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As the volume of sequence data from variable pathogens increases, means of analyzing, annotating and extracting specific taxa for study becomes more difficult. To meet these challenges for datasets with hundreds to thousands of taxa, 'Phylobook' was developed. Starting with a sequence alignment file, Phylobook generates and displays phylogenetic trees adjacent to highlighter plots showing the position of mutations, and allows the user to identify lineages and recombinants, annotate and export selected subsets of sequences for downstream analysis. Accurate lineage assignment, which is difficult to automate, is aided using annotations created by different clustering methods. Phylobook provides web-based display combined with automated clustering and manual editing to allow for expert assessment and correction of lineage assignments and extraction for downstream analysis.
Phylobook generates and displays phylogenetic trees adjacent to highlighter plots showing the position of mutations, allows the user to identify lineages and recombinants, annotate and export selected subsets of sequences for downstream analysis.
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Filogenia , Software , Alinhamento de Sequência/métodos , Biologia Computacional/métodos , Anotação de Sequência Molecular/métodos , Análise por ConglomeradosRESUMO
Aggregatibacter actinomycetemcomitans is a periodontal pathogen associated with periodontitis. This species exhibits substantial variations in gene content among different isolates and has different virulence potentials. This study examined the distribution of genomic islands and their insert sites among genetically diverse A. actinomycetemcomitans strains by comparative genomic analysis. The results showed that some islands, presumably more ancient, were found across all genetic clades of A. actinomycetemcomitans. In contrast, other islands were specific to individual clades or a subset of clades and may have been acquired more recently. The islands for the biogenesis of serotype-specific antigens comprise distinct genes located in different loci for serotype a and serotype b-f strains. Islands that encode the same cytolethal distending toxins appear to have been acquired via distinct mechanisms in different loci for clade b/c and for clade a/d/e/f strains. The functions of numerous other islands remain to be elucidated. JP2 strains represent a small branch within clade b, one of the five major genetic clades of A. actinomycetemcomitans. In conclusion, the complex process of genomic island acquisition, deletion, and modification is a significant force in the genetic divergence of A. actinomycetemcomitans. Assessing the genetic distinctions between JP2 and non-JP2 strains must consider the landscape of genetic variations shaped by evolution.
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BACKGROUND: Deep tissue culture specimens obtained at the time of revision shoulder arthroplasty are commonly positive for Cutibacterium. Clinical interpretation of positive cultures can be difficult. This was a multi-institutional study evaluating the accuracy of cultures for Cutibacterium using positive control (PC) and negative control (NC) samples. The relationship between time to culture positivity and strength of culture positivity was also studied. METHODS: Eleven different institutions were each sent 12 blinded samples (10 PC and 2 NC samples). The 10 PC samples included 2 sets of 5 different dilutions of a Cutibacterium isolate from a failed total shoulder arthroplasty with a probable periprosthetic infection. At each institution, the samples were handled as if they were received from the operating room. Specimen growth, time to culture positivity, and strength of culture positivity (based on semiquantitative assessment) were reported. RESULTS: A total of 110 PC samples and 22 NC samples were tested. One hundred percent of specimens at the 4 highest dilutions were positive for Cutibacterium. At the lowest dilution, 91% of samples showed positive findings. Cutibacterium grew in 14% of NC samples. Cutibacterium grew in PC samples at an average of 4.0 ± 1.3 days, and all of these samples showed growth within 7 days. The time to positivity was significantly shorter (P < .001) and the strength of positivity was significantly higher (P < .001) in true-positive cultures compared with false-positive cultures. CONCLUSIONS: This multi-institutional study suggests that different institutions may report highly consistent rates of culture positivity for revision shoulder arthroplasty samples with higher bacterial loads. In contrast, with lower bacterial loads, the results are somewhat less consistent. Clinicians should consider using a shorter time to positivity and a higher strength of positivity as adjuncts in determining whether a tissue culture sample is a true positive.
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Artroplastia do Ombro , Propionibacteriaceae , Infecções Relacionadas à Prótese , Articulação do Ombro , Humanos , Propionibacterium acnes , Infecções Relacionadas à Prótese/microbiologia , Ombro/cirurgia , Articulação do Ombro/microbiologia , Articulação do Ombro/cirurgiaRESUMO
BACKGROUND: Biofilm formation and hemolytic activity are factors that may correlate with the virulence of Cutibacterium. We sought to compare the prevalence of these potential markers of pathogenicity between Cutibacterium recovered from deep specimens obtained at the time of surgical revision for failed shoulder arthroplasty and Cutibacterium recovered from skin samples from normal subjects. METHODS: We compared 42 deep-tissue or explant isolates with 43 control Cutibacterium samples obtained from skin isolates from normal subjects. Subtyping information was available for all isolates. Biofilm-forming capacity was measured by inoculating a normalized amount of each isolate onto a 96-well plate. Planktonic bacteria were removed, the remaining adherent bacteria were stained with crystal violet, the crystal violet was re-solubilized in ethyl alcohol, and biofilm-forming capacity was quantitated by optical density (OD). Hemolytic activity was measured by plating a normalized amount of isolate onto agar plates. The area of the colony and the surrounding area of blood lysis were measured and reported as minimal, moderate, or severe hemolysis. RESULTS: Biofilm-forming capacity was significantly higher in the tissue and explant samples compared with the control skin samples (OD of 0.34 ± 0.30 for deep tissue vs. 0.20 ± 0.28 for skin, P = .002). Hemolytic activity was also significantly higher in the tissue and explant samples than in the control skin samples (P < .0001). Samples with hemolytic activity had significantly higher biofilm-forming capacity compared with samples without hemolytic activity (OD of 0.27 ± 0.29 vs. 0.12 ± 0.15, P = .015). No difference in biofilm-forming capacity or hemolytic activity was found between subtypes. CONCLUSIONS: Cutibacterium obtained from deep specimens at the time of revision shoulder arthroplasty has higher biofilm-forming capacity and hemolytic activity than Cutibacterium recovered from the skin of normal subjects. These data add support for the view that Cutibacterium harvested from deep tissues may have clinically significant virulence characteristics. The lack of correlation between these clinically relevant phenotypes and subtypes indicates that additional study is needed to identify genotypic markers that better correlate with biofilm and hemolytic activity.
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Artroplastia do Ombro , Propionibacteriaceae , Biofilmes , Hemólise , Humanos , PeleRESUMO
Many bacteria use the second messenger cyclic diguanylate (c-di-GMP) to control motility, biofilm production and virulence. Here, we identify a thermosensory diguanylate cyclase (TdcA) that modulates temperature-dependent motility, biofilm development and virulence in the opportunistic pathogen Pseudomonas aeruginosa. TdcA synthesizes c-di-GMP with catalytic rates that increase more than a hundred-fold over a ten-degree Celsius change. Analyses using protein chimeras indicate that heat-sensing is mediated by a thermosensitive Per-Arnt-SIM (PAS) domain. TdcA homologs are widespread in sequence databases, and a distantly related, heterologously expressed homolog from the Betaproteobacteria order Gallionellales also displayed thermosensitive diguanylate cyclase activity. We propose, therefore, that thermotransduction is a conserved function of c-di-GMP signaling networks, and that thermosensitive catalysis of a second messenger constitutes a mechanism for thermal sensing in bacteria.
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Proteínas de Bactérias/metabolismo , GMP Cíclico/análogos & derivados , Proteínas de Escherichia coli/metabolismo , Fósforo-Oxigênio Liases/metabolismo , Pseudomonas aeruginosa/metabolismo , Sistemas do Segundo Mensageiro/fisiologia , Transdução de Sinais/fisiologia , Algoritmos , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Cromatografia Líquida , GMP Cíclico/metabolismo , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Espectrometria de Massas , Fósforo-Oxigênio Liases/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/fisiologia , TemperaturaRESUMO
BACKGROUND: The skin of healthy shoulders is known to harbor multiple different subtypes of Cutibacterium (formerly Propionibacterium) acnes at the same time. C acnes can often be isolated from deep tissue and explant samples obtained during revision of a failed shoulder arthroplasty, presumably because the shoulder was inoculated with organisms from the patient's skin at the time of the index arthroplasty. It is possible that specific subtypes or distributions of subtypes may be associated with an increased pathogenic potential and that the skin of patients undergoing revision arthroplasty contains different distributions of the subtypes than in patients undergoing primary arthroplasty. We analyzed the subtype distribution of Cutibacterium from the skin of shoulders undergoing revision arthroplasty vs. primary arthroplasty. METHODS: Preoperative skin swabs were collected from 25 patients who underwent primary shoulder arthroplasty and 27 patients who underwent revision shoulder arthroplasty. The results of semiquantitative cultures of the skin and deep tissues were reported as specimen Cutibacterium values, and scores from all deep tissue samples were added to report the total shoulder Cutibacterium score. Single-locus sequence typing (SLST) of C acnes from the skin swabs was used to determine the subtype distribution for each patient. The percentage of each subtype for each patient was averaged in patients undergoing revision arthroplasty and then compared with that in patients undergoing primary arthroplasty. RESULTS: The C acnes subtype distribution on the skin of revision arthroplasty patients was different from that of primary shoulder arthroplasty patients, with a significantly higher percentage of SLST subtype A (36.9% vs. 16.0%, P = .0018). The distribution of SLST subtypes was similar between revision arthroplasty patients with strongly positive culture findings vs. those with weakly positive or negative culture findings. CONCLUSIONS: Significant differences in the skin Cutibacterium subtype distributions were found between shoulders undergoing revision shoulder arthroplasty and those undergoing primary shoulder arthroplasty. Future studies are needed to determine whether certain Cutibacterium subtype distributions are associated with an increased risk of arthroplasty revision.
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Artroplastia do Ombro , DNA Bacteriano/análise , Propionibacterium acnes/isolamento & purificação , Reoperação , Ombro/microbiologia , Pele/microbiologia , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Tipagem Molecular , Período Pré-Operatório , Propionibacterium acnes/genética , Análise de Sequência de DNA , Articulação do Ombro/cirurgiaRESUMO
Cutibacterium acnes is the most common bacterium associated with periprosthetic shoulder infections. Sequencing of C. acnes has been proposed as a potential rapid diagnostic tool and a method of determining subtypes associated with pathogenicity and antibiotic resistance patterns. When multiple deep samples from the same surgery are culture positive for the same species and the isolates show the same culture phenotype, it is typically assumed that these isolates are clonal. However, it is well-known that C. acnes is not clonal on the skin of most individuals. We hypothesized that the C. acnes bacteria recovered at the time of revision shoulder arthroplasty would often represent more than one subtype, and we tested this hypothesis in this work. For patients undergoing revision shoulder arthroplasty, multiple samples from the surgical field were taken. For those patients with multiple samples that were culture positive for C. acnes, isolates from each sample were subjected to full genome sequencing. Of 11 patients, 5 (45%) had different subtypes of C. acnes within the deep tissues even though the colony morphology was similar. One patient had four subtypes in the deep tissues, while four patients had two different subtypes. Up to four different subtypes of C. acnes were observed in the deep tissues of a single patient. Clonality of C. acnes isolates from deep specimens from a potential periprosthetic shoulder infection cannot be assumed. Sequence-based characterization of virulence and antibiotic resistance may require testing of multiple deep specimens.
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Artroplastia do Ombro/efeitos adversos , Genoma Bacteriano , Propionibacteriaceae/genética , Infecções Relacionadas à Prótese/microbiologia , Pele/microbiologia , Contagem de Colônia Microbiana , Humanos , Propionibacteriaceae/isolamento & purificação , Sequenciamento Completo do GenomaRESUMO
Aggregatibacter actinomycetemcomitans genome can be divided into an accessory gene pool (found in some but not all strains) and a core gene pool (found in all strains). The functions of the accessory genes (genomic islands and non-island accessory genes) are largely unknown. We hypothesize that accessory genes confer critical functions for A. actinomycetemcomitans in vivo. This study examined the expression patterns of accessory and core genes of A. actinomycetemcomitans in distinct growth conditions. We found similar expression patterns of island and non-island accessory genes, which were generally lower than the core genes in all growth conditions. The median expression levels of genomic islands were 29%-37% of the core genes in enriched medium but elevated to as high as 63% of the core genes in nutrient-limited media. Several putative virulence genes, including the cytolethal distending toxin operon, were found to be activated in nutrient-limited conditions. In conclusion, genomic islands and non-island accessory genes exhibited distinct patterns of expression from the core genes and may play a role in the survival of A. actinomycetemcomitans in nutrient-limited environments.
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A large amount of panomic data has been generated in populations for understanding causal relationships in complex biological systems. Both genetic and temporal models can be used to establish causal relationships among molecular, cellular, or phenotypical traits, but with limitations. To fully utilize high-dimension temporal and genetic data, we develop a multivariate polynomial temporal genetic association (MPTGA) approach for detecting temporal genetic loci (teQTLs) of quantitative traits monitored over time in a population and a temporal genetic causality test (TGCT) for inferring causal relationships between traits linked to the locus. We apply MPTGA and TGCT to simulated data sets and a yeast F2 population in response to rapamycin, and demonstrate increased power to detect teQTLs. We identify a teQTL hotspot locus interacting with rapamycin treatment, infer putative causal regulators of the teQTL hotspot, and experimentally validate RRD1 as the causal regulator for this teQTL hotspot.
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Algoritmos , Biologia Computacional/métodos , Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes/genética , Estudos de Associação Genética/métodos , Teorema de Bayes , Análise Multivariada , Locos de Características Quantitativas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de TempoRESUMO
We present here the draft genome sequence of Tannerella forsythia 9610, a clinical isolate obtained from a periodontitis patient. The genome is composed of 79 scaffolds with 82 contigs, for a length of 3,201,941 bp and a G+C of 47.3%.
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Aggregatibacter actinomycetemcomitans, a known pathogen causing periodontal disease and infective endocarditis, is a survivor in the periodontal pocket and blood stream; both environments contain serum as a nutrient source. To screen for unknown virulence factors associated with this microorganism, A. actinomycetemcomitans was grown in serum-based media to simulate its in vivo environment. Different strains of A. actinomycetemcomitans showed distinct growth phenotypes only in the presence of human serum, and they were grouped into high- and low-responder groups. High-responders comprised mainly serotype c strains, and showed an unusual growth phenomenon, featuring a second, rapid increase in turbidity after 9-h incubation that reached a final optical density 2- to 7-fold higher than low-responders. Upon further investigation, the second increase in turbidity was not caused by cell multiplication, but by cell death. Whole transcriptomic analysis via RNA-seq identified 35 genes that were up-regulated by human serum, but not horse serum, in high-responders but not in low-responders, including prominently an alternative sigma factor rpoE (σE). A lacZ reporter construct driven by the 132-bp rpoE promoter sequence of A. actinomycetemcomitans responded dramatically to human serum within 90 min of incubation only when the construct was carried by a high responder strain. The rpoE promoter is 100% identical among high- and low-responder strains. Proteomic investigation showed potential interactions between human serum protein, e.g. apolipoprotein A1 (ApoA1) and A. actinomycetemcomitans. The data clearly indicated a different activation process for rpoE in high- versus low-responder strains. This differential human serum-specific activation of rpoE, a putative extra-cytoplasmic stress responder and global regulator, suggests distinct in vivo adaptations among different strains of A. actinomycetemcomitans.
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Aggregatibacter actinomycetemcomitans/metabolismo , Soro/química , Fator sigma/genética , Aggregatibacter actinomycetemcomitans/genética , Aggregatibacter actinomycetemcomitans/crescimento & desenvolvimento , Apolipoproteína A-I/química , Apolipoproteína A-I/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Meios de Cultura/química , Meios de Cultura/farmacologia , Perfilação da Expressão Gênica , Genes Reporter , Humanos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Regiões Promotoras Genéticas , Proteômica , RNA Bacteriano/química , RNA Bacteriano/isolamento & purificação , RNA Bacteriano/metabolismo , Análise de Sequência de RNA , Sorogrupo , Fator sigma/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
The microbiome of plants is diverse, and like that of animals, is important for overall health and nutrient acquisition. In legumes and actinorhizal plants, a portion of essential nitrogen (N) is obtained through symbiosis with nodule-inhabiting, N2-fixing microorganisms. However, a variety of non-nodulating plant species can also thrive in natural, low-N settings. Some of these species may rely on endophytes, microorganisms that live within plants, to fix N2 gas into usable forms. Here we report the first direct evidence of N2 fixation in the early successional wild tree, Populus trichocarpa, a non-leguminous tree, from its native riparian habitat. In order to measure N2 fixation, surface-sterilized cuttings of wild poplar were assayed using both 15N2 incorporation and the commonly used acetylene reduction assay. The 15N label was incorporated at high levels in a subset of cuttings, suggesting a high level of N-fixation. Similarly, acetylene was reduced to ethylene in some samples. The microbiota of the cuttings was highly variable, both in numbers of cultured bacteria and in genetic diversity. Our results indicated that associative N2-fixation occurred within wild poplar and that a non-uniformity in the distribution of endophytic bacteria may explain the variability in N-fixation activity. These results point to the need for molecular studies to decipher the required microbial consortia and conditions for effective endophytic N2-fixation in trees.
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Microbiota , Fixação de Nitrogênio , Populus/metabolismo , Populus/microbiologiaRESUMO
⤠Propionibacterium is a slow-growing gram-positive rod that is part of the normal skin microbiome but can be found on culture of specimens from a large number of patients having revision shoulder arthroplasty performed for pain, stiffness, and component loosening. ⤠Propionibacterium infections do not present with obvious signs of infection, such as swelling, erythema, drainage, or tenderness, but rather are of the so-called stealth type, presenting with unexplained pain, stiffness, or component loosening months to years after the index arthroplasty. ⤠Not all propionibacteria are the same: certain subtypes of Propionibacterium are enriched with virulence factors that may enhance deep infection. ⤠Because propionibacteria typically reside in the pilosebaceous glands of the oily skin of the chest and back, standard surgical skin preparation solutions and even perioperative intravenous antibiotics are often inadequate at sterilizing the incision site; therefore, other prophylactic measures such as meticulous implant handling to avoid contact with dermal structures need to be considered. ⤠Recovery of Propionibacterium from the surgical wounds requires that multiple specimens for culture be taken from different areas of the shoulder to reduce sampling error, and cultures should be held for two weeks on multiple culture media. ⤠Future research efforts can be focused on reducing the risk of implant infection and point-of-care methods for identifying Propionibacterium infections.
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Artroplastia , Propionibacterium/isolamento & purificação , Articulação do Ombro/cirurgia , Infecções por Actinomycetales/diagnóstico , Infecções por Actinomycetales/imunologia , Infecções por Actinomycetales/prevenção & controle , Biofilmes/crescimento & desenvolvimento , Humanos , Reoperação , Pele/microbiologiaRESUMO
We present the draft genome ofPorphyromonas gingivalisMP4-504, a low-passage clinical isolate obtained from a periodontitis patient. The genome is composed of 92 contigs for a length of 2,373,453 bp and a G+C of 48.3%. ThetraA-Qconjugative transfer locus is genetically distinct from W83 but highly similar to ATCC 33277.
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We announce here a draft genome sequence of Veillonella parvula strain SHI-1, obtained from healthy human saliva, discovered to be active at low pH using metatranscriptomics within an in vitro oral biofilm model. The genome is composed of 7 contigs, for a total of 2,200,064 bp.
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Here, we present the draft genome sequence of Actinomyces odontolyticus subsp. actinosynbacter strain XH001, isolated from the human oral cavity. Uniquely, it was discovered as a host bacterium to the ultrasmall epibiont TM7x, which is the first cultivated member of "Candidatus Saccharibacteria" (formerly candidate phylum TM7).
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Genome editing by designer nucleases is a rapidly evolving technology utilized in a highly diverse set of research fields. Among all fields, the T7 endonuclease mismatch cleavage assay, or Surveyor assay, is the most commonly used tool to assess genomic editing by designer nucleases. This assay, while relatively easy to perform, provides only a semi-quantitative measure of mutation efficiency that lacks sensitivity and accuracy. We demonstrate a simple droplet digital PCR assay that quickly quantitates a range of indel mutations with detection as low as 0.02% mutant in a wild type background and precision (≤6%CV) and accuracy superior to either mismatch cleavage assay or clonal sequencing when compared to next-generation sequencing. The precision and simplicity of this assay will facilitate comparison of gene editing approaches and their optimization, accelerating progress in this rapidly-moving field.
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Exodesoxirribonucleases/química , HIV-1/genética , Mutação INDEL , Reação em Cadeia da Polimerase/métodos , Provírus/genética , Células HEK293 , HumanosRESUMO
OBJECTIVE: We have previously demonstrated robust control of simian/human immunodeficiency virus (SHIV1157-ipd3N4) viremia following administration of combination antiretroviral therapy (cART) in pigtailed macaques. Here, we sought to determine the safety of hematopoietic stem cell transplantation (HSCT) in cART-suppressed and unsuppressed animals. DESIGN: We compared disease progression in animals challenged with SHIV 100 days post-transplant, to controls that underwent transplant following SHIV challenge and stable cART-dependent viral suppression. METHODS: SHIV viral load, cART levels, and anti-SHIV antibodies were measured longitudinally from plasma/serum from each animal. Flow cytometry was used to assess T-cell subset frequencies in peripheral blood and the gastrointestinal tract. Deep sequencing was used to identify cART resistance mutations. RESULTS: In control animals, virus challenge induced transient peak viremia, viral set point, and durable suppression by cART. Subsequent HSCT was not associated with adverse events in these animals. Post-transplant animals were challenged during acute recovery following HSCT, and displayed sustained peak viremia and cART resistance. Although post-transplant animals had comparable plasma levels of antiretroviral drugs and showed no evidence of enhanced infection of myeloid subsets in the periphery, they exhibited a drastic reduction in virus-specific antibody production and decreased T-cell counts. CONCLUSIONS: These results suggest that virus challenge prior to complete transplant recovery impairs viral control and may promote drug resistance. These findings may also have implications for scheduled treatment interruption studies in patients on cART during post-HSCT recovery: premature scheduled treatment interruption could similarly result in lack of viral control and cART resistance.
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Antirretrovirais/farmacologia , Antirretrovirais/uso terapêutico , Farmacorresistência Viral , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Mutação de Sentido Incorreto , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Animais , Antirretrovirais/sangue , Anticorpos Antivirais/sangue , Progressão da Doença , Citometria de Fluxo , Trato Gastrointestinal/imunologia , Sequenciamento de Nucleotídeos em Larga Escala , Estudos Longitudinais , Macaca , Masculino , Plasma/química , Plasma/virologia , RNA Viral/genética , Subpopulações de Linfócitos T/imunologia , Carga ViralRESUMO
Massively parallel sequencing (MPS) technologies, such as 454-pyrosequencing, allow for the identification of variants in sequence populations at lower levels than consensus sequencing and most single-template Sanger sequencing experiments. We sought to determine if the greater depth of population sampling attainable using MPS technology would allow detection of minor variants in HIV founder virus populations very early in infection in instances where Sanger sequencing detects only a single variant. We compared single nucleotide polymorphisms (SNPs) during acute HIV-1 infection from 32 subjects using both single template Sanger and 454-pyrosequencing. Pyrosequences from a median of 2400 viral templates per subject and encompassing 40% of the HIV-1 genome, were compared to a median of five individually amplified near full-length viral genomes sequenced using Sanger technology. There was no difference in the consensus nucleotide sequences over the 3.6kb compared in 84% of the subjects infected with single founders and 33% of subjects infected with multiple founder variants: among the subjects with disagreements, mismatches were found in less than 1% of the sites evaluated (of a total of nearly 117,000 sites across all subjects). The majority of the SNPs observed only in pyrosequences were present at less than 2% of the subject's viral sequence population. These results demonstrate the utility of the Sanger approach for study of early HIV infection and provide guidance regarding the design, utility and limitations of population sequencing from variable template sources, and emphasize parameters for improving the interpretation of massively parallel sequencing data to address important questions regarding target sequence evolution.