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Article

Optimization of 16S RNA Sequencing and Evaluation of Metagenomic Analysis with Kraken 2 and KrakenUniq

by
Nasserdine Papa Mze
1,*,
Cécile Fernand-Laurent
1,
Sonnentrucker Maxence
2,
Olfa Zanzouri
1,
Solen Daugabel
1 and
Stéphanie Marque Juillet
1
1
Service de Biologie, Unité de Microbiologie, Hôpital Mignot, Centre Hospitalier de Versailles, 177 rue de Versailles, 78150 Le Chesnay, France
2
Faculté de Santé, Université Paris-Est Créteil, 61 Avenue du Général de Gaulle, 94010 Créteil, France
*
Author to whom correspondence should be addressed.
Diagnostics 2025, 15(17), 2175; https://doi.org/10.3390/diagnostics15172175
Submission received: 21 May 2025 / Revised: 29 July 2025 / Accepted: 26 August 2025 / Published: 27 August 2025
(This article belongs to the Section Pathology and Molecular Diagnostics)

Abstract

Background/Objectives: 16S ribosomal RNA sequencing has, for several years, been the main means of identifying bacterial and archaeal species. Low-throughput Sanger sequencing is often used for the detection and identification of microbial species, but this technique has several limitations. The use of high-throughput sequencers may be a good alternative to improve patient identification, especially for polyclonal infections and management. Kraken 2 and KrakenUniq are free, high-throughput tools providing a very rapid and accurate classification for metagenomic analyses. However, Kraken 2 can present false-positive results relative to KrakenUniq, which can be limiting in hospital settings requiring high levels of accuracy. The aim of this study was to establish an alternative next-generation sequencing technique to replace Sanger sequencing and to confirm that KrakenUniq is an excellent analysis tool that does not present false results relative to Kraken 2. Methods: DNA was extracted from reference bacterial samples for Laboratory Quality Controls (QCMDs) and the V2-V3 and V3-V4 regions of the 16S ribosomal gene were amplified. Amplified products were sequenced with the Illumina 16S Metagenomic Sequencing protocol with minor modifications to adapt and sequence an Illumina 16S library with a small 500-cycle nano-flow cell. The raw files (Fastq) were analyzed on a commercial Smartgene platform for comparison with Kraken 2 and KrakenUniq results. KrakenUniq was used with a standard bacterial database and with the 16S-specific Silva138, RDP11.5, and Greengenes 13.5 databases. Results: Seven of the eight (87.5%) QCMDs were correctly sequenced and identified by Sanger sequencing. The remaining QCMD, QCMD6, could not be identified through Sanger sequencing. All QCMDs were correctly sequenced and identified by MiSeq with the commercial Smartgene analysis platform. QCMD6 contained two bacteria, Acinetobacter and Klebsiella. KrakenUniq identification results were identical to those of Smartgene, whereas Kraken 2 yielded 25% false-positive results. Conclusions: If Sanger identification fails, MiSeq with a small nano-flow cell is a very good alternative for the identification of bacterial species. KrakenUniq is a free, fast, and easy-to-use tool for identifying and classifying bacterial infections.
Keywords: kraken 2; krakenUniq; Smartgene; 16S RNA; Sanger; MiSeq kraken 2; krakenUniq; Smartgene; 16S RNA; Sanger; MiSeq

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MDPI and ACS Style

Papa Mze, N.; Fernand-Laurent, C.; Maxence, S.; Zanzouri, O.; Daugabel, S.; Marque Juillet, S. Optimization of 16S RNA Sequencing and Evaluation of Metagenomic Analysis with Kraken 2 and KrakenUniq. Diagnostics 2025, 15, 2175. https://doi.org/10.3390/diagnostics15172175

AMA Style

Papa Mze N, Fernand-Laurent C, Maxence S, Zanzouri O, Daugabel S, Marque Juillet S. Optimization of 16S RNA Sequencing and Evaluation of Metagenomic Analysis with Kraken 2 and KrakenUniq. Diagnostics. 2025; 15(17):2175. https://doi.org/10.3390/diagnostics15172175

Chicago/Turabian Style

Papa Mze, Nasserdine, Cécile Fernand-Laurent, Sonnentrucker Maxence, Olfa Zanzouri, Solen Daugabel, and Stéphanie Marque Juillet. 2025. "Optimization of 16S RNA Sequencing and Evaluation of Metagenomic Analysis with Kraken 2 and KrakenUniq" Diagnostics 15, no. 17: 2175. https://doi.org/10.3390/diagnostics15172175

APA Style

Papa Mze, N., Fernand-Laurent, C., Maxence, S., Zanzouri, O., Daugabel, S., & Marque Juillet, S. (2025). Optimization of 16S RNA Sequencing and Evaluation of Metagenomic Analysis with Kraken 2 and KrakenUniq. Diagnostics, 15(17), 2175. https://doi.org/10.3390/diagnostics15172175

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