title: Human hand metagenome
design: For each sample, we amplified the 16S rRNA gene using a primer set similar to that described in Hamady et al. 2008 that was found to be well-suited for the phylogenetic analysis of pyrosequencing reads (Liu et al. 2008). The forward primer (5'-GCCTTGCCAGCCCGCTCAGTCAGAGTTTGATCCTGGCTCAG-3') contained the 454 Life Sciences primer B, the broadly conserved bacterial primer 27F, and a two-base linker sequence ('TC'). The reverse primer (5'-GCCTCCCTCGCGCCATCAGNNNNNNNNNNNNCATGCTGCCTCCCGTAGGAGT-3') contained the 454 Life Sciences primer A, the bacterial primer 338R, a 'CA' inserted as a linker between the barcode and the rRNA primer, and a unique 12-bp error-correcting Golay barcode used to tag each PCR product (designated by NNNNNNNNNNNN). PCR reactions consisted of 0.25 microliters (30 micromolar) of each forward and reverse primer, 3 microliters template DNA and 22.5 microliters Platinum PCR SuperMix (Invitrogen, Carlsbad, CA, USA).  Samples were initially denatured at 94 degrees C for 3 min, then amplified using 35 cycles of 94 degrees C for 45 sec, 50 degrees C for 30 sec, and 72 degrees C for 90 sec. A final extension of 10 min at 72 degrees C was added at the end of the program to ensure complete amplification of the target region. All samples were amplified in triplicate. Negative controls (both no-template and template from unused swabs) were included in all steps of the process in order to check for primer or sample DNA contamination. All aliquoting and diluting of  primers, as well as assembly of PCR reactions, was done in a PCR hood in which all surfaces and pipettors had been decontaminated with DNA Away (Molecular BioProducts, San Diego, CA, USA) and exposed to ultraviolet light for 30 min.