EV-D68 KP-step4-dpi3 experiment
Nucleic acid was extracted from 50 µL of spinal cord lysate with the Direct-zol™ RNA Kit (Zymo Research, Irvine, CA ) or 50 µL virus culture using the automated Qiagen EZ1 robotic instrument (Qiagen, Valencia, CA), followed by treatment with Turbo DNase, then reverse transcribed to cDNA with random hexamer primers. NGS libraries were constructed by using the Nextera XT kit (Illumina, San Diego, CA), followed by 161-bp, single-end sequencing on an Illumina MiSeq instrument,Metagenomic next-generation sequencing data were analyzed using the SURPI ("sequence-based ultrarapid pathogen identification") computational pipeline (Naccache, et al., 2014, Genome Research, 24(7):1180-92), which identifies viruses, bacteria, fungi, and parasites by computational subtraction of human host sequences followed by nucleotide and translated nucleotide (protein) alignment of remaining reads to all microbial sequences present in the National Center for Biotechnology Information (NCBI) GenBank database (as of December 2015). Raw SURPI outputs consisting of aligned microbial reads were taxonomically classified to the appropriate rank (family, genus, or species) by use of an in-house developed LCA (lowest common ancestor) algorithm incorporating the SNAP nucleotide aligner (v0.15.4). Summary read count tables were generated for viruses, bacteria, and eukaryotic organisms including fungi and parasites outside of the phylum Chordata or kingdom Viridiplantae ("non-chordate eukaryotes"). Reads aligning to enterovirus D68 (EV-D68) were automatically mapped using SURPI to the US/MO/14-18947 genome (GenBank accession KM851225.1) for determination of percent genomic coverage achieved.