Pyrosequencing of AD grain type
Two universal primers Y1 (5’ – TGG CTC AGG ACG AAC GCT GGC GGC– 3’) (position 20-43 on 16S rRNA gene, Escherichia coli numbering) and Y2 (5’ – CCT ACT GCT GCC TCC CGT AGG AGT – 3’) (positions 361-338) (Young et al., 1991), were used to amplify by the PCR technique a 348-bp stretch of DNA embracing the V1 and V2 variable regions of the prokaryotic 16S rDNA. 454-adaptors were included in both forward (5’ – CGTATCGCCTCCCTCGCGCCATCAG– 3’) and reverse (5’ – CTATGCGCCTTGCCAGCCCGCTCAG – 3’) primers, followed by 10-bp sample specific barcode sequences (5’ – ATATCGCGAG – 3’, 5’ – CGTGTCTCTA– 3’, 5’ – CTCGCGTGTC – 3’, respectively).   For each sample a 50 µl PCR mix was prepared using a Taq-DNA polymerase master mix ( 75 mM Tris-HCl pH= 8.5, 20 mM (NH4)2SO4, 1.5 mM MgCl2, 0,1% Tween 20®, 0.2 mM of each dNTP, and 1.25 units Taq polymerase) (Ampliqon ApS, Skovlunde, Denmark), with 100 ng of extracted DNA and 0.2 mM of each primer. The PCR conditions used were 95°C for 5 min, 30 cycles of 94°C for 40 s, 55°C for 1 min and 72°C for 10 min.   Pyrosequencing    Amplicons were purified and DNA concentration and quality was measured using a Nanodrop spectrophotometer (Thermo Scientific; Wilmington, DE, USA). Equal amounts of the four samples were pooled to a total amount of 100 ng. Pooled DNA was subsequently amplified in PCR-mixture-oil emulsions and sequenced in different lanes of a PicoTiterPlate on a 454 Genome Sequencer 20 system (Roche, Basel, Switzerland).
Universidade Federal do Rio de Janeiro
AD