Long reads from Nanopore sequencing as a tool for animal microbiome studies

Abstract

19In the era of bioinformatics and metagenomics, the study of the ruminal microbiome has gained considerable 20 relevance in the field of animal breeding, since the composition of the rumen microbiota significantly impacts 21 production and the environment. Illumina sequencing is considered the gold standard for the analysis of 22 microbiomes, but it is limited by obtaining only short DNA sequences to analyze. As an alternative, Oxford 23 Nanopore Technologies (ONT) has developed a new sequencing technique based on nanopores that can be 24 carried out in the MinION, a portable device with a low initial cost which long DNA readings can be obtained 25 with. The aim of this study was to compare the performance of both types of sequencing applied to samples of 26 ruminal content using a similar pipeline. The ONT sequencing provided similar results to the Illumina 27 sequencing, although it was able to classify a greater number of readings at the species level, possibly due to 28 the increase in the read size. The results also suggest that, due to the size of the reads, it would be possible to 29 obtain the same amount of information in a smaller number of hours. However, detection of archaeal and 30 eukaryotic species is still difficult to accomplish due to their low abundance in the rumen compared to 31 bacteria, suggesting different pipelines and strategies are needed to obtain a whole representation of the less 32 abundant species in the rumen microbiota.33 34 35 36 Background 37 38 The bovine rumen has been studied for years in an attempt to reveal functions and microorganisms associated 39 with nutritional features such as feed efficiency to use them in animal breeding programs for cattle (Knapp, 40 Laur, Vadas, Weiss, & Tricarico, 2014). Lately, methane emissions from ruminants are among the main 41 concerns in animal husbandry, given their contribution to global warming. Microbial cultures were essential 42 for the first descriptions of the rumen content but they are usually hard to achieve (Creevey, Kelly, 43 Henderson, & Leahy, 2014). Thanks to the Next Generation Sequencing (NGS) techniques it is now possible 44 to detect different microbial taxa in rumen samples avoiding culture (Seshadri et al., 2018). This has allowed 45 analyzing metagenomic samples in an easier way and to detect non-culturable microbes that broaden the 46 knowledge of complex microbial communities as well as allow quantifying the relative abundances of each 47 65 profiles in municipal sewage, obtaining similar results as Illumina, although it was still necessary to improve 66 the throughput of the sequencing runs. ONT has also been used to detected arbovirus in mosquitos (Batovska, 67 Lynch, Rodoni, Sawbridge, & Cogan, 2017) obtaining comparable results to Illumina sequencing, despite the 68 poorer quality of the reads was poorer. Other studies focus on the applicability of ONT for real-time viral 69 pathogen detection (Greninger et al., 2015) in human blood as it shows similar results to Illumina but provides 70 faster results, which ...

Aplicación de la secuenciación masiva paralela para el análisis de alta definición de regiones genómicas de interés en el ganado ovino y para el estudio de la microbiota de la leche de oveja = Appliction of massively parallel secuencing to the high-resolution analysis of relevant genomic regions in dairy sheep and the study the sheep milk microbiota

“…Esta tecnología está empezando a utilizarse como técnica de diagnóstico para la sospecha de enfermedades infecciosas, por ejemplo para la identificación y caracterización genética del enterovirus en vacuno (Beato et al, 2018), para la detección de patógenos causantes de enfermedades entéricas en cerdos (Theuns et al, 2018), o para el diagnóstico de la viruela aviar mediante secuenciación del genoma completo del avipoxvirus (Croville et al, 2018). Además, estudios del microbioma del rumen en el ganado bovino están revelando que la secuenciación ONT ofrece unos resultados similares a la secuenciación de Illumina, clasificando un mayor número de lecturas a nivel de especie, y por lo tanto puede posicionarse como una alternativa interesante para caracterizar microbiomas de interés en animales (Delgado et al, 2019b). En especies domésticas lecheras, como el búfalo, se ha puesto de manifiesto que la secuenciación utilizando la tecnología MinION para la identificación de patógenos potenciales y de bacterias resistentes en muestras de leche representa una de las futuras aplicaciones de esta tecnología (Catozzi et al, 2020).…”