TARGETING THE 16S RRNA GENE FOR BACTERIAL IDENTIFICATION IN COMPLEX MIXED SAMPLES: COMPARATIVE EVALUATION OF SECOND (ILLUMINA) AND THIRD (OXFORD NANOPORE TECHNOLOGIES) GENERATION SEQUENCING TECHNOLOGIES.

Raf Winand,Sigrid Cj De Keersmaecker,Nancy Hc Roosens,Julien Van Braekel,Loïc Lefevre,Maud Delvoye,Qiang Fu,Kevin Vanneste,Stefan Hoffman,Bert Bogaerts

doi:10.3390/ijms21010298

Raf Winand, Sigrid Cj De Keersmaecker + Show 8 more

Open Access

https://doi.org/10.3390/ijms21010298

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Abstract

Rapid, accurate bacterial identification in biological samples is an important task for microbiology laboratories, for which 16S rRNA gene Sanger sequencing of cultured isolates is frequently used. In contrast, next-generation sequencing does not require intermediate culturing steps and can be directly applied on communities, but its performance has not been extensively evaluated. We present a comparative evaluation of second (Illumina) and third (Oxford Nanopore Technologies (ONT)) generation sequencing technologies for 16S targeted genomics using a well-characterized reference sample. Different 16S gene regions were amplified and sequenced using the Illumina MiSeq, and analyzed with Mothur. Correct classification was variable, depending on the region amplified. Using a majority vote over all regions, most false positives could be eliminated at the genus level but not the species level. Alternatively, the entire 16S gene was amplified and sequenced using the ONT MinION, and analyzed with Mothur, EPI2ME, and GraphMap. Although >99% of reads were correctly classified at the genus level, up to ≈40% were misclassified at the species level. Both technologies, therefore, allow reliable identification of bacterial genera, but can potentially misguide identification of bacterial species, and constitute viable alternatives to Sanger sequencing for rapid analysis of mixed samples without requiring any culturing steps.

Highlights

Clinical microbiology laboratories and public health authorities have relied, traditionally, on phenotypic methods to identify bacterial pathogens, but these cannot be used for uncultivable bacteria
NGS technologies present an interesting alternative to traditional bacterial isolation and 16S rRNA gene amplification and Sanger sequencing for bacterial identification, especially for complex mixed samples, because they allow rapid orientation of samples by not requiring any intermediate culturing step(s)
We performed a comparative evaluation of the performance of both second and third generation NGS technologies for bacterial identification based on 16S rRNA gene targeting and sequencing of a well-characterized reference sample, and found that both technologies offer their own advantages and disadvantages

Summary

Introduction

Clinical microbiology laboratories and public health authorities have relied, traditionally, on phenotypic methods to identify bacterial pathogens, but these cannot be used for uncultivable bacteria. A potential alternative is the use of 16S rRNA gene sequencing, which has historically been used to identify known and new bacteria independently of cultivability and phenotype [2,3]. Since the 16S rRNA gene is present in all bacteria and subject to different evolutionary rates depending on the gene region considered, it has historically been used for classification of isolates [4], and more recently in complex samples coming from a variety of environments, such as the human gut [5], soil [6], and oceans [7] where it can be used to determine the bacterial number and composition of samples. Employing 16S rRNA gene sequencing for bacterial identification is, impacted by several factors, such as the 16S rRNA gene region(s) considered and their amplification efficiency, the sequencing technology itself, and the bioinformatics workflow(s) employed

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