Abstract
Nanopore sequencing is rapidly becoming more popular for use in various microbiota-based applications. Major limitations of current approaches are that they do not enable de novo species identification and that they cannot be used to verify species assignments. This severely limits applicability of the nanopore sequencing technology in taxonomic applications. Here, we demonstrate the possibility of de novo species identification and verification using hexamer frequencies in combination with k-means clustering for nanopore sequencing data. The approach was tested on the human infant gut microbiota of 3-month-old infants. Using the hexamer k-means approach we identified two new low abundant species associated with vaginal delivery. In addition, we confirmed both the vaginal delivery association for two previously identified species and the overall high levels of bifidobacteria. Taxonomic assignments were further verified by mock community analyses. Therefore, we believe our de novo species identification approach will have widespread application in analyzing microbial communities in the future.
Highlights
Third generation nanopore sequencing has revolutionized the field of analyzing microbial communities, with the promise of on-site high throughput analyses (Acharya et al, 2019)
We evaluated the nanopore de novo species identification approach, both by analyzing the human infant gut microbiota of 3-month-old children and a mock community
Genomic DNA from a mock community (HM-783D, BEI Resources, Manassas, Virginia, USA), containing genomic DNA from 20 bacterial strains mixed based on 16S rRNA gene copy number counts, was used as a control and followed the library preparation along with the genomic DNA isolated from the infant fecal samples as described below
Summary
Third generation nanopore sequencing has revolutionized the field of analyzing microbial communities, with the promise of on-site high throughput analyses (Acharya et al, 2019). Despite several recent advances in nanopore sequencing, the error rates are too high for de novo species identification (Shin et al, 2016). All current approaches are based on some kind of reference, or black-box systems for species identification How to cite this article Angell IL, Nilsen M, Carlsen KCL, Carlsen K-H, Hedlin G, Jonassen CM, Marsland B, Nordlund B, Rehbinder EM, Saunders C, Skjerven HO, Staff AC, Söderhäll C, Vettukattil R, Rudi K. De novo species identification using 16S rRNA gene nanopore sequencing.
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