Sequencing
Viral RNA was extracted using the ReliaPrep™ Cell Mini Kit (Promega)
using a protocol adapted for extraction of RNA from seasonal influenza
viruses (using qPCR positive specimens. First, 200 µl diluted specimen,
50 µl water and 250 µl freshly prepared buffer (BL+TG lysis buffer) were
mixed by vortexing. Next, 85 µl of isopropanol (100%, Roth) were added
and mixed by vortexing. All subsequent steps (including DNase digestion)
were performed according to manufacturer’s instructions. Finally, the
RNA was eluted in 60 μl of water. Next, multisegment reverse
transcription-PCR was performed according to Zhou et al. (10), using
viral 14 µl RNA and SuperScript™ III One-Step RT-PCR System with
Platinum™ Taq High Fidelity DNA Polymerase (Invitrogen).
One-Step RT-PCR products were purified using SPRIselect beads (Beckman
Coulter) according to the manufacturer’s instructions, with a ratio of
0.6. Then, DNA quantification was performed using Qubit dsDNA kit and a
Qubit Fluorometer (ThermoFisher).
NGS was performed using 1 ng purified One-Step RT-PCR product, Nextera
XT DNA Library Preparation Kit and subsequently the ISeq platform
(Illumina). Trimming, reference mapping and generation of consensus
sequences (threshold 90%) were done with Geneious software (11.1.5) and
influenza A(H1N1)pdm09 genome sequences (minority variants ≥10%) were
deposited in GISAID (www.GISAID.org).