NTC-RNA-title
Separate DNA and RNA metagenomic libraries were constructed and sequenced from the patient's CSF sample according to a standardized operating procedure (SOP) in a Clinical Laboratory Improvement Amendments (CLIA)-certified microbiology laboratory as previously described (Monkolrattanothai, et al., 2017, JPIDS,  https://doi.org/10.1093/jpids/piw066; Schlaberg, et al., 2017, Arch Pathol Lab Med. 2017 Feb 7. doi: 10.5858/arpa.2016-0539-RA). For each sequencing run, the SOP includes running two external controls: (1) a negative “no-template” control (NTC) sample consisting of elution buffer, (2) and a positive control (PC) sample consisting of a mixture of 7 representative pathogens (CMV, HIV, Streptococcus agalactiae, Klebsiella pneumoniae, Cryptococcus neoformans, Aspergillus niger, and Toxoplasma gondii) spiked at concentrations 1-2 log above the estimated limits of detection. Each sample also contains two internal spiked bacteriophage controls for RNA and DNA corresponding to M2 phage and T1 phage, respectively. Metagenomic NGS data was analyzed for pathogens using SURPI+, a clinical version of the SURPI (“sequence-based ultra-rapid pathogen identification”) computational pipeline for automated detection of pathogen sequences from mNGS data (Naccache, et al., 2014, Genome Research). The March 2015 build of NCBI GenBank was used as the reference database for comparison. The pre-established criteria for pathogen identification and reporting were as follows: (1) for reporting of bacteria, fungi, and parasites, an RPM (reads per million) ratio of 10, defined as the RPMsample / RPMNTC for any given taxon (species, genus, or family); (2) for reporting of viruses, coverage at least 2 non-contiguous / non-overlapping gene regions of size greater than the read length (140 bp).  Viruses corresponding to non-pathogenic flora (e.g. anelloviruses) or contaminants found in the NTC are not reported.       Deposited into SRA are nonhuman reads corresponding to RNA and DNA libraries from the cerebrospinal fluid sample from the patient with St. Louis encephalitis virus infection ("case patient") and from the negative (NTC) and positive control (PC) samples used for the clinical metagenomic next-generation sequencing (mNGS) assay.