Sequence-Independent Single-Primer Amplification (SISPA)

The double stranded cDNA was subjected to SISPA, using anchor primers corresponding to the respective random anchored primers (Table 1). The 50 μl SISPA reaction consisted of 5.0 μl ds-cDNA, 2.5 μl anchor primer, 1.0 μl 25mM dNTPs, 0.5μl AmpliTaq Polymerase (Invitrogen), 10X PCR buffer (Invitrogen) and 1.25 μl 25mM MgCl₂ (Invitrogen). The reaction was carried out at 95°C for 3 min, followed by 35 cycles at 95°C for 20 sec; 54°C for 45 sec; 72°C for 1 min and a final extension at 72°C for 7 min.

SISPA reaction was followed by a magnetic bead purification step using AMPure XP magnetic beads (Beckman-Coulter) to purify amplicons as per the manufacturer’s instructions. The amplicons were eluted in nuclease-free water and quantified using Qubit fluorometer. Only samples with quantities higher than 100 μg were taken forward for library preparation to ensure inclusion and representation of viral species from mosquito samples.