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Background Our understanding of the composition, function, and health implications of human microbiota has been advanced by high-throughput sequencing and the development of new genomic analyses. However, trade-offs among alternative strategies for the acquisition and analysis of sequence data remain understudied. Methods We assessed eight popular taxonomic profiling pipelines; MetaPhlAn2, metaMix, PathoScope 2.0, Sigma, Kraken, ConStrains, Centrifuge and Taxator-tk, against a battery of metagenomic datasets simulated from real data. The metagenomic datasets were modeled on 426 complete or permanent draft genomes stored in the Human Oral Microbiome Database and were designed to simulate various experimental conditions, both in the design of a putative experiment; read length (75–1,000 bp reads), sequence depth (100K–10M), and in metagenomic composition; number of species present (10, 100, 426), species distribution. The sensitivity and specificity of each of the pipelines under various scenarios were measured. We also estimated the relative root mean square error and average relative error to assess the abundance estimates produced by different methods. Additional datasets were generated for five of the pipelines to simulate the presence within a metagenome of an unreferenced species, closely related to other referenced species. Additional datasets were also generated in order to measure computational time on datasets of ever-increasing sequencing depth (up to 6 × 107). Results Testing of eight pipelines against 144 simulated metagenomic datasets initially produced 1,104 discrete results. Pipelines using a marker gene strategy; MetaPhlAn2 and ConStrains, were overall less sensitive, than other pipelines; with the notable exception of Taxator-tk. This difference in sensitivity was largely made up in terms of runtime, significantly lower than more sensitive pipelines that rely on whole-genome alignments such as PathoScope2.0. However, pipelines that used strategies to speed-up alignment between genomic references and metagenomic reads, such as kmerization, were able to combine both high sensitivity and low run time, as is the case with Kraken and Centrifuge. Absent species genomes in the database mostly led to assignment of reads to the most closely related species available in all pipelines. Our results therefore suggest that taxonomic profilers that use kmerization have largely superseded those that use gene markers, coupling low run times with high sensitivity and specificity. Taxonomic profilers using more time-consuming read reassignment, such as PathoScope 2.0, provided the most sensitive profiles under common metagenomic sequencing scenarios. All the results described and discussed in this paper can be visualized using the dedicated R Shiny application ( All of our datasets, pipelines and results are made available through the GitHub repository for future benchmarking.

Original publication




Journal article





Publication Date





e9688 - e9688