title: Clinical metagenomic identification of Balamuthia mandrillaris encephalitis and assembly of the draft genome: the critical need for reference strain sequences
design: Metagenomic Sequencing of CSF and Brain Biopsy  Total nucleic acid was extracted from 200 ?L of CSF using the Qiagen EZ1 Viral kit. Half of the nucleic acid from CSF was treated with Turbo DNase (Ambion). Total RNA was extracted from 2 mm3 brain biopsy tissue using the Direct-zol RNA MiniPrep Kit (Zymo Research), followed by mRNA purification using the Oligotex mRNA Mini Kit (Qiagen). Total RNA from CSF and mRNA from brain biopsy was reverse-transcribed using random hexamers and randomly amplified.  The resulting double-stranded cDNA or extracted DNA from CSF (the fraction not treated with Turbo DNase) was used as input into Nextera XT, following the manufacturer's protocol except with reagent volumes cut in half for each step in the protocol.  After 14-18 cycles of PCR amplfiication, barcoded libraries were cleaned using Ampure XP beads, quantitated on the BioAnalyzer (Agilent), and run on the Illumina MiSeq (1 x 160 bp run).  Metagenomic NGS data were analyzed for pathogens via SURPI using NCBI nt/nr databases from June 2014.  A rapid taxonomic classification algorithm based on the lowest common ancestor was incorporated into SURPI, and used to assign viral, bacterial, and non-chordate eukaryotic NGS reads to the species, genus, or family level.  For the SNAP nucleotide aligner, an edit distance cutoff of 12 was used for viral reads, but adjusted to a more stringent edit distance of 6 for bacterial and non-chordate eukaryotic reads to increase specificity.