OptiFit: an improved method for fitting amplicon sequences to existing OTUs
Authors: Sovacool Kelly L. , Westcott Sarah L. , Mumphrey M. Brodie , Dotson Gabrielle A. , Schloss Patrick D. 
Published: November 2021

AbstractAssigning amplicon sequences to operational taxonomic units (OTUs) is often an important step in characterizing the composition of microbial communities across large datasets. OptiClust, ade novoOTU clustering method, has been shown to produce higher quality OTU assignments than other methods and at comparable or faster speeds. A notable difference betweende novoclustering and database-dependent reference clustering methods is that OTU assignments fromde novomethods may change when new sequences are added to a dataset. However, in some cases one may wish to incorporate new samples into a previously clustered dataset without performing clustering again on all sequences, such as when comparing across datasets or deploying machine learning models where OTUs are features. Existing reference-based clustering methods produce consistent OTUs, but they only consider the similarity of each query sequence to a single reference sequence in an OTU, thus resulting in OTU assignments that are significantly worse than those generated byde novomethods. To provide an efficient and robust method to fit amplicon sequence data to existing OTUs, we developed the OptiFit algorithm. Inspired by OptiClust, OptiFit considers the similarity of all pairs of reference and query sequences in an OTU to produce OTUs of the best possible quality. We tested OptiFit using four microbiome datasets with two different strategies: by clustering to an external reference database or by splitting the dataset into a reference and query set and clustering the query sequences to the reference set after clustering it using OptiClust. The result is an improved implementation of closed and open-reference clustering. OptiFit produces OTUs of similar quality as OptiClust and at faster speeds when using the split dataset strategy, although the OTU quality and processing speed depends on the database chosen when using the external database strategy. OptiFit provides a suitable option for users who require consistent OTU assignments at the same quality afforded byde novoclustering methods.ImportanceAdvancements in DNA sequencing technology have allowed researchers to affordably generate millions of sequence reads from microorganisms in diverse environments. Efficient and robust software tools are needed to assign microbial sequences into taxonomic groups for characterization and comparison of communities. The OptiClust algorithm produces high quality groups by comparing sequences to each other, but the assignments can change when new sequences are added to a dataset, making it difficult to compare different studies. Other approaches assign sequences to groups by comparing them to sequences in a reference database to produce consistent assignments, but the quality of the groups produced is reduced compared to OptiClust. We developed OptiFit, a new reference-based algorithm that produces consistent yet high quality assignments like OptiClust. OptiFit allows researchers to compare microbial communities across different studies or add new data to existing studies without sacrificing the quality of the group assignments.