Following a July 2010 16S data freeze, data was downloaded from NCBI SRA projects SRP002395: Human Microbiome Project 16S rRNA Clinical Production Phase I, and SRP002012: Human Microbiome Project 454 Clinical Production Pilot. This dataset corresponds to over 5,700 samples and over 10,000 sequence preps. 16S variable region 3-5 (V35) was sequenced for the entire set of samples, and variable region 1-3 (V13) for a subset of samples.
The QIIME (Quantitative Insights Into Microbial Ecology) software package was used to process HMP 16S data using an OTU-binning strategy to which taxonomic classification is added.
Raw 16S sequence and metadata, available at HMR16S, were demultiplexed using QIIME. OTU picking was performed for the V1-3 and V3-5 region sequences using OTUPipe, which includes error correction, chimera checking through UCHIME, and clustering via UCLUST, and postprocessing by picking the optimal representative sequence centroid. Taxonomy was assigned using the RDP classifier version 2.2.
The resulting OTU tables were checked for mislabeling and contamination, as described in the SOP available below. Alpha and beta diversity for each sample and Procrustes analysis were established using QIIME with default parameters.
All QIIME output files are available here, for both the V1-3 and V3-5 variable regions, as well as Procrustes summary data. SOPs and custom scripts can be found below.
If you're interested in joint analysis of 16S and shotgun metagenomic datasets from the HMP, pairing up data from the same microbiome samples can initially seem tricky. The HMP Sample Flow Schematic indicates how these sample IDs are related experimentally, and provides tables joining 16S dataset "SN" and "PSN" identifiers with metagenomic dataset "SRS" identifiers.
Protocols and Tools