Targeted and untargeted nanopore sequencing approaches to profile the gut microbiota of mice infants exposed to ethanol in utero

The gut microbiome plays a vital role in host homeostasis and understanding of its biology is essential for a better comprehension of the etiology of disorders such as foetal alcohol spectrum disorders. Here we assessed the effectiveness of targeted and untargeted (metagenomic) nanopore sequencing approaches to profile the gut microbiota of infant mice exposed to ethanol in utero. DNA extracts from the gut content of 12 infant mice exposed to ethanol in utero were analysed using one untargeted and two targeted (full-length 16S rRNA gene and the 16S-ITS-23S region of the ribosomal RNA operon) nanopore sequencing approaches. The targeting of the full-length 16S rRNA gene provided the most comprehensive analysis of the mouse gut microbiota. The differences in diversity between approaches were accounted by the sequencing target (p-value < 0.001). Faecalibaculum rodentium and Duncaniella sp. were the two most prevalent taxa detected using targeted sequencing approaches, while bacterial taxa were more evenly represented when using the metagenomic approach. Full-length 16S rRNA gene nanopore sequencing provides the most discriminatory microbiota compositional analysis of mice faecal samples. However, using nanopore sequencing approaches targeting the metagenome or different taxonomically-informative DNA region appears to introduce significant target-related biases. Importance Current nanopore approaches have not been standardized which may confound the biological interpretations of hight-throughput sequencing datasets. Additionally, nanopore sequencing still present a high error-rate compared to other more mature sequencing technologies, such as Illumina sequencing. These technological handicaps create the need to study and optimize nanopore sequencing approaches to answer biological questions, such as interrogations of the microbial composition and abundance of clinical and environmental samples. In this work, three nanopore sequencing approaches were designed and attempted to optimize fungal and bacterial profiling sequencing methodologies. Two targeted methods based on the bacterial 16S rRNA gene, and 16S-ITS-23S rrn operon region, and one untargeted shotgun/metagenomic approach were tested. Despite potential experimental and/or bioinformatical biases were found, the 16S rRNA gene-targeted nanopore sequencing was the most comprehensive approach to study the microbial composition of the infant mice gut microbiotas.