known OTUs as described above, after which those that fail to match to a known OTU are then put through a de novo step.Table 1A comparison of OTU-picking strategiesStrategyProsConsData combination biasClosed-reference• Is extremely parallelizable• Is limited to finding diversity present in OTU reference• May show large bias if combining studies with differential representation in the reference• Computes reference assignments only once• Is highly unlikely to retain non-16S sequences• Supports and reads fragments from multiple loci• Gets the phylogeny and taxonomy for freeDe novo• Utilizes all of the sequences• Must hold all sequence data in memory• May generate spurious OTUs if combining studies with differential error profiles• Requires no OTU database• Is very complex to parallelize• Can group organisms distinct from anything seen before• Produces spurious OTUs without pre-filtering• May produce phylogenies sensitive to subtle differences in OTUs• Is infeasible if data are from multiple loci• Must redo OTU picking with all data being combinedOpen-reference• Leverages an OTU database but also utilizes sequences that do not match to that database• Produces spurious OTUs without pre-filtering• Shows less bias due to differential diversity representation than closed-reference• Is infeasible if data are from multiple loci• Is modestly parallelizable• Must redo OTU picking with
