Metagenomics (MiSeq-Illumina) biomass aerobic granular sludge
The genespecific sequences used in this protocol target the 16S V3 and V4 region.  They are selected from the Klindworth et al. publication (Klindworth A, Pruesse E, Schweer T, Peplles J, Quast C, et al. (2013) Evaluation of general 16S ribosomal RNA gene PCR primers for classical and nextgeneration sequencingbased diversity studies. Nucleic Acids Res 41(1).) as the most promising bacterial primer pair. Illumina adapter overhang nucleotide sequences are added to the genespecific sequences. The full length primer sequences, using standard IUPAC nucleotide nomenclature, to follow the protocol targeting this region are: 16S Amplicon PCR   Forward Primer = 5' TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG 16S   Amplicon PCR Reverse Primer = 5' GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC   (in red is the sequence of the adapters).   The sequencing was done in Miseq - Illumina, 2x250 cycles