design: Total DNA was extracted using theQIAamp DNA Stool Mini Kit (QIAGEN), following the manufacturer’sinstructions, except for the addition of a series ofmechanical disruption steps (11 x 45 s) using a FastPrepapparatus and Lysing Matrix B tubes (MP Biochemicals)(32).PCR amplification was carried out using two sets of primerstargeting the hypervariable regions (V) 1–3 (V123) and 4–6(V456) of the 16S ribosomal RNA gene. For the amplificationof the V123 region, a mixture of forward primerswas combined to ensure optimal coverage of bacterialphylogenetic diversity, as described previously(33). Theprimers were designed according to Hamady et al.(34):V123 forward primer 1, 50-CTATGCGCCTTGCCAGCCCGCTCAGTCAGAGTTTGATYMTGGCTCAG-30; V123 forwardprimer 2, 50-CTATGCGCCTTGCCAGCCCGCTCAGTCAGGGTTCGATTCTGGCTCAG-30; V123 forward primer3, 50-CTATGCGCCTTGCCAGCCCGCTCAGTCAGAGTTTGATCAGAGTTTGATCCTGGCTTAG-30; V123 forward primer 4,50-CTATGCGCCTTGCCAGCCCGCTCAGTCAGAATTTGATCTAGAATTTGATCTTGGTTCAG-30; V123 reverse primer,50-CGTATCGCCTCCCTCGCGCCATCAGNNNNNNNNGGTTACCGCGGCTGCTGGCAC-30; V456 forward primer, 50-CTATGCGCCTTGCCAGCCCGCTCAGGCCRRCACGAGCTGACGAC-30; and V456 reverse primer, CGTATCGCCTCCCTCGCGCCATCAGNNNNNNNNAGGCCAGCAGCCGCGGTAA-30 (wherethe adapter sequences for Roche 454 FLX Titanium sequencingare italicised, the linkers are underlined, NNNNNNNNsequences designate the sample-specific eight-base barcodesused to tag each PCR product, and bold sequences correspondto broadly conserved 16S ribosomal RNA gene regions). V123forward primers 1, 2, 3 and 4 were combined in 4:1:1:1 ratios.Amplifications were performed in 50 ml volumes with 2ml ofDNA extract, 50mM of deoxyribonucleotide triphosphate(dNTP), 200 nM of forward primers (mix of forward primersfor the V123 region), 200 nM of reverse primers, 1 x ExpandHigh Fidelity Reaction Buffer and 5 U/100 ml of Expand HighFidelityPLUS enzyme blend (Roche Applied Science). PCRconditions were as follows: 948C for 2 min followed bytwenty-five cycles of 948C for 30 s, 498C for 30 s, and 728Cfor 1 min, ending with a final step of 728C for 7 min. After purification,pooling in equimolar amounts, the PCR productswere sequenced using the 454 FLX Titanium technology(Microsynth AG).
library_name: Pool_R10_A.D301.D03.V123-Uni500R-linker_ACCACTAGGG.GGTTACCGCGGCTGCTGGCAC.sff.D301.D03.V123-Uni500R-linker_ACCACTAGGG.GGTTACCGCGGCTGCTGGCAC
library_construction_protocol: missing