title: MetageMetagenomic libraries used in ZIKV genome recovery: Bahia03_XGen
design: RNA metagenomic libraries were constructed from the patient's serum                 sample as previously described (Greninger, et al., 2015, Genome Medicine). Total                 nucleic acid was extracted using the QIAamp Viral RNA mini kit (Qiagen, Valencia                 CA). 20ul of extract was treated with Turbo DNase (Ambion, Waltham MA) and Baseline                 Zero DNase (Epicentre Bioscience) followed by cleanup using the RNA Clean               Concentrator kit (Zymo Research). Eluted RNA was reverse transcribed to cDNA using                 SuperScript III Reverse Transcriptase (Invitrogen) mediated by random hexamers, and                 second strand synthesis was conducted using SequenaseS Version 2.0 DNA Polymerase                 (Affymetrix) for 10 minutes at 37C following denaturation. Double stranded cDNA was                 cleaned up using the DNA Clean and Concentrator kit (Zymo Research) and NGS                 libraries were generated form the entirety of the eluate utilizing the Nextera XT                 DNA Library Prep Kit (Illumina, San Diego CA). The Nextera XT library was cleaned                 using Ampure XP beads (Beckman-Coulter). Dual-indexed, barcoded NGS libraries were                 quantitated on the Qubit 3.0 fluoremeter, mixed equally by concentration and                 quantified for size and concentration on the BioAnalyzer (Agilent, Santa Clara CA)                 using the high sensitivity dsDNA kit. Libraries were run on the Illumina HiSeq (1 x                 160 bp runs and 250 PE runs). Metagenomic NGS data was analyzed for pathogens using                 automated SURPI ("sequence-based ultra-rapid pathogen identification") computational                 pipeline and the updated National Center for Biotechnology Information (NCBI) nt                 database as of March 2015 (Naccache, et al., 2014, Genome                 Research).