Ensamblado y análisis comparativo de metagenomas de rumen vacuno
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Date
2022
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Facultad de Ciencias Agrarias-Universidad Nacional de Rosario
Abstract
Los rumiantes pueden transformar la energía almacenada en las plantas en productos
alimenticios que pueden ser utilizados por los humanos, como la carne y la leche.
La microbiota del rumen está compuesta por protozoos, bacterias, hongos y arqueas, que son
responsables de la degradación del material vegetal.
A pesar del fuerte interés industrial y científico, el rumen sigue siendo un hábitat poco
conocido, con muchas especies y cepas microbianas no cultivadas. La secuenciación
metagenómica del rumen produce secuencias muy novedosas, que pueden ser de gran interés
para las industrias de biocombustibles, alimentos y biotecnología. En este trabajo, los
metagenomas de muestras de rumen fueron obtenidos de vacas regionales jóvenes y adultas
alimentadas con una dieta rica o pobre con el objetivo de ensamblar nuevos genomas. El
ADN fue extraído y secuenciado por WGS. Luego, las lecturas se filtraron por calidad y se
ensamblaron con Megahit. La calidad de los contigs se evaluó con el software QUAST, y
BWA MEM se usó para asignar lecturas a los ensamblajes. El binning se realizó con
Metabat2 usando los contigs obtenidos, y archivos BAM correspondientes a alineaciones de
lecturas. Se recuperaron de 12 a 31 bins por muestra, y se evalúo su integridad y
contaminación mediante CheckM. El filtrado de estos por completitud ≥80% y
contaminación ≤10%, generó entre tres y cinco genomas ensamblados de metagenomas
(MAGs) por muestra. La asignación taxonómica se llevó a cabo utilizando el servidor MiGA,
lo cual permitió identificar organismos asociados al tracto gastrointestinal y la degradación
de material vegetal. La predicción de genes se llevó a cabo mediante Prodigal. Con esta
información, se determinó el perfil metabólico utilizando el programa Genomaple, el cual
permitió obtener un análisis global de las principales vías presentes en los metagenomas.
Debido a la importancia de las enzimas activas sobre carbohidratos (CAZymas) en el rumen,
se realizó un estudio de las CAZymas presentes en las muestras, identificándose
posteriormente aquellas enzimas no ortólogas a la base de datos de proteínas ruminales
RumiRef, lo cual sugeriría que estas proteínas son únicas en las muestras analizadas
Ruminants can transform the energy stored in plants into food products that can be used by humans, such as meat and milk. The rumen microbiota is composed of protozoa, bacteria, fungi and archaea, which are responsible for plant material degradation. Despite strong industrial and scientific interest, the rumen remains a poorly understood habitat, with many uncultivated microbial species and strains. Metagenomic sequencing of the rumen still produces highly novel sequences, which can be of great interest for biofuels, food and biotechnology industries. In this work, the metagenomes of rumen samples of regional young and adult cows fed with a rich or poor diet were obtained aiming to assemble novel genomes. DNA was extracted and sequenced by WGS. The reads were then filtered for quality and assembled with Megahit. Quality of the contigs was assessed with QUAST software, and BWA MEM was used to map reads back to the assemblies. Binning was carried out with Metabat2 using the obtained contigs, and BAM files corresponding to reads alignments. 12 to 31 bins were recovered per sample, and their integrity and contamination were evaluated by CheckM. Filtering these for completeness ≥80% and contamination ≤10% generated between three and five metagenome assembled genomes (MAGs) per sample. The taxonomic assignment was carried out using MiGA server, which allowed the identification of organisms associated with gastrointestinal tract and plant material degradation. Gene prediction was carried out using Prodigal. With this information, the metabolic profile was determined using Genomaple program, which allowed obtaining a global analysis of the main pathways present in the metagenomes. Due to the importance of carbohydrate-active enzymes (CAZymes) in the rumen, a study of the CAZymes present in the samples was carried out, subsequently identifying those enzymes not orthologous to the RumiRef ruminal protein database, which would suggest that these proteins are unique in the analyzed samples
Ruminants can transform the energy stored in plants into food products that can be used by humans, such as meat and milk. The rumen microbiota is composed of protozoa, bacteria, fungi and archaea, which are responsible for plant material degradation. Despite strong industrial and scientific interest, the rumen remains a poorly understood habitat, with many uncultivated microbial species and strains. Metagenomic sequencing of the rumen still produces highly novel sequences, which can be of great interest for biofuels, food and biotechnology industries. In this work, the metagenomes of rumen samples of regional young and adult cows fed with a rich or poor diet were obtained aiming to assemble novel genomes. DNA was extracted and sequenced by WGS. The reads were then filtered for quality and assembled with Megahit. Quality of the contigs was assessed with QUAST software, and BWA MEM was used to map reads back to the assemblies. Binning was carried out with Metabat2 using the obtained contigs, and BAM files corresponding to reads alignments. 12 to 31 bins were recovered per sample, and their integrity and contamination were evaluated by CheckM. Filtering these for completeness ≥80% and contamination ≤10% generated between three and five metagenome assembled genomes (MAGs) per sample. The taxonomic assignment was carried out using MiGA server, which allowed the identification of organisms associated with gastrointestinal tract and plant material degradation. Gene prediction was carried out using Prodigal. With this information, the metabolic profile was determined using Genomaple program, which allowed obtaining a global analysis of the main pathways present in the metagenomes. Due to the importance of carbohydrate-active enzymes (CAZymes) in the rumen, a study of the CAZymes present in the samples was carried out, subsequently identifying those enzymes not orthologous to the RumiRef ruminal protein database, which would suggest that these proteins are unique in the analyzed samples
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Keywords
Metagenómica, Rumen, Ensamblado