Human nasopharyngeal swab, Illumina sequencing
A nasopharyngeal swab, randomly selected from samples submitted to the Laboratory of Virology positive to 2009  pandemic influenza A H1N1 with conventional real time PCR, was analyzed (influenza H1N1pdm09 M gene load: 3.3x10 7  cp/ml). The amplification products, generated with a prior semi-random RT-PCR, after purification (AMPure beads)  and quantification (TBS 380 Fluorometer) were split in two aliquots to be sequenced with two platforms: Roche 454 GS  FLX+ and Illumina MiSeq.  The RNA was extracted by using a QIAmp viral RNA minikit (Qiagen) and eluted in 60 μl of RNase-free water. Reverse  transcription (RT) was performed with random tagged primer as previously described. More in details, 10 μl of the  extracted RNA was mixed with primer FR26RV-N (5’GCCGGAGCTCTGCAGATATCNNNNNN3’) at 10 μM, incubated  at 65°C for 5 min and chilled on ice. A mix containing 4 μl of 5×First-Strand buffer (Invitrogen), 2 μl of DTT (100 mM),  1μl dNTPs (10 mM), 0.2 μl (8U) of recombinant RNase inhibitor (Promega), and 0.5 μl (100U) of SuperScript III reverse  transcriptase (Invitrogen) was added, and then incubated at 25°C for 10 min and 42°C for 50 min. After the denaturation  step (94°C for 3 min and chilling on ice), 2.5U of 3′-5′ exo- Klenow DNA polymerase (New England Biolabs) was added.  The reaction was incubated at 37°C for 1 h, followed by 75°C for 10 min as enzyme inactivation step.  Five μl of cDNA from each sample were then used as a template for the PCR amplification, that was performed with  High Fidelity Taq Gold DNA polymerase (Applied Biosystems) with the FR20RV primer  (5’GCCGGAGCTCTGCAGATATC3’); cycling conditions were: 10 min at 94°C, 40 cycles of amplification (94°C for 1  min, 65°C for 1 min, and 72°C for 2 min).
Influenza metagenomic sample, Illumina sequencing