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Updated: Aug 8, 2025
Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing
Published on: October 15, 2019
Benchmarking of microbiome detection tools on RNA-seq synthetic databases according to diverse conditions
Francisco Jurado-Rueda1, Lola Alonso-Guirado1, Tomin E Perea-Cham-Blee2
1Genetic & Molecular Epidemiology Group, Spanish National Cancer Research Centre and CIBERONC, Madrid 28029, Spain.
Motivation:
Here, we performed a benchmarking analysis of five tools for microbe sequence detection using transcriptomics data (Kraken2, MetaPhlAn2, PathSeq, DRAC and Pandora). We built a synthetic database mimicking real-world structure with tuned conditions accounting for microbe species prevalence, base calling quality and sequence length. Sensitivity and positive predictive value (PPV) parameters, as well as computational requirements, were used for tool ranking.
Results:
GATK PathSeq showed the highest sensitivity on average and across all scenarios considered. However, the main drawback of this tool was its slowness. Kraken2 was the fastest tool and displayed the second-best sensitivity, though with large variance depending on the species to be classified. There was no significant difference for the other three algorithms sensitivity. The sensitivity of MetaPhlAn2 and Pandora was affected by sequence number and DRAC by sequence quality and length. Results from this study support the use of Kraken2 for routine microbiome profiling based on its competitive sensitivity and runtime performance. Nonetheless, we strongly endorse to complement it by combining with MetaPhlAn2 for thorough taxonomic analyses.
Availability And Implementation:
https://github.com/fjuradorueda/MIME/ and https://github.com/lola4/DRAC/.
Supplementary Information:
Supplementary data are available at Bioinformatics Advances online.
