wild red deer faecal microbiota
16S rRNA gene amplicon sequencing of red deer faecal samples collected from two Portuguese regions (Montesinho and Lousã).
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Six independent amplifications of 16S rRNA were performed for each one of the 24 faecal samples in a final volume of 25 μL containing 3mM of MgCl2, 1X green NZYTaq buffer, 0,2 mM dNTPs, 0,5 μg/μL bovine serum albumin (Calbiochem), 0,3 pmol/μL of each primer, 1U of NZYTaq DNA polymerase (NZYtech) and 5 ng of template DNA. The primer pair 27F and 1392R were used . The PCR conditions were 94 ºC for 4 min, followed by 30 cycles of 94 ºC for 30 s, 55 ºC for 30 s and 72 ºC for 90 s and a final elongation step at 72 ºC for 5 min. The six reactions of each sample were pooled together and DNA concentration was measured with Qubit fluorometer. Two pools corresponding to the 12 Lousã PCR and to the 12 Montesinho PCR were prepared in a total volume of 25 μL containing 530 ng of each reaction. The two samples were sent for 454 pyrosequencing at Biocant (Cantanhede, Portugal). The samples were prepared for 454 pyrosequencing by PCR amplification of the V3V4 hypervariable region with fusion primers containing the Roche-454 A and B Titanium sequencing adapters, an eight-base barcode sequence in fusion primer A, the forward primer 5’– ACTCCTACGGGAGGCAG-3’ and the reverse primer 5’– TACNVRRGTHTCTAATYC -3’ . PCR reactions were amplified in 40 μL reactions with Advantage Taq (Clontech) using 0.2 μM of each primer, 0.2 mM dNTPs, 1X polymerase mix and 6% DMSO. The PCR conditions were 94oC for 4 min, followed by 30 cycles of 94 ºC for 30 s, 44 ºC for 45 s and 68 ºC for 60 s and a final elongation step at 68 ºC for 10 min. The amplicons were quantified by fluorimetry with PicoGreen (Invitrogen, CA, USA), pooled at equimolar concentrations and sequenced in the A direction with GS 454 FLX Titanium chemistry, according to manufacturer’s instructions (Roche, 454 Life Sciences, Brandford, CT, USA). The raw pyrosequencing reads (fasta files) were processed using an automatic pipeline implemented at Biocant. In a first step, sequencing reads were assigned to the appropriated samples based on the respective barcode. Then, reads were quality filtered to minimize the effects of random sequencing errors, by elimination of sequence reads with <100 bp and sequences that contained more than two undetermined nucleotides (N). Sequences were additionally cut for the reverse primer if present.