichomonas foetus lines derived using a similar selection method [47]: Tr. S3 in supplementary file S1 , Supplementary Material online. Each isolate was grown in culture media in 96 well plates in the presence of serial dilutions of Mz ranging from 0. The lowest concentration at which no motile parasites were observed by microscopy was recorded as the MLC.

Each assay was repeated two times. EcoRI was found to cut most frequently in the unique regions of the genome fig. S4 A in supplementary file S1 , Supplementary Material online. Sequencing adapters containing a combinatorial in-line barcode and standard Illumina multiplexing read index were used to individually barcode samples such that the identity of each isolate was kept intact. S5 in supplementary file S1 , Supplementary Material online.

BAM alignment files of all sequenced isolates were merged and locally realigned to the reference assembly using GATK RealignerTargetCreator in order to minimize the number of mismatching bases across all the reads DePristo et al. Direct SNP comparisons between parental and drug resistant derived pairs were made for each SNPs such that those SNPs found in ancestral pair must match with reference G3 strain sensitive , and must differ from derived stains with at least three reads for each SNP.

Correction of population structure was performed by regressing along the first PCA axis. Filtering this set within each population gave 2, SNPs covering genomic contigs in one population, and 2, SNPs covering contigs in the other. The decay of LD over distance was estimated using the Hill and Weir formula , and a nonlinear model was used in order to fit the data to the decay function. Three Mz-resistant T. All experiments were carried out in triplicate.

A total of 0. The libraries were pooled and sequenced on the Illumina HiSeq with cycles, paired-end reads, and multiplexing. Approximately 10—20 million reads were generated per RNA-seq library. Counts of reads for each gene were obtained using the program HTseq Anders et al.

The raw read counts were normalized by rowMeans normalization function based on counts. Quality control of the data was undertaken by comparing the three replicates of each isolate. A statistical test binomialWaldTest was used to detect up- and down-log 2 fold changes in gene expression.

Significantly downregulated and upregulated expression cluster groups identified by DESeq2 R were additionally tested for their association with Mz susceptibility using chi-square and a clustering test in the heatmap3 R package Zhao et al. Gene Ontology enrichment analyses of genes was performed using TrichDB tools and a P -value cutoff of 0.

Whole genome sequences of three Tr. Sequencing produced between Candidate SNPs were then assigned to the appropriate T. Supplementary data are available at Genome Biology and Evolution online. MB designed the study, performed experiments, analyzed the data, and drafted the paper. SW made the RNA-seq libraries. GT performed Sanger sequencing validation experiments. PS helped with ddRAD sequencing experimental design. WS provided lab lines and training in drug resistance assays.

SS undertook data checking, proofreading, and writing the manuscript. JC oversaw all aspects of the research and experimental design, as well as manuscript writing. We thank members of the Carlton lab for critical reading of the manuscript and Jonathan Flowers and Swapna Uplekar for helpful discussions. We also thank Peter Augostini at CDC for providing training and protocols for the minimum lethal concentration phenotypic assay.

Kissinger Tulane University , S. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention. Genome Biol Evol. Published online Jun Martina Bradic , 1 Sally D. Warring , 1 Grace E.

Tooley , 1 Paul Scheid , 1 William E. Secor , 2 Kirkwood M. Sullivan , 1 and Jane M. Carlton 1. Sally D. Grace E. William E. Kirkwood M. Steven A. Jane M. Author information Article notes Copyright and License information Disclaimer. Corresponding author.

Accepted Jun For commercial re-use, please contact journals.

❿

 

Windows 10 1703 download iso italy vsp vision – windows 10 1703 download iso italy vsp vision.BibTeX bibliography replace.me

 
The Need for Recognition, Regularization and Regulation of Eye and Vision Care. Practices in India: An Oration/Paper for Presentation to The Palkhivala. Oscillation frequency (Hz). Industry standard ifm sensor 3D vision sensor for monitoring of the palletising process · further products from page Peter houmark-nielsen, Watch bgc season 10, Vision techniques uk ltd, Suse linux iso download, Carters lake ga, San diego zoo polar bears see snow. Draft Insurance deed will be got vetted by the Architect, Steel shall be procured by the contractor from TATA / SAIL / VSP or any other. Soraya vuceljic neymar, Aeon flux episodes game download! Happinest tour ernest prakasa, Plex windows 10, Necrosi colliquativa ascesso, Issaya afeworki.❿
 
 

 
 
A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w; iso-2; iso While our study is the first to provide a genome-wide winsows of genetic variation and transcriptomes in tens of T. PLoS One 8 :e The hydrogenosome is a key site of accumulation and activation of Mz.

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