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El <span class="elsevierStyleItalic">bootscan</span> ofrece un mismo valor de subtipado HIV<span class="elsevierStyleHsp" style=""></span>A para la secuencia Sanger y secuencia consenso NGS-20%, sin embargo se observa un subtipado CRF03_AB para secuencia consenso NGS-10%.</p>" ] ] ] "autores" => array:1 [ 0 => array:2 [ "autoresLista" => "Jose Ángel Fernández-Caballero Rico, Natalia Chueca Porcuna, Marta Álvarez Estévez, María del Mar Mosquera Gutiérrez, María Ángeles Marcos Maeso, Federico García" "autores" => array:6 [ 0 => array:2 [ "nombre" => "Jose Ángel" "apellidos" => "Fernández-Caballero Rico" ] 1 => array:2 [ "nombre" => "Natalia" "apellidos" => "Chueca Porcuna" ] 2 => array:2 [ "nombre" => "Marta" "apellidos" => "Álvarez Estévez" ] 3 => array:2 [ "nombre" => "María del Mar" "apellidos" => "Mosquera Gutiérrez" ] 4 => array:2 [ "nombre" => "María Ángeles" "apellidos" => "Marcos Maeso" ] 5 => array:2 [ "nombre" => "Federico" "apellidos" => "García" ] ] ] ] ] "idiomaDefecto" => "es" "Traduccion" => array:1 [ "en" => array:9 [ "pii" => "S2529993X1830011X" "doi" => "10.1016/j.eimce.2016.08.004" "estado" => "S300" "subdocumento" => "" "abierto" => array:3 [ "ES" => false "ES2" => false "LATM" => false ] "gratuito" => false "lecturas" => array:1 [ "total" => 0 ] "idiomaDefecto" => "en" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S2529993X1830011X?idApp=UINPBA00004N" ] ] "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S0213005X16302634?idApp=UINPBA00004N" "url" => "/0213005X/0000003600000002/v2_201802071725/S0213005X16302634/v2_201802071725/es/main.assets" ] ] "itemSiguiente" => array:18 [ "pii" => "S2529993X18300170" "issn" => "2529993X" "doi" => "10.1016/j.eimce.2016.08.005" "estado" => "S300" "fechaPublicacion" => "2018-02-01" "aid" => "1591" "copyright" => "Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica" "documento" => "article" "crossmark" => 1 "subdocumento" => "sco" "cita" => "Enferm Infecc Microbiol Clin. 2018;36:95-9" "abierto" => array:3 [ "ES" => false "ES2" => false "LATM" => false ] "gratuito" => false "lecturas" => array:2 [ "total" => 249 "formatos" => array:2 [ "HTML" => 190 "PDF" => 59 ] ] "en" => array:13 [ "idiomaDefecto" => true "cabecera" => "<span class="elsevierStyleTextfn">Brief report</span>" "titulo" => "First case of chromoblastomycosis due to <span class="elsevierStyleItalic">Phoma insulana</span>" "tienePdf" => "en" "tieneTextoCompleto" => "en" "tieneResumen" => array:2 [ 0 => "en" 1 => "es" ] "paginas" => array:1 [ 0 => array:2 [ "paginaInicial" => "95" "paginaFinal" => "99" ] ] "titulosAlternativos" => array:1 [ "es" => array:1 [ "titulo" => "Primer caso de cromoblastomicosis causado por <span class="elsevierStyleItalic">Phoma insulana</span>" ] ] "contieneResumen" => array:2 [ "en" => true "es" => true ] "contieneTextoCompleto" => array:1 [ "en" => true ] "contienePdf" => array:1 [ "en" => true ] "resumenGrafico" => array:2 [ "original" => 0 "multimedia" => array:7 [ "identificador" => "fig0010" "etiqueta" => "Fig. 2" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr2.jpeg" "Alto" => 1510 "Ancho" => 2003 "Tamanyo" => 504528 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0020" class="elsevierStyleSimplePara elsevierViewall">Culture on OA (a) and MEA (b) after 8 days of growth, showing pigmented, woolly, green olivaceous colonies. (c) Abundant pigmented chlamydoconidia (10í); insert: magnification of a chlamydoconidium (scale bar: 10<span class="elsevierStyleHsp" style=""></span>α/4m). (d) Globose picnidium (scale bar: 50<span class="elsevierStyleHsp" style=""></span>α/4m), showing three ostiolae (arrows). (e) Polymorphic cells of the picnidium wall (scale bar: 10<span class="elsevierStyleHsp" style=""></span>α/4m). 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Díaz, Yolanda Lladó" "autores" => array:12 [ 0 => array:2 [ "nombre" => "Begoña" "apellidos" => "de Dios" ] 1 => array:2 [ "nombre" => "Marcio" "apellidos" => "Borges" ] 2 => array:2 [ "nombre" => "Timothy D." "apellidos" => "Smith" ] 3 => array:2 [ "nombre" => "Alberto" "apellidos" => "del Castillo" ] 4 => array:2 [ "nombre" => "Antonia" "apellidos" => "Socias" ] 5 => array:2 [ "nombre" => "Leticia" "apellidos" => "Gutiérrez" ] 6 => array:2 [ "nombre" => "Jordi" "apellidos" => "Nicolás" ] 7 => array:2 [ "nombre" => "Bartolomé" "apellidos" => "Lladó" ] 8 => array:2 [ "nombre" => "Jose A." "apellidos" => "Roche" ] 9 => array:2 [ "nombre" => "Maria P." "apellidos" => "Díaz" ] 10 => array:2 [ "nombre" => "Yolanda" "apellidos" => "Lladó" ] 11 => array:1 [ "colaborador" => "Equipo de Sepsis" ] ] ] ] ] "idiomaDefecto" => "en" "Traduccion" => array:1 [ "es" => array:9 [ "pii" => "S0213005X16303913" "doi" => "10.1016/j.eimc.2016.11.011" "estado" => "S300" "subdocumento" => "" "abierto" => array:3 [ "ES" => false "ES2" => false "LATM" => false ] "gratuito" => false "lecturas" => array:1 [ "total" => 0 ] "idiomaDefecto" => "es" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S0213005X16303913?idApp=UINPBA00004N" ] ] "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S2529993X18300030?idApp=UINPBA00004N" "url" => "/2529993X/0000003600000002/v2_201802191141/S2529993X18300030/v2_201802191141/en/main.assets" ] "en" => array:20 [ "idiomaDefecto" => true "cabecera" => "<span class="elsevierStyleTextfn">Brief report</span>" "titulo" => "A safe an easy method for building consensus HIV sequences from 454 massively parallel sequencing data" "tieneTextoCompleto" => true "paginas" => array:1 [ 0 => array:2 [ "paginaInicial" => "91" "paginaFinal" => "94" ] ] "autores" => array:1 [ 0 => array:4 [ "autoresLista" => "Jose Ángel Fernández-Caballero Rico, Natalia Chueca Porcuna, Marta Álvarez Estévez, María del Mar Mosquera Gutiérrez, María Ángeles Marcos Maeso, Federico García" "autores" => array:6 [ 0 => array:4 [ "nombre" => "Jose Ángel" "apellidos" => "Fernández-Caballero Rico" "email" => array:1 [ 0 => "jose.angel.fernandez.caballero@gmail.com" ] "referencia" => array:2 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">a</span>" "identificador" => "aff0005" ] 1 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">*</span>" "identificador" => "cor0005" ] ] ] 1 => array:3 [ "nombre" => "Natalia" "apellidos" => "Chueca Porcuna" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">a</span>" "identificador" => "aff0005" ] ] ] 2 => array:3 [ "nombre" => "Marta" "apellidos" => "Álvarez Estévez" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">a</span>" "identificador" => "aff0005" ] ] ] 3 => array:3 [ "nombre" => "María del Mar" "apellidos" => "Mosquera Gutiérrez" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">b</span>" "identificador" => "aff0010" ] ] ] 4 => array:3 [ "nombre" => "María Ángeles" "apellidos" => "Marcos Maeso" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">b</span>" "identificador" => "aff0010" ] ] ] 5 => array:3 [ "nombre" => "Federico" "apellidos" => "García" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">a</span>" "identificador" => "aff0005" ] ] ] ] "afiliaciones" => array:2 [ 0 => array:3 [ "entidad" => "Servicio de Microbiología Clínica, Hospital Universitario San Cecilio, Complejo Hospitalario Universitario Granada e Instituto de Investigación IBS, Granada, Spain" "etiqueta" => "a" "identificador" => "aff0005" ] 1 => array:3 [ "entidad" => "Servicio de Microbiología Clínica, Centro de Diagnóstico Biomédico, Hospital Clínic, Universidad de Barcelona, Barcelona, Spain" "etiqueta" => "b" "identificador" => "aff0010" ] ] "correspondencia" => array:1 [ 0 => array:3 [ "identificador" => "cor0005" "etiqueta" => "⁎" "correspondencia" => "Corresponding author." ] ] ] ] "titulosAlternativos" => array:1 [ "es" => array:1 [ "titulo" => "Validación de un método seguro y sencillo para la elaboración de secuencias consenso del virus de la inmunodeficiencia humana a partir de los datos de secuenciación masiva 454" ] ] "resumenGrafico" => array:2 [ "original" => 0 "multimedia" => array:7 [ "identificador" => "fig0010" "etiqueta" => "Fig. 2" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr2.jpeg" "Alto" => 1265 "Ancho" => 3000 "Tamanyo" => 194961 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0050" class="elsevierStyleSimplePara elsevierViewall">Bootscan of the Sanger sequence (A), NGS-10% consensus sequence (B) and NGS-20% consensus sequence (C), using the REGA HIV-1 Subtyping Tool v. 3.0. In the Bootscan, the HIV A subtype value is the same in the Sanger and NGS-20% consensus sequence, however, a CRF03_AB subtype can be seen in the NGS-10% consensus sequence.</p>" ] ] ] "textoCompleto" => "<span class="elsevierStyleSections"><span id="sec0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0065">Introduction</span><p id="par0005" class="elsevierStylePara elsevierViewall">Many clinical microbiology departments have started to use next generation sequencing (NGS) techniques to study antiretroviral resistance in HIV patients. Several studies<a class="elsevierStyleCrossRef" href="#bib0080"><span class="elsevierStyleSup">1</span></a> have shown the capacity of NGS to detect low abundance viral variants by lowering sensitivity below the 1% threshold (minority variants). This can greatly improve therapeutic decision-making and prevent therapeutic failure.<a class="elsevierStyleCrossRefs" href="#bib0085"><span class="elsevierStyleSup">2,3</span></a> In Spain, the use of NGS for the detection of antiretroviral resistance has been prompted to a certain extent by the decision of some suppliers to discontinue supply of Sanger sequencing systems.</p><p id="par0010" class="elsevierStylePara elsevierViewall">Protease (PR) and reverse transcriptase (RT) sequences obtained from drug resistance tests are often used by researchers in molecular epidemiology studies, using phylogenetic and phylodynamic techniques.<a class="elsevierStyleCrossRef" href="#bib0095"><span class="elsevierStyleSup">4</span></a> With the introduction of NGS techniques, this information can be lost due the complexity of processing and storing the sequences for this type of study; in addition, incorrect processing of NGS sequences can yield incorrect results. Special training in sequence processing and high-performance computers capable of processing the vast amounts of data generated are needed in order to use NGS sequences in phylogenetic studies.<a class="elsevierStyleCrossRef" href="#bib0100"><span class="elsevierStyleSup">5</span></a> An alternative, in the case of molecular epidemiology studies, is to generate a single consensus NGS sequence; however, some studies do not clearly describe, or omit altogether, the method used to generate the sequence.<a class="elsevierStyleCrossRef" href="#bib0105"><span class="elsevierStyleSup">6</span></a> In addition, we cannot know for certain the extent to which this consensus NGS represents the sequence obtained by Sanger methods and the effect of the thresholds used to generate this consensus.</p><p id="par0015" class="elsevierStylePara elsevierViewall">The objective of this study was to determine the best threshold for obtaining a consensus NGS sequence that is representative of the Sanger sequence and can be used in molecular epidemiology studies.</p></span><span id="sec0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0070">Methods</span><p id="par0020" class="elsevierStylePara elsevierViewall">For the purpose of our study, we used sequences from 62 treatment naïve patients, newly diagnosed with HIV between 2014 and 2015 and referred for antiretroviral resistance studies. Sanger sequences were obtained using <span class="elsevierStyleItalic">Trugene</span><span class="elsevierStyleSup"><span class="elsevierStyleItalic">®</span></span> HIV-1 Genotyping (Siemens – [NAD]). For NGS, we used the GSV-type HIV-1 Drug Resistance Primer kit (Roche) for 454 GS-Junior, based on the same RNA. The NGS consensus sequences are generated using the Mesquite v. 2.75 software, setting thresholds at 10%, 15% and 20%. Prior to the use of Mesquite, the sequences were filtered using Usearch fastq_filter commands, according to the desired amplicon length and sequence quality (>30 Q). Mesquite<a class="elsevierStyleCrossRef" href="#bib0110"><span class="elsevierStyleSup">7</span></a> is a programme with an intuitive icon- and tab-based interface. Before processing data on Mesquite, the filtered sequences must be exported in <span class="elsevierStyleItalic">pfam</span> format and the threshold for the consensus sequence must be set before exporting in <span class="elsevierStyleItalic">fasta</span> format. Following this, the pol gene sequences (PR 4–99; RT 38–247) are processed, aligned by MUSCLE in MEGA 6.06 and phylogenetic trees are generated using the maximum likelihood method, using the General Time Reversible (GTR) model to calculate evolutionary distances, with a gamma distribution equivalent to 1.89 obtained with FindModel DNA, and using bootstrap resampling with 1000 replicas to build the consensus phylogenetic trees. To define a relationship between sequences, only the branches from clusters with a bootstrap value greater than 75% are taken into consideration. Finally, the trees are processed in FigTree v. 1.4.2. The viral subtype analysis was performed using the REGA HIV-1 Subtyping Tool v. 3.0.</p></span><span id="sec0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0075">Results</span><p id="par0025" class="elsevierStylePara elsevierViewall">Our study included 62 treatment naïve HIV-1 patients with a median age of 37 years (IQR 30–45), viral load (median) of 74,900<span class="elsevierStyleHsp" style=""></span>cp/ml (IQR 20,715–176,250), CD4 count (median) of 430<span class="elsevierStyleHsp" style=""></span>cells/ml (IQR 48.5–567.78); 82% were men.</p><p id="par0030" class="elsevierStylePara elsevierViewall">To evaluate concordance between the NGS consensus sequences with different thresholds and the original Sanger sequence, we analysed the number of sequences with inter-related pairs and the bootstrap values between the pairs. Using a 10% threshold, we observed that the Sanger sequence pairs were correlated with NGS from the same sample in only in 17/62 (27%) patients and in these, the median bootstrap value was 88% (IQR 83.5–95.5). Increasing the threshold to 15%, the sequence pairs were correlated in 36/62 (58%) patients, with a median bootstrap value of 94% (IQR 85.5–98). At 20%, sequences were correlated in 61/62 patients, with a median bootstrap value of 99% (IQR 98–100) (<a class="elsevierStyleCrossRef" href="#fig0005">Fig. 1</a>). A large number of differences between base pairs were detected in cases where the NGS sequence was not correlated with the Sanger sequence.</p><elsevierMultimedia ident="fig0005"></elsevierMultimedia><p id="par0035" class="elsevierStylePara elsevierViewall">Most patients were infected by subtype B (77.4%), followed by CRF02_AG (12.9%), A and F (3.2%) and C and G (1.6%). Using a consensus NGS threshold of 10% and 15%, we observed 2 cases that differed from the Sanger subtype: one case of subtype B-NGS and A1-Sanger and another one from subtype CRF03_AB-NGS and A1-Sanger. These differences disappeared when using the 20% threshold consensus NGS sequences (<a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a>). <a class="elsevierStyleCrossRef" href="#fig0010">Fig. 2</a> shows the bootscan plot of the subtype in the second discordant sample.</p><elsevierMultimedia ident="tbl0005"></elsevierMultimedia><elsevierMultimedia ident="fig0010"></elsevierMultimedia></span><span id="sec0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0080">Discussion</span><p id="par0040" class="elsevierStylePara elsevierViewall">Phylogenetic studies of HIV,<a class="elsevierStyleCrossRefs" href="#bib0115"><span class="elsevierStyleSup">8,9</span></a> specifically studies into relatedness, transmission dynamics of the HIV epidemic and molecular pol gene sequence subtyping, have been used for various purposes, among them to understand HIV transmission networks and clusters and the migratory networks of the different subtypes. Most studies published at the international<a class="elsevierStyleCrossRef" href="#bib0125"><span class="elsevierStyleSup">10</span></a> and local level<a class="elsevierStyleCrossRefs" href="#bib0130"><span class="elsevierStyleSup">11,12</span></a> have used Sanger sequencing. Some of these studies have used all the information obtained through NGS<a class="elsevierStyleCrossRef" href="#bib0140"><span class="elsevierStyleSup">13</span></a> but investigators generally try to generate a single consensus sequence, usually using complex computer commands. The transition from Sanger sequencing to NGS for pol gene analysis of resistance mutations has changed the type of sequences used in clinical microbiology departments and can, paradoxically, stand in the way of local HIV molecular epidemiology studies in Spain. In this study, we propose using Mesquite, an intuitive, user-friendly, software without the need for commands that simplifies the process of obtaining a consensus sequence from NGS-generated sequences. We have shown that using a threshold of 20% to generate this consensus yields safe, reliable information that is identical to that obtained using Sanger sequencing. This information can be used in molecular epidemiology studies and solves the current problems arising from the use of NGS sequences.</p><p id="par0045" class="elsevierStylePara elsevierViewall">As we have shown in this study, the threshold must be raised to 20% in order to safely use consensus sequences that are representative of Sanger sequences in HIV molecular epidemiology studies. This was the only threshold that yields a median bootstrap value of 99% (IQR 98–100) between the NGS consensus sequences and the Sanger sequence. Using thresholds of 10% or 15%, the percentage of correlated NGS-Sanger sequence pairs and the median bootstrap values are too low. Because of this variability, errors are made even in the determination of the viral subtype; this was corrected with the 20% consensus. These discrepancies are due to the multitude of ambiguous base pairs generated with the 10% and 15% thresholds, which made it impossible to correctly determine the viral subtype.</p><p id="par0050" class="elsevierStylePara elsevierViewall">An important part of molecular epidemiology studies is sequence alignment, in which homologous positions are aligned based on the true evolutionary history of the sequences.<a class="elsevierStyleCrossRef" href="#bib0145"><span class="elsevierStyleSup">14</span></a> The problem with using 10% and 15% NGS consensus sequences in such studies lies in the presence of ambiguous regions, which present substantial uncertainty and detract from the robustness of both phylogenetic<a class="elsevierStyleCrossRef" href="#bib0150"><span class="elsevierStyleSup">15</span></a> and subtype statistical analyses, yielding unexpected results.</p><p id="par0055" class="elsevierStylePara elsevierViewall">It is important to point out that the methodology presented here is appropriate for obtaining consensus sequences for use in HIV molecular epidemiology studies but not for the analysis of resistance mutations. The greater sensitivity of NGS to detect minority variants and its clinical utility have been studied in detail.<a class="elsevierStyleCrossRefs" href="#bib0080"><span class="elsevierStyleSup">1–4</span></a> NGS provides very valuable information on the relative proportion of a mutation with respect to the total circulating viruses. This information would be lost when obtaining the consensus sequence.</p><p id="par0060" class="elsevierStylePara elsevierViewall">In summary, we present a methodology for generating consensus sequences that are representative of the Sanger sequence for use in molecular epidemiology studies by processing sequences with a threshold of at least 20%.</p></span><span id="sec0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0085">Funding</span><p id="par0065" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleGrantSponsor" id="gs1">Health Research Fund</span> (<span class="elsevierStyleGrantNumber" refid="gs1">PI12/01053</span>, <span class="elsevierStyleGrantNumber" refid="gs1">PI15/00713</span>), RD12/0017/006 (National R+D+I Programme, European Regional Development Fund—<span class="elsevierStyleGrantSponsor" id="gs2">ERDF</span>). Federico García is part of a Research Intensification Programme from the Andalusian Health Service. José Ángel Fernández-Caballero has an RD12/0017/006 contract.</p></span><span id="sec0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0090">Conflicts of interest</span><p id="par0070" class="elsevierStylePara elsevierViewall">The authors declare that they have no conflicts of interest.</p></span></span>" "textoCompletoSecciones" => array:1 [ "secciones" => array:11 [ 0 => array:3 [ "identificador" => "xres986160" "titulo" => "Abstract" "secciones" => array:4 [ 0 => array:2 [ "identificador" => "abst0005" "titulo" => "Objective" ] 1 => array:2 [ "identificador" => "abst0010" "titulo" => "Material and methods" ] 2 => array:2 [ "identificador" => "abst0015" "titulo" => "Results" ] 3 => array:2 [ "identificador" => "abst0020" "titulo" => "Conclusion" ] ] ] 1 => array:2 [ "identificador" => "xpalclavsec953791" "titulo" => "Keywords" ] 2 => array:3 [ "identificador" => "xres986161" "titulo" => "Resumen" "secciones" => array:4 [ 0 => array:2 [ "identificador" => "abst0025" "titulo" => "Objetivo" ] 1 => array:2 [ "identificador" => "abst0030" "titulo" => "Material y métodos" ] 2 => array:2 [ "identificador" => "abst0035" "titulo" => "Resultados" ] 3 => array:2 [ "identificador" => "abst0040" "titulo" => "Conclusión" ] ] ] 3 => array:2 [ "identificador" => "xpalclavsec953792" "titulo" => "Palabras clave" ] 4 => array:2 [ "identificador" => "sec0005" "titulo" => "Introduction" ] 5 => array:2 [ "identificador" => "sec0010" "titulo" => "Methods" ] 6 => array:2 [ "identificador" => "sec0015" "titulo" => "Results" ] 7 => array:2 [ "identificador" => "sec0020" "titulo" => "Discussion" ] 8 => array:2 [ "identificador" => "sec0025" "titulo" => "Funding" ] 9 => array:2 [ "identificador" => "sec0030" "titulo" => "Conflicts of interest" ] 10 => array:1 [ "titulo" => "References" ] ] ] "pdfFichero" => "main.pdf" "tienePdf" => true "fechaRecibido" => "2016-02-29" "fechaAceptado" => "2016-08-29" "PalabrasClave" => array:2 [ "en" => array:1 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Keywords" "identificador" => "xpalclavsec953791" "palabras" => array:4 [ 0 => "Human immunodeficiency virus" 1 => "Phylogeny" 2 => "Next generation sequencing" 3 => "Thresholds" ] ] ] "es" => array:1 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Palabras clave" "identificador" => "xpalclavsec953792" "palabras" => array:4 [ 0 => "Virus de la inmunodeficiencia humana" 1 => "Filogenia" 2 => "<span class="elsevierStyleItalic">Next generation sequencing</span>" 3 => "Umbrales" ] ] ] ] "tieneResumen" => true "resumen" => array:2 [ "en" => array:3 [ "titulo" => "Abstract" "resumen" => "<span id="abst0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0010">Objective</span><p id="spar0005" class="elsevierStyleSimplePara elsevierViewall">To show how to generate a consensus sequence from the information of massive parallel sequences data obtained from routine HIV anti-retroviral resistance studies, and that may be suitable for molecular epidemiology studies.</p></span> <span id="abst0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0015">Material and methods</span><p id="spar0010" class="elsevierStyleSimplePara elsevierViewall">Paired Sanger (Trugene-Siemens) and next-generation sequencing (NGS) (454 GSJunior-Roche) HIV RT and protease sequences from 62 patients were studied. NGS consensus sequences were generated using Mesquite, using 10%, 15%, and 20% thresholds. Molecular evolutionary genetics analysis (MEGA) was used for phylogenetic studies.</p></span> <span id="abst0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0020">Results</span><p id="spar0015" class="elsevierStyleSimplePara elsevierViewall">At a 10% threshold, NGS-Sanger sequences from 17/62 patients were phylogenetically related, with a median bootstrap-value of 88% (IQR 83.5–95.5). Association increased to 36/62 sequences, median bootstrap 94% (IQR 85.5–98), using a 15% threshold. Maximum association was at the 20% threshold, with 61/62 sequences associated, and a median bootstrap value of 99% (IQR 98–100).</p></span> <span id="abst0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0025">Conclusion</span><p id="spar0020" class="elsevierStyleSimplePara elsevierViewall">A safe method is presented to generate consensus sequences from HIV-NGS data at 20% threshold, which will prove useful for molecular epidemiological studies.</p></span>" "secciones" => array:4 [ 0 => array:2 [ "identificador" => "abst0005" "titulo" => "Objective" ] 1 => array:2 [ "identificador" => "abst0010" "titulo" => "Material and methods" ] 2 => array:2 [ "identificador" => "abst0015" "titulo" => "Results" ] 3 => array:2 [ "identificador" => "abst0020" "titulo" => "Conclusion" ] ] ] "es" => array:3 [ "titulo" => "Resumen" "resumen" => "<span id="abst0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0035">Objetivo</span><p id="spar0025" class="elsevierStyleSimplePara elsevierViewall">Generar una secuencia consenso a partir de los datos de secuenciación masiva obtenidos en estudios de resistencias a antiretrovirales, que sea representativa de la secuencia Sanger y que sirva para estudios de epidemiología molecular.</p></span> <span id="abst0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0040">Material y métodos</span><p id="spar0030" class="elsevierStyleSimplePara elsevierViewall">En 62 pacientes se obtuvo la secuencia de transcriptasa reversa-proteasa, mediante Sanger (Trugene-Siemens), y NGS (454GSJunior-Roche). Las secuencias consenso NGS se generaron con Mesquite, seleccionando umbrales 10%, 15% y 20%. Para el estudio filogenético se empleó MEGA.</p></span> <span id="abst0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0045">Resultados</span><p id="spar0035" class="elsevierStyleSimplePara elsevierViewall">Utilizando el umbral 10%, 17/62 pacientes presentaron secuencias pareadas NGS-Sanger, con una mediana de <span class="elsevierStyleItalic">bootstrap</span> del 88% (IQR 83,5-95,5). La asociación aumenta a 36/62 pacientes y el <span class="elsevierStyleItalic">bootstrap</span>, a 94% (IQR 85,5-98), y alcanza el máximo al 20% en 61/62 pacientes, <span class="elsevierStyleItalic">bootstrap</span> 99% (IQR 98-100).</p></span> <span id="abst0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0050">Conclusión</span><p id="spar0040" class="elsevierStyleSimplePara elsevierViewall">Mostramos un método seguro para generar secuencias consenso NGS para su uso en estudios de epidemiología molecular procesadas con umbral 20%, de fácil uso y aplicación en los servicios de microbiología clínica.</p></span>" "secciones" => array:4 [ 0 => array:2 [ "identificador" => "abst0025" "titulo" => "Objetivo" ] 1 => array:2 [ "identificador" => "abst0030" "titulo" => "Material y métodos" ] 2 => array:2 [ "identificador" => "abst0035" "titulo" => "Resultados" ] 3 => array:2 [ "identificador" => "abst0040" "titulo" => "Conclusión" ] ] ] ] "NotaPie" => array:1 [ 0 => array:2 [ "etiqueta" => "☆" "nota" => "<p class="elsevierStyleNotepara" id="npar0005">Please cite this article as: Fernández-Caballero Rico JÁ, Chueca Porcuna N, Álvarez Estévez M, Mosquera Gutiérrez MM, Marcos Maeso MÁ, García F. Validación de un método seguro y sencillo para la elaboración de secuencias consenso del virus de la inmunodeficiencia humana a partir de los datos de secuenciación masiva 454. Enferm Infecc Microbiol Clin. 2018;36:91–94.</p>" ] ] "multimedia" => array:3 [ 0 => array:7 [ "identificador" => "fig0005" "etiqueta" => "Fig. 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 1718 "Ancho" => 3057 "Tamanyo" => 246008 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0045" class="elsevierStyleSimplePara elsevierViewall">Representation of phylogenetic trees in FigTree v. 1.4.2, formed by the Sanger and NGS sequences at different thresholds: (A) NGS-10%; (B) NGS-15%, and (C) NGS-20%. The bootstrap values are shown according to colour. A good association is 70% and over.</p>" ] ] 1 => array:7 [ "identificador" => "fig0010" "etiqueta" => "Fig. 2" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr2.jpeg" "Alto" => 1265 "Ancho" => 3000 "Tamanyo" => 194961 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0050" class="elsevierStyleSimplePara elsevierViewall">Bootscan of the Sanger sequence (A), NGS-10% consensus sequence (B) and NGS-20% consensus sequence (C), using the REGA HIV-1 Subtyping Tool v. 3.0. In the Bootscan, the HIV A subtype value is the same in the Sanger and NGS-20% consensus sequence, however, a CRF03_AB subtype can be seen in the NGS-10% consensus sequence.</p>" ] ] 2 => array:8 [ "identificador" => "tbl0005" "etiqueta" => "Table 1" "tipo" => "MULTIMEDIATABLA" "mostrarFloat" => true "mostrarDisplay" => false "detalles" => array:1 [ 0 => array:3 [ "identificador" => "at1" "detalle" => "Table " "rol" => "short" ] ] "tabla" => array:1 [ "tablatextoimagen" => array:1 [ 0 => array:2 [ "tabla" => array:1 [ 0 => """ <table border="0" frame="\n \t\t\t\t\tvoid\n \t\t\t\t" class=""><thead title="thead"><tr title="table-row"><th class="td" title="table-head " align="" valign="top" scope="col"> \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " colspan="7" align="center" valign="top" scope="col" style="border-bottom: 2px solid black">HIV subtype</th></tr><tr title="table-row"><th class="td" title="table-head " align="" valign="top" scope="col" style="border-bottom: 2px solid black"> \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">B \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">G \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">F \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">C \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">A \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">crf02_AG \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">crf03_AB \t\t\t\t\t\t\n \t\t\t\t</th></tr></thead><tbody title="tbody"><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">Sanger \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">48 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">1 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">2 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">1 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">2 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">8 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">0 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">NGS-10% \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">49 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">1 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">2 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">1 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">0 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">8 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">1 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">NGS-15% \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">49 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">1 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">2 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">1 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">0 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">8 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">1 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">NGS-20% \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">48 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">1 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">2 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">1 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">2 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">8 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">0 \t\t\t\t\t\t\n \t\t\t\t</td></tr></tbody></table> """ ] "imagenFichero" => array:1 [ 0 => "xTab1673992.png" ] ] ] ] "descripcion" => array:1 [ "en" => "<p id="spar0055" class="elsevierStyleSimplePara elsevierViewall">Distribution of HIV viral subtypes according to the Sanger and NGS consensus sequences at the different thresholds, using the REGA HIV-1 Subtyping Tool v. 3.0.</p>" ] ] ] "bibliografia" => array:2 [ "titulo" => "References" "seccion" => array:1 [ 0 => array:2 [ "identificador" => "bibs0015" "bibliografiaReferencia" => array:15 [ 0 => array:3 [ "identificador" => "bib0080" "etiqueta" => "1" "referencia" => array:1 [ 0 => array:2 [ "contribucion" => array:1 [ 0 => array:2 [ "titulo" => "A comparison of parallel pyrosequencing and Sanger clone-based sequencing and its impact on the characterization of the genetic diversity of HIV-1" "autores" => array:1 [ 0 => array:2 [ "etal" => true "autores" => array:6 [ 0 => "B. 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2024 October | 15 | 0 | 15 |
2024 September | 30 | 6 | 36 |
2024 August | 26 | 2 | 28 |
2024 July | 15 | 2 | 17 |
2024 June | 14 | 4 | 18 |
2024 May | 12 | 2 | 14 |
2024 April | 30 | 2 | 32 |
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2024 February | 24 | 3 | 27 |
2024 January | 22 | 5 | 27 |
2023 December | 33 | 5 | 38 |
2023 November | 30 | 4 | 34 |
2023 October | 33 | 2 | 35 |
2023 September | 32 | 2 | 34 |
2023 August | 12 | 6 | 18 |
2023 July | 15 | 1 | 16 |
2023 June | 8 | 1 | 9 |
2023 May | 27 | 6 | 33 |
2023 April | 18 | 3 | 21 |
2023 March | 35 | 1 | 36 |
2023 February | 19 | 2 | 21 |
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2022 December | 19 | 7 | 26 |
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2022 August | 32 | 11 | 43 |
2022 July | 16 | 8 | 24 |
2022 June | 12 | 6 | 18 |
2022 May | 12 | 7 | 19 |
2022 April | 7 | 11 | 18 |
2022 March | 12 | 7 | 19 |
2022 February | 26 | 4 | 30 |
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2021 November | 14 | 12 | 26 |
2021 October | 22 | 7 | 29 |
2021 September | 12 | 9 | 21 |
2021 August | 30 | 8 | 38 |
2021 July | 20 | 12 | 32 |
2021 June | 16 | 10 | 26 |
2021 May | 21 | 9 | 30 |
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2021 March | 17 | 7 | 24 |
2021 February | 13 | 5 | 18 |
2021 January | 13 | 9 | 22 |
2020 December | 8 | 7 | 15 |
2020 November | 16 | 5 | 21 |
2020 October | 13 | 4 | 17 |
2020 September | 14 | 7 | 21 |
2020 August | 21 | 6 | 27 |
2020 July | 20 | 3 | 23 |
2020 June | 5 | 6 | 11 |
2020 May | 14 | 9 | 23 |
2020 April | 12 | 1 | 13 |
2020 March | 16 | 2 | 18 |
2020 February | 11 | 5 | 16 |
2020 January | 19 | 1 | 20 |
2019 December | 16 | 9 | 25 |
2019 November | 13 | 8 | 21 |
2019 October | 14 | 6 | 20 |
2019 September | 13 | 6 | 19 |
2019 August | 11 | 1 | 12 |
2019 July | 12 | 6 | 18 |
2019 June | 33 | 14 | 47 |
2019 May | 93 | 23 | 116 |
2019 April | 26 | 4 | 30 |
2019 March | 0 | 1 | 1 |
2019 February | 6 | 3 | 9 |
2019 January | 4 | 2 | 6 |
2018 December | 3 | 1 | 4 |
2018 November | 5 | 0 | 5 |
2018 October | 7 | 14 | 21 |
2018 September | 4 | 1 | 5 |
2018 August | 5 | 0 | 5 |
2018 February | 4 | 0 | 4 |