title: Child with meningoencephalitis: untreated CSF
design: LIBRARY CONSTRUCTION For the TruSeq-adapted protocol, reverse transcription was performed using random nonamers attached to a 18 bp linker adapter (Greninger, et al., 2010, PloS ONE), followed by second-strand synthesis using Sequenase (Affymetrix, Santa Clara, CA) and 10-25 cycles of PCR amplification using high-fidelity KlenTaq LA DNA polymerase (Sigma-Aldrich, Carlsbad, CA). Linker adapter sequences were then cleaved using the Type IIs restriction endonuclease BpmI, followed by end-repair, A-tailing, truncated Illumina adapter ligation, and size selection using a 2% agarose gel or AMPure XP beads (New England Biolabs, Ipswitch, MA), targeting an insert size of 270–350 bp. Full-length Illumina adapters containing 6-bp barcodes were then added via an additional 17 cycles of PCR amplification. Libraries were validated using the Kapa qPCR kit (Kapa Biosystems) and Agilent Bioanalyzer (Agilent, Santa Clara, CA), and then sent to an outside company (Elim Biopharmaceuticals, Hayward, CA) for next-generation sequencing (NGS) on an Illumina MiSeq instrument. EXPERIMENTAL DESIGN A 14 year-old boy from Wisconsin with severe combined immunodeficiency presented 3 times to a medical facility over 4 months with fever and headache that progressed to hydrocephalus and status epilepticus necessitating a medically-induced coma. An exhaustive diagnostic workup for an infectious etiology including brain biopsy was unrevealing. Unbiased next-generation sequencing of the patient’s cerebrospinal fluid and serum was performed, revealing sequences corresponding to pathogenic Leptospira, confirmed by subsequent specific PCR and Sanger sequencing to be Leptospira santarosai. Conventional diagnostic assays for leptospirosis performed subsequently were all negative. The patient responded dramatically when treated with appropriate antibiotics for neuroleptospirosis. Nucleic acid for NGS was extracted from untreated and DNase-treated CSF (for virus enrichment) and DNase-treated serum using the Qiagen EZ1 robot (Qiagen, Carlsbad, CA). cDNA NGS libraries were constructed as described above, and 4 individually barcoded libraries corresponding to a (i) CSF sample without any pre-treatment (“untreated”), (ii) DNase-treated CSF sample, (iii) DNase-treated serum sample, and (4) control serum sample from another individual at the University of California, San Francisco (UCSF) hospital with acute febrile illness (a returning traveler from Australia, see Naccache, et al. 2013, submitted) were sequenced on an Illumina MiSeq instrument using 150/350 base pair (bp) paired-end (PE) sequencing. The Illumina MiSeq run yielded a total of 10,196,620 raw 150 / 350 bp paired-end reads. DATA ANALYSIS The run was analyzed using SURPI, a cloud-compatible bioinformatics pipeline for ultra-rapid pathogen identification from next-generation of clinical samples (Naccache, et al., 2013, submitted). First, only the read 1 (R1) data was analyzed using SURPI, followed by subsequent analysis of the combined R1 / R2 data. SURPI analysis was performed prior to debarcoding into individual samples. For SURPI analysis, either the R1 data was analyzed alone or R1 and R2 were combined into a single file and then computationally preprocessed. The preprocessing steps in SURPI consist of (1) quality trimming and removal of adapter sequences using cutadapt [please note that both Illumina adapters and a 18-linker adapter need to be removed; see Greninger, et al., PloS ONE, 2010], (2) removal of low-complexity sequences using the DUST algorithm in prinseq, and (3) read normalization for nucleotide SNAP alignment (Zaharia, et al., ArXiv 1111.5572) by cropping reads of length greater than 75 to 75 bp. Human reads were then removed (computational subtraction) by SNAP alignment against a human reference database. The sequences deposited here in SRA thus correspond to the full-length raw reads with human sequences removed ("human-subtracted") and thus represent only the nonhuman fraction of the NGS data. From a total of 10,196,620 raw PE reads (8,187,737 corresponding to CSF and serum samples from the patient with neuroleptospirosis from Leptospira santarosai), the number of reads remaining after human subtraction were as follows: (i) untreated CSF sample, 52,626 R1 and 93,187 R2 reads remaining out of 3,063,784 PE reads; (ii) DNase-treated CSF sample, 1,282,953 R1 and 1,295,036 R2 reads remaining out of 2,235,787 PE reads; (iii) DNase-treated serum sample, 2,806,178 R1 and 2,800,891 R2 reads remaining out of 2,888,166 PE reads; (iv) control serum sample, 16,670 R1 and 34,925 R2 reads remaining out of 2,008,883 PE reads.