metagenomic sequencing for polio viruses: acute flaccid paralysis
The complementary DNA product was produced using random hexamers. Barcodes were added to the DNA strands using  "NEBNext Ultra II End Repair/dA-Tailing Module (E7546) and NEBNext Quick Ligation Module. The barcoded fragments were pooled and cleaned using AMPure XP beads, Agencourt. AMII adapter ligation of the amplicon pools was performed using, NEBNext Quick Ligation Reaction Buffer (5X), AMII adapter mix and Quick T4 DNA Ligase 5. The pool was again cleaned using AMPure XP beads, Agencourt and Long fragment buffer wash and was then subjected to nanopore sequencing. The raw fastq sequence reads generated were basecalled and de-barcoded using guppy package and checked for sequence quality. The bad reads were trimmed using trimal and corrected reads were then de-novo assembled using canu.   The draft genomes were polished using medaka, homopolish and pilon package. The fasta sequence genomes produced were then annotated using genome detective tool available on https://www.genomedetective.com/app/typingtool/virus/. The final annotated fasta sequences have been submitted to NCBI repository.