Primer design.

<p>Panel A shows the primer design for amplifying the V6–V9 16S rRNA gene region analyzed by cloning and Sanger sequencing and 454 pyrosequencing. Regular (non-encoded) 10 bp barcodes were added to the 5′end of the forward PCR primer. Panel B shows the primers used to amplify the 16S V6 region, analyzed by Illumina sequencing. Hamming barcodes (8 bp in length) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040425#pone.0040425-Hamady1" target="_blank">[32]</a> and padding sequences were introduced to the 5′ ends of the forward and reverse PCR primers, different for each strand, so that reads from each strand could be distinguished. Note: the reverse primer sequences shown are the actual oligonucleotide sequences used in PCR amplification (i.e., the reverse complement of the 5′–3′ target DNA sequence).