bacterial 16S rRNA gene MiSeq amplicon sequencing of groundwater microcosm with 13C starch: SIP light fraction
Illumina MiSeq amplicon sequencing targeting bacterial 16S rRNA genes was performed on all samples using the primer combinations Bakt_785R/Bakt_341F. Illumina MiSeq amplicon sequencing was carried out by LGC Genomics GmbH (Berlin, Germany). The PCRs included about 5 ng of DNA extract, 15 pmol of each forward primer 341F 5 -NNNNNNNNNNTCCTACGGGNGGCWGCAG and reverse primer 785R 5 - NNNNNNNNNNTGACTACHVGGGTATCTAAKCC in 20 uL volume of 1 x MyTaq buffer containing 1.5 units MyTaq DNA polymerase (Bioline) and 2  l of BioStabII PCR Enhancer (Sigma). For each sample, the forward and reverse primers had the same 10-nt barcode sequence. PCRs were carried out for 30 cycles using the following parameters: 2 min 96 C pre-denaturation; 96 C for 15 s, 50 C for 30 s, 70 C for 90 s. DNA concentration of amplicons of interest was determined by gel electrophosesis . About 20 ng amplicon DNA of each sample were pooled for up to 48 samples carrying different barcodes. If needed PCRs showing low yields were further amplified for 5 cycles. The amplicon pools were purified with one volume AMPure XP beads (Agencourt) to remove primer dimer and other small mispriming products, followed by an additional purification on MinElute columns (Qiagen). About 100 ng of each purified amplicon pool DNA was used to construct Illumina libraries using the Ovation Rapid DR Multiplex System 1-96 (NuGEN). Illumina libraries were pooled and size selected by preparative gel electrophoresis.