EFECTO DEL FILTRADO DE SECUENCIAS EN EL ENSAMBLADO DEL GENOMA DE Bacillus altitudinis 19RS3 AISLADO DE Ilex paraguariensis / Effect of sequence filtering on the assembly of the Bacillus altitudinis 19RS3 genome isolated from Ilex paraguariensis

RESUMEN El filtrado de secuencias es un paso esencial sin importar el tipo de tecnología aplicada para la secuenciación de un genoma, en el cual las lecturas de baja calidad o una parte son eliminadas. En un ensamblado la construcción de un genoma se realiza a partir de la unión de lecturas cortas en cóntigos. Algunos ensambladores miden la relación que existe entre secuencias de una longitud fija (k-mer) que puede verse afectada por la presencia de secuencias de baja calidad. Un enfoque común para evaluar los ensamblados se basa en el análisis del número de cóntigos, la longitud del cóntigo más largo y el valor del N50, definido como la longitud del cóntigo que representa el 50 % de la longitud del conjunto. En este contexto, el presente estudio tuvo como objetivo evaluar el efecto del uso de lecturas crudas y filtradas en los valores de los parámetros de calidad obtenidos en el ensamblado del genoma de Bacillus altitudinis 19RS3 aislada de Ilex paraguariensis. Se realizó el análisis de calidad de ambos archivos de partida con el software FastqC y se filtraron las lecturas con el softwareTrimmomatic. Para el ensamblado se utilizó el software SPAdes y para su evaluación la herramienta QUAST. El mejor ensamblado para B. altitudinis 19RS3 se obtuvo a partir de las lecturas filtradas con el valor de k-mer 79, que generó 16 cóntigos mayores a 500 pb con un N50 de 931 914 pb y el cóntigo más largo de 966 271 pb.

ABSTRACT Sequence filtering is an essential step regardless of the type of technology applied for sequencing a genome, in which low-quality readings or a portion are eliminated. In an assembly, the construction of a genome is carried out from the union of short reads in contigs. Some assemblers measure the relationship between sequences of a fixed length (k-mer) that can be affected by the presence of low-quality sequences. A common approach to evaluating assemblies is based on the analysis of the number of contigs, the length of the longest contig, and the value of N50 defined as the length of the contig representing 50 % of the length of the assembly. In this context, the objective of this study was to evaluate the effect of the use of crude and filtered reads on the values of the quality parameters obtained from the genome assembly of Bacillus altituidinis 19RS3 isolated from Ilex paraguariensis. The quality analysis of both starting files was performed with the FastqC software and the readings were filtered with the Trimmomatic software. The SPAdes software was used for the assembly and the QUAST tool for its evaluation. The best assembly for B. altitudinis 19RS3 was obtained from the filtered readings with the value of k-mer 79, which generated 16 contigs greater than 500 bp with a N50 of 931 914 bp and the longest contig of 966 271 bp.

De novo genome assembly of Bacillus altitudinis 19RS3 and Bacillus altitudinis T5S-T4, two plant growth-promoting bacteria isolated from Ilex paraguariensis St. Hil. (yerba mate).

Plant growth-promoting bacteria (PGPB) are a heterogeneous group of bacteria that can exert beneficial effects on plant growth directly or indirectly by different mechanisms. PGPB-based inoculant formulation has been used to replace chemical fertilizers and pesticides. In our previous studies, two endophytic endospore-forming bacteria identified as Bacillus altitudinis were isolated from roots of Ilex paraguariensis St. Hil. seedlings and selected for their plant growth-promoting (PGP) properties shown in vitro and in vivo. The purposes of this work were to assemble the genomes of B. altitudinis 19RS3 and T5S-T4, using different assemblers available for Windows and Linux and to select the best assembly for each strain. Both genomes were also automatically annotated to detect PGP genes and compare sequences with other genomes reported. Library construction and draft genome sequencing were performed by Macrogen services. Raw reads were filtered using the Trimmomatic tool. Genomes were assembled using SPAdes, ABySS, Velvet, and SOAPdenovo2 assemblers for Linux, and Geneious and CLC Genomics Workbench assemblers for Windows. Assembly evaluation was done by the QUAST tool. The parameters evaluated were the number of contigs ≥ 500 bp and ≥ 1000 bp, the length of the longest contig, and the N50 value. For genome annotation PROKKA, RAST, and KAAS tools were used. The best assembly for both genomes was obtained using Velvet. The B. altitudinis 19RS3 genome was assembled into 15 contigs with an N50 value of 1,943,801 bp. The B. altitudinis T5S-T4 genome was assembled into 24 contigs with an N50 of 344,151 bp. Both genomes comprise several genes related to PGP mechanisms, such as those for nitrogen fixation, iron metabolism, phosphate metabolism, and auxin biosynthesis. The results obtained offer the basis for a better understanding of B. altitudinis 19RS3 and T5S-T4 and make them promissory for bioinoculant development.

Streptococcus agalactiae,medios de conservación accesibles a laboratorios de diagnóstico de baja y mediana complejidad / Streptococcus agalactiae and accessible conservation means for diagnostic laboratories of low and medium complexity

Introducción: Si bien Streptococcus agalactiae es comensal del tracto gastrointestinal y genitourinario, es la principal causa de enfermedades invasivas y de mortalidad en niños recién nacidos. La infección puede adquirirse a través de la aspiración de líquido amniótico infectado o durante el paso por el canal de parto. Objetivos: comparar la utilidad de diversos métodos de conservación de cepas de Streptococcus agalactiae que resulten reproducibles, accesibles a laboratorios de baja y mediana complejidad, y asegurar su estabilidad fenotípica y genotípica mediante el método de preservación llamado subcultivo continuo, para el mantenimiento del cultivo en medio adecuado con transferencias a medio fresco a intervalos variables. Métodos: Se seleccionaron al azar 40 cepas de Streptococcus agalactiae, las que se sometieron a verificación de viabilidad, pureza y caracterización fenotípica y genotípica antes y después de ser sometidas a conservación, utilizando idénticos medios, reactivos y metodología en ambas circunstancias. Se probaron diferentes medios de preservación de las cepas, que permitieran a laboratorios de baja y mediana complejidad su traslado a centros especializados para la vigilancia adecuada del organismo. Las cepas se conservaron durante nueve meses con subcultivos que mostraron las características originales. Resultados: Los medios más efectivos fueron ATS-TA y LD 4 %-SO-20 ºC, ya que garantizaron viabilidad, pureza y estabilidad fenotípica y genotípica de las cepas. Se demostró que el uso de este medio es una alternativa adecuada para la conservación de Streptococcus agalactiae en laboratorios donde la liofilización y la desecación no están disponibles y que son de bajo costo, rápidos y muy fáciles de usar en la práctica habitual. Conclusiones: los medios ATS-TA y LD 4 %-SO-20 ºC constituyen una buena alternativa para cortos períodos de preservación y transporte de cepas de Streptococcus agalactiae en laboratorios de baja y mediana complejidad.

Introduction: Although Streptococcus agalactiae is commensal of the gastrointestinal and genitourinary tract, it is the main cause of invasive diseases and mortality in newborns. The infection can be acquired through the aspiration of infected amniotic fluid or during the passage through the birth canal. Objectives: to compare the effectiveness of various methods for the conservation of Streptococcus agalactiae strains that can be reproducible and accessible to laboratories of low and medium complexity and guarantee their phenotypic and genotypic stability through a preservation method called continuous subculture, for the maintenance of the culture in an adequate environment with fresh medium transfers at variable interval periods. Methods: 40 strains of Streptococcus agalactiae were randomly selected, which were subjected to verification of viability, purity and genotypic and phenotypic characterization before and after being subjected to conservation, using identical environments, reactive and methodologies in both circumstances. Different means of preservation of strains were tested, which allowed laboratories of low and medium complexity their transfer to specialized centers for the proper surveillance of the organism. The strains were kept for nine months with subcultures that showed original characteristics. Results: The most effective environments were ATS-TA and LD-4 % -SO-20 ºC because they guaranteed viability, purity and genotypic and phenotypic stability of the strains. It was shown that the use of this environment is an adequate alternative for the conservation of Streptococcus agalactiae in laboratories where lyophilization and dessication are not available and are low cost, fast and very easy to use in regular practice. Conclusions: The ATS-TA and LD-4 % -SO-20 ºC environments are a good alternative for short periods of preservation and transportation of Streptococcus agalactiae strains in laboratories of low and medium complexity.