title: RT-PCR and amplicon sequencing of partial enterovirus VP1-coding segment
design: Viral genomes were extracted from 140 l of viral concentrate using the QIAamp Viral RNA Mini Kit (Qiagen) according manufacturers instructions. A RT-nested PCR method (Nix et al. 2006) was used to amplify a partial segment of VP1-coding segment [2,602 to 2,977 positions in the genome of PV1 Mahoney strain. GenBank accession number J02281], with primers AN32 to AN35 in the reverse transcription and primers 222-224 and AN88-AN8), in first and second PCR rounds respectively. For the second round, AN88 and AN89 primers (Nix et al. 2006) were modified by addition of Illumina Universal Adapter sequences at the 5ends, according to the protocol of library preparation for metagenomic sequencing in Illumina MiSeq platform (Illumina Inc) Sequencing libraries were prepared by Macrogen Next Generation Sequencing Service (South Korea). Samples were sequenced on Illumina MiSeq 2  300 bp, producing paired end reads. Libraries from 2011-2012 and 2017-2018 were sequenced in two independent runs.