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Challenges in benchmarking metagenomic profilers


Accurate microbial identification and abundance estimation are crucial for metagenomics analysis. Various methods for classification of metagenomic data and estimation of taxonomic profiles, broadly referred to as metagenomic profilers, have been developed. Nevertheless, benchmarking of metagenomic profilers remains challenging because some tools are designed to report relative sequence abundance while others report relative taxonomic abundance. Here we show how misleading conclusions can be drawn by neglecting this distinction between relative abundance types when benchmarking metagenomic profilers. Moreover, we show compelling evidence that interchanging sequence abundance and taxonomic abundance will influence both per-sample summary statistics and cross-sample comparisons. We suggest that the microbiome research community pay attention to potentially misleading biological conclusions arising from this issue when benchmarking metagenomic profilers, by carefully considering the type of abundance data that were analyzed and interpreted and clearly stating the strategy used for metagenomic profiling.

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Fig. 1: Comparison of profiling results.
Fig. 2: Correlation between sequence and taxonomic abundance in synthetic profiles based on different kingdoms.
Fig. 3: Quantitative and qualitative benchmarking results of four representative metagenomic profilers using 25 simulated communities.
Fig. 4: Alpha diversity based on sequence and taxonomic abundance.
Fig. 5: Ordination analyses of simulated profiles based on rJSD.

Data availability

All simulated datasets can be downloaded from data are provided with this paper.

Code availability

R scripts used in this paper are available at


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Research reported in this publication was supported by grant nos. R01AI141529, R01HD093761, R01AG067744, UH3OD023268, U19AI095219 and U01HL089856 from the National Institutes of Health. This work was also supported by IBM Research through the AI Horizons Network, UC San Diego AI for Healthy Living program in partnership with the UC San Diego Center for Microbiome Innovation.

Author information




Y.-Y.L. and R.K. conceived and designed the analysis. Z.S. and S.H. led the analysis. M.Z., Q.Z., N.H., A.P.C., Y.V.-B, L.P. and H.-C.K. contributed evaluation strategies. All authors analyzed the results. Z.S., S.H., Y.-Y.L. and R.K. wrote the paper. All authors edited the paper.

Corresponding authors

Correspondence to Rob Knight or Yang-Yu Liu.

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Competing interests

This work received support from IBM Research through the AI Horizons Network. Coauthors N.H., A.P.C., L.P. and H.-C.K. are employees of IBM. The authors declare no other competing interests.

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Peer review information Nature Methods thanks the anonymous reviewers for their contribution to the peer review of this work. Lin Tang was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Note, Figs. 1–6 and references.

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Supplementary Data

An Excel file that includes the source data used in each of the Supplementary Figures.

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Sun, Z., Huang, S., Zhang, M. et al. Challenges in benchmarking metagenomic profilers. Nat Methods 18, 618–626 (2021).

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