Gs flx titanium xlr70 sequencing

Barcoded primers Bact-515F (5′-GTGCCAGCMGCNGCGC-3′) and Bact-1061R (5′-CRRCACGAGCTGACGAC-3′) described by Klindworth et al. (2013) [46 (link)] were used for the initial amplification of the V4-V6 region of the 16S rRNA gene as previously described [3 (link)]. PCR reactions contained 2.5 U of Taq DNA Polymerase (Invitrogen, Cergy Pontoise, France), 5 µL of 5X buffer, 75 nmol MgCl₂, 1 µL of 10 mM dNTPs, 1 µL of each primer (50 µM) and 50 ng of DNA. Three PCR reactions were run for each sample as follows: 95 °C for 5 min, followed by 40 cycles at 95 °C for 45 s, 60 °C for 45 s, 72 °C for 45 s and a final extension at 72 °C for 5 min. PCR reactions from the same sample were pooled, purified using the QIAquick PCR purification kit (Qiagen, Courtaboeuf, France) and quantified using a Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) using the dsDNA HS Assay Kit (Life Technologies). To ensure equal representation of each sample in the sequencing run, each barcoded sample was standardized by calculating equimolar amounts (100 ng/sample) using the SequalPrep Normalization Plate Kit (Invitrogen) prior to pooling. Pooled samples of the 16S rRNA gene multiplexed amplicons were sequenced on a Roche 454 Genome Sequencer FLX Titanium instrument using the GS FLX Titanium XLR70 sequencing reagents and protocols (Beckman Coulter Genomics, Danvers, MA, USA).

Initial amplification of the V4-V6 region of the 16S rRNA gene was performed using barcoded primers Bact-515F (5′-GTGCCAGCMGCNGCGC-3′) and Bact-1061R (5′-CRRCACGAGCTGACGAC-3′) described by Klindworth et al.^{59 (link)} in which the barcode was an 11-bp sequence unique to each sample. PCR reactions contained 2.5 U of Taq DNA Polymerase (Invitrogen, Cergy Pontoise, France), 5 µl of 5X buffer, 75 nmol MgCl₂, 1 µl of 10 mM dNTPs, 1 µl of each primer (50 µM) and 50 ng of DNA. Three PCR reactions were run for each sample as follows: 95 °C for 5 min, followed by 40 cycles at 95 °C for 45 s, 60 °C for 45 s, 72 °C for 45 s and a final extension at 72 °C for 5 min. PCR reactions from the same sample were pooled, purified using the QIAquick PCR purification kit (Qiagen, Courtaboeuf, France) and quantified using a Qubit 2.0 Fluorometer (Invitrogen) using the dsDNA HS Assay Kit. To ensure equal representation of each sample in the sequencing run, each barcoded sample was standardized by calculating equimolar amounts (100 ng/sample) using the SequalPrep Normalization Plate Kit (Invitrogen) prior to pooling. Pooled samples of the 16S rDNA multiplexed amplicons were sequenced on a Roche 454 Genome Sequencer FLX Titanium instrument using the GS FLX Titanium XLR70 sequencing reagents and protocols (Beckman Coulter Genomics, Danvers, USA).