Comparing read recruitment, de novo, and insertion tree strategies for phylogenetic diversity computation. Comparing read recruitment, de novo, and insertion tree strategies for phylogenetic diversity computation. (A) Ideally, all short amplicon fragments (red) would have known full-length 16S sequences (black), which in turn would allow reconstruction of a phylogenetic tree. (B) In real-world experiments, only a minority of fragments have corresponding full-length 16S references. (C) The “read recruitment” strategy, also known as closed-reference OTU picking, assigns fragments to tips of a well-curated reference phylogeny, e.g., Greengenes, with a given sequence similarity threshold. Fragments of clades not covered in the reference are rejected. (D) In order to keep all fragments, the de novo strategy reconstructs the whole phylogeny based on the short fragments that do not carry as much evolutionary signal as full-length 16S sequences and thus often results in topologically very different trees. (E) The insertion tree strategy takes advantage of a well-curated phylogeny and extends it with fragments obtained by experiment. Only highly unrelated fragments are rejected, while the overall topology of the resulting phylogenetic trees remains stable. Stefan Janssen et al. mSystems 2018; doi:10.1128/mSystems.00021-18