June 13, 2022

Evaluation of taxonomic profiling methods for long-read shotgun metagenomic sequencing datasets

Author(s): Portik, Daniel M.and Brown, C. Titus and Pierce-Ward, N. Tessa

Long-read shotgun metagenomic sequencing is gaining in popularity and offers many advantages over short-read sequencing. The higher information content in long reads is useful for a variety of metagenomics analyses, including taxonomic profiling. The development of long-read specific tools for taxonomic profiling is accelerating, yet there is a lack of consensus regarding their relative performance. Here, we perform a critical benchmarking study using five long-read methods and four popular short-read methods. We applied these tools to several mock community datasets generated using Pacific Biosciences (PacBio) HiFi or Oxford Nanopore Technology (ONT) sequencing, and evaluated their performance based on read utilization, detection metrics, and relative abundance estimates. Our results show that long-read methods generally outperformed short-read methods. Short-read methods (including Kraken2, Bracken, Centrifuge, and MetaPhlAn3) produced many false positives (particularly at lower abundances), required heavy filtering to achieve acceptable precision (at the cost of reduced recall), and produced inaccurate abundance estimates. By contrast, several long-read methods displayed very high precision and acceptable recall without any filtering required, including BugSeq, MEGAN-LR using translation alignments (DIAMOND to NCBI nr) or nucleotide alignments (minimap2 to NCBI nt). Furthermore, in the PacBio HiFi datasets these long-read methods detected all species down to the 0.1% abundance level with high precision. Other long-read methods, such as MetaMaps and MMseqs2, required moderate filtering to reduce false positives to achieve a suitable balance between precision and recall. We found read quality affected performance for methods relying on protein prediction or exact kmer matching, and these methods performed better with PacBio HiFi datasets. Finally, for a given mock community we found that the long-read datasets produced significantly better results than short-read datasets, demonstrating clear advantages for long-read metagenomic sequencing. Our critical assessment of available methods provides recommendations for current research using long reads and establishes a baseline for future benchmarking studies.

Organization: PacBio
Year: 2022

View Conference Poster

Talk with an expert

If you have a question, need to check the status of an order, or are interested in purchasing an instrument, we're here to help.