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

International Journal of Molecular Sciences

Maud Delvoye¹  Stefan Hoffman¹  Loïc Lefevre¹  Kevin Vanneste¹  Julien Van Braekel¹  Raf Winand¹  Bert Bogaerts¹  NancyHC Roosens¹  Qiang Fu¹  SigridCJ De Keersmaecker¹ 

[1] Transversal activities in Applied Genomics, Sciensano, 1050 Brussels, Belgium;

关键词: orientation;    bacterial identification;    16s rrna;    targeted genomics;    public health;    illumina;    nanopore;    targeted metagenomics;   

DOI  :  10.3390/ijms21010298

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown