LotuS2: An ultrafast and highly accurate tool for amplicon sequencing analysis

Amplicon sequencing is an established and cost-efficient method for profiling microbiomes. However, many available tools to process this data require both bioinformatics skills and high computational power to process big datasets. Furthermore, there are only few tools that allow for long read amplicon data analysis. To bridge this gap, we developed the LotuS2 (Less OTU Scripts 2) pipeline, enabling user-friendly, resource friendly, and versatile analysis of raw amplicon sequences. In LotuS2, six different sequence clustering algorithms as well as extensive pre- and post-processing options allow for flexible data analysis by both experts, where parameters can be fully adjusted, and novices, where defaults are provided for different scenarios. We benchmarked three independent gut and soil datasets, where LotuS2 was on average 29 times faster compared to other pipelines – yet could better reproduce the alpha- and beta-diversity of technical replicate samples. Further benchmarking a mock community with known taxa composition showed that, compared to the other pipelines, LotuS2 recovered a higher fraction of correctly identified genera and species (98% and 57%, respectively). At ASV/OTU level, precision and F-score were highest for LotuS2, as was the fraction of correctly reconstructed 16S sequences. In conclusion, LotuS2 is a lightweight and user-friendly pipeline that is fast, precise and streamlined. High data usage rates and reliability enable high-throughput microbiome analysis in minutes.