title: HmmUFOtu: An HMM and phylogenetic placement-based tool for ultra-fast taxonomic assignment and OTU clustering of 16S rRNA gene sequencing data
design: In the last decade our understanding of host-associated and environmental microbiota has been dramatically improved by the joint advances in next-generation sequencing of target loci such as bacterial 16S microbiome sequencing, and bioinformatics tools that enable community analysis at a knowledge-independent resolution most often known as Operational Taxonomic Units (OTUs). Research employing these tools has revealed a multitude of significant roles bacteria play in human health and disease. The accuracy of such studies relies on properly assigning taxonomic annotations to high-throughput sequencing reads for subsequent community analysis. Present methods achieve this by first organizing reads into OTUs, then assigning a taxonomic assignment annotationtaxonomy to all reads in of an OTU based on the assignment of a representative read. Although straightforward in principle, present methods often rely on heuristics while constructing (or “picking”) OTUs to avoid computationally expensive algorithms, and ignore a the priori knowledge of microbial phylogeny to further reduce the computational timecomplexity. In this study, we present HmmUFOtu, a novel tool for processing bacterial 16S and other target-loci sequencing studies, which relies on rapid per-read phylogenetic placement followed by OTU organization picking and taxonomic assignment based on the phylogeny of known taxa. By reversing the traditional analysis order of OTU picking and taxonomic taxonomy assignmenttaxonomic assignment, this tool achieves high assignment accuracy, sensitivity, specificity and precision, even at species-level resolution. HmmUFOtu can perform taxonomic taxonomy assignmenttaxonomic assignment in a species-resolution reference tree with ~ 200,000 taxa nodes for 1 million 16S Next-Gen sequencing reads within 8 hours on a modest Linux workstation with 16 processors and 32 GB RAM. HmmUFOtu is written in C++98 and freely available at https://github.com/Grice-Lab/HmmUFOtu/
library_construction_protocol: The V4 region of the 16S rRNA gene was amplified using forward primer 27F and reverse primer 534R and the Accumprime kit. Primers contained unique error-correcting barcodes to facilitate multiplexing of samples. Due to the heterogeneous template mixture, PCR amplification was performed in duplicate and resulting products pooled to reduce amplification bias. PCR products were purified using Agencour AMPure kit, normalized, and then subject to Illumina MiSeq paired-end sequencing. See Gardner SE, et.al, Diabetes. 2013 62(3):923–30 for details