Nanopore-Based Approaches to Study Microbial Diversity in Complex Samples
This webinar will be looking at applications for the MinIONTM in new molecular approaches for the taxonomy characterization of microbial communities aiming resolution at species and strain level.
The webinar is available on-demand.
The miniaturised and portable DNA sequencer MinIONTM has demonstrated great potential in different analyses such as genome-wide sequencing, pathogen outbreak detection and surveillance, and human genome variability. In the last three years, we have been working on the development of new molecular approaches for the taxonomy characterization of microbial communities aiming resolution at species and strain level. In 2016, we described the first microbial diversity study using the portable nanopore sequencing device. Consequently, we demonstrated the feasibility to perform this kind of analysis despite the high error rate of the R7.3 chemistry and we also evidenced the throughput as a major handicap for its complete implementation at this regard. Later on, we tested the ability of the MinIONTM platform to perform long amplicon sequencing in order to design new approaches to study microbial diversity using a multi-locus approach. Using R9 and R9.4 chemistries and MinIONTM MkIb, we were able to sequence amplicons ~5kb in length obtained from amplification of bacterial 16S-ITS-23S genetic markers, the rrn region hereinafter. After compiling a robust database by parsing and extracting the rrn bacterial region from more than 67,000 complete or draft bacterial genomes, we were able to demonstrate that the data obtained during sequencing of the long amplicon in the MinIONTM device was sufficient to study two mock microbial communities in a multiplex manner and to almost completely reconstruct the microbial diversity contained in the HM782D and D6305 samples. Although nanopore-based sequencing produces reads with lower per-base accuracy compared with other platforms, we presented a novel approach consisting of a multi-locus and long amplicon multiplex sequencing that helps to overcome the main disadvantage of this portable sequencing platform. Improvements in nanopore chemistry, such as minimising base-calling errors and new protocols able to produce rapid 1D libraries, will provide more reliable information. Such data will be useful for more comprehensive and faster specific detection of microbial species and strains in complex ecosystems.
Alfonso Benítez-Páez, PhD
Dr. Benítez-Páez is a Biologist with postgraduate degrees M.Sc in Biochemistry and Sp. in Biotechnology. His PhD in Molecular Biology was gained at the UAM in Madrid, Spain. During his PhD, he worked in the analysis of the enzyme-dependent post-transcriptional modifications in bacterial rRNA and tRNA. He remained at FISBABIO-CSISP (Valencia, Spain) for his first post-doc, studying the human oral microbiome and the characterization of probiotics for oral health. Since 2014, he is involved in the MyNewGut project funded by the European Commission working in the development of dietary-based strategies to tackle XXI century pandemics such as obesity and co-morbidities at the Institute of Agrochemistry and Food Technology (IATA-CSIC) in Valencia, Spain. Dr. Benítez-Páez is currently coordinating metagenome analyses of dietary interventions in humans aiming the functional characterization of diet compounds able to modulate the human gut microbiota towards a healthy status. His research continues to focus on the development of new molecular approaches for the deep characterization of microbial communities, aiming the identification at species and strain level for direct association of gut microbes with health and disease.
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